Naturally Occurring Bioactive Compounds, Volume 3 (Advances in Phytomedicine)

Advances in Phytomedicine Series, Volume Three Editors: Mahendra Rai and Marıá Cecilia Carpinella Naturally Occurring...

Author: Mahendra Rai | Maria Cecilia Carpinella

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Advances in Phytomedicine Series, Volume Three Editors: Mahendra Rai and Marıá Cecilia Carpinella

Naturally Occurring Bioactive Compounds

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Advances in Phytomedicine 3

Naturally Occurring Bioactive Compounds Mahendra Rai Department of Biotechnology, SGB Amravati University, Amravati, Maharashtra, India

Marıá Cecilia Carpinella Laboratoria Quı´mica Fina y Productos Naturales, Facultad de Ciencias Quı´micas, Universidad Cato´lica de Co´rdoba, Co´rdoba, Argentina

Amsterdam – Boston – Heidelberg – London – New York – Oxford – Paris San Diego – San Francisco – Singapore – Sydney – Tokyo iii

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK First edition 2006 Copyright r 2006 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN-13: 978-0-444-52241-2 ISBN-10: 0-444-52241-7 ISSN: 1572-557X (series)

For information on all Elsevier publications visit our website at books.elsevier.com

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Contents Preface to the Series

vii

Preface

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1. Natural compounds as antioxidant and molting inhibitors can play a role as a model for search of new botanical pesticides Carlos L. Ce´spedes A., J. Guillermo Avila, J. Camilo Marin, Mariana Domıńguez L., Patricio Torres and Eduardo Aranda

1

2. Pesticides based on plant essential oils: from traditional practice to commercialization Murray B. Isman and Cristina M. Machial

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3. Natural substrates and inhibitors of multidrug resistant pumps (MDRs) redefine the plant antimicrobials George P. Tegos

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4. New concept to search for alternate insect control agents from plants Isao Kubo

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5. Role of Melia azedarach L. (Meliaceae) for the control of insects and acari: present status and future prospects Marıá C. Carpinella, Marıá T. Defago´, Graciela Valladares and Sara M. Palacios

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6. Bioactivity of fabaceous plants against food-borne and plant pathogens: potentials and limitations Deepak Acharya, Aniket Gade and Mahendra Rai

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7. Screening of plants against fungi affecting crops and stored foods Olı´via C. Matos and Caˆndido P. Ricardo

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8. Opportunities and potentials of botanical extracts and products for management of insect pests in cruciferous vegetables Tong-Xian Liu, Han-Hong Xu and Wan-Chun Luo

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9. The potential for using neem (Azadirachta indica A. Juss) extracts for pine weevil management in temperate forestry Jonathan R.M. Thacker, Wendy J. Bryan, Robin H.C. Strang and Stuart Heritage

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10. Plant allelochemicals in thrips control strategies Elisabeth H. Koschier

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11. Importance of plant secondary metabolites for protection against insects and microbial infections Michael Wink

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12. Naturally occurring house dust mites control agents: development and commercialization Young-Joon Ahn, Soon-Il Kim, Hyun-Kyung Kim and Jun-Hyung Tak

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13. The search for plant-derived compounds with antifeedant activity Monique S.J. Simmonds

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14. An overview of the antimicrobial properties of Mexican medicinal plants Diana Jasso de Rodrı´guez, Jose´ Luis Angulo-Sańchez and Francisco Daniel Hernańdez-Castillo

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15. Promissory botanical repellents/deterrents for managing two key tropical insect pests, the whitefly Bemisia tabaci and the mahogany shootborer Hypsipyla grandella Luko Hilje and Gerardo A. Mora

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16. Naturally occurring anti-insect proteins: current status and future aspects Tzi Bun Ng 405 17. Antifungal natural products: assays and applications Doris Engelmeier and Franz Hadacek

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Contributors

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Subject Index

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Preface to the Series The systematic study of herbal medicinal products and the investigation of the biologically active principles of phytomedicines, including their clinical applications, standardization, quality control, mode of action and potential drug interactions have emerged as one of the most exciting developments in modern therapeutics and medicine. Studies in phytomedicine have moved from purely descriptive analytical studies to conceptual inquiries on the pharmacodynamic advantages and limitations of plant medicines for the treatment of moderate or moderately severe diseases and prevention. Healthcare practitioners and medical scientists have come to accept herbal medicinal products as drugs that are different from the pharmacologically active molecules that they may contain. Several comparative clinical studies have been published to show that these plant medicines could have full therapeutic equivalence with chemotherapeutic agents, while retaining the simultaneous advantage of being devoid of serious adverse effects. Developments in molecular biology and information technology have now made it possible for us to begin to understand the mechanism of action of many herbal drugs and the associated phytomedicines, which differ in many respects from that of synthetic drugs or single chemical entities. Herbal medicinal products are now generally available in both industrialized countries and traditional societies. With the current lack of standardization and regulation of herbal products, it is important to develop common criteria for judging safety and efficacy of phytotherapeutic agents. This ‘new’ science demands different approaches to the classical methods of drug analysis, dosage formulations, manufacturing and claims substantiation. The therapeutic response observed with most herbs and phytomedicines are often not fully explainable using the currently available methods. Their activity usually characterized as polyvalent and interpreted as an aggregate or additive outcome of several constituents in the plant medicines are subjects of intense pharmacological studies. In most cases, a rationale does not even exist for the observed pharmacodynamic effects of very low doses of phytomedicines after prolonged or long-term application. The public press is replete with lay information and claims about the use of herbal remedies, however, there is scarcity of scientifically accurate reviews and guides on the efficacy and safety of plant medicines. The time therefore seemed ripe to broaden the communication on the use and benefits of phytomedicines as safe and useful natural health care products aimed at the health professionals and scientists. It is also important to provide a broader dissemination of the extant scientific literature on phytomedicines, to enable the conventional medical community to fully appreciate the fact that plant extracts specifically, and natural products generally, offer valuable and needed benefits in the treatment and prevention of diseases, especially for conditions where there is no effective or generally acceptable drugs. Although there are many papers published yearly on the use and analysis of plants as sources of biologically active molecules and some published materials on the use of plants as medicinal substances, volumes specifically addressing the needs of vii

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Preface to the Series

scientists and clinicians in the use of herbal products and standardized extracts as medicinal agents are far-in-between. We considered it therefore appropriate to fill this gap by producing an entirely new multi-volume series on the sourcing, selection, standardization, safety and clinical application of herbal medicinal products. As the name of the series implies, emphasis will be on those herbal medicinal products that are well characterized, standardized and substantiated as phytomedicines. The series will also provide timely reviews on the industrial production, regulatory and policy issues related to the use of phytomedicines. Although the literature in this field is evolving so rapidly that books on the subject become obsolete soon after they leave the press, the volumes in this series will aim at capturing the fundamental framework of each topic while remaining thoroughly up-to-date and comprehensive in scope. The aim of this series is to present to the scientists and clinicians the state of current knowledge in various fields of phytomedicine research, development and use. The approach is to provide the historical background to each topic, discuss methodological issues and illustrate current trends with case studies and critical examples, and when ever possible, the authors will indicate those plant medicines that are available for immediate use in clinical settings. The series is not intended to serve or replace the many excellent journals in this field but it will rather attempt to distill information from primary references and introduce elements of medicinal plants research and development that are in transition from speculative knowledge to standard practice. Advances in Phytomedicine is also not meant to be a textbook of the various topics covered in the series. It will, however, provide a guide to specialized articles and books on the topics that are relevant to scientific research and development of plant medicines, as well as information on the regulatory issues, clinical trials and application of phytomedicines for healthcare. Advances in Phytomedicine will therefore serve as a platform for reviewing recent developments in the use of herbal medicinal products. The coverage will include reviews of studies and use of all plant medicines, phytotherapeutic agents, nutraceuticals, plant cosmetics and therapeutically important molecules derived from these plant medicines. The first volume in this series has been devoted to exploring the ethnomedical approaches to drug discovery. It is indeed a very important starting point in addressing the relationship between plants and human health. Subsequent volumes will deal with other aspects of the use of herbal medicinal products. Selection of subjects will be through consultations with experts in the various fields of interest. We shall be guided by the principles of the so-called 6S, that is herb selection, sourcing, structure, standardization, safety and substantiation. Acknowledged experts and authorities in the various aspects of phytomedicine will be invited to assemble and edit specific volumes in the series. The series is the outcome of extensive consultations among several biomedical scientists and clinicians who participated in the workshops and conferences organized by the Bioresources Development and Conservation Program (BDCP) on related subjects. I am immensely grateful to these colleagues for their support in developing the original concept. Many thanks go also to Ms. Kim Briggs and Ms. Joke Zwetsloot of Elsevier Science for their suggestions and help in producing this series. I am indebted to my colleagues at BDCP and the International Centre for Ethnomedicine and Drug Development for their contribution; and to my wife, Kate for her love and support. I acknowledge the International Cooperative Biodiversity

Preface to the Series

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Group (ICBG) of the Fogarty International Centre, United States National Institutes of Health for providing financial support to my research group at the Walter Reed Army Institute of Research and BDCP. Maurice M. Iwu M.Pharm., Ph.D. Se´ries Editor


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Preface There has been a long history of success in discovering drugs from natural sources, particularly in tropical countries like Japan, China, India and Nepal. Unfortunately, natural products have fallen out of favour in current high-throughput screening. However, the diversity of three-dimensional shapes of natural molecules still surpasses that from synthetic compounds, and this ensures that natural products will continue to be important for drug discovery. Owing to the excessive use of the chemicals, the environment has been polluted. Consequently, there is a pressing need of search for new and natural chemicals for the sustainability of the environment. The plant-based drugs provide the natural and harmless system for the cure of various diseases. Majority of the drugs are prepared from the plants or natural products. There are a number of new and emerging diseases and new spectrum of microbes which has warranted the scientists to go for new natural drugs. Since time immemorial, herbal drugs have been in use all over the world in general and tropical countries like Japan, China and India, also Mexico and South Africa, in particular. Natural dyes and colours are health-friendly and responsible for maintaining the sanctity of the environment. The medicinal use of plant parts and extracts has been traditionally practiced since ancient times. However, a scientific approach to understand the relation between active chemicals and cure of diseases is recent. The use of plants in medicine has evolved because, from the biochemical point of view, they produce different chemical compounds against bacteria and fungi, the number of chemical structures is large and only a few are well known. Secondary metabolites obtained from plants have diverse functions into the plant such as defensive and protective process. The phytochemical studies are biodirected with an aim to find botanical origin biopesticides. Diterpenes, limonoids, triterpenes, sesquiterpene lactones, coumarins and flavonoids from Agavaceae, Asteraceae, Cactaceae, Celastraceae, Meliaceae and Zygophyllaceae families and also some chemical derivatives from them are important. These compounds possess antioxidant, antifungal, insect growth regulator (IGR) or insecticidal activities. The acetylated and carbonyl a-b-unsaturated derivatives showed mainly a low activity, while those that contain hydroxyl, carbonyl and oxy groups are more active as well as antifungal, IGR or insecticides. The pests and diseases of plants as well as animals have developed resistance as a result of which the existing antibiotics do not give the expected results. Therefore, there is a great need to develop the natural product-based drugs. The book will not only describe the effects of natural extracts and/or their isolated compounds, but also an update of its use and commercialisation. In some cases, the results related to the effects are new or they are an update of known data. The book will also help to make the use of effective and environmentally safe botanical pesticides, a new weapon to control these negative pests and diseases, which means a considerable alternative to the dangerous and excessive use of synxi

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thetic pesticides. Although there are several books on natural drugs, the present book covers multiple aspects of natural products, which is the need of the hour. We are indebted to Professor R.C. Rajak, director of Basic Sciences, Bundelkhand University, Jhansi, Uttar Pradesh; and to Professor S.C. Agrawal, head, Department of Applied Microbiology and Biotechnology, Dr. H.S. Gaur University, Sagar, Madhya Pradesh, who graciously offered suggestions for this book. We thank all the contributors for submitting their valuable manuscripts. Thanks are also due to my colleagues – Drs. Anita Patil, N.J. Chikhale, P.A. Wadegaonkar, P.V. Thakare, Aniket Gade and S.D. Kove. MKR thanks Deepak Acharya and Ravindra Ade, who checked manuscripts and helped in type-setting. Our special thanks to Drs. Susanna Zacchino, Donatella Mares, Michael Wink, George Tegos, N.P. Manandhar and Shubhangi Ingole, who went through the chapters and offered helpful suggestions. We are grateful to Dr. Kamal Singh, Vice Chancellor, SGB Amravati University, Amravati, for inspiration. Finally, and most importantly, MKR thanks Shivangi, Shivani and Aditya for their unconditional love, support and patience during the editing process. Mahendra Rai, Maria Cecilia Carpinella

Rai and Carpinella (eds.) Naturally Occurring Bioactive Compounds r 2006 Elsevier B.V. All rights reserved.

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CHAPTER 1

Natural compounds as antioxidant and molting inhibitors can play a role as a model for search of new botanical pesticides CARLOS L CE´SPEDES A, J GUILLERMO AVILA, J CAMILO MARIN, MARIANA DOMIŃGUEZ L, PATRICIO TORRES, EDUARDO ARANDA

Introduction Nowadays, pesticides of synthetic origin have been widely used, producing a strong impact on the environment with the emergence of resistant strains of microbes and insects to these types of compounds. New plant protection chemicals are needed for modern pest management due to insect resistance and ecological disorders associated with numerous currently used pesticides (Castillo et al., 1998). One area of investigation that is not so much an innovation as it is a return to an old approach with new technology is the characterization of secondary plant products (Berenbaum, 1989, 2002), and this can help in plant defense through new biotechnological approaches like metabolomic, genomic, and proteomic (Berenbaum, 1995, 2002; Kessler and Baldwin, 2002; Sumner et al., 2003). Most of them have potent effects on insect pests, low mammalian toxicity, lack of neurotoxic activity, low persistence in the environment, and biodegradability (Jacobson, 1989; Singh et al., 1997). Thus, organic molecules of botanical origin may offer a safe source of compounds for pest management, being environmentally friendly, and an excellent alternative to persistent synthetic insecticides (Berenbaum, 1989; Miyazawa et al., 1997; Castillo et al., 1998; Crowley et al., 1998). Plants in general produce a great variety of secondary metabolites that do not have apparent function in physiological or biochemical processes; these compounds (or allelochemicals) are important in mediating interactions between plants and their biotic environment (Berenbaum, 1989, 1991, 1995, 2002; Kessler and Baldwin, 2002). There is a widespread effort to find new pesticides, and currently it is focused on natural compounds such as flavonoids, coumarins, terpenoids, and phenolics from diverse botanical families from arid and semi-arid lands of Mexico and Americas. Additional experimental work has been carried out with natural products, which are potential models for defensive substances against insect and fungal predators (Kubo et al., 1994, 1995; Crombie, 1999; Kubo et al., 2000) and as enzyme inhibitors,

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Naturally occurring bioactive compounds

tyrosinase or acetylcholinesterase (AChE) for instance (Kubo, 1997; Keane and Ryan, 1999; Ortego et al., 1999; Kubo et al., 2000; Ce´spedes et al., 2001a, 2001b; Kubo et al., 2003a, 2003b). The increasing interest in the possible application of secondary metabolites for pest management has directed the investigation towards search for new sources of biologically active natural products, with new mode, sites, and mechanisms of action, selectivity, and specific action (Jacobson, 1989; Gonza´lezColoma et al., 1997; Singh et al., 1997; Conner et al., 2000; Eisner et al., 2000; Eisner and Meinwald, 1995); these characteristics may enhance their value as commercial pesticides (Gonza´lez et al., 1992; Isman et al., 1995, 1996; Valladares et al., 1997; Gonza´lez and Este´vez-Braun, 1998; Gonza´lez et al., 2000). Recent studies have demonstrated that many plant species produce and accumulate a large variety of secondary metabolites that provide defense against insect predators (Berenbaum, 1989; Guella et al., 1996; Marvier, 1996; Berenbaum, 2002). Among several efforts to find new pesticides, current research is focused on limonoids from the Meliaceae family due to their potent effects on insect pests and their low toxicity to non-target organisms (Koul and Isman, 1992; Kumar and Parmar, 1996; Singh et al., 1997). Some examples are Azadirachta indica (Meliaceae) and Derris elliptica (Fabaceae), which produce very well known insecticides azadirachtin and rotenone, respectively (Gomes et al., 1981; Kraus et al., 1993, 1995). The main characteristics that account for the successful use of these secondary metabolites as natural insecticides are mentioned above, which make them lesser aggressive to the environment than the synthetic ones (Camps, 1988; Berenbaum, 1989; Castillo et al., 1998). Although the members of the family Meliaceae are widely distributed in the world, only Melia, Toona, Cedrela, and Swietenia have been studied (Arnason et al., 1987; Champagne et al., 1992; Kubo, 1992; Arnason et al., 1993; Kraus et al., 1993; Kubo, 1993; Govindachari et al., 1995; Chan and Taylor, 1966; Ce´spedes et al., 2000), and have afforded some limonoids such as azadirachtin, gedunin, toosendanin, cedrelanolide, mexicanolide, odoratol, anthothecol, nomilin, bussein, entandrophragmin, among others. Azadirachtin is the best known example of these limonoids (Champagne et al., 1989; Ramji et al., 1996). This compound and their analogues are potent insect antifeedant and ecdysis inhibitors (Govindachari et al., 1995; Kraus, 1995). However, the structural complexity of this compound precludes its synthesis on a commercial scale (Isman et al., 1996). These facts have led us to search for new simple secondary metabolites with insecticidal activity from other families including plants of Agavaceae, Asteraceae, and Meliaceae, such as Yucca spp., Parthenium spp., Roldana spp., Tagetes spp., and Cedrela spp., respectively, specially from tropical and subtropical lands of Mexico. As mentioned earlier, tyrosinase also known as polyphenol oxidase (PPO) (Meyer, 1987) is a copper containing enzyme widely distributed in microorganisms, animals, and plants. It catalyzes two distinct reactions of melanin synthesis (Robb, 1984), the hydroxylation of a monophenol (monophenolase activity) and the conversion of an o-diphenol to the corresponding o-quinone (diphenolase activity). Tyrosinase is responsible for browning in plants and is considered to be deleterious to the color quality of plant-derived foods and beverages. In addition, sclerotization and molting regulation processes in insects show that tyrosinase is one of the key enzymes in the insect metamorphoses process (Andersen, 1990).

Natural compounds as antioxidant and molting inhibitors

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AChE, which is the enzyme contained in nerve tissues, plays an exceedingly important role in the transmission of a nerve impulse. The free acetylcholine in the inactive form bound to proteins accumulates in the ending of a nerve in vesicles. The consumed acetylcholine is constantly replenished by its synthesis (by the acetylation of choline). All these processes are occurring when an impulse is transmitted through a cholinergic synapse. Thus, the process of synaptic transmission is an involved biochemical cycle of acetylcholine exchange. AChE has a key role in this cycle because the inhibition of activity leads to the accumulation of free acetylcholine in the synaptic cleft, producing the disruption of nerve impulses, then convulsive activity of the muscles can be transformed into paralysis, and other features of selfpoisoning by surplus acetylcholine appear. It occurs when AChE is inhibited by some terpenoids (Ryan and Byrne, 1988; Miyazawa et al., 1997). On other hand, modifications to AChE can confer resistance to insecticides (Fournier et al., 1992). In addition to many chemicals (flavonoids, stilbenoids, phenylpropanoids, and phenolics, among others) that show tyrosinase inhibitory activity, these compounds also show a strong antioxidant (AOX) activity against diverse reagents as model of AOX measurements (i.e., DPPH, ABTS, TROLOX, TRAP, ORAC, etc.). Mainly the activity is due to the presence of diverse moieties of the chemical structure of the molecules, for instance, orcinol or catechol groups, or hydroxyl bonded to an aromatic system (Gallic acid, Gallates in general, resveratrol, quercetin, etc.); in these cases, it is possible to correlate the AOX activity with tyrosinase inhibition and insect growth regulatory (IGR) activity. All these data are important for allelopathic, antifungal, IGR, and insecticidal studies, and are being accepted as direct or indirect measures of different physiological processes affected by the assayed chemicals (Baldwin et al., 2001; Schultz, 2002; Kubo et al., 2003a, 2003b; Torres et al., 2003). Some investigations on sites and mechanism of action of insecticidal or IGR report that different phenolic compounds are enzymatic and metabolic inhibitors (Klocke and Kubo, 1982; Kubo and Klocke, 1986; Kubo et al., 1994, 1995; Hammond and Kubo, 1999; Kubo and Kinst-Hori, 1999a, 1999b; Kubo et al., 2000; Shimizu et al., 2000; Calderoń et al., 2001; Panzuto et al., 2002; Kubo et al., 2003a, 2003b). In addition, many of these compounds are polyphenolic secondary compounds that are ubiquitous in angiosperms and that have antifeedant effects on phytophagous insects (Feeny, 1968, 1976; Rhoades and Cates, 1976; Swain, 1979; Champagne et al., 1989, 1992; Simmonds, 2003). It has been assumed that phenols bind to proteins, acting as nutritional protein precipitating agents, thus reducing their digestibility (Feeny, 1976; Rhoades, 1979). We have previously demonstrated that diverse secondary metabolites have different sites of action and different molecular targets when they interact with enzymes and metamorphosis processes (Ce´spedes et al., 1999, 2000; Calderoń et al., 2001; Ce´spedes et al., 2001a, 2001b; Ce´spedes et al., 2002; Torres et al., 2003; Ce´spedes et al., 2004). Our field observations indicate that these botanical species from arid and semiarid lands possess a strong resistance to the insect attack. The aim of this work is to correlate this phenomenon between the phytochemical composition with the inhibitory behavior on growth and development of Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae). The role of these compounds as ‘‘chemical messengers’’ has proven important to our understanding of many ecological problems and has led to the development of ‘‘chemical ecology’’ (Seigler, 1997).

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Aspects such as insecticide and growth regulatory activities, rate of development, pupation time, adult emergence, and deformity were evaluated and compared with those of gallic acid 10, gedunin 19, toosendanin 21 (Chen et al., 1995), anisic acid 38, and Cedrela-MeOH extract known growth inhibitors of S. frugiperda and tyrosinase, respectively (Ce´spedes et al., 2000; Calderoń et al., 2001; Ce´spedes et al., 2001a, 2001b; Kubo et al., 2003a, 2003b). S. frugiperda is a very common insect pest in North- and South-American crops. These bioassays were used as model systems of insect pests. Our data also indicate that it is possible to correlate some AOX activities (i.e., crocin, 2,2-diphenyl-1-pycrilhydrazyl (DPPH)) with growth and development of fungus and insects; these data are important for allelopathic and chemical ecology studies (Ce´spedes et al., 2002). In addition to gedunin 19, toosendanin 21 (Figure 3), parthenolide 1, santamarin 2, 20 ,40 ,60 -trihidroxy acetophenone 3, rutin 4, and (7)-naringenin 5 were used as natural pattern substances and internal standard, together with THQ 6, BHA 7, quercetin 8, caffeic acid 9, gallic acid 10, and tocopherol 11 (Figure 1) in tests concerned with the reduction of DPPH and other AOX measurements. In the present chapter, we present a review of extracts and bioactive compounds isolated by our group from selected endemic botanical species, and their AOX and biological activities. The chapter includes antifeedant activities shown by assayed compounds, i.e., insecticidal or IGR activities and their activities on some enzymes.

Insecticidal or IGR (insect growth regulator) activity Agavaceae Yucca periculosa F. Baker, known commonly as ‘‘palmitos’’ or ‘‘izote’’ is a tree, endemic to Mexico, that grows in the semi-arid regions of Tehuacan–Cuicatlan Valley, Puebla–Oaxaca States. In the wild, these plants survive under different environmental stress conditions (Casas et al., 2001), with a lifespan of up to 100 years or so. Our field observations indicate that this specie has a strong resistance to insect attack. From methanol extract were isolated 4,40 -dihydroxstilbene 12, resveratrol 13, and 3,30 ,5,50 -tetrahydroxy-4-methoxystilbene 14 (Figure 2). The most active compound was stilbene 14 which showed the highest larval mortality together with MeOH extract of Y. periculosa (Me-Yuc), producing 100% of larval mortality at concentrations greater than 25.0 and 50 ppm, respectively, being noteworthy insecticidal. Thus, compound 14 and the Me-Yuc extract showed relative growth index (RGI) values of 0.25 and 0.45 at 10 and 15 ppm, respectively (Table 1). In addition, this compound was more active than gedunin and the MeOHCedrela extract used as positive control. The LD50 values for 14 and Me-Yuc extract were 5.4 and 7.18 ppm, respectively. In addition, compounds 12, 13, 14, gedunin 19, and the Me-Yuc and Me-Ced extracts inhibited each larval stage and diminished considerably the percentage of larvae that reached pupation; 14 and Me-Yuc showed the highest larval growth inhibitory activity, significant delay in pupation, and reduction of the percentage of emergence (Torres et al., 2003).


5

OH

O HO

OH

CH 2

O O

CH 2

H O

O

(1)

OH

O

(2)

(3)

OH OH HO

O OH O

HO

OH

O

O

CH 3 O

O OH

OH

O

OH

OH

OH

OH

O

OH

(5)

(4)

OH OH

O HO

HO

OH

O OH

HO

OH

OH O

OCH 3

(6)

(7)

OH

HO

O

HO

O

(8)

O HO

OH

HO

OH HO

(9)

(10)

CH 3 HO H H3C

O CH 3

CH 3

H

CH3

CH 3 CH 3

CH 3

(11)

Fig. 1. Chemical structures of pattern and standard compounds used for antioxidant and bioassays.

In order to establish correlation between IGR and acute toxicity activities with the AOX properties of these phenolic compounds, crocin and DPPH radical scavenging tests of these stilbenes were carried out. In 14 the presence of a methoxyl group increases the strength of these compounds upon inhibition of DPPH. We suggest that insect growth inhibitory activity of Me-Yuc extract could be caused by a


6 H HO

C

C

OH

H

12 HO H C

C

OH

H HO

13 HO

OH H C

CH3O

C H

HO

14

OH

Fig. 2. Stilbenes from Y. periculosa.

synergistic effect of the stilbene composition. The inhibition of DPPH activity by stilbenes is well known (Burns et al., 2000, 2002; Rimando et al., 2002). Therefore, the plant stilbenes may be considered as efficient IGR and radical scavengers (Stivala et al., 2001; Kim et al., 2002; Rimando et al., 2002). The AOX activity of these compounds was also evaluated spectrophotometrically on the bleaching of the H2Osoluble crocin (Bors et al., 1992). Compounds 12, 13, 14, and Me-Yuc extract were all active, with activities comparable to gallic acid (positive control). Resveratrol 13 and the methoxylated stilbene 14 had more potent insecticidal inhibitory activity. This shows that nature of the substituent at C-3 plays an important role for the insecticidal activity (Torres et al., 2003). Meliaceae Cedrela is a genus belonging to the Meliaceae family. This genus includes several species like C. odorata, C. oaxacensis, C. dugessi, and C. salvadorensis, among others. Particularly we were interested in these species, because it is known that several of these species contain limonoids of gedunin-type (i.e., gedunin, photogedunin, cedrelanolide, mexicanolide, etc.), which have been isolated from Mexican species of the Genus Cedrela, and limonoids are noted because of their potent insect antifeedant characteristics and its potential for the development of ‘‘green pesticides’’ in insecticidal and chemical ecology studies. C. salvadorensis Standley is a small tree that grows on the dry pacific slope ranging from Jalisco to Chiapas in Mexico, through Central America, and up to the north of Panama (Rzedowski, 1972; Huerta, 1981; Rzedowski, 1991, 1993). Previously, we reported the isolation of photogedunin epimers 15, 17 as a mixture from a CH2Cl2 extract of this plant and the synthesis of its acetate derivatives 16 and 18 (Figure 3) (Ce´spedes et al., 1998).


7

C. dugessi is also a small tree that grows on the dry pacific slope ranging from Michoacan to Oaxaca states in Mexico, in a similar form to C. salvadorensis. C. odorata is a tree of 4–5 m height that grows on the cost of Gulf of Mexico, throughout Veracruz and Tabasco states. C. oaxacensis is a tree of 4–5 m height that grows on the slope of Sierra Madre Mixteca, throughout Michoacan, Oaxaca, Veracruz, and Tabasco states (Rzedowski, 1972; Huerta, 1981; Rzedowski, 1991, 1993). Limonoids, photogedunins 15, 17, gedunin 19, cedrelanolide 20, and photogedunin epimeric mixture (15+17) were isolated from young trees of C. salvadorensis and C. dugessi (Ce´spedes et al., 1998, 2000). Photogedunin epimeric acetate mixture (16+18) showed the highest insecticidal activity at 10.0 ppm with 17% survival; the photogedunin epimeric mixture (15+17) with 50% survival at 10.0 ppm in a similar form to toosendanin 21 was next; gedunin 19 showed this effect at 39.0 ppm. Cedrelanolide 20 induced only moderate larval mortalities (o40%) while photogedunin epimeric mixture (15+17), gedunin 19, photogedunin acetates (16+18), and toosendanin 21 generally produced higher mortalities (>45%). The surviving larvae produce deformed pupae, which did not survive (Ce´spedes et al., 2000). Table 1 Insect growth regulatory activity of the isolated compounds, mixtures, and extracts, isolated from mentioned plants against S. frugiperda larvae in a no-choice bioassaya 7 Days Treatment 12 13 14 Epimers (15+17) Epimers (16+18) Gedunin 19 Cedrelanolide 20 Toosendanin 21 Agarofuran 22 Agarofuran 23 24 25 26 27 28 29 Argentatin A 30 Argentatin B 31 32 33 34 35 36 37

21 Days

Pupation

GWI50b

GLI50c

LD50d

GI50b

EI50b

pI50e

26.54 8.75 6.97 5.0 7.5 2.87 25.0 1.75 3.84 7.55 9.7 13.5 30.3 N.D. 3.1 4.0 o1.0 >5.0 5.1 24.7 5.9 6.8 31.7 57.8

32.74 11.6 7.24 10.0 15.0 5.53 75.0 29.0 o6.5 o12.0 5.28 27.3 8.14 N.D. 3.1 8.36 17.8 36.1 21.3 20.6 5.6 5.8 14.7 N.D.

27.6 6.4 5.4 10.0 8.0 30.08 N.D. 8.5 7.5 8.2 3.9 N.D. 27.8 N.D. 10.7 3.46 21.3 37.0 19.12 20.76 33.31 5.77 62.02 81.81

9.24 5.94 3.45 o5.0 o4.0 1.90 >5.0 2.68 0.37 0.43 o1.0 6.5 6.5 9.5 o1.0 >2.0 o5.0 5.0 8.07 14.65 4.20 4.90 18.08 24.41

N.D. 12.29 4.83 N.D. N.D. 3.95 N.D. N.D. o1.5 o2.5 0.55 4.68 4.46 6.91 0.74 3.05 o15.0 o15.0 4.32 22.60 4.59 10.54 39.91 7.07

0.96 0.77 0.54 N.D. N.D. 0.40 N.D. N.D. N.D. N.D. 0.26 0.67 0.64 0.84 0.13 0.48 N.D. N.D. 0.90 1.16 0.62 0.69 1.26 1.39

PI50f 38.26 7.19 9.02 10.0 o5.0 14.01 >60.0 o5.0 o1.5 o2.5 3.46 N.D. N.D. N.D. 2.11 4.62 o15.0 o5.0 3.84 24.27 6.04 14.21 23.01 7.61 (Continued )


8 Table 1 (continued ) 7 Days

21 Days

Pupation

Treatment

GWI50b

GLI50c

LD50d

GI50b

EI50b

pI50e

PI50f

M Ma Me-Yuc Hex-G. microcephala Me-G. microcephala Me-Ced Me-P. argentatum Me-Maytenus Hex-Maytenus

4.1 19.2 14.99 3.2 5.5 3.56 o10.0 14.0 7.3

33.4 33.2 13.22 12.82 14.45 10.87 6.4 13.5 7.5

17.76 27.51 7.18 7.5 7.95 8.22 6.9 9.4 6.0

2.44 5.17 5.13 10.0 12.0 14.17 7.5 4.8 0.37

8.92 3.60 5.79 10.37 13.85 1.43 3.5 o5.0 o5.0

0.39 0.71 0.78 1.01 1.14 0.99 N.D. N.D. N.D.

13.90 4.78 18.82 5.91 12.4 3.84 o0.5 o5.0 o5.0

Notes: M and Ma correspond to a mixture of the compounds 32, 35, and 38 and its acetylated form, respectively. a The parameters are in ppm values. b The GWI50 and GI50 correspond to the inhibitory concentrations for reduction of 50% of larval growth in weight at 7 and 21 days, respectively, in ‘‘no-choice’’ test (po0.05) and EI50 correspond to concentration producing 50% of emergence and were calculated as the dose corresponding to midpoint between complete inhibition (100% of control) and no effect by PROBIT analysis and ANOVA (po0.05) corresponding to the growth inhibition at 7 and 21 days, respectively, under Microcal Origin 6.0. c GLI50 correspond to the growth inhibition in length at 7 days, and was calculated as the dose corresponding to midpoint between complete inhibition (100% of control) and no effect by PROBIT analysis and ANOVA (po0.05) under Microcal Origin 5.1. d LD50 is the concentration producing 50% of lethal mortality at 7 days in ‘‘no-choice’’ test calculated by PROBIT analysis and ANOVA (po0.05). e pI50 correspond to log GI50. f PI50 correspond to concentration producing 50% of pupation, and was calculated as the dose corresponding to midpoint between complete inhibition (100% of control) and no effect by the computer program ANOVA (po0.05) under Microcal Origin 6.0.

We do not ignore the fact that these compounds could act as antifeedant because we did not do the election test, but as pupation did not take place (100% of inhibition above 52 ppm), we suggest that the mechanism by which these compounds act may be due to physiological effects in a similar form to other limonoids (Nihei et al., 2002). We are investigating to elucidate the target, mechanism, and mode of action of these compounds and probably corresponds to a combination of antifeedant action and/or postdigestive toxicity, as found for other limonoids (Champagne et al., 1992; Isman et al., 1995). Celastraceae Celastraceae family embraces shrubs and trees of moderate height occurring in southern Chile. Plants from this family belonging to Maytenus genus have antifeedant and anti-inflammatory effects. These species show high allelopathic and antifeedant effects on plant and insect species in their habitat, respectively (Gonza´lez et al., 2000; Ce´spedes et al., 2001a). Maytenus genus is characterized by the occurrence of different bioactive compounds, especially b-dihydroagarofurans (Itokawa et


9

OR

OR

O

O

O

O

O O

O O

O

O

O O

O

O

O

OAc

O

OAc

15 R = H 16 R = Ac

O

OAc

17 R = H 18 R = Ac

19

O OAc

H O O

O

OH

OAc COEt OHC

O O

O AcO

O

OH

O CO2Me

20

HO H

21

Fig. 3. Limonoids from Cedrela spp., Meliaceae.

al., 1993; Backhouse et al., 1994; Shirota et al., 1994). Until now, we focused mainly on two South-American species of this genus, Maytenus disticha (Hook) Urban and M. boaria. The former, commonly known as ‘‘maitencito’’ or ‘‘romasillo,’’ is a small tree that grows in rainfall forests in the South Pacific slope ranging from Araucanian Region to ‘‘Tierra del Fuego’’ in the Patagonian Region in Chile. M. boaria, the sole tree among the Celastraceae family in Chile, usually grows in the arid climate in the Slope Mountains. Considering that in Celastraceae family the main biologically active compounds are b-agarofuran type sesquiterpenoids (Figure 4), we selected these plants due to their high resistance to insect attack and no insecticidal work has been carried out on this plant. Moreover, we investigated the inhibition of AChE, a key enzyme in the insect nervous system in which the colynergic system is essential. From the aerial parts of M. disticha we isolated six agarofurans (Alarcoń et al., 1991, 1995, 1998) and from the seeds of M. boaria, four b-agarofuran polyesters were obtained and chemically characterized (Alarcoń et al., 1991; Ce´spedes et al., 1999). Insecticidal bioassays showed that a hex/EtOAc (HE) extract produced the highest mortality (79.1%) at 12.0 ppm, similar to 22, 23, and MeOH extract (>65%) and at 15.0 ppm, 23 and HE extract showed the highest larval mortality (100%). When larvae were fed with a diet containing 25.0 ppm or higher doses of all extracts and compounds tested, all the larvae died. Both b-agarofurans 22 and 23 inhibited larval growth after 7 days, and showed a significant growth reduction after 23 days. Compound 23 was more active than the positive control (toosendanin) and


10 CH2OAc

OAc

OBz

OAc AcO

OFu OAc

OAc

O

O OAc 22

OAc 23

Fig. 4. b-Agarofuran type sesquiterpenoids from Maytenus spps.

compound 22, decreasing larval growth, total length of the larvae, and percentage of larvae that reached pupation and emergence. It is noteworthy that b-agarofuran 22, although was not so effective as 23, showed an appreciable reduction on larval length growth compared with the control, for this reason the activity of these compounds is comparable to the commercial insecticide toosendanin 21 (Chen et al., 1995). In all cases the adult weight was clearly reduced. Moreover, these compounds and extracts showed dose-response dependent activity. In order to determine the site of action of compounds and extracts from both plants, we carried out an AChE activity assay. MeOH and HE extracts, and 22 and 23 showed inhibitory AChE activity in a dose-dependent manner confirming that these compounds are active inhibitors of AChE in Maytenus spp. The activity of extracts may be due to a synergistic effect more than an activity of a single compound, and the presence of a furanoxy ring substituent increases the strength of these compounds on inhibition of AChE (Ce´spedes et al., 2001a). Asteraceae Our interest is centered on the study of possible insecticides of desert shrubs belonging to the Asteraceae family, due to their strong resistance against insect attack observed in nature. We have worked on four species of this family – Gutierrezia microcephala, Parthenium argentatum, Roldana barba-johannis, and Tagetes lucida – and evaluated their insecticidal and IGR activities on FAW. Gutierrezia microcephala G. microcephala A. Gray is commonly known as broom wood and grows in arid regions of the Central and North of Me´xico and in the southwestern region of the United States (Roitman and James, 1985). The plant collected in Saltillo, Coahuila State, is rich in flavonoids (Wollenweber et al., 1997). Four flavones 24–27 were obtained from the acetonic and methanolic extracts that were isolated previously by Fang et al. (1985, 1986). n-Hexane extract afforded bacchabolivic acid 28 (Zdero et al., 1989, 1992), a new ent-clerodane reported on this plant, which was lately submitted to esterification to yield the methyl ester 29. Insecticidal effects of 24–29

Natural compounds as antioxidant and molting inhibitors HO

11 OH

OMe OMe

OMe HO

HO

O

O

OMe OMe

MeO OH

MeO

OMe OH

O 24

O 25

OMe HO OH

OMe

OMe

OMe MeO HO

O

O

OMe OH OMe

MeO

MeO

OMe OH

OH

O

O 26

27

O CO2R

28 R = H 29 R = Me

Fig. 5. Flavonoids and clerodane from G. microcephala.

(Figure 5), and hexane and methanol extracts against larvae were evaluated (Calderoń et al., 2001). Although 24, 28, and MeOH and Hex extracts produced significant larval mortalities (>49%) at a low concentration (10 ppm). Compound 29 produces higher mortality (81%). It is relevant that 29, 24, hexane, MeOH extracts, and 28 were more active than control (gedunin) with MC50 (50% lethal concentration of larvae at 7 days) of 3.46, 3.9, 7.5, 7.95, and 10.7 ppm, respectively. Compounds 24, 26, 28, 29, and hexane and MeOH extracts inhibited specifically each larval stage. Compounds 28, 29, and hexane and MeOH extracts produced higher inhibition (upto 90% of weight). Clerodane 28 showed the highest inhibition (100% of length and weight) at 50.0 ppm and flavonoids had lower larval inhibition at the same concentration. Growth reduction at 21 days was produced mainly by 24 and 28. Although 28 produced a significant delay in pupation, most important effect was observed by 24, 28, and hex extract. Significant delays in pupation time were observed at 10.0, 15.0, and 50.0 ppm for 28, 29, and hexane extract. Furthermore, hexane extract

12


significantly reduced pupae weights at 50.0 ppm. Related to percentage of emergence, all the compounds and extract tested showed further reductions. Nevertheless, 24, 29, and hexane extract drastically reduced the percentage of adult emergence to 0% at 25.0, 15.0, and 50.0 ppm. In many of the treatments, mean adult weight was significantly delayed in the average time to reach the adult stage relative to control larvae. Growth index (GI) and RGI clearly showed that the stronger effect was shown by 28, 29, and hexane extract. It is possible to infer that the substitution of polymethoxy flavones induces an increase on the activity of these flavones and a furan ring seems to be necessary for insecticidal activity of ent-clerodane type diterpenes. In order to determine the site of inhibition on the IGR activity and the acute toxicity of the compounds and extracts, the effect on AChE activity was studied. In a similar form to the acute toxicity, 28, 29, MeOH, and hexane extracts showed the greatest inhibitory effect at 50.0 ppm and flavonoids 24–27 displayed a lower inhibitory effects. At minor concentration (25.0 ppm), 28 and hexane extract showed stronger activity level than MeOH extract and 29. In the same way the last two were more active than flavonoids. In addition, both extracts and ent-clerodanes 28 and 29 inhibited AChE activity in a dose-dependent manner (Calderoń et al., 2001). In addition, the presence or absence of a methyl ester group increases or decreases, respectively, the strength of these compounds on inhibition of AChE in clerodanes. It is obvious that the nature of the ester substituent at C-8 plays an important role for the insecticidal activity of ent-clerodanes. The most active compound 28 contained a small and relative hydrophilic acid group at C-8, whereas 29 with a bulky and more lipophilic ester group exhibited a lower activity level. This is according to previously reported QSAR analysis performed by Rodriguez et al. (1999), where they proposed that antifeedant activity of the respective natural products depends on the polarity of ring B and the size of the ester substituents. With respect to the flavonoids, the presence of an extra methoxyl substituent in the A ring seems to be the cause of growth inhibitory activity (length and weight) shown by 24 and 25. In a similar form to polymethylated flavonoids where the introduction of a methyl ether excluding the B-ring in the flavonoids structure increased the antifeedant activity (Morimoto et al., 2000). Parthenium argentatum P. argentatum, commonly known as ‘‘guayule,’’ is a shrub endemic to the Chihuahua desert and is used as an important source of natural rubber. Plant was collected in Matehuala, State of San Luis Potosi, Me´xico. After collection, aerial parts were milled and extracted exhaustively with methanol and partitioned with n-hexane and ethyl acetate. Preliminary assays showed that MeOH extract had a considerable insecticidal activity. Chromatography purification afforded mainly two cycloarten-type triterpenes named Argentatin A and B (Rodrı´ guez-Hahn et al., 1970; Matsubara and Romo de Vivar, 1985; Martı´ nez-Va´zquez et al., 1994). As both compounds were purified in sufficient amount these were used in bioassays. Insecticidal effects of the Argentatin A 30, B 31, toosendanin 21 as a control, and methanolic extract against larvae of first instar of S. frugiperda were performed. Argentatin A produced higher mortality (80.2%) at 35 ppm; however, MeOH extract


13

shows the highest insecticidal activity (96.5%) at a lower concentration (10.0 ppm). It is important to note that when larvae were fed in a diet containing 15.0 ppm or higher doses of methanolic extract all larvae died. Argentatin A and B inhibited specifically each larval stage. At 23 days this growth reduction was clearly significant between 25.0 and 50.0 ppm. However, only toosendanin 21 showed the highest larval growth inhibition at the same concentrations. All tested compounds in comparison to control decreased the percentage of larvae that reached pupation. Although compounds 30 and 31 showed significant delay in survival after pupation, pupation time, and percentage of emergence, the most important effect was observed by MeOH extract and the control (Figure 6). Furthermore, 30 and MeOH extract significantly reduced pupae weights, but toosendanin 21 showed the greatest effect (Ce´spedes et al., 2001b). GI and RGI clearly showed that the stronger effect was shown by MeOH extract and toosendanin. These parameters together with LD50 values corroborate the highest effect that showed methanolic extract. Compounds 30 and 31 showed moderate acute toxicity, nonetheless toosendanin and MeOH extract showed a potent acute toxicity on larvae of last stage at 35.0 and 15.0 ppm, respectively. The LD50 values of 21 and MeOH extract were 1.5 and 3.1 ppm, respectively. In order to determine the site of inhibition on the IGR activity and the acute toxicity of compounds and extracts tested, AChE activity was studied. Argentatin B showed a moderate AChE inhibition activity, unlike Argentatin A, MeOH extract, and toosendanin that are strong inhibitors of AChE activity in a dose-dependent manner, confirming that these compounds are the active inhibitors of AChE. The presence of a ring with seven members, as in Argentatin B, decreases the strength of this compound on inhibition of AChE, opposite to the presence of a furan ring. Strong activity showed by MeOH extract may be due to a synergistic effect (Ce´spedes et al., 2001b). Roldana barba-johannis The methanol extract from the aerial parts of R. barba-johannis (Asteraceae) afforded sargachromenol, sargahydroquinoic acid, and sargaquinoic acid. These natural products and the corresponding acetylated and methylated derivatives showed

OH O OH OH

O

O

O

31

30

Fig. 6. Triterpenes from P. argentatum.


14

insecticidal and IGR activities against FAW. The most active compounds were sargachromenol 32, and its acetylated derivative 34; sargahydroquinoic acid 35 and its acetylated derivative 37; and a mixture of sargachromenol 32, sargahydroquinoic acid 35, and sargaquinoic acid 38 (6:3:1) and the acetylated form of this mixture; 33 and 36 showed a minor activity. All these compounds and mixtures had significant effects between 5.0 and 20.0 ppm in diets. Most compounds were insecticidal to larvae, with lethal doses between 20 and 35 ppm. In addition, these substances also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in TLC autographic and spectrophotometric assays. These compounds appear to have selective effects on the pre-emergence metabolism of the insect (Figure 7). The results from these compounds were fully comparable in activity to those known natural insect growth inhibitors such as gedunin and methanol extracts of C. salvadorensis and Y. periculosa (Ce´spedes et al., 2004). Tagetes lucida T. lucida known as ‘‘pericoń,’’ is a medicinal plant used from pre-Columbian times by Aztecs and other Mesoamericans. This specie is distributed from Northern Me´xico to Northern Nicaragua (Rzedowski, 1972, 1991). Their leaves and inflorescence are used for stomachache and for the treatment of diverse anti-inflammatory ailments. From a dichloromethane extract, we isolated several coumarins: scopoletin

O CO2R

R'O

32 R, R' = H 33 R = CH3 R' = H 34 R = H R' = Ac

CO2R

OR'

35 R, R' = H 36 R = CH3 R' = H 37 R = H R' = Ac OR'

O

CO2R

38

O M = 32 + 35 + 38 Ma = M methylated

Fig. 7. Plastoquinones and tocotrienols from R. barba-johannis.

Natural compounds as antioxidant and molting inhibitors CH3O

15 HO

HO

HO

O

O

HO

39

O

O

40

CH3O

O 42

O

O

O

O

O

41

AcO

CH3O

CH3O

CH3O

AcO

O

O

43

AcO 44

Fig. 8. Coumarins from T. Lucida.

(6-methoxy-7-hydroxycoumarin) 39, esculetin (6,7-dihydroxycoumarin) 40, 7-methoxy-6-hydroxycoumarin 41, scoparone (6,7-dimethoxy-coumarin) 42, and their derivatives 6,7-diacetoxy coumarin 43 and 6-methoxy-7-acetoxycoumarin 44 (Figure 8). The compounds 39, 40, 41, and dichloromethane extract showed high mortality effects between 5.0 and 15.0 ppm and an IGR activity between 1.0 and 5.0 ppm; 40 being the most active compound. The compounds 42, 43, and 44 showed IGR activity at concentrations higher than 70.0 ppm. A complete inhibition of the pupation and emergence above 70.0 ppm was observed. In the literature there are few information about insecticidal effects of coumarins. Nagaiah et al. (1992) reported about the IGR and antifeedant effects of some coumarins from the bark of Xeromphis uliginea on the growth of Spodoptera litura. These compounds are formed by the action of phenylalanine ammonia lyase (PAL) and are then converted to ortho-coumaric acid by the action of cinnamic acid o-hydrolase (Seigler, 1997). Coumarins are located within specialized structures such as secretory ducts called vittae (Berenbaum, 1991). Araucariaceae In the continuation with our search for natural products with possible insecticide activity, we have studied trees of the rain forest of southern Chile, due to their strong resistance against insect attack observed in nature. We have evaluated the insecticidal activity of Araucaria araucana and IGR activities on FAW. Araucaria araucana (Mol.) K. Koch This conifer is endemic to rain forest of southern Chile and Argentina. It has high commercial, ethnobotanical, taxonomic, and ecological value derived from its long biogeographical and remote occurrence. These characteristics offer a unique opportunity for the search of secondary metabolites, especially those that have a defensive role against pathological and phytofagous pests. Five lignans (secoisolariciresinol 45, lariciresinol 46, pinoresinol 47, eudesmin 49, and methyl-pinoresinol 48) were

16


isolated from MeOH extracts of bark and wood of A. araucana and their structures were determined with spectroscopic methods. In addition to antifeedant, insecticidal, and IGR activities against S. frugiperda (FAW), the antibacterial activity of these compounds was determined against Gram (+) and Gram () bacteria and the antifungal activity against Fusarium moniliforme, Aspergillus niger, Trichophyton mentagrophytes, Mucor miehei, Paecilomyces variotii, Ceratocystis pirifera, Trametes versicolor, and Penicillium notatum. These lignans exhibited antifungal and antibacterial activities (Ce´spedes et al., 2006a) and antifeedant and IGR activities on FAW (Ce´spedes et al., 2006b), in the range between 1.0 and 50.0 ppm. Against FAW the strongest compound was pinoresinol 47 with an LD50 19.0 ppm, the MeOH extract from wood shows an LD50 of 400.5 ppm, and the MeOH extract from bark shows an LD50 of 92.1 ppm, these values show the antifeedant action of A. araucana against a Lepidoptera as FAW. In addition, these results show that the larval mortality of all compounds – below 50 ppm – is stronger than the bark MeOH extract and this suggests that this effect could be attributed to the lignin occurrence in this extract, since these lignans were isolated from this extract and all these measurements were twice lower than natural positive control – MeOH extract from bark of Y. periculosa – that showed a 98% of mortality at 50 ppm, as was previously observed (Torres et al., 2003; Ce´spedes et al., 2004). In addition to the above effects these lignans showed a strong AOX activity against DPPH radical reduction, with very low concentrations, between 1 and 10 ppm; almost all lignans showed an I50 inhibition at around 4.5 ppm, except methyl pinoresinol 48 and eudesmin 49, which did not show inhibition (Figure 9).

Experimental part Bioassays with fall armyworm (FAW), Spodoptera frugiperda Larvae used for the experiments were obtained from the culture at the Centro de Investigacioń en Biotecnologı´ a at the Universidad Autońoma del Estado de Morelos, Cuernavaca, Morelos, Me´xico, maintained under previously described conditions (Aranda et al., 1996; Calderoń et al., 2001; Ce´spedes et al., 2000, 2001a, 2001b, 2004; Torres et al., 2003). Acute toxicity on FAW Acute toxicity was elucidated by topical application to last stage larvae of S. frugiperda. The larvae of S. frugiperda were iced to stop their movement and treated on their abdomens with each one of the test compounds. The solvent used was 10.5 ml of acetone in topical form with 50 ml microsyringe, and control was only treated with 10.5 ml of acetone. After 24 h, survival was recorded. Five larvae were used for each concentration, respectively (Torres et al., 2003; Ce´spedes et al., 2004). Relative growth index and growth index The RGI and GI were calculated according to Zhang et al. (1993).


17 OCH3 OH

OH

HO

O H 3CO

OCH3

HO

OH

45

H3CO

O

46

R3 COOH

O O

R1

OH

HO

R4

O O OCH3 R2

O

50 47 R1=R3=CH3; R2=R4=H 48 R1=R3=R4=CH3; R2=H 49 R1=R2=R3=R4=CH3

Fig. 9. Lignans from A. araucana and anisol from other sources.

Reduction of 2,2-diphenyl-1-picrylhydrazyl [ ¼ 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl; DPPH] radical TLC autographic assay After developing and drying, TLC plates were sprayed with a 0.2% DPPH solution in MeOH. The plates were examined 30 min after spraying. Active compounds appear as yellow spots against a purple background. In a similar form, TLC plates were sprayed with 0.05% b-carotene solution in CHCl3. The plates were examined under UV254 light until the background had become discolored (bleached). Active compounds appeared as pale yellow spots against a white background. Spectrophotometric assay (Bors et al., 1992; Cuendet et al., 1997, 2000): A solution (50 ml) containing the compound to be tested was added to 5 ml of a 0.004% MeOH solution of DPPH. The measurement was done after 30 min, and the percent of activity was calculated (Torres et al., 2003). Inhibition of acetylcholinesterase An enzyme extract containing AChE was obtained according to the method of Grundy and Still (1985). Inhibition of AChE was determined according to the Ellman’s procedure (colorimetric method) (Ellman et al., 1961) using both the control (MeOH) and test solutions (compounds and extracts). All AChE inhibition was carried out according to procedures described previously in Ce´spedes et al. (2001a, 2001b).

18


Discussion The sites and mode of action of these compounds is being investigated and probably correspond to a combination of diverse antifeedant actions as midgut esterase inhibition and postdigestive toxicity, as found for other limonoids (Champagne et al., 1992; Nakatani et al., 1994; Isman et al., 1996) and extracts (Feng et al., 1995) or as enzyme inhibitors such as estearases, proteases, tyrosinase, or AChE (Miyazawa et al., 1997; Ortego et al., 1999; Kubo et al., 2003a, 2003b). There are mainly three types of tyrosinase inhibitors based on their inhibition mechanisms. First, the inhibitors directly interact with the binuclear copper active site of the enzyme, known as either copper chelators or substrate analogues. The inhibitors belonging to this category are mostly aromatic compounds and usually act as competitive inhibitors; the inhibitors disrupt the tertiary structure of the enzyme rather than directly interact with the active site. These inhibitors are not only aromatic but are also non-aromatic compounds, and generally act as noncompetitive inhibitors. Second, some molecules have a hydrophobic part and this section interacts with the hydrophobic domain in the enzyme. These subunits associate in a geometrically specific manner, known as a quaternary structure. The hydrophobic moiety which is exposed on the other side of the molecule may disrupt tyrosinase’s quaternary structure. The low conformational stabilities of native proteins make them easily susceptible to denaturation by altering the balance of the weak non bonding forces that maintain the native conformation (Kubo et al., 2003c). Third, the inhibitors do not interact with the enzyme directly or indirectly but act as reducing agents for dopaquinone. In addition to these three main types, there are some additional types of inhibitors. For example, inactivators of the enzyme and some tannins belong to this type of inhibitors. Hence, tyrosinase inhibitors might ultimately provide clues to control insect pests by inhibiting tyrosinase, resulting in incomplete cuticle hardening and darkening (Kubo, 1997, 2000; Kubo et al., 2002). In the case of AChE, the action is directly related with the interaction between the inhibitors and acetylcholine, the inhibition of the enzyme occurs with detachment of the acid residue of the toxic compound, and the nature of acid residue does not affect the structure of the inhibited enzyme, but acts strongly on the process of its inhibition. Natural compounds carrying a charge in an acid residue are very active inhibitors of cholinesterase, such as alkylammonio, alcohols (Brestkin et al., 1992), alkaloids (physostigmine, phenserine, tolserine, among others) (Yu et al., 2002), some terpenoids (Ortego et al., 1999; Calderoń et al., 2001; Ce´spedes et al., 2001a, 2001b), and monoterpenoids (Gracza, 1985; Grundy and Still, 1985; Miyazawa et al., 1997). Some investigations on sites and mechanism of insecticidal or IGR action report that different phenolic compounds are enzyme and metabolism inhibitors (Hammond and Kubo, 1999; Kubo and Kinst-Hori, 1999a; Kubo, 2000; Kubo et al., 2000; Shimizu et al., 2000; Calderoń et al., 2001; Panzuto et al., 2002). It is important to note that similar IGR activity on S. litura (Common cutworm) was studied by Morimoto et al. (2000). These authors reported that flavonoids of Gnaphalium affine (Asteraceae) have insect antifeedant properties against this insect. It is possible to infer that the substitution pattern of flavonoids induce an increase in the activity


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of those phenolic compounds (Calderoń et al., 2001). Until our report (Torres et al., 2003), there were no reports about insecticidal activity of stilbenes, but only those having tyrosinase inhibitory (Shimizu et al., 2000), and antifeedant activities of similar phenolic compounds (Kubo and Kinst-Hori, 1999a; Kubo, 2000; Kubo et al., 2000, 2003a, 2003b). The presence of hydroxyl, methoxyl, and furan moieties seems to be necessary for insecticidal activity as in limonoids containing several of these chemical groups (Nakatani et al., 1994; Ce´spedes et al., 2000, 2001a, 2001b; Calderoń et al., 2001) and in other phenolic compounds as alkanols, tannic, gallic, and anisic acid, respectively (Hammond and Kubo, 1999; Panzuto et al., 2002; Kubo et al., 2003a, 2003b). It is worth mentioning that resveratrol 13, the methoxylated stilbene 14, photogedunin epimeric mixture (16+18), toosendanin 21, agarofurans 22 and 23, the flavonoid 24, the diterpenes 28 and 29, sargahydroquinoic acid 35, 6,7-dihydroxycoumarin 40, and pinoresinol 47 showed the most potent insecticidal activity. It is obvious that the nature of the substituents as well as hydroxyl and methoxyl groups in the aromatic ring plays an important role for the insecticidal activity. These results prove previous findings on the quantitative structure-activity relationship of stilbene derivatives for instance, namely that the inhibitory tyrosinase activity of the respective natural products depends on the polarity of ring A and on the size of the substituent (Gorham et al., 1995; Shimizu et al., 2000). These facts show that the antifungal activity showed by stilbenoids (Schultz et al., 1990, 1992; Pacher et al., 2002), acute toxicity, and growth inhibition may be due to the inhibition of tyrosinase, and this target was demonstrated also for other stilbenes and phenolics from natural sources, such as gallic 10 and anisic acid 50 (Shimizu et al., 2000; Gilly et al., 2001; Kim et al., 2002; Kubo et al., 2003a, 2003b). In addition to the enzymatic activities, IGR correlate very well with AOX measurements of the reduction of DPPH radical, in many of our cases mainly when the phenolic compounds show AOX activity they also show IGR activity. Our phenolic compounds could be considered as efficient IGR and radical scavengers (Ce´spedes et al., 2000, 2001a, 2002, 2003, 2004; Stivala et al., 2001; Kim et al., 2002; Rimando et al., 2002). Thus, it is possible to infer that when some compounds show AOX properties they can also show IGR or tyrosinase inhibitory activity. In our case the compounds that showed these activities were stilbenes, sargachromene and sargahydroquinoic acid, flavonoids such as quercetin, naringenin, and kaempferol, and coumarins. Thus, it can be said that natural compounds are AOX, antifungal, and molting inhibitors, which can play a role as a model for search of new botanical pesticides, and nutraceutical and bioactive compounds. The percentage of larvae that reached pupation decreased in all tested compounds in comparison to the control (Table 1). The most important effect was observed with stilbene 14, gedunin 19, toosendanin 21, b-dihydroagarofurans 22, and 23, clerodane 28, Argentatin A 30, sargachromenol 32, sargahydroquinoic acid 35, 6,7-dihydroxycoumarin 40, and pinoresinol 47, photogedunin epimeric mixture, photogedunin acetate mixture, which reduced successful pupation into a range between 0% and 25% and caused a significant delay in pupation time. The effects of these substances on reducing insect growth, decreasing the percentage of emergence, and increasing mortality of S. frugiperda are similar to those of other natural products (Arnason et al., 1987, 1993).

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Inspection of the structure of the most active compounds isolated from these plants suggests that the presence of oxygenated function was necessary for the activity displayed by the gedunin-type and phenolic molecules against S. frugiperda. Almost all tested compounds except cedrelanolide and methoxylated flavones showed comparable activity to the commercial insecticide toosendanin, which suggests potential for further development of these materials. However, none of these substances has been found with the outstanding activity of azadirachtin (Govindachari et al., 1995; Kraus, 1995).

Concluding remarks In summary, the insecticidal activity of extracts may be due to synergistic effects shown by the phenolic components of the mixtures in the test system used in this investigation. These facts are indicative of the potency of the methanol extracts. Thus, the effect of compounds on reducing insect growth, increasing development time, and increasing mortality of S. frugiperda is similar to that of gedunin and more potent than the MeOH extract from C. salvadorensis (Ce´spedes et al., 2000; Calderoń et al., 2001). The sites and mode of action of these compounds and extracts are being investigated and probably correspond to a combination of antifeedant action as midgut phenol oxidase, proteinase, AChE, tyrosinase, or other PPOs and cuticle synthesis inhibition, as well as molting, sclerotization, toxicity, and nerval system inhibition, as has been found for other phenolics and terpenoids (Miyazawa et al., 1997; Kubo and Kinst-Hori, 1999a, 1999b; Kubo, 2000; Kubo et al., 2000; Simmonds, 2003) and extracts (Feng et al., 1995). In addition, the presence of an orcinol or catechol group seems to be important for these activities as shown for the most potent compounds in this chapter. Furthermore, a great percentage of larvae that reached pupation decreased with the application of phenolics in comparison to control, which might be due to the inhibition by tyrosinase as well or to the accumulation of proteinase inhibitors (Tamayo et al., 2000). The activity of these plants, their metabolites, and MeOH extracts is comparable to the insect growth regulators gedunin and toosendanin, which suggests potential for further development of these materials.

Future perspectives The plant kingdom offers a rich source of a wide range of structural biodiversity of natural secondary metabolites. One of the most recent trends in fungal and insect pest control is to reduce heavy reliance on synthetic pesticides and to move towards biodegradable substances. Synthetic pesticides of broad spectrum have been widely used as the main tools for controlling weeds, and fungal and insect pest, which are highly toxic to many living organisms as well as to the environment. Hence, new biorational and specific trends to pest control should be developed. In this chapter, we presented an update of our findings in this field, those approaches resulting from our studies on inhibition of growth (including larval growth, pupation, and emergence) and of the enzymes involved in key processes of insect life, specially modifying the apolysis during molting, sclerotization, pupation, and


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emergence. Since these approaches refer to control of insect pests, many of them can be extrapolated and also considered suitable for medicinal chemistry studies, because the mechanism of action of these inhibitors is similar to that from human and other animals (Guerrero and Rosell, 2004, 2005). The study of AChE and other phenol oxidases inhibitors, as well as IGR substances, can help in the search of new natural compounds for the treatment of Alzheimer’s disease (Cummings, 2000) and Parkinson’s disease (Weinstock et al., 2003). Therefore, there is an increasing expectation about the research on enzymes inhibition by those compounds of botanical origin that could serve as lead compounds for the development of important substances with agrochemical and pharmacological properties. Thus, by studying the plant organisms that protect themselves against the pest attack, we can learn to control this attack in an ecological way and in addition can get pharmacologically active substances.

Acknowledgments We are very grateful to Prof. Isao Kubo (Department of ESPM, U. C., at Berkeley), Prof. Murray B. Isman (Faculty of Agricultural Sciences, U. British Columbia, Vancouver), and Prof. David S. Seigler (Plant Biology Dept. University of Illinois, at Urbana-Champaign), for the great and valuable help given to authors. The bioassays involving insecticidal to FAW larvae were skillfully conducted by Mrs. Laura Lina (Biological Control Lab., CEIB, UAEM, Cuernavaca, Morelos State) and in similar form the antifungal activity by Mrs. Ana Ma. Garcı´ a (Phytochemistry Lab, FESIztacala, UNAM) whose technical support is greatly appreciated. The works were partially supported by grants: IN243802 and IN211105 from DGAPA-UNAM.

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29

CHAPTER 2

Pesticides based on plant essential oils: from traditional practice to commercialization MURRAY B ISMAN, CRISTINA M MACHIAL

Introduction With very few exceptions, botanical insecticides in commercial use have been used in traditional practices dating back at least 150 years, and often much longer (Jacobson and Crosby, 1971; Philoge`ne et al., 2005). Not unlike pyrethrum, rotenone and neem, plant essential oils or the plants from which they are obtained have been used for centuries to protect stored commodities or to repel pests from human habitations. Unlike other botanicals however, plant essential oils have a long history of human uses apart from pest control, notably as fragrances, flavourings, condiments or spices, as well as medicinal uses. It is ironic then, that development of plant essential oils as commercial insecticides has only occurred within the past decade. In noting the disparity between the vast number of plants shown to poison, deter or repel insects and the actual number of botanical insecticides in commercial use, I previously suggested that there are three barriers to commercialization of botanicals (Isman, 1997). These are: (1) availability of the plant material on a sustainable basis; (2) standardization of extracts based on quantification of active ingredients; and (3) governmental approval, normally requiring costly toxicological evaluation of the candidate product. Given the worldwide use of essential oils in the perfume, food and beverage industries, oils from many species are available in abundance yearround, and their major constituents are either well characterized or are themselves available in a relatively high degree of purity. Thus the major barrier to their use as pesticides may be government approval, but in the United States, several plants and their derivatives are exempt from registration owing to their widespread use in foods and generally acknowledged safety to humans. In the present chapter, the chemistry of plant essential oils, their biological activities in arthropods and fungi, and their potential as botanical pesticides in commercial practice is discussed.


30

Sources and chemistry of essential oils Plant essential oils are obtained through steam distillation of the foliage or other plant parts of terrestrial plants in a limited number of taxa. Among the better-known families from which essential oils are frequently obtained are the mint (Lamiaceae), myrtle (Myrtaceae), citrus (Rutaceae) and carrot (Apiaceae) families. Most essential oils are highly complex mixtures of mono- (C10) and sesquiterpenes (C15), including biogenetically related phenols (phenylpropenes and cinnamates). These compounds are usually responsible for the characteristic odours and/or flavours of the plants from which they are obtained. The predominant group, the monoterpenes, are often cyclic, with a saturated (e.g. pulegone, Figure 1) or unsaturated hexacycle (e.g. limonene) or an aromatic system (e.g. thymol). Bicyclic (e.g. 1,8-cineole) and acyclic examples (e.g. citronellal) are not uncommon. CHO

O O

(+)-Limonene

(+)-Pulegone

1,8-Cineole

Citronellal

OH OCH3

OH

OH CHO Eugenol

Cinnamaldehyde

Thymol

Carvacrol

OCH3

O

Menthone

trans-Anethole

Fig. 1. Structures of some essential oil constituents with documented insecticidal and/or antifungal activities.

Pesticides based on plant essential oils

31

Functional groups include alcohols (e.g. thymol), aldehydes (e.g. citronellal) and ketones (e.g. menthone), but simple hydrocarbons also occur (e.g. limonene). Owing to their generally low boiling points (140–180 1C for monoterpenes, >200 1C for sesquiterpenes), all are relatively volatile, accounting for their use as fragrances and as insect repellents. While many of the monoterpenoids are chemically stable at ambient temperature, their volatility usually renders them nonpersistent in the environment. Among the best known essential oils with bioactivity against insects and other pests are clove oil (eugenol), thyme oil (thymol, carvacrol), mint oil (menthol, pulegone), lemongrass oil (citronellal, citral), cinnamon oil (cinnamaldehyde), rosemary oil (1,8-cineole) and oil of oregano (carvacrol). It is important to emphasize the intraspecific variability in chemical composition of plant essential oils. Sources of compositional variability can include the plant part extracted, phenological state of the plant, and time of year, as well as climatic and soil variations. Numerous studies have also confirmed that some species have distinct chemotypic races or populations, often separated geographically (Salgueiro et al., 1997; Pascual-Villalobos and Ballesta-Acosta, 2003; Angioni et al., 2004). In an investigation of the essential oils isolated from two rosemary ecotypes from Italy, the major constituent, 1,8-cineole, ranged from as low as 7.3% by weight to a high of 55.3% and a-pinene ranged from 11.5% to 30.3% (as isolated from apical, intermediate and lower foliage) (Flamini et al., 2002).

Biological activity Mode of action in arthropods The rapid onset of toxicity from essential oils or their constituents in insects and other arthropods points to a neurotoxic mode of action. In the American cockroach this is manifest as hyperactivity followed by hyperextension of the legs and abdomen culminating in rapid knockdown or immobilization, often in less than 30 s (Enan, 2001). Several essential oil monoterpenes have been shown to be weak inhibitors of acetylcholinesterase (AchE) in insect and mammalian preparations (Ryan and Byrne, 1988; Miyazawa et al., 1997), but toxicity in vivo did not correlate with AchE inhibition in vitro. More recently Enan (2001, 2005a) has provided evidence that many essential oil constituents poison insects by blocking octopamine receptors. Octopamine, synthesized from tyramine, is a neurotransmitter and neuromodulator in arthropods and may have neurohoromonal influences as well. Several essential oil constituents were demonstrated to be mild antagonists of octopamine binding activity in the cockroach nervous system, however, other such compounds were found to be agonists and yet others were without effect. Notable among the last group was thymol, one of the most toxic essential oil compounds to insects (Table 2). Enan (2005b) has also provided evidence that at least some essential oil constituents may act via the tyramine receptor cascade in the fruit fly Drosophila melanogaster. Physical effects, viz. dissolution and disruption of cell membranes, or blockage of the tracheal system in insects cannot be ruled out as alternative mechanisms of toxicity.


32

Efficacy of essential oils to insects Table 1 lists a number of studies in which plant essential oils have been screened for bioactivity against insects. It should be readily apparent that the majority of studies to date have utilized stored product pests, particularly beetles such as Sitophilus, Tribolium and Acanthoscelides species, in which case fumigant toxicity has been investigated (Shaaya et al., 1991; Regnault-Roger et al., 1995; Obeng-Ofori and

Table 1 Screening studies with plant essential oils against insects Essential oil

Insect tested

Reference

53 species 31 species 28 species

Choi et al. (2003) Singh et al. (1989) Shaaya et al. (1991)

11 species

Trialeurodes vaporariorum Sitophilus oryzae Sitophilus oryzae, Rhyzopertha dominica, Oryzaephilius surinamensis, Tribolium castaneum Drosophila auraria

22 species

Acanthoscelides obtectus

4 species

Sitophilus oryzae, Tribolium confusum, Ephestia kuehniella Pediculus humanus Cydia pomonella Musca domestica Acanthoscelides obtectus

5 species 27 species 31 species 22 species 13 species

23 species Vetiver, Vetiveria zizanioides Patchouli, Pogostemon cablin Chamaecyparis obtuse Minthostachys andina, Hedomea mandonianum Tansy, Tanacetum vulgare Garlic, Allium sativum Nutmeg, Myristica fragrans Tetradenia riparia Holy basil, Ocimum sauve

Oryzaephilus surinamensis, Rhyzopertha dominica, Sitophilus oryzae, Tribolium castaneum Lipaphis pseudobrassicae Coptotermes formosanus Coptotermes formosanus Callosobruchus chinensis, Sitophilus oryzae Rhodnius neglectus, Triatoma infestans Choristoneura rosaceana Tribolium castaneum, Sitophilus zeamais Tribolium castaneum, Sitophilus zeamais Zabrotes subfasciatus Sitophilus zeamais, Rhyzopertha dominica, Sitotroga cerealella

Konstantopoulou et al. (1992) Regnault-Roger and Hamraoui (1994) Sarac and Tunc (1995) Mumcuoglu et al. (1996) Landolt et al. (1999) Singh and Singh (1991) Regnault-Roger et al. (1993) Shaaya and Kostjukovsky (1998) Sampson et al. (2005) Maistrello et al. (2001) Zhu et al. (2003) Park et al. (2003) Fournet et al. (1996) Larocque et al. (1999) Ho et al. (1996) Huang et al. (1997) Weaver et al. (1994) Bekele et al. (1996)


33

Table 2 Toxicity of some essential oil constituents to insects Compound

Insect tested

Effect/dose

Reference

Eugenol

Spodoptera litura

LD50 ¼ 157.6 mg/larva

Sitophilus granaries

LD50~2.5 mg/beetle

Coptotermes formosanus Musca domestica Diabrotica virgifera Drosophila melanogaster Aedes aegypti

LD50~40 ppm (sand)

Hummelbrunner and Isman (2001) Obeng-Ofori and Reichmuth (1997) Cornelius et al. (1997)

Pulegone

Periplaneta americana Musca domestica Diabrotica virgifera Peridroma saucia Spodoptera litura Ostrinia nubilalis

Thymol

Citronellal

d-Limonene

Menthone

Drosophila melanogaster Musca domestica Drosophila melanogaster Drosophila melanogaster Spodoptera litura Culex pipiens molestus Spodoptera litura Callosobruchus maculatus Musca domestica Drosophila melanogaster Musca domestica Diabrotica virgifera Spodoptera litura Callosobruchus maculates Sitophilus oryzae Blattella germanica Musca domestica Sitophilus oryzae Drosophila melanogaster Peridroma saucia

LD50 ¼ 77 mg/fly LD50 ¼ 12 mg/beetle LD50 ¼ 4 mg/cm2 LD50~0.5 mg/43 cm2 paper LC50 ¼ 148 mg/cm2 paper LD50 ¼ 39 mg/fly LD50 ¼ 38 mg/beetle LD50 ¼ 753.9 mg/larva LD50 ¼ 51.6 mg/larva LC50 ¼ 26.3 ppm (in diet) LD50 ¼ 170 mg/4 mm dia paper LD50 ¼ 29 mg/fly LD50 ¼ 2600 mg/4 mm dia paper LD50 ¼ 5 mg/cm2 LD50 ¼ 25.4 mg/larva LC50 ¼ 36 mg/l LD50 ¼ 111.2 mg/larva

Lee et al. (1997) Isman (unpubl. Data) Bhatnagar et al. (1993) Ngoh et al. (1998) Lee et al. (1997) Harwood et al. (1990) Hummelbrunner and Isman (2001) Lee et al. (1999) Franzios et al. (1997) Lee et al. (1997) Franzios et al. (1997) Isman (unpubl. Data) Hummelbrunner and Isman (2001) Traboulsi et al. (2002)

LC50 ¼ 5.5 ml/l

Hummelbrunner and Isman (2001) Don-Pedro (1996)

LD50 ¼ 66 mg/fly LD50 ¼ 27 mg/cm2

Lee et al. (1997) Isman (unpubl. Data)

LD50 ¼ 50 mg/fly LD50 ¼ 88 mg/beetle LD50 ¼ 273.7 mg/larva

Lee et al. (1997)

LC50 ¼ 8.3 ml/l LC50 ¼ 61.5 ml/l LC50 ¼ 23 ml/l LD50 ¼ 98 mg/fly LC50 ¼ 12.7 ml/l LD50 ¼ 1290 mg/4 mm diameter paper LD50 ¼ 1735 mg/larva

Hummelbrunner and Isman (2001) Don-Pedro (1996) Lee et al. (2001) Coats et al. (1991) Lee et al. (1997) Lee et al. (2001) Franzios et al. (1997) Harwood et al. (1990)

(Continued)


34 Table 2 (continued ) Compound

Insect tested

Effect/dose

Reference

Trans-anethole

Tribolium castaneum Musca domestica

LC50 ¼ 48.1 ml/l LD50 ¼ 75 mg/fly

Drosophila melanogaster Spodoptera litura

LD50 ¼ 7 mg/cm2

Ho et al. (1997) Marcus and Lichtenstein (1979) Isman (unpubl. Data)

Blattella germanica Cinnamaldehyde

Tribolium castaneum Coptotermes formosanus Trichoplusia ni

LD50 ¼ 65.5 mg/larva 80% mortality at 159 mg/cm2 LC50 ¼ 0.70 mg/cm2 96% mortality at 1 mg/g paper LD50 ¼ 176.3 mg/larva

Hummelbrunner and Isman (2001) Chang and Ahn, 2001 Huang and Ho (1998) Chang and Cheng (2002) Isman (unpublished)

Reichmuth, 1997; Shaaya and Kostjukovsky, 1998; Lee et al., 2001). Blood-feeding insects (Pediculus, Triatoma) have also been screened (Muncuoglu et al., 1996). Surprisingly, fewer crop pests have been examined in this regard. When all the data are reviewed collectively, certain plant species emerge as being consistently active against insects, although their rankings by potency vary greatly between pest species. Among the more active essential oils are those from thyme, oregano, basil, rosemary and mint, but much more empirical testing using less common plant species and a wider array of pest species will undoubtedly reveal particularly valuable biological activities (Shaaya and Kostjukovsky, 1998; Isman, 2005). Efficacy of pure constituents to insects Eight of the more potent essential oil constituents (monoterpenes and phenylpropenes) are listed in Table 2, along with their toxicity to various insect species if reported. Structures of these compounds are shown in Figure 1. Comparisons of toxicity among insect species for any particular compound are seldom relevant because of the differences in route of administration or exposure method between investigations. On the other hand, some studies have made direct comparisons of different compounds in one or more pest species (Lee et al., 1997; Hummelbrunner and Isman, 2001; Sampson et al., 2005). Accordingly, thymol and pulegone emerge as the most potent individual compounds, at least based on bioassays against the house fly Musca domestica, the tobacco cutworm Spodoptera litura and the turnip aphid Lipaphis pseudobrassicae. However, relative toxicity of individual compounds among pests can also be idiosyncratic. For example, in comparing the phenols eugenol and carvacrol and the monoterpenes a-terpineol and terpinen-4-ol for toxicity against four insect pest species and the twospotted spider mite, eugenol was the most toxic to two species (fruit fly and corn rootworm), terpinen-4-ol to two others (rice weevil and the spider mite), and carvacrol to the fifth (tobacco cutworm) (Isman, 2000). Comprehensive investigation into structure–activity relations of the toxicity of monoterpenes to insects has yielded few insights (Rice and Coats, 1994a), and efforts to enhance potency of


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selected terpenes and phenols through derivitization produced very few analogues more potent than the parent natural products (Rice and Coats, 1994b; Tsao et al., 1995). In general though, phenols (e.g. thymol, eugenol) tend to be more active, both as insecticides and as fungicides, than the related monoterpenes. Essential oils as insect repellents The major use of insect repellents is for personal protection against biting arthropods, particularly mosquitoes. For almost 50 years the most heavily used and reliable insect repellent has been DEET (N,N-diethyl-m-touamide) (Peterson and Coats, 2001). One of the few viable alternatives to DEET for application to human skin has been preparations containing oil of citronella ( ¼ lemongrass oil). A number of such products containing citronella, often mixed with mint oil, are marketed in the United States, in spite of research indicating that they are far less efficacious than DEET (Fradin and Day, 2002). Other oils used in personal repellents include cinnamon oil, clove oil and soybean oil. Presumably these products, with modification, could be useful for protection of domestic animals (e.g. equestrian applications). Another use of repellents is for the removal of cockroaches and the prevention of reinfestation in human habitations and other buildings such as hospitals and restaurants. Certain essential oil constituents clearly repel cockroaches in a laboratory setting (Ngoh et al., 1998) and it is likely that at least part of the efficacy of commercial pest-control products based on essential oils is a consequence of ‘‘flushing’’ cockroaches from their harbourages and discouraging their return. Impregnating repellents into packaging to prevent insect infestation represents another potential use of essential oils that has yet to be realized. It is important to note that repellents could also prove useful in integrated pest management of agricultural crops, particularly in the context of a stimulodeterrent diversionary strategy (SDDS) or ‘‘pushpull’’ strategy (Miller and Cowles, 1990). Finally, it should be mentioned that certain essential oil constituents are effective attractants for some insects. For example, geraniol and eugenol are used as lures in traps for the Japanese beetle Popillia japonica and methyl eugenol has been used to trap Oriental fruit fly Dacus dorsalis (Vargas et al., 2000). Cinnamyl alcohol, 4-methoxy-cinnamaldehyde, cinnamaldehyde, geranylacetone and a-terpineol are attractive to adult corn rootworm beetles (Diabrotica spp.) (Hammack, 1996; Petroski and Hammack, 1998). Efficacy of oils and pure constituents to mites Some studies of the effects of essential oils or pure constituents against both parasitic and free-living mites are listed in Table 3. The tracheal mite Acarapis woodi and the varroa mite Varroa jacobsoni are economically important parasites of the honeybee Apis mellifera and there are few viable treatments for infested bee colonies. Menthol, thymol and some other compounds have been shown to have moderate efficacy as mite fumigants in beehives, although the difference in toxicity between the mites and their host bees (as little as 2–3 fold) requires that these substances be used precisely in practice. Essential oils rich in 1,8-cineole (tea tree oil, eucalyptus oil) have been shown to be effective against house dust mites. Several oils and their constituents are


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Table 3 Acaricidal activity of some essential oils and constituents Effective oil or compounds

Species tested

Reference

Menthol, thymol Blend of thymol, camphor, menthol and eucalyptus oil Citronellal, pulegone, terpin4-ol, piperitone Lavender oil, menthol, linalool Thymol, eugenol, menthol Wormwood oil, tansy oil Eucalyptus oil, Thuja oil, Melaleuca oil

Acarapis woodi Varroa jacobsoni

Ellis and Baxendale (1997) Calderone and Spivak (1995)

Ornithonyssus sylviarum

Carroll (1994)

Tyrophagus longior

Perrucci (1995)

Psoroptes cuniculi Tetranycus urticae Dermatophagoides pteronyssinus, D. farinae Tetranychus urticae

Perrucci et al. (1995) Chiasson et al. (2001) Yatagai (1997)

Tetranychus urticae, Phytoseilus persimilis Tetranychus urticae

Choi et al. (2004)

Carvomenthenol, terpinen-4ol, eugenol 53 plant essential oils Rosemary oil

Lee et al. (1997)

Miresmailli et al. (2006)

effective against the phytophagous spider mites, as commercial miticides based on rosemary or cinnamon oils attest (Isman, 2000; Miresmailli et al., 2006). Efficacy of oils and pure constituents to fungi Many plant essential oils and their major constituents have demonstrated antifungal activity against a range of plant pathogenic fungi including those responsible for both pre- and postharvest diseases. Some examples are listed in Table 4. Unlike insects, different fungal species yield more consistent results – thyme oil and its major phenolic constituents thymol and carvacrol are clearly active against most fungal species tested. The mechanism of action of these compounds against fungi is unknown but may be related to their general ability to dissolve or otherwise disrupt the integrity of cell walls and membranes. As a cautionary note, many of these oils and pure compounds show considerable phytotoxicity to plants at concentrations only slightly above those required for control of plant pathogenic fungi, presumably because plant cells are affected by a similar mechanism. Toxicity to mammals and other nontarget organisms Given the widespread use of plant essential oils in fragrances, as flavouring agents or condiments in food and in aromatherapy, we might expect these materials to be without appreciable toxicity in mammals. For most of the essential oil constituents toxic to insects and fungi this is the case (Table 5), although some may cause irritation of the skin, eyes and mucous membranes in concentrated form. But there are some notable exceptions. Pulegone is considerably toxic to rats (i.p. LD50 ¼ 150 mg/kg), although data on oral, acute toxicity has not been published. Pennyroyal oil, the source of pulegone, has been implicated in both human and animal poisonings


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Table 4 Antifungal activity of some essential oils and constituents Effective oil or compounds

Species tested

Reference

Palmarosa, red thyme, cinnamon, clove bud oils Thymol, carvacrol

Botrytis cinerea

Wilson et al. (1997)

Botrytis cinerea, Monilinia fructicola Rhizoctonia solani, Fusarium moniliforme, Sclerotinia sclerotiorum Fusarium oxysporum 22 species of fungi Aspergillus niger Aspergillus spp. Aspergillus flavus

Tsao and Zhou (2000)

Thymol, carvacrol

Clove oil Cymbopogon spp. oils Cymbopogon nardus oil Thyme, oregano oils Thyme, oregano, cinnamon, basil, peppermint oils Thyme, oregano, marjoram, dictamus oils Thymol, eugenol, citronellol, cinnamaldehyde Thyme, mint, lavender oils

Muller-Riebau et al. (1995)

Penicillium digitatum

Bowers and Locke (2000) Singh et al. (1980) De Billerbeck et al. (2001) Paster et al. (1995) Montes-Belmont and Carvajal (1998) Daferera et al. (2000)

Seven species of fungi

Kurita et al. (1981)

Fusarium solani, Rhizoctonia solani, Pythium ultimum, Colletotrichum lindemuthianum

Zambonelli et al. (1996)

Table 5 Toxicity of some essential oil constituents to rats Compound

LD50 (mg/kg)a

1,8-Cineole (eucalyptol) Cinnamaldehyde Citral (from citronella oil) Eugenol d-Limonene Menthol Pulegone Terpinen-4-ol Thymol trans-Anethole

2480 1160 (guinea pig) 4960 2680 4600 (lowest published lethal dose) 3180 150 (mouse, intraperitoneal) 4300 980 (mouse ¼ 1800) 2090

a

Oral, acute unless otherwise specified. Source: Food and Agriculture Organization of the United Nations, 1999.

(Anderson et al., 1996). a-Thujone, the toxic constituent of the liqueur absinthe (made from wormwood oil), is also very toxic to rats (i.p. LD5045 mg/kg) (Hold et al., 2000). On the other hand, an essential-oil-based insecticide produced no mortality in rats when fed at 2 g/kg, and the LD50 exceeded 5 g/kg, the normal upper limit for testing required by regulatory agencies (E. Enan, unpubl. data). Eugenol is approximately

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1500 times less toxic than pyrethrum and 15,000 times less toxic than the organophosphate insecticide azinphosmethyl to juvenile rainbow trout Oncorhynchus mykiss, based on 96-h static water tests (Stroh et al., 1998). Effects on other nontarget organisms, namely, pollinators and natural enemies, have yet to be evaluated under field conditions. In the laboratory, a rosemaryoil-based insecticide/acaricide was significantly less toxic to predatory mites (Phytoseilus persimilis) than to their spider mite prey (Tetranychus urtica), although the whitefly parasitoid Encarsia formosa was more susceptible than its whitefly hosts (Miresmailli and Isman, unpublished data). However, most essential-oil constituents degrade quickly in the environment or are rapidly lost from plant foliage through volatilization, minimizing residual contact and favouring immigration or reintroduction of biocontrol agents and the foraging activities of honeybees and other pollinators.

Commercial products and uses In spite of considerable research effort in many laboratories throughout the world and an ever-increasing volume of scientific literature on the pesticidal properties of essential oils and their constituents, surprisingly few pest control products based on plant essential oils have appeared in the marketplace. This may be a consequence of regulatory barriers to commercialization (i.e. cost of toxicological and environmental evaluations) or the fact that efficacy of essential oils toward pests and diseases is not as apparent or obvious as that seen with currently available products. Nonetheless, some U.S. companies have introduced essential-oil-based pesticides in recent years. Mycotech Corporation produced an aphidicide/miticide/fungicide for greenhouse and horticultural crops and for bush and tree fruits based on cinnamon oil with cinnamaldehyde (30% in the EC formulation) as the active ingredient. However, this product is no longer being sold. EcoSMART Technologies has introduced insecticides containing eugenol and 2-phenethyl propionate aimed at controlling crawling and flying insects, under the brand name EcoPCOR for pest control professionals. An insecticide/miticide containing rosemary oil as the active ingredient has recently been introduced for use on horticultural crops under the name EcoTrolTM. Another product based on rosemary oil is a fungicide sold under the name SporanTM, while a formulation of clove oil (major constituent: eugenol), sold as MatranTM, is used for weed control. All of these products have been approved for use in organic food production. Several smaller companies in the U.S. and the U.K. have developed garlicoil-based pest control products and in the U.S. there are consumer insecticides for home and garden use containing mint oil as the active ingredient. Menthol has been approved for use in North America for control of tracheal mites in beehives, and a product produced in Italy (Apilife VARTM) containing thymol and lesser amounts of cineole, menthol and camphor is used to control Varroa mites in honeybees.

Conclusion and future prospects Pesticides based on plant essential oils or their constituents have demonstrated efficacy against a range of stored product pests, domestic pests, blood-feeding pests and certain soft-bodied agricultural pests, as well as against some plant pathogenic fungi


39

responsible for pre- and post harvest diseases. They may be applied as fumigants, granular formulations or direct sprays with a range of effects from lethal toxicity to repellence and/or oviposition deterrence in insects. These features indicate that pesticides based on plant essential oils could be used in a variety of ways to control a large number of pests. In general, the efficacy of these materials falls short when compared to synthetic pesticides although there are specific pest contexts where control equivalent to that with conventional products has been observed. Essential oils also require somewhat greater application rates (as high as 1% active ingredient) and may require frequent reapplication when used out-of-doors. Additional challenges to the commercial application of plant essential-oil-based pesticides include availability of sufficient quantities of plant material, standardization and refinement of pesticide products, protection of technology (patents) and regulatory approval (McChesney, 1994; Isman, 2005). Although many essential oils may be abundant and available year round due to their use in the perfume, food and beverage industries, large-scale commercial application of essential-oil-based pesticides could require greater production of certain oils. Pesticides derived from essential oils of relatively rare plants would not be easily produced due to a lack of sufficient quantities of plant material, limiting the essential oils of use to those which are readily available or from those plants which can be easily cultivated (e.g. in plantations) (McChesney, 1994). In addition, as the chemical profile of plant species can vary naturally depending on geographic, genetic, climatic, annual or seasonal factors, pesticide manufacturers must take additional steps to ensure that their products will perform consistently. All of this requires substantial cost and smaller companies may not be willing to invest the required funds unless there is a high probability of recovering the costs through some form of market exclusivity (e.g. patent protection). Finally, once all of these issues are addressed, regulatory approval is required. Although several plant essential oils are exempt from registration in the United States, many more oils are not, and few countries currently have such exemption lists. Accordingly, regulatory approval continues to be a barrier to commercialization and will likely continue to be a barrier until regulatory systems are adjusted to better accommodate these products. Nonetheless, pesticides derived from plant essential oils do have several important benefits. Due to the rapid volatilization of these products, there is a much lower level of risk to the environment than with current synthetic pesticides. Predator, parasitoid and pollinator insect populations will be less impacted as a result of the minimal residual activity, making essential-oil-based pesticides compatible with integrated pest management programs. Further, while resistance development continues to be an issue for many synthetic pesticides, it is likely that resistance will develop more slowly to essential-oil-based pesticides owing to the complex mixtures of constituents that characterize many of these oils. Ultimately, it is in developing countries where the source plants are endemic that these pesticides may ultimately have their greatest impact. However, while commercial development of plant essential-oil-based pesticides may face a variety of problems, it is evident that where human and animal health are at a premium and minimal residues are desirable, it is worth the effort to surmount these challenges. It is therefore expected that these pesticides will find their greatest commercial application in urban pest control, public health, veterinary health, mosquito abatement

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and in stored product protection. In agriculture, these pesticides will be most useful for protected crops (e.g. greenhouse crops), high-value row crops and within organic food production systems where few alternative pesticides are available. As conventional, hazardous insecticides are removed from the marketplace and the cost of developing new products continues to rise, it is likely that pesticides based on plant essential oils will gain further acceptance.

Acknowledgment We thank Dr. E. Enan (Vanderbilt University, Nashville, TN, USA) and Mr. S. Miresmailli (University of British Columbia) for allowing us to include their unpublished data. Supported by grants from NSERC and EcoSMART Technologies Inc. to MBI.

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Park IK, Lee SG, Choi DH, Park JD, Ahn YJ. (2003) Insecticidal activities of constituents identified in the essential oil from leaves of Chamaecyparis obtuse against Callosobruchus chinensis (L.) and Sitophilus oryzae (L.). J Stored Prod Res 39:375–384. Pascual-Villalobos MJ, Ballesta-Acosta MC. (2003) Chemical variation in an Ocimum basilicum germplasm collection and activity of the essential oils on Callosobruchus maculatus. Biochem System Ecol 31:673–679. Paster N, Menasherov M, Ravid U, Juven B. (1995) Antifungal activity of oregano and thyme essential oils applied as fumigants against fungi attacking stored grain. J Food Protect 58:81–85. Perrucci S. (1995) Acaricidal activity of some essential oils and their constituents against Tyrophagus longior, a mite of stored food. J Food Protect 58:560–563. Perrucci S, Macchioni G, Gioni PL, Flamini G, Morelli I. (1995) Structure/activity relationship of some natural monoterpenes as acaricides against Psoroptes cuniculi. J Nat Prod 58:1261–1264. Peterson C, Coats JR. (2001) Insect repellents—past, present and future. Pestic Outlook 12:154–158. Petroski RJ, Hammack L. (1998) Structure activity relationships of phenyl alkyl alcohols, phenyl alkyl amines, and cinnamyl alcohol derivatives as attractants for adult corn rootworm (Coleoptera: Chyrsomelidae: Diabrotica spp.). Environ Entomol 27:688–694. Philoge`ne BJR, Regnault-Roger C, Vincent C. (2005) Botanicals: yesterday’s and today’s promises. In: Regnault-Roger C, Philoge`ne BJR, Vincent C, editors. Biopesticides of plant origin. Paris, France: Lavoisier, pp. 1–15. Regnault-Roger C, Hamraoui A, Holeman M, Theron E, Pinel R. (1993) Insecticidal effect of essential oils from Mediterranean plants upon Acanthoscelides obtectus Say (Coleoptera, Bruchidae), a pest of kidney bean (Phaseolus vulgaris L.). J Chem Ecol 19:1233–1244. Regnault-Roger C, Jamraoui A. (1994) Inhibition of reproduction of Acanthoscelides obtectus Say (Coleoptera), a kidney bean (Phaseolus vulgaris) bruchid, by aromatic essential oils. Crop Protect 13:624–628. Rice PJ, Coats J. (1994b) Insecticidal properties of monoterpenoid derivatives to the house fly (Dipera: Muscidae) and red flour beetle (Coleoptera: Tenebrionidae). Pestic Sci 41:195–202. Rice PJ, Coats JR. (1994a) Insecticidal properties of several monoterpenoids to the house fly (Diptera: Muscidae), red flour beetle (Coleoptera: Tenebrionidae), and southern corn rootworm (Coleoptera: Chrysomelidae). J Econ Entomol 87:1172–1179. Ryan MF, Byrne O. (1988) Plant–insect coevolution and inhibition of acetylcholinesterase. J Chem Ecol 14:1965–1975. Salgueiro LR, Vila R, Tomi F, Figueiredo AC, Barroso JG, Canigueral S, Casanova J, Proenca da Cunha A, Adzet T. (1997) Variability of essential oils of Thymus caespititius from Portugal. Phytochemistry 45:307–311. Sampson BJ, Tabanca N, Kirimer N, Demirci B, Baser KHC, Kahn IA, Spiers JM, Wedge DE. (2005) Insecticidal activity of 23 essential oils and their major compounds against adult Lipaphis pseudobrassicae (Davis) (Aphididae: Homoptera). Pest Manage Sci 61:1122–1128. Sarac A, Tunc I. (1995) Toxicity of essential oil vapours to stored-product insects. J Plant Dis Protect 102:69–74. Shaaya E, Kostjukovsky M. (1998) Efficacy of phyto-oils as contact insecticides and fumigants for the control of stored-product insects. In: Ishaaya I, Degheele D, editors. Insecticides with novel modes of action. Berlin: Springer. Shaaya E, Ravid U, Paster N, Juven B, Zisman U, Pissarev V. (1991) Fumigant toxicity of essential oils against four major stored-product insects. J Chem Ecol 17:499–504. Singh AK, Dikshit A, Sharma ML, Dixit SN. (1980) Fungitoxic activity of some essential oils. Econ Bot 34:186–190. Singh D, Siddiqui MS, Sharma S. (1989) Reproduction retardant and fumigant properties in essential oils against rice weevil (Coleoptera: Curculionidae) in stored wheat. J Econ Entomol 82:727–733. Singh D, Singh AK. (1991) Repellent and insecticidal properties of essential oils against housefly, Musca domestica L. Insect Sci Applications 12:487–491.

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Stroh J, Wan MT, Isman MB, Moul DJ. (1998) Evaluation of the acute toxicity to juvenile Pacific coho salmon and rainbow trout of some plant essential oils, a formulated product, and the carrier. Bull Environ Contam Toxicol 60:923–930. Traboulsi AF, Taoubi K, El-Haj S, Bessiere JM, Rammal S. (2002) Insecticidal properties of essential plant oils against the mosquito Culex pipiens molestus (Diptera: Culicidae). Pest Manage Sci 58:491–495. Tsao R, Lee S, Rice PJ, Jensen C, Coats JR. (1995) Monoterpenoids and their synthetic derivatives as leads for new insect-control agents. Am Chem Soc Symp Ser 584:312–324. Tsao R, Zhou T. (2000) Antifungal activity of monoterpenoids against postharvest pathogens Botrytis cinerea and Monilinia fructicola. J Essential Oil Res 12:113–121. Vargas RI, Stark JD, Kido MH, Ketter HM, Whitehand LC. (2000) Methyl eugenol and cuelure traps for suppression of male oriental fruit flies and melon flies (Diptera: Tephritidae) in Hawaii: effects of lure mixtures and weathering. J Econ Entomol 93:81–87. Weaver DK, Dunkel FV, van Puyvelde L, Richards DC, Fitzgerald GW. (1994) Toxicity and protectant potential of the essential oil of Tetradenia riparia (Lamiales, Lamiaceae) against Zabrotes subfasciatus (Col., Bruchidae) infesting dried pinto beans (Fabales, Leguminosae). J Appl Entomol 118:179–196. Wilson CL, Solar JM, El Ghaouth A, Wisniewski ME. (1997) Rapid evaluation of plant extracts and essential oils for antifungal activity against Botrytis cinerea. Plant Dis 81:204–210. Yatagai M. (1997) Miticial activity of tree terpenes. Curr Top Phytochem 1:85–97. Zambonelli A, D’Aulerjo AZ, Bianchi A, Albasini A. (1996) Effects of essential oils on phytopathogenic fungi in vitro. J Phytopathol 144:491–494. Zhu BCR, Henderson G, Yu Y, Laine RA. (2003) Toxicity and repellency of patchouli oil and patchouli alcohol against Formosan subterranean termites Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). J Agric Food Chem 51:4585–4588.


45

CHAPTER 3

Natural substrates and inhibitors of multidrug resistant pumps (MDRs) redefine the plant antimicrobials GEORGE P TEGOS

Introduction Efflux mechanisms have become broadly recognized as major components of resistance to many classes of antibiotics. Some efflux pumps selectively extrude specific antibiotics, while others referred to as multidrug resistance pumps (MDRs) expel a variety of structurally diverse compounds with differing modes of action. Based on sequence similarity, multidrug transporter systems are classified into six super-families (Paulsen et al., 2002) (i) ATP binding cassette transporters (ABC), (ii) major facilitators (MF), (iii) resistance nodulation division (RND), (iv) small multidrug resistance (SMR), (v) multidrug and toxic compound extrusion (MATE), and (vi) multidrug endosomal transporter (MET) family. The first five families of multidrug systems are found mostly in microorganisms (the MET family appears restricted to higher eukaryotes) but representatives of all these families are also present in mammalian cells. Multidrug resistant human pathogenic microorganisms are directly associated with serious recalcitrant infections such as cystic fibrosis, nosocomial infections, and infections in immunocompromised patients undergoing anticancer chemotherapy or infected with HIV. A novel and promising approach to deal with multidrug resistance is improving the clinical performance of various antibiotics by the inhibition of efflux pumps. Many companies and research laboratories have undertaken programs to discover and develop efflux pump inhibitors and in many cases their attempts were successful. In addition, it was clearly demonstrated that an efflux pump inhibitor (reserpine) inhibits effectively not only the bacterial but the mammalian MDR ABC-transporter P-glycoprotein (P-gp) as well (Beck et al., 1988; Wang et al., 2001), thus implying that the development of broad range MDR inhibitors is not a chimera. Plants produce an enormous array of secondary metabolites and it is commonly accepted that a significant part of this chemical diversity serves to protect plants against microbial pathogens (Dixon, 2001). These plant substances are classified as either phytoanticipins, compounds present constitutively, or phytoalexins, whose

46


levels increase strongly in response to microbial invasion (Morrissey and Osbourn, 1999). Plant compounds are routinely classified as ‘‘antimicrobials’’ on the basis of susceptibility tests that produce minimal inhibitory concentrations (MICs) in the range of 100–1000 mg/ml, orders of magnitude weaker than typical antibiotics produced by bacteria and fungi (MICs of 0.01–1 mg/ml) (Cowan, 1999). Simply because a compound is synthesized in response to pathogen invasion, is required for protecting the plant from a pathogen but shows little activity in an in vitro susceptibility test it is not necessarily an antimicrobial. Such a substance might have a regulatory function, which indirectly increases plant resistance. This analysis suggests that we currently lack a solid rationale for making a functional assignment for the vast majority of plant compounds that have been classified as antimicrobials. Helpful evidence regarding the possible function of plant secondary metabolites is that these compounds often show considerable activity against Gram-positive bacteria, but not against Gram-negative species or yeast (Lewis et al., 2001). Both yeast and Gram-negative bacteria have evolved significant permeability barriers. Gram-negative species have an outer membrane that is a fairly effective barrier for amphipathic compounds and a set of MDRs that extrude amphipathic toxins across the outer membrane. The single membrane of Gram-positive bacteria is considerably more accessible to permeation by amphipathic toxins in regions where MDRs provide limited protection. In yeast, the presence of ergosterol, which decreases permeability, combined with a set of broad-specificity MDRs also provides an effective barrier (Lomovskaya and Lewis, 1992; Nikaido, 1999; Lewis, 2001). Several Gram-positive bacteria invade plants, but the majority of plant pathogens are Gramnegative bacteria, or yeast and related fungi (Rogers et al., 2001). In this chapter we shall emphasize the rationale for characterization of natural substrates of MDR pumps, reporting the identification of the first MDR substrates and inhibitors from plants against bacteria and yeasts. We shall discuss the methodology for the isolation of these compounds as well as the specificity of their activity. Finally we shall analyze the current and future perspectives to natural drug discovery based on this classification of plant antimicrobials.

The quest for natural substrates – rationale Even though MDR pumps extrude structurally unrelated compounds, a general theme emerges if one considers the preferred artificial substrates of most MDRs. These substances are mostly amphipathic cations. It has been suggested that amphipathic cations represent the existing natural substrates of MDR pumps (Lewis, 1999). The simplest MDRs of the SMR family have amphipathic cations as their exclusive substrates, at least in Gram-positive bacteria (Chung and Saier, 2001). Most of the MDR members of the MF super-family extrude exclusively amphipathic cations. The QacA pump only extrudes cations (Mitchell et al., 1998); the NorA pump of the human pathogen Staphylococcus aureus extrudes cations and some quinolones (Kaatz et al., 2000); the BMR pump of Bacillus subtilis extrudes primarily cations and neutral chloramphenicol (Van Bambeke et al., 2000; VazquezLaslop et al., 2000). RND super-family MDRs have a broad substrate spectrum, including amphipathic cations (Elkins and Nikaido, 2002; Mao et al., 2002; Murata

Natural substrates and inhibitors of MDRs

47

et al., 2002) that are preferential substrates in the case of ABC transporters (Lage, 2003). Simple MDRs like SMRs only export amphipathic cations (Krupka, 1999); MF and MATE MDRs export mainly cations; RND and ABC have broad spectra of specificity that include amphipathic cations as preferred substrates. It appears that a similar need to protect the cell from amphipathic cations evolved in different groups of MDRs (and in different organisms) in spite of a lack of overall homology or similarity in the action mechanism. Quite surprisingly, one does not find amphipathic cations in a general list of natural antimicrobials. (The known cationic antibiotics of the aminoglycoside group such as streptomycin and kanamycin are hydrophilic substances that get smuggled into the cell via specific translocases and are not general substrates of MDRs).

MDR inhibitors – proof of principle Research efforts in a series of academic and industrial laboratories led to the identification of numerous inhibitors of the NorA pump of S. aureus (Aeschlimann et al., 1999; Markham et al., 1999; Gibbons et al., 2003; Kaatz et al., 2003) and MexABOprM, MexCD-OprJ, MexEF-OprN MDRs of Pseudomonas aeruginosa (Renau et al., 1999; Lomovskaya et al., 2001). The inhibitors INF271 (NorA-Influx Co., Chicago, Illinois) and MC207110 (MexAB-OprM, MexCD-OprJ, MexEF-OprN P. aeruginosa Essential Therapautics-Mountain View, CA) have been extremely valuable tools for drug discovery. INF271 identified by screening a synthetic chemical library using ethidium bromide (EtBr) as a substrate in a sub-inhibitory concentration. On the contrary, in the case of MC207110 an array of synthetic compounds and natural product extracts was assayed using strains of P. aeruginosa over-expressing each of the three pumps in the presence of levofloxacin. To qualify as MDR inhibitors hit compounds had to meet several criteria: (i) must enhance the activities of levofloxacin and other antibiotics that are effluxes in strains containing functioning pumps, (ii) must not significantly potentiate the activities of antibiotics in a strain that lacks efflux pumps, (iii) must not potentiate the activities of antibiotics that are not effluxes, (iv) must increase the level of accumulation and decrease the level of extrusion of efflux pump substrates, and (v) must not affect the proton gradient across the inner membrane (Table 1).

MDR mutants and inhibitors valuable tools for natural drug discovery It seems plausible that MDRs evolved in response to natural antimicrobial amphipathic cations and this fact makes these substances difficult to discover in standard screens that employ cells carrying MDR pumps. The development of MDR mutants for the NorA of S. aureus gives an answer to this apparent paradox (Hsieh et al., 1998) and serves as a sensitive tool for drug discovery. The plant isoquinoline alkaloids, berberine, and palmatine resemble artificial MDR substrates such as EtBr and are widely spread among the plant world especially in species of Ranunculales (Schmeller et al., 1997). A susceptibility test to identify MICs for these compounds against the wild type and the NorA mutant of S. aureus was performed. Palmatine


48 Table 1 Natural MDR inhibitors H N

O HN

H2 N

OMe O

O

H N

N H

O

HO

O

NH

OH OMe

O

OH

MeO

N H

INF271

NH2

OH O

MC207110

OMe

5-MHC Berberis sp., Mahonia sp. OH

O

MeO MeO OH

Pheophorbide a

R4O

OR5

2'

3' rha 1' 4'

R3O

OR OMe

O

Chrysoplenol D /Chrysoplenetin Artemisia annua

HO 3 2

O O

OR2 glu 4 1

O

5'

CH3

5

O 6

OR1

6'

1: R1 = R2 = R4 = R5 = H; R3 = CO(CH2)14 CH3 2: R1= -CO(CH2)14CH3; R2 = R4 = R5 = H; R3 = -COPr 3: R1 = R2 = R3 = -COiPr; R4 = -COPr 4: R1 = R2 = R3 = -COiPr; R4 = OAc; R5 = COPr

Neohesperidosides Geranium caespitosum

Dalea versicolor

Dalea versicolor

has very low (>200 mg/ml MIC) and berberine poor (250 mg/ml) activity against wild type S. aureus. The antimicrobial activity of the alkaloids increased sharply in a norA mutant, with an MIC of 50 mg/ml for palmatine and 7.5 mg/ml for berberine. Berberine resembles EtBr and binds to DNA (Jennings and Ridler, 1983). The DNA binding apparently contributes to the antimicrobial activity of berberine. Similar to EtBr, DNA-bound berberine has increased fluorescence and is used as a model plant antimicrobial for uptake studies. Efflux of berberine was more rapid than EtBr. Similarly, the differential rate of berberine accumulation in both strains indicated that these alkaloids represent the first identified natural plant substrates of the MFS MDRs of S. aureus (Stermitz et al., 2000a). The outer membrane of Gram-negative bacteria is a barrier to a broad range of amphipathic compounds, and to all large molecules, which allows for evolving of broadly specific MDRs that pump their various substrates across this barrier.


49

Similarly, Gram-positive species that lack an outer membrane have MDRs with a reported substrate spectrum largely limited to amphipathic cations. Apart from the usefulness of protecting the cell from this class of compounds that actively accumulate in the cell, the relatively slow permeation of amphipathic cations across the cytoplasmic membrane made the evolution of ‘‘cation-pumping’’ MDRs possible, as compared to neutral compounds of similar polarity.

A new series of promising plant antimicrobials Similar studies aimed at finding the basis of Gram-negative bacterial resistance to plant antimicrobials. In Rhizobium etli, there is an operon activated by a number of plant phytoalexins. The operon appeared to code for an RmrAB MDR pump (Gonzalez-Pasayo and Martinez-Romero, 2000). Mutant rmrAB had a diminished ability for root colonization, and increased susceptibility for phytoalexins naringenin and coumaric acid. However, the difference in susceptibility between wild type and mutant is very small, around 30%. In Agrobacterium tumefaciens, coumestrol, an antifungal phytoalexin of soybeans induces expression of an LfeAB MDR (Palumbo et al., 1998). Mutation in the pump increases accumulation of coumestrol in the pathogen, and the mutant was out competed by the wild type in colonizing the plant. Neither the wild type nor the mutant, however, is sensitive to coumestrol. In a slightly different approach a panel of plant antimicrobials was tested by using a set of bacteria representing the main groups of plant and human pathogens and applying a combination of MDR mutants and MDR inhibitors (Tegos et al., 2002). One of the main observations was the strong potentiation of antimicrobial action against strains with disabled MDRs. The activities of the majority of plant antimicrobials were considerably greater against the Gram-positive bacteria S. aureus and B. megaterium and disabling of the MDRs in Gram-negative species led to a striking increase in antimicrobial activity. Thus, the activity of rhein, the principal antimicrobial from rhubarb (Agarwal et al., 2000), was potentiated 100–2000-fold (depending on the bacterial species) by disabling the MDRs. A similar effect was observed with plumbagin (Didry et al., 1994), resveratrol, gossypol, coumestrol, and berberine. Direct measurement of the uptake of berberine confirmed that disabling of the MDRs strongly increases the level of penetration of berberine into the cells of Gram-negative bacteria (Figure 1). Recently the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16 was identified and characterized (Barabote et al., 2003). The gene encodes a 51-kDa protein with 70% identity to its Escherichia coli homolog. The E. chrysanthemi gene functionally complements the E. coli tolC gene in MDR efflux pumps. A tolC mutant of E. chrysanthemi is extremely sensitive to an array of plant-derived chemicals including berberine, rhein, plumbagin, pyrithione, genistein (40 ,5,7-trihydroxyisoflavone), p-coumaric acid and t-cinnamic acid (phenolic acids), (2-mercaptopyridine-1-oxide) and esculetin (6,7-dihydroxycoumarin). This mutant is unable to grow in planta and its ability to cause plant tissue maceration is severely compromised and is defective in the efflux of berberine. The E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue conferring resistance to plant antimicrobials.


50 200

A

150

RFU

RFU

150

100

50

0

B

200

100

50

0

5

10

15

20

25

0

30

0

5

Time (min)

C

20

25

30

D

200

150

150

RFU

RFU

15

Time (min)

200

100

50

0

10

100

50

0

5

10

15

20

25

30

Time (min)

0

0

5

10

15

20

25

30

Time (min)

200

E

RFU

150

100

50

0

0

5

10

15

20

25

30

Time (min)

Fig. 1. Accumulation of berberine by Gram-negative bacteria. Uptake of berberine without (o) or with MC207110 (i), INF271 (D), MC207110/INF271 (}) by cells of Salmonella typhimurium (A), Pseudomonas syringae (B), Xanthomonas campestris (C), Erwinia carotovora (D), and Sinorhizobium meliloti (E) was measured by fluorescence increase following binding to DNA and expressed as RFU (relative fluorescence units). Berberine was present at a concentration of 30 mg/ml; MC207110 and INF271 were added at the same final concentrations as for the MIC determinations.

Natural MDR inhibitors from plants – an increasing body of evidence, 50 methoxyhydnocarpin and synergy in Berberis plants The rationale behind detecting MDR inhibitory activity is to test the combined action of a plant extract (the nonalkaloid fraction) with a weak plant antimicrobial coming from the same plant added at a sub-inhibitory concentration (Table 2). Extracts that inhibit cell growth in the presence of the antimicrobial and have no


51

Table 2 Identified natural substrates or MDRs O

OCH H3 OCH H3

O

O OH

N +

N +

CH3O OCH3

OMe Berberine

OH O

O

Palmatine O

HO

OH

Plumbagin

O N

O

COOH

SH

O

O

OH

Rhein

Coumestrol

Zink-Pytothione OH

OHC

OH

OH

HO

HO CHO OH

HO

HO

O

O

Me

OH

Me

Resveratrol

Gossypol

Esculetin

O O

O

O

O O

Asarinin

activity when added alone are likely to contain an MDR inhibitor. A bioassay-driven purification is the most common technique used to detect MDR inhibitors. Several Berberis medicinal plants (Berberis repens, B. aquifolia, and B. fremontii) producing berberine were found also to synthesize an inhibitor of the NorA MDR pump S. aureus. The inhibitor 50 -methoxyhydnocarpin (50 -MHC), a flavonolignan, was previously identified in Hydnocarpus wightiana (Flacourtaceae) (Ranganathan and Seshadri, 1974) and reported as a minor component of chaulmoogra oil, a traditional therapy for leprosy (Norton, 1994). 50 -MHC-D is an amphipathic weak acid and is

52


distinctly different from the cationic substrates of NorA. 50 -MHC-D had no antimicrobial activity alone but strongly potentiated the action of berberine and other NorA substrates against S. aureus. MDR-dependent efflux of EtBr and berberine from S. aureus cells was completely inhibited by 50 -MHC-D. The level of accumulation of berberine in the cells was increased strongly in the presence of 50 -MHC-D, indicating that this plant compound effectively disables the bacterial resistance mechanism against berberine (Stermitz et al., 2000a, 2000b). This first identified natural MDR inhibitor is abundant in other Berberis sp. (Musumeci et al., 2003).

Inhibitors against MFS MDRs Identification of 50 -MHC-D intensified the search for natural MDR inhibitors of plant origin and made the idea of synergy in plant antimicrobials more plausible. Bioassay-driven purification and structural determination from various plant sources yielded a number of new MDR inhibitors acting against Gram-positive bacteria with activity similar to 5-MHC pheophorbide a from Berberis sp., and Mahonia (Stermitz et al., 2000a, 2000b, 2001), crysoplenol and crysoplenetin from Artemisia annua (Stermitz et al., 2002), dicaffeoylquinic acids from A. absinthium (Fiamegos et al., 2003), polyacylated neohesperidosides from Geranium caespitosum (Stermitz et al., 2003), diterpenoids from Pinus edulis, (F.R. Stermitz personal communication), and chalcones and a stilbene from Dalea versicolor (Belofsky et al., 2003). Additionally, isoflavones such as genistein, orobol, and biochanin exhibit MDR inhibitory activity against the NorA pump of Gram-positive bacteria (Morel et al., 2003) and aminoquinolines in the AcrAB-TolC pump of E. coli (Mallea et al., 2003).

Inhibitors against ABC transporters P-gp the mammalian ABC transporter is mainly responsible for the unsuccessful chemotherapy of cancers through occurrence of MDR (Leonard et al., 2003). P-gp shares high structural homology with yeast ABC transporters (Saier and Paulsen, 2001). The development of screening methodologies has significantly enhanced the number and the efficiency of available inhibitors (Hiraga et al., 2001; Varma et al., 2003). The inhibitory activity of numerous plant compounds against P-gp such as alkaloids, polymethoxyflavones, diterpenoids, camptothecins (Ulukan and Swaan, 2002), polyphenols (Jodoin et al., 2002), ascorbic acid, vitamins, and others has been extensively studied (Chen and Waxman, 1995; Ikegawa et al., 2000; Foster et al., 2001; Motohashi et al., 2001; Perloff et al., 2001; Deferme et al., 2002; Appendino et al., 2003; Corea et al., 2003; El-Masry and Abou-Donia, 2003). Likewise, many plant sources and products seem quite promising for the identification of new P-gp specific inhibitors: sweet pepper ‘‘Anastasia green’’ (Motohashi et al., 2001), St. John’s Wort (Hypericum perforatum) (Perloff et al., 2001), caper spurge (Euphorbia lathyris) (Appendino et al., 2003), and apricots (Deferme et al., 2003). A novel and interesting inhibitor from G. caespitosum (Figure 2) shows interesting properties. It potentiates berberine in S. aureus, erythromycin in P. syringae pv. maculicola and fluconazole against Saccharomyces cerevisae and Candida albicans.


53

Fig. 2. Geranium caespitosum a paradigm of synergy between plant antimicrobials.

In addition, a structurally similar compound from the same source has no antimicrobial action alone, but combined with the inhibitor is active against P. syringae (a designated pathogen of Geranium sp.) and S. cerevisae (Tegos et al., 2006 (unpublished results)).

Conclusions and future perspectives The experimental data available demonstrate and reassure that plants produce MDR inhibitors against Gram-positive bacteria and yeasts. An effective plant MDR inhibitor of Gram-negative bacteria is still a missing part of the puzzle. Currently, there is only preliminary evidence for this, but the identification of such a compound is more than plausible. A comprehensive library of extracts from the National Cancer Institute (NCI-USA), which consists of over 50,000 extracts from approximately 35,000 plants from all over the world is currently being screened (Tegos et al., 2006 (unpublished results)). The collection represents most genera of vascular plants found in the tropics. The extracts are tested against a panel of representative microorganisms (S. aureus, E. coli, S. cerevisiae) for direct antimicrobial activity. Furthermore, they are tested for MDR inhibitory activity with a sub-inhibitory amount of antibiotic. A pilot screen produced extremely promising hit rates for direct and MDR antimicrobial activity (Figure 3). It is significant to find MDR inhibitory activity against Gram-negative bacteria of which no known natural inhibitors have


54 0.19 0.57 0.76

0.57 0.38 0.19

1.32 2.65 6.25 7.95 10.41

68.75

Inactive

S. cerevisae[MDR]

S. aureus[D], E. coli[D]

S. aureus [D]

S. aureus[D], S. cerevisae[D][D]

S. aureus[D], E.coli[MDR]

S.aureus[MDR]

S. aureus[D], S. cerevisae[MDR]]

S. aureus[MDR], E. coli[MDR]]

S. serevisae [D]

S. aureus[MDR], S. cerevisae[MDR]]

S. aureus[D], E. coli[D], S.cerevisae [MDR]

Fig. 3. Hit rates of extracts from the Natural Products Repository Program (National Cancer Institute-USA, http://dtp.nci.nih.gov/branches/npb/repository.html). A pilot screen of 524 extracts (352 organic/176 aqueous) produced very promising hit rates for the identification of new MDR inhibitors and plant antimicrobials (see text). The majority of hits came from organic extracts. [D] – Direct, [MDR] – MDR-inhibitory activity.

been identified. Extracts with MDR inhibitory activity and without direct detectable activity suggest that antimicrobials from these plants exist. Interestingly, a number of instances exist where a specific part of the same plant produced direct activity against S. aureus; while an extract from another part exhibits MDR inhibitory activity. Closer examination may demonstrate both activities operating at different levels in the entire plant. Similar examples of synergistic activities coming from the same plant are observed in the case of E. coli and S. cerevisiae. The potential activity of plant antimicrobials when they gain access to the microbial cell is only part of the intriguing puzzle. Even if a plant uses a synergistic combination of an antimicrobial and an MDR inhibitor, this does not in itself explain why plants do not come up with self-sufficient antibiotics like aminoglycosides or tetracycline. Plants are stationary, slowly evolving organisms that confront mobile, rapidly evolving pathogens. Resistance to antibiotics such as streptomycin or tetracycline can be gained by a single mutation leading to target modification, or to over-expression of an MDR pump (Lewis, 2001). It is possible that plants employ an antimicrobial strategy aimed at limiting the probability of resistance development. This is apparently the case when plants produce MDR inhibitors, directly targeting the microbial resistance mechanism. Importantly, disabling MDRs also results in a dramatic decrease in acquisition of resistance based on target modification (Zheleznova et al., 1999; Lomovskaya and Watkins, 2001; Markham and Neyfakh, 2001; Lomovskaya et al., 2003). There is another intriguing possibility that plants may employ to limit resistance development. Resistance will not be easily achieved against a substance that hits


55

more than one target. Apparent low activity of an antimicrobial could then be a result of a trade-off – penetration capability is sacrificed in favor of additional functionality, an ability to hit two different targets, while access to the pathogen cell is ensured by a separate molecule, an MDR inhibitor. Do plant antimicrobials hit more than one target? Answering this question might be highly significant for combating the rapid rise of multidrug resistance. Antimicrobials able to act against more than one target would be extremely valuable as therapeutics. Interestingly, a large-scale ‘‘experiment’’ on resistance development to this type of compounds has been performed unwittingly. Synthetic fluoroquinolones act against two different targets – DNA gyrase and topoisomerase (Hooper, 2001, 2002). Resistance by target modification occurs in a series of steps and is a low-probability event. It was shown that blocking MDR pumps of pathogens by an inhibitor decreases resistant mutation development to fluoroquinolones below the limit of detection (o1011). Blocking MDRs increases the effective concentration of fluoroquinolone in the cell and makes the first step in the target modification process ineffective. In retrospect, this means that were fluoroquinolones applied originally in combination with an MDR inhibitor, we might have had a case of largely resistance-proof therapy. It is too late for fluoroquinolones, the resistance to this class of compounds has already developed. But we might still have a chance, or many chances, with potentially dual/multiple target acting plant antimicrobials combined with an MDR inhibitor. There is not sufficient information concerning the plant antimicrobial action mechanisms. Some useful indirect evidence, however, does point to more than one target for at least some compounds. Rhein is effective at mg/ml levels against yeast and Gram-positive bacteria (Cyong et al., 1987; Hatano et al., 1999) and was found specifically to inhibit NADH dehydrogenase in mitochondria (Kean et al., 1970). It is assumed that microbial NADH dehydrogenase is the target of action. At the same time, rhein was reported to be very effective against Gram-positive oral pathogens under anaerobic conditions (Didry et al., 1994) and is very effective against E. coli tolC (defective in major MDRs) under both aerobic and anaerobic conditions. Since NADH dehydrogenase in not functional under anaerobiosis, there should be a second target. Rhein is currently used as a systemic drug against arthritis-administered in the form of a prodrug diacerein (Spencer and Wilde, 1997).

Acknowledgments The author thanks Mahendra Rai for his invitation to participate in this text as well as Kim Lewis (Northeastern University) for continuous support and critical supervision and Frank R. Stermitz (Colorado State University) as a colleague and a mentor in the organic chemistry of natural products. Most of the author’s work was carried out in Kim Lewis Molecular Microbiology Laboratory, Department of Biology at Northeastern University and supported by the NIH grant RO1GM59903 (In search of Natural Substrates and Inhibitors of MDR pumps). Gil Belofsky (University of Tulsa) and John Fiamegos (University of Ioannina) offered valuable feedback information. Susan Harvey and Jennifer Freedman provided significant help revising this manuscript.

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Stermitz FR, Cashman K, Halligan KM, Morel C, Tegos GP, Lewis K. (2003) Polyacylated neohesperidosides from Geranium caespitosum: bacterial multidrug resistance pump inhibitors. Bioorg Med Chem Lett 13:1915–1918. Stermitz FR, Lorenz P, Tawara JN, Zenewicz LA, Lewis K. (2000a) Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 50 -methoxyhydnocarpin, a multidrug pump inhibitor. Proc Natl Acad Sci USA 97:1433–1437. Stermitz FR, Scriven LN, Tegos G, Lewis K. (2002) Two flavonols from Artemisa annua which potentiate the activity of berberine and norfloxacin against a resistant strain of Staphylococcus aureus. Planta Med 68:1140–1141. Stermitz FR, Tawara-Matsuda J, Lorenz P, Mueller P, Zenewicz L, Lewis K. (2000b) 50 -Methoxyhydnocarpin-D and pheophorbide A: Berberis species components that potentiate berberine growth inhibition of resistant Staphylococcus aureus. J Nat Prod 63:1146–1149. Tegos G, Higginbotham A, Ball A, Stermitz FR, Lewis K. (2006) Neohesperidosides from Geranium caespitosum; versatile multidrug pump inhibitors Chemistry & Biology, submitted. Tegos G, Stermitz FR, Lomovskaya O, Lewis K. (2002) Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials. Antimicrob Agents Chemother 46:3133–3141. Ulukan H, Swaan PS. (2002) Camptothecins: a review of their chemotherapeutic potential. Drugs 62:2039–2057. Van Bambeke F, Balzi E, Tulkens PM. (2000) Antibiotic efflux pumps. Biochem Pharmacol 60:457–470. Varma MV, Ashokraj Y, Dey CS, Panchagnula R. (2003) P-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement. Pharmacol Res 48:347–359. Vazquez-Laslop N, Zheleznova EE, Markham PN, Brennan RG, Neyfakh AA. (2000) Recognition of multiple drugs by a single protein: a trivial solution of an old paradox. Biochem Soc Trans 28:517–520. Wang EJ, Casciano C, Clement RP, Johnson WW. (2001) Active transport of fluorescent P-glycoprotein substrates: evaluation as markers and interaction with inhibitors. Biochem Biophys Res Commun 289:580–585. Zheleznova EE, Markham PN, Neyfakh AA, Brennan RG. (1999) Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter. Cell 96:353–362.


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61

CHAPTER 4

New concept to search for alternate insect control agents from plants ISAO KUBO

Introduction There is no doubt that many plant secondary metabolites affect insect behavior, development and reproduction. Identifying these substances is an important first step in understanding the effect of plants on insect life at the molecular level. Besides its benefits to basic science, accumulation of this kind of knowledge may provide us with a more rational and scientific approach to insect pest control. Continuing search for alternate insect control agents from plants resulted in characterization of many active phytochemicals. Despite their excellent and interesting activities, the use of these botanical insect control agents is still limited. The reason for this limitation is caused mainly by the lack of availability of materials and/or complex molecules for synthetic optimization. In addition, plant defenses to insect attack usually involve multifunctional multichemicals (Kubo and Hanke, 1985). Preliminary artificial diet-feeding assay has indicated that the methanol extract of a medicinal plant Fuchsia tetradactyla (Onagraceae) contains two hydrolyzable tannins in varying quantities. Both tannins inhibited Pectinophora gossypiella larvae when ingested in small amounts. P. gossypiella larvae became much smaller in size from the control due to the ingestion of the tannins, and this activity is likely due to the prooxidant action. Tannins are estimated to be the fourth most abundant biochemical produced by vascular plant tissue after cellulose, hemicellulose and lignin. Tannins are known to play a role in a number of ecological processes in addition to herbivore defense. Because tannins are complex and energetically costly molecules to synthesize, their widespread occurrence and abundance suggests that application of tannins as insect control agents needs to be investigated from practical points of view rather than as leading compounds for synthetic optimization. In addition, both mosquito larvicidal triterpene saponins isolated from the root bark of Pachyelasma tessmannii (Leguminosae) (Nihei et al., 2005) and insect antifeedant limonoids characterized from Croton jatrophoides (Euphorbiaceae) (Kubo et al., 1990; Nihei et al., 2002; 2004) are other examples. The saponins isolated from

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P. tessmannii were found to exhibit potent mosquito larvicidal activity. However, these triterpene saponins have not been further studied as practical mosquito control agents because of the lack of availability. The limonoids isolated from C. jatrophoides showed potent antifeedant activity against two lepidopteran larvae: pink bollworm, P. gossypiella; and fall armyworm, Spodoptera frugiperda. However, these limonoids are complex and energetically costly molecules to synthesize, and are available in minute amounts. Hence, the application of natural products as alternate insect control agents needs to be investigated from a different aspect. The data have been reported as the result of sporadic research, so it is timely to review and synthesize in one paper all that is currently known.

Tannins Gallae Rhois is a unique natural medicine that is used in the manufacture of tannin, tannic acid and gallic acid. It is a kind of gall produced by the gall-forming aphid Melaphis chinensis on the leaves of Rhus javanica. Eggs of M. chinensis are artificially deposited on the fresh leaves of R. javanica to make commercial Gallae Rhois. After the aphid is colonized, the plant tissue undergoes rapid cell division and enlargement. The aphid, during the wingless stage of its complex life cycle, forms galls along the midrib of the leaves from which the aphid parthenogenetically produces winged offspring. The galls are highly irregular and lobed shape, grayish-brown in color, and have a thin but hard wall and large hollow interior (Figure 1). The wall is the source of gallic acid, tannin and tannic acid, and used as the commercial Gallae Rhois. This gall formation may also be a plant defense mechanism to minimize its damage by aphid invasion. The cavity contains numerous dead winged aphid bodies in white

Fig. 1. Gallae Rhois.

New concept to search for alternate insect control agents

63

powder that was removed prior to commercial extraction of tannins. It should be noted that the dead aphid bodies are a result of the heating process of Gallae Rhois before their escape. There has been little study to verify the biological activities and chemical analysis of this white powder. In a preliminary thin-layer chromatography (TLC) analysis, this white powder was found to contain mainly two types of chemicals: both nonpolar and polar compounds without anything in between. Non-polar compounds were found to consist mainly of fatty acids and di- and tri-glycerides by gas chromatography–mass spectroscopy (GC–MS) analysis. Interestingly, the ethanol extract of the white powder was found to exhibit growth inhibitory activity against the pink bollworm P. gossypiella in an artificial diet-feeding assay (Chan et al., 1978; Kubo, 1993). The ethanol extract of the commercial Gallae Rhois showed much more potent activity. Plants can defend themselves against insect attack by means of their secondary metabolites, which interfere with many biological activities. Since there is no way to anticipate a priori what effects specifically involve in the growth inhibitory activity against P. gossypiella larvae, the artificial diet-feeding assay (Kubo, 1991) was performed to detect as much of these activities as possible. Importantly, careful observation of the insects throughout the assay is essential. Any difference from the control should not be overlooked (Kubo, 1993). In the current experiment, P. gossypiella larvae became much smaller in size from the control caused by the ingestion of the ethanol extract, but any additional noticeable difference was not observed. Fractionation of the ethanol extract of the white powder of Gallae Rhois was guided by the artificial diet-feeding assay against the pink bollworm. Successive partitioning of the crude extract with n-hexane, CH2Cl2, EtOAc and H2O revealed the biological activity to be retained in the EtOAc layer. Further fractionation of the EtOAc fraction, using droplet counter-current chromatography (DCCC), gave three major compounds. Further purification with Sephadex LH-20 column chromatography gave three purified phenolic compounds (1–3) (see Figure 2 for structures). The 1H-NMR (nuclear magnetic resonance) spectrum of 1 is rather unusual in that it shows only a single peak, corresponding to the aromatic protons near 6.9 ppm, and the other two peaks present are due to the solvent (DMSO and H2O). The simplicity of the spectra reveals the highly symmetrical nature of the compound. The 13 C-NMR data confirm that compound (1) corresponds to gallic acid. Confirmation was also obtained by comparing 1 with an authentic sample. The 1H-NMR spectra of the two remaining compounds, 2 and 3, show the presence of a methyl and ethyl

1, R=H 2, R=CH 3 3, R=CH 2CH3

Fig. 2. Structures of gallic acid and its methyl and ethyl esters.

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group, respectively. In conjunction with their 13C-NMR and EI-MS data, they are characterized respectively as methyl gallate (2) and ethyl gallate (3). Since ethanol was used in the initial extraction, the latter compound is probably an artifact. This was confirmed by the fact that ethyl gallate was not detected when methanol was used for extraction. Similarly, methyl gallate may also be an artifact since methanol was used as a solvent during purification procedure, although this was not confirmed. As a result, the polar part of the ethanol-extractable fraction of the white powder probably consists almost exclusively of gallic acid (Kubo et al., 2003). Interestingly, gallic acid characterized in the white powder could not be detected in the fresh leaves of the original plant R. javanica by high-performance liquid chromatography (HPLC), prior to attack by the gall-forming aphid M. chinensis. In contrast, hydrolyzable tannin is abundant in the fresh leaves. More importantly, this tannin rapidly gets accumulated in the wall after an attack by M. chinensis and produces Gallae Rhois. This process can be termed as a proteinase inhibitor inducing factor (PIIF) (Ryan, 1979), since the aqueous extract of this Gallae Rhois much more strongly inhibited two digestive proteinases tested, chymotrypsin and leucineaminopeptidase, than that of the fresh leaves of R. javanica. It may be logical to assume that gallic acid was derived from hydrolyzable tannin during the gall-forming process in R. javanica by the insect, or by accompanying microorganisms within the parasites’ body, or the plant itself. It appears, therefore, that the releasing mechanism of gallic acid may be one of the key processes to understanding plant defense and/or gall formation. It should be pointed out that the ethanol extract of the white powder did not significantly inhibit the aforementioned two digestive enzymes. The chemicals involved in gall formation are undoubtedly numerous. However, as an initial step to understanding this process, we focused the current study on the biological activity of gallic acid, especially its effects on insects. To facilitate it, tannic acid isolated from Gallae Rhois was also studied for comparison. It should be noted that the structure of tannic acid has not yet been established, although it was reported as the mixture of at least seven related galloylglucoses by HPLC analysis. The basic structure is 1,2,3,4,6-penta-O-galloyl-b-D-glucose to which the depside galloyl group is randomly distributed among C-2, C-3 and C-4 positions (Nishioka, 1983). In the artificial diet-feeding assay against P. gossypiella larvae, both gallic acid and tannic acid showed growth inhibitory activity and the ED50 of gallic acid was found to be 2000 ppm while that of tannic acid was 50 ppm. In other words, tannic acid showed 40-fold more potent growth inhibitory activity in this feeding assay, indicating that P. gossypiella larvae are sensitive to ingested tannic acid but somewhat tolerate gallic acid. It appears that the gallic acid characterized is responsible for the original insect growth inhibitory activity of the ethanol extract of the white powder observed in the preliminary screening. Similarly, tannic acid is responsible for that of the Gallae Rhois ethanol extract. It should be borne in mind that plant secondary metabolites can act in general by a variety of different mechanisms in insects. The sensitivity of P. gossypiella to ingested tannic acid may be a consequence of its extensive chemical modification in the midgut and oxidation is the first thinkable chemical modification. If so, quinones should be the first oxidized structure. Needless to say, quinones are usually highly toxic to insects as well as to many other organisms. For example, quinones may readily react with biologically important


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nucleophilic groups such as sulfhydryl, amino or hydroxyl. Sulfhydryl groups in proteins and lower molecular weight compounds such as glutathione are known to play an important role. The quinones may cause depletion of cytoplasmic and mitochondrial glutathione which functions in eliminating reactive oxygen species (ROS). This depletion can be caused by a direct interaction between the enone moiety and the sulfhydryl group of glutathione by a Michael-type addition. Neither benzoic acid nor 2,4-dihydroxybenzoic acid (b-rosorcylic acid) inhibited insect growth. The result obtained is consistent with the previous work (Elliger et al., 1980). This observation may support that the oxidized products are responsible for the activity. In addition, oxidation also produces ROS in the gut lumen, and this ROS damages biological systems (Pardini, 1995). In fact, alkyl gallates are known to induce ROS production thereby triggering apoptosis pathway in the human leukemia HL60RG cells (Serrano et al., 1998; Sakaguchi, 1999). It seems that the detrimental effects of tannic acid on the non-adapted pink bollworm are the result of ineffective defenses in a consequence of chemical modification in the midgut and/or against ROS generated in the gut lumen (Barbehenn and Martin, 1994; Barbehenn et al., 1996). The possibility that their adverse effects are a consequence of their potential to act as a prooxidant may need to be considered. In fact, gallic acid is known to produce superoxide anion (Serrano et al., 1998). Furthermore, tannic acid may somewhat irreversibly inactivate enzymes (proteins) in the midgut by cross-links, a process known as tanning, prior to being oxidized. This process also needs to be taken into consideration since tannic acid may bind with proteins in the gut and, as a result, inhibit digestive enzymes as well as protein digestion (Feeny, 1976). This can be supported by the observation that tannic acid showed significant inhibitory activity against fungal protease. At the concentration of 3 mg/mL, tannic acid inhibited the enzyme activity 68%, while gallic acid did not show any inhibitory activity up to 6 mg/mL. In addition, tannic acid inhibited the two digestive enzymes, chymotrypsin and leucine-aminopeptidase. Gallic acid also inhibited the same enzymes but much weaker compared to tannic acid. In connection with this, tannic acid is also known to form complexes with heavy metal ions (Scalbert, 1991). For example, gallic acid is known to inhibit iron absorption to the same extent as tannic acid, per mol galloyl groups (Brune et al., 1989) (Table 1). The possibility of involving tanning effect is unlikely but cannot be entirely ruled out since tannic acid is relatively stable compared to other hydrolyzable tannins such as agrimoniin and davidiin (Kubo et al., 1995). Despite numerous investigations, the precise mechanisms of tannin–herbivore interactions are still largely unknown.

Table 1 Scavenging activity of gallic acid and tannic acid Compounds tested Gallic acid Methyl gallate Ethyl gallate Tannic acid

DPPH consumption 6.5170.30 6.3470.12 6.1870.18 78.874.8

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On the other hand, tannic acid isolated from Gallae Rhois was found to inhibit tyrosinase activity with an IC50 of 19 mM that is about 230-fold more potent than that of gallic acid (Kubo et al., 2003). Tyrosinase (EC 1.14.18.1), also known as phenol oxidase (PO), is a copper-containing enzyme widely distributed in microorganisms, animals and plants. In our continuing search for alternative insect control agents from plants (Kubo, 1993), tyrosinase inhibitors have recently been targeted (Kubo, 1997) because tyrosinase is one of the key enzymes in the insect-molting process (Andersen, 1979, 1990). Hence, tyrosinase inhibitors might ultimately provide clues to control insect pests by inhibiting tyrosinase, resulting in incomplete cuticle hardening and darkening (Kramer and Hopkins, 1987). For example, this enzyme is previously reported to highly correlate with aphid resistance of the Solanum plants (Ryan et al., 1982). Finding alternate insect control agents by inhibiting tyrosinase is one of our goals in our continuing search for tyrosinase inhibitors from plants. However, the in vitro results using fungal tyrosinase described is still far from our goal. For example, mushroom tyrosinase used for the initial screening differs somewhat from insects (Andersen, 1990). More importantly, it has not been intentionally overlooked but, as a result, the dynamic function of tyrosinase in insect cuticle has not been thoughtfully taken into account. Thus, tyrosinase does not always exist as the active form in insects (Gupta et al., 2005) and also its inhibitors cannot always reach to tyrosinase in cuticle in sufficient concentration. Tannic acid is a rather bulky polar compound and hence, it unlikely penetrates insect cuticle. Moreover, the reaction time and the amount of available oxygen need to be considered from a practical point of view since insect tyrosinase is an aerobic oxidase. As far as the artificial diet-feeding assay using P. gossypiella larvae is concerned, some tyrosinase inhibitors characterized inhibit insect growth but some do not. The fact that plant secondary metabolites affect by a variety of different mechanisms in insects needs to be kept in mind. The relevance of the results of in vitro experiments in simplified systems to the in vivo situation should be carefully considered. The further study using more appropriate bioassay methods (Londershausen et al., 1996) is currently under investigation (Li and Kubo, 2004). The gall-forming process in which the aphid M. chinensis makes the white powder still cannot be clearly explained on a molecular level, but it may not be illogical to assume that gallic acid is an end product resulting from the defense of the gallforming aphid against the induced defense mechanisms of R. javanica. This may be supported by the previous report that gallic acid is known to be the metabolite of tannic acid when the latter acid was administered to insects (Bernays and Chamberlain, 1980) and sheep (Zhu et al., 1995) orally. Although the effects of tannic acid against M. chinensis could not be directly tested, it should be reasonable to conclude that this aphid can detoxify, at least in part, ingested toxic tannic acid to relatively non-toxic gallic acid. This ability to tolerate ingested tannic acid may rely on its rapid and extensive hydrolysis, similar to being reported to the tolerance of polyphagous grasshoppers (Bernays and Chamberlain, 1980). This can be supported by the finding of gallic acid in large quantities in the white powder, indicating that hydrolysis is the predominant of possible chemical fates of ingested tannic acid. It seems that tannic acid plays an important role in the defense mechanisms of R. javanica as an antimetabolite, resulting from PIIF. Nevertheless, understanding the


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gall-forming mechanism or the defense mechanisms of R. javanica needs to be investigated not only from one aspect but also from a whole and dynamic perspective. In our earlier studies, gallic acid and its methyl ester were often characterized in large quantities from several plants such as a Guatemalan medicinal plant F. tetradactyla (Onagraceae) locally known as ‘‘patayuc’’ and Bersama abyssinica (Melianthaceae) (Kubo and Matsumoto, 1985). However, their weak inhibitory activity against insect’s growth was usually overlooked in the presence of more potent active compounds (Kubo and Matsumoto, 1984). On the other hand, tannins have seen limited success as speciality chemicals. Previous attempts to develop tanninbased chemicals have suffered because of our failure to appreciate fully economic requirements, purity and stability factors and application needs of industrial process. In addition, tannins may not be good leading compounds to develop more effective insect control agents through synthetic optimization because of their complicate structures. Thus, chemical structures of tannins were started to clarify not long ago but the mechanisms of tannin–herbivore interactions has not yet been much studied, especially on a molecular basis. However, tannins are still available as pest control agents for individual farmers, and need to be investigated from more practical points of view. F. tetradactyla contains two hydrolzsable tannins as well as gallic acid in quantities. Both the tannins also inhibited P. gossypiella larvae when being ingested. Tannins are known to play a role in a number of ecological processes in addition to herbivore defense (Kraus et al., 2003). Because tannins are complex and energetically costly molecules to synthesize, their widespread occurrence and abundance suggests that application of tannins as pest control agents needs to be investigated from practical points of view rather than as leading compounds. Extraction and isolation The white powder (350 g) used for chemical analysis was obtained from the galls and extracted with 95% aqueous EtOH for a week (3 ) at an ambient temperature. The solvent was concentrated in vacuo at 40 1C to give a slightly yellowish residue that weighed 41.8 g. The crude extract was successively partitioned between n-hexane CH2Cl2, EtOAc and H2O in this order and subsequent bioassay revealed the biological activity to be retained in the EtOAc fraction. The solvent was evaporated from the bioactive EtOAc fraction in vacuo to give a residue (11.3 g). Then, 2.02 g of this residue was dissolved in 5 mL of the mobile phase and were injected onto DCCC. The ascending method was used with one fraction equivalent to 300 drops. The solvent system consisted of CHCl3–MeOH–H2O–AcOH–MeCoEt (23:42:26:3:6, v/v/v/v/v). Fractions were collected and checked with SiO2 TLC plates using the stationary phase as the developing solvent. Three active compounds were obtained and the further purification by LH-20 column chromatography yielded, gallic acid (1) (83 mg), methyl gallate (2) (17 mg) and ethyl gallate (3) (134 mg). They were identical in all respects including spectroscopic data to authentic samples. Insect feeding assay The artificial diet-feeding assay was carried out as previously described (Chan et al., 1978) with some modifications (Kubo, 1991). The homogeneous incorporation of

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plant extracts or purified chemicals into the diet with the addition of as little heat as possible is an important concern in the artificial-feeding assay. Since there is no way to anticipate a priori what effects will appear, careful observation of the insects throughout the assay is important. Any difference from the control should not be overlooked. For example, P. gossypiella larvae failed their molting cycle caused by the ingestion of the crude extracts of some plants such as Ajuga (Kubo et al., 1981), Podocarpus (Kubo et al., 1984), and Vitex (Kubo et al., 1985; Zhang et al., 1992) species; the insect underwent normal apolysis, but failed to complete ecdysis (Kubo et al., 1983). If materials permit, plant extracts should be fractionated, at least between organic solvent-soluble and -insoluble fractions. In general, water-soluble substances, especially primary metabolites such as sugars and amino acids, are usually feeding stimulants, so they often mask interesting biological activity.

Saponins Despite significant advances in the techniques used to control mosquitoes during recent decades, they still pose a serious public health problem. In addition to the persistent irritation they cause to humans and animals simply by virtue of their bloodsucking behavior and the itching it causes, mosquitoes are also the principal vector of a variety of serious diseases, including malaria, yellow fever, dengue and encephalitis. Worldwide, approximately 2.7 million human deaths occur each year solely as a result of malaria transmitted by mosquitoes (Butler, 1997). By bioassay-guided fractionation using Aedes aegypti larvae, four novel saponins, pachyelaside A, B, C and D, were isolated as potent mosquito larvicidal principles from the root bark of a west African medicinal plant P. tessmannii (Leguminosae) (Nihei et al., 2005) by using recycling HPLC (Kubo and Nakatsu, 1990). Despite their excellent mosquito larvicidal activity, these triterpene saponins are difficult to obtain in quantities and energetically costly complex molecules to synthesize (Figure 3). To cross these hurdles, a series of aliphatic primary alkanols were tested as a model for their mosquito larvicidal activity, since saponins are usually considered as surface-active agents (surfactants). It should be noted that alkanols are among the most versatile of all organic compounds; free and esterified alkanols occur widely in nature. The use of a series of aliphatic primary alkanols applied to water as soluble solutions for the control of mosquitoes in their larval stage. The species used for this study is Culex tarsalis, the principal vector of the Western equine and St. Louis encephalitis viruses common throughout the western US, but preliminary assays against Culiseta incidens suggest that the alkanols tested show similar activity against members of other mosquito genera too (Hammond and Kubo, 1999). In an effort to understand the molecular basis of alkanol toxicity, a variety of naturally occurring unsaturated long-chain alcohols were also tested for comparison. Of these, farnesol was previously reported as acting in some insects as juvenile hormone mimics (Schmialek, 1961). The toxicity of the homologous series of primary alkanols against C. tarsalis larvae was recorded as mortality after 24 h and the results show that larvicidal activity at dodecanol, with an LD50 of 30 mM against the 1st instar larvae and 28 mM against mixed 3rd and 4th instar larvae. Methanol and ethanol were very


Pt 72A C 54 H 88 O 23 Mol. Wt.: 1105.26

H COOH Glc I O

HO HO HO HO HO HO HO HO HO

HO HO O O

H O

H

O O

Glc III

OH Glc II

O

Pt 75A

O Glc IV

69

O

C 63 H 94 O 25 Mol. Wt.: 1251.41

OH

Glc I O

HO HO HO HO HO HO

Glc III

HO HO HO

HO HO O O

H

O COOH

H O

H

O O

OH Glc II

O O

Glc IV

OH O

Pt 741 C 68 H 102 O 29 Mol. Wt.: 1383.52

H

O COOH

Glc I HO O O O HO OH O HO O Glc III HO HO O O OH HO Glc II HO O HO O HO HO OH Glc IV Pt 751 C 67 H 100 O 28 Mol. Wt.: 1353.49 Xyl

HO HO HO HO HO HO HO HO

H

O

HO HO

Xyl II HO O O OH HO

Glc II HO O O

Glc I O O

H

O H

O COOH

H O

H

O Xyl I OH

O O

Glc III OH

Fig. 3. Structure of potent mosquito larvicidal saponins characterized from P. tessmannii.

weak agents and mortality did not reach 50% at the highest dose tested, 500 mM. Activity increases through the series as chain length is lengthened from propanol to dodecanol, tapers off slightly from dodecanol to pentadecanol, and then cuts off at hexadecanol. Alkanols with greater than 16 carbons were ineffective as mosquito larvicides because they did not display biochemical toxicity and did not consistently


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prevent larvae from surfacing; consequently, they did not produce 50% mortality up to the maximum practical dosage, even when assays were extended to 4 days. The values cited in Table 2 for tridecanol through eicosanol exceed literature values for their maximum solubility in water (Bell, 1973) but experiments at high doses were included because these compounds are far more soluble in cell membranes – the putative active site – than in water. By creating a supersaturated suspension in the aqueous medium, even temporarily, we were able to deliver a high dose to the mosquitoes themselves. Addition of the surfactant Tween-80 to improve solubility increased the toxicity of tridecanol and tetradecanol solubility, and of pentadecanol by nearly a full order of magnitude (Table 2). Activity of alkanols with less than 13 or more than 15 carbons was unaffected by the use of surfactant or sonication. Tween itself had no noticeable toxicity to larvae up to 400 mg/mL, four times the concentration used here. Sublethal doses provoked symptoms of anaesthesia in mosquitoes (e.g. unresponsiveness to outside stimuli such as tapping on the container and cessation of characteristic diving and feeding behaviors).

Table 2 LD50 values of alkanols against C. tarsalis larvae Alkanols tested

LD50 (mM) 1st instar

Methanol Ethanol Propanol Butanol Pentanol Hexanol Heptanol Octanol Nonanol Decanol Undecanol Dodecanol Tridecanol Tetradecanol Pentadecanol Hexadecanol Heptadecanol through Eicosanol Tridecanol+Tweena Tetradecanol+Tween Pentadecanol+Tween Hexadecanol through Eicosanol +Tween 1,10-Decanediol a

>500 >500 171 (727.8) 74.6 (73.66) 20.0 (74.10) 7.55 (71.18) 2.64 (70.322) 1.12 (70.081) 0.260 (70.061) 0.057 (70.015) 0.038 (70.005) 0.030 (70.004) 0.034 (70.003) 0.049 (70.010) 0.302 (70.058) Inactive up to 0.6 Inactive up to 0.6 0.029 (70.003) 0.034 (70.005) 0.034 (70.007) Inactive up to 0.6 Inactive up to 4.6

In combination with Tween-80 added at 100 mg/mL.

3rd and 4th instar

1.02 (70.139) 0.278 (70.026) 0.081 (70.007) 0.046 (70.004) 0.028 (70.002) 0.042 (70.006) 0.039 (70.005) 0.088 (70.028) 0.222 (70.047)


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Whereas there was no appreciable difference between the patterns of physiological toxicity of alkanols to larvae of different instars, the lethal effects of reduced surface tension were most evident against 3rd and 4th instar larvae, which were less able than 1st instars to tolerate prolonged period without air – note the potency of pentadecanol and hexadecanol against late instars as compared to 1st instar. In short, the effectiveness of hexadecanol against late instars only is apparently the result of its physical properties for temporarily lowering surface tension, and not of inherent biochemical toxicity. Statistical analysis of the data showed that the difference in potency of alkanols differing in length by one carbon was highly significant (Po0.01) for C3–C10 and for C14–C16. The difference between pentadecanol alone and pentadecanol+Tween was also highly significant (Po0.01). However, the differences among the most potent homologues (C10–C14) and the effects of combining each of these with Tween were not significant for this data set (n ¼ 5). In order to better understand the significance of the cutoff in activity after pentadecanol, several other alcohols of relevant structure and chain length were tested as well, all in combination with Tween to ensure maximum solubility. It was interesting to note that, whereas hexadecanol was not toxic to 1st instar mosquitoes at the highest concentrations tested, cis-11-hexadecanol was nearly as potent as the strongest of the saturated alkanols. Similarly, the introduction of two (in the case of linoleyl alcohol) or three (linoenyl alcohol) double bonds to octadecanol converted the compound from completely inactive into being among the most potent. The isoprenoid sesquiterpenoid farnesol, which has an overall length of 12 carbons, also showed toxicity comparable to that of dodecanol, leading us to infer the hydrophobic and hydrophilic moieties. The head—tail-type structure is apparently essential for larvicidal activity because decanol was toxic to mosquitoes in the range of 60 mM, while decane-1,10-diol was not toxic even at 4.6 mM. Although the differences in relative toxicity were consistent among the four unsaturated alkenols tested, they were not statistically significant (Po0.01 and n ¼ 5). The objective of the surface tension experiments was to test the hypothesis that the lethal concentration of a given alkanol can be positively correlated to the concentration required to lower surface tension enough to prevent larvae from surfacing for air. Dilute solutions of Ivory soap produce low, yet stable surface tension values, and were used to confirm the results of earlier researchers that some mosquito larvae have difficulty surfacing for air when surface tension (g) – normally about 72 dynes/cm – is decreased to 27–36 dyne/cm. For C. tarsalis we found variability among individuals, but most 1st- and 2nd instar larvae were unable to attach to the surface g when decreased to the range of 28–32 dyne/cm; 4th instar larvae were able to tolerate lower surface tensions, but none were able to surface when g decreased to about 26 dyne/cm. In contrast, the concentration of C3–C10 alkanol that caused 50% mortality in 1st instar larvae produced surface tensions of 62–47 dyne/cm, which allowed even the smallest larvae to surface without difficulty. The concentration of each C3–C10 alkanol necessary to lower g to 30 dyne/cm was on an average eight times than its respective LD50 concentration. Furthermore, for chain lengths up to 10 carbons, many of the dead larvae were found with their respiratory siphons still firmly suspended from the water’s surface, unequivocally eliminating surface phenomena as the cause of death.

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Alkanols with 11–16 carbons, on the other hand, were found to be superior surface tension-reducing agents and even at low doses, larvae attempting to surface for air simply bounce off the air–water interface and sink back downward. Nevertheless, the swiftness of death hinted that even these larvae were succumbing to a biochemical phenomenon long before they could be expected to display the effects of suffocation. For these long-chain alkanols, the role of a biochemical mode of action was demonstrated by using a method that prevented contact with the surface for the course of the assay. Mosquito larvae, especially early instars, can survive for extended periods without access to air, apparently by cutaneous respiration of dissolved oxygen in the water. A glass ceiling such as a Petri dish can be used to trap larvae without air. By creating a test system free of air and its associated interfaces, the container ceiling is effectively made identical to its walls and bottom and, most importantly, the larvae are specifically isolated from any surface-related phenomenon that might wet their siphon opening, reduce its hydrofuge properties or allow leaks into their respiratory siphons during attempts to surface. When we trapped 20 1st instar C. tarsalis larvae under glass without air as a control, 100% were alive and active 16 h later. Fourth instar populations survived only about 3–4 h, apparently due to their higher oxygen requirements and lower surface area-to-volume ratio. Survival time of 1st instar larvae under glass was approximately doubled to 32–48 h by lowering the temperature of water to 8–10 1C. In contrast, 1st instar larvae trapped without air in jars of water treated with decanol or undecanol were immobilized on the bottom and made no further efforts to surface after 5 min; the same result was observed with dodecanol after about 12 min, with tridecanol after an hour, with tetradecanol after 3 h, and with pentadecanol after approximately 9 h. Extrapolation of this time-dependency curve predicts activity for hexadecanol after about 18 h, but no mortality due to hexadecanol nor heptadecanol treatments occurred before control larvae themselves began to die of anoxia, after about 24–30 h. Nevertheless, we could not rule out the possibility that hexadecanol may act by a mechanism similar to – but weaker than – shorter chain homologues, for example as a partial anesthetic. In an effort to extend the survival of larvae trapped without air and thereby allow sufficient time for the possibility that hexadecanol might still show activity at longer exposures, the same air-free experiments were also carried out under refrigeration, at 6 1C, 120 1C and 14 1C. Control larvae were thus able to survive up to 48 h, but we were surprised to find that 260 mM pentadecanol+Tween, which had caused 100% mortality at 20 1C, caused just 20% mortality at 14 1C, and toxicity disappeared entirely at 10 1C or less. Comparatively, the toxicity and time dependency of tetradecanol and the shorter-chain alkanols were unaffected by these lower temperatures, suggesting that the loss of activity is at least partially a function of declining solubility. Subsequent experiments, in which larvae treated with lethal doses of C10–C15 alkanols were transferred to clean water well after the onset of anesthesia had become evident, confirmed that the effects were completely reversible. Within minutes or hours, again depending on chain length, larvae returned to behavior indistinguishable from that of untreated larvae.


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The significance of our findings are twofold: First, they challenge the previously accepted belief that alkanols act as mosquitocides only via suffocation provoked by surface phenomena; second, for studies measuring biological activity of the alkanol series, this is the first-documented instance of cutoff occurring in an animal at a chain length beyond C12. The speed with which alkanols pC15 act as mosquito larvicides is in sharp contrast to the conclusions of previous researchers using insoluble monolayers of dodecanol (McMullen and Hill, 1971), hexadecanol (McMullen and Hill, 1971), and lecithins (McMullen et al., 1977), which were specifically described as producing larval death only after the dissolved oxygen content of the water was depleted, usually overnight. The increasingly time-dependent nature of toxicity as chain length is increased offers further evidence of the biochemical mode of action for alkanols, independent of any suffocation mechanism resulting from reduced surface tension at the air–water interface. Action of farnesol was also rapid, as far as mosquito larvicidal activity is concerned, the rapid lethality may eliminate the possibility of previous conclusion that farnesol is a juvenile hormone mimics (Schmialek, 1961) since hormonal disturbances take time. The toxicity of farnesol against mosquito larvae is apparently via the same mechanism as for unbranched, saturated alcohols. When, for comparison purposes, mosquitoes were treated with Golden Oil, a commercial product sold as a surface-active mosquitocide, 4th instar larvae died after a period of several hours and 1st instar larvae were weakened, but still alive, 24 h later – both results consistent with an explanation of death via suffocation. Alkanols, in contrast, were generally observed to act more quickly on the 1st instar than on the 4th instar larvae. Also, whereas pupae have been shown previously to be more susceptible to monolayers than larvae (McMullen and Hill, 1971; Levy et al., 1982), our preliminary findings indicate that solutions of long-chain alkanols are more toxic to Culex larvae than to the pupae, possibly owing to the pupae’s thicker cuticle and concomitant capacity to resist penetration by foreign substances. Tests with animals in vivo are somewhat limited, but long-chain alkanols are known to produce anesthesia in fathead minnows (Veith et al., 1983), tadpoles (Alifimoff et al., 1989; Miller et al., 1989) and brine shrimp (Chiou et al., 1990), and to cause growth impairment in the ciliate protozoan Tetrahymena pyriformis (Schultz et al., 1990). In our own earlier studies, they showed activity against a variety of Gram-positive bacteria and fungi, but not against Gram-negative bacteria (Kubo et al., 1995). A persistent quandary facing researchers has been the ‘‘cutoff’’ phenomenon in the homologous series of alkanols, whereby potency increases with chain length until reaching a maximum, and the alkanol containing a single additional carbon shows no potency at all, even when duration of exposure lasts several days (Miller et al., 1989). Although attention has been focused on instances where cutoff occurs immediately after dodecanol, the exact length of carbon chain where cutoff occurs clearly varies among genera (Figure 4). Our review of the literature found no other animal (only microorganisms and a protozoan) for which cutoff has been reported at a chain length beyond dodecanol, which may be the result of differences in the membrane composition of test organisms. By comparison, tadpoles were fully anesthetized by nonanol after 30 min, by

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Fig. 4. Comparison of biological activity of alkanols against various organisms. Cutoff in activity against fish and tadpoles occurs after C12, but in mosquitoes not until after C15. Although the endpoints measured are not identical in all test systems, sublethal doses provoked symptoms of anaesthesia in mosquitoes and growth inhibition in microorganisms. &, Tadpole (Rana pipiens), loss of righting reflex, EC50 (Alifimoff et al., 1989); J, Minnow (Pimephales promelas), 96 h LC50 (Veith et al., 1983); r, Protozoan (T. pyriformis), inhibitory growth concentration, EC50 (Schultz et al., 1990); D, S. aureus, minimum bactericidal concentration (MBC) (Kubo et al., 1995); , Mosquito (C. tarsalis), 24 h LD50, Tween used to maximize solubility of alkanols ZC13; B, P. acnes, MBC (Kubo et al., 1995); +, Clostridium botulinum (Huhtanen, 1980), minimum inhibitory concentration.

decanol and undecanol after 60 min, by dodecanol after 120 min, but not by tridecanol, even when exposed for 96 h (Alifimoff et al., 1989). Mosquitoes are also unusual in that the loss of activity is gradual, tapering off from the strongest compounds before disappearing, whereas for most organisms, maximum activity occurs at a given chain length of n, and absolutely no activity is present at the alkanol of chain length n+1. For example, the minimum bactericidal concentration against Propionibacterium acnes was 1.56 mg/mL for hexadecanol, but heptadecanol was completely inactive, even when tested at 800 mg/mL (Kubo et al., 1995). The precise anesthetic mechanism is still not well understood, and attempts to explain it have covered a wide array of biological functions, mostly related to structure and function of lipid membranes and/or proteins (Franks and Lieb, 1994). The increase in activity with chain lengthening has been widely correlated to each compound’s octanol– or lipid–water partition coefficient, and thereby its relative tendency to accumulate in lipid regions of the cell membrane at concentrations


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sufficient to interfere with basic nutrient or iron transport processes (Treistman and Wilson, 1987; Elliott and Elliott, 1994; Weingart and Bukauskas, 1998). Nevertheless, it has been difficult for lipid-based theories to account for the cutoff effect and with this deficiency in mind, other researchers used alkanol inhibition of the lipid-free luciferase enzyme to build a case for anesthetics acting by building directly to a protein pocket of circumscribed dimension (Franks and Lieb, 1994; Abraham et al., 1991). More recently, the effects of alkanols on ligand-gated ion channels in wildtype and mutated neurotransmitter receptors have also led some to conclude that alkanols act directly on membrane proteins, and do not depend on lipid–protein interactions (Mascia et al., 1996; Mihic et al., 1997; Wick et al., 1998). Still, doubt remains as to whether these systems adequately model the site of general anesthesia in animals (Alifimoff et al., 1989; Moss et al., 1991), or for that matter whether anesthetic activity of alkanols is limited to a single mechanism. The fact that sonication, temperature and combination with a surfactant could be used to manipulate mosquitocidal activity nearly 10-fold around the cutoff, supports the notion that the solubility of alkanols in biological membranes and their capacity to perturb membrane lipids at the lipid–protein interface have some bearing on explanation of the cutoff phenomenon. Indeed, there are several recent studies to support this hypothesis. Anesthetic chain lengths were shown to cause perturbation of membrane lipids whereas non-anesthetic alkanols dissolved in membrane lipids without perturbing them (Miller et al., 1989). Fourier transform infrared spectroscopy (FTIR) studies of lipid membrane vesicles in D2O showed that hydrogen bonding of the alkanol hydroxyl to the phosphate moiety in reversed micelles increases up to decanol, and then declines sharply at tetradecanol (Chiou et al., 1990). If alkanols bond to the phosphate moiety in biological membranes the way they do in membrane vesicle, then certainly they would affect the conformation and function of proteins normally held in place by hydrogen bonds at the lipid–protein interface. The surfactant concept can be extended to answer many other questions related to membrane-bound proteins. For example, there has been a long-standing debate on whether alkanols produce their effects in the central nervous system by acting on lipids or on proteins. Alkanols are known to act as general anesthetics, with increasing potency correlated to increasing chain length until a point of cutoff is reached, usually at dodecanol, after which activity disappears entirely. Since anesthesia involves many membrane-bound proteins such as synaptosomal ATPases and acetylcholine receptor, alkanols may alter these membrane-bound proteins in similar fashion, by disrupting and disorganizing the hydrogen bonds at the lipid bilayer–protein interface nonspecifically. Knowledge that introduction of a double bond shifts the cutoff to a longer-chain length in tadpoles (Pringle et al., 1981) – and now in mosquitoes – is further evidence in support of explanations which consider the structural role of lipids rather than isolated action on a protein-binding site, because if cutoff were due to a compound’s hydrophobic moiety exceeding the limited size of a protein pocket, then since the addition of a cis double bond makes the compound even bulkier, it should theoretically provoke cutoff at shorter, rather than longer, alkyl chain lengths. We speculate that hydrogen bonding plays an important role in determining the molecular basis of alkanol activity in mosquitoes and other organisms.

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Alkanols are stable, colorless, inexpensive, biodegradable (Swisher, 1970), and essentially nontoxic to humans (Opdyke, 1973). The fact that they are already approved for use in food products at concentrations comparable to the doses used here may facilitate their approval as insecticides. If further experience bears out the conclusions of earlier workers who reported a cutoff in activity after dodecanol for fish and amphibians, then the narrower spectrum activity of tridecanol, tetradecanol, and pentadecanol against mosquitoes makes these promising candidates for environmentally sensitive pest management programs. The cutoff in anesthetic potency occurs at different chain lengths in different organisms, lending new perspective to the molecular basis of anesthesia. In brief, the mosquito larvicidal activity of alkanols against C. tarsalis larvae depends on the hydrophobic alkyl chain length from the hydrophilic hydroxyl group. The hydrophilic hydroxyl group can be replaced by any hydrophilic groups as long as the ‘‘head and tail’’ structure is balanced. On the basis of this surfactant concept, various phytochemicals previously isolated were tested for their mosquito larvicidal activity against C. tarsalis larvae. As expected, some phytochemicals showed even more potent and selective larvicidal activity compared to aliphatic alkanols. For example, phytochemicals possessing the phenolic moiety as the hydrophilic portions can be expected to exhibit mosquito larvicidal activity. Anacardic acids and cardols isolated from the cashew nut shell liquid (CNSL) are an excellent example for the surfactant concept (Kubo et al., 1986). Although CNSL is available in far greater tonnage, it is neglected in commercial terms, and there is considerable potential for its exploitation. Extraction and isolation of saponins The air-dried root bark (500 g) of P. tessmannii (Leguminosae) was cut into small pieces and extracted with MeOH (500 mL 3) at ambient temperature for 2 weeks. The MeOH extract (70 g) was evaporated to dryness and suspended in 500 mL of H2O, and then it was extracted successively with n-hexane, ethyl ether, CHCl3, EtOAc and n-BuOH to yield four fractions, n-hexane and ethyl ether (3 g), CHCl3 (14 g), EtOAc (10 g), and n-BuOH (30 g). A portion (4 g) of the n-BuOH fraction was subjected by a recycling HPLC (Kubo and Nakatsu, 1990). Column: 50 cm 2.5 cm Asahipack GS-320, 5 mm; mobile phase: MeOH; flow rate: 3 mL/min; detection: refractive index; amount of sample loaded: 500 mg (Nihei et al., 2005). Bioassays Bioassays were conducted at room temperature (2072 1C) using distilled water (20 mL) in 1 oz clear plastic containers made by Plastics Inc. (St. Paul, MN). Alcohol was added by solubilizing them first in acetone, then diluting in water to the appropriate concentration and briefly shaking to ensure mixing. Ten larvae were then pipetted into each 20 mL volume and observed for a minimum of 24 h, when mortality was recorded. Controls were treated with the maximum amount of acetone applied in each alcohol assay, usually 0.1 mL. Acetone itself was found to have no effect on the larvae up to a concentration of 1%.


77

All compounds were assayed against 1st instar larvae and octan-1-ol through hexadecane-1-ol were also tested against mixed 3rd and 4th instar larvae. Assays were repeated at least five times for each compound. Compounds were tested at a minimum of five concentrations following range finding tests. Owing to their excessive clumping and general insolubility in water, alkanols with 15 or more carbons were tested only up to 0.6 mM. Although this dose exceeds the measured solubility of long-chain alkanols in water (Bell, 1973), the application of excess material was used as a way to supersaturate the water and thereby deliver a higher dose to the mosquito itself, in whose cell membranes these compounds are much more soluble. For alkanols greater than 10 carbons, bioassays were repeated with the addition of the commercial surfactant Tween-80 (100 mg/mL) to see if improved solubility might increase activity. For assays employing surfactant, shaking was impractical, so solutions were added while sonicating the test water, which helped to maximize solubility and mixing. Larvae were considered dead or moribund if they stopped moving for a prolonged period, were unable to resurface after sinking to the bottom of the container, and were still unable to respond after gentle probing with a small spatula. Mortality among controls was zero for more than 95% of the assays, and in no instance did it exceed 10%. In assays where larvae were trapped without air and prevented from any contact with the surface, each test solution was prepared as described above, then used to fill a 250 mL glass jar above its brim so as to form a meniscus; a Petri dish was then slid horizontally across the mouth of the jar so that no air bubbles remained. The edge was sealed with petroleum jelly to prevent drying and leakage. Time required to manifest toxicity is expressed as the time at which the last bodily movement could be detected from the last survivor among 10 larvae, and values cited are the mean of at least three experiments (Hammond and Kubo, 1999).

Acknowledgments The work was partly reported at the 15th International Plant Protection Congress in Beijing, China. I am indebted to Dr. D. G. Hammond, Dr. J. A. Klocke and Dr. S. Asano for performing mosquito larvicidal and artificial diet-feeding assays, Mr. S. Kawamichi for supplying Gallae Rhois; Dr. M. Takasaki for chemical analysis of Gallae Rhois at an earlier stage of the work, Dr. N. Masuoka for obtaining scavenging antioxidant data and Dr. C. Ce´spedes for assisting the final manuscript preparation.

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Andersen SO. (1990) Sclerotization of insect cuticle. In: Ohnishi E, Ishizaki H, editors. Molting and metamorphosis. Tokyo: Japan Scientific Society Press, pp. 133–155. Barbehenn RV, Martin MM. (1994) Tannin sensitivity in larvae of Malacosoma disstria (Lepidoptera): roles of the peritrophic envelope and midgut oxidation. J Chem Ecol 20:1985–2001. Barbehenn RV, Martin MM, Hagerman AE. (1996) Reassessment of the roles of the peritrophic envelope and hydrolysis in protecting polyphagous grasshoppers from ingested hydrolyzable tannins. J Chem Ecol 22:1901–1919. Bell GH. (1973) Solubilities of normal aliphatic acids, alcohols, and alkanes in water. Chem Phys Lipid 10:1–10. Bernays EA, Chamberlain DJ. (1980) A study of tolerance of ingested tannin in Schistocerca gregaria. J Insect Physiol 26:415–420. Brune M, Rossander L, Hallberg L. (1989) Iron absorption and phenolic compounds: importance of different phenolic structures. Eur J Clin Nutr 43:547–558. Butler D. (1997) Time to put malaria control on the global agenda. Nature (Lond) 386:535–536. Chan BG, Waiss Jr. AC, Stanley WL, Goodban AE. (1978) A rapid diet preparation method for antibiotic phytochemical bioassay. J Econ Entomol 71:366–368. Chiou JS, Ma SM, Kamaya H, Ueda I. (1990) Anesthesia cutoff phenomenon: interfacial hydrogen bonding. Science 248:583–585. Elliger CA, Chan BC, Waiss Jr. AC. (1980) Flavonoids as larval growth inhibitors. Naturwissenschaften 67:358–360. Elliott JR, Elliott AA. (1994) The effects of alcohols and other surface-active compounds on neuronal sodium channels. Progr Neurobiol 42:611–683. Feeny P. (1976) Plant apparency and chemical defense. In: Wallace JW, Masell RL, editors. Biochemical interactions between plants and insects. New York: Plenum Press, pp. 1–40. Franks NP, Lieb WR. (1994) Molecular and cellular mechanisms of general anesthesia. Nature (Lond) 367:607–614. Gupta S, Wang Y, Jiang H. (2005) Manduca sexta phenoloxidase (proPO)-activating proteinase (PAP) and serine proteinase homologues (SPHs) simultaneously. Insect Biochem Mol Biol 35:241–248. Hammond DG, Kubo I. (1999) Structure–activity relationship of alkanols as mosquito larvicides with novel findings regarding their mode of action. Bioorg Med Chem 7:271–278. Huhtanen CN. (1980) Inhibition of Clostridium botulinum by spice extracts and aliphatic alcohols. J Food Protec 43:195–196. Kramer KJ, Hoplins TL. (1987) Tyrosine metabolism for insect cuticle tanning. Arch Insect Biochem Physiol 6:279–301. Kraus TE, Dahlgren RA, Zasoski RJ. (2003) Tannins in nutrient dynamics of forest ecosystems – a review. Plant Soil 256:41–66. Kubo I. (1991) Screening techniques for plant–insect interactions. In: Hostettmann K editor. Methods in plant biochemistry. London: Academic Press, Vol. 6, pp. 179–193. Kubo I. (1993) Insect control agents from tropical plants. In: Downum KR, Romeo JT, Stafford HA, editors. Recent advances in phytochemistry, phytochemical potential of tropical plants. New York: Plenum Press, Vol. 27, pp. 133–151. Kubo I. (1997) Tyrosinase inhibitors from plants. In: Hedin P, Hollingworth R, Masler E, Miyamoto J, Thompson D, editors. Phytochemicals for Pest Control, ACS Symposium Series 658. Washington, DC: American Chemical Society, pp. 310–326. Kubo I, Hanke FJ. (1985) Multifaceted chemically based resistance in plants. In: Conn E editor. Recent advances in phytochemistry. Chemically mediated interactions between plants and other organisms. New York: Plenum Press, Vol. 19, pp. 171–194. Kubo I, Hanke FJ, Asaka Y, Matsumoto T, Clardy J, Cun-Heng H. (1990) Insect antifeedants from tropical plants I. Structure of dumsin. Tetrahedron 46:1515–1522. Kubo I, Kinst-Hori I, Nihei K, Soria F, Takasaki M, Calderoń JS, Ce´spedes CL. (2003) Tyrosinase inhibitors from galls of Rhus javanica leaves and their effects on insects. Z Naturforsch C: J Biosci 58c:719–725.


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Kubo I, Klocke JA, Asano S. (1981) Insect ecdysis inhibitors from the East African medicinal plant, Ajuga remota (Labiatae). Agric Biol Chem 45:1925–1927. Kubo I, Klocke JA, Asano S. (1983) Effects of ingested phytoecdysones on the growth and development of two lepidopterous larvae. J Insect Physiol 29:307–316. Kubo I, Komatsu S, Ochi M. (1986) Molluscicides from the cashew Anacardium occidentale and their large-scale isolation. J Agric Food Chem 34:970–973. Kubo I, Matsumoto T. (1984) Abyssinin, a potent insect antifeedant from an African medicinal plant, Bersama abyssinica. Tetrahedron Lett 4601–4604. Kubo I, Matsumoto T. (1985) Potent insect antifeedants from the African medicinal plant Bersama abyssinica. In: Hedin PA editor. Bioregulators for Pest Control, ACS Symposium Series 276. Washington, DC: American Chemical Society, pp. 183–200. Kubo I, Matsumoto A, Hanke FJ, Ayafor JF. (1985) Analytical DCCC isolation of 20-hydroxyecdysone from Vitex thyrsiflora (Verbenaceae). J Chromatogr 321:246–248. Kubo I, Matsumoto T, Klocke JA. (1984) Multichemical resistance of the conifer Podocarpus gracilior (Podocarpaceae) to insect attack. J Chem Ecol 10:547–559. Kubo I, Muroi H, Himejima M, Kubo A. (1995) Structural functions of antimicrobial longchain alcohols and phenols. Bioorg Med Chem 3:873–880. Kubo I, Nakatsu T. (1990) Recent examples of recycling preparative scale high performance liquid chromatography in natural products chemistry. LC GC 933–939. Levy R, Chizzonite JJ, Carrett WD, Miller TW. (1982) Efficacy of the organic surface film isostearyl alcohol containing two oxyethylene groups for control of Culex and Psorophora mosquitoes: laboratory and field studies. Mosq News 42:1–11. Li W, Kubo I. (2004) QSAR and kinetics of the inhibition of benzaldehyde derivatives against Sacrophaga neobelliaria phenoloxidase. Bioorg Med Chem 12:701–713. Londershausen M, Turberg A, Spindler-Barth M, Peter MG. (1996) Screening test for insecticides interfering with cuticular sclerotization. Pestic Sci 48:315–323. Mascia MP, Machu TK, Harris RA. (1996) Enhancement of homomeric glycine receptor function by long-chain alcohols and anesthetics. Br J Pharmacol 119:1331–1336. McMullen AI, Hill MN. (1971) Anoxia in mosquito pupae under insoluble monolaters. Nature (Lond) 234:51–52. McMullen AI, Reiter O, Phillips MC. (1977) Mode of action of insoluble monolayers on mosquito pupal respiration. Nature (Lond) 267:244–245. Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, Mascia MP, Valenzuela CF, Hanson KK, Greenblatt EP, Harris RA, Harrison NL. (1997) Site of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors. Nature 389:385–389. Miller KW, Firestone LL, Alifimoff JK, Streicher P. (1989) Nonanesthetic alcohols dissolve in synaptic membranes without perturbing their lipids. Proc Natl Acad Sci USA 86:1084–1087. Moss GWJ, Lieb WR, Franks NP. (1991) Anesthetic inhibition of firefly luciferase, a protein model for general anesthesia does not exhibit pressure reversal. Biophys J 60:1309–1314. Nihei K, Asaka Y, Mine Y, Ito C, Furukawa H, Ju-ichi M, Kubo I. (2004) Insect antifeedants from tropical plants: Structures of dumnin and dumsenin. J Agric Food Chem 52:3325–3328. Nihei K, Hanke FJ, Asaka Y, Matsumoto T, Kubo I. (2002) Insect antifeedants from tropical plants II: Structure of zumsin. J Agric Food Chem 50:5048–5052. Nihei K, Ying BP, Murakami H, Matsuda N, Hashimoto M, Kubo I. (2005) Pachyelasides A, B, C, and D; novel molluscicidal triterpene saponins from Pachyelasma tessmannii. J Agric Food Chem 53:608–613. Nishioka I. (1983) Chemistry and biological activities of tannins. Yukugaku Zasshi 103:125–142. Opdyke DLJ. (1973) Monographs on fragrance new materials. Food Cosmet Toxicol 11:1011–1181. Pardini RS. (1995) Toxicity of oxygen from naturally occurring redox-prooxydants. Arch Insect Biochem Physiol 29:101–118. Pringle MJ, Brown KB, Miller KW. (1981) Can the lipid theories of anesthesia account for the cutoff in anesthetic potency in homologous series of alcohols? Mol Pharmacol 19:49–55.

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Ryan JD. (1979) Proteinase inhibitors. In: Rosenthal GA, Janzen DH, editors. Herbivores: their interaction with secondary plant metabolites. New York: Academic Press, pp. 599–618. Ryan JD, Gregory P, Tingey WM. (1982) Phenolic oxidase activity in glandular trichomes of Solanum berthaultii. Phytochemistry 21:1885–1887. Sakaguchi N, Inoue M, Isuzugawa K, Ogihara Y, Hosaka K. (1999) Cell death-inducing activity by gallic acid derivatives. Biol Pharm Bull 22:471–475. Scalbert A. (1991) Antimicrobial properties of tannins. Phytochemistry 30:3875–3883. Schmialek P. (1961) Double identification in Tenebrio faces and in yeast of substances with juvenile hormone activity. Z Naturforschg 16b:461–463. Schultz TW, Arnold LM, Wilke TS, Moulton MP. (1990) Relationships of quantitative structure-activity for normal aliphatic alcohols. Ecotox Environ Safe 19:247–253. Serrano A, Palacios C, Roy G, Cespoń C, Villar ML, Nocito M, Gonza´lez-Porque´ P. (1998) Derivatives of gallic acid induce apoptosis in tumoral cell lines and inhibit lymphocyte proliferation. Arch Biochem Biophys 350:49–54. Swisher RD. (1970) In: Swisher RD, editor. Surfactant biodegradation. New York: Marcel Dekker. Treistman SN, Wilson A. (1987) Alkanol effects on early potassium currents in Aplysia neurons depend on chain length. Proc Natl Acad Sci USA 84:9299–9303. Veith GD, Call DJ, Brooke LT. (1983) Structure-toxicity relationships for the fathead minnow, Pimephales promelas: narcotic industrial chemicals. Can J Fish Aquat Sci 40:743–748. Weingart R, Bukauskas FF. (1998) Long-chain n-alkanols and arachidonic acid interfere with the Vm-sensitive gating mechanism of gap junction channels. Eur. J. Physiol. 435:310–319. Wick MJ, Mihic SJ, Ueno S, Mascia MP, Trudell JR, Brozowski SJ, Ye Q, Harrison NL, Harris RA. (1998) Mutations of gamma-aminobutyric acid and glycine receptors change alcohol cutoff: evidence for an alcohol receptor? Proc Natl Acad Sci USA 95:6504–6509. Zhang M, Stout MJ, Kubo I. (1992) Isolation of ecdysteroids from Vitex stricheri using RLCC and recycling HPLC. Phytochemistry 31:247–250. Zhu J, Filippich LJ, Ng J. (1995) Rumen involvement in sheep tannic acid metabolism. Vet Hum Toxicol 37:436–440.


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CHAPTER 5

Role of Melia azedarach L. (Meliaceae) for the control of insects and acari: present status and future prospects MARIÁ C CARPINELLA, MARIÁ T DEFAGO´, GRACIELA VALLADARES, SARA M PALACIOS

Introduction One of the biggest challenges that humanity confronts is providing enough food to the fast growing population of the planet, which is calculated to reach approximately 8 billions of people in the year 2020 (Knight et al., 1997). In order to materialize this, it is necessary to increase food production, which mainly depends on plants. These are often affected by many pests and diseases among which insects play a preponderant role. Commercially available synthetic pesticides are currently the most effective means of pest control. However, the continuous and indiscriminate use of these substances have not only caused adverse effects on mammals health, but have also affected other benefic members of the ecosystem (Theiling and Croft, 1988; C - elik et al., 2005; Chauhan and Gupta, 2005) and the environment in which they are immersed. They are also responsible for the development of resistance in pathogens (Thompson et al., 1993; Bass et al., 2004). Under these circumstances, new ways of pest control must be studied and established. There are many options, but the most promising is the one that involves the secondary metabolites produced by plants, many of which are highly toxic to a wide spectrum of insects and microorganisms. There is a great variety of families of plants that possess potent anti-insect compounds. From the Meliaceae family, strong insecticide molecules have been isolated, the limonoid azadirachtin obtained from Azadirachta indica or Melia azadirachta (neem) being the most potent and studied (Govindachari, 1992; Schmutterer, 1995). Another tree belonging to the Meliaceae family, far less studied than the previously mentioned, is Melia azedarach L., commonly named Paraı´ so, Chinaberry or Persian Lilac. Our research group has been studying the activity of extracts obtained from different structures of M. azedarach on a wide variety of pest insects, either in laboratory or in field experiments. A limonoid (compound 1) has been isolated from this tree as the most potent substance for controlling insects of agronomic importance. In

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the present chapter, information about the active principles isolated by various investigators from M. azedarach is provided together with their respective bioactivities. Moreover, a description of the effects on feeding, growth, larval and pupal development, fecundity, fertility, toxic effects, as well as alterations in the insect morphology of different extracts and some pure compounds from the tree is reported either in laboratory or in field trials.

Phytochemistry Many compounds have been isolated from different vegetal structures of M. azedarach collected in different places of the world. Most of them have been assayed as antiinsect and cytotoxic compounds showing excellent results. In relation to the first activity, most assays have been carried out on insects belonging to Spodoptera genus and in less measure on Epilachna. Other activities such as antifungal, antibacterial and anti-inflammatory have been less studied (Table 1). Phytochemical analysis of Argentinian M. azedarach ripe fruit extract revealed that alkaloids are absent (Carpinella et al., 1999a); however, Khan et al. (2001), reported the presence of this family of compounds in extracts from leaves, root and stem bark of M. azedarach from Papua New Guinea. Khan et al. (2001) also found tannins and flavonoids in the three mentioned tree structures. These last compounds were also found in the fruit extract from Argentinian M. azedarach (Carpinella et al., 1999a). Riba et al. (1996) followed a bioguided assay purification from a methanolic extract from M. azedarach seeds collected in Spain. After carrying out an HPLC purification, three main fractions were obtained. The first fraction (Fr. 1) obtained at the first 10 min with methanol/water 50:50 did not show any effect on Sesamia nonagrioides (Lepidoptera: Noctuidae) survival while the one obtained between 10 and 20 min of running (Fr. 2) exhibited the highest percentage of mortality till it reached pupation. The fraction obtained with methanol 100% (Fr. 3) showed a 46% of accumulated mortality till pupation on S. nonagrioides. From this fraction four majority products were obtained but only one showed effectiveness (Riba et al., 1996). From Fr. 2, a subfraction which exhibited the major activity was obtained. This subfraction contained two products in a relation 3:1. These products could not be separated; although successive purifications were performed similar chromatograms were obtained (Figure 1; Riba et al., 1996). The authors concluded that it was a decomposition of the majority compound. Carpinella et al. (2002) afforded the same mixture of compounds (compound 1) after carrying out a bioassay-guided isolation from the ethanolic ripe seed kernel extract from M. azedarach from Argentina. The fact that was interpreted as decomposition by Riba et al. (1996) was elucidated by Carpinella et al. (2002) as a mixture of two interchangeable isomeric limonoids, which occur/exist as an equilibrium between isomers A and B. The last one was named ‘meliartenin’ (Carpinella et al., 2002). Compound A was favoured in CH3CN (A/B ¼ 80–70:20–30) while the molar fraction of B increased in CHCl3 (A/B ¼ 53:47) (Figure 2a and b). Note the similarity between chromatograms of Figures 1 and 2a.

Table 1 Pure compounds isolated from extracts obtained from different structures of M. azedarach and their respective bioactivities Origin

Activity

Reference

1-cinnamoyl-3-hydroxy11methoxymeliacarpinin

EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.

Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 1.5 mg/ml

Takeya et al. (1996b)

1-(2-methylpropanoyl)-3acetyl-11methoxymeliacarpinin

MeOH ext. of root of M. azedarach L. var japonica

No lethal activity on Artemia salina at 100 mg/ml

Fukuyama et al. (2000)

1,12-di-O-acetyltrichilin B

Et2O ext. of root bark of Chinese M. azedarach L.

Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2

Nakatani et al. (1994b); Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)



Takeya et al. (1996a)

1,3-dicinnamoyl-11hydroxymeliacarpin

Me2CO and MeOH ext. (CH2Cl2 fr.) of leaves of Indian M. azedarach L.

Insecticide on S. littoralis, LC50 ¼ 2.36 ppm; EC50 ¼ 0.57 ppm

Bohnenstengel et al. (1999)

11-methoxy-20acetylmeliatinin

MeOH ext. of fruit of M. azedarach from Greece

12 a-acetoxyfraxinellone

Et2O ext. of stem bark of Japanese M. azedarach

Kraus et al. (1985) Antifeedant against S. littoralis (Boisduval) at 10 mg/cm2

Nakatani et al. (1998)

Ichthyotoxic on Oryzias latipes at 50 ppm 12-acetoxyamoorastatin ¼ toosendanin ¼ 29deacetylsendanin

Cytotoxic against human tumour cells, ED50 ¼ 0.0007–0.04 mg/ml

Ahn et al. (1994)

Bark of Chinese M. azedarach

Antifeedant against S. litura

Chiu (1989)

Et2O ext. of root bark of Chinese and Japanese M. azedarach

Antifeedant against S. eridania (Boisduval), MIC ¼ 6 mg/cm2

Nakatani et al. (1998); Huang et al. (1995b)



Itokawa et al. (1995)

83

MeOH ext. (EtOAc fr.) of stem bark of M. azedarach var. japonica

Role of Melia azedarach L. (Meliaceae) for the control of insects and acari

Compound

84

Table 1 (continued ) Origin

Activity

Reference

1-O-deacetylochinolide A

MeOH ext. of fruits of Brazilian M. azedarach L.

Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 1449 mg/ml

Zhou et al. (2004)

1-O-deacetylochinolide B


Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 58 mg/ml

Zhou et al. (2004)

1-O-deacetyl-1-Obenzoylochinolide B


Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 22,638 mg/ml

Zhou et al. (2004)

1-O-deacetyl-1-Otigloylochinolide A



Zhou et al. (2004)

1-O-deacetyl-1-Otigloylochinolide B



Zhou et al. (2004)

12-deacetyltrichilin I




12-hydroxyamoorastatin ¼ 12-deacetyltoosendanin



Ahn et al. (1994)




Et2O ext. of root bark of Chinese M. azedarach

Antifeedant S. eridania (Boisduval) 3 mg/cm2


EtOH ext. (CH2Cl2 fr.) of seed kernel of M. azedarach from Argentina

Antifeedant and insecticide against E. paenulata at 4 mg/cm2 and antifeedant against S. eridania at 1 mg/cm2

Carpinella et al. (2002)

Isomer A of compound 1


Compound




Ahn et al. (1994) Nakatani et al. (1998)



Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)

12-O-acetylazedarachin B




12-O-acetyltrichilin B



Nakatani et al. (1994a); Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)

1-acetyl-2-deacetyltrichilin H




1-acetyl-3-deacetyltrichilin H




1-acetyl-3-tigloyl-11methoxymeliacarpinin




1-acetyltrichilin H




1-cinnamoilmelianolona

MeOH ext. (CH2Cl2 fr.) of fruit of M. azedarach from USA

Insecticide against Heliothis virescens, EC50 ¼ 0.12 ppm; LC50 ¼ 1.50 ppm and S. frugiperda, EC50 ¼ 0.04 ppm; LC50 ¼ 1.30 ppm

Lee et al. (1987); Kraus et al. (1989); Lee et al. (1991) 85

12-O-acetylazedarachin A


12-hydroxyamoorastatone

86

Table 1 (continued ) Compound

Origin

Activity

Reference

1-cinnamoyl-3-11 dihydroxymeliacarpin

MeOH ext. (CH2Cl2 fr.) of fruit of M. azedarach from USA

Insecticide against H. virescens, EC50 ¼ 0.18 ppm; LC50 ¼ 3.50 ppm and S. frugiperda, EC50 ¼ 0.04 ppm; LC50 ¼ 1.60 ppm

Lee et al. (1991)

1-cinnamoyl-3-acetyl-11hydroxymeliacarpin


Insecticide against S. littoralis, EC50 ¼ 0.27 ppm; LC50 ¼ 0.48 ppm


1-cinnamoyl-3-acetyl-11methoxymeliacarpinin

Root bark of Japanese M. azedarach L.

Antifeedant against S. eridania (Boisduval), MIC ¼ 1 mg/cm2

Nakatani et al. (1994a); Huang et al. (1995b)



MeOH ext. of fruit of M. azedarach from Greece

Growth regulator on E. varivestis

Growth regulator on E. varivestis No antifeedant against E. varivestis

Kraus et al. (1985, 1987)

Effect on moulting on Tessaratoma papillosa at 50 mg/ml

Chiu (1989)

Stem bark of Okinawan M. azedarach L. EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L. 23bmethoxymeliacarpin Kraus et al. (1985, 1987) MeOH ext. of fruit of M. azedarach from Greece

1-cinnamoyl-3methacrylyl-11hydroxy-meliacarpin


Insecticide against S. littoralis, EC50 ¼ 0.57 ppm; LC50 ¼ 1.19 ppm


1-deoxy-3-methacrylyl-11methoxymeliacarpinin





1-cinnamoyl-3-feruloyl11-hydroxy-22,23dihydroNo antifeedant against E. varivestis 1-cinnamoyl-3-feruloyl11-hydroxymeliacarpin

Antifeedant against S. exigua (Boisduval) at 3 mg/cm2 and S. eridania (Boisduval), MIC ¼ 1 mg/ cm2


1-desacetylnimbolinin B

PE and Et2O ext. of fruit of M. azedarach var. japonica

Antifeedant against E. varivestis

Kraus and Bokel (1981)

1-O-detigloyl-1-Obenzoylohchinolal


No cytotoxic activity against HeLa S3 human epithelial cancer cells

Zhou et al. (2004)

1-O-detigloyl-1-Ocinnamoylohchinolal



Zhou et al. (2004)

1-methacrylyl-3-acetyl-11methoxymeliacarpinin

MeOH ext. root of M. azedarach L. var japonica

Lethal on A. salina, LC50 ¼ 19 mg/ml


1-tigloil-11-methoxy-20acetylmeliacarpin

Fruit

1-tigloyl-3,20-diacetyl-11methoxylmeliacarpinin


Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 100 mg/ml


1-tigloyl-3-acetyl-11methoxymeliacarpinin




1,5-dihydroxy-8-methoxy2-methylanthraquinone3-O-a-Lrhamnopyranoside

Stem bark

Srivastava and Mishra (1985)

1,8-dihydroxy-2methylanthraquinone-3O-b-Dgalactopyranoside

Stem bark

Srivastava and Mishra (1985)

Kraus et al. (1987)

87



1-deoxy-3-tigloyl-11methoxymeliacarpinin


Activity

Reference

29-isobutylsendanin




2b,3b-dihydroxy-5apregn-17(20)-(2)-en-16one

MeOH ext. of root of M. azedarach L. var japonica

Lethal on A. salina, LC50 ¼ 2.1 mg/ml


30-hydroxy-fraxinellone

AcOEt ext. of roots of Italian M. azedarach

3-deacetyltrichilin H




3-tigloyl-1,20-diacetyl-11methoxylmeliacarpinin




4,8-dimethoxy-1-vinyl-bcarboline

MeOH ext. (EtOAc fr.) of cortex of M. azedarach L. var japonica

Anti-inflammatory activity (suppression inducible nitric oxide synthase and cyclooxygenase-2 induction) 1.5–10 mM

Lee et al. (2000)

EtOH ext. (CH2Cl2 fr.) of seed kernels of Argentinian M. azedarach L.

Antifungal against Fusarium verticillioides, MIC ¼ 0.4 mg/ml

Anti-inflammatory activity (suppression inducible nitric oxide synthase and cyclooxygenase-2 induction) 2–10 mM

Lee et al. (2000)

Carpinella et al. (2003b) 4-methoxy-1-vinyl-bcarboline 6-acetoxy-3b-hydroxy-7oxo-14b, 15bepoxymeliac-1,5-diene3-O-b-Dglucuronopyranoside

methoxycinnamaldehyde

Cortex of M. azedarach L. var japońica

Roots of M. azedarach L. from India

D’Ambrosio and Guerreiro (2002)

Srivastava and Gupta (1985)


4-hydroxy-3-

88

Compound

6-acetoxy-11-a hydroxy 7oxo 14 b, 15 b epoxymeliacin 1, 5 diene 3-O-a-L rhamnopyranoside

EtOH ext. (C6H6 fr.) of seeds of M. azedarach L. from India

6-hydroxy-7methoxycoumarin

EtOH ext. of leaves

Khalil et al. (1979)

6b-hydroxy-4-campesten3-one

PE extract of bark

Nair and Chang (1973)

6b-hydroxy-4stigmasten3-one

PE extract of bark

Nair and Chang (1973)

7a-acetoxy-14b, 15bepoxy-gedunan-1-ene-3O-b-D-glucopyranoside

Stem bark

Saxena and Srivastava (1986)

7-deacetyl-7-oxogedunin

Light PE ext. of stem wood

Okogun et al. (1975)

9a-hydroxyfraxinellone



9b-hydroxyfraxinellone

AcOEt ext. roots of Italian M. azedarach


Aesculetin

EtOH ext. of leaves


Amoorastatone



Aphanastatin


Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval), MIC ¼ 4 mg/cm2

Nakatani et al. (1995b); Huang et al. (1995b)

Azedarachin A




Srivastava and Gupta (1985) Antibacterial activity

Srivastava (1986)

89

Roots of M. azedarach L. from India


6-acetoxy-7a-hydroxy-3oxo-14b, 15bepoxymeliac-1,5-diene


Activity

Reference

Azedarachin C



Nakatani et al. (1995b); Huang et al. (1995a, 1995b)

Azedarachol

Et2O ext. of root bark of M. azedarach var. japonica

Antifeedant against Ajrotis sejetum Denis at 10 mg/cm2


Azedaralide


Antifeedant against S. littoralis (Boisduval) at 10 mg/cm2 Ictiotoxic O. latipes at 50 ppm


Azedaric acid

Light PE ext. stem wood


Campesterol

Fruit

Hanifa Moursi and AlKhabitib (1984)

EtOH ext. of fruit of M. azedarach


Cinamodiol

MeOH ext. (EtOAc fr.) of seeds of Brazilian M. azedarach

Cinnamic acid

EtOH ext. of leaves


Cycloeucalenone



D-Glucuronic

EtOH ext. (CHCl3 fr.) of root of M. azedarach from India

Keshri et al. (2003)

acid

Kelecom et al. (1996)

Deacetylsalannin


Antifeedant against S. eridania (Boisduval) at 20 mg/cm2

Huang et al. (1996a, 1996b)

Fraxinellone


Antifeedant against S. littoralis (Boisduval) at 10 mg/cm2



Ictiotoxic on O. latipes at 10 ppm


PE ext. of trunk wood

Ekong et al. (1969)




Little antimoulting activity on R. prolixus at 10 mg/ml

90

Compound


No antifeedant against S. littoralis (Boisduval)

Gedunin




Ekong et al. (1969)


Ictiotoxic O. latipes at 50 ppm.


Prevents the pregnancy and the number of implants at 50 mg/kg of rat/day


PE ext. of bark M. azedarach from Taiwan

Chiang and Chang (1973); Chang and Chiang (1969)

Kulinone


Chiang and Chang (1973)

Kulolactone


Chiang and Chang (1973); Chang and Chiang (1969)

Melain

Buffer ext. of fruit M. azedarach var. japonica

Protease

Kaneda et al. (1994)

Melain G

Buffer ext. green fruit M. azedarach var. japonica

Protease

Uchikoba et al. (1999)

Melazolide A



Meldenin

EtOH ext. (C6H6 fr.) seeds M. azedarach L. from India

Srivastava (1986)

Meliacarpinin A



Nakatani et al. (1995b)

Meliacarpinin B



Nakatani et al. (1995b) 91

Kulactone


Fraxinellonone

92


Activity

Reference

Meliacarpinin C




Meliacarpinin D





No lethal activity on A. salina at 100 mg/ ml


Meliacarpinin E



Huang et al. (1996b)

Meliandiol

Fruit

Han et al. (1991)

Melianin A



Melianin B



Melianin B



Melianol

CHCl3 ext. of fruits

Lavie et al. (1967a); Han et al. (1991)

Melianolide

Root bark of Chinese M. azedarach L.

Huang et al. (1996a)

Melianone


Lavie et al. (1967a); Han et al. (1991)

Melianoninol

Fruit

Antifeedant against Pieris rapae L.

Han et al. (1991)

Meliantriol


Repellent against S. gregaria at 8 mg/ cm2

Lavie et al. (1967b); Ascher (1986)


Compound

EtOH ext. (CH2Cl2 fr.) of seed kernel of M. azedarach from Argentina

Meliatoxin A1 ¼ trichilin D

EtOH ext. (Et2O fr.) of fruit of M. azedarach var. australasica

Meliatoxin A2

Meliatoxin B1

Antifeedant and insecticide against E. paenulata at 4 mg/cm2 and antifeedant against S. eridania at 1 mg/cm2

Carpinella et al. (2002, 2003a) Oelrichs et al. (1983); MacLeod et al. (1990)


Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2 (400 ppm)

Nakatani et al. (1994b, 1995b); Huang et al. (1994, 1995b)





Antifeedant against S. litura at 400 ppm (480 mg/cm2)

Oelrichs et al. (1983); MacLeod et al. (1990)



Huang et al. (1994); Nakatani et al. (1994b, 1995b); Huang et al. (1995b)


Growth inhibitory effect against S. litura at 400 ppm







Methyl kulonate

PE ext. of bark of M. azedarach from Taiwan

Chiang and Chang (1973)

Methylene cycloartanone

Fruit and roots of M. azedarach L.

Schulte et al. (1979)

Myristic acid


Prevents the pregnancy and the number of implants at 50 mg/kg of rat/day


Nimbolidin A

PE and Et2O ext. of fruit of M. azedarach var. japonica and from Yugoslavia and Greece


Kraus et al. (1980); Kraus and Bokel (1981)

93

Meliatoxin B2


Meliartenin (isomer B of compound 1)

94

Table 1 (continued ) Compound

Origin

Activity

Reference

Nimbolidin B

PE and Et2O ext. of fruit of M. azedarach var. japonica from Yugoslavia and Greece



Et2O ext. of root bark of Chinese M. azedrach L.

Antifeedant against E. varivestis at 0.005%

Bokel (1980)


Huang et al. (1996a, 1996b)

Nimbolin A



Ekong et al. (1969)

Nimbolin B


Ekong et al. (1969)

Nimbolinin B




Et2O ext. of root bark of Chinese M. azedrach L.


Nakatani et al. (1995a, 1995b) Huang et al. (1996a, 1996b)






Zhou et al. (2004)





Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ mg/ml

Zhou et al. (2004)

Ochinolid A

Ochinolid B



Fruit of M. azedarach L. var. japonica Makino


Ohchinolal

Fruit of M. azedarach L. var. japonica Makino


Pinoresinol



Zhou et al. (2004)

MeOH ext. (EtOAc fr.) seeds of Brazilian M. azedarach

Inhibited moulting R. prolixus, ED50 ¼ 84 mg/ml

Cabral et al. (1995); Cabral et al. (1999)

EtOH ext. of seed kernels (CH2Cl2 fr.) of Argentinian M. azedarach L.

Antifungal against F. verticillioides, MIC ¼ 1.0 mg/ml

Carpinella et al. (2003b)

Pyroangolensolide



Rutin



Salannal


Nakatani et al. (1995a); Huang et al. (1996a, 1996b)

Salannin

EtOH ext. (Et2O fr.) of seeds of M. azedarach L. from India

Srivastava (1986)



Nakatani et al. (1995a, 1995b); Huang et al. (1995b, 1996a, 1996b)

Scopoletin




Sendanin

MeOH ext. of bark and fruit of M. azedarach L. var. japonica

Sitosterol

Fruit

Ochi and Kotsuki (1976); Ochi et al. (1978) Reduce the incidence of induced gastric ulcers in rats

Hanifa Moursi and AlKhabitib (1984) Khalil et al. (1979)

95

EtOH ext. of fruit of M. azedarach


Ohchinin

96


Activity

Reference

Teracrylmelazolide A



Teracrylmelazolide B



Trichilin B



Huang et al. (1994); Nakatani (1994b, 1995b); Huang et al. (1995b)

Trichilin H



Nakatani et al. (1994b); Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)




Vanillic acid

Fruit

Han et al. (1991)

Vanillin

Fruit

Han et al. (1991)




EC50: effective concentration 50. LC50: lethal concentration 50. MIC: minimum inhibitory concentration. IC50: inhibitory concentration 50. ED50: effective dosage 50. Ext.: extract. Fr.: fraction. AcOEt: ethyl acetate. Et2O: diethyl ether. CH2Cl2: dichloromethane. EtOH: ethanol. MeOH: methanol. PE: petroleum ether.


Compound


97

0.030

C

0.025

AU

0.020 0.015 0.010 0.005 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes

Fig. 1. HPLC chromatogram of a subfraction from Fr. 2. Reversed-phase C18 column; mobile phase: 0–20 min 50% MeOH in water; 20 min 100% MeOH. UV detection at 220 nm.(From Riba et al., 1996, with permission.) 23

22

O

OH 18 20

O

12 11 30

OH

9

1

O 3

14

5

H

AcO

HO OH

21

17 16

O

O

12

O

H

AcO

OH 29

11

7

OH H

H HO

O

O

HO

28

A

B 1

It must be noticed that no other compound with similar activity than 1 was found in the seed kernel extract. According to this fact, toosendanin was ruled out as the most active anti-insect compound. Toosendanin which exhibits a similar structure than isomer A was previously reported in bark, root and stem bark extracts of M. azedarach (Chiu, 1989; Huang et al., 1995b; Nakatani et al., 1998). Azadirachtin (aza), whose presence was previously described in paraı´ so tree by Morgan and Thornton (1973) and Luo et al. (1995), was not found (Palacios et al., 1993). Its occurrence was also denied by many other authors (Lee et al., 1991; Ascher et al., 1995; Wandscheer et al., 2004). Reports about aza in M. azedarach extracts could be due to the confusion between M. azedarach and A. indica trees (Schmutterer, 1995; Ho¨rdegen et al., 2003).

Laboratory assays Antifeedant activity on insects of different orders For measuring the antifeedant activity on insects belonging to different orders, extracts obtained from different structures of Argentinian M. azedarach were prepared

98


Fig. 2. HPLC chromatograms of compound 1 (a) dissolved in CH3CN and (b) dissolved in CH3Cl. Each peak corresponded to each isomer A and B. Reversed-phase C18 column; mobile phase: 32% acetonitrile in water. UV detection at 210 nm.

(Valladares et al., 1997, 1999; Carpinella et al., 2002; Banchio et al., 2003; Carpinella et al., 2003a; Valladares et al., 2003). The antifeedant activity of the ethanolic fruit extract measured through choice test was very high showing an antifeedant index [AI (%)] higher than 75% in most studied insects such as adults of the Coleoptera Pantomorus leucoloma Boheman,


99

Priocyphus bosqui (Hustache), Tribolium confusum Duval, Diabrotica speciosa (Germar), Chrysodina sp., Epitrix argentiniensis Bryan, Eumolpinae sp., Plagioneda erythroptera (Blanchard), Xanthogalleruca luteola (Mu¨ller) and adults and larvae of Epilachna paenulata (Germ). The same results were observed on larvae of the Lepidoptera Spilosoma virginica (Fabricius), Anticarsia gemmatalis (Hubner) and Colias lesbias (Fabricius) (Carpinella et al., 2003a). Only Rachiplusia nu Gueneé did not respond to M. azedarach fruit extract. Coleoptera species appeared particularly sensitive to this extract, in contrast with the observations made after treatments with A. indica extracts in which case Lepidoptera was the most affected order (Mordue and Blackwell, 1993). Studies on the above-mentioned insects using senescent leaf extract from M. azedarach showed a potent antifeedant effect on Coleoptera and Lepidoptera (Valladares et al., 2003), being a little more effective on the former ones. Although there is previous information about the activity of extracts or isolated terpenoids from paraı´ so on some species of Spodoptera (Chiu, 1983; Khadr et al., 1986; MacLeod et al., 1990; Schmutterer, 1995; Carpinella et al., 2003a), S. ornithogalli (Guen) (Lepidtera: Noctuidae) was not affected by the leaf extract. In the case of Sitophilus oryzae (L.) (Coleoptera: Curculionidae), the strong rejection towards the leaf extract of M. azedarach observed here disaffirmed the lack of activity of extracts from paraı´ so reported by Imti and Zudir (1997), but corresponded with the positive effects registered on the control of other curculionide (Oroumchi and Lorra, 1993; Fernandes et al., 1996). Many studies on different pest insects have been carried out with M. azedarach extracts obtained from different vegetal structures of the tree, mainly from fruits. Aqueous extracts from fruits showed antifeedant activity against Authonomus grandis Boh. (Coleoptera: Curculionidae) (Fernandes et al., 1996). Adults of Dicladispa armigera and Callosobruchus chinensis, both Coleoptera, were greatly deterred from feeding on rice plants and mung beans; they were respectively treated with leaves and seed extract from M. azedarach collected in Bangladesh as reported by Islam (1986). The effect on D. armigera is in concordance with the activity found in other Chrysomelidae confronted to ethanolic fruit and leaf extracts from Argentinian M. azedarach (Valladares et al., 1997; Carpinella et al., 2003a). Schmutterer (1989) found that M. azedarach bark extracts were inactive up to a concentration of 800 ppm in the E. varivestis larvae leaf-disc feeding test. However, leaf extracts were considerably active as antifeedant at 200 ppm (70% activity) and 400 ppm (100% activity), more than analogous extracts from A. indica. Tewary and Moorthy (1985) tested a petroleum ether extract of drupes of M. azedarach against larvae and adults of Epilachna vigintioctopunctata (Coleoptera: Coccinellidae). The extracts at 0.1% gave 100% protection of leaves. Freshly treated larvae were less parasitized by Pediobius foveolatus (Hymenoptera: Eulophidae) than the control, but on exposure to parasitation 24 h after treatment, larvae were normally parasitized. The parasites that emerged from treated hosts were normal. A choice test with the leaf-disc method on the poliphagous larvae V of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) using methanolic extract of seed kernel of Chinese M. azedarach showed an antifeedant concentration 50 (AFC50, concentration that gives 50% of AI), 100 times lower than the one found in no choice test (Chiu et al., 1984). According to the authors, the difference could be attributed to the fact that the full grown larvae were highly sensitive to the extract.


100

Antifeedant activity measured in an artificial diet bioassay on larvae III of Spodoptera littoralis (Lepidoptera: Noctuidae) was related to extract concentration, varying from 11.2% to 56.9% at 10 to 100 ppm respectively. In Agrotis ipsilon (Lepidoptera: Noctuidae) same concentrations produced 7.7–51.1% of deterrency (Schmidt et al., 1997). A 1% methanolic extract from seed kernels of M. azedarach from China showed more than 80% of feeding inhibition on 1st and 2nd instar larvae from the Oriental armyworm Mythimna separata (Walker) (Lepidoptera: Noctuidae) (Chiu et al., 1987). In choice tests performed with Plutella xylostella L. (Lepidoptera: Yponomeutidae) where cabbage leaf discs were dipped in different solutions of methanolic kernel extracts from M. azedarach, the larvae consumed less or no food compared to untreated leaf discs at 24 h from the beginning (Dilawari et al., 1994). Methanolic extracts from fruits and seeds of M. azedarach collected in Spain showed a strong antifeedant activity against larvae II of S. nonagrioides Lefe`bre (Lepidoptera: Noctuidae) (Joan Serra et al., 1998; Juan et al., 2000). Extracts obtained from seed kernels of M. azedarach with petroleum ether showed antifeedant effect against 2nd and 3rd instar nymphs of Nilapavarta lugens Stal. (Hemiptera: Delphacidae). The AC50 (concentration at which 50% of insect were deterred from the plant) corresponded to 1.16% (Chiu, 1983). The activity of meliartenin (as compound 1) was studied under a choice test in comparison with aza and toosendanin (Carpinella et al., 2002). Both compound 1 and aza exhibited similar activity on E. paenulata (Coleoptera: Coccinellidae) larvae showing an AI (%) higher than 90% at 4 mg/cm2, while toosendanin showed same level of effectiveness at 10 mg/cm2. On larvae III of the southern armyworm Spodoptera eridania (Lepidoptera: Noctuidae), same rate of activity was observed at 1 mg/cm2 for compound 1 and aza and at 14 mg/cm2 for toosendanin. MacLeod et al. (1990) reported that the antifeedant activity of meliatoxin A2 measured in choice tests carried out with S. litura is in concordance with the activity of trichilin D reported by Nakatani et al. (1981) on S. eridania showing in both cases high activity at 400 ppm. It is important to say that, even when the concentrations are the same, in accordance with the methodology used by each author, the real quantity of compounds applied on the leaves were different (480 mg/cm2 calculated according to MacLeod et al., 1990 data, against 8 mg/cm2 according to data reported by Nakatani et al., 1995b and Huang et al., 1995a, 1995b). Meliatoxin B1 reduced feeding to a half with respect to control at 400–500 ppm (480–600 mg/cm2, MacLeod et al., 1990). 22

22

O

O

21

21 12

OH O

11 9

19

1

AcO

(CH 3)2CHOCO

H

29

28

H

Meliatoxin A2

OHO

17 30

19 15

O

H

O AcO

18

OH

1

AcO

CH3CH2CHCH3OCO

12 11 9

H

O AcO

H

29

18

28

H

Meliatoxin B1

17 30

15

H OH

O


101

Bentley et al. (1988) showed that the epoxide and furan groups are essential for high antifeedant activity in citrus limonoids. Besides, these substituents in ring D and independent of substitution patterns in ring A and C-28 ester moieties, the 12-OH translate in most potent activity, followed by 12a-acetoxy (Huang et al., 1994; Nakatani et al., 1994b). Acylation of C-29 also reduced the activity (Zhou et al., 1998). According to our results, it seems that 11-OH also ensures a high feeding inhibition. Compounds without the 14-15 epoxide group exhibited weak antifeedant action (Nakatani et al., 1999). Effects on deterrence, survival and development of insects from different orders Order Coleoptera Epilachna paenulata Germ. E. paenulata was used as reference insect for the isolation of the most effective anti-insect compound from the fruits of Argentinian M. azedarach L. Differences in the antifeedant rate were observed when larvae III of E. paenulata were fed with different concentrations of compound 1 in choice or no choice tests (Carpinella et al., 2003a). Results were measured at 24 h from beginning, being the larval stage the same for both tests. The ED50 (effective dosage 50, dosage in which an AI of 50% was obtained) in choice tests corresponded to 0.80 mg/cm2, while in no choice test the ED50 was lower: 0.25 mg/cm2. In no choice test, at 24 h, larvae of E. paenulata fed with cotyledons treated with 1 ate approximately four times less than larvae confronted to aza at all dosages, or to the control. Aza showed an abrupt reduction in food consumption 24 h later. After 6 days of treatment, larvae fed with both compounds exhibited almost null values of food consumption. Compound 1 would act as a primary antifeedant, demonstrated by an immediate rejection of treated food, probably via the gustatory pathway regulated by sensory organs of the mouthparts. Body weight was also affected reaching, depending on concentration, half of weight than those larvae receiving untreated food. Larval mortality showed at 24 h after treatment, 20% of dead larvae fed with 1 while the larvae confronted to aza, to untreated food or the starved ones were all alive. The fact that the larvae confronted to 1 died earlier, even compared to the starved group, suggests a toxic effect. Mortality was complete at 192 h with 0.5 mg/cm2 of 1, while at the same concentration of aza this level of mortality was reached at 240 h. This effect was probably more related to the strong feeding inhibiting effect of the compounds, mortality being due to the reduced food consumption previously mentioned. Epilachna varivestis Muls. A leaf extract from M. azedarach inhibited growth and disrupted metamorphosis of larvae IV of the Mexican bean beetle, E. varivestis (Coleoptera: Coccinellidae). A compound, whose structure was not elucidated, was isolated. A complete mortality was observed with the pure compound at a concentration below 2 ppm (Zhu and Ermel, 1991). Strong growth-disrupting effects were demonstrated by Steets (1975) on 4th larvae E. varivestis fed for 2 days with bean leaves sprayed with 5% and 2.5% extract. None of the larvae pupated and they died within 11 days after the start of the experiment. Similar results were obtained with the first stage of the larvae: only 5%

102


and 20% of larvae reached the second stage when feeding with 2% and 4% extract respectively. € Xanthogalleruca luteola ðMullerÞ. The elm beetle X. luteola (Coleoptera: Chrysomelidae) is the most important pest of the elm tree (Ulmus spp.) in America. Complete larval and adult mortality was observed in no choice test after feeding the insects with 2%, 5% and 10% fruit extract concentration. Development in surviving insects was delayed (Valladares et al., 1997). When the ripe fruit extract was applied as a spray twice with 24 h intervals, no apparent effect on survival of adults was observed (Valladares et al., 1997), whereas in larvae III treated with 5% extract concentration, mortality values were higher than 80%. It was also observed that only a 40% of the treated larvae reached the pupal stage with a delay of 2–6 days compared to control ones (Valladares et al., 1997). Considering the above-mentioned facts, the anti-insect effect was greater when the extract was ingested than when it was topically applied; this indicates that the skin is a strong barrier to be trespassed. This fact must be taken into account in the preparation of formulations. The lack of effect by contact and effectiveness by ingestion was also detected after treatments with neem extracts (Isman, 1994). Hypera postica Gyllenh. An investigation of the activity of aqueous extracts of leaves from M. azedarach collected in Iran was carried out on the cosmopolitan pest of the alfalfa weevil Hypera postica (Coleoptera: Curculionidae) in laboratory assays (Oroumchi and Lorra, 1993). In studies on larvae II–IV high larval-pupal mortality was obtained with leaf extracts at 2.5–10 g of powdered vegetal/100 ml.The feeding activity was reduced and the affected larvae were reduced in size, became darker, refused feeding even on the untreated leaves and gradually lost movement, which resulted in death. In some cases black spots appeared on the body surface of the insects (Oroumchi and Lorra, 1993). A similar reaction also occurs in larvae IV of E. varivestis after treatment with A. indica, which would be attributed to a massive degeneration of the tissue with the resulting liberation of high quantities of cellular debris which stimulates the formation of nodules (Schlu¨ter, 1995). In larval IV period high quantities of phenoloxidase (this enzyme catalyses the cuticular tanning and melanin formation) are available to react with the resulting cellular debris producing, together with the following haemocytic reactions, blackish nodules (Schlu¨ter, 1995). Some survival larvae developed to pupae and adults with normal or abnormal appearance, showing the latter, deformation in wings, legs and parts of the head (Oroumchi and Lorra, 1993). They often did not eliminate the pupal skin, being unable to move and therefore dying. Five percent of parasitism was observed, showing that the active principles did not affect the natural enemies of the weevil (Oroumchi and Lorra, 1993). Diabrotica undecimpunctata howardi Barber. In the search for new active substances that protect seedling corn from injury produced by southern corn rootworm Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae), 88 crude extracts obtained by pressure from plants were studied under no choice tests (Landis and Gould, 1988). The only extract that significantly reduced the feeding of the rootworm was the leaf extract from M. azedarach collected in North Carolina.


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Diabrotica speciosa ðGerm:Þ. Aqueous extracts of leaves, flowers, stems and fruits of M. azedarach collected in Brazil were evaluated against D. speciosa (Coleoptera: Chrysomelidae) (Ventura and Ito, 2000). The most efficient extracts in decreasing food consumption were the ones obtained from flowers and fruits followed by stems and leaf extracts which exhibited an intermediate activity. In a greenhouse experiment in which the flower extract was applied on plants of the common beans Phaseolus vulgaris L. protected from light, the leaf consumed by the beetle was null but when plants were exposed to light, the percentage of consumption increased to about 40%, probably due to the adverse effect of UV rays on the extract (Ventura and Ito, 2000). Tribolium confusum Duv. A population of T. confusum (Coleoptera: Tenebrionidae) was fed with wheat flour to which crushed fruits of M. azedarach collected in Spain were added. Larvae fed with 10–25% of crushed fruit showed an important delay in reaching pupal and larval stage (Del Tı´ o et al., 1996). This delay could be due to the strong antifeedant effect [AI (%) ¼ 88.4] observed by Carpinella et al. (2003) after feeding the weevil with 10% fruit extract. At higher concentrations, a demographic reduction of the insect was observed (Del Tı´ o et al., 1996). Dicladispa armigera Olivier and Callosobruchus chinensis Lucas. Fresh leaves and seeds of ripe fruits of paraı´ so were Soxhlet extracted with various solvents and studied against adults of rice hispa D. armigera (Coleoptera: Chrysomelidae) and the pulse beetle C. chinensis (Coleoptera: Bruchidae) allowed to feed on treated rice plants or mung beans respectively (Islam, 1986). Ovipositional and adult emergence of both insects were respectively deterred and reduced by 1% extracts obtained with methanol, methyl-tert-butyl-ether, methyltert-butyl-ether and methanol, ethanol, n-butanol, n-hexane and acetone being the oviposition most affected in the case of D. armigera. The newly emerged pulse beetle adults were small in size, did not survive more than 6 days and were less active in feeding (Islam, 1986). Order Diptera Liriomyza huidobrensis ðBlanchardÞ. With the aim of measuring the translaminar effect that the ethanolic fruit extract of M. azedarach possesses, larvae I of the leafminer Liriomyza huidobrensis (Diptera: Agromyzidae) were placed on Cucurbita ma´xima seedlings (Banchio et al., 2003). Results showed no significant mortality when the extracts were applied on leaves containing larvae I and III of the leafminer, but an increase in mortality rate of about 60% was observed in pupal stage (Banchio et al., 2003) even at 10% concentration. These results demonstrated the translaminar effect of the extracts even without the addition of surfactant, in accordance with that found by Brunherotto and Vendramim (2001) who suggest that the active principles present in extracts of M. azedarach exhibited translaminar action at producing mortality and delay in larval and pupal phase of the tomato pinworm Tuta absoluta, when the extracts were applied on the outer surface. If the plants were treated with the same extracts at 10% before being infected with females of the miner, the leaves received up to 90% fewer punctures than the control. On the other hand, these plants exhibited a lower number of pupae, while no larval or pupal mortality was observed. The fact that a high pupal mortality was absent

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when the seedlings were treated before exposing them to the adults suggests a shorter residual activity of the extract. In field assays plants of Vicia faba were treated four times with weekly intervals with ripe fruit extract of paraı´ so at 10% at neutral pH and with the addition of 0.5% of Tween 20 (Banchio et al., 2003). A 50% fewer pupae than in plants treated with water (control) and those treated with the synthetic insecticide Tamaron was observed. Pupal survival was also affected (30% of survival), this value being lower than in treatments with Tamaron (80% of survival). Parasitism rates were slightly affected, the difference with the control being much lower than with the use of Tamaron. This result could be due to the delayed action of the extracts, which allows the pupation of the larvae killing them slowly (Banchio et al., 2003). In this way the population of parasitoids is conserved as was observed by Oroumchi and Lorra (1993) and Breuer and Devkota (1990). Aqueous extracts of fruits and leaves of paraı´ so were tested for their efficacy against L. huidobrensis (Abou-Fakhr Hammad et al., 2000b) on naturally infested swiss chard, Beta vulgaris var. cicla L. and in greenhouse experiments on artificially infested cucumber, Cucumis sativus L. Results indicated that Melia fruit extract significantly lowered the number of larvae per swiss chard plant causing deformed larvae, partially brown, rooting and oozing, indicating a growth regulating activity. Leaf extract did not exhibit any effect. In greenhouse experiments, the fruit extract significantly decreased the number of live larvae of the leafminer per cucumber leaf throughout the period of the experiment and with comparable effect to that of synthetic insecticides such as abamectin, cyromazine, imidacloprid and pyrazophos. On its own, leaf extract kept the number of larvae at a significant lower density with respect to control at 20 days after a second application (Abou-Fakhr Hammad et al., 2000b). Anastrepha fraterculus ðWied:Þ. The South American fruit fly, Anastrepha fraterculus (Diptera: Tephritidae) is one of the most important pests in Brazil where the insect has developed resistance to synthetic insecticides (Salles and Rech, 1999). The use of a formulation obtained by aqueous maceration of dry dust of fruits of M. azedarach was studied for the control of the pest. Treatments with doses of the dust at 25–150 g/l produced a decrease in the number of eggs, larvae, pupae or adults. At 150 g/l, malformed pupae and adults were obtained (Salles and Rech, 1999). Orseolia oryzae ðWood-MasonÞ. Orseolia oryzae (Diptera: Cecidomyiidae) is a rice pest that causes important losses in the production of the crop. Studies on the rice gall midge using methanolic extracts of seed kernels of M. azedarach from China showed an oviposition repellence of 83.1% at 1% extract concentration when a choice test was carried out and a 70.7% of repellence with 2% concentration in no choice test (Chiu et al., 1984). Aedes aegypti Linnaeus. Studies of ethanolic extracts from endocarps of M. azedarach collected in Brazil on late larvae III and earlier larvae IV of the mosquito vector of dengue fever, Aedes aegypti 1762 (Diptera: Culicidae) showed an LC50 (lethal concentration 50) of 0.034 g% in an artificial diet assay. This value was lower than that observed when the larvae were not fed at all (Wandscheer et al., 2004).


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Order Lepidoptera Spodoptera litura ðFabriciusÞ. Through a no choice test, MacLeod et al. (1990) studied the effect of meliatoxins A2 and B1, isolated from the ethanolic fruit extract from M. azedarach var. australasica on the 2nd instar of S. litura. Meliatoxin A2 significantly reduced the ingestion of food at 400 ppm (480 mg/cm2), while meliatoxin B1 inhibited the feeding in only 50% at the same concentration. However, this last compound significantly reduced the weight gain at 400 ppm, showing an inhibitory growth effect rather than an antifeedant action. Meliatoxin A2 also reduced the growth but it is apparently due to the inhibition of food consumption. According to the authors, this difference in the activity between both compounds could be attributed to the structure of ring D, showing that the epoxi function confers higher antifeedant activity, and the C-15 keto group would be responsible for the growth inhibition. Spodoptera frugiperda ðJ:E: SmithÞ. The effects of extracts obtained with methanol from fruits of M. azedarach L. collected in Greece were studied in S. frugiperda (Lepidoptera: Noctuidae) (Breuer and Schmidt, 1995, 1996). The larvae fed with 1% and 10% solutions of extract sprayed on lettuce leaves formed smaller pupae and the emerged adults showed deformations and morphogenetic defects. A decrease in feeding was observed and a retarded larval growth and development was detected. When increasing the concentrations of Melia, the ingesta decreased and the larvae digested and/or metabolized the food with less effectiveness. The reduction in growth was not only due to starvation but also due to ingestion of toxic substances (Breuer and Schmidt, 1995). When the experiment was done by topic application no significant differences were observed compared to control (Breuer and Schmidt, 1995). The number of eggs laid by adult females treated from the larval stage was reduced. Adult emergence was not affected. The larval and pupal mortality was increased while increasing the dosages, mainly due to alterations in moulting which could be caused by defects in the neuroendocrine system (Breuer and Schmidt, 1995, 1996). Breuer and De Loof (2000a) obtained different fractions from a methanolic extract of green fruits of M. azedarach collected in Greece. For the assays, the fractions were incorporated into an artificial diet. The growth of S. frugiperda was completely retarded and the larval mortality reached 100% with the ethyl acetate fraction at 1000 ppm, while the petroleum ether and water ones did not show differences with the control. A significant delay in larval development was observed at 10 ppm and upwards and body weights were highly reduced after treatment with 50 ppm of the ethyl acetate fraction (Breuer and De Loof, 2000a). Artificial diet containing 0.01% of an ethyl acetate fraction obtained from unripe fruit of M. azedarach collected in Greece and extracted with methanol resulted in an inhibition of the cholinesterase activity of the larvae of S. frugiperda (Breuer et al., 2003). This effect together with the lower food intake was responsible for the diminished locomotory activity of the larvae. The activity of the NADPH cytochrome c reductase was significantly higher (34%) to that of the control. In the cockroach Leucophaea maderae the effect of 1% extract on the activation of this enzyme was 43% (Breuer et al., 2003). It is known that this detoxification system becomes more

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activated as larvae develop (Breuer et al., 2003), which would explain the lower sensitivity to treatments of the bigger larvae (Breuer and Schmidt, 1996). Rodriguez Hernandez and Vendramim (1998) compared the effect of aqueous fruit extracts obtained from M. azedarach from Brazil with extracts equally obtained from other Meliaceae species on 1st instar larvae of S. frugiperda. Larvae fed with M. azedarach extract, suffered from a longer larval phase with respect to control, being this extract more effective than the extracts obtained from the other Meliaceae. Melia extract also resulted in lower food consumption and body weight compared to other extracts. Less efficient food conversion into biomass was also observed (Rodriguez Hernandez and Vendramim, 1998). Since S. frugiperda consume less food and this was metabolized with lower efficacy after treatments with extract from fruits of Melia (Breuer and Schmidt, 1996; Rodriguez Hernandez and Vendramim, 1998), the content of haemolymph protein was decreased (Schmidt et al., 1998). The reduction in the reserve of this material (necessary for the development of eggs in the adult females) would be the reason for a lower number of eggs laid by S. frugiperda (Breuer and Schmidt, 1996). On the other hand, protein biosynthesis could be affected by M. azedarach compounds. This alteration and the subsequent alteration in neuropeptide production have also been reported for aza (Fritzsche and Cleffmann, 1987; Meurant et al., 1994). Leaves from M. azedarach L. were extracted with hexane, chloroform, methanol and water. Resulting extracts were incorporated to a base of agar in order to feed S. frugiperda (McMillian et al., 1969). Hexanolic and aqueous extracts did not exhibit any effect on larval growth; however, the chloroformic extract at 3 mg equivalents of extract based on dry leaf weight per gram of diet showed smaller larvae. Methanolic extract was less active. With the chloroformic one a decrease in weight of about 100 times compared to control was observed at 30 mg equivalents/g of diet. All larvae died when they were fed with this mentioned concentration. At 10 mg equivalents/g of diet, the mortality of S. frugiperda was 90% and although the remaining 10% pupated, they did not develop into adults. A delay in time till pupation and adult emergence was also observed showing some deformities in the adults at intermediate concentrations (McMillian et al., 1969). When S. frugiperda larvae were fed with corn seedlings treated with the chloroformic extract, a reduction in weight was observed (McMillian et al., 1969). As is observed in this study, the most active extracts were not those obtained with solvents of extreme polarity. This is in accord with the data exposed in Table 1 and by Breuer and De Loof (2000a) where most active principles were those extracted with medium polarity solvents. Spodoptera littoralis ðBoisd:Þ and Agrotis ipsilon ðHufn:Þ. The effectiveness of methanolic extracts enriched by fractionation with petroleum ether and ethyl acetate obtained from fruits of M. azedarach collected in Greece (Schmidt et al., 1997) was studied on 3rd instar larvae of the cotton leafworm S. littoralis (Boisd.), and the black cutworm, A. ipsilon (Hufn.) (Schmidt et al., 1997). After the addition of the methanolic extract at 50 ppm to an artificial diet, consumption was decreased. Weight gain was significantly reduced with the use of the extract at 10 ppm.


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Complete mortality of S. littoralis and A. ipsilon at 50 and 100 ppm respectively was observed (Schmidt et al., 1997). At lower concentrations, pupal weight and development were affected. In both, insect malformations and moulting defects were detected. Adults emerged from larvae previously treated with extract showed a reduction in fecundity and fertility (Schmidt et al., 1997). S. littoralis was slightly more sensitive than A. ipsilon in terms of pupation and adult emergency. Decreasing of body weight may be due to the reduction in food consumption and this is in agreement with the histological findings where destroyed epithelial cells were observed; thus digestion and absorption of the food may be inhibited (Schmidt et al., 1997). This effect was also observed on Schistocerca gregaria and Locusta migratoria after treatments with aza (Mordue and Blackwell, 1993). The fact that larval and pupal intermediates were found, after treatments with methanolic green fruit extract of M. azedarach was due to an increase of juvenile hormone II, S. littoralis being more affected than A. ipsilon (Schmidt et al., 1998). In both insects, the content of haemolymph protein was diminished after treatments with 50 ppm of extract. Heliothis zea ðBoddieÞ. Extracts obtained by McMillian et al. (1969) were incorporated to a base of agar in order to feed larvae I of Heliothis zea (Lepidoptera: Noctuidae) The chloroformic extract at 3 mg equivalents/g of diet was responsible for smaller larvae. All larvae died when they were fed with 10 mg equivalents/g of diet (McMillian et al., 1969). Mythimna separata ðWalkerÞ. Most of the larvae I and II of M. separata did not develop into larvae V and some of them lost weight after treatment with 1% methanolic extract from seed kernels of M. azedarach collected in China. The larvae which reached the last instar were more resistant since the same extract at 2% exhibited only a 60% of feeding inhibition to the 5th and 6th instar although some of them lost weight and died (Chiu et al., 1984) suggesting the presence of toxic substances in M. azedarach extracts. Busseola fusca ðFullerÞ. The level of infection of the maize stalk borer Busseola fusca (Lepidoptero: Noctuidae) in field tests was significantly reduced after two applications of solutions obtained from a macerate in water of dried fruits of M. azedarach L. collected in Ethiopia (Gebre-Amlak and Azeferegne, 1999). An 8.6% of decrease in the number of plants with leaf infestation resulted in a 47.9% of increase in the crop yield. With the same application schedule, solutions of fresh and dried leaves and fruits produce significant reduction in the number of live larvae in each plant after 72 h of the first treatment. After 72 h from the second treatment, results were similar to those obtained with the use of Lamdacyhalotrin (positive control). However, the number of larvae recorded at harvest was not significantly different from the number of larvae obtained from the control, indicating that the protection of the Melia extract did not last for the entire period of growing (Gebre-Amlak and Azeferegne, 1999) in contrast with Lamdacyhalotrin which showed lower degradability. Tuta absoluta ðMeyrickÞ. Aqueous extracts from leaves, branches and unripe and ripe fruit of M. azedarach collected in Brazil were used for controlling the tomato

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pinworm T. absoluta (Lepidoptera: Gelechiidae) (Brunherotto and Vendramim, 2001). Newly emerged larvae of the insect were fed with tomato leaves (Lycopersicon esculentum) treated with extracts from different tree structures until adult emergence. Larval and pupal survival was affected with 0.1% leaf extract. Surviving larvae and pupae showed extended larval development. Male and female pupae weight was lower than the control group even at 0.1%. When the activity of leaf extract was compared to extracts from different structures of the tree, it was observed that the former was the most effective in delaying larval phase. All extracts produce similar larval and pupal mortality, being ripe fruit extract the less effective (Brunherotto and Vendramim, 2001). This similarity in the action is not frequent since, in accordance with what was previously stated by Rodriguez Hernandez and Vendramim (1996), effect on the plants is more drastic in larvae than in pupae because the former are the ones that ingest the treated food. The authors suggested that larvae treated with M. azedarach extracts were not capable of degradating, at least in part, the active principles present in the extracts also affecting the pupal development. This is not in accordance with the findings of Banchio et al. (2003) where larvae of L. huidobrensis treated with complete ripe fruit extract of M. azedarach showed highest mortality percentages in pupal stage. Plutella xylostella L. With the aim of finding an alternative for controlling the diamondback moth, P. xylostella, which has exhibited resistance to different synthetic insecticides (Mehrota, 1991) and even to Bacillus thuringiensis (Tabashnik et al., 1990), Dilawari et al. (1994) studied the effect of a methanolic extract from kernels of M. azedarach. The number of eggs measured when the inner surface of a recipient was sprayed with the crude extract was significantly reduced. The same tendency was also observed for emergency. When the larvae hatching from eggs treated with 10% solution were fed with fresh food, they suffered a higher larval and pupal mortality than controls. Up to 17.7% of the emerged adults suffered malformations (Dilawari et al., 1994). In another assay, chloroform and hexane fractions from kernel extracts of Melia in acetone were topically applied on larvae of P. xylostella at 0.1 mg/3.7 mg of larval weight resulting in 30% and 60% of mortality respectively (reference in Dilawari et al., 1994). In a similar way, the extract was applied on cauliflower cotyledons before placing moth adults on them. There was no effect on the number of laid eggs but hatching was 90% lower (reference in Dilawari et al., 1994). These results suggest that the extracts would also act by contact on P. xylostella eggs, in opposition to what was found by Breuer and Schmidt (1995) on S. frugiperda. With the aim of studying the effect on oviposition, extracts of fruits of M. azedarach were obtained by extraction with diethyl ether (Chen et al., 1996a). Choice and no choice tests were carried out with newly emerged and mated female P. xylostella placed together with rape seedlings (Brassica campestris L.) dipped in the extract solutions. In choice tests, oviposition in treated seedlings was reduced in 49.6–93.5% compared to control at 0.5–4%, while the reduction was lower in no choice test. The number of moths found on the treated seedlings was drastically reduced (Chen et al., 1996a). The effectiveness of the extract was maintained throughout the experiment, indicating that its activity lasts by at least 48 h under laboratory conditions. Besides, there were no signs of phytotoxicity (Chen et al., 1996a).


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When the above-mentioned extracts were applied by dipping plants of Brassica olereaceae L. (Chinese Kale), mortality values over 90% were obtained even when the larvae were treated in 1st, 2nd, 3rd or 4th instar. Some of them died before moulting, while the majority did it for the inability to eliminate the old skin. The diary consumption was significantly reduced at 2% and 4% extract concentration in treatments of all instars. Pupal weight, adult emergence and adult longevity decreased when the larvae were continuously reared with rape leaves treated with 0.05% solution or above. Egg hatchability was significantly reduced when the eggs were directly dipped in 1% extract concentration (Chen et al., 1996b). Studies carried out on two species of parasitoids attacking P. xylostella, Cotesia plutellae (Hymenoptera: Braconidae) and Diadromus collaris (Hymenoptera: Ichneumonidae) showed that aqueous leaf extracts from M. azedarach did not negatively affect the longevity of either parasitoids or the percentage of parasitism (Charleston et al., 2005). Thaumatopoea pityocampa ðDen: & Schiff:Þ. Larvae of the needle devouring caterpillar Thaumatopoea pityocampa (Lepidoptera: Thaumatepoeidae) were put on pine branches of Pinus mugo Turra treated with 1% and 10% of an extract obtained by grounding in methanol ripe fruits of M. azedarach collected in Greece (Breuer and Devkota, 1990). Larvae fed on treated twigs consumed less food than controls and produced very low quantity of faeces. Mortality increased up to 100% depending on the concentration and stage of the larvae. The early death of the caterpillar might be due to the intake of toxic substances, deduced by typical symptoms of poisoning and the lethargic behaviour, or due to the diminished food ingestion (Breuer and Devkota, 1990). The insects on treated branches neither gathered nor built their nest as the control animals did, which could be associated to the repellent effect. As expected, young stage larvae are more sensitive to M. azedarach extract. This monophagous insect seems to be quite sensitive to paraı´ so extract as compared with other poliphagous Lepidoptera such as S. frugiperda or A. ipsilon, perhaps because the later possess a broad spectrum of detoxification mechanism due to its larger nutritional spectrum (Breuer and Devkota, 1990). Field trials on T. pityocampa were undertaken in a pine forest (Breuer and Devkota, 1990) in Greece. In the night of spraying the trees with M. azedarach methanolic ripe fruit extract, the insects rejected feeding on the pine needles. In the case of partially sprayed trees, only the untreated needles were consumed. In the following nights, the caterpillars did not come out of the nests sprayed with 10% Melia extract. A number of T. pityocampa could be seen lethargic or dead hanging on twigs. Inside the nests the larvae were found either dead or lethargic (Breuer and Devkota, 1990). Natural enemies such as the wasp Erigorgus femorata Aubert (Hymenoptera: Ichneumonidae) and the flies Phryxe caudata Rondani and Phorocera grandis Rondani (both Diptera: Tachinidae) were alive and trying to parasitize the lethargic caterpillars (Breuer and Devkota, 1990). These results indicate that M. azedarach extract is highly specific on pest insects. Thaumatopoea processionea L. Thaumatopoea processionea is a Lepidoptera of the Thaumetopoeidae family, whose larvae completely defoliate the oak trees (Breuer

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and De Loof, 1998). Unripe fruits of paraı´ so collected in Greece were extracted with methanol defatted with petroleum ether, and partitioned in water/ethyl acetate. The organic fraction was applied on oak twigs (Quercus robur L.) where larvae of 2nd and 4th instar of T. processionea were placed. In 2nd instar, the faecal production was decreased indicating a strong antifeedant effect of the botanical extracts on the insect. Surviving treated larvae weighed the same throughout the experience (Breuer and De Loof, 1998). Those larvae which have ingested treated twigs were inactive and from day 8 after beginning of experience, the mortality was complete in the concentration of 0.1% or above. Larvae IV fed with twigs sprayed with Melia at 1% stopped its feeding and excretion, and body weight suffered a decrease. Contrary to the effect observed with larvae II, in larvae IV there would be a more important antifeedant effect which masks a possible toxic effect. While control larvae aggregated in one place, the treated young insects separated from each other. The treated larvae were inactive, finally dying at 1% concentration (Breuer and De Loof, 1998). Death was related to problems in moulting probably by the influence of the active principles in the hormonal system and due to the reduced ingestion. The insects could not remove the old skin and were often ‘caged’ in their exuviae. Younger larvae reacted more sensitively to the extracts than the older ones (Breuer and De Loof, 1998). The same organic fraction was tested against T. processionea on Q. robur plantations sprayed with 1% extract (Breuer and De Loof, 2000b). One week after the treatment, the consumed areas in the trees sprayed with the extract were small or almost in traces. In the control trees the caterpillars gathered in groups, while in the treated trees, the insects were often found distributed all over the oak and the larvae did not construct their nest. The insects in the treated trees appeared inactive, and only moved if touched, in coincidence with those previously found in the laboratory assays (Breuer and De Loof, 1998). Two weeks after the beginning, only 10–40% of the caterpillars survived and at 4 weeks, 100% of mortality was registered. The mortality was mainly due to problems during ecdysis. It was also observed that spiders, carabids, scorpionflies and tachinids were fed on weak or death larvae in the treated trees showing that these natural predators were not affected by the extracts (Breuer and De Loof, 2000b). Phthorimaea operculla Zeller. Kroschell and Koch (1996) studied the effect on Phthorimaea operculla (Lepidoptera: Gelechiidae) of aqueous seed extract of M. azedarach at 20% applied on potatoes. The repeated treatment with extract resulted in a lower larval development towards the adult stage when the potatoes were treated before infecting with the eggs of the moths. It was observed that in experiments made with potatoes placed in storage during 3 months and treated before releasing the adults, extract of M. azedarach reduced the frequency of infection (number of infected tubers) in 35.2% compared to controls. The number of points of infection per tuber (intensity of infection) was decreased in treatments with Melia extract (Kroschell and Koch, 1996). Sesamia nonagrioides Lef e bre. Seeds of M. azedarach L. collected in Spain were first extracted with methanol and then defatted with hexane. The resulting extract and fractions obtained from it through the use of preparative HPLC were studied against one of the most important pests of the corn in the Mediterranean area, the


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Mediterranean corn borer S. nonagrioides (Riba et al., 1996). The assays were made with 2nd instar larvae fed either on corn leaves dipped in aqueous solution of the extract at 10 ppb and then transferred to untreated food or with an artificial diet mixed with extract at 450 ppm (prepared with 0.16 g of extract). The isolated fractions were also added to an artificial diet in a concentration equivalent to process 0.16 g of extract. Azadirachtin was used for comparison purposes. While control larvae increase their body weight, those larvae confronted to the extract or to aza maintained the same weight throughout the study (Riba et al., 1996). At the end of the experience, some of the treated larvae increased their weight and then pupating, contrary to those confronted to aza which died at 42 days. Mortality produced by the Melia extract until they reached the pupal stage was 77% while in aza treatments it was 100%, taking into account the very low concentration of M. azedarach extract. While the larvae were fed with the Melia extract, a mortality percentage of 65% was observed whereas the mortality percentage with aza corresponded to 5%; however, this last compound showed a staggering mortality even during or after the treatment until reaching a 100% (Riba et al., 1996). These results are in concordance with those found by Carpinella et al. (2003a) between compound 1 and aza where the first compound acted earlier than the second one. In the artificial diet assay the mortality of the corn borer fed with the M. azedarach extract till pupation was 86% and a delay in development was observed. Larval development was prolonged after treatments with Fr. 2 and Fr. 3 obtained from methanolic seed extract from M. azedarach by HPLC (Riba et al., 1996). The growth speed for larvae confronted to diet with extract or Fr. 2 was significantly different than in the controls, while treatment with Fr. 3 exhibited an intermediate behaviour (Riba et al., 1996). These results showed that the compound present in Fr. 2 was more potent than the one found in Fr. 3. The activity of methanolic extracts from fruits and seeds of M. azedarach collected in Spain at 1000 and 2000 ppm was compared with aza and a commercial product of neem called Mubel, which contained 1.5 ppm of aza using an artificial diet. At 30 days after the ending of the study, only aza, Mubel and M. azedarach seed extracts showed significant differences in weight. Surviving larvae that had recovered at this time pupated with similar weights than the control but with longer development time (Joan Serra et al., 1998; Juan et al., 2000). The difference in the results between both Melia extracts indicates that the active principles of the tree are concentrated in the seed. These differences were also observed in S. littoralis and A. ipsilon (Schmidt et al., 1997). Mubel and seed extract at 2000 ppm showed values of ingest 10 times lower than the ones of the control. The seed extract, Mubel and aza showed high levels of deterrency, being the first two the most effective. This suggests that in Mubel there are other compounds besides aza that increase the activity of the product (Juan et al., 2000). This fact is one of the reasons why the use of complete extracts is recommended over the use of pure compounds. Mortality was complete only in case of Mubel at 75 ppm, while in other assays it corresponded to 6–27%. Order Hemiptera Triatoma infestans Klug. Experiments oriented to study the repellence of ethanolic ripe and unripe fruit and green leaf extracts of M. azedarach collected in Argentina

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were carried out with the hematophagous insect Triatoma infestans (Hemiptera: Reduviidae) or ‘vinchuca’, the vector of Chagas disease (Valladares et al., 1999). For this purpose, treated filter paper refuges were used and the distribution of the insects on them was assessed. The unripe fruit extract was the most effective exhibiting 93% and 80% repellency index on 1st and 4th instar nymphs respectively at environ 11% extract concentration. These results coincide with those reported by Arias and Schmeda Hirschmann (1988) who found high repellency from unripe fruit extract and oil from M. azedarach of Paraguay at 0.5% in contact with 4th instar nymphs of T. infestans. Ovicidal effects measured on eggs sprayed with unripe fruit extract were not observed, nor were alterations in mortality or moulting when placing 1st instar nymphs in contact with treated refuges (Valladares et al., 1999). However, nymphs were smaller and lighter when they were reared on the extract treated refuges for one intermoulting period suggesting that a stronger effect could be observed if the study was carried out along the insect life cycle. Arias and Schmeda Hirschmann (1988) did not find insecticide or moulting activity by contact, but a slight increase in mortality was observed when 4th instar nymphs were topically treated with unripe fruit oil. The lack of effectiveness of the extract by contact could be due to the extract not being ingested, since when feeding 4th instar nymph of Rhodnius prolixus with the lignan pinoresinol, isolated from seeds of M. azedarach collected in Brazil (Cabral et al., 1995) at 100 mg/ ml, a 58% inhibition in the ecdysis of the nymph was observed (Cabral et al., 2000). Pinoresinol was also obtained from ripe fruit of Argentinian M. azedarach (Carpinella et al., 2003b). This compound also produced a high antifeedant effect on R. prolixus instar IV when it was ingested at 500 mg/ml of feeding blood (Cabral et al., 1999). When the compound was applied on the surface of a Petri dish, a low mortality was detected at the highest studied concentration: 100 mg/cm2, with no significant effects on ecdysis (Cabral et al., 1999), but a prolonged intermoulting period was observed in 4th instar larvae of the insect (Cabral et al., 2000). The highest negative effects on pest insects were observed again when extracts or pure compounds were ingested instead of applying them topically. Diaphorina citri Kuwayama. Assays made by spraying the Asiatic citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae), with seed oil from M. azedarach at 2% even after a rain, showed a repellency of 69.7% at 5 days after treatments. Preliminary experiments carried out in large cages showed that the incidence of the yellow shoot disease of citrus of South China, a disease transmitted by the psyllid, was significantly reduced by nearly 70% in young trees sprayed with the previously mentioned emulsion (Chiu et al., 1984). Nilapavarta lugens Stal; Sogotella furcifera ðHorvathÞ; Nephotettix virescens ðDistantÞ. The quantity of food ingested by newly emerged females of N. lugens (Hemiptera: Delphacidae), Sogotella furcifera (Hemiptera: Delphacidae) and Nephotettix virescens (Hemiptera: Cicadellidae) was significantly reduced when they were fed with seed oils of neem and methanolic extract of M. azedarach L., being Melia oil relatively more effective than neem oil (Saxena et al., 1983). When neem oil and Melia extracts were topically applied on S. furcifera, an almost complete mortality was reached at 50 mg/female. Neem oil was more effective on N. lugens than Melia extract. Neither neem oil nor Melia extract was active against


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N. virescens up to 50 mg/female. Even at this last concentration the extracts were slightly toxic on the predator Cyrtorhinus lividipennis (Hemiptera: Miridae) and on the predatory mirid bug Lycosa pseudoannulata (Araneae: Lycosidae). These results indicate that the active principles present in these extracts are rather pest specific (Saxena et al., 1983). Schizaphis graminum ðRondaniÞ. In order to study the systemic effect of methanolic extract obtained from seed kernel of M. azedarach collected in USA or in China, the potting soil of sorghum plants was drenched with emulsions of the extract (Hu et al., 1998). The plants were infected with adults of Schizaphis graminum (Hemiptera: Aphididae). With the addition of 150 mg of Melia extract, the population was slightly reduced (Hu et al., 1998). According to this experience it could not be determined if the poor effect of the M. azedarach extract was due to the lack of systemic translocation or if this extract was not toxic to the insect (Hu et al., 1998). Order Orthoptera Locusta migratoria migratorioides ðReiche & FairmaireÞ. Aqueous, methanolic and petroleum ether extracts of fruits and leaves of M. azedarach collected in Dominican Republic and Germany respectively were assayed in the laboratory against L. migratoria migratorioides (Orthoptera: Acrididae) (Wen and Schmutterer, 1991). Treatments of 1st instar nymphs with aqueous fruit extract at 3% showed 93% of cumulative mortality and when most nymphs were still in instar II, control insects had reached the third stage. Aqueous extract of leaves at 3% applied on 1st instar nymphs and food resulted in a total mortality, while less than 60% mortality was observed when the same concentration was applied only to nymphs. The effect produced by the ingestion was stronger than by contact, this is expected for any substance due to the barrier imposed by the cuticle. Nymphs fed on treated food ate only once and then they rejected the food, even the untreated one. When 1st instar nymphs were treated, lower weights and a delay in reaching the following instar were observed. Treatment with aqueous extract of leaves on nymph III exhibited 93.2% mortality at 3% concentration and a reduction of feeding activity. When nymph IV was treated with methanolic fruit extract at 5% only 23.5% of mortality was observed, increasing to 64.7% when the same concentration was applied to nymphs and food. The weight of treated nymphs was reduced. With the petroleum ether phase, there were no different results to control, suggesting that the activity is due to the presence of polar substances (Wen and Schmutterer, 1991). Schistocerca gregaria ðForsk:Þ. Cabbage leaves were sprayed with different concentrations of an ethyl acetate extract obtained from a methanolic extract from unripe fruits of paraı´ so collected in Greece and confronted with control leaves to 2nd instar nymphs of S. gregaria (Orthoptera: Acrididae) (Breuer and De Loof, 2000a). The development of the larvae was only possible in treatments with extract concentrations below 0.001%. At higher concentrations a strong antifeedant effect was observed. A complete mortality was obtained even at 0.01%. The weight of surviving nymphs treated with 0.01% was threefold lower than the control group (Breuer and De Loof, 2000b).

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The effect of a methanolic extract from fruits of paraı´ so was studied by applying them at 20 and 50 mg/ml on wheat seedlings (Schmidt and Assembe-Tsoungui, 2002). Oral ingestion of the extract reduced the feeding in 3rd instar as in adults of the locust. The larvae showed a decrease in the length of hind femora and elytra and width of head as well as in weight gain. The development of the larvae was prolonged and the moulting process was altered. The oviposition was strongly reduced and the maturation of oocytes was retarded only reaching the stage of previtellogenesis. The normal yellow colour of the males was affected as a step to gregarization (Schmidt and Assembe-Tsoungui, 2002). Order Homoptera Bemisia tabaci ðGenn:Þ. Tests under screen-house conditions were carried out with aqueous extracts of equal parts of leaves and ripe fruits of M. azedarach from Brazil on the vector of the bean golden mosaic virus, Bemisia tabaci (Homoptera: Aleyrodidae) (Nardo et al., 1997). With the aim of measuring toxic effects, adults of the insect were placed together with two plants of P. vulgaris L. cv. carioca sprayed with the extract. A percentage of mortality of 70%, 90% and 100% at 48, 72 and 96 h, respectively, was observed with no significant differences with respect to starved adults (Nardo et al., 1997). These results suggest an antifeedant action of the extract instead of a toxic effect (Nardo et al., 1997). To evaluate the effect on oviposition, adults fed with soybean were confronted to treated or untreated bean plants. The number of pupae found in sprayed plants was half than in water treated plants. However, the extract did not affect the development. On this basis the authors suggest that the extract would affect the number of eggs laid probably due to the antifeedant action and this would be explained because the insect laid the egg during feeding (Gamell, 1974). Aqueous extracts of fresh fruits and leaves of M. azedarach from Beirut were applied at 40% concentration on a single bean leaf. Whole fruits were also extracted with water and methanol and diluted to 20% (higher concentrations of the extracts caused phytotoxicity on tomato leaf). Other fruit extracts were obtained extracting the vegetal material with acetone or methanol after defatting with petroleum ether. The dried extracts dissolved in distilled water were applied on tomato plants (AbouFakhr Hammad et al., 2000a). The fruit and leaf aqueous, methanolic and acetone extracts showed a lower number of B. tabaci adults on treated plants (Abou-Fakhr Hammad et al., 2000a). Mortality of about 23–49% was observed on 1st and 2nd instar of the whiteflies with the use of 20% extracts, the ether and water extracts being the less effective. The highest concentration of methanol extract caused the highest pupal mortality (24%) comparable to Azatin (25%) and water plus Triton 0.2% (23%). These studies indicated that earlier instar was more susceptible to M. azedarch extracts than pupal stage (Abou-Fakhr Hammad et al., 2000a). This could be attributed to the fact that late stages are highly protected by fat bodies (Schlu¨ter, 1995) that may either detoxify or sequester toxins. Extracts of fruit and leaves of M. azedarach collected in Beirut (AbouFakhr Hammad et al., 2001) and from callus obtained from leaf branches of the mentioned tree, even fresh or frozen, were extracted with methanol or water. The resulting extracts (at 20%) were applied to tomato plants (L. esculentum)


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under glasshouse compartment together with adult whiteflies of B. tabaci in a no choice test. Either methanolic or aqueous extracts of leaves, fruits and callus, fresh or frozen, developed significant repellent activity (58–68%) (Abou-Fakhr Hammad et al., 2001). In accordance with the author there would not be differences in using methanol or water as extractive solvents of the fresh or frozen vegetal material. It was found that the mortality of adults confronted to sprayed plants was not significantly different from that of unfed adults, but in both cases it was higher than in the controls. This fact suggests that a possible antifeedant effect would be responsible for death. At the end of the assay, surviving adults in treatments with methanolic and aqueous extracts were transferred to untreated plants. In 1 week the number of eggs laid in treatments was 19.1–22.1 eggs/adult, a value significantly different from the one in controls: 41 eggs/adult. The percentage of emergence was not affected (AbouFakhr Hammad et al., 2001). Order Thysanoptera Thrips palmi Karny. The effectiveness of an extract from seeds of M. azedarach was compared with a sunflower oil and an agricultural oil used at 2% against Thrips palmi (Thysanoptera: Thripidae), a pest that affected the eggplant (Solanum melongena) (Pinõń et al., 1999). With non-conventional products the population of the insects was reduced with higher plant yields registered with M. azedarach extract and sunflower oil. On the other hand, in these plots a high presence of natural enemies of the pest was observed. Order Acari Boophilus microplus ðCanestriniÞ. The efficacy of ripe fruit of M. azedarach L. obtained in Brazil was evaluated against the thick, Boophilus microplus (Acari: Ixodidae), a parasite with economic importance in cattle, dogs and horses (Borges et al., 2003). The fruit extracts were extracted with hexane, chloroform and ethanol and applied on filter papers, which remain in contact with the larvae for 10 min.All the studied extracts were toxic against the larvae, the chloroform extract being the most potent exhibiting 100% of mortality, followed by the hexanic (97%) and the ethanolic one (50%) at a concentration of 0.125%. Hexanic and chloroformic extracts showed more acaricide effectiveness than the ethanolic extract against engorged females of the thick immersed in the solutions of extracts. Egg production and eclosion were inhibited with the hexanic and chloroformic extract and in a lower degree with the ethanolic one (Borges et al., 2003). The same tendency of activity was observed by McMillian et al. (1969) on S. frugiperda. Panonychus citri ðMc GregorÞ. On spraying fields in China with an emulsion of seed oil of M. azedarach at 0.25% a reduction of 85.6% was observed in the population of the citrus red mite Panonychus citri (Acari: Tetranychidae), 1 day posttreatment (Chiu et al., 1984). The effectiveness was comparable to the treatment with Amitraz. A second application on the same trees produced a 98.8% reduction either with the vegetal extract or with Amitraz (Chiu et al., 1984). A spray of seeds oil at 0.5% on citrus trees proved to be comparatively safe to the predator thick Amblysius newsami (Evans), which controlled the citrus red mite, P. citri (Chiu et al., 1984).

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Conclusion and future prospects Today, there is an urgent need of finding new ‘weapons’ for controlling insect attack on plants which are the basis of our food in a direct or indirect way. Extracts obtained with solvents of different polarities from different structures of M. azedarach tree produced a decrease in insects and acari food consumption. This fall in feeding resulted in a diminished weight of the insect and posterior death, even when in some cases this last effect was due to the consumption of toxic compounds. Longer larval and pupal phases were observed after feeding insects with Melia extracts. Alterations in moulting, fecundity and fertility were often observed. These effects resulted in a decrease in the number of pest individuals and therefore a decrease in the damage of crops. According to the above-mentioned results, the extracts and pure compounds isolated from the paraı´ so tree, M. azedarach, could be used for controlling insects of agronomic importance showing effectiveness, availability and seemingly no adverse effects on human and animal health (Carpinella et al., 1999b) or on the environment. The high availability of the renewable resources of the tree such as leaves and fruits as well as their high specificity and potency as antifeedants make it possible to use this natural product for the control of insects which negatively affect plants. In this way, high yields in crops could be obtained guaranteeing a sustainable agriculture.

Acknowledgments This work was supported by FONCyT. MCC gratefully acknowledges receipt of a fellowship from CONICET.

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Uchikoba T, Yonezawa H, Shimada M, Kaneda M. (1999) Melain G, a cysteine proteasa from green fruit of the bead tree, Melia azedarach: a proteasa affected by specific amino acids at P3 position. Biochim Biophys Acta 1430:84–94. Valladares G, Defago´ MT, Palacios S, Carpinella MC. (1997) Laboratory evaluation of Melia azedarach (Meliaceae) extracts against the Elm Leaf Beetle (Coleoptera: Chrysomelidae). J Econ Entomol 90:747–750. Valladares G, Garbin L, Defago´ MT, Carpinella MC, Palacios SM. (2003) Actividad antialimentaria e insecticida de un extracto de hojas senescentes de Melia azedarach (Meliaceae). Rev Soc Entomol Argent 62:53–61. Valladares GR, Ferreyra D, Defago MT, Carpinella MC, Palacios S. (1999) Effects of Melia azedarach on Triatoma infestans. Fitoterapia 70:21–424. Ventura MU, Ito M. (2000) Antifeedant activity of Melia azedarach (L.) extracts to Diabrotica speciosa (Genn). (Coleoptera: Chrysomelidae). Braz Arch Biol Technol 43:15–219. Wandscheer CB, Duque JE, da Silva MAN, Fukuyama Y, Wohlke JL, Adelmann J, Fontana JD. (2004) Larvicidal action of ethanolic extracts from fruit endocarps of Melia azedarach and Azadirachta indica against the dengue mosquito Aedes aegypti. Toxicon 44:829–835. Wen J-H, Schmutterer H. (1991) Effects of extracts from fruit and leaves of Melia azedarach L. on Locusta migratoria migratorioides (R & F). Anz Scha¨dlingskde, Pflanzenschutz, Umweltschutz 64:128–133. Zhou H, Hamazaki A, Fontana JD, Takahashi H, Esumi T, Wandscheer CB, Tsujimoto H, Fukuyama Y. (2004) New ring C-seco limonoids from Brazilian Melia azedarach and their cytotoxic activity. J Nat Prod 67:1544–1547. Zhou JB, Tadera K, Minami Y, Yagi F, Kurawaki J, Takezaki K, Nakatani M. (1998) New limonoids from Melia toosendan. Biosci Biotechnol Biochem 62:496–500. Zhu J, Ermel K. (1991) Isolation of a substance with growth-disturbing properties on the Mexican bean beetle Epilachna varivestis Muls. from the leaves of Melia azedarach L. Z Pfl Kkrankh PflSchutz J Plant Protect 98:422–427 (in German).


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CHAPTER 6

Bioactivity of fabaceous plants against foodborne and plant pathogens: potentials and limitations DEEPAK ACHARYA, ANIKET GADE, MAHENDRA RAI

Introduction There are various plant diseases caused by pathogenic microbes, which are responsible for reduction in production. The continuous and irrational use of chemicals has resulted into several problems like residues in edible plant parts, resistant strains, and environmental pollution, etc. In the present day agriculture, a lot of emphasis is being given on using eco-friendly means for controlling diseases and pest problems affecting various crops (Thind et al., 2005). There is general growing trend among consumers for more natural rather than synthetic products in a whole range of industries, including food and drink, cosmetic, agricultural, and pharmaceuticals (Bauer et al., 1997; Svoboda and Deans, 1998; Glaser, 1999; Traffic International, 1999; Walton and Brown, 1999; Bansal and Gupta, 2000; Singh et al., 2000; Kamanzi Atindehou, 2002). The use of botanicals for the management of the phytopathogens is gaining ground. Plant extracts and volatile oils may have an important role to play in the preservation of foodstuffs against fungi, in fungicidal application against plant diseases, and in the fight against various human fungal infections. Recent literature has shown the biological activities of plant-extracts, essential oils and their individual pure components, and has documented the inhibitory activity of these substances against the growth of various fungi (Maruzzella and Balter, 1959; Maruzzella and Logeuri, 1959; Maruzzella et al., 1959; Maruzzella et al., 1960; Maruzzella, 1963; Sarvamangala et al., 1993; Awuah, 1994; Bhat et al., 1994; Mwosu and Okafor, 1995; Arellanes et al., 1996; Ezer and Abbasoglu, 1996; Pattnaik et al., 1996; Arras et al., 1997; Singh and Handique, 1997; Dwivedi and Singh, 1999; Abraham and Prakasam, 2000; Jantova et al., 2000; Rao et al., 2000; Rawal and Thakore, 2000; Tsao and Zhou, 2000; Wang and Ng, 2000; Agarwal et al., 2001; Agnese et al., 2001; Alvarez-Castellans et al., 2001; Ye et al., 2001; Abou-Jawdah et al., 2002; Locke, 2002; Pistelli et al., 2002; Muhammad et al., 2003; Rai and Mares, 2003; Ezra et al., 2004; Fukai et al., 2004; Kelemu et al., 2004; Kim and Fung, 2004; Kim et al., 2004; Kone et al., 2004; Randhir et al., 2004; Sato et al., 2004; Usui et al., 2004; Xie et al., 2004; Lee et al., 2005; Romagnoli et al., 2005; Talas-Ogras et al., 2005).

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The family Fabaceae is the third largest family of flowering plants, with approximately 650 genera and nearly 20,000 species (Doyle, 1994). Leguminous plant natural products have been important sources and models of forage, gums, insecticides, fungicides, phytochemicals, and other industrial, medicinal, and agricultural raw materials. Since most of these legume species have not been screened for chemical or biologically active components, it is logical to expect that new sources of valuable substances remain to be discovered (Morris, 1999). The present chapter focuses mainly on antimicrobial potential of plants of family Fabaceae, their bioactive compounds and role in sustainable plant disease management. In fact, a little work has been done in the field of antimicrobial potential of plants of Fabaceae against phytopathogens.

Antimicrobial activities of some important fabaceous plants A review of literature reveals that a significant contribution has been made regarding the antimicrobial potential of family Fabaceae (Mahajan, 1971; Umalkar et al., 1976; Allen and Allen, 1981; Pandey et al., 1982; Lal et al., 1998; Samy, 2000). Antimicrobial activities of some plants are as follows: Abrus precatorius L: A woody twinning plant of the Leguminosae family with characteristic red and black seeds. The leaves are pinnate and glabrous, with many leaflets arranged in pairs. The plant bears orange-pink flowers. The plant produces short and stout brownish pods, which curl back on opening to reveal pendulous red and black seeds, four to six peas in a pod. It grows wild in thickets, farms and secondary clearings, and sometime in hedges. It is most common in rather dry areas at low elevation throughout the tropics and subtropics. The chemical constituents of the plant include: arachidic acid, docosadienoic acid, docosatetraenoic acid, docosatrienoic acid, docosenoic acid, eicosadienoic acid, eicosanoic acid, eicosatrienoic acid, hexadecenoic acid, lauric acid, lignoceric acid, linoleic acid, linolenic acid, myristic acid, octadecadienoic acid, octadecatrienoic acid, oleic acid, palmitic acid, pentadecanoic acid. Antimicrobial activity Dhawan et al. (1977) reported that ethanol/water (1:1) extract of the aerial parts at the concentration of 25.0 mg/ml on agar plate was inactive on Bacillus subtilis, Escherichia coli, Salmonella typhosa, Staphylococcus aureus, Agrobacterium tumefaciens, Microsporum canis, Trichophyton mentagrophytes, and Aspergillus niger. Ether extract of seeds on agar plate demonstrated activity against Staphylococcus aureus (Desai and Sirsi, 1966). The ethanol (95%) extract was active on Escherichia coli and Staphylococcus aureus. Cassia alata L: Ornamental shrub with large pinnate leaves. Flowers are bright yellow in spikes. These are called as ‘‘Roman candle tree’’. The fruit is a legume with two prominent,

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wavy wings extending along its full length. It grows in wet habitats from sea level to 250 m but also cultivated in gardens. The main chemical constituent of this plant is tannin. Antimicrobial activity Dhawan et al. (1977) reported that ethanol/water (1:1) extract of the aerial parts, at a concentration of 25.0 mcg/ml on agar plate was inactive on Bacillus subtilis, Escherichia coli, Salmonella typhosa, Staphylococcus aureus, Agrobacterium tumefaciens, Microsporum canis, Trichophyton mentagrophytes, and Aspergillus niger. Cassia alata is known for antibacterial and antifungal activities (Fuzellier, 1982; Ibrahim, 1995; Selvamani and Latha, 2004). Ogunti et al. (1991) evaluated chloroform extract of dried leaves at a concentration of 5.0 mcg/ml on agar plate and found that it was active on Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus. But the extract showed weak activities on Aspergillus fumigatus, Botrydiplodia theobromae, Penicillium italicum, and Trichophyton mentagrophytes. Palacinchamy et al. (1991) reported that ethanol (85%) extract of dried leaves at a concentration of 10.0% on agar plate was active on Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, and Staphylococcus aureus. Ibrahim and Osman (1995) found that ethanol (95%) extracts of dried leaves at a concentration of 500 mg/ml on agar plate was active on Microsporum canis, M. gypseum, Trichophyton mentagrophytes, and T. rubrum; weakly active on Aspergillus niger, Cladosporium werneckii, Fusarium solani, and Penicillium species; inactive on Candida albicans, Rhodotorula rubra, and Saccharomyces cerevisiae. Cassia occidentalis L: It is found in many tropical areas of South America, including the Amazon. Indigenous to Brazil, it is also found in warmer climates and tropical areas of South, Central, and North America. The oil of C. occidentalis contains lignoceric acid, linoleic acid, linolenic acid, and oleic acid. Antimicrobial activity Samy (2000) reported that extract of C. occidentalis leaves showed activity against the Gram-negative and Gram-positive bacteria. C. occidentalis leaves and seeds extract varies in activity against pathogenic fungi (Caceres et al., 1991). Cassia tora L: In Sanskrita, it is called as ‘‘Chakramard’’ meaning there by killer of ringworm. It is known as one of the well known fungal agents in Ayurveda. C. tora is an annual indigenous herb. It has yellow flowers. The leaves are applied to ulcers and itching eruptions. The powdered seeds are recommended to keep in water overnight and the ointment formed is suggested to apply on scabies and ringworm infections. The root is also used topically in ringworm (Chakraberty, 1983). Extracts of C. tora was also found to be active against plant pathogenic fungi (Damayanti et al., 1996; Kim et al., 2004).

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Lopez Abraham et al. (1981) reported that acetone and water extracts of dried stem at a concentration of 50% were inactive and ethanol (95%) extract at a concentration of 50% on agar plate demonstrated activity on Neurospora crassa. Ray and Majumdar (1976) reported that ethanol (95%) extract of fruits on agar plate was active on Trichophyton mentagrophytes and T. rubrum. Caceres et al. (1991) found that hot water extract of dried fruit pulp at a concentration of 1.0 ml in broth culture was active on Microsporum canis, Epidermophyton floccosum, and Trichophyton mentagrophytes var. algodonosa. Singh and Pathak (1984) reported that water extract of fresh leaves on agar plate produced strong activity on Ustilago nuda. Kim et al. (2004) reported fungicidal activities of Cassia tora extracts and their active principles against Botrytis cineria, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia grisea, and Rhizoctonia solani using a whole plant method in vivo. At 1 g/L, the chloroform fraction of C. tora showed a strong fungicidal activity against B. cinerea, E. graminis, P. infestans, and R. solani. Emodin, physcion, and rhein were isolated from the chloroform fraction using chromatographic techniques which showed strong and moderate fungicidal activities against B. cinerea, E. graminis, P. infestans, and R. solani. Moreover, aloe-emodin showed strong and moderate fungicidal activities against B. cinerea and R. solani, respectively, but did not inhibit the growth of E. graminis, P. infestans, P. recondita, and Py. grisea. Mucuna pruriens L: A vine of the Papilionaceae family. The seeds of Mucuna pruriens are black in color with pale-brown specks, uniform in shape. The seed coat is hard, thick, and glossy. It originated in India, and is now commonly found throughout the tropics. The chemical constituents include threo-12-octadec-trans-9-enoic acid, threo-12-1, octadec-cis-9-enoic acid, vernolic acid 4.0%, in seed oil. Antimicrobial activity Dhar (1968) evaluated some Indian plants for biological activity and reported that Mucuna pruriens showed the maximum inhibition against pathogenic fungi and bacteria. Pongamia pinnata ðL:Þ Pierre The tree is smooth throughout, reaches a height of 8–25 m. Leaves imparipinnate, shiny; young leaves pinkish-red, mature leaves glossy, deep green; leaflets 5–-9, the terminal leaflet larger than the others. Flowers fragrant, white to pinkish, paired along rachis in axillary, pendent, long racemes or panicles. Calyx campanulate or cup-shaped. Pod short stalked, oblique-oblong, flat, smooth, thickly leathery to sub woody, indehiscent, 1-seeded; seed thick, reniform (Allen and Allen, 1981). P. pinnata is commonly found throughout the Philippines along the seashore. In some localities it extends inland near the borders of the lakes. It also occurs in the Mascarene Islands, and in tropical Asia, across Malaya to Australia. The chemical constituents include – palmitic 3.7%–7.9%, stearic 2.4%–8.9%, arachidic 2.2%–4.7%, behenic 4.2%–5.3%, lignoceric 1.1%–3.5%, oleic 44.5%–71.3%, linoleic 10.8%–18.3%, and eicosenoic 9.5%–12.4%.


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Antimicrobial activity The seed-oil of P. pinnata is generally used in eczema (Sebastian and Bhandari, 1984). Chaurasia and Jain (1978) reported that Pongam oil, a bitter, red brown, thick, nondrying, non-edible oil, 27%–36% by weight, which is used for lubrication and indigenous medicine. Pongam oil showed inhibitory effect on broad-range of microbes, viz., Bacillus anthracis, B. mycoides, B. pulilus, Escherichia coli, Pseudomonas mangiferae, Salmonella typhi, Sarcina lutea, Staphylococcus albus, S. aureus, and Xanthomonas compestris, but did not inhibit Shigella sp. Flavonoids present in the extracts of Pongamia glabra were found responsible for the inhibition of spore germination and mycelial growth of Alternaria solani, Macrophomina phaseolina, and Fusarium udum (Pan et al., 1985). Simin et al. (2002) found that pongarotene, a new rotenoid and karanjin, a known flavonol were isolated from the methanolic extracts of the seeds of P. pinnata demonstrated antifungal activity. Tamarindus indica L: A large tree up to 30 m high, having spreading branches; bark brownish grey, flaked. Leaves are even-pinnate consisting 10–18 pairs of small leaflets, rather closed together, seeds are glossy, dark-brown, embedded in a thick, sticky, and acid-brown envelope. It is cultivated for the edible fruits and as ornamental and shed tree. It contains tannin 7%, arachidic acid, behenic acid, lauric acid, lignoceric acid, linoleic acid, 2.7%–3.4%, linolenic acid, stearic acid (SD OL), geranial, geraniol (EO). Antimicrobial activity Ross et al. (1980) reported that the ethanol (70%) extract was active against several fungi and bacteria, viz., Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus albus, and S. aureus. Ethanol 95% extract of fruit on agar plate was active on Escherichia coli and inactive on Staphylococcus aureas. Laurens et al. (1985) found that methanol extract of dried stem-bark at a concentration of 10.0 mg/ ml was active on several Gram-negative organisms and inactive on several Grampositive organisms. A concentration 15.0 mg/ml was active on Sarcina lutea. Trigonella foenum-graecum L: Fenugreek is an erect annual herb, growing about 2 feet high, similar in habit to Lucerne. The seeds are brownish, about 1/8 inch long, oblong, rhomboidal, with a deep furrow dividing them into two unequal lobes, ten to twenty together, in long, narrow, sickle-like pods. Indigenous to the countries on the eastern shores of the Mediterranean. Cultivated in India, Africa, Egypt, Morocco, and occasionally in England. Abdo and Al-Kafawi (1969) reported that oil contains 3-hydroxy-4, 5-dimethyl-2furanene, dihydrobenzofuran, dihydroactinidiolide, muurolene, elemene, selinene. Saponins, based mainly on the sapogenins diosgenin and its isomer yamogenin, gitogenin, and tigogenin. Antimicrobial activity Khanna et al. (1971) demonstrated that Trigonella foenum-graecum can inhibit Candida albicans. Omolosa and Vagi (2001) reported that T. foenum-graecum was

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screened against 26-pathogens and all exhibited broad-spectrum antibacterial activity. Methanol extracts of seeds showed activity against Staphylococcus aureus, Bacillus anthracis, B. mycoides, B. pulilus, Escherichia coli, Pseudomonas mangiferae, Salmonella typhi, Sarcina lutea, and Staphylococcus albus. Bhatti et al. (1996) noted that hexane, chloroform, ethanol, and aqueous extracts of the seeds of T. foenum-graecum showed significant activity. The aqueous and ethanol extracts showed significant antibacterial activity against nine pathogenic bacteria at 50 and 100 mg/ml concentration.

Antimicrobials for management of phytopathogens There are many reports which vouch the antimicrobial activities of fabaceous plants. Maruzzella and Liquroi (1958) and Maruzzella et al. (1960) reported the toxicity of the oil of Cassia sp. against Lentinus lepideus, Lenzites trabea, and Polyporus versicolor. Growth of Aspergillus flavus and Trichoderma viride was inhibited by embryos of green peanut seeds (Lindsey and Turner, 1975). Antimicrobial activity against Agrobacterium tumefaciens and Aspergillus niger was shown by the ethanolic extract of Crotalaria verrucosa (Dhawan et al., 1977). The toxicity of the oil of Caesalpinia sappan was effective against Aspergillus nidulans and methicillin-resistant Staphylococcus aureus (Yadav et al., 1978; Kim et al., 2004b). Jain et al. (1979) reported antimicrobial activity of leaf oil of Dalbergia sissoo against Alternaria solani, Aspergillus flavus, A. fumigatus, A. nidulans, A. niger, Cladosporium herbarum, Cunninghmella sp., Fusarium monoliforme, Helminthosporium sp., Penicillium digitatum, P. italicum, Rhizopus nigricans, and Trichothecium roseum. Grover and Rao (1979) and Sharma and Singh (1979) noted toxicity of the oil of Psoralea corylifolia against Alternaria sp., Aspergillus candidus, A. flavus, A. fumigatus, A. nidulans, A. niger, Cladosporium herbarum, Cunninghmella echinulata, Fusarium monoliforme, F. oxysporum, Helminthosporium sacchari, Microsporum cocci, M. gypseum, Mucor mucedo, Penicillium digitatum, Rhizopus stolonifer, Trichophyton mentagrophytes, T. rubrum, and Trichothecium roseum. Acetone, benzene, and ethanol extracts of the groundnut were active against Alternaria alternata and Curvularia spp. (Kalaichelvan and Mahadevan, 1988). Porwal et al. (1988) reported that alcoholic extract of Butea monosperma demonstrated antifungal activity against plant pathogens, viz., Geotrichum candidum and Penicillium notatum. Methanolic and water extracts of the plant parts and cotyledons of peanuts showed efficacy against Aspergillus parasiticus (Azaizeh et al., 1990). The antifungal activity of the bean chitinase on R. solani was observed by Benhamou et al. (1993). Cajanas cajan was found to be effective against Fusarium oxysporum (Singh et al., 1994). Jurzysta and Waller (1996) reported the antifungal effect of alfalfa (Medicago sativa). Gao et al. (2000) demonstrated that the alfalfa antifungal peptide (alfAFP) defensin isolated from seeds of M. sativa displayed strong activity against the agronomically important fungal pathogen Verticillium dahliae. An antifungal protein designated sativin was isolated from the legumes of the sugar snap (also known as honey pea) Pisum sativum var. macrocarpon. Sativin exerted antifungal activity against Fusarium oxysporum, Coprinus comatus, and Pleurotus ostreatus (Ye et al., 2000). Saxena and Mathela (1996) isolated iridodial beta-monoenol acetate


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from the essential oil of Nepeta leucophylla, and actinidine from N. clarkei and screened for antifungal activities against Aspergillus flavus, A. ochraceus, Penicillium citrinum, and P. viridicatum, all known mycotoxin-producing taxa, and Sclerotium rolfsii and Macrophomina phaseolina, potential soybean pathogens. It was found that iridodial beta-monoenol acetate was most effective against S. rolfsii, while actinidine was highly active against M. phaseolina. Extracts of Gliricidia inhibited the germination of Drechslera oryzae by only 6%. However, in another study, 50 mg of stem chloroform extracts inhibited the growth of Cladosporium cucumerinum and slightly inhibited the growth of Candida albicans (Herath et al., 1998). Esimone et al. (1999) found antibacterial and antifungal activities of leaves of Indigofera dendroides. Fakhoury and Woloshuk (2001) isolated a 36-kDa alphaamylase inhibitor from Lablab purpureus (AILP). AILP inhibited the alpha-amylases from several fungi. The protein inhibited conidial germination and hyphal growth of Aspergillus flavus, which is a fungal pathogen of maize causing an important ear rot disease when plants are exposed to drought and heat stress. The antimicrobial and antifungal properties of several crude extracts and pure saponins, astraverrucins I–VI, from the aerial parts of Astragalus verrucosus were investigated by Pistelli et al. (2002). Simin et al. (2002) reported the antifungal and antibacterial activities of a new rotenoid from Pongamia pinnata. Similarly, a new prenylisoflavone from Ulex jussiaei showed antifungal activity against Cladosporium cucumerinum (Maximo et al., 2002). Chloroform extract of Erythrina vogelii inhibited the growth of Cladosporium cucumerinum (Atindehou et al., 2002). Rao and Joseph (1971) reported that the oil from seed husk of Cicer arietinum was toxic to Cephalosporium sacchari, Ceratocystis paradoxa, Curvularia lunata, Fusarium moniliforme, Helminthosporium sacchari, Physalospora tucumanensis, Rhizopus arrhizus, and Sclerotium rolfsii. Chu et al. (2003) isolated a peptide cicerarin from Cicer arietinum which exerted antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, and Physalospora piricola. The fungicidal activities of Cassia tora extracts and its active principles were determined against Botrytis cinerea, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia grisea, and Rhizoctonia solani by Kim et al. (2004). The chloroform fraction of C. tora showed a strong fungicidal activity against B. cinerea, E. graminis, P. infestans, and R. solani. Emodin, physcion, and rhein were isolated from the chloroform fraction which showed fungicidal activities against B. cinerea, E. graminis, P. infestans, and R. solani. Furthermore, aloe-emodin demonstrated fungicidal activities against B. cinerea and R. solani, respectively. This study suggests the fungicidal actions of emodin, physcion, and rhein present in C. tora. According to Kelemu et al. (2004), the tropical forage legume Clitoria ternatea is resistant to a number of pathogens and pests. A highly basic small protein was purified from seeds of the plant. The protein, designated ‘finotin’, has broad and potent inhibitory effect on the growth of various important fungal pathogens of plants, namely Rhizoctonia solani, Fusarium solani, Colletotrichum lindemuthianum, Lasiodiplodia theobromae, Pyricularia grisea, Bipolaris oryzae, and Colletotrichum gloeosporioides. It also inhibits the common bean bacterial blight pathogen Xanthomonas axonopodis pv. phaseoli. Flavonol glycoside isolated from the stems of Teramnus labialis showed antibacterial and antifungal activities against plant

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pathogens (Yadava and Jain, 2004). Deepa et al. (2004) reported that the extracts of leaf, root, stem, and the callus obtained from Pseudarthria viscida showed significant inhibitory activity against some fungal pathogens causing major diseases in crop plants and stored food grains. Ng (2004) reported a profile of antifungal proteins and peptides of leguminous plants. The crude methanolic extract of Zuccagnia punctata was active against the fungal pathogens of soybean Phomopsis longicolla and Colletotrichum truncatum (Svetaz et al., 2004). Mandal et al. (2005) reported antibacterial and antifungal activities of saponins from Acacia auriculiformis. They found complete inhibition of conidial germination of Aspergillus ochraceous and Curvularia lunata at 300 mg/ml or less, whereas Bacillus megaterium, Salmonella typhimurium, and Pseudomonas aeruginosa were inhibited at 700 mg/ml or higher concentrations of the mixture. Abbas and Zayed (2005) isolated seven saponins from the aerial parts of Astragalus suberi. These saponins exhibited antibacterial activities against Gram-positive and Gram-negative bacteria and also showed antifungal activity. The heartwood extracts from Acacia mangium and A. auriculiformis was examined on the growth of wood rotting fungi with in vitro assays. A. auriculiformis heartwood extracts had higher antifungal activity than A. mangium (Mihara et al., 2005). Study carried out by Wong and Ng (2005) showed that seeds of Phaseolus vulgaris gave rise to a peptide vulgarinin that manifested an antifungal activity toward Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola, and Botrytis cinerea. It also exhibited the antibacterial action on Mycobacterium phlei, Bacillus megaterium, B. subtilis, and Proteus vulgaris. Peraza-Sanchez et al. (2005) isolated a new Pimarene from Acacia pennatula, which showed in vitro inhibition of growth, sporulation, and germination of Colletotrichum gloeosporioides. A chitinase with antifungal activity was isolated by Wang et al. (2005) from mung bean (Phaseolus mungo) seeds. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Mycosphaerella arachidicola, Pythium aphanidermatum, and Sclerotium rolfsii. D’Souza et al. (2005) evaluated 15 native Western Australian legumes for their potential to biologically control Phytophthora cinnamom. Acacia extensa, A. stenoptera, and A. alata along with A. pulchella, were identified with the highest potential for biological control of P. cinnamomi. Acacia urophylla and Viminaria juncea exhibited the least potential for biological control of P. cinnamom.

Future directions and conclusions The search for antimicrobial agents from higher plants, microbes, insects, and vertebrates is continued. Family Fabaceae is known for its antimicrobial potential since time immemorial. Plants like Pongamia pinnata, Cassia tora, Psoralea corylifolia, Caesalpinia sapan, Mucuna pruriens are described in ancient literature for their antimicrobial uses. There is a pressing need to search for new antimicrobial agents for control of pathogenic enemies of crops for the sustainable future. Although, a significant contribution has been made in the field of search for fabaceous-derived natural drugs against human pathogenic fungi, phytopathogens are woefully neglected. Therefore, the pathogens causing diseases in economic plants should be given priority. The peptides play an important role in inhibition of the microbial


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growth. The structural, biochemical, and functional diversity of the proteins found in nature provides an exceptional opportunity for future research (O’Keefe, 2001). The future research should be directed towards search for antimicrobial molecules from the marine microbes and the lower plants like bryophytes and pteridophytes. Unfortunately, microbial resistance is increasing with fast pace. To fight with emerging microbial infections, a strategic ‘natural drug discovery programme’ should be developed, which may open up new vistas in antimicrobial drug research by giving a new generation of broad spectrum, natural drugs/botanicals.

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CHAPTER 7

Screening of plants against fungi affecting crops and stored foods OLI´VIA C MATOS, CAˆNDIDO P RICARDO

Introduction Nature is a rich source of products with interesting and useful biological activities. The use of plants for non-edible purposes is a practice dating back to prehistory. Besides the primary metabolites plants contain a diversity of secondary metabolites related to mechanisms of adaptation and interaction with environment. Collectively, more than 10,000 low-molecular mass compounds are known as secondary metabolites of plants (Cowan, 1999; Dixon, 2001). Simple phenols, phenylpropanoid derivatives, coumarins, flavonoids, tannins, quinones, ethereal oils, isoprene derivatives (iridoids, sesquiterpene lactones, diterpenes, triterpene saponins, steroid saponins), cardiotonic heterosides, alkaloids (pirolidone, pirolysine and piperidine alkaloids, kinolisidine alkaloids, kinoline alkaloids, isokinoline, indole, purine, diterpene and steroide alkaloids), and cyanogene glycosides are examples of such compounds (Mann, 1970; Marston and Hosttetman, 1987; Colegate and Molyneux, 1993; Cowan, 1999). This diversity results in part from an evolutionary process driven by selection for acquisition of improved defence against pests and diseases (Dixon, 2001). Plant secondary metabolites evidencing therapeutic, flavouring, odoriferous, stimulant, poisoning, hallucinogenic or colouring properties still play an important role in human activities. As estimated, there are 250,000–500,000 plant species, but only a small percentage (less than 10%) have ethnobotanical use (Borris, 1996; Moermann, 1996; Cowan, 1999). Several reports on the history of plant applications in folk medicine are known. In the secondhalf of the 20th century, after the development and generalised utilization of synthetic antibiotics, bioactive products derived from plants for antimicrobial application have lost importance. Crop protection from competing weeds, insects and diseases has been necessary for the higher yields in agriculture. Whereas in the early days manual labour was used to solve the weed problem, the manual control of insect infestations was in most cases an impossible task and plant diseases were even more difficult to control.

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Plant diseases, pests and weeds are responsible for crop losses in all continents in spite of the wide search of science and industry for better controlling agents and improved methods (Knight et al., 1997). The first generation of crop protection products consisted of inorganic arsenic, sulphur, copper and mercury compounds. Conventional pesticides, obtained from organic synthesis, which were considered a key for pest and disease control risks to lose importance due to problems of public health, environmental pollution, preservation of ecosystems and wild life habitats (Farrel, 1990). The generalized use of this type of pesticides conduced to serious problems of toxicity, water contamination, persistence in the ecosystems, residues in foods, as well as accumulation in fat tissues of man and animals, and increased pest resistance to them (Lyon and Zalon, 1990). The levels of pollution caused by atrazine and diazinon, an herbicide and an insecticide respectively, measured by Morley (1977), conduced to the definition of rules and restrictions to the use of pesticides, and to the development of strategies to reverse the pollution process (Kuc, 1990; Matos, 2000). Socio-economic and environmental factors, such as, those caused by resistance of pests and microorganisms to synthetic pesticides emphasize the importance of new ways and new strategies for the control of pathogenic agents. The present concept is based on the idea that agro-ecosystem should be managed in ways to hold pest to tolerable levels, making use of natural products, integrated pest management, and the use of pest-resistant crop varieties or even transgenic plants. Natural chemical substances can be useful for crop and food protection. Extracts from Pyrethrum flowers, containing piretrine, made their de´but as household insecticides in the first half of the 19th century and were the first pesticide commercialized under the designation of ‘‘Caucasian insect powder’’ (Banergi et al., 1985), while nicotine, extracted from tobacco, was the first natural insecticide used in crop protection (Casida, 1983; Pumarola-Batlle and Xirau-Vayreda, 1983; Matos, 2000). From the 1970s an increased interest on the study of the plants autoprotective capacity against pathogenic agents or adverse environmental conditions was noted (Bailey et al., 1992). Studies on the discovery of ways of manipulating plants in order to increase or to produce novel chemical molecules have been developed by different research teams. The isolation of several substances synthesized by several plants as a response to biotic or abiotic stresses evidencing insecticidal, fungicidal, bactericidal or herbicidal action have been reported (Lamb and Lawton, 1989; Bouguerra, 1990; Keen, 1990; Matos et al., 1998). In Table 1 some examples of plants analysed for compounds with fungicidal activity are provided. Table 2 illustrates antifungal compounds detected in plant cell cultures and in the respective plant (Constabel and Vasil, 1987; Kutney, 1997).

Fungi as contaminants of crops and foods Phytopathogenic fungi can cause local or general symptoms on their hosts. According to Agrios (1997) the most common necrotic symptoms are leaf-spots (localized lesions on leaves consisting of death and collapsed cells), blight (general and extremely rapid browning death of leaves, branches, twigs, and floral organs), canker (localized necrotic lesion), die-back (extensive necrosis of twigs beginning on their tips and advancing toward their bases), root-rot (disintegration or decay of part of

Screening of plants against fungi affecting crops and stored foods

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Table 1 Plant substances with activity against phytopathogenic fungi Botanical name

Compound

Fungi

References

Ammi visnaga

Khelin Visnagin

Averbeck (1989), Borges et al. (1998)

Arabidopsis thaliana

Ferrulic acid Vanillic acid trans-Cinnamic acid o-Coumaric acid

Brassica napus

Cylobrassin Methoxybrassinin

Carum carvi

Hydroxiacetophenone

Cassia tora

Miscellaneous

Fusarium culmorum Cladosporium cucumerinum Fusarium oxysporum Phytophthora dreshleri Rhizoctonia solani R. solani Alternaria brassicae Botrytis cinerea C. Cucumerinum Fusarium nivale Sclerotinia sclerotium Leptosphaeria maculans Pythium ultimum Rhizoctonia solani F. oxysporum f.sp.dianthi Aspergillus niger

Chelidonium majus

Alkaloids

Aspergillus fumigatus, Fusarium sp.

Chenopodium ambrosioides Coriandrum sativum

Essential oils

Aspergillus flavus

Essential oils

C. cucumerinum Fusarium sp.

Essential oils Dihydroresveratrol

Aspergillus sp. Botrytis cinerea C. cladosporioides

Essential oil

Rhizoctonia solani

Pulegone

C. cucumerinum

Essential oil

Sclerotinia sclerotium

Cuminum cyminum Dioscorea bulbı´fera, D. dumentorum Satureja thymbra Thevetia peruviana Thyma spicata

Walker et al. (2003)

Dahiya and Rimmer (1988)

Curir et al. (1996) Mukherjee et al. (1996) Hejtmankova et al. (1984), Matos et al. (1999) Varma and Dubey (1999) Delgado (1993), Matos and Ricardo (2003) Matos (2001), Matos and Ricardo (2003) Adesanya et al. (1989) Mu¨ller-Riebau et al. (1995) Gata-Gonc- alves et al. (2003) Mu¨ller-Riebau et al. (1995)

all root system of a plant), damping-off (rapid death and collapse of very young seedlings), basal stem rot (disintegration of the lower part of the stem), soft rots and dry rots (maceration and disintegration of fruits, roots, bulbs, tubers, and fleshy leaves), anthracnose (necrotic and sunken ulcerlike lesions of the stem, leaf, fruit, or flower), scab (localized lesions with a scabby appearance on fruit, leaves, etc.), and decline (plants growing poorly, leaves small and yellowish or red, and some


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Table 2 Efficacy of production of secondary metabolites by plant cell culture Plant species

Metabolite

Cell culture media

Cassia tora Catharanthus roseus

Anthraquinones Ajmalicine

Agar gel Liquid

6.0 1.0

Catharantine Caffeine Berberine Berberine Rosmarinic acid Diosgenine

Liquid Agar gel Liquid Agar gel Liquid Liquid

Chiconine

Coffea arabica Coptis japonica Coleus blumei Dioscorea deltoidea Lithospermum erythrorhizon Macleaya cordata Morindia citrofolia Nicotiana tabacum Panax ginseng Papaver somniferum

Content in the cell culture (% dry weight)

Content in the plant (% dry weight)

Content ratio (cell cultures/ plant)

0.6 0.3

10 3

0.24 1.6 13.0 7.4 15.0 2.0

0.002 1.6 7.0 3.0 2.0

77 1 1.1 5 1

Agar gel

12.0

1.5

8

Chiconine Protopine

Liquid Agar gel

14.0 0.4

1.5 0.32

9.3 1.25

Anthraquinones

Liquid

18.0

2.5

7.2

Ubiquinone-10

Liquid

Ginsengoside Sanguinarine

Agar gel Liquid

0.18 27.0 2.9

0.003 4.5 –

60 6.0 –

Source: Constabel and Vasil (1987) and Kutney (1997).

defoliation can occur). Fungi can also cause excessive enlargement or induced growth and distortion of plant parts, such as: clubroot (enlarged roots appearing like spindles or clubs), galls (enlargement portions of stems, leaves, blossoms or roots), warts (wartlike protuberances on tubers and stems), witches’-brooms (profuse, upward branching of twigs), leaf curls (distortion, thickening, and curling of leaves). Additional symptoms can also occur, like wilt (generalized loss of turgidity and drooping of leaves or shoots), rust (many small lesions on leaves or stems, usually of rusty colour), smut (seed or gall filled with the mycelium or black spores of the smut fungi), mildew (areas on leaves, stems, blossoms, and fruits, covered with whitish mycelium and the fructifications of the fungus). In Table 3, examples of fungi responsible for important crop diseases are presented. The genera Aspergillus and Fusarium are examples of known phytopathogenic fungi reported to be widespread and harmful toxin-producers (Harris and Mantle, 2001; Harvey et al., 2001; Hussein and Brasel, 2001). Aspergillus flavus is a common mold that grows on many substances, especially on grains and nuts, producing the dangerous aflatoxins (Vargas et al., 2001). A. flavus is also referred as being responsible for paranasal invasive aspergillosis, a rare but chronic diseases affecting man (Alrajhi et al., 2001).


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Table 3 Fungi responsible for important crop disease Pathogen

Diseases

Crops

References

Aspergillus flavus A. niger

Synthesis of aflatoxin Grey mold rot or botrytis blight Scab

Peanut

Vargas et al. (2001)

Vegetable and fruit crops Cucumber Pecan Peach

Sherf and MacNab (1986) Agrios (1997)

Ripe rot Antracnose

Blueberry Cucumber

Bailey et al. (1992)

Botrytis cinerea Cladosporium cucumerinum C. effusum C. carpophylum Colletotrichum acutatum Colletotrichum magna Fusarium culmorum F. graminearum F. oxysporum f.sp. conglutinans F. oxysporum f.sp. cubense F. oxysporum f.sp. cumini F. oxysporum f.sp. dianti F. oxysporum f.sp. gladioli F. oxysporum f.sp .lentis F. oxysporum f.sp. lycopersici Leptospheria maculans

Crown rot Corn damping-off Wilt or Fusarium yellows Fusarium wilt Fusarium wilt Fusarium wilt Fusarium yellows, corm rot Fusarium wilt Fusarium wilt

Barley Maize Crucifers Banana Cumin Carnation Gladiolus Lentil Tomato

Mes et al. (1994), Lodha (1995), Singh and Tripathi (1995), Mao et al. (1997), Davanlou et al. (1999), Vargas et al. (2001)

Blackleg

Phoma lingam

Blackleg

Pseudoscorporella herpotrichoides Pythium violae P. Ultimum Phytophthora cinnamomi, P. infestans

Eyespot

Crucifers, cabbages Crucifers, cabbages Winter cereals

Root rot

Carrot

Shafer and Wostemeyer (1994) Shafer and Wostemeyer (1994) Dewey and Priestley (1994) Campion et al. (1998)

Root rot Late blight

Rhizoctonia solani

Soil rot Sheath blight Brown patch Stem canker Southern blight

Ornamental trees, shrubs, wheat Potato Tomato Rice Turfgrass Potato

Sclerotium rolfsii Spongospora subterranea Verticillium dahliae, V. albo-atrum

Powdery scab Vascular wilt

Tomato, pepper, apple Potato Dicotyledoneous crops, potato

Agrios (1997), Drenth and Govers (1994) Thornton et al. (1994)

Jones et al. (1991) Harrison et al. (1994) Carder et al. (1994), Robb et al. (1994)

Infections by Fusarium are responsible for destroying crops or dramatically reducing production yields. The economic importance of Fusarium species is enhanced by their ability to synthesize harmful mycotoxins, such as zearalenone, fusarins, fumonisins or trichothecenes (Koopmann et al., 1994; Blat et al., 1997; Desjardins

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et al., 2000). Despite intensive research, efforts to control Fusarium infections and prevent or eliminate the presence of its mycotoxins in foods have not met with a great deal of success. Fusarium mycotoxins found in foods are generated primarily in the field although some toxin synthesis may occur during storage. Temperature and moisture conditions during the growing season and insect infestations are critical factors affecting fungal infection and toxin synthesis. Toxins like deoxynivalenol (DON) were found in grains of wheat and barley evidencing symptoms of the Fusarium blight (scab). Wet and cool weather during flowering of wheat is conducive to infection with F. graminearum, which produces DON, while late season rainfall increases infection of corn silk with F. verticillioides (F. moniliforme), the main producer of fumonisins (Koopmann et al., 1994; Blat et al., 1997; Desjardins et al., 2000). Even normal-looking wheat kernels may contain DON although concentrations are generally quite low (Munkvold and Desjardins, 1997; Sinha and Savard, 1997). Surveys have demonstrated the worldwide occurrence of fumonisins in cereal grains like corn, barley, oats or rice (Ghebremeskel and Langseth, 2001; Vargas et al., 2001).

History of the use of antifungal compounds Fungi are the major cause of plant diseases and so, most of the phytopharmacological products are aimed at fungal pathogens. Even before the identification of fungi as causal organisms of plant disease, dusting plants with sulphur was a generalized practice. The mixture of copper sulphate and calcium hydroxide (Bordeaux mixture) in 1885 can be considered the beginning of the development of chemical disease control. Fungicides similar to those of sulphur and copper, considered the first generation of fungicides, are still in wide use (Koller, 1999). The synthesis of carbamates and dithiocarbamates, two groups of broad-spectrum fungicides dates back to 1940s. The use of the second generation of fungicides was restricted in several countries due to the possibility of conversion of the most dithiocarbamates into potential carcinogens. Trichloronitrobenzene, quinines, chlorothalonil or pentachloronitrobenzene are other examples of the second generation of fungicides. Despite the restrictions in their use most fungicides of the second generation are still widely used, due to their effectiveness, convenience of handling, low harmful effects on plants and low resistance problems (Borges, 2002). Systemic compounds that can be absorbed by plants and translocated in it to reach the growing pathogen and control the infection constitute a third generation of fungicides. Implemented in the late 1960s, the most representative are the carboxamides and hydroxypyrimidines, highly specific to Basidiomycetes and powdery mildews (Ruberson, 1999). A major focus on the development of systemic fungicides still remains, and families of compounds such as sterol demethylation inhibitors (DMIs) and the morpholines are still of much interest. DMIs current importance lays in the control of cereal diseases in Europe and morpholines are mostly used for controlling cereal powdery mildews. Phenylamide fungicides that include benalaxyl, of urace and oxadiocyl are both used as soil and foliar fungicides for the control of potato late blight and downy mildews. These compounds are fungicides specific for the Oomycete fungi family (Ruberson, 1999; Borges, 2002).


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At the present, studies are focused on the development of a fourth generation of fungicides consisting of chemicals that interfere with specific steps in host–pathogen interactions, either by preventing fungal penetration into the plant or by enhancing the defence system of the pathogens. They are considered as disease control agents with indirect modes of action. The side-effects of these compounds on non-target organisms will be less severe than those caused by the conventional fungicides. Thus far only a few commercial products with such indirect modes of action have been introduced. Examples of fungicides to inhibit host penetration are thalide and carpropamid, developed for the control of rice blast. Probenazole and acibenzolarS-methyl are two examples of compounds that act on the reduction of plant defence reaction.

Interaction of plant products with fungi The term natural fungitoxins has been sometimes used to refer substances of plant origin that cause a significant toxic or inhibitory action on the fungus growth at relatively low concentrations. Flavonoids, isoprenoids and alkaloids with these properties are essential for plant adaptation, participating in a broad range of physiological processes (Harborne, 1987; Mayer, 1989). They can be present in healthy and infected tissues and are considered to be important in the protection of plants even if they do not prevent the infection (Harborne, 1987; Mayer, 1989). Postinhibitins are a group of fungitoxic vascular substances co-existing with the cytoplasmic specific hydrolytic enzymes. Seven classes of these toxins are known, most of them being involved in the plant resistance to one or more phytopathogenic fungi. Some of these compounds are saponins, of sulphurated aminoacids derivatives and several types of glycosides (Harborne, 1987). Examples of antifungal compounds secreted by plant leaves as a response to the attack of a fungus are diterpenoides isolated from Nicotiana glutinosa, isoterpenoides extracted from several species of Lupinus, and a sesquiterpenic lactone produced by Chrysanthemum parthenium (Harborne et al., 1976; Thara et al., 1984; Matos, 2000). Studies on plant organs other than leaves are also common. Roots are, in general, resistant to infection, which means that fungitoxic substances, present or exuded by the epidermis of the roots may play a protective role. In fact, more than twenty substances belonging to the group of isoflavones have been isolated from the roots of Lupinus albus. Borbonol-2, a hydrocarbonated lactone with antifungal activity, was also isolated from the roots of Persea borbonica but was not found in P. indica, which is sensitive to the disease (Harborne et al., 1976; Thara et al., 1984). The area of soil surrounding a plant root, the rhizosphere, is a unique physical, biochemical, and ecological interface between the plant and the environment known. Walker et al. (2003) analysed root exudates of Arabidopsis thaliana elicited by salicylic acid, jasmonic acid and chitosan as well as by two fungal cell-wall elicitors and profiled the secondary metabolites subsequently secreted. Among the several compounds detected 10 were structurally characterized and successfully used on the inhibition of Fusarium oxysporum, Phytophthora drechsleri and Rhizoctonia solani. Studies of Whips (2001) on the relation between plant growth and rhizosphere competence in biocontrol refer the extreme complexity of interactions that can occur

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at the rhizosphere level and provide an extensive list of vascular phytopatogenic fungi (such as Fusarium sp., Rhizoctonia sp. or Pythium sp.) that are controlled through those interactions. One of the most important defence systems of the plant consists in the synthesis de novo of substances generally called phytoalexins, that are not present in healthy plant tissues but are synthesized when plant cells are exposed to microorganisms or other stressing stimulus (Darvill and Albersheim, 1984; Ebel, 1986; Fagboun et al., 1987; Adesanya et al., 1989; Osbourn, 1996). Phytoalexins are compounds of low molecular weight of a heterogeneous group of compounds like isoflavonoids, sesquiternes, diterpenes, polyacetilens and coumarins. Beier (1990) reports the presence of several phytoalexins in the following food plants: alfalfa (vesitol, sativan, medicarpin), pea (pisatin, cinnamylphenols and a methylchalcone), bean (phaseolin), broadbean (wyerone), lima bean (5-deoxikievitol, 8,20 -dihydroxygenistein), soybean (glyceollin), grapevine (viniferin, resveratrol), cotton (gossypol, cadalenes, lacinilenes, hemigossypol), peanut (resveratrol), celery, parsley and parsnip (furanocoumarins), rice (momilactones, oryzalexins), castor bean (casbene), potato (rishitin, hydroxilubimin, phytuberin, a-solanine, a-chalcone, lubimin, solavevitone, phytuberol), pepper (capsidiol), sweet potato (ipomeamarone), carrot (6-methoxymellein, falcarinol), tomato (a-tomatine, rishitin, falcarindiol, falcarinol), tobacco (rishitin, lubimin, phytuberin, phytuberol, solavetivone, capsidiol, glutinosone), eggplant (lubimin). Phytoalexin have long been inferred to be important in the defence of plants against fungal infection (Brooks et al., 1986, 1987). For instance, stilbene phytoalexins are formed in several unrelated plant species, such as peanut (Arachis hypogaea), grapevine (Vitis vinifera) and pine (Pinus sylvestris). Hain et al. (1993) investigated the potential of stilbene biosynthetic genes in a strategy of engineering pathogen resistance. They isolated stilbene synthase genes from grapevine and observed an increased disease resistance (based on the production of a foreign phytoalexin) against Botrytis cinerea in transgenic tobacco expressing the gene at a high level. In Table 4, several phytoalexins of crops elicited by fungi or bacteria are indicated. Since the in vitro plant tissue cultures can synthesize substances different from those of the plant grown in natural conditions, the development of in vitro studies are also important. DiCosmo and Misawa (1985) referred several examples of phytoalexins produced in tissues of different plant species, elicited in vitro by some chemical compounds or extracts of fungi (Table 5).

Plant synthesis of light-activated compounds It has been shown that several compounds when irradiated (photoactive compounds or photosensitizers) can cause toxic reactions in living cells (Matos and Ricardo, 2003). This can be especially important when the photoactive compounds react with arthropods, bacteria or fungi affecting human, animal health or crops. The main classes of plant photosensitizers are polyetilenes, thiophenes and related compounds, coumarins and furanocoumarins, furanocromones, b-carbolines and other alkaloids and complex quinines (Heitz and Downum, 1987; Arnason et al., 1992). By absorbing light photosensitizers trigger chemical modifications of a substrate or

Family

Species

Plant organ

Pathogenic agent

Phytoalexin

Compositae Cruciferae

Artium lappa L. Brassica napus L.

Roots Leaves and stems

Polyacetylenes Methoxibrassinine, cyclobrassinine

Dioscoriaceae

Dioscorea bulbifera L. D. rotundata Poir. Oryza sativa L.

Bulbs and tubers Whole plant Whole plant

Bacteria Leptosphaeria maculans (Desm.) Ces de Nat. Botryodiplodia theobromae (Pat.) Griff. and Maubl. Pyricularia oryzae Carv.

Lupinus albus L. Pisum sativum L. Glycine sp. Glycine max (L.) Merr. Capsicum annum L. Nicotiana tabacum L. Solanum tuberosum L. S. tuberosum L.

Leaves and roots Whole plant Roots Cotyledons Whole plant Leaves Tubers Whole plant

Daucus carota L.

Roots

Gramineae Leguminosae

Solanaceae Umbelliferae

Nectaria haematococca Berk. Phytophthora megasperma Drechsler f.sp. glycine Mucor ramosissimus Samutsevitsch Giberella pulicaris (Fr.) Sacc. Phytophthora infestans (Mont.), Caspary Chaetomium globosum Kunze

Dimethyl batatasine IV Dihydrostilben Carboxylic acid, 2,2-dichloro-3,3-dimethyl cyclopropane Isoflavenoids Pterocarpen Glyceolin Glyceolin Sesquiterpenoids, capsidiol Sesquiterpenoids, diterpenoids

Lubimin Araquidonic and icosapentaenoic acids


Table 4 Induction of phytoalexins in crop plants

6-Methoximeleine

Robeson et al. (1980), Darvill and Albersheim (1984), Amin et al. (1986), Brooks et al. (1986, 1987), Ebel (1986), Zeringue and Hampden (1987), Dahiya and Rimmer (1988), Therelfall and Whitehead (1988), Whitehead et al. (1988), Desjardins et al. (1989), Laks and Pruner (1989), Felicice et al. (2000) and Matos (2000).

147


148 Table 5 Production of phytoalexins by cultures of plant cells Class of compounds

Phytoalexins

Plant species

Elicitor

Alkaloids

Acridon Codein Sanguinarine Morphine Ajmalicine Catharantine

Ruta graveolens L. Papaver somniferum L. Catharanthus roseus G. Don.

Benzoquinones

Equinofuran

Coumarins

Psoralen Xanthotoxin Graveolone Diosgenin

Lithospermum srythrorhizon Sieb. and Zucc. Petroselinum crispum (Miller). A.W. Hill

Quitosan, polylysine Products from the fungus cell wall Ribonuclease (denatured) Fungal glycoprotein Fungal polysaccharides Activated charcoal

Steroids Phenylpropanoids

Glyceolinol Kievitone Phaseolin

Naftoquinones

Chiconine

Polyacetylenes

Unspecific Phenylheptatrine

Terpenoids

Phytuberine Sesquiterpenoids

Dioscorea deltoide Wall. Glycine maxima, (L.) Merr. Phaseolus vulgaris L.

Lithospermum erythrorhizon, Sieb. and Zucc. Carthamus tinctorius L. Bidens spinosa L. Solanum tuberosum L. Nicotiana tabacum L.

Fungal polysaccharides Mycelium of fungus Quitosan, polylysine Products from the fungus cell wall Ribonuclease (denatured) Fungal glycoprotein Fungal polysaccharides Agaropectine Fungal polysaccharides Filtered fungus extract Fungus suspension Mycelium of the fungus

Source: DiCosmo and Misawa (1985).

target. Absorption of light generates highly reactive electronic excited states, which can either interact with biomolecules or with oxygen, generating the highly toxic reactive oxygen singlet excited state. UV radiation elicits the synthesis of endogenous photosensitizers by the plant defence mechanisms (Heitz and Downum, 1987; Heitz, 1995). It is considered most relevant the development of new pesticides environmentally safe and harmless to non-target microorganisms for crop protection. The first utilization of light effects was for the control of insects. Presently photosensitizers are used to control weeds, algae, insects, nematodes, viruses, bacteria, yeast and fungi or even tumour cells (Berenbaum, 1987). Plant photosensitizers emerged as relevant compounds for agricultural applications. In the last decades, there has been an increased interest in the plant screening for


149

photopesticides. Several plant extracts and a diversity of natural compounds were analysed for their light-dependent antifungal properties (Towers and Champaghe, 1986; Matos and Ricardo, 2003). Compounds known as bioactive in the ground state, such as most of the flavones, can become photoactive by structural modifications (Becker et al., 1993; Borges et al., 1995, 2002).

Factors affecting plant synthesis of bioactive compounds The use of plants for therapeutic purposes has a long history, but the utilization of crude preparations can have some drawbacks, like variation in the amount of the active component with geographic, climate and ecological conditions or with the plant organs and morphology. Interconnection of living organisms, interrelations between biotic and abiotic factors and environmental conditions can induce changes in the synthesis of biologically active metabolites by plants. The diversity of plant compounds also suggests a high probability that synergistic, antagonistic or other undesirable effects can also occur. A synergistic interaction occurs when the combined activity of two or more compounds is greater than that of their individual activities. On the other hand, antagonist interactions are present when a combined activity of two or more chemicals is less than that of their individual activities (Mckey, 1979; Berenbaum, 1985). Although the increased attention on synergistic interactions in the last decade, in bibliography only few studies are reported (Down, 1989; Hay et al., 1994), and one case of antagonistic interactions is known (Terras et al., 1993). Synergy is an adaptive strategy in plant defence that can provide insight into diversity and function of secondary metabolites. Synergic effects can be due to the inhibition of a toxin detoxification, to the modification of an inactive compound rendering it toxic, to an enhanced penetration, transport or accessibility of a toxin to a certain target, or when two independent steps of biosynthetic pathways were affected (Vaara, 1992; Nelson and Kursar, 1999). Variability in collection, treatment and storage of raw material can also cause losses in bioactivity. Although there are several reports of the use of crude extracts in therapy and crop protection the isolation of the bioactive substances can be advantageous. Pure bioactive compounds are easy to formulate and to dose (Colegate and Molyneux, 1993). In Table 3, representative examples of compounds extracted from plants exhibiting activity against phytopathogenic fungi are presented.

Experimental Plant secondary metabolites are synthesized in specialized cells at distinct developmental stages and the complexity of their structures makes their extraction and purification difficult. The plant screening for bioactive compounds require appropriated assays all along the extraction procedure and along the subsequent purification steps. The development of efficient methods for the detection and characterization of natural products of crude plants extracts should consider their preliminary identification at an early stage of separation as a strategic element for guiding selective isolation procedures. Plant screening process can be shorted

150


by the development of bioguided plant fractionation towards a certain group of pathogens. Crude extracts were obtained with the adequate solvent that solubilizes a certain group of compounds present in the sample of the plant material to be analysed. The extracts were then analysed by common chromatographic techniques, immediately or after being fractionated with a range of solvents of increased polarity and the active principles were labelled and visualized under UV light (254 and 366 nm). TLC analysis is a rapid and efficient method that combines sensitivity and simplicity and allows the bioguided fractionation analysis. This procedure enables the differentiation between inactive and active compounds directly in the crude extracts or organic fractions. Isolation of no active compounds can be avoided and constituents with novel activity can be isolated. Preparative chromatography was helpful for high throughout purification of the compounds. Separation of the extracts was performed on reversed phase (CC/TLC RP18) with broad gradient mixtures of two or three solvents. Compounds exhibiting interesting antifungal activity were identified by chemical analysis using NMR, UV, GC–MS or by comparison with the correspondent standards.

Bioassays Biological targets The 12 phytopathogenic fungi, viz., Aspergillus flavus, A. niger, Botrytis cinerea, Cladosporium cucumerinum, Fusarium culmorum, F. oxysporum f.sp. cubense, F. oxysporum f.sp. melonis (F.o.m.), F.o.m. race 1,2, F.o.m. race 0, Phytophthora cambivora, Rhizoctonia sp. and Roselinea necatrix were analysed for their sensitivity to plant extracts, and to plant cell culture or to pure compounds, detected or isolated from those extracts. Fungi were supplied by the Centro de Estudos da Ferrugem do Cafeeiro (Center of Coffee Rust Studies) and Departament of Phytopathology of Estac- aõ Agrono´mica Nacional (E.A.N.), Oeiras, Portugal.

Plant analysis Portuguese native plants were studied for the activity of their constituents against fungi affecting crops or foods. The species Biscutella lusitanica Jordan (Cruciferae), Luzula lactea Link (Juncaceae), Picris spinifera Franco (Asteraceae) and Silene foetida Sprengel (Caryophilaceae) as well as Chelidonium majus L. (Papaveraceae), were obtained by seed germination and grown in the Estac- aõ Agrono´mica NacionalE.A.N’s green-house. The leaves of Sambucus nigra L. (Caprifoliceae) were collected in the Northeast of Portugal. Thevetia peruviana Schum (Apocynaceae), collected in Macau, was also included in the present studies. Extracts were prepared with organic solvents and tested on several phytopathogenic fungi. Extracts (whole plant) of B. lusitanica, L. lactea, P. spinifera and S. foetida prepared in methanol (MeOH) (1:5 weight/volume) were fractionated in n-hexane (n-hex), chloroform (CHCl3), ethyl


151

acetate (EtOAc) and MeOH. Extraction procedures of C. majus (roots and shoots) in MeOH and of T. peruviana (leaves) in EtOAc, MeOH or H2O were performed as described in previous work (Matos et al., 1999; Gata-Gonc- alves, 2003). The leaves of S. nigra were fully dried (in the dark at 35 1C, for 10 days). The extracts were prepared (300 g dry weight/1500 ml of solvent) in water (boiling for 30 min), or in EtOAc, or in EtOH (throughout contact and stirring at 200 rev./min, for 3 days at 12 1C). Aqueous extracts were frozen at 80 1C and lyophilized; those obtained in the organic solvents were dried, by evaporation under vacuum at 40 1C, and resuspended in adequate volume of the solvent used for the extraction to a final concentration of 10–20 mg/ml. All the extracts were submitted to bioautographic fractionation techniques (Matos, 2001). Bioactive fractions were recovered from preparative thin-layer chromatography and concentrations of 1:5 (p/v) were used in the biological tests. Extracts of S. nigra and T. peruviana were tested against B. cinerea, C. cucumerinum and F. culmorum, while the extracts of C. majus were tested on F. oxysporum f.sp. cubense, C. cucumerinum, P. cambivora, Rhizoctonia sp. and Roselinia necatrix. The extracts in n-hexane, CHCl3, EtOAc and MeOH of B. lusitanica, L. lactea, P. spinifera and S. foetida were used for testing C. cucumerinum and F. culmorum.

Cell culture analysis Cell systems can produce different secondary metabolites or in different concentration of those produced by the complete plant. The cell culture of the Portuguese native species B. lusitanica, L. lactea, P. spinifera and S. foetida were included in these studies with phytopathogenic fungi. For the cell cultures, plantlets obtained from seeds aseptically germinated were used. Cells were developed in solid media MS or GB5 (Murashigue and Skoog, 1962; Gamborg et al., 1968) with 1 or 2 mg/l of dichlorophenoxiacetic acid (2,4-D), and incubated at 25 1C. After 3–5 weeks of growth cells were removed to fresh media and the initial media were transferred to Erlenmeyers and MeOH was added to a ratio of 1:2 (weight/volume), stirred on an orbital shaker at 180–200 rev./min, for 24 h, at room temperature. The mass extracted was centrifuged at 3000 g, for 15 min, the supernatant was recovered and fully dried in a rotary evaporator under vacuum. These crude extracts were fractionated by successive direct washing with n-hex, CHCl3, EtOAc and MeOH. Fractions were evaporated to dryness, weighed and re-suspended in the corresponding solvent to a final concentration of 10 mg/ml, and tested on A. flavus, B. cinerea, C. cucumerinum, F. culmorum and F. oxysporum f.sp. melonis. The cells of C. majus were grown in MS liquid culture medium, supplemented with five different combinations of hormones: 20 mg/l of 2,4-dichlorophenoxiacetic acid (2,4-D), 0–0.1 mg/l of kinetin (KIN) and 2.5–5.0 mg/l of gibberelic acid (GA3), for 6 weeks with agitation on an orbital shaker (100 rev./min). The media, after being frozen and lyophilized, were extracted by contact in methanol (1:5 weight/volume). Extracts were filtered and sterilized using a Millipore filter and used for the evaluation of their effectiveness on the control of F. oxysporum f.sp. cubense, C. cucumerinum, P. cambivora, Rhizoctonia sp. and Roselinia necatrix.

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Isolated compounds Active compounds were preliminarily characterized by their reaction on the TLC plates with dyeing reagents (vanillin, MFA, KOH/EtOH, H2SO4/EtOH and Dragendorf reagent). Analysis by capillary gas chromatography followed by mass spectrometry (GC–MS) was performed for T. peruviana, using an Autosystem XL gas chromatograph with a vaporization injector in split mode (1:50) interfaced to a Turbomass Perkin–Elmer mass spectrometer. High-purity helium was used as carrier gas (25 cm/s), with a oven temperature program of 40 1C for 3 min, increased at 5 1C/min to 180 1C, followed by 15 1C/min to 240 1C and finally to 300 1C at 10 1C/min (isothermal 15 min), using a fused-silica capillary column, 30 m 25 mm i.d. 0.32 mm (DB-1; 100% dimethylpolysiloxane) (Gata-Gonc- alves et al., 2003). The characterization of bioactive compounds was also achieved by subjecting chemical standards to similar TLC tests. After elution with the same solvent mixtures and testing against the same fungi, the Rf of the standards and those of the active compounds were compared. Compounds that are frequently present in the plants studied were included in our assays. They were tested together with some of the extracts contributing to an approach to the chemical characterization of the extracts’ components. Stock solutions (102 M) of benzoic acid, caprilic acid, palmitic acid, linoleic acid, oleic acid, (+)-pulegone, isopulegol, ergosterol, citronellol, carvacrol, thymol, flavone (6-hydroxyflavone), coumarin (7-hydroxy-4-methylcoumarin), psoralen, chelerithrine and sanguinarine were prepared. The solvents used for the preparation of these solutions were selected from the solvents used for the extraction of plant material considering their ability to dissolve each of the compounds. For the bioassay the solutions were diluted to concentrations of 1045 104 M. The fungi A. flavus, A. niger, B. cinerea, C. cucumerinum, F. culmorum, F. oxysporum f.sp. melonis (F.o.m.), F.o.m. Y race 1,2, F.o.m. 26 race 1 and F.o.m. MC2 race 0 were the biological targets used in the assays here included. Standards were supplied by Dr. Maria Joaõ Marcelo Curto from Instituto Nacional de Engenharia e Tecnologia Industrial of Lisbon, by Dr. Antońio Mac- anita from Instituto Superior Tećnico of Lisbon, or were purchased in the market from Aldrich and Sigma.

Assay procedure Fungi were grown and maintained in culture on PDA (Potato Dextrose Agar, Difco Laboratories) at 25 1C in the dark. For the present antifungal tests, 5-, 7- or 10-dayold fungus cultures were used for harvesting the mycelium. Fungi suspensions were prepared by disintegrating the mycelium in a specific liquid medium (Allen and Kuc, 1969). Each fungus suspension was filtered under vacuum through nylon net (100 mm pores) on a Buckner funnel, adding the liquid media until a spore count of about 57.5 104 per ml was achieved for each fungus. Assays in chromatographic plates For the antifungal tests performed on TLC plates, 30 ml of each plant crude extract or fraction, 50 ml of each culture media extract or 20 ml of the solution of the natural


153

compounds (104, 2 104 and 5 104 M) were applied. The extracts, the organic fractions and the standard compounds were applied directly into spots of 5–7 mm. The fractions were also chromatographed. After the elution in CHCl3:MeOH (97.5:2.5), the plates were visualized at 254 and 366 nm to visualize the compounds (Matos, 2001; Matos and Ricardo, 2003). Control spots with the solvents were also applied. Fungi were inoculated by spraying the TLC plates with the fungal suspension and these plates were incubated in humid chamber at 23 7 2 1C under continuous dark for 4–5 days. Inhibition areas were revealed as white spots, and qualitatively evaluated by the diameter of the area free of fungus growth. Replicates of each TLC were performed in order to be treated with the dying reagents [H2SO4/EtOH (10%), KOH/EtOH, molybdophosphoric acid (MFA), vanillin, Dragendorff reagent. The standards of benzoic acid, octanoic acid, palmitic acid, linoleic acid, oleic acid, (+)-pulegone, isopulegol, ergosterol, citronellol, carvacrol, thymol, were submitted to similar tests. Assays in liquid media Stock solutions of the standard compounds 3,6-dihydroxyflavone and 7-hydroxy-4methylcoumarin, chelerithrine and sanguinarine and 8-methoxypsoralen of 102– 103 M were previously prepared. From these stock solutions 100–200 ml were taken and added to 20 ml of the liquid fungus suspension. Compounds were solubilized in MeOH (20 ml/ml) to guarantee their solubility in the aqueous media. The samples were firstly incubated for 24 h at 25 1C, in dark, with agitation, in order to allow the fungus adaptation to these conditions. Subsequent to this initial growth, the compounds were added to the samples, with a micropipette directly into the suspension. In the photobiological tests the concentrations of each compound were established by estimating the overlap integral between the absorption spectrum of the compound and the lamp emission spectrum, to assure that, in the final fungus suspension, about the same concentration of species in the excited state was present. Samples containing the 20 ml of spore suspension and 100–200 ml of each of the compounds were placed on an orbital shaker (100 rev./min). This procedure was essential to oxygenate the fungus suspension, to obtain a good contact between the fungus and the compounds that are dissolved in the suspension and, in the case of assays performed under light irradiation, to achieve the best irradiation conditions to activate the compounds to be tested. Six replicates of each sample were prepared. Blank samples, either without any compound or with methanol or each solvent–methanol mixture were also tested. The samples without compound were used to evaluate whether the fungi were growing properly. Results were evaluated 3–5 days after inoculation at 25 1C as the difference between dry weights (Balance Mettler AB204-S, d ¼ 0.1 mg) observed in the samples having the compounds and the samples only with the solvent. Photobiological assays When phototoxic activity was tested the plates or liquid samples were prepared in duplicate, one for testing in the dark and the other for testing under light exposure.

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Incubation was performed also at 2372 1C and the plates, after an initial period (12–24 h) of continuous dark, were exposed to light (8 h/day) for 2–3 days and kept in dark till the end of the assay. A set of five fluorescence lamps (Osram L18w/10 Daylight, emission range 350–730 nm, lmax ¼ 485 nm with 0.62 W/m2 fluence) was used as light source. The intensity per unit area was measured as previously described (Matos, 2001; Borges et al., 2002). The distance between the fluorescent lamps and the samples to be analysed was 30–40 cm. Photoactivity was determined by comparing the results achieved in the dark to those of the correspondent sample submitted to irradiation.

Results and discussion Promising results were obtained with most of the several plant extracts and fractions. The presence of antifungal compounds in the different culture media synthesized by the plant cells was also confirmed. In these experiments the higher efficacy with the lowest quantity of active substance was attempted, in order to avoid or to reduce to a minimum the problems of toxicity to plants, to beneficial microorganisms or insects or even to the environment. In the biological tests we take in consideration that for a natural compound to be considered a fungicide it is necessary that an inhibition of 50% (ED50) of the mycelium’s growth was obtained, with concentrations between 5 and 50 mg/ml. For extracts the concentrations corresponding to the ED50 were between 5 103 and 15 103 mg/ml. In an attempt to characterize the plant constituents responsible for the antifungal activities, the extracts or the extract fractions were chromatographed by liquid chromatography and/or by preparative thin-layer chromatography. Antifungal activity was detected on the TLC plates by the diameter of the halos corresponding to fungus growth inhibition. Antifungal photoactivity was detected by comparing the plates kept in the dark with those submitted to light irradiation (Matos and Ricardo, 2003). Results of assays in liquid media were evaluated by the difference between the mycelium dry weights produced by the fungus in the presence of the active compounds or only the solvent.

Plant extracts Extracts prepared from leaves of S. nigra and T. peruviana with ethyl acetate, water and ethanol or either methanol were tested against B. cinerea, C. cucumerinum and F. culmorum using the liquid media method. As illustrated in Figure 1, for S. nigra, and Figure 2, for T. peruviana, fungi were inhibited by the extracts of both plants. Aqueous extracts were less active then those in EtOAc and in EtOH. With the extracts in EtOAc the level of fungus inhibition reached about 60% for both plants. In the case of S. nigra the extract in EtOH induced inhibitions of about 70–80%, while for T. peruviana the extract in MeOH reduced the fungi growth around 60–70%. As a whole F. culmorum was the fungus that showed higher resistance to all the extracts of both plants.

Screening of plants against fungi affecting crops and stored foods Sambucus nigra

155

B. cinerea C. cucumerinum F. culmorum

Inhibition (%)

100 80 60 40 20 0 EtOAc

EtOH

H2O

Fig. 1. Effect of the leaf extracts of Sambucus nigra obtained in EtOAc, EtOH or H2O on the growth of Botrytis cinerea, Cladosporium cucumerinum, and Fusarium culmorum. Growth inhibition (affected by standard deviation) observed 5 days after inoculation is expressed as a percentage of the growth inhibition observed for the control with the correspondent solvent.

Thevetia peruviana

B. cinerea C. cucumerinum F. culmorum

100

Inhibition (%)

80 60 40 20 0 EtOAc

MeOH

H2O

Fig. 2. Effect of the leaf extracts of Thevetia peruviana obtained in EtOAc, MeOH or H2O on the growth of Botrytis cinerea, Cladosporium cucumerinum and Fusarium culmorum. Growth inhibition (affected by standard deviation) observed 5 days after inoculation is expressed as a percentage of the growth observed for the control with the correspondent solvent.

Results achieved in previous work on the antifungal properties of C. majus (Matos et al., 1999) led to perform with this plant antifungal assays using different genus of fungi as biological targets. Figure 3 presents the sensitivity of the fungi C. cucumerinum, P. cambivora, R. necatrix, Rhizoctonia sp. and Fusarium oxysporum f.sp. cubense (used as control) to the shoot and root methanolic extracts. For the five fungi the mycelium growth reached in the presence of the shoot extracts was quite similar, the inhibitions ranging from 53.5% (Rhizoctonia sp.) to 35.9% (R. necatrix). P. cambivora (26.7%) was the most resistant fungus to shoot extracts while R. necatrix and Rhizoctonia sp. exhibited a reduction in mycelium growth of 48.1%


156

Root

Chelidonium majus L

Leaf Fungus growth (%)

100 80 60 40 20 0 F1

F2

F3

F4

F5

Fig. 3. Effect on the growth of Fusarium oxysporum f.sp. cubense (F1), Cladosporium cucumerinum (F2), Phytophthora cambivora (F3), Roselinea necatrix (F4) and Rhizoctonia sp. (F5) of root and shoot extracts of C. majus obtained in MeOH. Growth inhibition (affected by standard deviation) observed 5 days after inoculation is expressed as a percentage of the growth observed in the presence of the control.

and 53.5%, respectively. F. oxysporum f.sp. cubense and C. cucumerinum were the most sensitive fungi to C. majus root extracts, showing a growth reduction corresponding to 92.6% and 94.3%, respectively. In another experiment, the whole plant material from B. lusitanica, L. lactea, P. spinifera and S. foetida, were extracted in n-hex, CHCl3, EtOAc and MeOH and analysed for their antifungal properties against C. cucumerinum and F. culmorum. The extracts of these plants were less active than those obtained from the cell cultures of same plants (see Table 6). In fact, when assayed without light exposure, the extracts in CHCl3 EtOAc and MeOH were active or slightly active, except in the case of L. lactea, which EtOAc extract was very active against C. cucumerinum (see results included in Table 7).

Cell cultures We also report here assays performed to evaluate the potentiality of different plant cell cultures for the synthesis of antifungal substances. Because bioactive compounds are excreted into the media by the undifferentiated cells, the antifungal properties of each plant cell culture was evaluated based on the concentration of antifungal compounds extracted from the corresponding media. Results of the antifungal assays performed with cells of C. majus, grown in five different liquid media, extracted in methanol are presented in Figure 4. In these assays, a similar pattern of sensitivity of the five fungi to the extracts from the different media was observed. The fungi were insensitive or only slightly affected by the components present in the culture media M4 and M5. A growth reduction of less than 50% was observed with the media M1 and M2. All the fungi were severely affected by M3, which conduced to a growth reduction over 60% to C. cucumerinum, R. necatrix and Rhizoctonia sp., over 65% to F. oxysporum f.sp. melonis and over 80% to P. cambivora. The results achieved in these assays are much relevant since

Plant cells

Media

Extracts

Aspergillus flavus Botrytis cinerea

Cladosporium cucumerinum

Fusarium culmorum

F. oxysporum f.sp. melonis

Biscutella lusitanica

GB5

Luzula lactea

GB5

n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH

+ ++ + ++ ++ + + T + + + +++ ++ ++ T + ++ +++ + ++ +

++ +++ +++ +++ +++ + ++ + + ++ +++ +++ +++ +++ ++ + ++ +++ + ++ +

+ + ++ ++ ++ + + + + ++ +++ ++ ++ T + ++ +++ + + +

+ ++ ++ ++ +++ ++ + + + ++ +++ +++ ++ +++ + + ++ +++ + ++ +

MS

Picris spinifera

GB5

MS

Silene foetida

GB5

MS

+ ++ +++ ++ +++ ++ + + + ++ +++ +++ +++ +++ + + ++ +++ + ++ +

157

Bioautographic assays performed in chromatographic plates. Fungus inhibition observed 3 days after incubation at 2372 1C: , inactive; t, traces; +, slight active; ++, active; +++, very active.


Table 6 Sensitivity of Aspergillus flavus, Botrytis cinerea, Cladosporium cucumerinum, Fusarium culmorum and F. oxysporum f.sp. melonis to plant cell media


158

Table 7 Sensitivity of Cladosporium cucumerinum and Fusarium culmorum to plant extracts Plant

Extracts

Biscutella

n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH

lusitanica Luzula lactea

Picris spinifera

Silene foetida

Cladosporium cucumerinum

Fusarium culmorum

Dark

Light

Dark

Light

t ++ + + ++ + +++ + + + ++ + + ++ + ++

+ +++ ++ + ++ + +++ + + +++ +++ ++ + ++ t ++

+ + + + ++ + + + + + + + + + +

+ ++ ++ + ++ + + + ++ +++ ++ t + t

Bioautographic assays performed in chromatographic plates. Fungus inhibition observed 3 days after incubation in continuous dark or irradiated with VIS light (8 h/day) at 2372 1C: , inactive; t, traces; +, slight active; ++, active; +++, very active.

M1 M2 M3 M4 M5

Fungus Growth (%)

120 100 80 60 40 20 0 F1

F2

F3

F4

F5

Fig. 4. Effect of the substances produced by cells of C. majus grown in liquid media MS with 2,4-D (20 mg/l) and 2.5 mg/l of GA3 (M1), 5.0 mg/l of GA3 (M2), 0.1 mg/l of KIN (M3), 0.1 mg/l of KIN and 2.5 mg/l of GA3 (M4), 0.1 mg/l of KIN and 5.0 mg/l of GA3 (M5), on the growth of Fusarium oxysporum f.sp. cubense (F1), Cladosporium cucumerinum (F2), Phytophthora cambivora (F3), Roselinea necatrix (F4) and Rhizoctonia sp. (F5). Growth affected by standard deviation, observed 5 days after incubation at 25 1C, is expressed as a percentage of the growth observed with the solvent control.

they indicate that the presence of kinetin promotes the synthesis of most active antifungal compounds. Conversely, the presence of GA3 conduced to the decrease in the synthesis of those compounds. It would be much informative to analyse the several compounds released to those different cell cultures.


159

In another experiment, a set of Portuguese endemic wild plant species cultured in vitro in MS or GB5, were also analysed for their ability to synthesize compounds useful for antifungal purposes. The effects of the extracts obtained in n-hex, CHCl3, EtOAc and MeOH of the cell culture of Biscutella lusitanica, Luzula lactea, Picris spinifera and Silene foetida on the growth of A. flavus, B. cinerea, C. cucumerinum, F. culmorum and F. oxysporum f.sp. melonis were evaluated. Results, based on a non parametric evaluation, showed significant differences on the antifungal properties of the cells of the four plants analysed against the five fungi (Table 6). Antifungal effects were highly dependent on the composition of the cell culture media and on the solvent used for the preparation of the extracts. ‘‘As expected, as a whole, the less affected fungi were F. culmorum and A. flavus and C. cucumerinum were the most affected fungi.’’ In the case of B. lusitanica, cells survived only in GB5 medium, and no significant differences were detected on the reduction of fungus growth in the presence of extracts in CHCl3, EtOAc and MeOH. The extracts obtained in n-hexane were quite inactive for most of the fungi, probably due to the low hydro solubility of the substances dissolved in this solvent, L. lactea GB5 cell growth media was an exception since n-hex extracts were active or very active. The components extracted by EtOAc from the MS medium of the L. lactea cells were also very active but only against B. cinerea, C. cucumerinum and F. oxysporum f.sp. melonis. In the case of P. spinifera no significant differences were detected for the CHCl3 extracts of both media, while the EtOAc extracts of the GB5 medium were much more active then those of MS. For this set of plants the methanolic extracts were usually less active, but the GB5 medium of B. lusitanica and S. foetida were active or very active. The assays just described reinforce the idea that in vitro cultures can be useful for the production of antimicrobial compounds as a substitute of the plant. Despite being costly this procedure can avoid the destruction of genetic resources mainly when studies are performed with plants existing in restricted areas or risking extinction.

Photoactivity The extracts obtained from the whole plants of B. lusitanica, L. lactea, P. spinifera and S. foetida, prepared with n-hex, CHCl3, EtOAc and MeOH, were also analysed for their antifungal photoactivity against C. cucumerinum and F. culmorum (Table 7). Only the extracts in CHCl3 and EtOAc of B. lusitanica and those of P. spinifera in CHCl3, EtOAc and MeOH showed increased antifungal activity against both fungi when exposed to light, relatively to the control tested in the dark. The cell culture media of these same plants were also analysed for their photoactivity, and as observed with the plant extracts only the cell culture media of B. lusitanica and P. spinifera showed the presence of antifungal photoactive principles. Results of the antifungal photoactivity against C. cucumerinum of the cell culture media of B. lusitanica extracted in n-hex, CHCl3, EtOAc and MeOH are shown in Figure 5. The extract in CHCl3 showed a good efficacy and photoactivity against C. cucumerinum and the MeOH extract was active mainly in the dark. As in the case of the extracts from the whole plant, the extracts obtained from the cell culture media of P. spinifera in CHCl3 and EtOAc (Figure 6) evidenced the


160

Biscutella lusitanica Inhibition spot (mm in diameter)

12 10 8 6 4 2 0 n-Hexane

CHCl3

EtOAc

MEOH

Fig. 5. Antifungal activity of Biscutella lusitanica cell culture medium (GB5) treated with n-Hex, CHCl3, EtOH and MeOH. Results of the assays performed against Cladosporium cucumerinum observed 4 days after incubation at 2372 1C, in continuous dark ’ or irradiated with VIS light (8 h/day) &. Picris spinifera

Inhibition spots (mm)

15

GB5 (Dark) GB5 (Light) MS (Dark) MS (Light)

10

5

0 CHCl3

EtOAc

Fig. 6. Antifungal activity of Picris spinifera cell culture media treated with CHCl3 and EtOAc. Results of the assays performed against Cladosporium cucumerinum observed 4 days after incubation at 23 1C, in continuous dark ’ or irradiated with VIS light (8 h/day) &.

highest levels of fungus photoinhibition. No significant differences of photoactivity were detected related to the composition of the media.

Isolated compounds T. peruviana, C. majus and P. spinifera were studied in order to identify their bioactive compounds. Capillary GC–MS analysis of the main antifungal fraction of T. peruviana showed to be a complex mixture of several classes of components. More than 25 compounds were identified, representing the terpenes 25.2% and the fatty


161

acids and derivatives 24.8%. In a previous work, it was found that antifungal activity T. peruviana against C. cucumerinum was mainly due to pulegone (14.3%) the major compound identified (Gata-Gonc- alves et al., 2003). Additional components of T. peruviana, like benzoic, octanoic, palmitic and linoleic acids, isopulegol, ergosterol, citronellol, carvacrol and thymol, were also studied for their antifungal properties. Results of tests performed with those substances against Aspergillus flavus, A. niger, B. cinerea, C. cucumerinum, F. culmorum, F. oxysporum f.sp. melonis, F. oxysporum f.sp. melonis Y race 1,2 and F. oxysporum f.sp. melonis MC2 race 2 are summarized in Table 8. Palmitic, linoleic and oleic acid as well as ergosterol were inactive or slightly active to the tested fungi. Isopulegol and thymol also exhibited a scarce activity. Pulegone and citronellol were active against this set of fungi. Very good inhibitions were achieved with benzoic and octanoic acids as well as with carvacrol, mainly when tested against B. cinerea. In fact, Mu¨ller-Riebau et al. (1995) in studies performed with Satureja thymbra and Thymbra spicata reported that thymol and carvacrol were the main active constituents of those plants. The importance of octanoic acid was also demonstrated in inhibiting the growth of B. cinerea and Penicyllium expansum on stored pears (Matos and Barreiro, 2004). Chelerithrine and sanguinarine, that were detected as responsible for the antifungal activity of the C. majus root and shoot extracts (Matos et al., 1999). Flavone and coumarin were referred as being synthesized by P. spinifera (Matos and Ricardo, 2003), so 6-hydroxyflavone and 7-hydroxy-4-methylcoumarin were analysed for their action against C. cucumerinum, F. culmorum, F. oxysporum f.sp. cubense, F. oxysporum f.sp. melonis 26 race 1 and F. solani. In order to evaluate the potentialities of these compounds as photopesticides they were tested either in the dark or after irradiation (UVA or visible spectrum) (Table 9). Flavone was active against the tested fungi mainly in dark conditions, while coumarin was active or slightly active when irradiated, evidencing higher inhibition than psoralen (Averbeck, 1989) under visible light irradiation (Borges et al., 1995). Chelerithrine and sanguinarine were active against F. oxysporum f.sp. cubense, F. oxysporum f.sp. melonis 26 race 1 and F. solani, mainly when irradiated with UV light.

Conclusions and future perspectives There is a relatively recent knowledge on opportunistic infections in man and animals caused by Fusarium strains (Rebell, 1981; Desjardins et al., 2000) and several reports suggest that diseases caused by Fusarium are responsible for important economic losses in crops (Matos et al., 1999). F. culmorum is a serious pathogen of cereals that infects seedlings, roots and stems (Koopmann et al., 1994). F. solani is responsible for potato wilt causing fusariosis in other vegetables. F. oxysporum is responsible for several vascular wilts (Cook, 1968, 1978; Nelson et al., 1981; Jaspal and Tripa´thi, 1995). Aspergillus infections besides promoting commercial losses in crop yields also produce fungitoxins which can be very dangerous to man and animals. Botrytis cinerea is responsible for fungal attack of foods which in addition to causing rots can make them unfit for consumption by producing mycotoxins (Tripathi and Dubey, 2004).

162 Table 8 Sensitivity of Aspergillus flavus, A. niger, B. cinerea, Cladosporium cucumerinum, Fusarium culmorum, F. oxysporum f.sp. melonis, F. oxysporum f.sp. melonis Y race 1,2 and F. oxysporum f.sp. melonis MC race 0 to isolated compounds Aspergillus flavus

Benzoic acid Octanoic acid Palmitic acid Linileic acid Oleic acid (+)-Pulegone Isopulegol Ergosterol citronellol Carvacrol Thymol

++ ++ ++ + ++ ++

Aspergillus niger

++ + ++ ++

Botrytis cinerea

Cladosporium Fusarium cucumerinum culmorum

+++ +++ t t ++ + + ++ +++ +

++ ++ + + + + t ++ + +

+ ++ ++ + ++ t

F. oxysporum F.o.m. Y race f.sp. melonis 1,2

++ + + ++ ++

+ ++ ++ + ++ ++

F.o.m. MC2 race 0 + ++ ++ + ++ ++

Autobiographic assays performed in chromatographic plants. Fungus inhibition observed after incubation for 3 days at 2372 1C:, inactive; t, traces; +, slight active; ++, active; +++, very active.


Compounds

Fungi

Irradiation condition

Flavone

Coumarin

Psoralen

Chelerithrine

Sanguinarine

C. cucumerinum

Dark UV light VIS light Dark UV light VIS light Dark VIS light Dark VIS light Dark VIS light

++ ++

+ ++ ++ + ++ ++

+ +++ t + +++

+ ++ + + ++ t ++

+ ++ + ++ ++

++ + ++ +

+ ++ + ++

+ + + +

++

++

++

++

F. culmorum F. oxysporum f.sp. cubense F. oxysporum f.sp.melonis 26 race 1 F. solani


Table 9 Sensitivity of Cladosporium cucumerinum, Fusarium culmorum, F. oxysporum f.sp. melonis Y race 1,2 and MC race 0 to isolated compounds

Assays performed in liquid media. Fungus inhibition observed 3 days after incubation in continuous dark or irradiated with UV or VIS light (8 h/ day): , inactive; t, traces; +, slight active; ++, active; +++, very active.

163

164


Fungicides are essential for plant diseases control. The efficacy of the present available fungicides is satisfactory, but a continuous effort to respond to the acquisition of resistance is required. Enhancement of crop quality is an advantage brought on by fungicides to the agricultural output. However, these advantages imply serious economic and environmental costs. As an alternative, the extracts of plants or of plant cell culture or the compounds they contain could be quite effective in the control of fusariosis, aspergillosis and crop infections caused by B. cinerea and C. cucumerinum. In the future many more new natural products useful for crop protection will certainly be identified from diverse natural sources. It is foreseeable, that biotechnology will expand its influence on crop protection. Important research areas for the development of new strategies for the protection of crops and foods will focus in the future on the utilization of natural products, including those that trigger defence mechanisms in plants (Knight et al., 1997; Cowan, 1999), the synergistic effects involving those compounds and synthetic ones as well as diseases control by means of biological interaction (Wink, 1993; Mari and Guizardi, 1998; Carpinella et al., 2003; Qin et al., 2004). Random gene shuffling in plants can be the source of new ‘‘natural’’ products with interesting biological activity and the transgenic technology will also contribute with new tools for crop protection. Even if, at present, synthetic chemicals remain as strong pillars of crop protection the search for natural compounds and new classes of fungicides with novel modes of action will be of increasing importance.

Acknowledgments We are grateful to Professor Antońio Mac- anita and Dr. Maria Joaõ Marcelo Curto for providing facilities and laboratory support. A gift of T. peruviana plant material, from Dr. L. Gata-Gonc- alves and Dr. S. Baptista, and support on the identification of bioactive compounds from Dr. J. M. Nogueira and Dr. C. Moiteiro are also thankfully acknowledged.

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CHAPTER 8

Opportunities and potentials of botanical extracts and products for management of insect pests in cruciferous vegetables TONG-XIAN LIU, HAN-HONG XU, WAN-CHUN LUO

Introduction Cruciferous vegetables include cabbage, Chinese cabbage, broccoli, brussels sprouts, cauliflower, kale, collards, and kohlrabi, and are economically important to vegetable production and consumers around the world. Insect pests that attack these crops include diamondback moth (Plutella xylostella L.), cabbage looper (Trichoplusia ni Hu¨bner), beet armyworm (Spodoptera exigua Hu¨bner) and other armyworm species, (imported) cabbageworm (Pieris rapae L.), corn earworm [Helicoverpa zea (Bodddie)], cross-striped cabbageworm [Evergestis rimosalis (Guenee)], and cutworms in Lepidoptera; cotton aphid (Aphis gossypii Glover), turnip aphid [Lipaphis erysimi (Kaltenbach)], green peach aphid [Myzus persicae (Sulzer)], cabbage aphid [Brevicoryne brassicae (L.)], and whiteflies (Bemisia spp.) in Homoptera; vegetable leaf miners (Liriomyza sativae Blanchard) in Diptera, and others (Capinera, 2001). Due to the importance of crucifer vegetables, synthetic insecticides have been intensively used to control those pests. Although many novel insecticides have been registered to control crucifer pests around the world in recent years, their management still relies heavily on broad-spectrum synthetic insecticides (Talekar and Shelton, 1993). Many insect pests of crucifers have developed resistance to synthetic insecticides in many countries; those insects include P. xylostella, including Btinsecticides (Tabashnik et al., 2003), spinosad (Zhao et al., 2002) and others. In addition, the economic, health, and environmental costs of synthetic insecticides, negative effects on beneficial arthropods, and increasingly stringent government regulation of pesticides has resulted in more interest in the development and use of botanical pest-management products (Ascher, 1993). The role of botanical insecticides in pest management in most industrialized countries is still minimal (Isman, 1999). In recent years, there have been considerable interests in natural products derived from plants that have been registered to control numerous agricultural insect pests (Schmutterer, 1997; Haseeb et al., 2004; Xu et al., 2004).

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For thousands of years, people have recognized that plants are efficient producers of chemical compounds that are used in defense against herbivore attack. Botanical compounds have been used in several parts of the world long before the arrival of synthetic pesticides. For example, farmers in India and China have been using botanical extracts to battle major agricultural pests (Chiu, 1993, 1995; Singh and Singh, 1996), and those botanical insecticides include pyrethrum obtained from the flower of Chrysanthemum cinerariifolium [(Trevir.)Vis.], nicotine from tobacco (Nicotiana tabacum L.), and rotenone from Lonchocarpus nicou (Aublet), Derris indica (Lam.) Bennet, Amorpha fruticosa L., and Tephrosia purpurea L. Whereas plant-derived pesticides or extracts containing active compounds can be used directly, they can also form the basis for synthesizing products with similar or even better insecticidal properties. For example, pyrethrum has been used as the basis for the synthesis of the synthetic pyrethroids, which are important in modern chemical crop protection. The synthetic carbamates are also derived from physostigmine, a natural carbamate from the legume Physostigma venenosum Balf. (Fabaceae). In addition, nontoxic allelochemicals with co-occurring chemicals have been used to control insect pests. Most widespread among these synergists are inhibitors of different insect enzymes, including mixed-function oxidases and esterases (Feyereisen, 1999). In this chapter, we focus on the effects of some crude extracts from plants and some commercially available botanical insecticides on important insect pests of cruciferous vegetables (Table 1). The botanical materials include crude extracts and isolated or purified compounds from various plants species and commercial products. The insect pests tested include Trichoplusia ni, Spodoptera exigua, S. litura, S. spp., Pieris rapae, Helicoverpa spp., Evergestis rimosalis, and Mythimna separate Walker in Lepidoptera, Aphis gossypii, Lipaphis erysimi, Myzus persicae, Brevicoryne brassicae, and Bemisia spp. in Hompotera, Phyllotreta vittata in Coleoptera, and L. sativae in Diptera. The advantages and disadvantages of using those materials, and the potential of these botanical materials in the integrated pest management of cruciferous vegetables are discussed.

Azadirachtin and related products from Azadirachta indica A. Juss Azadirachtin or neem-based insecticides containing azadirachtin derived from extracts of neem tree (Azadirachta indica A. Juss, Meliaceae) have played important roles in crop protection. Azadirachtin, a complex tetranortriterpenoid, has been effectively used against >400 species of insects, including many key crop pests, and has proved to be one of the most promising plant ingredients for integrated pest management at the present time (Schmutterer, 1990, 1992, 1995; Isman, 1999; Walter, 1999). Neem-based compounds display an array of effects on insects, acting inter alia as a phago- and oviposition deterrent, repellent, antifeedant, growth retardant, molting inhibitor, sterilant, and preventing insect larvae from developing into adults (Schmutterer, 1990, 1995). Liang et al. (2003) evaluated three commercial neem-based insecticides in the laboratory for oviposition deterrence, antifeedant effects on larvae, and toxicity to eggs of P. xylostella. The three commercial neem-based insecticides were: AgroneemTM (0.15% azadirachtin, Agro Logistic Systems, Diamond Bay, CA) at

Botanical extracts and products for management of insect pests

173

Table 1 Botanical materials (extracts or commercial products) and their active ingredients that have been tested against major insect pests of crucifer vegetables Plants

Extracts or formulation

Active ingredients

References

Azadirachta indica A. Juss

Neemixs, Agroneems, Ecozins, Neepels

Azadirachtins and many others

Chrysanthemum cinerariaefolium Vis.

Extracts, isolates

Spiro enol ether, pyrethrin, pyrethrum

Daphne tangutica Maxim

Benzene extract, methanol extract, crude extracts

Lignans

Derris trifoliate Lour., D. elliptica (Wallich), D. chinensis (L.) Melia azedarach L. and M. toosendan Sieb. et Zucc.

Extracts, isolates

Rotenone

Wu (1997), Isman (1999), Schmutterer (1990, 1992, 1995), Walter (1999) Xu et al. (2000b, 2000c), Zhang et al. (2001a, 2001b, 2004) Perez-Izequierdo et al. (1992), PerezIzequierdo and Ocete (1994), Chen et al. (2000a, 2000b), Xu (2000a) Chiu (1993), Visetson and Milne (2001), Zeng et al. (2002)

Methanol extracts

Toosendanin

Parthenium argentatum Gray, P. hysterophorus L., P. schottii Greenman, and P. tomentosum DC Rhododendron molle G. Don

Methanol extracts, cold alcohol extract, isolates, petroleum ether extract

Parthenin, argentitatins

Isolates, ethanol extracts

Rhodojaponin III, many others compounds

Sophora alopecuroids L.

Purified alkaloids

Stellera chamaejasme L.

Ethanol extract

Aloperine, matrine, oymatrin, sophoramine, sophocarpine, cytisine, sophoradine 7-Hydro-coumarin, daphnoritin and chamaechromone

Zhang and Chiu (1991, 1992), Wang et al. (1992, 1994, 1999), Chiu (1993, 1995), Ce´spedes et al. (2000) Gajendran and Gopalan (1981), Isman and Rodriguez (1984), Patil et al. (1993), Ce´spedes (2001), Sohal et al. (2002), Sundarajan (2002) Hu et al. (1993, 2000b), Zhong et al. (2000b, 2001a, 2001b, 2001c), Cheng et al. (2002), Zhong (2002) Luo (1995), Luo and Li (1996), Luo and Zhang (2003), Luo et al. (1996, 1997, 1999) Ji et al. (2000), Zhang et al. (2000a, 2000b, 2000c, 2000d, 2000e), Wang et al. (2002), Su (2003)

(Continued)


174 Table 1 (continued ) Plants

Extracts or formulation

Active ingredients

References

Strophanthus divaricatus (Lour.)

Water extract, 0.05% strophanthin

Huang and Renwick (1994), Hu et al. (2000a)

Tripterygium wilfordii Hook. f. and T. hypoglaucum (level) Hutch.

Acetone extracts, chloroform extracts Ethanol extract

Caudoside, Fcaudostroside, divaricoside, sinlside, isosinotrodise, sarmutodise and Dstrophanthin Rotenoids (rotenone, rotenolone, and 5-,or 6-hydroxylrotenone) wilforine, alkaloids, non-alkaloids, wilfordine, wilforine, wilfortrine

Xanthium sibiricum Patrin ex Widder

Methanol and ethanol extracts

Xanthostrumarin, carboxyatractyloside, xanthatin, atractyloside, xanthin, xanthunin, xanthanodiene, strumaroside

Chiu (1987), Tong and Chiu (1988), Yang et al. (2000), Zhang et al. (2000e, 2000f), Xu et al. (2001, 2002), Zhang et al. (2001c), Luo et al. (2004) Peng (1984), Wang et al. (1999), Zhou et al. (2002)

4.8 mg azadirachtin/l, manufactured by Ajay Bio-Tech (India) Ltd., Erandwane, Pune 411004, Maharastra, India), EcozinTM (3% azadirachtin, AMVAC Chemical Corporation, Los Angeles, CA) at 20 mg azadirachtin/l, and NeemixTM (0.25% azadirachtin, Certis, Columbia, MD) at 20 mg azadirachtin/l. All three neem-based insecticides did not cause significant oviposition deterrence on P. xylostella. Numbers of eggs oviposited by P. xylostella adults on the cabbage leaves treated with the three neem-based insecticides were not significantly different from those treated with water. However, when aluminium foil sheets coated with cabbage juice residue were treated with the three insecticides and were used as egg-laying substrates, significantly fewer eggs were found on the aluminium foil sheets compared with those treated with water. When eggs were treated with Agroneem, Ecozin, and Neemix, 61.6, 66.2, and 75.2% of P. xylostella eggs developed to neonates, respectively, although the larval hatching rates in the treatment of Neemix were not significantly different from that in water control (81.2%). All larvae of P. xylostella fed on the leaves treated with the three neem-insecticides died on or before day 7 compared with 70–74% larvae in the water control surviving to adults. All three neem insecticides exhibited a significant antifeedant effect, and the larvae quickly stopped feeding and dropped off treated leaves, resulting in no or minimal foliar damage. Plutella xylostella larvae that fed on neem-based insecticide-treated leaves were significantly smaller (0.012–0.016 mg per larva, 13.5–14.8 mm in length, and 2.0–2.5 mm in diameter) compared with those fed on water-treated leaves (0.058 mg per larva, 30.2 mm in length, and 4.8 mm in diameter).


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Greenberg et al. (2005) evaluated the same three commercial neem-based insecticides, Agroneem, Ecozin, and Neemix, and a noncommercial neem leaf powder for oviposition deterrence, antifeedant effect on larvae, and toxicity to eggs and larvae of S. exigua in the laboratory. They found that in a no-choice assay, the proportion of eggs laid on the leaves was 2.5- to 9.3-fold higher in the control compared with other treatments. In a two-choice assay, significantly more eggs were deposited on the control leaves than on neem-treated leaves. Significant differences were also detected between pairs of neem treatments, and the treatments could be ranked in the following order of oviposition deterrence: neem leaf powder>Agroneem>Ecozin>Neemix. In a five-choice assay, oviposition preference was significantly lower on neem-treated leaves than on the control. No differences were observed between the neem treatments. Significantly more leaf area was consumed by each beet armyworm instar in the control than any of the neem-based treatments, but differences among the neembased treatments were not detected. Agroneem, Ecozin, and Neemix caused S. exigua egg 78, 77, and 72% of mortality compared with the nontreated control, but neem leaf powder caused 55% mortality, significantly lower than the commercial neem-based insecticides. Three days after treated leaves were offered to the beet armyworm larvae, mortality was not significantly different between neem treatments and the nontreated control. After 5 days of feeding, larval survival in the Agroneem- and Neemix-treatments was 1.5-fold greater than that in the Ecozin treatment and 1.3-fold greater than in the neem leaf powder treatments. After 7 days, larval survival was 61, 60, 33, and 27% in the Agroneem, Neemix, Ecozin, and neem leaf powder treatments, respectively, compared with 93% in the control. Neem-based insecticides have been tested and used for management of P. xylostella and other pest insects on cabbage (Leskovar and Boales, 1996; Perera et al., 2000). The neem-based insecticide, AlignTM (3% azadirachtin, formerly AgriDyne, Salt Lake City, UT), was tested in the field against lepidopterous pests, mainly P. xylostella and T. ni, on cabbage in Texas by Leskovar and Boales (1996). They found that Align significantly reduced larval densities of P. xylostella and T. ni on cabbage plants and foliage damage, and significantly increased marketable cabbage head weights. Neempel 0.3% EC (0.3% azadirachtin) is commercially produced using a new extraction technology developed by the Laboratory of Insect Toxicology, South China Agricultural University in China, and now commercially manufactured by Neemtech (Haikou, Hainan, China). The azadirachtin extraction and isolation process was modified by using a supercritical-fluid chromatography and microwave extraction method as described by Wu (1997). In the new azadirachtin extraction and isolation process, stabilizing agents and some specific epoxidized vegetable oil compounds have been incorporated into the final product, Neempel 0.3% EC, which restrains decompounding of azadirachtin in conditions of sunlight or heat. Neempel 0.3% EC has been tested against P. xylostella and turnip flea beetle, Phyllotreta vittata Fab. (Coleoptera: Chrysomelidae) under field conditions. Xu (unpublished data) found that Neempel was effective against P. xylostella and P. vittata larvae. At the dilutions of 800, 1000, and 1200 , P. xylostella populations were reduced by 81–91% 7 days after application and 81–86% 15 days after application. Percentage of control was 90–92% 7 days after application, and 86–89% 15 days after application. These results were similar to abamectin (1.8% EC, dilution 2000 ) (81 and 91% population reduction and control, respectively). At the dilutions of 1600,


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1800, and 2000 , P. vittata populations declined by 67–84% 7 days after application, and percentage of control was 69–86% 7 days after application, compared with cypermethrin (10% EC) with 52% population reduction and 56% control. In addition to controlling pest insects, many azadirachtin-based insecticides have negligible effects on natural beneficial insects and low environmental impact (Schmutterer, 1990, 1995). Because the neem-based insecticides are not toxic to humans and many beneficial arthropods, and the target pests are unlikely to become resistant, these neem-based insecticides become more amenable for use in pest management programs (Feng and Isman, 1995; Immaraju, 1998; Walter, 1999). However, we need to realize that neem-based formulations also contain numerous bio-active compounds other than azadirachtin, and azadirachtin itself might not always be the most important, or only compound causing the insect responses.

Spiro enol ether analogues and extracts from Chrysanthemum coronarium L. Spiro enol ether isolated from crown-daisy, Chrysanthemum coronarium L. (Asteraceae), has distinct bioactivity against many species of insect pests (Bohlmann and Fritz, 1979; Wu and Wang, 1994). Because of their bioactivity, a series of analogues of spiro enol ethers were synthesized by Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China (Figure 1). The two of the most promising compounds of spiro enol ether, named after the sequence as Nos. 12 and 20, have been extensively studied in China (Xu et al., 2000b, 2000c; Zhang et al., 2001a, 2004). Xu et al. (2000b) found that the LC50 values of compounds No.12 and No. 20 to 3rd instar S. litura were 945.25 and 1295.76 mg/ml, respectively. Xu et al. (2000c) also found that the LC50s of the compounds No. 12 and No. 20 against the 2nd instar P. xylostella were 498.7 (95% FL: 489.4–558.0) mg/ ml and 299.8 (273.8–325.7) mg/ml, respectively, 5 days after treatment, and that of compound No. 20 is more toxic to the larvae than No. 12. Zhang et al. (2004) found that compound No. 20 prolonged the hatching time of eggs of P. rapae, and the egg hatching rates 3 days after treatment were 49, 36, and 25% at concentrations of 200, 400, and 800 mg/ml, respectively. Zhang et al. (2001b) showed that both analogues of spiro enol ethers had repellent activity against 4th instar larvae of P. rapae, with AFC50 (the concentration causing 50% antifeedant effect) values at 398.88 mg/ml for No. 12 and 280.54 mg/ml for No. 20 24 h after application. The analogues also reduced the weight of larvae and pupae O2N

O O

O O

Compound No. 12

N

Compound No. 20

Fig. 1. Chemical structure of compounds No. 12 and No. 20 isolated from Chrysanthemum coronarium L. (Wu and Wang, 1994).


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of P. rapae and decreased the emergence of adults. Zhang et al. (2004) screened 19 spiro enol ether analogues against the larvae of P. rapae for antifeedant activity. They found that the antifeedant activities of compounds No. 20 and No.12 were higher compared with the other 17 analogues. In a no-choice test, AFC50 values of No. 20 and No.12 against the 3rd instar P. rapae were 226.93 and 370.00 mg/ml, whereas in a choice test against the 4th instars, the AFC50 values were 280.54 and 398.88 mg/ml, respectively, 24 h after the start of the treatment. Compound No. 20 protected cabbage leaves and controlled the larvae of P. rapae effectively. Xu et al. (2000b) determined the antifeedant effects of spiro enol ether’s 19 analogues against the larvae of S. litura. Among the 19 analogues, the highest antifeedant rate of 75.3% was recorded in compound No. 12, followed by 70.2% in compound No. 20. The AFC50 of No. 12 against 3rd instar larvae in preference tests and 4th instar larvae in nonpreference tests were 340.39 and 403.83 mg/ml, respectively. The 4th instar larvae injected with compounds No.12 and No. 20 at 10 mg/ larva, and 66.7 and 87.5% of these larvae died in 24 h, respectively. Meanwhile, 4th instars injected with the two compounds had significantly higher mortality than that of larvae injected with acetone+water (1:1) as controls. Xu et al. (2000b) also found that larvae at 7 days after treatment with compounds No. 12 and No. 20 were significantly smaller than those of the controls, especially in those treated with compound No. 12. Xu et al. (2000c) also found that compounds No. 12 and No. 20 exhibited significant oviposition deterrent activities and larval hatching of P. xylostella, and females oviposited 25.6 and 47.2% fewer eggs on the plants treated with compounds No. 12 and No. 20 at 1000 mg/ml, respectively.

Extracts from Daphne tangutica Maxim. Daphne tangutica Maxim. (Thymelaeaceae) is a local endemic plant, widely distributed in the Qinghai-Tibet plateau of China. Chen et al. (2000a, 2000b) and Xu (2000a) studied the insecticidal activities of the methanol, acetone, ethyl acetate, chloroform, benzene, and petroleum ether extracts from the air-dried, powdered plant tissues. These crude extracts exhibited strong antifeedant effects when applied to 3rd instar S. litura. The AFC50s of the 3rd instar S. litura were 294.77 (130.2–667.6), 111.69 (60.48–206.25), 108.27 (60.10196.65), 102.14 (52.97–195.97), 71.75 (31.65–162.63), and 65.6 (28.4–151.3) g dried powder/l, respectively. Of the extracts, the benzene extract possessed the highest insecticidal activity against the 5th instar P. rapae, followed by the chloroform extract. The petroleum ether extract at a concentration of 500 mg dried powder/ml exhibited 62.3% antifeedant effects. Further bioassay revealed strong stomach toxicity along with antifeedant activity of the extracts against the 5th instar P. rapae at the concentration of 100 mg dried powder/ ml. Moreover, the methanol extract of D. tangutica exhibited high antifeedant and stomach poison effects. Fraction 9 of silica-gel column chromatography proved to be the most active fraction among those partitioned from the methanol extract. Larvae fed on leaf discs treated with fraction 9 grew slowly, and their bodies were significantly smaller than those of the control. Histological study indicated that the midgut tissue of the treated larvae was destroyed and fat bodies atrophied. Preliminary

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physiological reaction studies showed that fraction 9 reduced the content of protein in haemolymph and inhibited the activity of esterase in the midgut (Xu et al., 2000a). Chen et al. (2000a, 2000b) also reported that among root bark, root xylem, stem bark, xylem in stem and leaf had strong antifeedant and stomach poison properties. Residues of different plant parts extracted with benzene were re-extracted with methanol; the leaf extracts showed the most insecticidal activity, implying that the content of active ingredients in the leaf is the highest. Wang et al. (2000) found that the alcohol extract from D. genkwa is a strong antifeedant to P. rapae. Perez-Izequierdo et al. (1992) and Perez-Izquierdo and Ocete (1994) conducted choice tests of ethanol extracts of dried leaves of D. gnidium and Anagyris foetida L. for their antifeedant activity against 5th instar S. littoralis. They found satisfactory antifeedant activity with doses of 30 and 40 mg/cm2 of both extracts. They also found that the ethanol extract of dried D. gnidium leaves exhibited significant antifeedant activity against other insects.

Rotenoids and extracts from Derris spp. The most common bioactive chemicals extracted from genus Derris (Fabaceae) and related genera are rotenoids, which exhibit strong insecticidal activities, including strong contact, fumigant, and stomach poison, and growth inhibition to numerous insect pests, including lepidopteran larvae and aphids (Chiu, 1993; Zeng et al., 2002). Rotenoids include rotenone and other chemicals, such as tephrosin, deguelin, and toxicarol. Besides Derris spp. [Derris elliptica (Wallich) Benth, D. chinesis (L.), and a few others], other plants that possess rotenone include Milletia pachycarpa Baker, Milletia reticulate L., Tephrosia vogelii Hook, and Lonchocarpus spp. (Chiu, 1993). It was found that rotenone inhibits the conversion of nutrients into energy at the cellular level (cellular respiration). After exposure to rotenone, insects quickly stop feeding, and death occurs several hours to a few days. It was believed that rotenone is acting between NADH dehydrogenase and coenzyme Q (Ware, 1994). When the enzymes were inhibited, the ATP level of the insect decreases, resulting in the deficiency of energy and consequently, the insect shows paralysis and slowly dies. Rotenone also inhibits the assembly of spindle body canaliculus and the formation of canaliculus of insects in vitro by reversible conjugation with canaliculus protein. It may change the components of protein in the integument of the lepidopteran larvae, and decrease the content of whole protein. It could also affect succinic acid, mannitol, and some other compounds. It could inhibit the epithelial–mesenchymal transition (EMT) of mitochondrial membranes, thus indirectly affecting the activity of Nicotinamide adenine dinucleotide (NADH) dehydrogenase (Bienen et al., 1991). Rotenone could inhibit 8090% of the reducing enzyme between NADH and CytC, and between NADH and coenzyme Q (Obungu et al., 1999). Elliptone, isolated from the roots of D. elliptica, is an active insecticide in the same chemical family as rotenone. Differences in toxicity, antifeedant properties, inhibition of growth and development, and oviposition deterrence between elliptone and rotenone were evaluated in P. rapae, P. xylostella, Brevicoryne brassicae L., and P. vittata (Zeng et al., 2002). Elliptone exhibited strong toxicity against P. xylostella larvae and P. vittata adults, but significantly lower toxicity against aphids and


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P. rapae larvae compared to rotenone (based on comparison of LC50). Both elliptone and rotenone displayed antifeedant activity. Elliptone, and to a lesser extent, rotenone, had some inhibitory effect on growth, development, and oviposition. Elliptone had a lesser inhibitory effect on NADH–ubiquinone oxidoreductase activity (50% inhibition, IC50, at 5.27 nmol/ml) than rotenone (IC50 at 2.58 nmol/ml), suggesting that the former is a less effective respiratory inhibitor than the latter. It is possible that the higher toxicity of elliptone in leaf-disc immersion trials reflects its lower antifeedant activity and higher ingestion rate. Xie et al. (2004) compared the antifeeding, contact poisoning, and growth-inhibiting activities of the crude extract of the calluses from young leaves and the crude extract of roots of D. elliptica against the 5th instar larvae P. rapae. They found that the AFC50 value at 1 and 2 days after treatment was 2.207 and 1.482 mg/ml, respectively, compared with 2.375 and 1.690 mg/ml from the crude extract of roots. The LD50s of the crude extract of the calluses by topical application was 0.283 mg/larva after 1 day while the crude extract of roots and the standard rotenone showed little toxicity to the larvae. The LC50s of the crude extract of the calluses were 2.050 mg/ml after 2 days, respectively, whereas those of the crude extract of roots and the standard rotenone were 1.239 and 1.190 mg/ml, respectively. They also found that 87.5% of pupae were manifestly inhibited by the crude extract of the calluses, compared with 82.5% by the standard rotenone and 72.5% by the crude extract of roots. Visetson and Milne (2001) used two types of ethanol extraction methods, Soxhlet and stirring soaking from the D. elliptica roots to determine their effects on 3rd instar larvae of P. xylostella. They determined the LD50 values of 24.25 and 89.07 mg/ ml for the Soxhlet and stirring methods, respectively, a 3.7-fold difference.

Rotenoids and extracts from Tephrosia vogelii (Hook f.) Tephrosia vogelii (Hook f.), a perennial shrub, is traditionally used for its ichthyotoxic and insecticidal properties. It originated from tropical Africa and is now widely distributed in Western and Eastern Africa, the Philippine, Cuba, Java, Puerto Rico, Malaysia, and the Southeast US. The purple and white varieties of T. vogelii have been introduced to China from the Philippine and Tanzania where the fast-growing plants with flourish branches and leaves produce high levels of legume. The chloroform extracts were isolated and purified by quick column chromatography, thinlayer chromatography (TLC), and liquid chromatography (LC), and the structures were identified by IR (infrared), MS, GC–MS, and nuclear magnetic resonance spectroscopy (NMR). The results reveal that the main active components of T. vogelii are rotenoids such as rotenone, rotenolone, and 5-, or 6-hydroxylrotenone (Zhang et al., 2000f). The crude extracts and the isolated bioactive ingredients of T. vogelii had strong antifeedant, growth-inhibition, and ovicidal activities against several lepidopteran species. Zhang et al. (2000e) evaluated the antifeedant properties of the acetone extracts of T. vogelii in both choice and no-choice bioassays involving four lepidopteran species, Piers rapae, Spodoptera litura, Mythimna separata Walker, and Plutella xylostella. In choice bioassays, the extract at 5 dilution reduced the feeding of 5th instar P. rapae larvae by 87.3%, which is similar to the antifeeding effect

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caused by 1000 mg toosendanin (12 alpha-acetoxyamoorastatin)/l (88.9%). The same extract dilution reduced feeding by 65.8 and 100.0% for the 5th instar S. litura and M. separata, respectively, and still caused 60.5% feeding reduction at the 200 dilution. In no-choice bioassays, the 100 dilution reduced feeding up to 94.2% for the 5th instars of P. rapae within 48 h, while the 10 dilution reduced feeding of 4th instar P. xylostella by 100%. In another study by Zhang et al. (2001c), the diluted acetone extracts of T. vogelii were applied topically on the dorsal shields of intermediate thoraxes of P. rapae larvae and by feeding P. rapae larvae with cabbage leaves treated with diluted acetone extracts. The results show that in the topical treatments of 100–250 mg/head, the 3–4 instar larvae had a mortality of 100%. Prepupae of P. rapae treated at 200 mg/head at early and late stages had a mortality of 96.1 and 52.4%, respectively, but there were no significant residual effects on their pupae. They also found that topical treatment at 20 mg/head resulted in a mortality of 7.9% to the larvae of S. litura and 8.3% to the larvae of M. separata. In feeding treatments, the growth and development of P. xylostella were inhibited significantly. In a study conducted to determine the effect of extracts from T. vogelii leaves and rotenone on the cuticle of P. rapae larvae and pupae, Xu et al. (2001, 2002) found that the larvae topically treated with acetone extracts from the leaves or rotenone caused malformations on the larvae and pupae. The 5th instars and pupae of P. rapae become malformed pupae exhibiting six different symptoms after treatment with topical application of acetone extracts of T. vogelii (Figure 2). When acetone extracts were diluted 50 to treat the earlier stage of the 5th instar larvae (1–8 h after ecdysis), 74.4% of larvae died, and the others turned into malformed pupae. It was found that the deformation caused by rotenone and its analogues mainly resulted from the influences on decomposition of the old cuticle and synthesis of new cuticle, and significant differences in content of the chitin, total fat, and total protein between the treatment and the check occurred.

Fig. 2. Variations of the malformed pupae of 5th-instar larvae of Pieris rapae after being treated by topical application with acetone extracts from leaves of Tephrosia vogelii (a–f) as compared with untreated control (ck).


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The acetone extract of T. vogelii also caused 65, 93, and 100% antifeedant effects to 4th instar P. xylostella at 0.01, 0.05, and 0.1 mg extract/l (0.114, 0.57, and 1.14 ml/ cm2), respectively, measured as cabbage leaf area eaten, compared with 71, 84, and 85% antifeedant to a 20% EC AZT-VR-K at the same concentrations, respectively (Xu unpublished data).

Toosendanin and extracts from Melia azedarach and M. toosendan Melia azedarach L. and M. taosendan Sieb. et Zucc. (Meliaceae) are widely distributed in China and elsewhere, and the former has been naturalized in the southeastern U.S. Both species contain toosendanin, the most important active ingredient of M. azedarach and M. toosendan, M. azedarach also contains a compound of two interchangeable isomers, the new isomer was called meliartenin. It possesses similar activity to that of azadirachtin and greater than the one showed by toosendanin (Carpinella et al., 2002), which was first isolated and identified in China and was mainly used as anthelmintic against ascariasis (Chiu, 1995; Wang et al., 1999). It possesses strong antifeedant activity at high concentrations against the larvae of Tryporyza incertulas (Walker), Spodoptera venalba, P. xylostella, and Ostrinia furnacalis (Guenee), and acts as a stomach poison at low concentration against larvae of T. incertulas and P. rapae, and it can also inhibit growth (Wang et al., 1992). Methanol extracts of the seed kernels of M. azedarach and M. toosendan, as well as toosendanin were very effective against S. litura as an antifeedant (Chiu, 1993). Fifth instar S. litura larvae were so sensitive to these materials that the AFC50 was found to be as low as 0.0027% in the leaf disc choice test. The 2nd instar larvae reduced feeding by 99.6% in choice tests when the leaves were treated with 1% methanol extract of M. azedarach. The acetone extracts of the leaves, bark, and stems of the two species of chinaberry also were effective as feeding deterrents at a concentration of 1–2% (weight of plant tissue/volume of the solvent). Both the methanol and ethanol extracts showed promising antifeedant activity against the larvae of P. rapae in both choice and no-choice tests. However, it was interesting to find that the larvae almost stopped eating either the treated or untreated leaves after feeding on the bioactive materials for several hours. The larvae were killed when treated with high concentrations of the plant extracts. Growth inhibition, oviposition deterrence, and egg-hatching disruption from treatments with lower concentrations were observed. Mehta et al. (2002) also found that petroleum ether extracts of M. azedarach had significant antifeedant activity on 3rd instar larvae of Pieris brassicae L. Experiments with radioactive isotopes indicated that 3H-toosendanin eaten by P. rapae was found in the blood and then transported to various organs. The phenomenon of antifeeding occurred only after 3H-toosendanin reached the blood. There seemed to be a correlation between the fluctuations of 3H-toosendanin in blood and the response of the insects (Zhang and Chiu, 1991, 1992; Ce´spedes et al., 2000). Zhang and Chiu (1991, 1992) found that by feeding or injecting 5th instars at the dosage of E1–3 mg toosendanin per P. rapae larva, pathological changes in histology could be observed, indicating that the midgut tissues were destroyed. The peritrophic membrane disappeared, so that the food in the gut contacted and damaged the ventriculus tissue.

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The results of field plot trials demonstrated that the vegetables were effectively protected from the injury of P. rapae larvae by spraying these plant extracts. Toosendanin at a concentration of 0.1% gave an effecticeness approximate to that of 0.01%. Therefore, a commercial formulation of toosendanin, 0.5% Toosedarin EC has been recently developed recently by The Botanical Pesticide Research and Development Center, Northwest A&F University at Yangling, Shaaxi, China. Wang et al. (1994) reported isolation of melianoninol, melianone, melianol, meliandiol, meliantriol, vanillic acid, vanillin, and toosendanin from the fruits of M. azedarach and their biological activities toward P. rapae. They found that the triterpenes melianone, melianol, meliandiol, meliantriol, and toosendanin possessed antifeedant activities against P. rapae larvae and only toosendanin showed considerable toxicity to the insect on consumption. The bio-activities of melianoninol, vanillic acid, and vanillin were much lower than the other compounds.

Parthenin, argentatins, and extracts from Parthenium spp. The potentials of extracts in the genus Parthenium (Asteraceae or Compositae), including P. argentatum Gray, P. hysterophorus L., P. schottii Greenman, and P. tomentosum DC. for pest management have been causing considerable attention in recent years. The extracts and chemical compounds from these plants that exhibit insecticidal activities include parthenin, argentatins, and many others. Parthenium argentatum Parthenium argentatum, or guayule, is a shrub containing high amounts of natural rubber. It grows wild in northern Mexico and southwestern US. The plant’s trichomes and pollen contain toxins called sesquiterpene lactones. The major component of these toxins are parthenin and other phenolic acids such as caffeic acid, vanillic acid, anisic acid, chlorogenic acid, parahydroxy benzoic acid, and p-anisic acid, which are lethal to humans and animals (Bauer, 1998). The de-rubberized resin from P. argentatum has been tested for potential as an ovicidal and feeding deterrent against S. exigua (Allan Showler, USDA-ARS, personal communication). Ce´spedes et al. (2001) evaluated argentatin A and B from P. argentatum for their effect on armyworm S. frugiperda (J.E. Smith) larvae. In nochoice diet bioassays with neonate larvae, argentatins A and B and methanol extract of P. argentatum significantly inhibited growth and increased development time. Also, a significant delay in time of pupation, adult emergence, and deformities was observed in most treated groups. Acute toxicity to adults of S. frugiperda was also found, with the methanol extract having an LD50 of 3.10 ppm. Methanol extract and argentatin A inhibited acetylcholinesterase by 93.7 and 90.0% at 5.0 and 50.0 ppm, respectively. Parthenium hysterophorus Although P. hysterophorus is one of the most undesirable weeds in the world (Aneja et al., 1991), its extracts have been studied for feasible botanical insecticides against cruciferous and other vegetable pests, including A. gossypii and L. erysimi, S. litura, and H. armigera.


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Patil et al. (1993) tested the cold alcohol extracts of P. hysterophorus against A. gossypii in the laboratory. Extracts of P. hysterophorus were more toxic against the aphids than the neem extracts. Sohal et al. (2002) tested the petroleum ether extracts of leaves, stems and inflorescence of P. hysterophorus at 500, 1000, 2000, and 5000 mg/ml concentrations in the laboratory for their toxic effects on the mean life span and progeny production of adults of the mustard aphid, L. erysimi. They found a significant decrease in life span and progeny production with all treatments. Among the three plant parts tested, the leaf extract showed the most significant effect in decreasing life span and progeny production in a dose-dependent manner. Gajendran and Gopalan (1981) evaluated the ovicidal activity of various extracts of P. hysterophorus against eggs of S. litura, and they found that the extracts of leaves of young plants, old leaves, inflorescence, stem, and roots applied topically all inhibited egg hatch; the leaf and inflorescence extracts completely prevented hatching when applied to eggs 0–24-h old, while the root and stem extracts inhibited 62.5 and 40.0% hatch, respectively, when applied at a rate of 50 mg/10 eggs. Eggs (48–72-h old) were least affected by low doses (2, 5, and 10 mg) of the extracts. In another laboratory study, Gajendran and Gopalan (1982) found the antifeedant activity of 3% extract from young leaves of P. hysterophorus against the 3rd instar larvae of S. litura could be as high as 55.58%. They also found that the extract from young leaves was superior to the other extracts (inflorescence, leaves, root, and stem from mature plants). Datta and Saxena (2001) studied 11 sesquiterpene lactone derivatives of parthenin from P. hysterophorus, including a pyrazoline adduct of parthenin, its cyclopropyl and propenyl derivatives, anhydroparthenin, a dihydro-deoxygenated product, a formate and its corresponding alcohol and acetate derivatives, a rearranged product, lactone, and hemiacetal. They found that all these derivatives, along with parthenin, had antifeedant action against 6th instar S. litura. They also found that pyrazoline adduct to be the most effective as an insecticide, with LC50 values after 24, 48, and 72 h of 96, 43, and 32 mg/l, respectively. Sundararajan (2002) tested the methanol extract of P. hysterophorus for its insecticidal activity against the fourth instar H. armigera by applying dipping method of the leaf extracts at various concentrations (0.25, 0.5, 1.0, 1.5, and 2.0%). The highest concentration (2%) caused >50% larval mortality 48 h after treatment. Sundararajan (2003) further evaluated the aqueous leaf extracts of P. hysterophorus and two other plant species for their biological activities against the larvae of H. armigera using a dipping method of the leaf extracts at 2, 4, 6, 8, and 10% concentrations. A larval mortality of more than 50% at higher concentrations (8 and 10%) was observed in all the extracts. A reduction in the rate of food consumption and growth was observed in the fourth instar larvae of H. armigera 48 h after treatment in all the extracts at 10% level. Parthenium schottii and P: tomentosum Isman and Rodriguez (1984) used artificial diets incorporating powdered foliage or foliar extracts of P. tomentosum or P. schottii and observed inhibited feeding, growth and dietary use by 3rd-instar larvae of S. exigua relative to larvae feeding on diets incorporating foliage extracts from P. argentatum or an F1 hybrid with P. tomentosum. A mixture of sesquiterpene lactones isolated from P. schottii added to an artificial diet

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inhibited feeding, but did not deter growth or dietary use by larvae. Foliage or foliar extracts of P. schottii added to diets also significantly inhibited growth and dietary use by 3rd-instar larvae of Heliothis zea (Boddie), although there was no reduction in consumption rate. In contrast, powdered inflorescences of P. schottii or extracts thereof, both rich in sesquiterpene lactones, deterred neither feeding nor growth of H. zea when added to artificial diets. Confertin, the principal sesquiterpene lactone of P. schottii foliage and inflorescences, had no effect on feeding or growth of 3rd-instar larvae, even at a concentration which strongly inhibits growth of neonate H. zea larvae. Chemical factors in P. schottii and P. tomentosum foliage appear to inhibit growth of both S. exigua and H. zea, although a contribution to that inhibition by sesquiterpene lactones is uncertain.

Rhodojaponin and extracts from Rhododendron molle G. Don Rhododendron molle G. Don (Ericaceae) is commonly known as yellow azalea, and is well known for its medical and insecticidal uses in China. Several isolates from R. molle have been identified and tested for insecticidal activities against insect pests (Hu et al., 1993; Zhong et al., 2000a, 2001a, 2001b, 2001c; Cheng et al., 2002). Cheng et al. (2000) found that rhodojaponin-III (R-III) was the main toxic component of R. molle and the content of R-III in flower, leaf, base, and stem was about 0.20, 0.14, 0.11, and 0.08%, respectively. Its ethanol extract (1%) of the flower, leaf, and base had antifeedant effects against the larvae of P. rapae and S. litura. The antifeedant rates of flower, leaf, and base to 5th instar P. rapae larvae were 92.8, 86.8, and 74.9%, respectively, and to 3rd instar S. litura were 89.9, 80.1, and 73.5%, respectively. However, the extract of the stems had antifeedant effects. R-III exhibited feeding deterrence against larvae and adults of L. sativae (Hu et al., 2000b), and 500 mg/ml of R-III provided 77.3 and 67.7% of feeding inhibition on 2nd and 3rd instars, respectively. The LC50 values were 208.65 and 300.62 mg/ml against the 2nd and the 3rd instars of L. sativae, respectively. The LC50 value of RIII against L. sativae adults was 159.07 mg/ml 24 h after treatment. Zhong et al. (2000b) determined the activities of R-III, ethyl acetate (EtOAc) extract of R. molle flower extracts as oviposition deterrents and ovicides against P. xylostella. Application of R-III at 0.5 g/l reduced oviposition by 77.5%, and egg eclosion rate by 80.1%, respectively, compared with 5 g EtOAc extract/l which resulted in 55.9% reduction of oviposition and 66.0% of egg mortality 72 h after treatment. Zhong et al. (2001a) determined the insecticidal activities of R-III against S. litura with different extractions. Rhododendron molle flowers were first extracted by methanol and the extract was partitioned with petroleum ether, dichloromethane, and EtOAc with two different extraction methods. The results showed that the EtOAc partitions had the highest activity among the four partitions. R-III isolated from EtOAc extract was the most active constituent compared with other fractions. The AFC50 of R-III against the 4th instar S. litura was 16.48 mg/ml, the LC50 was 16.42 mg/ larva, and the IC50 was 41.70 mg/ml. R-III also had significant antifeeding activity on S. litura (Cheng et al., 2002). The AFC50 values of R-III against the 5th instar S. litura were 16.93, 19.31, and 16.96 mg/ml 24, 48, and 72 h after treatment, respectively. Cheng et al. (2002) studied that antifeeding and toxicity of R-III to S. litura larvae. They found that the AFC50s of R-III against the 5th instar S. litura were 16.93,


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19.31, and 16.96 mg/ml 24, 48, and 72 h after treatment, respectively. After application of R-III at 100 mg/ml, the development of S. litura larvae was as long as 22.4 days, the larval mortality was as high as 39.7%, and the pupating rate was only 34.0%. The weight of the female pupa was reduced to 0.296 g/pupa. They also found that after application of R-III at 500 mg/ml on 3rd instar P. xylostella, the mortality was 53.3% at 4 days after treatment and the pupating rate was only 16.7%. Zhong et al. (2001b) determined the insect growth regulating properties of R-III and EtOAc extracts from R. molle flowers against P. rapae. The IC50 of R-III and EtOAc extract were 6.78 and 70.29 mg/ml against 3rd instars and 13.72 and 346.00 mg/ml against 5th instars, respectively. R-III and EtOAc extract also reduced pupating rate, pupal weight, emergence rate, and extended the duration of development. Thus, development of insects was inhibited significantly and development index decreased. These results also showed that the R-III was superior to toosendanin as an insect growth regulator. Hu et al. (2002) found that ingestion of R-III (1, 3, and 5 mg/dish) by the 5th instar larvae of P. rapae reduced the total haemolymph volume by 40–50%, protein content by 40–50%, carbohydrate content by 14–30%, and glyceride content by an average of 77%, compared with the controls. These results indicated that application of R-III inhibited larval growth and development. Besides R-III, 13 other compounds (numbered from 1 to 13) were isolated from R. molle (Zhong, 2002). Testing with P. rapae and P. xylostella, Zhong (2002) found that the 13 compounds had different insecticidal activities on the two pests: compounds 7, 8, and 10 possessed strong oviposition deterrent activities with 53–75% reduction of P. rapae eggs and 58 to 77% reduction of P. xylostella eggs; compounds 8 and 10 had high antifeeding activities of both P. rapae and P. xylostella; compounds 8, 10, and 12 had insect growth regulating properties (inhibiting larvae’s weight increase and growth, reducing the generation potency and affecting growth rate) on P. rapae; compounds 5 and 10 reduced pupating rate and emergence rate and sublethal effects on oviposition; compounds 4, 8, and 10 were strongly toxic to P. rapae larvae; compounds 5, 6, 7, 8, 9, 10 had strong stomach toxicity to 5th instar P. rapae; and compounds 7, 8, 10, and 11 significantly reduced egg-hatching rates of P. xylostella by 40–58%. To understand the mode of action and optimize rhodojaponin structure, Zhong et al. (2004) also determined the effects of 14 active compounds, previously isolated from R. molle on the cuticle components of S. litura larvae and their structure–activity relationship. The structures of three new compounds, rhodomolin A (R-A), B (R-B), and C (R-C), were elucidated based on IR, UV, NMR, and MS spectroscopic data. When the 4th instar larvae of S. litura were treated with 50 mg/l of R-I, R-III, R-A, R-B, R-C, R-XVIII, grayantoxin III, and azadirachtin, the weight of larvae and pupae and the emergence rate were significantly less than the control, exhibiting greater insect growth inhibition activities than the tested compounds from R. molle. When treated with R-I, R-III, R-A, R-B, R-C, R-XVIII, and azadirachtin, the contents of total larval cuticle protein significantly increased and those of total fats decreased than those of the control, respectively. Furthermore, the contents of water-soluble protein, hydrogen bond combined protein and covalent bond combined protein in the cuticle decreased significantly, while the content of weak bond combined protein increased significantly. However, the content of electrovalence bond

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combined protein was not significantly different from that of the control when larvae were treated with R-III and azadirachtin. They concluded that the mode of action of insect growth inhibition in rhodojaponins is not to inhibit chitin syntheses, but to disturb internal secretion, and significantly decreasing the content of water-soluble protein in cuticle. Their preliminary analysis of the structure–activity relationship showed that substitute group structures of C-2,3-epoxy, C-6, C-10, and C-14 were most important to these grayanoid diterpenids with insect growth-inhibition activity.

Alkaloids and extracts from Sophora alopecuroids L. Sophora alopecuroids L. (Papilionaceae) is known as kudouzi (means ‘‘bitter (ku) bean (dou) seed (zi)’’ in Chinese). It is a perennial leguminous plant with a wide distribution in northwest China. Sophora alpecuroids is also well known as having an abundance of alkaloids with a wide range of useful biological activities including insecticidal activity against some of vegetable pests (Luo, 1995; Luo and Li, 1996; Luo et al., 1996, 1997, 1999; Luo and Zhang, 2003). In the past few years, seven alkaloids (sophoramin, cytisin, matrine, oxymatrine, sophoradine, sloperine, and sophocarpine) were found in methanol and chloroform extracts from S. alopecuroids (Luo and Li, 1996). The major alkaloids in the methanol extracts include 4.29% of matrine, 6.32% of oxymatrine, 1.40% of sophocarpine, 4.29% of sophoramin, and 0.70% of cytisin. The chloroform extract had a slightly different amount of alkaloids, 4.50% of matrine, 8.56% of oxymatrine, 0.68% of sophocarpine, 2.70% of sophoramin, and 0.63% of cytisin. Luo et al. (1996) conducted bioassays of the two extracts from S. alopecuroids against L. erysimi. In the dipping bioassays, the LC50 values for the apterous adults of L. erysimi were 0.8 to 1.2 g/l for both extracts, and no significant difference was detected. In the spray bioassays, the LC50 values for L. erysimi nymphs were 0.7 to 1.1 g/l for both extracts, and, again, there were no significant differences between the two extracts. In another experiment, Luo et al. (1997, 1999) found that the LC50 values against L. erysimi for the three alkaloids, cytosine, anabasine, and nicotine (the latter two as controls), were 432.59, 648.70, and 1090.65 mg/ml, respectively, 48 h after treatment using the cage-dip method, and the results again indicate that cytisine is the most toxic alkaloid against the aphid. Luo et al. (1997) also tested the efficacies of the eight alkaloids from the two extracts of S. alopecuroids on apterous L. erysimi. Results indicated that cytisine was the most effective alkaloid with 96.7 and 100% mortality at 1000 and 2000 P(A)(mg/ml) 48 h after treatment, and the second most effective alkaloid is aloperine which gave much lower mortality, 43.3 and 45.0% at the two concentrations, respectively. All others had little activity on the aphids with 5.0–36.7% mortality. In a field trial on cabbage, Luo and Li (1996) found that the 50% methanol extract effectively reduced the population of L. erysimi when applied on cabbage plants at field rates. The controls were 64.8, 81.1, and 88.7% at 1.0, 5.0, and 10 g/l field rates, respectively, 3 days after application, compared with the untreated control. Similarly, the controls were 78.8, 83.3, and 89.1% at the three field rates, respectively, 5 days after application, compared with the untreated control. At 7 days after application,


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percentages of control were similar to those 6 days after application, ranging from 36.4 to 84.1%. In another field study on cabbage plants, Luo and Li (1996) also found that the methanol extract at 1.0, 5.0, and 10 g/l concentrations exhibited significant repellent activity. Percentages of population reduction were 53.8, 78.1 and 74.2 at 1.0, 5.0 and 10 g/l concentrations, respectively, 3 days after application compared with the untreated control. Similarly, the percentages of reduction were 73.5, 64.5, and 74.3 at 0.05, 0.10, and 0.20% 6 days after application, compared with untreated control. At 9 days after application, percentages of population reductions were similar to those at 6 days after application, ranging from 64.5 to 74.2%. The alkaloids from the extracts from S. alopecuroids exhibited significant synergistic activities when mixed with three commonly used insecticides, profenofos, abamectin, and methomyl (Zhang and Luo, 2002). Abemectin exhibited greatest synergistic effects when it was mixed with cytosine, sophoradine, aloperine, and oxymatrin, followed by matrine, and no synergistic effects when it was mixed with sophocarpine or sophoramine. Methomyl displayed synergistic effects when it was mixed with all alkaloids, although the synergism was not strong when it was mixed with matrine. Profenofos showed distinct synergistic effects when it was mixed with sophoradine, oxymatrine, sophoramine, or cytisine, and lower synergism when it was mixed with aloperine, matrine, or sophocarpine. Luo and Zhang (2003) studied the effects of the seven alkaloids from S. alopecuroids on metabolic esterases of the larvae of P. xylostella. Their results indicate that cytisine and aloperine could inhibit carboxylesterase activity through noncompetitive inhibition. Sophoramine, sophoradine, matrine, oxymatrine, and cytisine could inhibit the activity of acid phosphoresterase, and cytisine could also weakly inhibit the activity of alkaline phosphoresterase. In addition, three alkaloids, cytisine, sophoramine, and sophocarpine could inhibit the activity of glutathione-Stransferase [glutathione transferase] in P. xylostella larvae.

Extracts from Stellera chamaejasme L. Stellera chamaejasme L. (Chinese Starwort or Chinese stellera) is a poisonous plant and is reputed to have medicinal and insecticidal value. It is also a repellant and contact-poison, possibly acting as a stomach poison (Ji et al., 2000; Zhang et al., 2000a, 2000b, 2000c, 2000d, 2002). The antifeedant rate to 5th instar P. rapae was 56.07% by topical application 24 h after at the dosage of 25 mg/larva (Zhang et al., 2000a). Furthermore, the root extract of S. chamaejasme with ethanol also exhibited oviposition deterrent activity and ovicidal activity against P. rapae (Zhang et al., 2000c). Zhang et al. (2000d, 2002) determined the insecticidal activities of the ethanol extract of root of S. chameajasme against P. rapae and Myzus persicae, and they found that the LD50 for the 5th instar P. rapae by stomach poison using the leafdisc sandwich method was 12.32 mg/larva at 2 days after treatment. Leaf dipping in 5 mg/ml extract, the corrected mortality for the 5th instar P. rapae was 100% at 7 days after treatment, and the value of AFC50 for the same larvae was 0.3813 mg/ml. Numerous chemicals have been isolated from the plant (Su et al., 2003), and of these, 7-hydro-coumarin, daphnoritin, and chamaechromone have strong antifeedant

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and growth inhibition activities to 5th instar P. rapae (Wang et al., 2002). The AFC50 of daphnoritin and chamaechromone to the 5th instar P. rapae were 160.57 mg/ml and 229.49 mg/ml 24 h after the treatment and 76.94 mg/ml and 131.30 mg/ml 48 h after the treatment, respectively (Zhang et al., 2000b). Physio-biochemical studies revealed that daphnoritin increased the content of sugar, while it decreased the content of protein in haemolymph. In contrast, chamaechromone decreased the content of sugar and increased the esterase activity in midgut significantly (Zhang et al., 2000d). Wang et al. (2002) studied the biological activity of an ethanol extract from the root of S. chamaejasme collected from Qinghai, China against, P. rapae, P. xylostella, O. furnacalis, S. litura, and M. persicae. Applied at 2.5, 5.0, and 50.0 mg/ml of the ethanol extract, the mortality of the 5th instar P. rapae, the 3rd instar P. xylostella, and the 3rd instar S. litura was 100, 31.0, and 16.7% 7 days after treatment, respectively. The LC50 of the ethanol extract against M. persicae was 0.599 2 mg/ml 2 days after treatment using a leafdipped method. With the ethanol extract at 10 mg/ml for the 3rd instar O. furnacalis, the mortality was 65.5 and 85.7% 7 and 14 days after treatments, respectively. These results showed that the ethanol extract had strong biological activity against P. rapae, O. furnacalis, and M. persicae, whereas it had weak biological activities against S. litura and P. xylostella.

Extracts from Strophanthus divaricatus (Lour.) Hooker & Arnnott Strophanthus divaricatus (Lour.) Hooker & Arnnott (Apocynaceae) [(called ‘‘yang jiao niu’’ in Chinese (goat horn)], is widely distributed in south China. The water extract of S. divaricatus showed contact poison to insects including Coptosoma cribraria ( ¼ cribrarium) (Fabricius) and Tryporyza incertulas (Walker). The extracts of fruit, leaves, and stems of S. divaricatus have strong antifeedant and insecticidal properties on the 4th instar P. rapae. The main secondary metabolites in this plant are caudoside, F-caudostroside, divaricoside, sinlside, isosinotrodise, sarmutodise, and D-strophanthin. Hu et al. (2000a) found that the LC50 and LC95 values of D-strophanthin isolated from this plant were 32.0469 and 379.7181 mg/ml, respectively, against the 4th instar P. rapae. Results from filed trials showed that the commercial product, 0.05% strophanthin was useful for controlling P. rapae. Huang and Renwick (1994) studied the oviposition responses of P. rapae and P. napi oleracea to 18 cardenolides. They found that most of the compounds were deterrent to oviposition by both insects, but to significantly different degrees. P. rapae was strongly deterred by K-strophanthoside, K-strophanthin- beta, cymarin, convallatoxin, oleandrin, erysimoside, erychroside, and gitoxigenin. The most deterrent compounds for P. napi oleracea were erychroside, cymarin, erysimoside, convallatoxin, and K-strophanthoside. They also found that strophanthidinbased glycosides were more of a deterrent than digitoxigenin-based ones, and the number and type of sugar substitutions had profound effects on activity. Cymarin was equally acting as a feeding deterrent to both pest species at all concentrations tested. However, when compared with P. rapae, P. napi oleracea was less sensitive to most cardenolides. P. napi oleracea was insensitive to K-strophanthin-beta and oleandrin at 0.5 104 M, which were an excellent feeding deterrent to P. rapae.


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Extracts from Tripterygium wilfordii Hook. F. and T. hypoglaucum (level) Hutch. Tripterygium wilfordii Hook. F. and T. hypoglaucum (level) Hutch. (Celastraceae) are widely distributed in China and have been used as traditional botanical insecticides and medicinal plants because they contain numerous bioactive chemicals (Yang et al., 2000). Tong and Chiu (1988) determined the toxicity of the extracts of T. wilfordii and T. hypoglaucum against P. rapae larvae in the laboratory and under field conditions. They found that the extracts from the root bark of the two plant species have strong antifeedant, stomach poisons, and growth inhibition properties. Larvae fed on the extracts were paralyzed, and the mid-gut epithelium of their alimentary canal was affected. The larvae finally died of starvation. Tong and Chiu (1988) also found that both alkaloids and nonalkaloids from T. hypoglaucum had high antifeedant activities against the 5th instar P. rapae, and the activities are in the following order: wilforine>total alkaloids>nonalkaloids. The activities of alkaloids from T. wilfordii are in the order: wilfordine>wilforine>wilfortrine. After feeding with extracts from T. wilfordii or wilforine, black spots appeared on the body of larvae (Tong and Chiu, 1988). Microscopic examination of the black area of the larval cuticle shows that the cuticle became thickened and tumors appeared between the epidermis and endocuticle. The epidermis was destroyed, the endocuticle separated from the epidermis and vacuoles formed. Wilforine affected the respiration of the larvae of P. rapae and their body weight was decreased. After eating cabbage leaves treated with wilforine, the nervous system of the larvae was poisoned and showed paralysis; their respiratory rhythm-waves became lower and the CO2 output decreased. The larvae treated with wilforine and those fully starved showed the same change trends in body weight and respiratory rates. It was found that the alimentary canal and the fat bodies were shriveled, and the epithelium of mid-gut and the peritrophic membrane were destroyed. Luo et al. (2004) studied the ethanol extract of the root bark of T. wilfordii and found it had strong insecticidal activities against larvae of M. separata. Three active compounds were isolated by bioassay-guided fractionation of the extract and characterized as triptolide, triptonide, and euonine by IR, 1 H, and 13C NMR and mass spectral analysis. They found that triptolide and triptonide had strong contact activities against 3rd or 5th instar M. separata (LD50 values: 1.6 and 2.9 mg/larva for triptolide and triptonide, respectively), but euonine had no contact activity against the larvae. Antifeedant activity against the 3rd instar M. separata 24 h after treatment was demonstrated; triptolide, triptonide, and euonine gave EC50 (the concentration that produces a level of effect that equals to 50% of the maximum effect) values of 0.25, 0.35, and 0.02 mM, respectively. Activities of triptolide and triptonide were inferior to the positive control represented by toosendanin, whereas euonine was superior to toosendanin. In ingestion bioassays against M. separata, triptolide had the more potent activity with a KD50 (the time taken to knockdown 50% of insects) value of 13.5 mg/g (insect body weight) than toosendanin. Field trials demonstrated that cabbage was well protected from the damage of the larvae of P. rapae after application of 3% ethanol extract from the root bark of T. wilfordii, and the natural enemies of the larvae were not affected (Chiu, 1987; Tong and Chiu, 1988).

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Extracts from Xanthium sibiricum Patrin ex Widder Xanthium sibiricum Patrin ex Widder (Asteraceae) have been used to control some insects for over a thousand years, and its extract was recorded as an effective pesticide against P. rapae in ‘‘Chinese Indigenous Pesticides’’ in 1959 (Peng, 1984). Recent studies showed that it contained many chemicals, such as xanthostrumarin, carboxyatractyloside, xanthatin, atractyloside, xanthin, xanthunin, xanthanodiene, strumaroside, etc. Bioassays indicated that xanthatin and atractyloside exhibited potent stomach-poison and growth inhibition activities as well as antifeedant activity against many insects. He et al. (unpublished data) found that the LD50 of xanthatin against the 5th instar P. rapae was 2.07 mg/larva 24 h after treatment. When treated at the dosage of 50 mg xanthatin/ml, larval pupation density of P. rapae was reduced by 73.3% 4 days after application treatment compared with untreated control. They also studied the mode of action of xanthatin against insects, and found that the contents of sugar and protein in haemolymph of P. rapae larvae decreased significantly, and the activity of esterase in the mid-gut was significantly inhibited. In addition, Zhou et al. (2002) found that the semiochemicals extracted from X. sibiricum deterred feeding and reduced reproduction of M. persicae and L. erysimi. The deterrence rates were 48.1 and 55.7% to M. persicae and L. erysimi, respectively. They also found that the reproduction of the females was reduced by 39.0 and 63.5% for to M. persicae and L. erysimi, respectively. Wang et al. (1999) studied 12 plant species collected from Jiangsu Province, China and tested their extracts for their insecticidal properties against P. rapae. They found that the extracts of X. sibiricum inhibited larval growth and was a stomach poison, but did not act as an antifeedant.

Others Besides the insecticidal plants mentioned above, there is a large number of Chinese medicinal plants that seem to have insecticidal properties. Preliminary studies showed that the following 23 species of plants possessed high antifeedant rates against either P. xylostella or S. litura (Table 2) (Chen and Xu unpublished data). All 23 plant species were extracted with a mixture of methanol, acetone and chloroform (23:30:47) except Osmunda japonica Thunb., Aconitum hemsleyanum Pritz, Picrasma quassioides (D. Don) Benn.(root), Sapium sebiferum (L.) Roxb (stem), Davidia involucrate Baill. var. vilmoriniana (Dode) Hemsl., Pharbitis purpurea (L.) Voigt and Emmenopterys henryi Oliv. (stem), which were extracted with petroleum ether. The results indicate that most of tested plant extracts have strong antifeedant activities on P. xylostella and S. litura with a few exceptions. These results also suggested that more studies are needed to test those with promising insecticidal activities.

Final remark: the pros and cons of botanical insecticides Botanically-derived insecticides have gained favor in recent years, due in part to the perception that, because they originate from plant material, and are considered more safe or ‘‘natural.’’


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Table 2 The antifeedant effect of extracts of selected plants against Plutella xylostella and Spodoptera litura Plants

Antifeedant rate (%)a Plutella xylostella Spodoptera litura

Aconitum hemsleyanum Pritz Aesculus wilsonii Rehd. (branches and leaves) Aesculus wilsonii Rehd. (root bark) Artemisia shennongjiaensis Ling et Y. R. Ling Caesalpinia sepiaria Roxb. (leaves) Caesalpinia sepiaria Roxb. (root) Cephalotaxus fortunei Hook. f. Cephalotaxus sinensis (Rehd. et Wils.) Li (root) Cephalotaxus sinensis (Rehd. et Wils.) Li (stem) Davidia involucrate Baill. var. vilmoriniana (Dode) Dendrathema indicum (L.) Des Monl. var. aromaticum Emmenopterys henryi Oliv. (stem) Liriodendron chinese (Hemsl.) Sarg. (stem) Lycoris aurea (L’Herit) Herb. Matteuccia intermedia C. Chr. (root) Matteuccia intermedia C. Chr. (stem and leaves) Matteuccia orientalis (Hook.) Trev. (root) Osmunda japonica Thunb. Paeonia papaveracea Andr. Periploca sepium Bunge (branches and leaves) Periploca sepium Bunge (root) Pharbitis purpurea (L.) Voigt Picrasma quassioides (D.Don) Benn. (root) Sapium sebiferum (L.) Roxb (stem) Sophora viciifolia Hance (root bark) Sophora viciifolia Hance (seed) Stylophorum lasiocarpum (Oliv.) Fedde Torreya fargesii Franch. (stem bark) Zanthoxylum armatum DC. (branches and leaves) Zanthoxylum armatum DC. (root)

43.59 82.61 92.71 79.66 81.93 88.92 100.00 100.00 97.08 80.06 89.11 80.09 91.80 94.09 83.33 76.59 97.77 2.11 92.18 91.26 100.00 80.59 77.85 77.16 75.43 94.14 89.16 75.21 94.36 98.09

83.10 0.05 16.65 19.30 18.25 35.55 87.48 98.57 91.18 12.73 – 62.80 78.54 – 21.26 72.68 39.98 85.76 20.64 60.24 88.26 26.30 73.65 80.89 83.33 72.39 31.48 73.85 82.36 88.84

a

Antifeedant rate was calculated as: antifeedant rate (%) ¼ (leaf area consumed by the insects in the control leaf area consumed by the insects in the treatment)/leaf area consumed by the insects in the treatment 100.

Botanical insecticides have many advantages and disadvantages. Generally, botanical insecticides do not persist in the environment and are quickly degraded by temperature, ultraviolet light, rainfall, and other environmental factors. Synthetic chemical insecticides are needed because they are not always as effective as other synthetic insecticides, and repeated applications may be needed to achieve the desired result. Because some botanical insecticides are insect growth regulators, they are only effective against the immature stages of insects. They normally do not exhibit an immediate knockdown effect and the insect pests might continue to feed.

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However, due to their repellent effects, insect feeding may be reduced. The presence of some insects on the plants may hide the real effectiveness of some botanical insecticides. For instance, Walter (1999) observed that numbers of lepidopteran larvae (P. xylostella, T. ni, and S. exigua) on broccoli treated with Neemix in a field trial were not significantly different from those on untreated plants. However, the damage caused to the plants was significantly reduced in the treated plants. In addition, most botanical insecticides or plant extracts are compatible with other insecticides, and are useful in integrated pest-management programs. Some botanical extracts have significant synergism when combined with other insecticides (Elzen and James, 2002; Zhang and Luo, 2002 ). Botanical insecticides or extracts, however, also possess many disadvantages, including rapid breakdown, being toxic to fish, bees, or other animals, being generally more expensive than synthetic insecticides for effective pest controls, and generally lacking in sufficient and steady supplies for large-scale commercial applications. Also, the potency of some botanicals can differ from one source or batch to the next. For example, we recently found that great variation in activity of two batches of Neemixs 4.5 (Certis, Columbia, Maryland, USA) on P. xylostella; batch A (Lot 00104563 H) had stronger adverse effects on the development and survival of P. xylostella larvae but little repellent effect on the adults, whereas, batch B (Lot NX220-1 H) had a weaker effect on the larvae but a substantial repellent effect on the adults (Liu and Liu, 2005). Although simple crude extracts from plants have been used as insecticides, they have not played a major role in pest control. Both the lack of knowledge of their mechanisms of action and the relatively laborious methods needed to prepare these natural pesticides has mitigated their widespread use. At present, only a limited numbers of botanical insecticides are commercially available for vegetable growers worldwide, and most are neem- or azadirachtin-based. Large-scale production at competitive prices is not currently feasible for most commercial products, but it is likely that major breakthroughs in botanical pesticide technology will accelerate the use of botanical pesticides for fresh vegetable production. Nevertheless, like other biopesticides, botanical insecticides might not completely replace synthetic chemical insecticides. As anticipated by Hall and Menn (1999), the botanical pesticide market will grow at an annual rate of 10% as compared with 1–2% increase for synthetic chemical insecticides. We anticipate that botanical and biopesticides will be used at least as supplements to synthetic insecticides or as part of pesticide rotations to retard the onset of resistance.

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CHAPTER 9

The potential for using neem (Azadirachta indica A. Juss) extracts for pine weevil management in temperate forestry JONATHAN RM THACKER, WENDY J BRYAN, ROBIN HC STRANG, STUART HERITAGE

Introduction There is now a substantial volume of scientific literature which reports that extracts from Azadirachta spp. may have a role to play in the management and/or control of a diversity of coleopterous pest species. These data are primarily associated with bioassays of extracts from Azadirachta indica (Table 1); however, there are also reports of active extracts from the related Malaysian tree species, colloquially known as setang, Azadirachta excelsa (Schmutterer and Doll, 1993; Ng et al., 2003) and from the related species the chinaberry tree, Melia azedarach (Ventura and Ito, 2000). In this chapter however we concentrate on extracts from the neem tree. In particular, we consider recent data that have been collected with respect to one particular coleopterous pest, viz. the most important pest of forestry within the UK, the large pine weevil Hylobius abietis (L.). At present, this pest species is believed to cause economic losses of approximately £10 million per annum in the UK alone (Anon., 2000a). These are losses that are likely to escalate as the area devoted to reforestation within the UK is predicted to increase over the next decade. For the time being, damage by weevils is managed and/or controlled in the UK with synthetic pyrethroid insecticides. However, for a variety of reasons (that are described in this chapter) it seems prudent to assay possible alternative chemical control measures. We begin this chapter with an introduction to UK forestry and the pest species that threaten its commercial success. The bulk of the review is then given over to the data we have collected to date both from laboratory experiments and from field trials that were designed to assay the utility of neem-based extracts for pine weevil management. We conclude with some speculative comments concerning the potential of neem-based insecticides for weevil pest management within UK forestry. In our conclusion, we attempt to draw together some of the economic aspects that are associated with timber management in the UK and some of the aspects that are associated with the development and registration of plant protection products in general.

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Table 1 Recent papers that have assayed the utility of neem extracts for the control of Coleopterous pests. All of the papers cited produced positive resultsa Pest species assayed

Citation

Pine weevil (Hylobius abietis) Root weevil (Diaprepes abbreviatus) Colorado Potato beetle (Leptinotarsa decemlineata) Japanese beetle (Popillia japońica) Lesser grain borer (Rhyzopertha dominica) White pine weevil (Pissodes strobi) Bark beetle (Dendroctonus ponderosae) Pine engraver (Ips pini) Australian Carpet beetle (Anthrenocerus australis) Bean weevil (Sitona lineatus) Larger grain borer (Prostephanus truncatus) Pulse beetle (Callosobruchus chinensis)

Thacker et al. (2003) Weathersbee and Tang (2002)

a

Zabel et al. (2002) Held et al. (2001) Muda and Cribb (1999) Helson et al. (1999) Naumann and Rankin (1999) Duthie-Holt et al. (1999) Gerard and Ruf (1995) Smart et al. (1994) Niber (1994) Khaire et al. (1992)

Data collated from Science Citation Index. Papers are in chronological order.

UK forestry In 2002, there were 2.8 million hectares of afforested land within the UK, a figure that represents approximately 12% of the UK land area (Anon., 2002a). Approximately 50% of this land area is planted with coniferous species, most notably Sitka spruce Picea sitchensis (Bongard) (Anon., 2002b). Figure 1 shows the breakdown of tree species that are planted within Scotland, both by area and as a percentage of the total land area. Overall, the Forestry Commission (UK) predicts that this area is expected to increase during the next decade (Anon., 2003). The range of pest species that are regarded as key pests of forestry within the UK is shown in Table 2. Of these pests, only H. abietis is considered a serious threat. Outbreaks of Panolis flammea and Dendroctonus micans have occurred, but these have been locally restricted and dealt with rapidly, often by the destruction of infected material. The most recent outbreak of D. micans occurred in Northern England in 2002 however, at the time of writing the results of more extensive monitoring (by the Forestry Commission UK) that had been carried out for this pest were not yet available (Anon., 2003). The species H. abietis, however, is not only the single most important threat to restocking sites within the UK, but it is also regarded as the most important pest of forestry throughout Northern Europe. A more detailed description of the biology and pest status of this species is provided in the next section of this chapter.

The pine weevil Hylobius abietis – life cycle and pest status H. abietis (Coleoptera: Curculionidae) or the large pine weevil is a widespread Palearctic forest insect. It is found throughout the UK, Scandinavia, mainland Europe and Asia (Langstrom, 1982), and can cause extensive damage to conifer seedlings throughout its range (Tilles et al., 1986a). The UK average for losses of unprotected

The potential for using neem extracts for pine weevil management

600

60.00

500

50.00

400

40.00

300

30.00

200

20.00

100 Corsican pine

Douglas fir

Mixed conifer

Norway spruce

Scots pine

Sitka spruce

0.00

Larch

10.00

0

hectares planted (000's)

hectares (thousands)

70.00

Lodgepole pine

percent of total

percentage

201

tree species

Fig. 1. Dominant tree species planted in Scotland. The total area planted with conifers is 916,000 ha or 72% of the total woodland area in Scotland. Scottish woodland accounts for approximately 50% of all woodland trees planted within mainland UK. Overall, woodland accounts for approximately 12% of the UK land area. Source: Data collated from the Forestry Commission UK Forestry Statistics 2003 (Anon., 2003, see reference list for more details). Table 2 Key pests of UK forestrya Common name

Latin name

Damage status

Pine weevil

Hylobius abietis

Pine beauty moth

P. flammea

Spruce bark beetle

Dendroctonus micans

Polyphagous pest of conifer and broadleaf trees throughout Northern Europe. Key pest of transplant or naturally regenerating trees. Unprotected trees in the UK suffer losses of approximately 50%. Damage can be catastrophic (100% seedling mortality) in some areas. Native to, but not damaging to, Scots pine. Caterpillars can however defoliate lodgepole pine. Appears to have outbreaks in Northern Scotland every 6 to 7 years. Eurasian beetle discovered in the UK in 1982. Tree mortality generally takes a number of years to occur, primarily as a result of larval feeding in galleries.

a

Data collated from Forestry Commission UK booklet entitled Reducing pesticide use in forestry (Willoughby et al., 2004), see reference list for further details.

seedling trees is estimated to be 50% (Heritage and Moore, 2001). However, on some restocking sites with large weevil populations seedling trees can experience catastrophic failure, with losses reaching 100%. It is one of the largest weevils found in Britain with the adults ranging in length from 10 to 20 mm.Typically, adults will live for up to 4 years. Older weevils are

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Fig. 2. Feeding damage caused by the large pine weevil Hylobius abietis. Source: Reproduced with permission from the Forestry Commission UK.

pitchy-black in colour with longitudinal ridges and furrows bearing oblong indentations, whereas young weevils are coloured purple-brown. A photograph of H. abietis feeding is provided in Figure 2. H. abietis recognises breeding sites primarily as a result of its olfactory orientation towards the volatile monoterpenes that are released from their host tree stumps, mainly pine and spruce, in clearfell areas (Nordlander et al., 1986). The exact combination of chemicals and their precise mode of action are unknown. It is known that the main cortical monoterpene hydrocarbon components of Scots pine and Norway spruce are a-pinene, b-pinene, 3-carene, myrcene, limonene and b-phellandrene (Nordlander, 1990). Behavioural studies in the laboratory (Mustaparta, 1975) and in the field (Tilles et al., 1986a) have shown that H. abietis can use a-pinene to recognise its breeding substrate, with the presence of ethanol signifying host deterioration. As a result it has now been clearly established that when the two compounds are combined, the attraction to host trees is synergised (Nordlander et al., 1986; Tilles et al., 1986b; Nordlander, 1990; Nordenhem and Eidmann, 1991). Both sexes respond similarly to host volatiles and males are as likely as females to be the first individual locating an underground odour source. Clear felling can happen at any time throughout the year and although mass migration of weevils is known to occur in the spring (Solbreck, 1980; Orlander et al., 1997), adult weevils may invade adjacent fresh clearfell sites at any time during their active period. It is known that outwith the period of mass migration, the beetles will typically not move further than ca. 20 m (S. Heritage, personal communication). At clearfell sites both sexes mate repeatedly prior to oviposition by females. It was initially believed that eggs were inserted by ovipositing females into niches chewed in


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the inner bark of underground parts of freshly killed coniferous trees (Scott and King, 1974), with most oviposition in the stump occuring in the upper regions just under (0–10 cm) the soil surface, with little or no oviposition below 40 cm (Pye and Claesson, 1981). However, it has subsequently been found under laboratory conditions that eggs are also readily laid in sand surrounding host material (Nordenhem and Nordlander, 1994). This work has since been reinforced by a field study that later showed that eggs are predominantly deposited in the soil near the stump or its major roots (Nordlander et al., 1997). The results of this research indicated that H. abietis eggs are placed in the bark of roots only when the surrounding material is too dry or is likely to dry out. Otherwise, the eggs are primarily deposited in the adjacent soil. After hatching, larvae tunnel downward between the bark and timber of the breeding material (Scott and King, 1974) to depths from 20 to 50 cm below the soil surface where they continue to develop (Nordenhem and Nordlander, 1994). Larval stages then develop as subcortical feeders on the inner cambial zone (Nordenhem and Nordlander, 1994) during which they construct larval tunnels, which run with the grain of the wood, usually away from the stem (Pye and Claesson, 1981). When the final larval instar is reached, temperature becomes critical. At this point, larvae are cream-coloured with a light-brown head and measuring 15–20 mm in length (Scott and King, 1974). If the temperature is high enough, larvae pupate between mid-June and late July (Nordenhem, 1989); if not, larvae enter a period of diapause until the following spring when they resume their development as the temperature rises. In laboratory experiments, temperatures between 10 and 20 1C can produce diapause in the last larval instar lasting 60–220 days. Temperature is important in deciding the length of the life cycle with the critical temperature governing this process appearing to be between 20 and 25 1C. When pupation occurs, larvae construct pupal chambers that are plugged with small strips of wood fibre (Scott and King, 1974). Depending on the thickness of bark the chamber may be located within the bark itself or at the sapwood and bark interface. Pupae metamorphose into the immature adults. New adults usually emerge around 14 days after pupation or alternatively, depending on the temperature they may overwinter as adults within pupal chambers prior to their emergence in early spring (Munro, 1928; Nordlander, 1987). At 20 1C, the overall developmental time from egg to virgin adult is 8–9 months (Selander et al., 1976), while at lower temperatures (15 1C) development time is longer at 1 and a half to 3 years or more. In the UK, completion of a life cycle generally takes from 1 to 2 years, though the rate of development from egg to emergent adult largely depends on prevailing climatic conditions and the exact time of year that the eggs are laid (Scott and King, 1974). In the UK, the length of generation between eggs is estimated at 2–4 years. Adults have been observed to leave hibernation sites early in spring at temperatures exceeding 8–9 1C, whereas new generation adults typically have an extended period of emergence from late April until the end of June (Nordenhem, 1989). It has been estimated that 5 months must elapse before ovaries mature so that a female weevil appearing in March or April cannot reproduce until September or October (Munro, 1914). In laboratory experiments, newly emerged male weevils have also been reported to start copulation after a similar period of 4–5 months (Selander, 1978). Following emergence weevils begin to feed almost immediately (Guslitz, 1969). Feeding itself is part of the process of maturation and is typically a

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prerequisite for the successful development of flight muscles and reproductive organs (Nordenhem, 1989). Overall, the population dynamics of H. abietis are still not entirely clear, and there may be marked differences between populations in the UK and Scandinavia, but it is believed that adults arriving at a fresh clear-cutting will remain on the clear-cut site for the summer and overwinter in the soil until the following year (Langstrom, 1982; Nordenhem, 1989). In contrast, new generation adults emerging from pupal chambers in the autumn feed for about a month before overwintering while their flight muscles develop. Thereafter, they fly away to find alternative suitable areas for breeding. At any given clear-cut area there will therefore be a mixed population of weevils consisting of new arrivals and newly emerged individuals. At any given site, it has been estimated that significant numbers of adults can emerge from a single stump and its associated root system, resulting in populations ranging from 150,000 adults per hectare (Heritage, 1997) up to 220,000 adults per hectare (Leather et al., 1995). Higher weevil populations are often found in dry pine-dominated forest sites compared with spruce-dominated areas (Langstrom, 1982). In Scandinavia, weevil abundance per hectare tends to decrease from south to north (Langstrom, 1982) due to the climate; whereas in Britain this appears to be reversed as weevils are much more frequent in pine-dominated areas in the northern regions. A schematic diagram of the complete life cycle of H. abietis is provided in Figure 3. It is feeding by adults that destroys seedling trees. Adults will feed on the living bark of most woody or herbaceous plants but they prefer the bark of young conifers and broadleaves (Heritage and Moore, 2001) particularly pine (Leather et al., 1994). Although olfactory orientation is less well-documented for feeding, it is known that the compounds that are present in the seedling act as attractants to weevils (Nordlander et al., 1986). Both male and female adults feed on conifers before, during and after the breeding period, though there tends to be two peak periods of damage in the UK. The first peak may start in early April, whilst the second peak can occur at any time between August and November (Heritage et al., 1997). The first of these peaks, in early spring, corresponds with the emergence of hibernating weevils and an influx of migrating weevils (Orlander et al., 2000). The autumn feeding peak by contrast corresponds with weevils that have newly emerged from pupal chambers (Nordlander, 1987). A period of intense feeding prior to winter hibernation ensures that females enter that stage of their life cycle with replenished fat reserves. Typically, feeding occurs at night and is often concentrated on the lower part of the tree’s main stem. Feeding damage scars the phloem of the seedlings, and girdling at the stem base can rapidly kill a tree (Von Sydow, 1997; Orlander and Nilsson, 1999). Although a seedling can survive a large amount of feeding damage as long as it is not girdled, other stresses to the seedling may interact with the damage resulting in a decline in tree vigour. Non-lethal injury can result in reduced seedling growth or leave the seedling weak and vulnerable to other damaging agents, e.g. drought will probably result in increased tree mortality in combination with feeding damage (Selander and Immonen, 1992). Overall damage by weevils will vary between years, within growing seasons and will also depend on the age and vigour of tree species present. In addition, the period during which plants remain vulnerable to damage varies considerably, depending on plant size and species as well as upon the size and activity of local pine weevil populations. For example, Douglas fir may remain susceptible to


205

Fig. 3. Pine weevil life cycle. Source: Reproduced with permission from the Forestry Commission UK.

serious damage for several growing seasons, while 3-year-old transplants of Sitka spruce are less likely to suffer fatal damage by 2 years after replantation (Heritage and Moore, 2001). As indicated above, overall losses of unprotected seedlings within the UK approximate to 50% of the newly replanted trees. It is because of this substantial damage potential that trees need protection from weevils.

Pine weevil pest management techniques – insecticides Since the 1940s the treatment of seedling trees (destined for a restock site) with chemical insecticides has been the most common method of seedling protection (Eidmann, 1979; Langstrom, 1982). However, as concerns have grown about the human and environmental impact of many of these chemicals their use has been gradually discontinued, the most recent example being the withdrawal of permethrin. The synthetic pyrethroid permethrin, formulated and marketed under the trade names Permit and Permasect, was used within the UK until December of 2003 for application to young trees for protection against weevil damage. Prior to 2003 this chemical was also used in large areas of mainland Europe. Approval to use permethrin within forestry was however withdrawn in December 2003 under the European


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Plant Protection Products Directive (Directive 91/414/EEC [Anon., 1991; Anon., 2000b]) because the relevant data holders failed to support this active ingredient through the reregistration process. From the spring of 2004, two formulations containing another pyrethroid (a-cypermethrin) were approved as replacements for treatment of plants under the product names Alphaguard 100EC and Alpha C 6ED. The approval of these pyrethroids supplements pre-existing approvals for both carbamate and organophosphorus products. A full list of the chemical insecticides approved for use within UK forestry is given in Table 3. The recommended treatments to reduce annual losses of transplants from weevil damage in the UK comprise a combination of insecticide treatments pre- and post-planting (Heritage, 1997). Of the options listed in Table 3, it is economically most sensible to use a pyrethroid insecticide. Pre-planting insecticide applications in the UK are carried out at centralised treatment facilities. Until recently, dipping had been the main form of pesticide application for pre-treatment planting. However, the use of dipping in the UK was phased out at the end of the 20th century and pre-planting treatment since July 2000 has most often been carried out using the electrodyn sprayer conveyor for barerooted stock. The electrodyn application principle was originally developed by Syngenta (formerly ICI), as a hand-held system to treat cotton crops in the tropics. However this system was subsequently adapted by the Forestry Commission UK in conjunction with Syngenta during the 1990s to enable electrostatic insecticide applications to be made to seedling coniferous transplants (Heritage and Jennings, 1997). The electrodyn system uses a specially formulated oil-based insecticide that is gravity fed through a plastic nozzle impregnated with carbon. A 20,000-volt electric current is applied across the nozzle resulting in highly charged ligaments that break up into even-sized droplets of less than 10 mm in diameter. As the plants pass through the spray booth fitted with twin electrodyn spray heads, the highly charged droplets travel towards the nearest earthed surface (the seedling) and coat a 150 mm band of the plant stem in pesticide (Heritage and Jennings, 1997) with a wrap-around effect. Table 3 Insecticides approved for use in forestry in the UKa Active ingredient

Product names

Type of use

Carbosulfan Chlorpyrifos

Marshal SuSCon Alpha Chlorpyrifos 48EC Ballad Barclay Clinch Choir Cyren Dursban 4 Equity Greencrop Pontoon Lorsban T Dimilin Flo Alpha C 6ED Alphaguard 100EC

Weevil control in restocking Weevil and beetle control in cut logs

Diflubenzuron a-Cypermethrin

Control of defoliating caterpillars Weevil control in restocking

a Data collated from Forestry Commission UK booklet entitled Reducing pesticide use in forestry (Willoughby et al., 2004) and from the UK website of the Pesticides Safety Directorate (www.pesticides.gov.uk); also see reference list for further details.


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The equipment is designed to apply approximately 0.1 ml of liquid per bare-rooted seedling and when permethrin was used, it provided protection for the main part of the first growing season. Extensive trials have shown that Alpha C 6ED (6% a.i. a-cypermethrin) is as effective as permethrin. A photograph of the electrodyn system for the pre-treatment of seedling plants is shown in Figure 4. The protection provided by pre-planting treatment is generally only effective for about 6 months, so to provide further protection plants can be treated after planting using the product Alphaguard 100EC (0.1% a-cypermethrin). Post-planting spray application or ‘top-up’ spraying involves the insecticide being applied as a spot treatment to each plant on the site using a conventional knapsack sprayer, a forest spot gun or a motorised knapsack sprayer. Approximately 10 ml spray of insecticide is directed at the middle of the plant, which allows sufficient solution to flow down the stem to treat around the root collar (Heritage, 1997). This is aimed at providing the seedlings with 12 weeks of protection. The alternative, but more expensive long-term option for plant protection is based on the use of a slow-release systemic carbamate insecticide (carbosulfan) formulated as a granule. Marshal SuScon granules are applied to the root zone at the time of planting. The active ingredient is then absorbed by the plant roots and transported to the stem and leaves. Although these granules provide protection for at least 2 years (Heritage et al., 1997), their primary drawbacks include higher costs and the fact that the granules have an initial delay in protection whilst the active ingredient is transported up the stem. The use of granules to minimise weevil damage, which has also received recent approval for use in the UK (August 2003) is at present a common control method in many parts of mainland Europe. Carbosulfan, the active ingredient in these granules is the only insecticide approved by Pesticide Safety Directorate (PSD) in the UK as a slow release formulation for the treatment of forest plants at the time of planting.

ED nozzles

Fig. 4. Conveyor belt system for the application of charged insecticide sprays to seedling trees. Note the yellow line on the conveyor belt indicating the point for placement of the tree root collar. The electrodyn (ED) nozzles are as indicated. Source: Reproduced with permission from the Forestry Commission UK.

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In addition to chemical methods of weevil management and/or control, a number of alternative methods are also under investigation. These include the use of cultural techniques such as scarification (Orlander et al., 1999) and mechanical or chemical barriers (Eidmann et al., 1996; Petersson and Orlander, 2003); the use of various biocontrol agents particularly nematodes (Brixey, 1997) and parasitoids (Leather et al., 1999); and, the use of more integrated approaches that utilise blends of management techniques within a forecasting-based pest management framework (Heritage and Moore, 2001). To date, however, none of these alternative approaches has proven to be as effective as chemical protection. There is currently a desire to replace many synthetic insecticides with more environmentally benign chemicals which some plant extracts, including neem, may represent. To date, a variety of plant extracts that have been assayed in relation to the control/management of H. abietis include various monoterpenoids, verbenone and ethyl compounds. For example, Klepzig and Schlyter (1999) assayed various plant-derived and insect-produced allelochemicals against H. abietis feeding in a series of laboratory assays, including limonin from Citrus spp. which was found to have substantial antifeedant effects. Additional research in Estonia assayed 20 plant-based extracts including garden rhubarb Rheum rhapunticum, yarrow Achillea millefolium, certain tree species and various neem preparations (Sibul et al., 2001). The results of this research indicated strong antifeedant and repellent effects, particularly with the neem preparations throughout the season, at azadirachtin concentrations of 10% and 20%. Laboratory-based studies elsewhere with neem have documented strong antifeedant effects with azadirachtin concentrations of 6% (Beitzen-Heineke and Hofman, 1994). Lastly, several other limonoids related to azadirachtin have also been shown to have antifeedant, growth-inhibitory and/or moult-disrupting properties in insects. For example, trichilins from Trichilia spp., cedrelone from Cedrela spp. and limonin from Citrus spp. have these properties, though only the latter has been tested against H. abietis (Klepzig and Schlyter, 1999). In short, there is now a substantial amount of evidence to indicate that plant-based extracts, particularly neem may have a role to play in the management of this pest. This is what we sought to explore and report on in relation to the research that is described in this chapter.

The potential of neem extracts for pest management The chemical composition of extracts from the neem tree A. indica, particularly that of the seed kernels and seed oil, has been extensively investigated (Jones et al., 1989) and an array of complex chemicals, primarily limonoids, have been identified that can cause diverse biological effects on insects. So far, more than 100 highly bioactive compounds (Kraus, 1995) have been recorded though the main active component of the neem tree appears to be the highly oxidised limonoid azadirachtin (Arnason, 1989; Schmutterer, 1990). It is known that the seeds of the neem tree contain the highest concentrations of azadirachtin as well as other biologically active chemical compounds (Koul et al., 1990; Ascher, 1993). In the introduction we listed some of the recent papers that have described adverse effects of neem-based extracts on coleopterous species (Table 1). In total however, and by 2004, more than 400


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pest species had been shown to be affected by neem extracts in some way or the other. The effects that neem extracts have on insects are diverse and include physiological and behavioural manifestations such as strong antifeedant activity, insect growth regulatory effects as well as sterility effects (Jacobson, 1988; Saxena, 1989; Schmutterer, 1990; Kraus, 1995). Most modern interest in neem as an insecticide stemmed from studies that investigated the antifeedant effects of neem extracts on the desert locust Schistocerca gregaria. One of the first descriptions (Anon., 1992) of the adverse affects of neem extracts reported that plots of different crops, which had been sprayed with aqueous suspensions of neem seed kernel, were effectively protected during an invasion by S. gregaria. Both primary and secondary antifeedant effects have been observed with azadirachtin (Ascher, 1993). Primary effects relate to interference with the process of chemoreception by the organism (e.g. sensory organs on mouthparts which stimulate the organism to begin feeding) whereas secondary effects relate to processes such as gut motility disorders following exposure to extracts (Schmutterer, 1990; Ascher, 1993). Antifeedant effects have been found to be very effective against larvae, nymphs and adults of numerous species belonging to different orders (Schmutterer, 1995). Lepidoptera appear to be particularly sensitive to azadirachtin’s antifeedant effects (Mordue and Blackwell, 1993; Mordue et al., 1998). In addition to antifeedant effects, various developmental, post-embryonic, reproductive and growth inhibitory effects have been observed, often causing malformation and mortality in a dose-dependent manner (Ascher, 1993). Growth regulatory effects are often manifested in growth and moulting abnormalities, and are believed to result from azadirachtin disrupting the hormonal system within arthropods (Schmutterer, 1990; Mordue et al., 1998), particularly by blocking the release of neurosecretory peptides, which regulate the synthesis and release of ecdysteroids and juvenile hormone. Growth and moulting abnormalities are also known to occur as a result of the direct effects of azadirachtin on dividing cells (Mordue et al., 1998; Salehzadeh et al., 2003). Finally, many of the other compounds present in neem seed extracts besides azadirachtin exhibit biological activity including oviposition deterrence, egg sterility and an inhibition of chitin biosynthesis (Ascher, 1993). Neem pesticides containing azadirachtin are now registered for use in a number of food and feed crops in North America (Sundaram, 1996). In addition, several formulations containing azadirachtin are now available for use in a number of countries in mainland Europe, e.g. France, Germany and Denmark (Ascher, 1993). One particularly valuable quality of many neem formulations is their systemic activity in certain plant species. For example, Isman (1991) in a review article cited examples where success was reported against certain species of foliar-feeding insects in agriculture. Root drenches of potted plants with neem seed extracts appear to be efficacious against foliar insects in certain cases, e.g. Chrysanthemum cuttings dipped in neem extract and exposed to the leafminer Liriomyza trifolii (Larew, 1988). Further studies by Sundaram have highlighted azadirachtin’s systemic qualities in other species of plants including Aspen and Sitka spruce (Sundaram and Sloane, 1995; Sundaram et al., 1995; Sundaram, 1996). Good quantitative information is sparse on the uptake, distribution and persistence of azadirachtin in plants. However, this still remains an active research area.

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Neem extracts, pine weevil management and forestry Specific research in Canada on the efficacy of neem led to its registration for use in forestry. In particular, neem has shown great promise against most of the major defoliating insects and has been found to be very active against at least 13 species of Lepidoptera, as well as sawfly larvae. For example, ground-based foliar applications of neem by motorised backpack mistblower or compressed air sprayer have proven effective against white pine weevil, pine false webworm and introduced pine sawfly on pines, and acceptable protection was achieved from spruce budworm damage on spruce and fir. Ultra-low volume aerial applications of EC formulations were effective against balsam fir sawfly on balsam fir and pine false webworm on red pine (Sundaram, 1996). As a result of knowledge concerning the systemic activity of neem, a novel device, the ‘systemic tree injection tube’, was developed in Canada to inject neem formulations into trees under pressure, quickly, easily and inexpensively. Using the systemic tree injection tube Duthie-Holt et al. (1999) investigated the extent of translocation of neem extracts in the pole of lodgepole pines, Pinus contora. Their results indicated that the active ingredients were translocated at least 9 m up the pole and that they persisted in active form for at least 6 weeks afterwards (Duthie-Holt et al., 1999). It was as a result of these and other successful applications of neem-based extracts that we began to investigate the utility of extracts for pine weevil management in Scotland. Our approach was to undertake both laboratory and field experiments. In 2002, a series of trials were initiated to evaluate whether neem-based formulations may have a role to play in plant protection in commercial coniferous forestry in the UK. The results of these first experiments were positive (Thacker et al., 2002, 2003). As a result, an application was made to the PSD (UK) for an experimental permit to undertake more extensive field trials with neem-based formulations. A 3year experimental permit was subsequently granted in October of 2002 and the first field trials with the commercial neem formulations Nivaar (Dawnlight Group) and NeemAzal (Trifolio Gmbh) began in March 2003. In this chapter, we present some preliminary data from our first field and laboratory experiments that were carried out with these and other neem-based formulations during 2002 and 2003.

Laboratory experiments with neem-based extracts Laboratory experiments in 2002 and 2003 comprised assaying the effects of neem extracts on weevil feeding activity by confining weevils in 9 cm diameter Petri dishes with Sitka spruce twigs. Two beetles were confined with two twigs that were either both untreated (control), both treated (no-choice) or only one twig treated (choice). Eight replicate Petri dishes were used per treatment. In the experiments, 6 cm long Sitka spruce twigs with 1 cm diameter were used. In the experiments, the twigs were treated with 1–2 ml of neat extract using a paintbrush. Needles were first removed from the twigs and the ends dipped in melted wax. The twigs were left to dry for 1 h after treatment application and were then placed on a wet filter paper in the Petri dish. One or two field-collected weevils were placed in the Petri dish with the twigs. The gender of the weevils was not determined. The Petri dishes were stored at a room


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temperature that varied from 10 to 15 1C. The percentage bark removed from the twigs was assayed weekly for 1 or 2 months by using a visual scoring system with 10% increments. In this system a score of 0% would indicate no feeding damage, while a score of 100% would indicate that all the bark had been stripped from the twig. In 2002, the twigs were treated with 2 ml of the neem extract Bugban (Gayne Prospero Ltd.) while in 2003 the twigs were treated with 2 ml of the formulations Nivaar (Dawnlight Group) and NeemAzal T/S (Trifolio Gmbh). These formulations contain approximately 1%, 0.15% and 1% azadirachtin, respectively. Figure 5 shows the 2002 results in terms of the mean percentage bark removed from twigs in control, no-choice and choice situations when undiluted Bugban (100%) extract was used. The data represent the means for each of the twigs within Petri dishes through time. Over the 8-week assessment period the percentage bark removed from control twigs increased to approximately 70%. The percentage bark removed from treated twigs (no-choice) increased to approximately 10%. The most obvious deterrent effect of the neem extract can be seen in the choice situation. Untreated twigs were fed upon and the percentage bark removed increased to approximately 80% by the end of the experiment. In contrast, treated twigs in the choice experiments were fed upon as per the no-choice experiment, i.e. the percentage bark removed increased to approximately 10%. Overall these data, therefore, indicate that the neem extract is having a significant deterrent effect in relation to weevil feeding activity. Figures 6 and 7 show the results of 2003 experiments. The percentage bark removed when beetles were confined with treated twigs in a no-choice situation is shown in Figure 6. The figure shows that both of the neem formulations had a significant deterrent effect upon weevil feeding activity. The percentage bark removed from control twigs increased to approximately 40% during the 3-week assessment period. In comparison, the percentage bark removed from twigs treated with either of the neem formulations increased to approximately 10% (Figure 6).

Mean percentage bark removed

100.00 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 control

no choice Experiment category

choice

Fig. 5. Mean percentage bark removed from twigs in Petri dishes through time. Treatment assessment dates were weekly over an 8-week sampling period. Each experiment category therefore has 16 means representing the two twigs in each Petri dish. In the choice experiment, treated twigs are indicated by unshaded bars. Extract was applied neat, i.e. 100%. Source: Reproduced with permission from Elsevier Science from Thacker et al. (2003). Crop Protect 22:753–760.


212 NeemAzal

Nivaar

Control

Percent Bark Removed

60 50 40 30 20 10 0 7/3/03

3/3/03

30/2/03

26/2/03

23/2/03

22/2/03

21/2/03

19/2/03

18/2/03

-10

Date

Fig. 6. Percentage bark removed in laboratory bioassays for beetles in a no-choice situation. Source: Reproduced with permission from Proceedings of Science and Application of Neem Conference, Glasgow 2003 (Cole and Strang editors, (2003), see reference list for further details). NeemAzal (untreated) Nivaar (treated)

NeemAzal (treated) Control

Nivaar (untreated) Control

60 50 40 30 20 10 0 7/3/03

3/3/03

30/2/03

26/2/03

23/2/03

22/2/03

21/2/03

19/2/03

-10

18/2/03

Percentage Bark Removed

70

Date

Fig. 7. Percentage bark removed in laboratory bioassays for beetles in a choice situation. Source: Reproduced with permission from Proceedings of Science and Application of Neem Conference, Glasgow, 2003 (Cole and Strang editors, (2003), see reference list for further details).

The percentage bark removed when beetles were confined with treated twigs in a choice situation is shown in Figure 7. As above, the figure shows that both of the neem formulations had a significant deterrent effect upon weevil feeding activity. The percentage bark removed from control twigs and untreated twigs in choice experiments increased, as above, to approximately 40% during the 3-week assessment period. In comparison, the percentage bark removed from twigs treated with


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either of the neem formulations increased to less than 10% (Figure 7). Overall, therefore, the laboratory experiments in both 2002 and 2003 appear to indicate that neem formulations assayed can act as significant feeding deterrents to the large pine weevil on Sitka spruce.

Field experiments with neem-based extracts To support our laboratory work, we also carried out a series of field trials throughout 2002–2003. Here we present some sample data from our field trials in 2003. These trials were established in the spring and summer of 2003 to assess the efficacy of three neem-based formulations in protecting trees from weevil damage. These formulations were (as above) NeemAzal-T/S, Nivaar and Bugban. The field trials were situated approximately 35 km west of Stirling, Central Scotland, in restock sites where the conifer forest had been previously clear-cut in December of 2001. Trials were set out in a randomised block design using standard planting methods. Threeyear old Sitka spruce (P. sitchensis) seedlings, 30–40 cm height, were used. Treatments were targeted to treat the main stem 0–15 cm up from the root collar. Two controls were used in the experiments, an untreated control and a positive control, viz. permethrin in the form of Permit (12% a.i., see earlier). The permethrin treatment was applied prior to planting using an electrostatic application system at a dose of 0.1 ml per tree (see also earlier). The neem extracts were applied before planting using a paintbrush to apply 5 ml per plant. The field trial consisted of 5 treatments 12 plants per plot 5 replicates giving 240 plants in total. In addition, an assessment of the efficacy of the product Bugban was made with respect to concentration. In this experiment, treatments of 1000 (no dilution), 800, 600, 400 and 200 g/l were made using vegetable oil as a diluent. The applications were made using a forestry spot gun fitted with a narrow cone nozzle calibrated to deliver a 10 ml dose. Overall, this latter field trial comprised 7 treatments 36 plants per plot 5 replicates, which gave 1160 plants in total. In the field trials two types of assessment were used. First, the percentage bark removed (0–15 cm from root collar) was recorded in 10% increments (as described above) throughout the season between May and November. Second, an assessment at the end of the field trial of the percentage survival of trees was made. In this assessment, the number of plants of each score of A, B, C, D or X was recorded where the scores are classified as follows: A B C D X

No damage Slight damage (may be extensive) with no girdling Severe damage (may be little) but girdled and likely to die Dead (not due to H. abietis) Missing

In this assessment scheme N ¼ A+B+C+D+X, where N is the total number of trees allocated to a particular treatment. Using this assessment scheme it is then possible to calculate the percentage survival of trees as ‘survival ¼ (A+B)/(N D X) 100’.


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Figures 8 and 9 plot the mean percentage bark removed through time and at the end of the assessment period, respectively. The latter, with the associated error term. The figures show that using the treatments NeemAzal-T/S and Nivaar increases the seedling trees’ chances of survival to a level comparable to that of the protection provided by Permit and it is significantly different from the untreated control where substantial damage has occurred. This level of protection is also highlighted in Figure 9 at the end of the assessment period. Figure 10 however shows the mean percentage survival rates for the seedling trees. The figures show that both NeemAzal-T/S and Permit were able to significantly increase the rate of survival in comparison to that of the untreated control. However trees that were treated with Nivaar all died, possibly as a result of some phytotoxic effects that may be associated with this formulation. Overall, therefore, the data indicate that neem-based extracts may protect trees from attack by H. abietis. Figures 11 and 12 show the percentage bark removed from seedling trees treated with variable doses of the extract Bugban. The figures show that all of the concenPermit

Nivaar

NeemAzal

5-Dec

26-Oct

16-Sep

7-Aug

28-Jun

19-May

45 40 35 30 25 20 15 10 5 0 9-Apr

Mean % bark removed (0 - 15 cm)

Control

Assessment Date

Fig. 8. Percentage bark removed over a 26-week period in 2003 from seedling trees treated with a range of neem-based formulations. 60 Mean % bark removed (0 - 15 cm)

50 40 30 20 10 0 -10

Control

Permit

Nivaar

NeemAzal

Treatment

Fig. 9. Percentage bark removed after 26 weeks in 2003 from seedling trees treated with two neem-based formulations. The graph gives the mean and associated confidence limits. Analysis of variance indicated statistically significant differences between treatments.


215

120

% Survival

100 80 60 40 20 0 Control

Permit

Nivaar

NeemAzal

Treatments

100%

80%

60%

20%

permit

control

40%

50 40 30 20 10 25-Nov

15-Nov

5-Nov

26-Oct

16-Oct

6-Oct

26-Sep

16-Sep

6-Sep

27-Aug

17-Aug

0 7-Aug

Mean % bark removed (0 - 15 cm)

Fig. 10. Percentage survival of trees after 26 weeks, treated with two neem-based formulations and a pyrethroid positive control. The graph gives the mean and associated confidence limits. Analysis of variance indicated statistically significant differences between the treatments.

Assessment Date

Fig. 11. Percentage bark removed over a 10-week period in 2003 from seedling trees treated with a range of concentrations of the neem-based formulation, Bugban.

trations assayed provided comparable protection to that of Permit and that they were significantly different to that of the control. The figures also hint at a degree of dose-response in relation to dilution, despite the fact that all dilutions were equally effective. As above, therefore, these data also indicate that neem extracts may protect trees from damage by the large pine weevil.

Future perspectives and conclusions Overall, the laboratory and field data that we have collected to date indicate that neem extracts may have a role to play in pine weevil management with UK forestry. Whether it will be economic to register neem extracts for use, given the relatively small size of the UK forestry industry, is still not clear and it may be dependent on finding


216 70 Mean % bark removed (0 - 15 cm)

60 50 40 30 20 10 0 -10

100%

80%

60%

40%

20%

Permit

Control

Treatment

Fig. 12. Percentage bark removed after 10 weeks in 2003 from seedling trees treated with a range of concentrations of the neem-based formulation, Bugban. The graph gives the mean and associated confidence limits. Analysis of variance indicated statistically significant differences between treatments.

other (possibly agricultural) uses for such extracts. In addition, prices for lumber have been falling in recent years, making the economics of restocking a difficult activity for forest managers within the UK. That said, within the European Union the neembased formulation NeemAzal has now been registered for use in a number of Mainland European countries including Germany and Austria for the control of a range of pests of both forestry and agriculture. This may make the registration of this product for use in UK forestry a relatively straightforward process. In the face of the declining availability and popularity of conventional chemical insecticides (such as pyrethroids), there is a need to find sound and effective options for managing the large pine weevil. Although there have been developments in biological control within an integrated pest management programme, these are aimed at reducing the H. abietis population over protracted time periods. What is needed in UK forestry in particular is a product that can be used with almost immediate effect. The results of several studies, including the data reported here, suggest that neem preparations may have potential for forest insect control (Helson, 1999; Thacker et al., 2003). Only time will tell whether the use of neem becomes widespread in European forestry for the management of this and possibly other, pest species.

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CHAPTER 10

Plant allelochemicals in thrips control strategies ELISABETH H KOSCHIER

Introduction Integrating the non-toxic mechanisms deriving from natural plant compounds into insect pest management seems quite appealing – the more so because such mechanisms are present and available wherever plants grow. The reasons range from general environmental concerns to the very practical problem of the increasing resistance of insect pests against chemical insecticides (Lewis, 1999; Isman, 2000; Jensen, 2000). Plants produce a wide range of secondary compounds that may act as signals, i.e. allelochemicals, mediating interactions between insects and plants. Among those, two functional categories of allelochemicals are particularly relevant to insect–plant interactions: kairomones are used by insects to locate a host plant and allomones confer protection to the plants that produce them (Whittaker and Feeny, 1971; Swain, 1977; Ryan, 2002). The following pages deal with aspects of using such secondary plant compounds as attractants, repellents or deterrents to manipulate the host selection and acceptance behaviour of thysanopteran pest species in the framework of either existing or novel biological control or Integrated Pest Management (IPM) strategies. The chapter aims at giving the reader an overview of past and current research in this field, showcasing perspectives and problems frequently met in the development of environment friendly pest control methods. Control of thysanopteran (thrips) pests is a particularly interesting proving ground for pest control because in the course of the last decade thrips have become a major world-wide problem in agriculture and horticulture (Lewis, 1999), with their pest status seemingly increasing on a wide range of crops (Mound and Teulon, 1995). The scale of damage caused by thrips has been described in detail by Childers (1997). Currently about 5000 thrips species within the two sub-orders Terebrantia and Tubulifera are known. Among those about 25 species, mainly in the family Thripidae (Terebrantia), are serious pests causing regular economic damage, both by direct feeding and as vectors of mainly viral plant diseases (Lewis, 1998, 1999).

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Monophagous as well as oligo- and polyphagous species occur among Thysanoptera (Ananthakrishnan and Gopichandran, 1993). The status of thrips pest species differs significantly between crops and between geographical regions (Lewis, 1997). Yet a steady increase in the international trade of fruits, vegetables and ornamentals has, together with opening up of new regions for crop production, globalised the threat of thrips pests: many species have spread to favourable new environments, sometimes continents apart from their natural habitats and hosts – for instance the polyphagous thrips species Frankliniella occidentalis Pergande, the western flower thrips and Thrips palmi Karny (Lewis, 1997, 1998). Not surprisingly, much research is currently directed to control of these species. Management of thrips pests is problematic due to their minute size and their cryptic habits, as they feed hidden in crevices of flowers and leaf sheaths. Their high reproductive capacity leads quickly to great numbers infesting individual plants (Lewis, 1999). Control strategies in the glasshouse as well as in the field have often relied on repeated application of chemical insecticides that not only produced environmental risks, but also resulted in widespread development of resistance in some species, e.g. in F. occidentalis (Robb et al., 1995). This, combined with the increasing economic impact of thrips pests, put considerable urgency on the development of novel control strategies. In protected crops, several successful programmes using biological control have been developed. Release of natural enemies provides adequate control in some crops (Lewis, 1999), but presents specific problems in other crops (e.g. Bennison et al., 2002, 2003). Furthermore, the incompatibility of many pesticides with concurrent use of natural enemies in IPM programmes is a source of problems (Van Lenteren and Loomans, 1998). For all these reasons, thrips pests make an especially interesting case study in the field of plant allelochemicals as crop protection agents serving as alternatives to conventional pesticides that have essentially toxic modes of action.

The role of plant allelochemicals in host selection of Thysanoptera Host plant selection and host plant acceptance are a complex of simultaneous and/or sequential decision steps, primarily mediated by vision, olfaction and/or contact chemoreception. It has been observed that orientation to and landing on a potential host plant is followed by a phase of testing whether the plant is suitable for feeding and as a reproductive host (Terry, 1997). In this decision-making process, attractants or repellents can play a role as olfactory cues that determine positive or negative orientation to a plant. Even before physical contact they can give olfactory cues for landing or not landing. Upon contact with the plant, allelochemicals present on the plant surface or in the plant interior can act as stimulants or deterrents for feeding and/or oviposition (Renwick, 1990, 1999). Attractants can trigger various behavioural sequences. Dethier et al. (1960) defined them as chemicals that cause insects to make oriented movements towards the odour source. Indeed, many insect species are guided to host plants by the olfactory stimuli emanating from them, often in addition to visual cues. Such volatiles are almost always food lures, although in some cases they can also act as sole or additional ovipositional lures.

Plant allelochemicals in thrips control strategies

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With thrips, several studies about their chemical ecology suggest that they are guided by odours of flowers, buds and/or foliage to locate a host plant over a distance and towards special parts of a plant at close range. It must be noted that, depending on their degree of specialisation, host plant preferences vary strongly from species to species. The biochemical environment of flowers is generally responsible not only for the attraction of thrips to floral parts for feeding but also for pollination by thrips (Ananthakrishnan and Gopichandran, 1993). Floral scents are often complex blends of substances that may belong to different chemical classes. Monoterpenes, phenylpropanoids and other floral volatiles are released, governing the attraction of flowers (Knudsen et al., 1993). But other secondary compounds such as flavonoids or carotenoids also contribute to the attractiveness of flowers as they compose the colour of most flowers (Ananthakrishnan and Gopichandran, 1993). For instance, F. occidentalis, a generalist flower-inhabiting thrips species, was attracted to flowers of sweet pepper, but not to its leaves in an olfactometer experiment (Gerin, 1994). Limothrips cerealium Haliday and Haplothrips aculeatus Fabricius preferably live in spikelets of cereals. Surprisingly, they did not respond to the odour of a whole oat plant or of oat leaf sheaths, but they were strongly attracted by the odour of oat spikelets and their extract (Holtmann, 1963). The alligator weed thrips Amynothrips andersoni O0 Neill is a monophagous thrips species and solely attracted to alligatorweed. Its host specificity is suggested to be at least in part chemical in nature (Maddox et al., 1971). Behavioural observations revealed that Kakothrips pisivorus Westwood, a specialist flower thrips species mainly on Vicia and Pisum, can distinguish clearly between pollen from its host plants and from nonhosts, probably involving olfactory attraction. It has been observed that on pollen from a preferred host plant sustained feeding is stimulated, resulting in higher ingestion rates and an increased oviposition rate (Kirk, 1985b). However, the single chemical stimuli required to initiate thrips feeding (Kirk, 1997) or oviposition have not yet been identified. Whether feeding preferences of thrips are determined by secondary compounds in their host plants or by nutritional factors, i.e. primary metabolites, is not yet entirely elucidated (Ananthakrishnan and Gopichandran, 1993; Scott Brown et al., 2002). In principal, primary metabolites such as sugar, nitrogen and carbohydrates are the basis for host selection and host utilisation of insects. They stimulate feeding and influence life history parameters of thrips (Ananthakrishnan, 1993; Ananthakrishnan and Gopichandran, 1993). Making use of this effect, growers added white sugar to insecticide tank mixtures against F. occidentalis to enhance insecticide uptake and thus the toxicity to the western flower thrips (Parrella, 1995). Nitrogenous compounds have been demonstrated to be related to foliar feeding of thrips. Mollema and Cole (1996) found that low concentrations of aromatic amino acids in leaf proteins of cucumber, tomato and peppers were correlated with a reduction in feeding damage caused by F. occidentalis on leaves, and Brodbeck et al. (2001) confirmed this finding. Protein contents influenced the suitability of plants as hosts for F. occidentalis and Heliothrips haemorrhoidalis Bouche´ more than the levels of carbohydrates, though it is not clear whether the thrips were responding to the proportion of protein, carbohydrate or both. However, plant species containing similar levels of protein and carbohydrates vary in their ability to support thrips, leading to the suggestion that secondary compounds in the leaves of these plants are

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likely to play a decisive role in thrips–plant interactions (Scott Brown et al., 2002). Moreover, the effect of individual chemicals on insect behaviour can be very different from the effect of the same chemicals when combined with other compounds present in plant material (Lewis and Van Emden, 1986). The observation that various generalist thrips species feed and even oviposit through artificial membranes led Terry (1997) to the conclusion that feeding stimulants are apparently not needed. In fact, particularly polyphagous insects are generally believed to rely on the presence of repellents or deterrents to ensure avoidance of unsuitable plants (Renwick, 2001). In contrast to attractants, repellents are olfactory stimuli that cause insects to move away from an odour source (Dethier et al., 1960), especially at close range (Visser, 1986). While repellents may prevent insects from contacting a plant (Norris, 1990), deterrents act after the landing of an insect on a plant surface. The function of deterrents involves contact chemoreception (Renwick, 1990) and inhibits feeding or oviposition on a plant (Dethier et al., 1960). However, repellents and deterrents are sometimes difficult to discriminate, because continued olfactory input after contact with the leaf surface caused by volatile stimuli might contribute to the rejection of a substrate (Renwick, 1990). Sometimes volatiles and contact stimuli may exhibit synergy in specific steps within the decision process of host plant selection of an insect (Staedler, 1992). Because of their potential to interfere with steps in the host selection process (Visser, 1986) prior to or after alighting on a potential host, many secondary plant metabolites are considered to have evolved as part of the chemically based defence system against phytophagous insects (Renwick, 1999). Plant defensive secondary compounds such as tannins or alkaloids have been shown to increase resistance (Seigler, 1998). Therefore, some breeding programmes for resistant cultivars focus on the detection of specific phytochemicals that are associated with host plant resistance against thrips. However, evidence for host plant resistance to thrips deriving from repellent volatiles is scarce. Apart from some morphological resistance factors, non-contact cues in Lycopersicon hirsutum f. glabratum were suggested to repel F. occidentalis nymphs (Kumar et al., 1995). Mollema et al. (1995) analysed feeding patterns of F. occidentalis on resistant cucumber genotypes and observed the thrips. They found dispersed punctures and restless movements of the thrips and suggested that resistance may be caused at least partly by some repellent factors. Fung et al. (1999) tested the olfactory responsiveness of F. occidentalis to susceptible and resistant chrysanthemum varieties in olfactometer experiments without finding any differences. But using a metal oxide sensor-based electronic nose, McKellar et al. (2005) were able to discriminate among western flower thrips-resistant chrysanthemum cultivars suggesting that volatile chemicals released from leaf discs are involved in resistance. Yet it is also recognised that non-volatile compounds play an important role in thrips resistance of number of plant species. Rice varieties that are resistant to the rice thrips Stenchaetothrips biformis Bagnall show a higher content of phenolic compounds, in particular chlorgenic and gallic acids, than susceptible varieties (Thayumanavan et al., 1990). Cotton varieties susceptible to Retithrips syriacus Mayet had higher concentrations of primary metabolites, but lower concentrations of gossypol, tannins and flavonoids than resistant varieties (Gopichandran et al.,


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1992). Even though being highly polyphagous, T. palmi landed and walked on tomato leaves, but did not feed on them (Hirano et al., 1993). T. palmi is also unable to pupate on the leaves of strawberry and tomato, and adult survival is reduced by about 70–75% compared to survival on the optimal host cucumber (Kawai, 1986). Yasumi et al. (1991) applied methanol extracts of tomato, eggplant or cucumber leaves on sucrose-impregnated filter paper leaf discs for testing their effects on the survival of T. palmi. Adult females died on the tomato leaf extract within a short period, but not on the extracts of the other two plants. Further fractionation of the tomato leaf extract showed a bioactive component in the n-butanol fraction. As the fraction was only active during direct exposure to the thrips, an antifeedant activity was suggested to be more likely than toxicity (Yasumi et al., 1991). Hirano et al. (1993) concluded that the immunity of tomato against T. palmi expresses itself shortly after the period of initial settlement, and that this thrips species does not use airborne phytochemical cues – neither attractants nor repellents – for location of a host plant at short range. Continuing their studies, Hirano et al. (1994) isolated the steriodal glykalkaloid a-tomatine from the tomato leaf n-butanol fraction and demonstrated that it has a strong antifeedant activity against T. palmi on tomato leaves. The performance of F. occidentalis on leaf saps of resistant chrysanthemum cultivars was affected by their chemical composition, indicating that they contained deleterious secondary metabolites: these were unfortunately not analytically identified (De Jager et al., 1995, 1996). Fung et al. (1999) analysed the amino acid composition of different chrysanthemum varieties and found that the active compounds in resistant varieties consisted of substances of both low and high molecular weight. Furthermore, Tsao et al. (2003, 2005) investigated possible biochemical resistance mechanisms against F. occidentalis in different chrysanthemum varieties. They found the concentration of an unsaturated fatty acid isobutylamid to correlate with the degree of resistance against the thrips.

Perception of plant allelochemicals In most insects, antennal receptors perceive volatile olfactory stimuli, whereas contact chemoreceptors are responsible for reception of non-volatile stimuli (Renwick, 1990). Apart from the fact that chemical receptors for the detection of olfactory and gustatory stimuli are located on antennae and mouthparts of thrips, not much is known yet about the chemosensory structures that are essential for their host plant recognition. The antennal segments 3 and 4 of several terebrantian and tubuliferan species have emergent – forked or simple – sense cones. A few families within the Terebrantia have differently shaped antennal sense organs, namely transverse or longitudinal sensory areas (Mound and Kibby, 1998). Some other structures such as antennal setae are also believed to be sense organs. Antennal receptors presumably have a function as chemoreceptors as they seem to be stimulated by chemicals (Moritz, 1997) and were found to contribute to the orientation behaviour of thrips to a plant in leaf-disc bioassays (Ananthakrishnan and Gopichandran, 1993). Sensilla with chemosensory function are found on the maxillary palps, the labial palps and the paraglossae, e.g. in F. occidentalis. The paraglossal sensilla have not

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only chemosensory but also mechanosensory function. The existence of these structures adds evidence to the suggestion that plant morphology and chemistry play an important role in host selection in thrips (Hunter and Ullman, 1992, 1994). Hunter and Ullman (1994) proposed a possible behavioural sequence for thysanopteran host selection and feeding. They observed that, before a thrips starts feeding, it waves its antennae while it walks over the plant surface. They furthermore described the contact of the chemosensilla with the surface as ‘labial dabbing’ and suggested that this behaviour is involved in examining the surface for chemical cues. In the presence of inappropriate stimuli the thrips sometimes moved on without probing and repeated the labial dabbing, whereas appropriate stimuli led to test probing by inserting the stylets in the plant tissue. During these probes the thrips takes up plant fluid. With its precibarial and cibarial sensilla, which are suggested to be gustatory chemosensilla, it is able to discriminate between chemicals in the food before it is ingested. The presence of inappropriate stimuli leads to the termination of the probe (Hunter and Ullman, 1994) and, in consequence thereof, presumably also to the rejection of the plant as a host.

Methods for testing the biological activity of plant allelochemicals on thrips It seems promising to evaluate both volatile and non-volatile plant compounds for their potential in future plant protection strategies. Therefore, the established methods applied in such studies are briefly described here, covering the evaluation of both volatile compounds as olfactory stimuli and non-volatile compounds as feeding or oviposition deterrents. Whole intact plants, excised fresh plant material, plant essential oils and plant extracts as well as plant volatiles (pure chemicals) have been used as basic material for biological testing of olfactory responses of Thysanoptera. Volatile plant allelochemicals can be isolated from fresh plant material using, for instance, techniques of air entrainment and headspace analysis (e.g. Pow et al., 1998; Chermenskaya et al., 2001). Volatiles released by the sample are trapped by a polymer, from which they can be eluted using a solvent. Subsequently, the chemical components can be identified from the liquid extract by means of a gas chromatograph and a mass spectrometer (Agelopoulos et al., 1999). Similarly, the chemical composition of plant extracts or essential oils can be analysed using gas chromatography/mass spectrometry (GC/MS). In other studies, plant material for bioassays has been extracted using for instance a Soxhlet apparatus with hexane as a solvent, or distilled for the derivation of essential oils (e.g. Chermenskaya et al., 2001; Koschier et al., 2002). In laboratory studies, thrips responses to plant volatiles can be detected using electroantennography (EAG) coupled with GC: the effluent of a gas chromatograph is linked to a preparation of thrips antennal receptors, which reacts to a stimulating compound in the form of an electrical signal. The signals are recorded as electroantennograms, with the active compounds again identified by means of GC/MS. While electrophysiological activity of a compound cannot be translated into behavioural responses of a living insect, and does not even indicate attractiveness or repellency, olfactometer experiments give at least some information on the behavioural


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role of a component. Therefore, compounds identified by using coupled EAG/GC should be further evaluated in behavioural bioassays (Finch, 1986; Agelopoulos et al., 1999). Odours emanating from the whole variety of basic materials listed above were tested in olfactometer experiments. Thrips responses to short distance olfactory stimuli were investigated using the Pettersson olfactometer (Manjunatha et al., 1998; Pow et al., 1999; Chermenskaya et al., 2001; Shamshev et al., 2003), a V-shaped olfactometer tube (Gerin, 1994), a dual-choice olfactometer tube (Shamshev et al., 2003), a Y-tube olfactometer (Holtmann, 1963; Gaum et al., 1994; Koschier et al., 2000; De Kogel and Koschier, 2003) for walking thrips, and using different kinds of flight chamber olfactometers or wind tunnels (Teulon et al., 1993b, 1999; Frey et al., 1994; Hollister et al., 1995; De Kogel and Koschier, 2003; Shamshev et al., 2003) for flying thrips. For observations of long distance responses of flying thrips to attractive volatiles, most greenhouse and field trials are simple bioassays comparing trap catches from paired coloured or non-coloured water or sticky traps. The case study of the olfactory attractant p-anisaldehyde in the next section gives an overview on the variety of experiments combining odours, colours and trap designs. In most studies investigating non-volatile plant factors, sustained settling, feeding and oviposition of thrips is indicative for the acceptability of a host plant. For an evaluation of plant allelochemicals applied on crop plants, techniques used in research on chemical resistance factors to thrips in or on plants (e.g. Van Dijken, 1992; Mollema et al., 1995; Soria and Mollema, 1995; Bergh and Le Blanc, 1997) and techniques used in thrips alarm pheromone evaluation (e.g. Teerling et al., 1993; Teerling, 1995) are often applicable. The plant material on which the test substances are applied for such bioassays are leaf discs, excised leaves or whole potted plants. Leaf-disc bioassays were conducted to determine the effects of essential oils and their constituents on the feeding activity of adult Thrips tabaci Lindeman (Koschier et al., 2002; Sedy and Koschier, 2003) or F. occidentalis (Sedy and Koschier, 2003) females. The percentage of feeding damage area on leaf discs sprayed with the test compounds and on control leaf discs caused by a defined number of individuals after 24 h was measured using an image analysis system consisting of a digital camera mounted on a microscope coupled with a special image analysis software. In similar leaf-disc bioassays, the influence of plant allelochemicals on the reproduction success of T. tabaci and F. occidentalis was determined by counting the number of eggs laid or the number of larvae emerging from the eggs (Koschier and Sedy, 2003a, 2003b; Sedy and Koschier, 2003). Both the bioassays for determination of feeding or egglaying activity may be designed as choice or no-choice experiments. In the former, single leaf discs are used, in the latter a treated and an untreated control are provided. But as results of choice tests may be very different from those obtained in nochoice situations (Harrewijn et al., 2001), the effects of plant compounds on feeding or oviposition of thrips should be verified in no-choice tests. This is more so since nochoice situations are representative for crops cultivated in monocultural systems (Lewis and Van Emden, 1986): in large field cultures, for example onions, thrips may not have any alternative host plants. With regard to a practical application in the future, bioassays used in antifeedant research (Koul, 1993) and any other laboratory assay testing effects of natural plant compounds should match field conditions as far

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as possible. For instance, bioassays or experimental set-ups that permit volatilisation of volatile compounds from treated plant surfaces where thrips are feeding or laying eggs should be considered. A method that may be useful in antifeedant research against Thysanoptera in the future is the direct current (DC) electrical penetration graph (EPG) technique, which is particularly suited to the study of factors affecting feeding behaviour (Kindt et al., 2003).

Search for phytochemicals attractive to thrips Logically, an important focus in the search for potent kairomones for thrips attraction has been placed on host plants and their preferred parts. Whole plants or plant parts were tested for thrips responses, and secondary metabolites – mostly volatile constituents – were isolated from the plant material and identified for subsequent testing as pure chemical compounds, attributing thrips responses to single constituents or constituent complexes with quite interesting results. Numerous studies were carried out with chrysanthemum, a highly preferred host plant of F. occidentalis, a species more attracted by unopened flower buds than by flowers, leaves or the extracts of the respective plant part. Chrysanthemum volatiles were collected by air entrainment and their active components were sought by coupled GC-EAG and identified by GC/MS. The results revealed that the increased attraction of F. occidentalis to flower buds can be attributed to a higher amount of (E)-b-farnesene in buds than in chrysanthemum flowers or leaves (Manjunatha et al., 1998). The attractiveness of (E)-b-farnesene to F. occidentalis was confirmed in olfactometer experiments with walking thrips (Manjunatha et al., 1998; Pow et al., 1999; Koschier et al., 2000) and in greenhouse experiments with flying thrips (Manjunatha et al., 1998; Pow et al., 1999). The attraction of flowers from different Verbena hybrida cultivars was investigated in olfactometer studies to evaluate them as a trap crop for attracting thrips away from profitable ornamental crops. The main volatile compound, to which the attractiveness of the flowers could be attributed, was identified as a linalool oxide pyran (Pow et al., 1998; Hooper et al., 1999). Although meadowsweet (Filipendula ulmaria Maxim.) has not been recorded as a host plant of F. occidentalis, thrips were strongly attracted to their fresh flowers (Shamshev et al., 2003) as well as to the extract (Chermenskaya et al., 2001; Shamshev et al., 2003), the volatiles entrained from the extract (Chermenskaya et al., 2001) and to most of the fractions of the extract (Shamshev et al., 2003). The composition of the volatiles in the extract was analysed using GC/MS, and coupled GC-EAG showed that the pure volatile compounds methyl salicylate and 1, 8-cineole (eucalyptol) elicited responses. Further olfactometer tests revealed eucalyptol to be somewhat attractive, and methyl salicylate to be repellent to the thrips (Chermenskaya et al., 2001). In the face of these results we can assume that the attractiveness of F. ulmaria flowers is caused by the complex mixture of all odour components, with repellent compounds being masked by attractive compounds. The New Zealand flower thrips Thrips obscuratus Crawford is strongly attracted to ripe peaches, a fact that led to investigations by Penman et al. (1982) on the


229

attractiveness of various chemicals that are possibly associated with fruit ripening. Ethyl nicotinate, an ester of nicotinic acid, was found to be an especially potent attractant to T. obscuratus and more than competitive with actual ripening peaches and apricots. Moreover, ethyl nicotinate is also attractive for F. occidentalis (Teulon et al., 1993b; Frey et al., 1994; Koschier et al., 2000), T. tabaci and L. cerealium (Teulon et al., 1993b), although with this component, not present in floral fragrances, the significance for host finding by flower-inhabiting thrips species is not yet clear. Teulon et al. (1993b) suggested as a possible explanation that either small quantities of ethyl nicotinate might be present in host plants, or even though this compound is not normally encountered by thrips, it elicits a strong response when detected by thrips’ olfactory receptors. A different approach to the selection of phytochemicals for evaluating their attractiveness to thrips may be based on chemotaxonomic considerations, or on botanical and entomological data. In some studies compounds were selected either randomly, or because they had previously been found to elicit positive responses from other thrips species or even other insect or mite species. Table 1 presents a list of all chemicals that up to now have been found to attract one or more thysanopteran species significantly. Phenylpropanoids and terpenes make up the larger part of the chemicals tested in this respect. They are volatiles found in essential oils (Seigler, 1998), with some of them particularly abundant in floral scents (Knudsen et al., 1993). These chemicals are listed as eliciting positive responses mainly in flower-inhabiting generalist thrips species, adding credibility to the suggestion that flower thrips use floral scents for host location. Only few non-floral chemicals such as ethyl nicotinate or organic acids have been tested yet for their attractiveness to thrips. It must be stressed that results obtained in olfactometer experiments with walking thrips are often difficult to relate to findings of greenhouse or field trials with flying thrips. Most olfactometer set-ups leave thrips only with the binary choice of walking towards the odour source or not, inhibiting the full display of the behavioural repertoire in host selection. Furthermore, most of them do not assess the mechanisms of the scent responses (e.g. anemotactic, chemokinetic or odour-induced visual responses) and only allow responses at close range, whereas in the field flying thrips orient towards coloured and/or scented traps at relatively longer distances. Therefore, olfactory attractiveness found in olfactometer experiments has to be reproduced in greenhouse and/or field experiments, especially with regard to a future application in practical plant protection. Beginning in the early 20th century, various attempts have been made to examine the attractiveness of floral fragrances and various pure chemical substances to thrips. First experiments in 1912 and observations by Howlett (1914) indicated chemotropic responses of thrips to a number of aldehydes. Morgan and Crumb (1928) published results of trials in oat fields infested with the two cereal thrips species L. cerealium and Frankliniella tritici Fitch. They used white pan traps filled with water, adding a wide range of different chemicals such as aldehydes, acids, esters, essential oils or petroleum derivatives to attract thrips to the traps. The authors cite in order of decreasing attractiveness cinnamaldehyde, salicylaldehyde, anisaldehyde, benzyl and cinnamyl alcohol, benzaldehyde among others. p-Anisaldehyde (4-methoxybenzaldehyde), a volatile phenylpropanoid component of the seed oil of anise (Pimpinella anisum L.) (Seigler, 1998) is very common in floral

Chemical class

Compounda

Thrips speciesb

Exp.c

Thrips walking (W) or flying (F)

Phenylpropanoids / Hydroxybenzoic Acid compounds (C6-C1)

p-anisaldehyde

Frankliniella occidentalis

F G

F F

Teulon and Ramakers (1990), Teulon et al. (1993b) Brødsgaard (1990), Baranowski and Gorski (1993), Teulon et al. (1993a), Hollister et al. (1995), Roditakis and Lykouressis (1996), Manjunatha et al. (1998), Teulon et al. (1999)

O, W

F

Teulon et al. (1993a), Frey et al. (1994), Hollister et al. (1995), Teulon et al. (1999)

Frankliniella intonsa Frankliniella tritici Limothrips cerealium Thrips fuscipennis Thrips hawaiiensis Thrips imaginis Thrips major Thrips obscuratus Thrips pillichi Thrips tabaci

O F F F F F F F F F F

W F F F F F F F F F F

Thrips vulgatissimus

F

F

Koschier et al. (2000), Katerinopoulos et al. (2005) Kirk (1985a), Teulon et al. (1993b) Morgan and Crumb (1928) Morgan and Crumb (1928) Teulon et al. (1993b) Murai et al. (2000) Kirk (1987) Kirk (1985a) Teulon et al. (1993b) Kirk (1985a) Kirk (1985a), Teulon and Ramakers (1990), Teulon et al. (1993b), Murai et al. (2000) Kirk (1985a)

o-anisaldehyde


O

W

Koschier et al. (2000)

benzaldehyde

Frankliniella intonsa Frankliniella occidentalis

F F G O F

F F F W F

Teulon and Ramakers (1990), Teulon et al. (1993b) Teulon and Ramakers (1990), Teulon et al. (1993b) Baranowski and Gorski (1993) Koschier et al. (2000) Morgan and Crumb (1928)

References


Frankliniella tritici

230

Table 1 Attractiveness of volatile secondary plant compounds to adult Thysanoptera in laboratory, greenhouse, or field experiments. Classification of compounds according to Seigler (1998)

Monoterpenes

F F F

F F F

Morgan and Crumb (1928) Teulon et al. (1993b) Teulon and Ramakers (1990), Teulon et al. (1993b)

benzyl alcohol

Frankliniella tritici Limothrips cerealium

F F

F F

Morgan and Crumb (1928) Morgan and Crumb (1928)

salicylaldehyde

Frankliniella occidentalis Frankliniella tritici Limothrips cerealium Thrips tabaci

G O F F O

F W F F W

Roditakis and Lykouressis (1996) Katerinopoulos et al. (2005) Morgan and Crumb (1928) Morgan and Crumb (1928) De Kogel and Koschier (2003)


F

F

Morgan and Crumb (1928)

Limothrips cerealium

F

F


eugenol


O O

F W

Frey et al. (1994) Koschier et al. (2000)

3-phenylpropionaldehyde


G

F

Baranowski and Gorski (1993)

O

W


cinnamaldehyde, cinnamyl alcohol


O

W


1,8-cineole (eucalyptol)


O

W

Katerinopoulos et al. (2005)

geraniol


O G O

F F W

Frey et al. (1994) Frey et al. (1994) Koschier et al. (2000)

Thrips hawaiiensis

F

F

Murai et al. (2000)

linalool


O

W

Koschier et al. (2000), Katerinopoulos et al. (2005)

linalool oxide pyran


O

W

Hooper et al. (1999)

nerol


O

W


231

(+)-citronellol


Volatile Phenylpropanoid Compounds (C6-C3)

Limothrips cerealium Thrips obscuratus Thrips tabaci

232

Table 1 (continued ) Chemical class

Compounda

Thrips speciesb

Exp.c

Thrips walking (W) or flying (F)

Sesquiterpenes

(E)-X-farnesene


O

W

Manjunatha et al. (1998), Pow et al. (1998), Koschier et al. (2000)

G

F

Manjunatha et al. (1998), Pow et al. (1998)

Haplothrips aculeatus

O

W

Holtmann (1963)


O

W

Holtmann (1963)

Thrips coloratus

F

F

Murai et al. (2000)

Thrips hawaiiensis

F

F

Murai et al. (2000)


Limothrips cerealium Thrips obscuratus Thrips tabaci

O F G O F F F

F F F W F F F

Frey et al. (1994) Teulon and Ramakers (1990), Teulon et al. (1993b) Teulon et al. (1993b) Koschier et al. (2000) Teulon et al. (1993b) Penman et al. (1982), Teulon et al. (1993b) Teulon and Ramakers (1990), Teulon et al. (1993b)


F

F



F

F


(also in combination with ethyl nicotinate) Organic acids and their derivatives

lactic acid, capric acid, caproic acid

methyl anthranilate (derivative of anthranilic acid)

nitronbenzoic acid and its derivatives a

at any concentration. females or males or both sexes. c F ¼ field trials; G ¼ greenhouse experiments; O ¼ olfactometer experiments; W ¼ wind tunnel experiments. b


ethyl nicotinate (ester of nicotinic acid)

References


233

scents (Knudsen et al., 1993). This volatile is an appropriate case study for the various possibilities of combining an attractant with different coloured traps, and the potential of a single volatile of being attractive over a wide range of concentrations for different flower-inhabiting thrips species. On non-flowering green, p-anisaldehyde has been found to attract five generalist flower thrips species (Thrips vulgatissimus Haliday, T. tabaci, Thrips major Uzel, Thrips pillichi Priesner and Frankliniella intonsa Trybom) to white water pan traps (Kirk, 1985a) and Thrips imaginis Bagnall, a polyphagous flower thrips, to white sticky traps (Kirk, 1987), while producing no results with cereal thrips species (L. cerealium, Limothrips denticornis Haliday and Frankliniella tenuicornis Uzel) or the predatory specialist aeolothripid species Aeolothrips intermedius Bagnall. In similar set-ups, Teulon et al. (1993b) added anisaldehyde to different kinds of traps and caught various generalist flower-inhabiting thrips species such as Thrips fuscipennis Haliday, T. obscuratus, T. tabaci, F. occidentalis and F. intonsa, but not the grass and cereal-inhabiting species Anaphothrips obscuratus Mu¨ller. In American basswood (Tilia americana L.) stands, p-anisaldehyde was combined with sticky traps of various colours to increase catches of Thrips calcaratus Uzel, a host-specific pest damaging buds and foliage, the polyphagous pest species Taeniothrips inconsequens Uzel, which feeds on buds, flowers and leaves, and Neohydatothrips tiliae Hood, a monophagous species living on basswood foliage. Interestingly, none of these species responded to the odorous compound, not even the two bud and flower-inhabiting species. The authors of the study, Rieske and Raffa (2003), suggest that geographic distribution, forest stand composition or thrips population size might be influential factors. In greenhouse trials F. occidentalis, also being mainly a flower-inhabiting thrips species, has been attracted to yellow (Roditakis and Lykouressis, 1996) or blue (Brødsgaard, 1990; Baranowski and Gorski, 1993) sticky traps and to yellow water traps (Teulon et al., 1993a, 1999; Hollister et al., 1995), which were scented with p-anisaldehyde. The attractiveness of this volatile compound to western flower thrips has been further confirmed in various kinds of flight chamber olfactometers in the laboratory (Teulon et al., 1993a; Frey et al., 1994; Hollister et al., 1995) and in wind tunnel assays (Teulon et al., 1999). Additionally, positive responses of walking western flower thrips to p-anisaldehyde at several concentrations ranging from 10% to 0.001% were observed in a Y-tube olfactometer. Because of their similar chemical structure, m-anisaldehyde and o-anisaldehyde were also expected to be attractive, but clear attraction was found only at 10% concentration of o-anisaldehyde (Koschier et al., 2000). In contrast to the field studies mentioned above, T. tabaci was not attracted by p-anisaldehyde at 1% concentration in the olfactometer (De Kogel and Koschier, 2003). So, as of now, only a few compounds (Table 1), with p-anisaldehyde ranking prominently among them, have been confirmed as very potent attractants for flying as well as walking F. occidentalis in different environments.

Strategies for use of attractants in thrips control An increasing number of studies deal with integrating different plant protection approaches into push–pull strategies (e.g. Pickett et al., 1997; Pickett, 1998; Bennison

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et al., 2003), which involve ‘pushing’ away the pest from the valuable main crop and ‘pulling’ it onto a sacrificial or trap crop. On trap plants the pest population can be controlled by a biological control agent or a selective pesticide. In such systems the amount of control agents and, as a consequence, the costs of labour can be minimised. Preferred host plants of F. occidentalis such as Verbena hybrida or pot chrysanthemums have been evaluated in greenhouses for use as trap plants in a push–pull strategy, luring thrips away from profitable ornamental crops (Pow et al., 1998; Hooper et al., 1999; Bennison et al., 2003). During the flowering period of the crop plants, the attractiveness of the ‘trap’ chrysanthemums was successfully increased by additionally baiting them with a field-stable formulation of the thrips attractant (E)-b-farnesene (Bennison et al., 2003). Based on extensive studies on the spectral responsiveness of thrips pest species such as F. occidentalis (e.g. Vernon and Gillespie, 1990), coloured sticky traps are commonly used for monitoring thysanopteran pest populations. As demonstrated in the case study of p-anisaldehyde (section on ‘Search for phytochemicals attractive to thrips’), several attempts have been made to combine an attractive colour cue with an attractive odour cue in order to improve trapping of different thrips pest species. Still, depending on the combination of thrips species, colour, scent, type of trap, dispenser, crop and location (field or greenhouse), the reported increase of catch results in numerous studies varied markedly, possibly influenced by variations in odour concentration and release rates, factors discussed in detail in section on ‘Concentration of allelochemicals’. Teulon et al. (1993b) considered the distance between traps to be a possible source of variation, because if distances are too small, capture rates might be underestimated. Further research will be needed to fully exploit the potential of olfactory attractants in combination with coloured traps, and to devise a composite trapping method that will reliably represent the intensity of thrips infestation in a crop and that will, in consequence, be more costeffective. However, traps baited with attractive volatile chemicals may be useful for control-trapping of thrips, this particularly in protected environments where wind does neither quickly disperse the volatiles nor interfere with the movement of thrips towards the traps (Murai et al., 2000). They may be a useful tool for the determination of the initial presence of thrips infestation in a crop (Teulon et al., 1993b; Frey et al., 1994), with this information being crucial for the prediction of outbreaks and the successful timing of control measures for some species (Teulon et al., 1993b). Another approach is the use of traps with colour and odour cues as a sole or contributory control measure. Mass trapping of F. occidentalis on greenhouse cucumber was reported to be effective using blue sticky traps in combination with the monoterpene nerol, but only at low thrips densities during the autumn/winter growing period (Roditakis et al., 2002). To further increase efficacy, an even more sophisticated trapping system could combine coloured traps baited with attractants in association with, for instance, entomopathogenic fungi in an ‘attract and infect’ strategy: thrips are lured into specific traps where they are infected with spores of an entomopathogenic fungus and pick up fungal spores to spread them through the rest of the population after leaving the trap.


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Search for repellent plant volatiles Repellents are olfactory stimuli that cause insects to move away from an odour source (Dethier et al., 1960) and may prevent insects from landing on a plant (Norris, 1990; Renwick, 1999). Thus, volatility is supposed to be essential for the repellent activity of a plant compound (Peterson and Coats, 2001). Not much is known about naturally occurring repellents in either host or non-host plants. Recent results do, however, present some indications. Koschier et al. (2000) expected to achieve clear attraction using diluted essential oils of geranium and rose, both preferred host plants of F. occidentalis, but recorded no significant reaction. With essential oils consisting of various different plant compounds, an explanation for this phenomenon could be that the presence of other odour compounds in the essential oils suppressed the effects of the attractants in these oils. Similar examples for such masking effects have been reported, for example for aphids (Nottingham et al., 1991) or beetles (Thiery and Visser, 1986; Yamasaki et al., 1997). Also, Gaum et al. (1994) observed that F. occidentalis was repelled by concentrated rose odours emanating from a mixture of six rose cultivars. Furthermore, western flower thrips avoided medium and strongly scented rose varieties, even susceptible ones, in olfactometer experiments. An interesting aspect in these findings seems to relate to odour concentration, an aspect which will later be addressed in detail. F. occidentalis proved to be repelled by one specific extract fraction of meadowsweet, although it was strongly attracted to several other fractions, the whole extract and fresh flowers (Shamshev et al., 2003). The repellent fraction may have contained the phenylpropanoid methyl salicylate, a compound that has been found to repel F. occidentalis in olfactometer bioassays (Chermenskaya et al., 2001). Methyl salicylate is a common compound in essential oils, in particular in wintergreen (Gaultheria procumbens L.) oil (Seigler, 1998). A related compound, salicylaldehyde, was found to repel the same thrips species at different concentrations in olfactometer experiments (Koschier et al., 2000). Many higher plants have evolved chemical protection against herbivores based on phenylpropanoids and/or monoterpenes. These are dominant compounds in their volatile oils, and have proved to be important not only as olfactory repellents (Norris, 1990; Rice and Coats, 1994), but also as deterrents. In particular, essential oils from plant species within the Lamiaceae family and their volatile constituents have been found to have a broad spectrum of biological activities against a number of agricultural pests (e.g. Mansour et al., 1986; Dube et al., 1989; Hori and Komatsu, 1997; Lee et al., 1997; Tunc- and S- ahinkaya, 1998; Hori, 1999a; Isman, 2000). Harrewijn et al. (2001) reported that some thrips species are repelled by crushed basil (Ocimum basilicum L.) leaves, suggesting that linalool, eugenol, caryophyllene or 1,8-cineole are the active compounds. In an extensive screening programme, Koschier and her coworkers (Koschier et al., 2002; Koschier and Sedy, 2002, 2003a, 2003b; Sedy and Koschier, 2003) investigated the biological activity of labiate essential oils against T. tabaci and F. occidentalis. Moreover, they analysed the essential oils by means of GC/MS to attribute the bioactive properties of the oils to specific pure volatile compounds—mainly monoterpenes, which constitute the corpus of many of the essential oils of Lamiaceae plants (Hallahan, 2000). But solely rosemary oil (Koschier and Sedy, 2003a) and the monoterpene carvacrol at 10% concentration (Koschier and Sedy, 2002) showed clear repellency in olfactometer experiments, and this only to T. tabaci.


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Table 2 Antifeedant and oviposition deterrent properties of labiate essential oils and pure chemical compounds against T. tabaci and F. occidentalis found in leaf-disc bioassays regardless of concentration applied on the discs (Koschier et al., 2002; Koschier and Sedy, 2003a, 2003b; Sedy and Koschier, 2003) Thrips species

Chemical class

Compound

Feeding deterrent

Oviposition deterrenta


monoterpenes

carvacrol thymol

(x)b (x)b

x x

Thrips tabaci

essential oils

lavender marjoram mint rosemary sage

x x x x -

x x x x

monoterpenes

1,8-cineole carvacrol thymol

-

(x)b x x

a

Deterrence confirmed in choice (oviposition preference) and/or no-choice (oviposition rate) bioassays. b Results not statistically significant.

In an earlier study, Holtmann (1963) screened numerous diluted organic acids for olfactory reactions of the two cereal thrips species L. cerealium and H. aculeatus. Thrips responded negatively to ammonium acetate and ammonium phosphate, and moreover to different organic acids: acetic acid, tartaric acid, malic acid, butyric acid and its derivative butyric ether were all found to be repellent at various specific concentrations. Harrewijn et al. (2001) suggested that butyric acid additions to terpenes to have some repellent effects against thrips that still have to be investigated. Please note that olfactory repellency must not be equated with gustatory deterrence involving contact chemoreception. L. cerealium and H. aculeatus were repelled by diluted acetic acid in the olfactometer, but when they got in contact with a droplet of diluted acetic acid, the negative olfactory response was overlapped by a positive gustatory response (Holtmann, 1963). In this context, although being no plant compound, a two-component alarm pheromone identified in the anal fluid of second-instar F. occidentalis (Teerling et al., 1993) is worth mentioning. The pheromone, in particular its component dodecyl acetate, caused nymphs as well as adults to move away from its source (Teerling et al., 1993), increased the flight activity of adults (MacDonald et al., 2002), but also reduced oviposition by adult F. occidentalis females (Teerling et al., 1993; Kirk et al., 1999).

Feeding and oviposition deterrents Dethier et al. (1960) defined deterrents as ‘chemicals which inhibit feeding or oviposition when present in a place where insects would, in their absence, feed or


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oviposit’. For many insects the avoidance of a non-host plant is based on initial attempts at colonisation or feeding, which are terminated by inappropriate physiology, nutritional factors, or by the detection of deterrent or even toxic secondary metabolites (Agelopoulos et al., 1999). Only few studies have investigated naturally occurring deterrents against thrips. a-Tomatine, a strong feeding deterrent in tomato leaves against T. palmi (Hirano et al., 1994), is one of these few compounds and has been discussed in detail earlier (section on ‘The role ofy.selection of Thysanoptera’). Unfortunately, the investigation of the bioactivity of a-tomatine against T. palmi was not continued and none was conducted on the factors responsible for the inhibition of this thrips species on strawberry (Kawai, 1986). As mentioned above, Koschier and her co-workers (Koschier et al., 2002; Koschier and Sedy, 2002, 2003a, 2003b; Sedy and Koschier, 2003) evaluated the effects of essential oils and/or their volatile main compounds when applied on host plants of T. tabaci and F. occidentalis (Table 2). Several of the oils and/or compounds were found to affect feeding activity and reproductive success. But even series of bioassays examining single steps in the decision process of host selection of thrips are sometimes not adequate to fully understand the mechanism(s) by which secondary metabolites act. Here, rosemary essential oil may serve as a good example: it has been demonstrated that the oil effectively interrupts the host selection behaviour of aphids (Hori and Komatsu, 1997; Hori, 1998, 1999a, 1999b). Volatile constituents in rosemary oil may be responsible for its repellency against T. tabaci at a high concentration, but also for significant settling inhibition found over a 4-h period, and feeding inhibition over a 24-h period. Interestingly, rosemary oil application did not affect the egg-laying activity of T. tabaci (Koschier et al., 2002; Koschier and Sedy, 2003a, 2003b). Further investigation of volatile and non-volatile main constituents may increase the insight into the biological activity of rosemary oil. In some cases the bioactive properties of the essential oils were successfully attributed to one of their pure compounds. It could for instance be established that the antifeedant property of lavender oil to T. tabaci is due to its main component linalool (Koschier et al., 2002). Tannin, a widespread plant defensive compound, when added to a pollen diet reduced oviposition of F. occidentalis females. It was suggested to be a locally acting egg-laying deterrent, probably reinforced by interfering with the feeding activity of the thrips (Whittaker and Kirk, 2004). But even with such results obtained in end point experiments, behavioural observations will be needed for correct interpretation and clarifying the exact mode of action of these natural products on thrips.

Strategies for use of repellents and deterrents in thrips control It is an intriguing perspective to make use of naturally occurring secondary compounds that defend plants against phytophagous insects, incorporating them in the protection of crop plants in an agricultural setting. A repellent and/or deterrent compound that is capable of irritating the insect–plant contact will cause the insect to spend a shorter time period in a treated area (Peterson and Coats, 2001). For this reason alone the incidence of economic damage might be reduced. But the precondition for any

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successful utilisation of biologically active secondary plant compounds in crop protection strategies is a profound knowledge about the behavioural reactions of the target pest to the compound. There are promising examples: mixed cropping systems can make use of the fact that repellent odours emanating from non-host plants are able to mask the normal attractiveness of a host plant. They may even repel thrips and prevent them from locating or contacting the valuable crop plants. In field trials, Kyamanywa et al. (1993) observed the bean flower thrips Megalurothrips sjostedti Trybom to orient to and to settle on sole cowpea crops in higher densities than on cowpea plants mixed with the non-host plant maize. Olfactometer assays revealed that volatiles from maize leaves interfered with the attractiveness of cowpea leaf buds. Besides olfactory masking, also other mechanisms such as visual camouflage of crop plants or changes in host plant quality have been suggested to be responsible for lower thrips infestations on intercropped plants in the field. Intercropping of pea with tansy phacelia and white mustard interfered with the search of the pea thrips Kakothrips robustus Uzel for its preferred host plant and thus reduced thrips infestation (Wnuk, 1998). Onions intercropped with carrots reduced T. tabaci infestation, possibly because of the reduced ‘apparency’ of the onion plants (Uvah and Coaker, 1984). Undersowing of leek with clovers suppressed populations of T. tabaci (Theunissen and Schelling, 1998), and Theunissen and Schelling (1999) found some evidence that flying adults prefer monocropped to undersown leek fields. However, Den Belder et al. (2000) suggest a relation between intercropping and plant quality as influencing T. tabaci densities in leek undersown with clover. Making use of the odour-masking mechanism, repellent volatiles applied on crop plants in the field in a way similar to an insecticide treatment could decrease the landing rates of thrips, especially during sensitive growth periods of the plants. Repellents might reduce immigration, for instance of T. tabaci, the onion thrips, to white cabbage or leek, where they are well-protected against insecticides, and consequently reduce infestation levels to a degree that prevents qualitative crop losses. Another aspect is that certain compounds may incite thrips to increased activity on the plant surface, thus enhancing the uptake of contact insecticides or spores of fungal insect pathogens. For example, the F. occidentalis alarm pheromone was suggested to have potential for improving the insecticide contact in the field (Cook et al., 2002). However, some behavioural observations have revealed that repellent phytochemicals incited thrips to increased flight movement of adults between plants, as was found for the F. occidentalis alarm pheromone in another study (MacDonald et al., 2002). Hence their application would have to be limited to crops where virus transmission by thrips is irrelevant. This might for example prevent the use of such an application in a greenhouse infested with F. occidentalis, because it would very likely increase the spread of tomato spotted wilt viruses (TSWV). Various antifeedant compounds from plants have been identified as potentially useful against aphid colonisation and feeding (Griffiths et al., 1989). The use of antifeedants against thrips adults may prevent direct feeding damage caused by the ingestion of plant sap, which frequently produces yield losses in vegetable, fruit and grain crops. But even more appealing is the potential use of oviposition deterrents that could inhibit the build-up of high infestation levels and the resulting quality


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losses caused by feeding damage from the larvae: particularly in ornamental crops, larvae disfigure foliage and flowers, making them unmarketable (Lewis, 1999). Finally, repellent and/or deterrent plant compounds may provide the ‘push’ element of a push–pull strategy. As F. occidentalis cause serious damage to nectarine buds in spring, Pearsall (2000) noted the preference of this thrips species for naturally occurring ground-cover blooms, and examined the potential of using such flowers as trap crops under nectarine trees. But even highly attractive blooms did not attract enough thrips to reduce densities on nectarine blooms, and the author then suggested increasing the trap crop efficiency by combining trap crops with a powerful deterrent spray on the nectarine blooms. As rosemary volatiles repelled F. occidentalis and reduced the attractiveness of chrysanthemum buds after application, they were considered as the ‘push’ component of a push–pull strategy in pot chrysanthemums. Unfortunately, the rosemary volatiles were also repellent to Orius laevigatus Fieber, a predatory bug used for biological control of western flower thrips on chrysanthemums in this strategy (Bennison et al., 2003). This leads to the conclusion that, if plant compounds are evaluated for their application against thrips pests within a push–pull strategy, their effects on the beneficial arthropods that are used in biological control have to be investigated at the same time.

Concentration of allelochemicals Without knowing the actual odour concentration around plants, it is difficult to relate the concentration of pure chemicals used in experiments to those that occur naturally (Staedler, 1992). Yet an increasing number of studies provide evidence that optimal concentration of volatile or non-volatile secondary metabolites is critical for attractiveness, repellency and even deterrence for thrips. Dosage-dependent attraction or repellence of F. occidentalis at close range has been established for both plant extracts and pure chemical compounds in olfactometer experiments (Manjunatha et al., 1998; Pow et al., 1999; Koschier et al., 2000; Chermenskaya et al., 2001; Shamshev et al., 2003), and also for the attractive extract of F. ulmaria in a tunnel olfactometer in the laboratory. Interestingly, at higher concentrations thrips preferred to leave the release point in the olfactometer by takeoff rather than by walking (Shamshev et al., 2003). Holtmann (1963) described the concentration-dependent attractiveness of lactic acid to the cereal thrips species L. cerealium and H. aculeatus. Sedy and Koschier (2003) noted that feeding as well as oviposition of F. occidentalis and T. tabaci were affected in dependence on the concentration of essential oils or their pure main compounds applied on their host plants. In these bioassays the oils/compounds were effective at both 1% and 0.1% concentrations, but not at a lower concentration (Koschier et al., 2002; Koschier and Sedy, 2003b; Sedy and Koschier, 2003). In most of these laboratory investigations, some phytochemicals were found to evoke responses solely at specific concentrations, whereas others proved effective at a considerably broader range of concentrations. Another interesting aspect is that high concentrations of otherwise attractive volatile compounds may have a repellent action at close range (Visser, 1986), a

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phenomenon recognised with kairomones that originally had a function as allomones (Harrewijn et al., 1995). Pure p-anisaldehyde, confirmed to be a potent attractant for F. occidentalis in many experiments, clearly repelled western flower thrips in the olfactometer (Koschier et al., 2000). Release rates of volatiles, or quantities or concentrations of chemicals determined to have a specific effect in an experimental set-up in the laboratory are difficult to correlate with greenhouse trials. Although using the same odour release systems for the F. occidentalis attractant geraniol, Frey et al. (1994) were not able to reliably reproduce their laboratory results in the greenhouse. They suggested changes in temperature and/or humidity in the greenhouse as causing variations in release rates. Attractiveness of (E)-b-farnesene to F. occidentalis was elicited exclusively at one specific quantity or release rate in the respective experiment. A quantity of 1 mg (E)b-farnesene applied on filter paper in olfactometer experiments, a quantity of 500 ng filled in a glass vial with a hole in the cap and a release rate of 2 mg/day of the chemical applied on foam squares in polyethylene sachets in greenhouse trials each significantly attracted western flower thrips (Pow et al., 1999). Most release systems used in greenhouse and field experiments are simple dispensers, for instance a glass vial containing the test substance stoppled with a cotton wool wick (Teulon et al., 1993b), or the substance mixed with a solvent applied on a cotton wool plug attached to a coloured trap (e.g. Roditakis and Lykouressis, 1996). Such differences in release methods and the resulting different diffusion rates may well have contributed considerably to the discrepancies in the trapping efficiency when using volatiles for thrips attraction. It may be safely concluded that proper evaluation of different odour concentrations or release rates used, particularly in greenhouse and field trials, is as crucial as the development of suitable odour dispensers that will ensure a long-term odour release.

Acute and chronic toxicity of plant allelochemicals to thrips The main focus of this chapter has been on secondary plant compounds as ‘infochemicals’ working by non-toxic mechanisms for management of thrips pests. Yet, the insecticidal properties of many of these plant compounds must not be neglected. Actually, any plant compound considered for utilisation as crop protection agent should be tested for its toxicity to the target pest, particularly when the intended application on crop plants is similar to an insecticide treatment. Bioassay procedures for plant compounds are quite similar to those applied for synthetic insecticides, and need not to be discussed in detail here. Acute toxicity of a substance to thrips can be assessed in residual and/or direct topical bioassays (Immaraju et al., 1990; Helyer and Brobyn, 1992). For the latter, the test substance is applied directly to the insect, either by using a spraying apparatus, a microapplicator or, more simply, by total immersion of thrips in dilutions of the substance. Chiasson et al. (2004) treated F. occidentalis adults and third and fourth instars topically with a botanical and compared its insecticidal efficacy to commercially available insecticides. In laboratory bioassays, this botanical with the active ingredient (a.i.) being an essential oil extract of Chenopodium ambrosioides variety near ambrosioides caused 95.7% mortality at 0.5% a.i. concentration 24 h after treatment and thus, proved to be more


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effective than neem oil (0.7% a.i.) or insecticidal soap (1% a.i.). For comparison only, in a similar bioassay direct topical treatment with the bioinsecticide spinosad (60 mg a.i. per litre) (Jones et al., 2005) – currently a common insecticide used to manage this pest in greenhouses (Loughner et al., 2005) – resulted in 100% mortality of western flower thrips adults (Jones et al., 2005). Also Helyer and Brobyn (1992) evaluated the direct contact toxicity of several insecticides from different chemical classes against F. occidentalis adults in comparable assays and found e.g. the pyrethroid deltamethrin (17.5 mg a.i. per litre) and the organochlorine endosulfan (500 mg a.i. per litre) to kill 34.1% and 86.6% thrips, respectively. It should be noted that an isolated comparison of dose rates in different bioassays may be a problematic tool for evaluating the merits of a substance in the framework of a pest control strategy. In residual bioassays the plant material is either sprayed with or dipped in serial dilutions of the test substance and, after allowing it to dry, thrips are introduced. Residual bioassays with T. tabaci females on leek leaves treated with serial dilutions of several labiate essential oils, monoterpenes or phenylpropanoids showed only marjoram (Origanum marjorana L.) essential oil and its main constituent terpinen-4ol to reduce adult survival on a treated plant surface, and that only slightly and at a relatively high (1% a.i.) concentration (Koschier et al., 2002). No other oil or plant compound tested caused increased mortality of T. tabaci. In contrast, in residual bioassays spinosad (60 mg a.i. per litre) was toxic to 100% F. occidentalis adults (Jones et al., 2005). It must be noted that sub-lethal or chronic effects of secondary compounds appear to be more important in the plant’s defence system than any acute toxicity (Rice and Coats, 1994), and should therefore also be evaluated. Especially terpenes have the potential to inhibit stages of the developmental process of insects such as moulting and adult emergence. For instance, pulegone retarded the development and inhibited reproduction of the lepidopteran species Spodoptera eridania Cramer, although it did not prove toxic even to the more sensitive larval stages (Gunderson et al., 1985). Bisabolene significantly decreased the total offspring production of the aphid species Myzus persicae Sulzer when applied to host plant leaves (Gutie´rrez et al., 1997). While long-term effects of secondary compounds on the reproductive fitness of thysanopteran females and on the duration of developmental stages, i.e. growth inhibition, have not yet been investigated, such sub-lethal effects could well contribute to reducing the build-up of thrips populations on crops.

Conclusions and future prospects In the light of the increasing economic impact of thrips pests and widespread demands to reduce the ecological side-effects of conventional pest control strategies, the search for alternatives is under way, and the prospects of plant protective agents extracted from renewable herbal raw materials as control agents are convincing: they are natural, many of them are inexpensive to produce and they are generally regarded as environment friendly (Isman, 1999). Harrewijn et al. (2001) emphasise the importance of additive or synergistic effects with volatile compounds and strongly suggest running comparative experiments with multicomponent blends versus single components. Isman (2000) points out that one of the most promising aspects is that

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plant compounds can be blended to obtain a special spectrum of biological activity and efficacy against pests. Moreover, mixtures, particularly of antifeedants, can mitigate the problem of insects becoming rapidly habituated to plant compounds (Isman, 2002). Besides, knowledge of plant compounds that deter feeding or oviposition can be extremely valuable to plant breeders in their search for resistant varieties (Renwick, 1990). Yet several key questions remain to be answered when seeking future integration of plant allelochemicals in thrips management strategies. Many natural plant compounds are used as fragrances and flavouring agents in food, some even in medical preparations. Therefore, while a good deal of toxicological data on vertebrates are available for most of these compounds, their effects on mortality, life history parameters and behaviour of beneficial arthropods, especially on those that are used in biological control, have not yet been evaluated. For instance, rosemary volatiles were found to be effective against F. occidentalis, but also to repel the predatory anthocorid O. laevigatus, making them valueless as an element of a push–pull strategy (Bennison et al., 2003). O. laevigatus did not show any olfactory responses to the monoterpene nerol (Kornherr and Blu¨mel, 2005), which is highly attractive to F. occidentalis (Koschier et al., 2000) and considered as a potential ‘pull’ element (Kornherr and Blu¨mel, 2005; Kornherr et al., 2005). With carvacrol, which deters the thrips (Sedy and Koschier, 2003), significant attraction to the predatory bug was observed (Kornherr and Blu¨mel, 2005). Neither lethal nor sub-lethal effects on reproduction of O. laevigatus of either monoterpenes were detected (Kornherr et al., 2005). Additionally, a full evaluation of the role of biologically active plant compounds in the chemical ecology of Thysanoptera calls for behavioural observations of, for instance, flight responses to odours or feeding and oviposition responses to deterrents. A profound knowledge of behavioural patterns and activities of the targeted thrips species is the precondition for successful behaviour manipulation in control strategies. Another problem specific to plant secondary metabolites is that some of them are phytotoxic (Lee et al., 1997), a fact that has to be considered when commercial products are formulated. Moreover, research has to be directed to formulations for volatile compounds and odour dispensers that ensure long-term odour release, as well as to formulations that improve the efficacy and persistence of nonvolatile compounds. These problems, together with availability and quality of the natural source material, and the high costs of registration form obstacles to the commercial development of plant compounds as control agents. Furthermore, legislative changes may alter the existing situation. For instance, the US Environmental Protection Agency may exempt ‘minimum risk pesticides’, including plant volatile constituents and essential oils for use as pesticides, from registration when these phytochemicals are employed within the scope of control strategies (Stewart and Weatherstone, 2002). While the collected results do not suggest that an application of plant compounds can be sufficiently effective as the sole control measure against thysanopteran pests, they are promising synergists for various biological or chemical control measures within the framework of both biological pest management strategies and IPM. Integration of behaviour-modifying natural products into control strategies is widely seen as the most promising approach to improvement of thrips control (Pickett et al., 1997; Pickett, 1998; Hooper et al., 1999). Mound and Teulon (1995) also point out


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that a successful thrips management strategy has to include a broad range of approaches. Various authors have stated rightly that achieving this will call for a fair amount of innovative thinking. Isman (2002) points out that ‘given that many antifeedants do not kill pests outright, and even their behavioural effects may be ephemeral under field conditions, their utility may ultimately depend on deploying them with more creative strategies.’ Which, of course, applies to all repellents, attractants and deterrents as well. In conclusion, it appears to me that an important way forward in this area of research would be to develop standardised procedures for laboratory, glasshouse and field experiments in order to be able to compare meaningfully and convincingly the effectiveness of different allelochemicals and their application in various control strategies.

Acknowledgments I would like to thank Michael Lynn for his encouragement and assistance in preparing the manuscript. And I thank Dr. William D.J. Kirk for his valuable suggestions and comments on the manuscript.

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CHAPTER 11

Importance of plant secondary metabolites for protection against insects and microbial infections MICHAEL WINK

Introduction A typical feature of plants is the production and accumulation of secondary metabolites (SMs). More than 50,000 structures have been determined by NMR, MS, and X-ray analysis (Harborne, 1993; Seigler, 1998; Wink, 1999a, 1999b). Since less than 20% of all plants have been studied in some depth so far, it is very likely that the real number of SMs that exist in the plant kingdom exceeds 100,000 structures. We can distinguish between N-containing and N-free SMs. Table 1 gives an overview of known classes and numbers of SM structures. Whereas primary metabolites are present in all plant species, SMs occur in mixtures that differ between species and systematic units. They are not essential for energy metabolism and life but, as discussed later, they are important for the ecological fitness and survival of plants (Wink, 1988, 1993, 1999a; Harborne, 1993; Seigler, 1998). SMs are produced in specific pathways that involve substrate-specific biosynthetic enzymes (Luckner, 1990; Dewick, 2002). Sites of synthesis can differ between types of compounds and between plant species. Some compounds can be produced by all tissues, whereas most others are produced in a tissue- or even cell-specific fashion. It is likely that the corresponding genes are regulated by specific transcription factors similar to the situation of other genes that are differentially regulated. The site of synthesis is not necessarily the site of accumulation. For several compounds it has been shown that they are transported within a plant via either the phloem or the xylem. Whereas hydrophilic compounds (such as alkaloids, amino acids, glucosinolates, cyanogenic glycosides, flavonoids, tannins, carbohydrates, and saponins) are stored in the vacuole, the lipophilic SMs (such as many terpenoids) are sequestered in resin ducts, laticifers, or in special (usually dead) cells such as oil cells, trichomes, or in the cuticle. Often epidermal cells that have to ward off enemies in the first instance are especially rich in SMs (Wink, 1988, 1999a). It is a typical feature of SMs that many of them are stored at relatively high levels in sink tissues that are often those which are important for the survival and

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Table 1 Structural types of secondary metabolites and known structures Class With nitrogen Alkaloids Non-protein amino acids (NPAA) Amines Cyanogenic glycosides Glucosinolates Alkamides Lectins, peptides Without nitrogen Monoterpenes (including iridoids) Sesquiterpenes Diterpenes Triterpenes, steroids, saponins Tetraterpenes Phenylpropanoids, coumarins, lignans Flavonoids, tannins Polyacetylenes, fatty acids, waxes Polyketides (anthraquinones) Carbohydrates

Number of structures 29,000 700 100 60 100 150 800 2500 5000 2500 5000 500 2000 4000 1500 750 200

reproduction of a plant, such as flowers, seeds, seedlings, or the bark of perennial plants. Several SMs are not end products of metabolism but can be recycled. For example, nitrogen-containing SMs such as alkaloids, non-protein amino acids (NPAAs), or lectins are often accumulated as toxic nitrogen storage compounds in seeds of legumes whose nitrogen is remobilized during germination and seedling growth (Wink, 1988, 1999a; Rosenthal and Berenbaum, 1991, 1992; Harborne, 1993).

Function of secondary metabolites Plants cannot run away when attacked by herbivores nor do they have an immune system against infecting bacteria, fungi, or viruses. Similar to the situation in other sessile organisms (such as marine animals), plants have developed biologically active SMs during evolution that help them to defend themselves against herbivores (insects, mollusks, vertebrates), microbes, viruses, and other competing plants (Figure 1) (Swain, 1977; Wink, 1988, 1999a; Harborne, 1993; Roberts and Wink, 1998). In order to be effective, SMs need to be present at the right site, time, and concentration in a plant. The biosynthesis of several SMs is constitutive, whereas in many plants it can be induced and enhanced by biological stress conditions, such as wounding or infection. This activation can be biochemical, e.g., through hydrolysis of glycosides that are stored as ‘‘prodrugs’’ (Table 2) or via the activation of genes responsible for synthesis, transport, or storage of SMs. Signal transduction pathways that lead to gene activation include the pathway leading to jasmonic acid or salicylic acid that have been found to trigger defense reactions in plants (Kessler et al., 2004).

Importance of plant secondary metabolites for protection

253

Plant secondary metabolites UV-Protection N-storage

Function

Attraction

Defence

Herbivores •insects •molluscs •vertebrates

•repellence •deterrence •toxicity •growth inhibition

Microbes/ viruses

Competing plants

•bacteria •fungi

•growth inhibition •toxicity

•pollinating insects •seed dispersing animals •root nodule bacteria •adapted herbivores (specialists)

inhibition of •germination and •growth of seedlings

Fig. 1. Function of secondary metabolites in plants. Table 2 Typical ‘‘prodrugs’’ present in plants that are activated by wounding or infection SM of undamaged tissue

Active allelochemical

Cyanogenic glycoside Glucosinolate Alliin Coumaroylglycoside Arbutin Salicin, methylsalicylate Gein Bi-desmosidic saponins Cycasin Ranunculin

HCN Isothiocyanate Allicin Coumarin Quinone Saligenin, salicylic acid Eugenol Mono-desmosidic saponins Methylazoxymethanol (MAM) Protoanemonine

Plants use SMs (such as volatile essential oils and colored flavonoids or tetraterpenes) also to attract insects for pollination or other animals for seed dispersion. In this case SMs serve as signal compounds. In addition, some SMs concomitantly carry out physiological functions, for example alkaloids, NPAAs, and peptides (lectins, protease inhibitors) can serve as mobile and toxic nitrogen transport, and storage compounds or phenolics, such as flavonoids, can function as UV-protectants (Wink, 1988, 1999a, 1999b; Harborne, 1993). Besides chemical defense a number of plants use mechanical and morphological features for protection, such as thorns, spikes, glandular, and stinging hairs


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Adaptations to chemical defence Herbivores

Microorganisms

avoidance of toxic plants, except host plants

inactivation of SM

non-resorption orfastintestinalfoodpassage

evolution of insensitivity

resorption followed by detoxification •hydroxylation; conjugation; •elimination (via faeces, urine) resorption and accumulation •specific compartments/cells/tissues for sequestration •evolution of insensitivity utilisation of dietary secondary products •defence against predators (e.g., cardiac glycosides, iridoid glycosides, cyanogenic glycosides, pyrrolizidine alkaloids, quinolizidine alkaloids, aconitine, phorbol esters) •signal molecule; pheromones (e.g., pyrrolizidine alkaloids) •morphogen (e.g., pyrrolizidine alkaloids; retinoic acid)

Fig. 2. Adaptation strategies of specialists.

(often filled with noxious chemicals), or develop a hardly penetrable bark (especially woody perennials). The defense strategy works against generalists, but usually not against specialists that have adapted to their host plants and their defense chemicals. A diverse collection of adaptations has already been detected and described (Figure 2). In general, it was observed that mono- and oligophagous insects can tolerate the defense chemicals of their host plants but are susceptible to those of non-host species (Wink, 1988, 1999a, 1999b). The observed multiple functions are typical and do not contradict the main role of many SMs as chemical defense and signal compounds. If a costly trait can serve multiple functions (and the maintenance of the biochemical machinery to produce and store SMs is energetically costly; Wink, 1999a), it is more likely that it is maintained by natural selection as it provides a selective advantage for its carrier.

Modes of action In order to fulfill the role of defense substances against herbivorous animals and microbes, SMs must be able to interfere with molecular targets in their organs, tissues, and cells. Figure 3 summarizes the major types of molecular targets in pro- and eukaryotes that are relevant in this context. Major targets include (1) the biomembrane, (2) Proteins, and (3) nucleic acids (DNA, RNA), which has been discussed in the following (Wink, 1988, 1999a, 2000; Roberts and Wink, 1998; Teuscher and Lindequist, 1998; van Wyk and Wink, 2004).


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(A) Proteins •Transporter •Enzymes •Structural proteins •Regulatory proteins

DNA Replication Transcription gyrase

Cell wall

biomembrane

polysomes

ribosomes

plasmid

(B) Receptors Ion channels Signal transduction Microtubules Y Y

Transporters Ribosomes Protein biosynthesis Nucleus

Vesicles

Actin filaments Biomembrane •Enzymes •Structural proteins •Regulatory proteins

ER Lysosomes Golgi

DNA Replication Transcription Repair

Fig. 3. Major molecular targets of cells. (A) Bacterial cell and (B) animal cell.

The biomembrane surrounds every living cell and functions as a permeation barrier. It prevents the leakage of polar molecules out of the cell or the entry of unwanted molecules into a cell. Several SMs exist in nature that interfere with membrane permeability (Table 3). Most famous are saponins that occur widely in the plant kingdom; mono-desmosidic saponins (Table 2) are amphiphilic and function as detergents. With their lipophilic moiety they are anchored in the lipophilic membrane bilayer, whereas the hydrophilic sugar part remains outside and interacts with other glycoproteins or glycolipids (Figure 4). As a result, pores are generated in the membrane and makes them leaky. This can be shown easily with red blood cells. If saponins are present, hemolysis can be seen, i.e., hemoglobin flows out of the cells. Also other lipophilic SMs such as mono-, sesqui-, and diterpenes can disturb membrane fluidity at higher concentration. These compounds will interact with the lipophilic inner core of biomembranes represented by phospholipids and cholesterol (Figure 4). Such a membrane disturbance is unselective and therefore SMs with such


256 Table 3 Molecular interaction of secondary metabolites Target Unselective targets Biomembrane

Proteins Non-selective interactions

Activity

SM (examples)

Membrane disruption Disturbance of membrane fluidity Inhibition of membrane proteins (change of protein conformation)

Saponins Small lipophilic SM

Non-covalent bonding (change of conformation)

Phenylpropanoids, polyphenols such as flavonoids, catechins, tannins, lignans, quinones, anthraquinones, isoquinoline alkaloids Isothiocyanates, sesquiterpene lactones, allicin, protoanemonine, furanocoumarins, iridoids (aldehydes), SM with aldehydes, SM with exocyclic CH2 group, SM with epoxide group HCN from cyanogens, many structural mimics Phorbol esters, caffeine

Covalent bonding (change of conformation)

Specific interaction

Inhibition of enzymes Modulation of regulatory proteins

DNA

Inhibition of microtubule formation Inhibition of ion pumps Inhibition of protein biosynthesis Inhibition of transporters Modulation of hormone receptors Modulation of neuroreceptors Modulation of ion channels Modulation of transcription factors Covalent modifications (point mutations) Intercalation (frame-shift mutations)

Inhibition of DNA topoisomerase I Inhibition of transcription

Small lipophilic SM

Vinblastine, colchicine, podophyllotoxin, Taxol Cardiac glycosides Emetine, cycloheximide Non-protein amino acids Genistein, many other isoflavonoids Nicotine, many alkaloids Aconitine, many alkaloids Cyclopamine, hormone mimics Pyrrolizidine alkaloids, aristolochic acids, furanocoumarins, SM with epoxy groups Planar, aromatic and lipophilic SM, sanguinarine, berberine, emetine, quinine, furanocoumarins, anthraquinones Camptothecin Actinomycin D


sugars O

O

wounding

A

O

B

C

H

2

3

5 4

257

Fig. 4. Interactions of secondary metabolites with biomembranes and membrane proteins. (1) Sarsaparilloside (a steroidal saponine), (2) ocimene (linear monoterpene), (3) alpha-pinene (bicyclic monoterpene), (4) cadinene (sesquiterpene), (5) phyllocladene (diterpene); (A) ion channel, (B) transporter, (C) membrane receptor.

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properties are toxic to bacterial, fungal, and animal cells. Some of them even affect viral membranes. Biomembranes contain a wide set of membrane proteins, including ion channels, transporters for nutrients and intermediates, receptors, and proteins of signal transduction, and the cytoskeleton. These proteins can only work properly, if their structures are in the right conformation. Membrane proteins with transmembrane domains are stabilized by the surrounding lipids. If lipophilic SMs are dissolved in the biomembrane, they disturb the close interaction between membrane lipids and proteins thus changing the protein conformation (Table 3, Figure 4). Usually a loss of function is the result. This property can be shown with anesthetics that are small and lipophilic compounds. They inactivate ion channels and neuroreceptors and thus block signal transduction. Several of the small terpenoids can react in a comparable way. Proteins have multiple functions in a cell, ranging from enzymes, transporters, ion channels, receptors, microtubules, histones to regulatory proteins (signal molecules, transcription factors, etc.). As mentioned above, proteins can only work properly if they have the correct conformation. Probably most SMs that have been found in nature interact with proteins in one or other way. Most SMs interfere with proteins in an unselective way, i.e., they affect any protein that they encounter (Figures 5 and 6). Such unselective interactions can be divided into those that involve noncovalent bonding and those with covalent bond formation. A major class of SMs includes structures with phenolic hydroxyl groups such as phenylpropanoids, flavonoids, catechins, tannins, lignans, quinones, anthraquinones, and several alkaloids (Table 3, Figure 6). The phenolic hydroxyl groups can partly dissociate under physiological conditions resulting in O– ions. The polyphenols have in common that they can interact with proteins by forming hydrogen bonds and ionic bonds with electronegative atoms of the peptide bond or the positively charged side chains of basic amino acids, respectively. A single of these noncovalent bonds is quite weak. But because several of them are formed concomitantly, a change in protein conformation is likely to occur leading mostly to protein inactivation. However, the formation of covalent bonds also occurs (Figure 5). Several types of SMs carry reactive substituents that can bind to amino and SH groups or to double bonds of proteins. Also this alkylation leads to a conformational change and thus loss of activity. SMs with reactive functional groups that are able to undergo electrophilic or nucleophilic substitutions are represented by isothiocyanates, allicin, protoanemonin, iridoid aldehydes, furanocoumarins, valepotriates, sesquiterpene lactones, and SMs with active aldehydes, epoxide, or terminal and/or exocyclic methylene groups (Table 3, Figure 5). In several instances the reactive metabolites are not natively present in plants. They can be converted to active metabolites either by the wounding process (releasing metabolizing enzymes) or in the animal body (transformation in intestine or liver). In addition to the unselective interactions, several SMs are known to modulate protein activities in a specific way in that they can bind as a ligand to the active site of a receptor or enzyme (Figure 7); this phenomenon has been termed ‘‘induced fit’’. In this case, the structure of a given SM is often a mimic of an endogenous ligand. Well-studied examples include several alkaloids that are structural analogues of

CH

CH 2 N

C

CH2

S

10 NH 2

C O

2

S

S

1

S

SH

H

S

O

9

CH2 H

HS

HO CH3

O

3

O

O

O O

CH

O

O

CH

O

OH

8

4


CH 2

HO O

O

OCH3

OCH3 O

N

O

N

5

O

O

7

O CH3

O

6 OH

O

259

Fig. 5. Interactions of secondary metabolites with proteins (covalent modifications). 1. allylisothiocyanate (mustard oil), 2. citral (linear monoterpene), 3. iridoid with opened lactol ring, 4. safrole (phenylpropanoid), 5. dictaminine (furoquinoline alkaloid), 6. plumbagin (naphthoquinone), 7. psoralen (furocoumarin), 8. helenaline (sesquiterpene lactone), 9. allicin (SH reagent), 10. BBT (polyine).

260

OH OH

O

1

O

O

HO

O

O O

OH

HN

OH

HO

O

OH

O

O

-

OH

OH

O

O HO

O

OH

O

O

HO

OH OH

OH

OH

O

OH

-

H O

OH HO

O

N

+

H H

OH

HO OH

OH O

OH

2

OH OH

OH

O

OH

OH OH

HO

CH3

HO

CH3

4 HO

COOH

OH

O

OH

OH

O

3 OH

OH

5

OH

O

OH

O

O

Fig. 6. Interactions of polyphenols with proteins (non-covalent interactions). 1. Pentagalloylglucose (gallotannine), 2. dimeric procyanidin B4 (catechol tannin), 3. hypericine (dimeric anthraquinone), 4. kaempherol (flavonoid), 5. rosmarinic acid (phenylpropanoid).


OH HO

-


261

neurotransmitters, e.g., nicotine binds to nAChR or hyoscyamine to mAChR. The various steps in neuronal signaling and signal transduction provide central targets that are affected by several amines and alkaloids. Target can be the neuroreceptor itself. Agonists mimic the function of a neurotransmitter (acetylcholine, dopamine, noradrenalin, adrenaline, serotonin, GABA, glutamate, glycine, endorphines, peptides) by binding to its receptor and causing the normal response. Antagonists (often called ‘‘blocker’’) also bind to the receptor but act as an inhibitor of the natural ligand by competing for binding sites on the receptor, thereby blocking the physiological response. Further targets are voltage-gated Na+, K+, and Ca2+ channels and the enzymes, which deactivate neurotransmitters after they have bound to a receptor, such as acetylcholine esterase, monoamine oxidase, and catechol-O-methyltransferase. Also relevant are transport processes, which are important for the uptake and release of the neurotransmitters in the presynapse or synaptic vesicles. Also Na+, K+, and Ca2+-ATPases, which restore the ion gradients, must be considered in this category. Furthermore, the modulation of key enzymes of signal pathways, including adenylyl cyclase (making cAMP), phosphodiesterase (inactivating cAMP or cGMP), phospholipase C (releasing inositol phosphates such as IP3 and diacylglycerol (DAG)), and several protein kinases such as protein kinase C or tyrosine kinase (activating other regulatory proteins or ion channels) are important steps for which inhibitors from nature are known. Another example of a more specific inhibitor is HCN released from cyanogenic glycosides. HCN is highly toxic for animals or microorganisms due to its inhibition of enzymes of the respiratory chain (i.e., cytochrome oxidase) because it blocks the essential ATP production. HCN also binds to other enzymes containing heavy metal ions. In case of emergency, i.e., when plants are wounded by herbivores or other organisms, the cellular compartmentation breaks down and vacuolar cyanogenic glycosides come into contact with an active b-glucosidase of broad specificity, which hydrolyses them to yield 2-hydroxynitrile (cyanohydrine). 2-Hydroxynitrile is further cleaved into the corresponding aldehyde or ketone and HCN by a hydroxynitrile lyase. A number of diterpenes are infamous for their toxic properties, such as phorbol esters of Euphorbiaceae and Thymelaeaceae. They activate protein kinase C that is an important key regulatory protein in animal cells. Or another diterpene forskolin acts as a potent activator of adenynyl cyclase. Another diterpene is taxol A (paclitaxel, taxols) that can be isolated from several yew species (including the north American Taxus brevifolia and the European Taxus baccata). Taxol stabilizes microtubules and thus blocks cell division in the late G2 phase; because of these properties taxol has been used for almost 10 years with great success in the chemotherapy of various tumors. Microtubule formation is a specific target for the alkaloids vinblastine (from Catharanthus roseus), colchicine (Colchicum autumnale), or the lignan podophyllotoxin (from Podophyllum and several Linum species). A special case of steroidal saponins are cardiac glycosides that inhibit Na+, K+ATPase, one of the most important target in animal cells responsible for the maintenance of ion gradients. Cardiac glycosides can be divided into two classes: cardenolides have been found in Scrophulariaceae (Digitalis), Apocynaceae (Apocynum sp., Nerium sp., Strophanthus sp., Thevetia sp.), Asclepiadaceae (Periploca sp.,

2 H

N

OH

O

N O

H

3

H NH2

4 H3C O N N

CH3

5

N

N H

OCOCH3

H3COOC

COOCH3 N

CH3O

CH3

1

HOOC C CH2 CH2 O

N O

O

OH

N

CH3

262

H

H3C N

OH

H3C

NH

OH

C NH2

O O O O

6 -

H3CO

OCH3 OCH3

H

-

O3SO O3SO

CH2OH O O

C O

7

H COOH

O O

Ligand – receptor interactions

OH HO

OCO(CH2)12CH3 OC OCH3

OH

OH

HO COOH

10

9

OH

O

OH

rha O

OH

8

CH2OH

Fig. 7. Examples for ligand–receptor interactions. 1. Canavaline (non-protein amino acid), 2. hyoscyamine (tropane alkaloid), 3. lupanine (quinolizidine alkaloid), 4. physostigmine (indole alkaloid), 5. vinblastine (dimeric monoterpene indole alkaloid), 6. podophyllotoxin (lignan), 7. atractyloside (diterpenes), 8. ouabain (cardiac glycoside), 9. TPA (phorbolester), 10. salicylic acid (phenolic acid).


O

R O

O N

N

11

CH3

N H

H3CO


R

1

R

R H3CO

O

O N

H3CO

N

CH3

2 OCH3

HN

OCH3

10

O O

OCH3

CH3

O

+

N

O

O

O

O

3

glucose

O

N

O

N N

+

O

8

CH3 H3C OH

9 H3CO

O

H

N

CH3

H

OCH3

-

N

HO

O

O

H3C

4

O O

O

6

COOH

N

NO2

H3C OH

OH O CH3

intercalation

5

OCH3

H3C

O

7O

O glucose

O

O

modification 263

Fig. 8. Interactions (intercalation, covalent modifications) of secondary metabolites with nucleic acids. 1. Harmaline (beta-carboline alkaloid), 2. emetine (isoquinoline alkaloid), 3. sanguinarine (protoberine alkaloid), 4. quinine (quinoline alkaloid), 5. aristolochic acid, 6. ptaquiloside (sesquiterpene), 7. safrole (phenylpropanoid), 8. dictamine (furoquinoline alkaloid), 9. cycasine (azoxyglycoside), 10. psoralen (furocoumarin), 11. pyrrolizidine alkaloid.

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Xysmalobium sp.), Brassicaceae (Erysimum sp., Cheiranthus sp.), Celastraceae (Euonymus sp.), Convallariaceae (Convallaria sp.), and Ranunculaceae (Adonis sp.). Bufadienolides occur in Crassulaceae (Kalanchoe sp.), Hyacinthaceae (Urginea sp.), and Ranunculaceae (Helleborus sp.). A further example of specific protein inhibitors can be found in the class of NPAAs that often figure as anti-nutrients or anti-metabolites. Many NPAAs resemble protein amino acids and quite often can be considered to be their structural analogues that may interfere with the metabolism of a herbivore: for example, in ribosomal protein biosynthesis NPAAs can be accepted in place of the normal amino acid leading to defective proteins. NPAAs may competitively inhibit uptake systems for amino acids. NPAAs can inhibit amino acid biosynthesis by substrate competition or by mimicking end product mediated feedback inhibition of earlier key enzymes in the pathway. An important target in all organisms is DNA and the enzymes involved with DNA replication, DNA repair, DNA topoisomerase, and transcription (Figures 3 and 8). Also the translation of mRNA into protein in ribosomes is a basic target that is present in all cells. Inhibitors to these systems are often active against bacteria, fungi, and animal cells. The DNA itself can be modified by compounds with reactive groups, such as epoxides. Infamous are pyrrolizidine alkaloids, aristolochic acid, cycasin, furanocoumarins, and SMs with epoxy groups (often produced in the liver). Covalent modifications can lead to point mutations and deletion of single bases or several bases if the converted bases are not exchanged by repair enzymes. Other SMs with aromatic rings and lipophilic properties intercalate DNA, which can lead to frame-shift mutations. Intercalating alkaloids include emetine, sanguinarine, berberine, quinine, b-carboline, and furoquinoline alkaloids (Wink and Schimmer, 1999). Furocoumarins combine DNA alkylation with intercalation. Furocoumarins can intercalate DNA and upon illumination with UV light can form cross-links not only with DNA bases but also with proteins. They are therefore mutagenic and possibly carcinogenic. These compounds are not only abundant in Apiaceae (contents up to 4%) but also present in certain genera of the Fabaceae and Rutaceae. Because frame-shift mutations and non-synonymous base exchanges in proteincoding genes alter the amino acid sequence, such mutations are usually deleterious for the corresponding cell. If they occur in germline cells such as oocytes and sperm cells even the next generation is negatively influenced either through malformations or through protein malfunctions responsible for certain kinds of health disorders or illnesses. Interference with DNA, protein biosynthesis, and related enzymes can induce complex chain reactions in cells. Among apoptosis, a process that leads to programmed cell death, figures as an important process. Several alkaloids, flavonoids, allicin, and cardiac glycosides have been shown to induce apoptosis in primary and tumor cell lines. Summarizing, structures of allelochemicals appear to have been shaped during evolution in such a way that they can mimic the structures of endogenous substrates, hormones, neurotransmitters, or other ligands; this process can be termed ‘‘evolutionary molecular modeling.’’ Other metabolites intercalate or modify DNA, inhibit DNA and RNA related enzymes, protein biosynthesis, other metabolic enzymes, and functional proteins, or they disturb membrane stability and membrane proteins.


265

In general, we find a series of related compounds in a given plant species; often a few major metabolites and several minor components are present, which differ in the position of their chemical groups. The metabolic profile usually varies between plant organs, within developmental periods, and sometimes even diurnally. Also marked differences can usually be seen between individual plants of a single population, even more so between members of different populations. Even small changes in chemistry can be the base for a new pharmacological activity. It is evident that SMs are often multifunctional compounds and most SMs carry more than one pharmacologically active chemical group (pharmacophor). In addition, SMs usually occur in complex mixtures of various types. As a consequence, the SMs present in a given plant affect always several molecular targets. Therefore, it is likely that several targets are modulated concomitantly in herbivores or microbes. It is likely that synergistic effects enhance the activity of individual components.

The dilemma of crop plants Under natural conditions when herbivores and microbes are present, plants would hardly survive without defensive SMs. This was basically also true for the ancestors of our crop plants and vegetables. Since the SMs would either have a bad taste or would be toxic for humans, an important step in human evolution was the selection and domestication of wild plants. During domestication plant breeders selected varieties without noxious SMs or with reduced levels. Table 4 lists some of the SMs that have been selected away in crop plants. As a consequence, most crop plants have thus lost their natural resistance against herbivores and/or microbes. As a result, farmers can only grow these plants if external defense compounds are applied, Table 4 Strategies to remove secondary metabolites from food plants Strategy

SM

Food plant

Breeding of SM-free plants

Quinolizidine alkaloids Steroid alkaloids Cyanogenic glycosides Glucosinolates

Lupin Potato Almond, cassava Rape, cabbage, broccoli, cauliflower, raddish Rape Lettuce Legumes Quinoa Cucumber Beans, peas Beans, peas Potato Cabbage, cauliflower Quinoa Cucumber Citrus fruits Potato

Boiling Leaching Peeling

Erucic acid Sesquiterpenes NPAAs Saponins Cucurbitacins Lectins Protease inhibitors Steroid alkaloids Glucosinolates Saponins Cucurbitacins Essential oil Steroid alkaloids


266

the pesticides (Wink, 1988; Roberts and Wink, 1998). The extensive use of synthetic pesticides in modern agriculture provides known ecological and economic problems. In some instances, humans have chosen a different strategy to get rid of noxious SMs in food items (Table 4). Since SMs are stored in epidermal or outer cell and tissue layers, peeling is an obvious solution. This is an established practice for potatoes, cucumbers, or citrus fruits. A number of SMs can be destroyed by heat; therefore, cooking is another way to eliminate unwanted SMs, e.g., lectins, protease inhibitors, or other toxic peptides and proteins. If plants are boiled in water, SMs (e.g., potato alkaloids, glucosinolates) will leak out into the cooking water. As the latter is discarded, levels of SMs can be significantly reduced (Wink, 1988).

Biorational control of herbivores and microbial infections The reduction of SM levels could also be carried out in an industrial process (Figure 8). Since many food items are processed anyhow by food industries, such a procedure would only require little additional steps. This option was not available at a time when most of our food plants had been selected. With our present knowledge we might consider it wise to go back to wild plants with their natural defensive compounds. They would certainly need much less pesticides than present crops. The elimination of SMs during food processing would then become a necessary but technologically feasible step. However, the SMs removed might even be useful products that could be employed in medicine or in agriculture as natural biorational plant protectants (Figure 9) (Wink, 1988, 1999b). If a noxious SM occurs in several plant tissues, one could try to select for mutants that maintain such a trait in most part of a plant but reduce it in the part that is needed for nutrition. Quinolizidine alkaloids are made in the leaves and exported all over the plant via the phloem. Sink tissues include the protein-rich seeds that are harvested. Since these alkaloids cross biomembranes by specific transporters, it should be possible to block such transporters in the fruits. Such plants will probably maintain their natural chemical defense in leaves, stems, and roots. The idea to use

isolated active metabolite

biorational pesticide medical use pharmaceutical

extracts

nutraceutical

Plant removal of unwanted SM

Induction & stimulation of secondary metabolism

food

Introduction of new genes for SM biosynthesis

Fig. 9. Utilizations of bioactive plant metabolites.


267

such a strategy has been discussed previously (Wink, 1988). Such a strategy would preserve part of the natural defense system. Alternatively, one could introduce genes that encode biologically active SMs (lectins, protease inhibitors, peptides) or the pathways leading to them by genetic engineering into crop plants and place them under a promotor that is activated by infection or wounding. The already practiced introduction of Bt-genes into some crop plants could be regarded as a pilot experiment that shows the general feasibility of such an approach.

Conclusions and future perspectives Plants produce a wide variety of bioactive metabolites that have been partly studied so far. Understanding the physiology, biochemistry, and ecology of secondary metabolism offers an opportunity to breed plants with a better protection against microbes and herbivores. Understanding their molecular pharmacology is a key to exploit bioactive plant chemicals in a rational way in medicine and agriculture (biorational pesticides). The human genome project will identify a large number of new molecular targets. Already today industry is employing high throughput screening (HTS) to search for interesting compounds that interact with such a target. SMs from plants, animals, and microbes, which have been preselected during evolution offer a very interesting chance to obtain relevant ‘‘hits.’’ The search for new active compounds or leads has been correctly termed ‘‘bioprospection,’’ the search for biological gold.

References Dewick PM. (2002) Medicinal natural products. A biosynthetic approach. New York: Wiley 507pp. Harborne JB. (1993) Introduction to ecological biochemistry, 4th edition. London: Academic Press 380pp. Kessler A, Halischke R, Baldwin IT. (2004) Silencing the jasmonate cascade: Induced plant defences and insect populations. Science 305:665–668. Luckner M. (1990) Secondary metabolism in microorganisms, plants and animals. Heidelberg: Springer 563pp. Roberts MF, Wink M. (1998) Alkaloids – biochemistry, ecological functions and medical applications. New York: Plenum 486pp. Rosenthal GA, Berenbaum MR. (1991) Herbivores: their interactions with secondary plant metabolites. The chemical participants, Vol. 1. San Diego: Academic Press 452pp. Rosenthal GA, Berenbaum MR. (1992) Herbivores: their interactions with secondary plant metabolites. Ecological and evolutionary processes, Vol. 2. San Diego: Academic Press 493pp. Seigler DS. (1998) Plant secondary metabolism. Boston, Dordrecht, London: Kluwer Academic Publishers 759pp. Swain T. (1977) Secondary compounds as protective agents. Annu Rev Plant Physiol 28:479–501. Teuscher E, Lindequist U. (1998) Biogene gifte. Stuttgart: Wissenschaftliche Verlagsgesellschaft 681pp. van Wyk B-E, Wink M. (2004) Medicinal plants of the world. Pretoria: Briza 480pp.

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Wink M. (1988) Plant breeding: importance of plant secondary metabolites for protection against pathogens and herbivores. Theor Appl Gen 75:225–233. Wink M. (1993) Allelochemical properties and the raison d’eˆtre of alkaloids. In: Cordell G editor. The alkaloids. Orlando: Academic Press, Vol. 43, pp. 1–118. Wink M. (1999a) Biochemistry of plant secondary metabolism. Annual plant reviews, Vol. 2. Sheffield: Academic Press and CRC Press 358pp. Wink M. (1999b) Function of plant secondary metabolites and their exploitation in biotechnology. Annual plant reviews, Vol. 3. Sheffield: Academic Press and CRC Press 362pp. Wink M. (2000) Interference of alkaloids with neuroreceptors and ion channels. In: Atta-UrRahman editor. Bioactive natural products. Amsterdam: Elsevier, Vol. 11, pp. 3–129. Wink M, Schimmer O. (1999) Modes of action of defensive secondary metabolites. Annual plant reviews, Vol. 3. Sheffield: Academic Press and CRC Press.


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CHAPTER 12

Naturally occurring house dust mites control agents: development and commercialization YOUNG-JOON AHN, SOON-IL KIM, HYUN-KYUNG KIM, JUN-HYUNG TAK

Introduction The house dust mites refer to mites of the family Pyroglyphidae, of which ten species have been reported to occur in house dust (Platts-Mills et al., 1992). Pyroglyphid mites usually account for >90% of the mite populations in houses. Many other species, including storage mites [e.g., Acarus siro (L.) and Tyrophagus putrescentiae (Schrank)], can occur in houses and become the predominant population. It has been proposed that the term domestic mites should be used to include both pyroglyphid and nonpyroglyphid mites found in house dust (Platts-Mills et al., 1992). The American house dust mite, Dermatophagoides farinae (Hughes), and the European house dust mite, D. pteronyssinus (Trouessart), are two of the most important pyroglyphid mites because of their cosmopolitan occurrence and abundance in homes (Pollart et al., 1987; Arlian, 2002), because they are a major source of multiple potent allergens (Platts-Mills et al., 1992; Stewart, 1995; Arlian, 2002), and due to their causal association with sudden infant-death syndrome (Helson, 1971). Changes in living environments such as a rise in the number of apartment households with centrally installed heating, space heating, tighter windows, and fitted carpets have provided improved conditions for dust mite growth (Pollart et al., 1987). Control of the house dust mite populations has been principally through development of synthetic chemical substances such as g-benzene hexachloride (g-BHC), pirimiphos-methyl, benzyl benzoate, diethyl-m-toluamide (deet), dibutyl phthalate, and pyrethroids (Pollart et al., 1987; Platts-Mills et al., 1992; Schober et al., 1992). Although effective, their repeated use has sometimes resulted in the development of resistance (van Bronswijk and Sinha, 1971), has undesirable effects on nontarget organisms, and fosters environmental and human health concerns (Pollart et al., 1987; Hayes and Laws, 1991). These problems have highlighted the need for the development of selective dust mite control alternatives, particularly with fumigant action for an easier and more effective application to the house dust mite nests because the currently available formulations such as foams, powders, solutions, or sprays have contact action and are inconvenient to use.

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Plants have been suggested as an alternative source for dust mite control because they constitute a range of bioactive chemicals (Wink, 1993) and some of them are selective, often biodegrade to nontoxic products, and have little or no harmful effects on nontarget organisms (Arnason et al., 1989; Barton, 1999; Isman, 2001). They can be applied to dust mite nests such as mattresses, carpets, and sofas in the same way as other conventional acaricides. Additionally, they also provide useful information on resistance management because certain plant extracts or phytochemicals can be highly effective against insecticide-resistant insect pests (Lindquist et al., 1990; Ahn et al., 1997). Because of this, much effort has been focused on plant extracts, essential oils, and/or their constituents as potential sources of commercial dust mite control agents. In spite of enthusiastic research effort for the discovery and development of new botanical acaricides with acceptable efficacy and minimal health impacts, very few botanical products have succeeded in reaching the marketplace, mostly because of availability (supply problem) and regulatory concerns (e.g., product standardization and change in quality arising from microbial contamination). This chapter is particularly focused on the acaricidal or repellent activities of plant extracts, essential oils, and their constituents against the house dust mites as well as their acaricidal route of action. Examples of commercialized cassia (Cinnamomum cassia) oil-containing acaricidal products are presented. Additionally, denaturing agents of dust mite allergens are also discussed in relation to the development of plant-based protein denaturing agents.

Human health importance of house dust mites Most allergens are usually found in pollen (40%), house dust mite-excretions (30%), animals (20%), molds (5%), insects (2%), foods (2%), and miscellaneous (1%) (UK BIVDA, 2002). Allergic disease is a common disorder affecting 40% of the world population (Johansson, 2000). The house dust mites are known to be vectors or causative agents of diseases that impact millions of people worldwide (Arlian, 2000). They produce a variety of allergens causing allergic symptoms to sensitive humans such as atopic dermatitis, asthma, rhinitis, and conjunctivitis (Platts-Mills et al., 1992; Stewart, 1995; Arlian, 2002). Allergens are contained not only in the bodies of living and dead mites, but also in dust mite body fragmenta and feces. Additionally, dust mite allergens may follow various allergic reactions showing clinical symptoms with swelling, itchy and watery eyes and nose, erythema, difficulty in breathing, headaches, skin rash, and itching (Arlian, 2002). Fourteen groups of house dust mite allergens have been isolated and biochemically characterized (Arlian, 2002). Among the diseases by dust mite excretion, asthma is the most important because of its high mortality. Approximately 5–10% of all adults and 10–20% of all children worldwide suffer from asthma (UK BIVDA, 2002). The overall rate of asthma incidence increased 75% since 1980, while the incidence among children increased 160%. Exposure to 2 mg of group I dust mite allergen (Der p I, Der f I, and Der m I) per gram of dust (100 mites per gram or 0.6 mg of guanine per gram) is considered to increase the risk of sensitization and bronchial hyper-reactivity (Peat et al., 1987; Wood et al., 1989; Arruda et al., 1991; Charpin et al., 1991). Especially, exposure to 10 mg of group I mite allergen per gram of dust (500 mites per gram) acts as an important

Naturally occurring house dust mites control agents

271

factor in the development of asthma in children (Sporik et al., 1990). Arbes et al. (2003) reported that over 45% of the U.S. housing stock, or approximately 44 million homes have bedding with dust mite allergen concentrations that exceed 2 mg per gram of dust. Of these, over 23% of U.S. homes or about 22 million dwellings, are estimated to have bedding with dust mite allergen concentrations that exceed 10 mg per gram dust.

House dust mite habitat The house dust mite habitats are very important in relation to the development of appropriate formulations of mite control agents (e.g., contact, stomach, or fumigant). Amount of dust mite allergens according to their number and species depends on variances of home environments (Fernandez-Caldas et al., 1994). The house dust mite habitats include bedroom, carpet, furniture, soft toys, and clothing. Homes with mite allergic patients contain significantly higher levels of dust mite density than those with nonallergic subjects in bedclothes (Konishi and Uehara, 1995). The most abundant two species in dust samples collected at 13 agricultural settlements of 9 geoclimatic subregions (northern coastal regions) in Israel were D. farinae (86%) and D. pteronyssinus (71%), and they were collected frequently from carpets (37%), sofas (34%), and bed rooms (29%) (Mumcuoglu et al., 1999). Most house dust mites generally prefer mattresses as living sites (Rao et al., 1975) but the number of D. farinae was seven times larger in a living room carpet than in the mattress (Arlian et al., 1982). Additionally, dust mite density is greater in frequently used furniture likely containing higher amount of human skin debris and moisture content than in occasionally used one (Mitchell et al., 1969). Dust mite allergens often contaminate various public facilities. Floors of inn and chairs of theater contain higher level of allergen than other facilities, such as hospital, hotel, and office (Konishi and Uehara, 1999).

Market of house dust mite control agents It is very difficult to directly estimate costs spent annually to prevent or control the house dust mites or dust mite-borne diseases because available informations are limited. Therefore, marketsize of dust mite control agents may be estimated from allergic incidences or anti-allergen products market. According to estimation of World Health Organization (2000), asthma affects nearly 150 million people worldwide and causes more than 180,000 deaths annually. Expenses used by allergic rhinitis globally each year are approximately 20 billion USD including medications, time off work and physician consultations, and the cost of allergy drugs alone is estimated to be 8 billion USD annually (World Allergy Organization, 2004). U.S. National Institute of Allergy and Infectious Disease (2001) estimates that students are off more than 10 million school days due to asthma annually, and economic production loss by working parents caring for the missing school children reaches about 1 billion USD. Anti-allergen products market is worth more than 200 million USD in the United States, in addition to the cost of pharmaceuticals and medical services, and the sale

272


of anti-allergen chemistry (exclusive of pharmaceuticals) will exceed 1 billion USD by 2010 (Groseclose, 2001). Estimating an economic burden by asthma, 10.7 billion USD was spent for direct and indirect medical expenses in 1994 (U.S. National Institute of Allergy and Infectious Disease, 2001). Especially, allergic asthma affects about 3 million children (8–12% of all children) and 7 million adults in the United States at a cost estimated at 6.2 billion USD a year (Health Goods, 1998). In EU, total costs for allergic asthma are approximately 22 billion ECU corresponding to the expense of 853 ECU per asthmatic patient (Storms et al., 1997).

Acaricidal activity of plant-derived materials Many plant extracts and essential oils possess acaricidal activity against the house dust mites (Table 1). Most promising botanical dust mite control agents are found in plants of the families Annonaceae, Apiaceae, Brassicaceae, Cupressaceae, Lamiaceae, Lauraceae, Myrtaceae, Pinaceae, and Poaceae. However, the differential responses of the house dust mite species might be influenced by extrinsic and intrinsic factors such as the plant species, the parts of the plant, the solvents used for extraction, the geographical location where the plants were grown, and the application methods. The acaricidal activity of very few plant extracts and essential oils against the house dust mites was compared with those of benzyl benzoate, a widely used acaricide, or an active ingredient of many commercial acaricides. On the basis of EC50 values, a dichloromethane extract (EC50, 0.028 g/m2) from Uvaria pauci-ovulata bark has more potent activity against D. pteronyssinus than benzyl benzoate (EC50, 0.06 g/m2) (Raynaud et al., 2000). A cyclohexane extract (EC50, 0.075 g/m2) from tonka bean (Dipterix odorata) is less active than benzyl benzoate (EC50, 0.025 g/m2) against D. pteronyssinus (Gleye et al., 2003). The acaricidal activity of certain plant extracts or essential oils against adults of D. farinae and D. pteronyssinus is comparable to that of benzyl benzoate. Variation in the house dust mite response to the plant extracts or essential oils related to plant genus has been noted. Differences in the acaricidal effects on D. pteronyssinus among extracts from three Uvaria species have been reported (Akendengue et al., 2003). A methanol extract from U. versicolor stem are more effective than that from either U. klaineana stem or U. mocoli bark against D. pteronyssinus. EC50 values of methanol and hexane extracts from U. versicolor stem are 0.095 and 0.12 g/m2, respectively. Interestingly, benzyl benzoate (EC50, 0.045 and 0.06 g/m2) is contained in U. versicolor stem hexane extract and U. pauci-ovulata bark ethanol extract. Similar differential susceptibility of adults of D. farinae and D. pteronyssinus to the essential oils from the same plant species was reported in seven Citrus species [bergamot (C. bergamia), bitter orange (C. aurantium), grapefruit (C. paradisi), lemon 10Fold (C. limonum), lime dis 5F (C. aurantifolia), mandarine (C. reticulata), and orange (C. sinensis) oils], three Cymbopogon species [citronella java (C. nardus), lemongrass (C. citratus), and palmarosa (C. martinii) oils)], and three Mentha species [pennyroyal (M. pulegium), peppermint (M. piperita), and spearmint (M. spicata) oils] (Kim, 2002). Various compounds, including phenolics, terpenoids, and alkaloids, exist in plants. Jointly or independently, they contribute to a variety of bioefficacy such as


273

Table 1 List of plants and essential oils with acaricidal activity Family

Plant species

Typea

Miteb

Source

Annonaceae

Uvaria klaineana Engler et Diels Uvaria pauci-ovulata Hooker Uvaria versicolor Pierre ex Engler et Diels Anethum graveolens L. Carum carvi L. Cnidium officinale Makino Cuminum cyminum L. Foeniculum vulagrec P. Miller Pimpinella anisumc L. Brassica junceac L. Cocholearia armoraciac L. Chamaecyparis obtusa Siebold et Zuccarini Chamaecyparis pisifera (Siebold et Zuccarini) Endlicher Chamaecyparis taiwanensis Masamune et Suzuki

Ex

DP

Ex

DP

Akendengue et al. (2003) Raynaud et al. (2000)

Ex

DP

Eo Eo Ex Eo Eo

DF/DP DF/DP DF/DP DF/DP DF/DP

Akendengue et al. (2003) Watanabe et al. (1989) Watanabe et al. (1989) Kwon and Ahn (2002) Watanabe et al. (1989) Lee (2004a)

Eo Eo Eo Sd

DF/DP DF/DP DF/DP DF/DP

Lee (2004b) Kim (2001) Kim (2001) Ando (1994)

Ex Eo

DF/DP DF

Jang et al. (2005) Miyazaki (1996a)

Eo Eo

DF/DP DP

Eo Eo Eo Eo

DF/DP DF DP DF

Eo

DP

Ando (1994) Oribe and Miyazaki (1997) Chang et al. (2001) Asada et al. (1989) Miyazaki (1996b) Morita et al. (2003, 2004) Oribe and Miyazaki (1997) Watanabe et al. (1989) McDonald and Tovey (1993) Soonthornchareonnon et al. (2005) Gleye et al. (2003)

Apiaceae

Brassicaceae Cupressaceae

Taiwania cryptomerioides Hayata Thujopsis dolabrata var. hondai Makino

Ericaceae

Gaultheria procumbens L.

Eo Eo

DF/DP DP

Euphorbiaceae

Trigonostemon reidioides (Kurz) Craib Dipterix odoratac (Aublet) Willdenow Dalbergia nigra Allem Pelargonium graveolensc L. Mentha spicata L.

Ex

DP

Ex

DP

Eo Eo Eo Eo

DF/DP DF DF/DP DP

Eo Eo

DF/DP DF/DP

Ando (1994) Kim (2002) Watanabe et al. (1989) McDonald and Tovey (1993) Nomura et al. (1993) Watanabe et al. (1989)

Eo

DF/DP

Kim (2002)

Eo Eo

DF DF/DP

Kim (2002) Watanabe et al. (1989)

Fabaceae Geraniaceae Lamiaceae

Lauraceae

Perilla frutescens (L.) Britton Pogostemon patchoulic Pelletier Thymus vulgarisc L. Cinnamomum camphora (L.) J. Presl

(Continued)


274 Table 1 (continued ) Family

Plant species c

Myrtaceae

Myristicaceae Paeoniaceae Pinaceae

Poaceae

Rosaceae Taxodiaceae

Cinnamomum cassia Blume Machilus thunbergii Siebold et Zuccarini Neolitsea sericea (Blume) Koidzumi Eucalyptus citriodora Hooker Eucalyptus globulus Labillardie´re Eugenia caryophyllatac Thunberg Melaleuca alternifolia (Maiden et Betche) Cheel Melaleuca bracteata F. Mueller Melaleuca symphyocarpa F. Mueller Pimenta racemosac (D. Mill.) J.W. Moore Myristica fragransc Houttuyn Paeonia suffruticosac Andrews Abies homolepis Siebold et Zuccarini Picea abies (L.) H. Karsten Pinus densiflora Siebold et Zuccarini Pinus monticola Douglas ex D. Don Tsuga heterophylla (Rafinesque) Sargent Tsuga sieboldii Carrie´re Cymbopogon citratusc (DC. ex Nees) Stapf Cymbopogon martiniic (Roxb.) J.F. Watsonooke Cymbopogon nardusc (L.) Rendle Vetiveria zizanoidesc Stapf Prunus dulcis var. amara (Duhamel) H.L. Moore Cryptomeria japonica (L. f.) D. Don

Typea

Miteb

Source

Ex Eo Eo

DF/DP DF/DP DF/DP

Kim (2001) Ahn and Kim (2005) Furuno et al. (1994)

Eo

DF/DP

Furuno et al. (1994)

Eo Eo

DF DP

Eo

DP

Eo

DF/DP

Miyazaki (1996a) McDonald and Tovey (1993) Tovey and McDonald (1997) Kim et al. (2003)

Eo

DP

McDonald and Tovey (1993)

Eo

DP

Yatagai et al. (1998)

Eo

DP


Ex

DF/DP

Kim (2002)

Eo

DF/DP

Kim (2002)

Ex

DF/DP

Kim et al. (2004b)

Eo

DF

Miyazaki (1996a)

Eo

DF

Miyazaki (1996a)

Sd

DF/DP

Ando (1994)

Eo

DF/DP

Ando (1994)

Eo

DP

Miyazaki et al. (1989)

Eo Eo

DF DF

Miyazaki (1996a) Kim (2002)

Eo

DF/DP

Kim (2002)

Eo

DP

Eo Eo Eo

DF/DP DF/DP DF/DP

McDonald and Tovey (1993) Kim (2002) Kim (2002) Watanabe et al. (1989)

Ex Eo Eo

DP DP DP

Morita et al. (1991) Miyazaki et al. (1989) Morita and Yatagai (1994)


275

Table 1 (continued ) Family

Plant species

Typea

Miteb

Source

Eo/ Ex Eo Sd

DF/DP


DF/DP DF/DP

Ando (1994) Ando (1994)

a

Ex, plant extract; Eo, essential oil; Sd, sawdust. DF, Dermatophagoides farinae; and DP, D. pteronyssinus. c Acaricidal activity of test plants is comparable to that of benzyl benzoate. b

killing, ovicidal, repellent, and antifeeding activities against arthropod pests. For example, neem (Azadirachta indica) oil containing azadirachtin as an active principle is found to have a variety of biological activities including insecticidal activity against nearly 200 species of insects without any adverse effects on most nontarget organisms (Saxena, 1989). The plant natural acaricidal compounds against the house dust mites include alkaloids, aldehydes, ketones, benzofuranoids, benzoic acids, benzpyranoids, diterpenoids, monoterpenoids, phenylpropanoids, polyketides, and sesquiterpenoids (Table 2). Because toxicity of plant compounds varied according to the experimental conditions, potencies are expressed as relative toxicity. p-Anisaldehyde is 7.9- and 6.7-fold more active than benzyl benzoate against D. farinae and D. pteronyssinus, respectively (Lee, 2004a). The phenylpropanoid eugenol is 1.7 and 1.8 times more toxic than benzyl benzoate against adults of D. farinae and D. pteronyssinus, respectively (Kim et al., 2003). Susceptibility of the house dust mites to certain plant extracts, essential oils, or their constituents has been noted. Susceptibility to some plant essential oils such as caraway (Carum carvi), perilla (Perilla frutescens), and shirodamo (Neolitsea sericea) oils is greater in adult D. farinae than in adult D. pteronyssinus (Watanabe et al., 1989; Furuno et al., 1994). However, adult D. farinae is more tolerant to the wood oils of Tsuga heterophylla and Cryptomeria japonica than adult D. pteronyssinus (Miyazaki et al., 1989). Similar results have been also reported for eugenol (Kim et al., 2003). However, there are no significant differences in toxicity of (E)-cinnamaldehyde, cinnamyl alcohol, and salicylaldehyde (Kim, 2001), butylidenephthalide (Kwon and Ahn, 2002), paeonol (Kim et al., 2004b), and b-thujaplicin (Jang et al., 2005) between two Dermatophagoides species. In addition to killing effects of plant-derived materials, the breeding and immobilizing effects of wood microingredients on the house dust mites have been noted. The house dust mites (D. farinae and D. pteronyssinus) that were fed diets mixed with small pieces of hinoki (Chamaecyparis obtusa), cedar (C. japonica), red pine (Pinus densiflora), and lauan (Shorea spp.) did not breed normally (Ando, 1994). Wood that had no essential oil had reduced or no breeding control effects. The essential oils of rosewood (Dalbergia nigra), white pine (Pinus monticola), and Formosan cypress (Chamaecyparis taiwanensis) had strong immobilizing effects against D. farinae and D. pteronyssinus (Ando, 1994). Especially, geranyl acetate, linalool, and a-terpineol contained in hinoki oil produced strong mite-immobilizing effects (Ando, 1994). Miyazaki (1996b) found that a mixture of 2% hiba (Thujopsis dolabrata var. hondai) wood sawdust oil, equivalent to the original oil concentration in hiba sawdust,


276

Table 2 List of acaricidal components derived from plant extracts and essential oils Class

Compound

Plant species

Alkaloid Aldehyde

caffeine p-anisaldehyde

Foeniculum vulgare Pimpinella anisum

salicylaldehyde

Prunus dulcis var. amara Cinnamoum cassia

Aryl ketone

Paeonol

Paeonia suffruticosa

Benzofuranoid

butylidenephthalide

Cnidium officinale

Benzoic acid

benzoic acid

Paeonia suffruticosa

benzyl benzoate

Uvaria versicolor

benzaldehyde

methyl salicylate Benzopyranoid Diterpenoid

coumarin ferruginol O-methyl pisiferic acid pisiferal pisiferic acid

Monoterpenoid

rediocides A, C, E, F bornyl acetate

camphene 2-carene 3-carene d-carvone l-carvone cuminaldehyde dipentene fenchone geranyl acetate limonene linalool

Uvaria pauciovulata Gaultheria procumbens Dipteryx odorata Taiwania cryptomerioides Chamaecyparis pisifera Chamaecyparis pisifera Chamaecyparis pisifera Trigonostemon reidioides Chamaecyparis obtuse Pinus abies Pinus pumila Pinus abies Pinus pumila Chamaecyparis obtusa Pimpinella anisum Carum carvi Anethum graveolens Mentha spicata Cuminum cyminum Pinus pumila Foeniculum vulgare Chamaecyparis obtusa Pinus abies Chamaecyparis obtusa Cinnamomum camphora Mentha spicata

Mitea

RTb

DP DF DP DF DP DF/DP

7.9 6.7 8.4 6.7

DP DF DF DP DF DP DF DP DP

Source Russell et al. (1991) Lee (2004a) Lee (2004b) Watanabe et al. (1989) Kim (2001)

1.0 1.0 1.3 1.0 1.2 1.0

Kim et al (2004b) Kwon and Ahn (2002) Kim et al (2004b)

DP

Akendengue et al. (2003) Raynaud et al. (2000)

DF/DP

Watanabe et al. (1989)

DP DF/DP

0.8

DP DP DP DP

1.2–8.5

DF/DP DP DP DP DP DF DP DF DP DF/DP DF/DP DF/DP DF/DP DP DF DP DF/DP

0.2 0.2 0.2 0.2

2.3 1.6

Gleye et al. (2003) Chang et al. (2001) Yatagai and Nakatani (1994) Yatagai and Nakatani (1994) Yatagai and Nakatani (1994) Soonthornchareonnon et al. (2005) Ando (1994) Miyazaki et al. (1989) Miyazaki et al. (1989) Miyazaki et al. (1989) Miyazaki et al. (1989) Jang et al. (2005) Lee (2004b) Watanabe et al. (1989) Watanabe et al. (1989) Watanabe et al. (1989) Watanabe et al. (1989) Miyazaki et al. (1989) Lee (2004a) Ando (1994)

DP DF/DP

Miyazaki et al. (1989) Ando (1994)

DF/DP

Watanabe et al. (1989)

DF/DP

Nomura et al. (1993)


277

Table 2 (continued ) Class

Compound

Plant species

Mitea

menthone perillaaldehyde a-pinene b-pinene

DF/DP DF/DP DP DF/DP

Nomura et al. (1993) Watanabe et al. (1989) Miyazaki et al. (1989) Ando (1994)

DP DF/DP

Miyazaki et al. (1989) Ando (1994)

DF/DP

Ando (1994)

DF

Morita et al. (2004)

thymol

Mentha spicata Perilla frutescens Pinus pumila Chamaecyparis obtuse Pinus abies Chamaecyparis obtusa Chamaecyparis obtusa Thujopsis dolabrata var. hondai Chamaecyparis obtusa Thujopsis dolabrata var. hondai Foeniculum vulgare

cinnamaldehyde cinnamyl alcohol estragole

Cinnamomum cassia Cinnamomum cassia Foeniculum vulgare

a-terpinene a-terpineol a-thujaplicin b-thujaplicin

Phenylpropanoid

Pimpinella anisum eugenol Polyketide

squamocin

Sesquiterpenoid

a-cadinol

caryophyllene oxide cedrol

cryptomerione b-eudesmol isosericenine T-muurolol sericealactone

Eugenia caryophyllata Uvaria pauciovulata Neolitsea sericea Taiwania cryptomerioides Neolitsea sericea Chamaecyparis obtuse Cryptomeria japonica Cryptomeria japonica Cryptomeria japonica Neolitsea sericea Taiwania cryptomerioides Neolitsea sericea

RTb

DF DP DF

1.3 1.2 2.1

DF DP DF/DP DF/DP DF DP DF DP DF DP DP

1.0 1.0

0.2 0.2 0.2 0.2 1.7 1.8 0.1

DF DF/DP DF/DP DF DP DF/DP DP

Source

Jang et al. (2005) Morita et al. (2003) Lee (2004a) Kim (2001) Kim (2001) Lee (2004a) Lee (2004b) Kim et al. (2003) Raynaud et al. (2000) Fruno et al. (1994) Chang et al. (2001)

0.9 0.9

Fruno et al. (1994) Jang et al. (2005) Yatagai et al. (1991)

DF/DP

Morita and Yatagai (1994) Yatagai et al. (1991)

DF/DP DF/DP

Fruno et al. (1994) Chang et al. (2001)

DP

Sharma et al. (1993)

a

DF, Dermatophagoides farinae; and DP, D. pteronyssinus. RT, relative toxicity ¼ LD50 or EC50 value of benzyl benzoate/LD50 or EC50 value of an active compound. b

with culture media gave 100% immobilizing effect on D. pteronyssinus. When D. pternonyssinus was exposed to diet containing 0.2% hiba wood oil or 0.2% Formosan cypress wood oil, the population growth of D. pternonyssinus was significantly decreased in 30 days after exposure compared with control group (Oribe and Miyazaki, 1997). The immobilized dust mites can be collected easily using vacuum cleaner.

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Mixtures of laundry detergent and certain plant essential oils are also available for the control of the house dust mites. More than 95% of dust mites exposed for 30 min to water solution containing 0.8% tea tree (Melaleuca alternifolia) oil at 30 1C were killed (McDonald and Tovey, 1993). The 0.8% oil solution was as effective as 0.5% benzyl benzoate solution. In addition, most mites were killed with this procedure when blankets were soaked for 30 min before washing in mixture solution composed of eucalyptus (Eucalyptus globulus) oil (100 ml), a specific kitchen detergent concentrate (25 ml), and warm (30 1C) water (50 l) (Tovey and McDonald, 1997). Eucalyptus oil left an odor in clothing that lasted for 2–3 days (Tovey and McDonald, 1997). These studies suggest that dilute solutions of essential oils are potentially an effective, acceptable, and inexpensive method of controlling house dust mites.

Poisoning symptoms and mode of action of plant-derived materials Elucidation of the poisoning symptoms and modes of action of acaricidal natural products and acaricides is of practical importance for mite control because it provides useful information on the most appropriate formulation and delivery means. Five types of poisoning symptoms have been reported in dust or storage mites: a knockdown-type death caused by Neolitsea sericea leaf oil in adults of D. farinae and D. pteronyssinus (Furuno et al., 1994); death related with uncoordinated behavior without knockdown by (E)-cinnamaldehyde, cinnamyl alcohol, salicylaldehyde, benzyl benzoate, eugenol, and deet in adults of D. farinae, D. pteronyssinus, and T. putrescentiae (Kim, 2001; Kim et al., 2003, 2004a); death associated with dessication by several monoterpenes such as fenchon, linalool, menthone, and pulegone in adult T. putrescentiae (Sanchez-Ramos and Castanera, 2001); death associated with lethargy by butylidenephthalide, isoeugenol, and methyleugenol in adults of D. farinae, D. pteronyssinusa, and T. putrescentiae (Kwon and Ahn, 2002; Kim et al., 2003); and death related with a characteristic depression of the dorsal surface of the idiosoma by tricalcium phosphate and ferric phosphate in adult T. putrescentiae (Ignatowicz, 1981). Certain Mentha species such as M. aquatica (water mint) inhibit acetylcholinesterase (AChE) from bovine erythrocytes in vitro (Miyazawa et al., 1998). Certain monoterpenoids (e.g., pulegone and menthone) and sesquiterpene alcohols (e.g., elemol and viridiflorol) derived from essential oils of Mentha species competitively inhibit AChE in vitro (Miyazawa et al., 1997, 1998). As suggested by Isman (2001), this inhibitory action does not appear to correlate with overall toxicity to insects in vivo. The octopaminergic nervous system has been suggested as a novel target site of the compounds by Enan (2001), who examined inhibition of 3H-octopamine binding in a cockroach nerve cord preparation in the presence of essential oil compounds (e.g., trans-anethole and eugenol). Formamidine pesticides such as chlordimeform target the octopaminergic nervous system, and the major difference between the amidines and other conventional pesticides lies in the potentially important behavioral and physiological effects at sublethal doses (Hollingworth and Lund, 1982). It has been reported that behavioral effects (feeding deterrence and repellency) of essential oils and their constituents are consistent with this mode of action (Hummelbrunner and Isman, 2001; Isman, 2001). However, very few studies on


279

acaricide mode of action of plant-derived compounds have been done. N. sericea leaf essential oil has knockdown effect on adults of D. farinae and D. pteronyssinus (Fruno et al., 1994). However, many plant extracts, essential oils, and their constituents do not cause knockdown effect (Kim, 2001, 2002; Kwon and Ahn, 2002; Kim et al., 2003, 2004a). Knockdown effect is not a common characteristic of AChE inhibitors carbamates and organophospates, but is the general symptom of arthropods affected by pyrethrins and pyrethroids (Miller and Adams, 1982). Therefore, an other mechanism rather than AChE inhibition might be involved in toxicity of most acaricidal natural products. Their exact mode of action remains to be proven. The acaricidal effect of certain plant extracts and essential oils is likely by vapor action via the respiratory system, whereas many commercial synthetic acaricides have contact toxicity. Fumigant action is reported for methanol extracts from Cnidium officinale rhizome (Kwon and Ahn, 2002) and Paeonia suffruticosa root bark (Kim et al., 2004b), as well as essential oils of Brassica juncea, Cocholearia armoracia (Kim, 2001), and Eugenia caryophyllata (Kim et al., 2003). Volatile compounds of plant extracts and essential oils are composed of alkanes, alcohols, aldehydes, and terpenoids, particularly monoterpenoids (Visser, 1986; Coats et al., 1991; Isman, 2001). Fumigant activity against adults of D. farinae and D. pteronyssinus has been reported for butylidenephthalide (Kwon and Ahn, 2002), (E)-cinnamaldehyde, cinnamyl alcohol, and salicylaldehyde (Kim, 2001), eugenol (Kim et al., 2003), and benzoic acid and paeonol (Kim et al., 2004b).

Plant-based mite repellents For the house dust mites killed with acaricide there are others in the area that survive. The adverse effects of acaricides call for alternative control agents such as repellents rather than attempts to kill the house dust mites. Unfortunately, there are a few researches on plant- or chemical-based repellents to the house dust mites compared with the other insects such as mosquitoes or moths, and most informations are supplied as patent documents. Plants or essential oils with repellency to dust mites are listed in Table 3. The repellency of plant odors on the house dust mites may provide a promising control strategy for people struggling to control arthropods. The wood odors from C. obtusa, P. densiflora, and C. japonica have repellency against D. farinae and D. pteronyssinus, and no significant differences in responses between males and females of these dust mites have been recognized (Ando, 1993). The monoterpenoid nepetalactone, derived from catnip (Nepeta cataria), or the sesquiterpenoid elemol, derived from the fruit of the osage orange tree (Maclura pomifera), have repellency against domestic mites (Coats et al., 2003). The monoterpenoid l-menthone exhibits acaricidal and repellent activity when applied to Japanese mats, carpets, food containers, and human body against house dust mites (Inagaki and Ishida, 1988).

Plant-based denaturing agents of dust mite allergens Unfortunately, dead mites and dust mite fecal pellets containing allergens persist months after eradication of live mites (Sears et al., 1989). Tannic acid is used to


280

Table 3 List of plants and essential oils with repellency against house dust mites Common name Anise Cajeput Calamus Cananga Cedar Cinnamon Citronella Clove Cornmint Cubeb Fennel Ginger Gurjun Hinoki Lemongrass Mace Nutmeg Patchouli Pepper Pine Sandalwood Thymus Vetiver a

Plant species Pimpinella anisum Melaleuca cajeputi L. Acorus calamus L. Cananga odorata (Lam.) Hook. F. et Thomson Cryptomeria japonica Cinnamomum zeylanicum Blume Cymbopogon nardus Eugenia caryophyllata Mentha arvensis L. Piper cubeba L. Foeniculum vulgare Zingiber officinale Roscoe Dipterocarpus alatus Roxb. Ex G. Don. Chamaecyparis obtusa Cymbopogon citratus Myrisica frangrans Myristica fragrans Pogostemon patchouli Piper nigrum Pinus densiflora Santalum album L. Thymus vulgaris Vetiveria zizanoides

Tissue/type a

Source

seed/Eo leaf/Eo rhizome/Eo flower/Eo

Kobayashi Kobayashi Kobayashi Kobayashi

sawdust bark/Eo

Ando (1993) Kobayashi et al. (1992)

grass/Eo bud/Eo seed/Eo fruit/Eo seed/Eo root/Eo balsam/Eo

Kobayashi Kobayashi Kobayashi Kobayashi Kobayashi Kobayashi Kobayashi

sawdust leaf/Eo husk/Eo seed/Eo leaf/Eo peppercorn/Eo sawdust root/Eo leaf/Eo root/Eo

Ando (1993) Kobayashi et Kobayashi et Kobayashi et Kobayashi et Kobayashi et Ando (1993) Kobayashi et Kobayashi et Kobayashi et

et et et et

et et et et et et et

al. al. al. al.

(1992) (1992) (1992) (1992)

al. al. al. al. al. al. al.

(1992) (1992) (1992) (1992) (1992) (1992) (1992)

al. al. al. al. al.

(1992) (1992) (1992) (1992) (1992)

al. (1992) al. (1992) al. (1992)

Eo, essential oil.

denature mite allergens, which results in reducing the allergenicity of house dust (Ehnert et al., 1992; Woodfolk et al., 1994). The acid is marketed in 1% and 3% solutions. Mite allergens are denatured by the phenol groups of tannic acid polymerizing them and making them more hydrophobic (Green, 1984). A 3% tannic acid solution (wt/vol) denatures group I allergens but is somwewhat less effective for group II allergens (Der p II and Der f II). Application of 3% tannic acid solution (wt/ vol) to mattress casings and carpets reduces bronchial hyper-reactivity in patients at 8 months after treatment, but this reducing effect was not shown at 4 or 12 months post-treatment (Ehnert et al., 1992). Although tannic acid gives the reduction of mite-borne allergen levels, rigorous allergen reduction treatment in typical home setting is very difficult and tannic acid stains fabrics in a practical application.

Commercialization of botanical mite control agents Although a number of plant preparations meet the criteria for efficacy, there are some formidable barriers to commercialization for many prospective botanical dust mite control agents as indicated by Isman (1997, 2001). Barriers to commercialization


281

are: (1) registration, (2) availability of the starting material on a sustainable and consistent basis, (3) the need for chemical standardization and quality control, and (4) costs of the raw materials and their refinement. The procedures and guidelines for expedited review of conventional pesticides are provided by requirement of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) as amended by the Food Quality Protection Act (FQPA) in 1996. U.S. Environmental Protection Agency (EPA) has established the Reduced Risk Initiative and the Biopesticides and Pollution Prevention Division to encourage the development, registration, and use of reduced-risk pesticide products to meet reduced risk criteria including low toxicity to humans and nontarget organisms, low contamination to groundwater, low pest-resistance potential, and compatibility with integrated pest management. Plant essential oils have potential as products for the house dust mite control because many of them are commonly used as fragrances and flavoring agents for foods and beverages. Additionally, essential oils are widely available and some are relatively inexpensive (10–25 USD/kg) (Isman, 2001). Plant extracts also have a qualification as reduced-risk pesticides, but they are not so widely used because of their high cost and limited availability. Neem, derived from the seeds of A. indica, and pyrethrum, derived from the flowers of Chrysanthemum cinerariefolium, are relatively expensive (125–200 and 45–60 USD/kg, respectively) (Isman, 2001). Many plant essential oils and their constituents are exempt from toxicity data requirements by the U.S. EPA. These regulatory, cost, and availability merits lead many venture companies to develop the dust mite control agents based on essential oils and/or their constituents against arthropod pests. Considering the house dust mite habitats, two types of formulations, contact and/or fumigant, are needed. In laboratory tests with adults of D. farinae and D. pteronyssinus, methanol extract and essential oil of cassia (C. cassia) bark are highly effective in the residual dry film and fabric diffusion bioassays (Kim, 2001; Ahn and Kim, 2005). The acaricidal activity of cassia oil was comparable to that of benzyl benzoate. The biologically active constituents of Cinnamomum bark were characterized as (E)-cinnamaldehyde, cinnamyl alcohol, and salicylaldehyde by spectroscopic analyses (Kim, 2001). Cassia oil and its constituents act as fumigants (Kim, 2001; Ahn and Kim, 2005). In a fabric diffusion bioassay to determine the acaricidal activity of cassia oil according to different drying regimes in air (10, 30, and 60 min), all treatments with 51 mg/cm2 of cassia oil showed 100% mortality within 3 h after treatment. Recently, two types of formulations (spray and fumigant type) of Allerzeros containing C. cassia oil have been developed by our group and released by NaturoBiotech Co., Ltd. (Republic of Korea). Sprays containing 2 and 5% cassia bark oil were formulated with 2 or 5 g C. cassia bark oil, 79 or 76 g distilled water, respectively, 9 g emulsifier, and 10 g ethanol in a 150 ml of polyethylene container. Both spray-type products produce >95% mortality against adult D. farinae at 2 h after treatment when applied to six different square (50 50 cm) substrates (fabric, glass, paper sheet, plastic, tin, and wood board). In a 0.125 m3 space treatment, the 5% spray product produced 92% and 100% mortality at 1- and 2 times-spray at 24 h after treatment, respectively. For a 0.255 m3 space treatment, 2and 3-times spray showed 85% and 97% mortality, respectively. In a 1.728 m3 space treatment, 90% and 100% mortality was obtained from 7- and 10-times spray,

282


respectively. Considering these results, approximately 50–60 mg of cassia oil was necessary to provide house dust mite control in a space of 1 m2 or in a volume of 3.4 m3. Spray-type product has excellent control effect with contact and fumigant actions. Unlike conventional acaricides with high toxicity, cassia bark oil can be applied as sprays to homes or other facilities, such as hospitals, hotels, offices, and theaters, etc., or directly to dust mites on mattresses, carpets, sofas, or toys in the same way as conventional acaricides. The fumigant device consisted of 3 g C. cassia bark oil confined in a sealed polypropylene plastic bag (7 8 0.7 cm), which was enclosed in a nonwoven fabric covering (40, 45, and 50 mm thickness; 7 7 cm). When the central part of the fumigant device is pressed, volatile components of the bark oil are released into the air. A single device enclosed in a 40 mm nonwoven fabric cover gave 85% mortality against adult D. pteronyssinus at 96 h post treatment in a space of 0.182 m3, whereas neither 45 nor 50 mm-film device was effective (Ahn and Kim, 2005). However, two devices resulted in 100% mortality at 24 h post treatment in a 0.182 m3 space regardless of thickness of their covers but 92% and 88% mortality in 0.255 and 1.728 m3 spaces, respectively, at 96 h after treatment. Initial volatility may be a very essential factor in exerting toxicity of cassia oil. In an experiment to practically determine control effect of the fumigant devices in six test spaces of differing volume (0.024, 0.05, 0.097, 0.182, 0.255, and 1.728 m3), groups of 30–40 D. pteronyssinus adults were individually placed into a cylindrical container (1.5 ml). Both ends were covered with 200-mesh screen to allow free air flow. Containers were placed directly on the fumigant devices or placed randomly in the test spaces. When the cylindrical containers containing adults were placed directly on the devices, a single device gave 100% mortality in the 0.05 m3 space. The lethal activity of the device varied, however, according to the volume of the space and exposure time when placed randomly. One fumigant device was needed to give X94% mortality at 72 and 96 h after treatment in 0.024 and 0.05 m3, respectively. One fumigant device gave ca 75% mortality in 0.097 and 0.182 m3 and ca 24% mortality in 0.255 and 1.728 m3 at 96 h after treatment. Two fumigant devices were needed to give 88% mortality at 96 h of exposure in 1.728 m3. The active ingredient of AllerZero device needs more time to reach a lethal concentration to exert effective toxicity in a wider space. These fumigant devices are useful for managing the house dust mites in enclosed spaces such as wardrobes, pet house, storage bins, factories, or buildings. It can be also placed under sofa or bedclothing, or in a mattress, a pillow, or a laundry box. Thus such fumigant devices have a great advantage in situations where staining is an issue, with carpet, sofa, and clothing. Particularly, carpets accumulate large quantities of residual dirt, which can act to protect mites from dust mite control agents (acaricide and repellent) and denaturing agents of mite allergens. Therefore, it is essential to optimize the conditions of application to reach dust mites. In this regard, certain essential oils, such as cassia bark oil, may be best used as fumigants. On the basis of efficacy and cost, 2% cassia oil-containg spray-type product and 3 g cassia oil-containg fumigant device are the most appropriate. Quality control of both formulations is done as (E)-cinnamaldehyde content in the oil. This commercial success based on plant essential oil may be able to supply a motive for venture companies to develop new botanical acaricides.


283

Mixture of laundry detergent and essential oil can be one of the tools for the control of dust mites. Mixture solution composed of cassia oil (60 ml), a specific detergent concentrate (40 ml), and tap water (65 l) gives complete control effect when operating for 40 min in an electric washing machine (Ahn, unpublished data). This solution does not stain blanket, cover sheet, or clothing. This product can reduce dust mite population and can be released in the market.

Future perspectives The pesticide regulation status will restrict the use of many conventional pesticides such as organophosphates and carbamates depended on for decades. By 2003, U.S. government banned or severely restricted 64 pesticides belonging to the categories of UN PIC (Prior Informed Consent), UN Severely Hazardous Pesticide Formulations (SHPF), and U.S. PIC lists such as chlordimeform and ethyl parathion (U.S. Environmental Protection Agency, 2003). In spite of the widespread public concerns for long-term health and environmental effects of conventionally used pesticides, natural pesticides based on a plant origin have not yet occupied much portion in the pesticide marketplace. Due to the improved conditions for mite growth by changes in living environments mentioned above, the house dust mites worldwide are prevalent. At the same time, consumers’ demands for new reduced-risk mite control products will be increasing. Considering these situations, plant-based house dust mite control products might replace some synthetic ones in the near future. The lack of octopamine receptors in vertebrates at least in part accounts for the selective mammalian toxicity of essential oils or their monoterpenoid constituents (Isman, 2001). But there are few studies in this regard. Many of these compounds have rat oral acute LD50 values in the 2–3 g/kg range, whereas commercial insecticides consisting of mixtures of essential oil compounds produce o50% mortality in rat (often no mortality) at 5 g/kg, the upper limit required for acute toxicity tests by most pesticide regulatory agencies (Isman, 2001). Mites have developed resistance to various acaricides, but resistance mechanism associated with octopamine receptors has not been reported with dust mites. Commercial acaricidal natural products and acaricides are listed in Table 4. The most currently available formulations such as foams, powders, solutions, or sprays have contact toxicity but the penetration rate of these acaricides into dust mite nests is not fully studied. Chemical powders are present in treated sites after treatment and should be eliminated using vacuum cleaner or others. Acaricidal solutions stain carpets and mattresses. Additionally, applying shampoo-type products will increase the humidity of the carpet and mite growth (Platts-Mills et al., 1992). Essential oilcontaining products with contact and fumigant action are ease to apply to homes or the house dust mite nests. In this respect, commercial success of the house dust mite control agents based on essential oils will be dependent on the development of more efficacious formulations. In general, plant extracts, essential oils, and their constituents are not as potent as most synthetic dust mite control products. In other words, they are not cure-all for dust mite control. They are effective against arthropod pests for a relatively short period because of their relatively high volatility. They could be of practical use as


284

Table 4 Commercial acaricides and denaturing agents of house dust mite allergens Trade name

Chemical

Formulation

Mechanism

Source

Acardust (Actomite)

0.4% esbiol, 3% piperonyl butoxide 3% benzyl benzoate

aerosol

contact acaricide

Geller-Bernstein et al. (1995)

foam

contact acaricide contact acaricide contact acaricide contact acaricide, allergen denaturant allergen denaturant

Schober et al. (1992)

Acarosan

powder Actellic50 Allerbiocid

Allergy Control Solution Allersearch DMS Allerzero

5% pirimiphosmethyl 3% benzyl benzoate, 1% tannic acid

spray

tannic acid

spray

alcohol based purified benzyltannate

spray

Cinnamomum cassia oil

spray

spray

Microstop Paragerm AK

Tymasil

benzyl benzoate, imidazole Permethrin, natural, pyrethrum benzoic acid, terpineol and salolthymol (in total 24%), 0.2% chlorophenol 2.17% natamycin, 0.2% benzylalkonium

Bird (1990) Green et al. (1989), Warner et al. (1993)

contact acaricide contact acaricide

Ahn and Kim (2005) Ahn and Kim (2005) Schober et al. (1992) Chen and Hsieh (1996)

aerosol

contact acaricide

Schober et al. (1992)

spray

contact acaricide

De Saint GeorgesGridelet (1988)

fumigant Artilin 3A

contact acaricide, allergen denaturant contact & fumigant fumigant

Schober et al. (1992) Hart et al. (1992)

paint spray, shampoo

fumigants for the house dust mites, provided that a carrier producing a slowrelease effect can be selected or developed. For example, Lantana camara L. flower extract in coconut oil provides 94.5% protection from Aedes albopictus (Skuse) and A. aegypti (L.), without adverse effects on the human volunteers for 3 month-period after the application (Dua et al., 1996). In addition to acaricidal products, new safer and more effective plant-based repellents or denaturing agents of dust mite allergens could be useful for protection of humans from mite-borne diseases.


285

Conclusions Certain plant essential oils rather than plant extracts have potential as the house dust mite control agents because they are widely available, relatively inexpensive, relatively nontoxic to mammals, and nonpersistent in the environment, but toxic to mites through contact and/or fumigant actions. These properties of the essential oils can lead to the development of appropriate types of formulations applying to a variety of the house dust mite habitats. Certain essential oils such as cinnamon, clove, and thyme oils are found as minimum risk pesticide products under FIFRA Section 25(b), and are exempt from toxicity data requirements by the U.S. EPA. Novel safer and more effective plantbased dust mite control agents or denaturing agents of dust mite allergens can enhance the possibility of commercial success in the near future.

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CHAPTER 13

The search for plant-derived compounds with antifeedant activity MONIQUE SJ SIMMONDS

Introduction Biochemical processes in plants have evolved to produce compounds often called the ‘‘secondary metabolites’’ that are involved in a multitude of functions including plant defence. These compounds often influence the behaviour of phytophagous insects, resulting in plant acceptance or rejection. Some of these compounds, such as the limonoid, azadirachtin, not only act as a deterrent but also have insecticidal activity if absorbed or eaten by insects (Simmonds and Blaney, 1996). The diversity of these ‘‘defence’’ compounds is high and many are very effective at protecting plants from being eaten. However, only a few have been developed into commercial products such as insecticides or ‘‘behaviour modifying deterrents’’. In contrast, thousands of plant-derived compounds and extracts have been developed as pharmaceutical drugs or as herbal medicines. Why is it that we have not been able to harness these natural feeding deterrents or antifeedants to assist us protect plants, whereas we have been able to develop them as drugs? Does this lack of success reflect the fact that finding a pesticide is harder than finding a pharmaceutical drug or is it simply a case that fewer compounds have been evaluated against insects compared to the high number screened for medicinal properties? In the late 1980s and early 1990s there was an increased interest in natural product chemistry as companies searched for the next generation of pesticides. However, the interest in screening natural products faded as the emphasis on genetic modifications of plants increased. We are now in a position to benefit from both approaches if they could come together. Molecular biologists and biochemists have advanced our understanding of the biosynthetic pathways in plants and this information could assist scientists manipulate the expression of defence compounds already in the plant rather than via the introduction of ‘‘foreign’’ genes. Many of the enzymes involved in the production of compounds with potential insecticidal/behaviour modifying activity are in a diversity of plants, but often these pathways are not switched on. Once the value of a specific compound in modulating insect behaviour is known then in theory there is the potential to stimulate its release. However, this is complex as the manipulation of an enzyme could influence the expression of an array of compounds as well as the target molecule.

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Whatever approach is taken in the search for novel pest control methods it is clear that as more insects become resistant to our present arsenal of insecticides, new leads are needed. Currently, very few commercial leads are being synthesised from computational chemistry programmes thus surely a case can be made for evaluating more plant-derived products. This review highlights the fact that there are many plant families and groups of compounds that justify further research. There are also a range of criteria that can be used to select the plants rather than randomly screening plant collections. The use of ethnobotanical information helped guide the discovery of many of the existing plant-derived leads such as the pyrethrins, carbamates, rotenoids and nicotine as well as the herbicide, leptospermone, from the bottlebrush (Callistemon lanceolatus). There are still examples of species, such as species of Plectranthus, Tephrosia and Salvia, with traditional uses in the area of pest control that still remain poorly studied (Grainge and Ahmed, 1988). However, there are ethical issues associated with the use of indigenous knowledge in selecting plants and this is an area that some companies could consider high risk. The ratification of the 1992 Convention on Biological Diversity has resulted in many countries restricting access to their genetic resources, including plants (Ten Kate and Laird, 1999). Access for research usually requires prior informed consent from a government body in the source country but also permission for the use of information about the traditional uses of a specific species, especially if this information is to be used in drug discovery. Recently, a group of 18 biodiversity rich countries got together to produce the 2005 Delhi Declaration that requires anyone wanting to patent a product derived from genetic resources to confirm that they had considered and obtained the permission of the stakeholders if any traditional knowledge had been used in the discovery, before they file the patent. It could be very difficult to ensure all the relevant stakeholders had been asked, even though a company might have the best intentions to honour this request. Thus the use of traditional knowledge, especially primary traditional knowledge, in selecting plants for agrochemical leads has complex practical implications. Another approach to decreasing insect damage on crops is to place more emphasis on the compounds present in wild relatives of crop plants that can confer resistance and thus decrease the susceptibility of some crops to attack. This could involve more traditional breeding methods as well as taking advantage of the advances in molecular biology. The challenges here are also complex as in many cases natural resistance involves an array of compounds and it is difficult to characterise the value of each specific compound. The benefits to the growers are that if an array of antiinsects compounds can be transferred into a crop it makes it more difficult for resistance to develop. Whatever strategy is used to select species for testing, account needs to be taken of the influence secondary metabolites have, not only on the pest but on the beneficial insects that are associated with the ecology of the specific crop and associated insect–plant relationships. The presence of a specific group of compounds in a plant can elicit behavioural responses from insects that vary from a stimulant to a deterrent. The deterrents or antifeedants usually signals the unsuitability of a plant, allowing the insect to reject on contact, thus avoiding the ingestion of compounds that could be toxic. In most cases it is the ovipositioning female that selects the host plant for the larvae, but for

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very mobile insects like locusts the nymphs select their food as they move through the vegetation. When the insect encounters a potential host plant, its selection behaviour will be influenced by its ability to detect compounds in the plant. Despite the amount of research already undertaken on insect selection behaviour we still do not fully understand which taste sensilla used by an insect to taste its food are of primary importance in enabling an insect detect antifeedants. When the compounds in the plant stimulate the taste neurones they produce electrical impulses that transmit the neural ‘‘code’’ that can result in the modification of insect behaviour. The behavioural and neural response of an insect to a specific compound is not always fixed. For example, exposure to a compound can influence subsequent encounters, thus giving the impression that an insect can learn to associate what might be a non-toxic compound with another toxic compound present in a plant. Thus host selection is not a fixed response and the variability in the behaviour of an individual will be influenced by its own experience as well as genetic or functional factors such as the expression of receptors on taste neurones that will be stimulated by specific compounds. Induction experiments have been used to show that the behavioural responses of insects to plant-derived antifeedant can be variable. For example, the Colorado potato beetle (CPB) Leptinotarsa decemlineata is deterred from feeding on varieties of potatoes with high levels of glycoalkaloids and breeders have tried to increase the levels of these compounds in the foliage of some potatoes to see if this could result in increased resistance. Research by Mitchell (1987) showed that the CPB did not appear to have specialised deterrent neurones. He tested a range of alkaloids and showed that they evoked a burst of neural firing from neurones in the taste sensilla and this response was associated with a deterrent behavioural response. This is in contrast with the classical dose-dependent neural response recorded from specific ‘‘deterrent’’ neurones in the taste sensilla of Spodoptera littoralis in response to a range of compounds (Simmonds et al., 1990). However, the CPB have the ability to give a dose-dependent neural response, as shown by the classic single neurone response they give to the glycoalkaloid leptine (Hollister et al., 2001). Adults of the western corn rootworm, Diabrotica virgifera virgifera gave similar responses to the deterrents b-hydrastine and strychnine (Chyb et al., 1995). Messchendorp et al. (1998) reported that the CPB also responded to sinigrin and drimanes, deterrents to the beetle. Some of these compounds not only stimulate deterrent neurones but also reduced the neural responses to stimulants such as sucrose. Thus compounds can elicit a deterrent behavioural response using different neural responses or ‘‘codes’’. Whatever the mechanisms it appears that specialist insects have a narrow host range and are more responsive to a greater range of plant-derived compounds than a polyphagous insect that has evolved to accept food that contains a greater diversity of compounds. For example, Bernays et al. (2000) showed that the monophagous species (Heliothis subflexa) was more likely to reject a non-host plant on first contact compared to the generalist insect (Heliothis virescens). Sometimes insects do not reject on first contact and this could be because compounds do not stimulate receptors on the external contact sensilla but via a post-ingestive feedback mechanism.

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To date more attention has been given to volatile compounds that either repel or attract insects than non-volatile antifeedants. This review provides examples of the less-volatile compounds that have potential as antifeedants, a term used to describe those compounds that deter insects from feeding. Schoonhoven et al. (1998) identified 11 criteria that should be considered when looking for a potential antifeedant. These included: no or low toxicity to beneficial arthropods, vertebrates and plants; active at low concentrations; effective against a range of pest species of insects; able to penetrate plant surface or taken up by roots; compatible with other pest control strategies; limited persistence in the environment; easily available, commercially viable and a long shelf life. Very few of the compounds and plant extracts covered by this review have been evaluated against all these criteria.

Classes of compounds with antifeedant activity Terpenoids The majority of terpenoids are derived from mevalonic acid (1) and their diversity depends in part on the position and number of isoprene units they have as well as the position and composition of the functional groups. The neo-clerodane diterpenoids (clerodin (2), jodrellin B (3) and ajugarin 1 (4)) isolated from species of Lamiaceae and Asteraceae have potent antifeedant activity at 100–1000 ppm (10–100 mg/treatment disc) against a range of insects, including species of Spodoptera (Lepidoptera). The activity of these molecules is significantly modulated by small changes in the composition of the functional groups on the molecule and the responses to a specific structure vary among different species of insects (Simmonds et al., 1989; Caballero et al., 2001). Despite the research on this group of compounds it is still unclear as to what is/are the key functional groups (Gebbinck et al., 2002). The presence of an epoxy group on the decalin portion of the molecule and a b-substituted a,b-unsaturated g-lactone are important for Lepidoptera, whereas a furan ring is important for the activity against Leptinotarsa decemlineata (Coleoptera). Furan and butenolide side chains are usually present in active molecules (Simmonds et al., 1989; Gebbinck et al., 2002). The classic example of an antifeedant is the triterpenoid, azadirachtin (5), isolated from the neem tree Azadirachta indica. There are many examples of active limonoids but this group of compounds has been considered too complex for a synthetic lead. Azadirachtin (5) has been tested against a number of agricultural pests such as locusts and lepidopteran larvae and has activity at concentrations less than 100 ppm. Another limonoid, toosendanin (6) has potent antifeedant at similar low concentrations (Schoonhoven et al., 1998). The active functional groups on these molecules were extensively studied in the early 1990s, but today there is more financial support for this type of research on compounds with pharmaceutical rather than agrochemical activity. The cucurbitacins, as illustrated by cucurbitacin B (7), are another group of biologically active triterpenoids (Chen et al., 2005a). They are very bitter to humans and deter many insects.


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HO CH3 O HOH2C

OH 1 O

O

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4

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5

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AcO MeO2C

OAc OAc

3

2

O

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OAc OCOCHMe2

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O 7

Saponins There are two main classes of saponins. Aescin (8) from the horsechestnut (Aesculus hippocastaneum) is an example of a triterpenoid saponin and dioscin (9) from yams (species of Dioscorea) of a steroidal saponin. Saponins are thought to have antifeedant activity but experiments suggest this is not always a direct effect on feeding but indirect as they decrease food utilisation (Adel et al., 2000). Recent research has shown that they are present in some crucifer species that are resistant to attack by flea beetles, but susceptible to attack by lepidopteran species such as Pieris (Agerbirk et al., 2003). Whether the difference in the responses of these two groups of insects is due to Pieris larvae being able to detoxify the saponins by enzymatic hydrolysis of the glycosidic bonds is not known. However, some saponins are deterrent, for example, 3-O-[O-b-D-glucopyranosyl-(1-4)–b-D-glucopyranosyl]-hederagenin (10) isolated from winter cress Barbarea vulgaris (Brassicaceae) is a feeding deterrent to


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the diamondback moth Plutella xylostella when tested in a no-choice test on cabbage leaf discs at 25 mg or more per disc (ED50 20 mg/disc (0.07 mg/mm2); Shinoda et al., 2002). This saponin is as active against P. xylostella as some quassinoids isolated from Picrasma ailenthoides (Simaroubaceae) that were active at 50–200 mg/disc (0.16–0.64 mg/mm2) (Daido et al., 1993). O O H H Glc O GlcA O O Glc

OAc CH2OH OH

H CH2OH 8

O

H

O

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H

COOH

H

Glc O O Rha

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10

HOH2C

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OH

HO OH

Phenolics It is highly unlikely that the major group of phenolics in plants, the flavonoids, will provide a novel lead that could be commercially developed. However, the different groups of flavonoids including the flavones (e.g. luteolin (11)), flavonols (e.g. kaempferol (12)), flavanones (e.g. naringenin (13)) as well as anthocyanins (e.g. cyanin (14)) and chalcones (e.g. isoliquiritigenin (15)) clearly play an important role in insect–plant interactions (Harborne and Grayer, 1994; Simmonds, 2001, 2003). Phaseolin (16) is an example of a potent antifeedant pterocarpan isoflavonoid. Increased levels of these compounds within a plant could be used to provide an increased level of natural resistance. However, we do not know what, if any, detrimental activity these changes would have on other herbivores. Flavonoids are present in most plants, although their profile varies among different species. Many flavonoids or groups of flavonoids are common to a range of different species, but the proportions of the compounds vary among the species. However, as yet there are very little experimental data with insects to show experimentally how these proportions


297

influence host-selection. Experiments with Helicoverpa armigera and different varieties of pigeon pea (Cajanus) and related wild relatives have shown that the proportion of isoquercitrin (17), quercetin (18) and quercetin-3-methyl ether (19) as well as a stilbene, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid (20), can influence the host selection behaviour of larvae (Green et al., 2003). The results of these experiments using plant extracts and isolated compounds on glass-fibre discs suggest that it is the proportion of the compounds that modulates the selection behaviour of the larvae rather than the amount of any specific compound. However, as yet we do not have enough experimental data to explain why the response of larvae to mixtures of compounds differs from the additive response to specific compounds in the mixture. Other research has shown that apigenin-C-glycosides, such as schaftoside (21), detected in extracts from varieties of rice resistance to pests modify the feeding behaviour of the brown planthopper, Nilaparvata lugens in parafilm sachet tests at between 250 and 500 ppm (Stevenson et al., 1996). To date one of the most studied flavonoid in insect–plant interactions is the flavonol glycoside rutin (quercetin-3-O-rhamnosyl[1-6]glucoside (22)) this compound occurs in many species and is also commercially available. When tested in a choice glass-fibre discs bioassay, the activity of rutin varies depending on the concentration encountered. For example, at concentrations greater than 10–3 M it deters 5th instar larvae of Heliothis zea and Helicoverpa armigera from feeding, whereas at concentrations of less than 10–4 M it stimulates these larvae to feed (Simmonds, 2003). Once rutin is ingested the glycosidic bond can be hydrolysed to release the aglycone quercetin (18). Quercetin is known to inhibit cytochrome P450-dependent mixed function oxidase (Michell et al., 1993) and mitochondrial ATPase and these actions could explain why ingestion of rutin could negatively influence the growth and feeding behaviour of insects, the action being greater in early instars compared to later stages. This difference in activity could be because the production of mixed function oxidase increases as the larvae mature and the 5th instar larvae might have enough enzymes to detoxify the quercetin. Knowledge about the role flavonoids play in insect–plant interactions is growing since the review by Harborne and Grayer (1994). However, it is still very difficult to predict how an insect will respond to a specific flavonoid. Overall, methoxylation of the hydroxyl groups on flavonoids results in a decrease in activity (Elliger et al., 1980). Of the 36 flavonoids tested for activity against the Bertha armyworm Mamesta configurata, only two (flavone (23) and the flavonol isorhamnetin-3-Osophoroside-7-O-glucoside (24) isolated from a host species Brassica napus L.) gave a Feeding Index (FI) ((C–T)/(C+T))%; where C and T represent the amount eaten of the control and flavonoid-treated cabbage discs, respectively of greater than 40% in a choice test (Onyilagha et al., 2004). In these assays a FI of greater than 40% indicates an antifeedant compound. Their data supported the earlier findings that the presence of a methoxy group on the B-ring and the substitution of the hydroxyl at C-3 enhance the antifeedant activity of flavonoids. Coumarins have been isolated from many different plant families (Murray et al., 1982). Of the different classes of coumarins the furanocoumarins have been reported to have the most potent antifeedant activity against a range of insects including Spodoptera littoralis (Stevenson et al., 2003). Most species that produce these furanocoumarins contain a diversity of structures and experiments with S. littoralis


298

have shown that the compounds have an additive toxicity but not antifeedant action (Calcagno et al., 2002). Of the most frequently encountered furanocoumarins, bergapten (25) and xanthotoxin (26) have more activity as antifeedants than psoralen (27), imperatorin (28) and angelicin (29). R1 OH HO

O

OH HO

O

OH HO

R OH O

OH O

OH O

13

15

11 R = H, R1 = OH 12 R = OH, R1 = H OH

HO

OH

O H

HO

O H O HO

HOH2C

O

O

O

OH

HO HO

OH OH CH2OH

14

O

O

16

OR2 OH RO

O

HO OR1

O

O

OH O

O

Glc

OH O 17 18 19 22 24

OH

Ara

R = R2 = H, R1 = β-Glc 21 R = R1 = R2 = H, R = R2 = H, R1 = CH3 R = R2 = H, R1 = α-Rha(1−>6)-β-Glc R = β-Glc, R1 = β-Glc(1−>2)-β-Glc, R2 = CH3

23

R

OH CO2H O

H3CO

O

O

O

O

O

R1 20

25 26 27 28

R = OCH3, R1 = H 29 R = H, R1 = OCH3 R = R1 = H R = H, R1 = OCH2CH=C(CH3)2

The expression of phenolics in plants is often influenced by environmental conditions and there is mounting evidence to show that climate change could


299

significantly influence the expression of these compounds in plants. Ozone has been shown to have a greater influence on plants than insects, but as yet there is little data to predict how insects will evolve to any changes in the chemistry of plants (Jackson et al., 2000). This is an area of research that is of increasing interest and importance. Alkaloids Only a few of the 10,000+ alkaloids characterised from plants have been tested for antifeedant activity. The pyridine alkaloid nicotine (30), produced from ornithine and nicotinic acid, is an example of one of the few plant-derived alkaloids that has been developed as an insecticide. Nicotine accumulates in species of Nicotiana (Solanaceae) and a few other genera in the tribe Nicotianoideae of the Solanaceae (Hegnauer, 1973). The concentration of nicotine in foliage can be induced and it has been shown to increase by 286% in leaves exposed to Lepidopteran larvae, compared to control leaves (Baldwin, 1988). Many alkaloids can be toxic to humans and thus cannot be developed as antifeedants. For example, strychnine (31) is one of the most potent antifeedant against the adult western corn rootworm (Diabrotica virgifera virigifera). The indole alkaloids strychnine (31) and quinine (32) are deterrent to many insects as are the purine alkaloid caffeine (33) and the pyrrolizidine alkaloid, senecionine N-oxide (34). H

HO

H

H N

N CH3

N

N

H

O

H H

30

H

H3CO

O

N

N 32

31 HO

O O H3C O

N

CH3 N

N N H CH3 33

O

H O H

O

N O 34

Diversity of angiosperms with antifeedant activity (Figure 1) Families within the angiosperms (flowering plants) differ in the diversity of compounds they contain and thus vary in the strategies used to protect themselves from herbivory. The families highlighted below are those that are known to contain compounds with potent anti-insect activity often because extracts from these plants have been shown to modify insect feeding behaviour. As part of a research programme into the antifeedant compounds in plants, researchers at the Royal Botanic

300


Gardens Kew have studied over 9000 species from 74 different families. The following list of active families is in part based on the results from this research and from research published after an earlier review by Simmonds et al. (1992). Emphasis is placed on those families with potent antifeedant rather than insecticidal activity. The families have been listed in the order presented in a recent phylogeny of the flowering plants based on DNA data (Chase, 2005; Figure 1). This shows that although there are active species scattered among the different families, a high proportion of extracts and compounds with antifeedant activity are in the Eurosids and Euasterid I groups of plant families. Magnoliids Magnoliales Annonaceae. The insecticidal tetrahydrofuran acetogenins associated with this family continue to be isolated (Araya et al., 2002) and there are now about 417 isolated from species of Annonaceae (Bermejo et al., 2005). These compounds are known to have potent antifeedant activity and extracts from species such as Annona squamosa L. are used in parts of Asia to treat seed to protect them from attack by bruchids (Dharmasena et al., 1995). There is increased interest in studying the chemistry of this group of plants because of their traditional use in the treatment of diabetes and their anti-cancer activity (Chen et al., 2004). Because the acetogenins are potent inhibitors of mitochondrial respiratory chain complex 1, they could have limited potential as commercial insecticides. Magnoliaceae. There is very little ecological information about the compounds found in species in this family. The compounds associated with the antifeedant activity of extracts from the tulip tree Liriodendron tulipifera (L.) against the gypsy moth Lymantria dispar have still to be characterised (Shields et al., 2003). Laurales Lauraceae. Persea indica contains diterpenes with insecticidal and antifeedant activity (Fraga et al., 2001). Fraga et al. (2001) tested a range of these diterpenoids against the Lepidoptera, Spodoptera littoralis and the CPB, Leptinotarsa decemlineata and showed that the activity of the compounds changes with the polarity of the cyclohexane ring. Garajonone (35) was less active (EC50 >25 nmol/cm2) than the ryanodol monoacetate (36), which had a EC50 0.01 nmol/cm2 against S. littoralis. The anhydro-compounds anhydrocinnzeylanone (37) (EC50 >27 nmol/cm2) and anhydrocinnzeylanine (38) were less potent antifeedants compared to the more polar cinnzeylanone (39) (EC50 1.46 nmol/cm2 against S. littoralis; EC50 0.22 nmol/cm2 against L. decemlineata) and cinnzeylanine (40) (EC50 0.01 nmol/cm2 against S. littoralis). Mode of action studies suggest that these compounds could mediate their antifeedant activity by acting as sodium channel antagonists on protein receptors on the taste sensilla, this action is similar to that seen with aconitine (Gonza´lezColoma et al., 1998).

angiosperms

Magnoliales Laurales Canellales Piperales

magnoliids Acorales

monocots

Alismatales Pandanales Dioscoreales Liliales Asparagales Arecales

Poales commelinids

Dasypogonaceae Commelinales

Zingiberales Ceratophyllales Ranunculales

eudicots

Sabiaceae Proteales Buxaceae Trochodendraceae

core eudicots

Gunnerales Aextoxicaceae Berberidopsidaceae Dilleniaceae Caryophyllales Santalales Saxifragales Crossosomatales

rosids

Geraniales Myrtales Celastrales Malpighiales

Oxalidales Rosales

Fabales Fagales Cucurbitales Brassicales Sapindales

eurosid I


Amborellaceae Nymphaeaceae Austrobaileyales Chloranthaceae

eurosid II

Malvales

Cornales

Ericales asterids

Garryales

Lamiales Solanales Gentianales

euasterid I

Asterales

euasterid II

Aquifoliales Apiales

301

Dipsacales Fig. 1. Phylogenetic distribution of species with potent anti-feedant activity. The phylogeny of the flowering plants is based on molecular data (Chase, 2005). Arrows indicate groups (clades) of families that contain species with potent antifeedant activity.


302

OH OH

OH HO AcO HO

HO O OH

O

HO AcO HO

O OH

35

36

OH R

OH R HO

HO

HO O

HO

O

O HO

OH 37 R = O 38 R = α-OH, H

OH 39 R = O 40 R = α-OH, H

Overall, the ryanodol diterpenoids are more potent antifeedants than the ryanodine-type diterpenoids. (Fraga et al., 2001). Canellales Canellaceae. The sesquiterpenoid dialdehyde, warburganal (41), isolated from Warburgia, disrupts the feeding behaviour of locusts and Lepidoptera, at concentrations as low as 0.1 ppm in a glass-fibre choice bioassay (Blaney et al., 1987). Although the antifeedant activity of chemically modified drimanes has been researched, there is very little information available about the distribution and activity of other drimanes from genera related to Warburgia. When tested in electrophysiological assays, warburganal caused bursts of rapid firing (‘‘bursting’’) from neurones in the taste sensilla of insects. It seemed to disrupt the functioning of the neurones that respond to deterrents as well as those that respond to phagostimulants. This type of reaction is often associated with potent antifeedants and it would be interesting to know more about the functional groups on the drimane molecule associated with the ability to cause this bursting reaction. This response is similar to that observed when the CPB was stimulated with glyoalkaloids (Mitchell, 1987). Winteraceae. The drimane polygodial (42) when tested in a choice bioassay on glass-fibre discs at 100 ppm deters lepidopterous from feeding (Blaney et al., 1987) and in no-choice bioassays disrupts the feeding of aphids (Messchendorp et al., 1998). It was isolated from Drimys lanceolata. This compound stimulates neurones in the sensilla positioned at the tip of the aphid’s antennae, but not taste neurones in the taste sensilla on their tarsi or mouthparts. CHO

CHO OH

CHO H 41

H 42

CHO


303

Piperales Aristolochiaceae. Aristolochic acids, such as aristolochic acid I and II (43 and 44), isolated from species of Aristolochia have been shown to have potent antifeedant activity against lepidopterous larvae, whereas some species such as the swallowtail larvae feed on plants containing the acids and sequester them as defence compounds in the adult (Fordyce, 2001). These compounds cause renal failure in humans (Martinez et al., 2002), so could not be developed as commercial products. However, it would be of interest to identify whether the aristolochic acids vary in their antifeedant activity and to assess the importance of the different functional groups to the potency of the molecule. CO2H

O

NO2

O

R 43 R = OCH3 44 R = H

Piperaceae. This family contains isobutylamide alkaloids with antifeedant and insecticidal activity. For example, pipercide (45) (LD50 ¼ 0.004 ppm) isolated from the fruits of Piper nigrum, was highly toxic against larvae of the mosquito Culex pipiens pallens (Park et al., 2002). Other amides have been shown to be as active as pyrethrin I (46) against the bruchid beetle, Callosobruchus chinensis (Miyakado et al., 1989). These compounds do not act in the same way as the pyrethrins and thus have potential for development. However, as with many natural product research projects, it is the recent interest in the medicinal properties of these compounds that is targeting their discovery, although insect assays are being used to test for the presence of active amides (Scott et al., 2005). The antifeedant activity of these molecules has not been well documented. O H N

O O

45 O H3C CH3 O H H H O 46

CH3


304

Monocots Alismatales There is very little recent research on the antifeedant activity of compounds from species in the families that belong to this group of plants. The families are known to contain a diversity of alkaloids. Araceae. The insecticidal and antifeedant activity of asarones from species of Acorus could be associated with the fumigant properties of extracts from this genus (Park et al., 2003). Pandanales Stemonaceae. Extracts from the roots of Stemona collinae Craib contain rotenoids that are known to have insecticidal activity as well as alkaloids. One of these alkaloids 16,17-didehydro-16(E)-stemofoline (47), had potent antifeedant as well as insecticidal activity against 3rd instar larvae of a pyrethroid-resistant strain of the diamondback moth (Plutella xylostella). The alkaloid was more active than rotenone (48) (Jiwajinda et al., 2001). OMe

H

H

O

N O

O

O

H O

O O O

H OCH3 OCH3

48

47

Velloziaceae. Species within this family contain neo-clerodanes and these compounds could explain the antifeedant activity of extracts. However, there is very little research undertaken on the antifeedant activity of diterpenoids from these plants. For example, species of Aylthonia contain aylthonic acid (49) (Pinto et al., 2004) but we do not know whether this compound has antifeedant activity. CO2H

H

OH 49

Dioscoreales Species within the family have a range of biological activities, especially in the area of cancer. However, there is little data on the antifeedant activity of compounds from the family, including the different saponins. There was an early report of antifeedant compounds being isolated from yam leaves that were active against ants (Febvay et al., 1985). Liliales Extracts from Lilium longiflorum deterred Plutella xylostella from feeding (Grainge and Ahmed, 1988), but the active compounds were not characterised.


305

Alkaloids from sabadilla, Schoenocaulon officinale, have potent insecticidal activity against a range of insects (Ujvary and Casida, 1997) and could also have antifeedant activity. Asparagales Alliaceae. The antifeedant activity of Allium is usually associated with the volatile compounds, so it is beyond the scope of this review. However, this is one of the few example of ‘‘leads’’ based on traditional use that is currently been developed commercially for use by the horticultural trade. Feeding experiments at Kew have indicated that the antifeedant activity of extracts from species of Allium could be associated with non-volatile compounds, so the chemistry of these extracts justifies further study. Amaryllidaceae. To date about 500 alkaloids have been isolated from the Amaryllidaceae (Jin, 2005). Many of these compounds have pharmaceutical uses, but their role in the plant, especially in the ecology of insect–plant interactions has not been studied. Preliminary studies have shown that the alkaloids lycorine (50) and galanthamine (51) have antifeedant activity when applied at 100 ppm to glass-fibre discs in a choice bioassay against Spodoptera littoralis, and the activity varies among closely related analogues of these compounds (Simmonds, unpublished). HO H

H

H OH

H O

H

H3CO

O H O

OH

N N

50

51

CH3

Agavaceae. Very few studies have been undertaken on the antifeedant activity of extracts from species in this family. Many Yucca species are sold by small companies in areas near where they grow. As a group of plants they show some resistance to attack by insects. Research on Yucca periculosa Baker identified some known stilbenes, including resveratrol (52), that regulated growth of the Fall armyworm Spodoptera frugiperda at 25 ppm (Torres et al., 2003). However, the stilbenes did not explain all the activity seen in the methanol extract of Y. periculosa. The growth inhibitory activity of a 25 ppm methanol extract of Y. periculosa was comparable to that of the limonoid, gedunin (53) at 25 ppm. O HO

O O OAc

OH HO

O 52

53

O


306

Hyacinthaceae. A group of alkaloids known as the polyhydroxyalkaloids are widely distributed within the Hyacinthaceae, including homoDMDP (54), a polyhydroxyalkaloid that appears to be restricted to the Hyacinthaceae (Asano et al., 2004). This compound is a potent inhibitor of b-glucosidase. Related compounds, including DMDP (55), isolated from different plant families have antifeedant activity against a range of insects including the nymphs of the locusts Locusta migratoria and Schistocera gregaria, the larvae of the Lepidoptera, Spodoptera littoralis, S. exempta and Heliothis virescens, the aphids, Acyrthosiphon psium and Myzus persicae and the Coleoptera, Callosobruchus manulatus and Tribolium confusum at concentration in the range 100–1000 ppm (Simmonds et al., 1990). Many of these polyhydroxyalkaloids are glycosidase inhibitors and appear to modulate the ability of insects to perceive sugars by interacting with sugar receptors on neurones in taste sensilla. The insect stops responding to sugars and stop feeding. Whether homoDMDP and the other polyhydroxyalkaloids isolated from species of Hyacinthaceae have antifeedant activity still needs to be established. HO HOH2C

OH

N H 54

CH2OH

HO HOH2C

OH

N H

OH OH

55

Iridaceae. Species contain isoflavonoids that along with the phytoecdysteroids could have antifeedant activity and contribute to the ability of extracts from species of this family to decrease insect growth of Lepidoptera such as Pieris rapae and the migratory milkweed Hemiptera bug Oncopeltus fasciatus (Grainge and Ahmed, 1988). Asphodelaceae. There is very little data on the antifeedant activity of extracts and compounds from species in this family despite the fact that many are resistant to attack by a wide range of insect species. The genus Aloe contains about 420 species and over 130 compounds have been identified from the aloes including anthrones, pyrones, coumarins, alkaloids, glycoproteins, naphthalenes and flavonoids (Dagne et al., 2000). As species of Aloe rarely suffer damage by leaf feeding insects, a study of the extracts of 47 species was undertaken. Extracts from eight species deterred Spodoptera littoralis from feeding when tested in a choice bioassay at 1000 ppm, whereas no extracts at this concentration deterred Locusta migratoria (Simmonds, 2004). The compounds associated with the antifeedant activity have not been characterised. Although in an earlier study a dihydroisocoumarin, 3,4-dihydro-6,8-dihydroxy-3-(20 -acetyl-30 -O-b0 D-glucopyranosyl-5 -hydroxyphenyl) methyl-2(1 H)-benzopyran-1-one (56), isolated from Aloe hildebrandtii was shown to have potent antifeedant activity in a choicebioassay using glass-fibre discs at 100 ppm, against Pieris brassicae and Plutella xylostella (Veitch et al., 1994). It stimulated larvae of Spodoptera littoralis to feed and was not active against Helicoverpa armigera. Thus the search for the antifeedant compounds in other species of Aloe continues.

The search for plant-derived compounds with antifeedant activity OH O

307

OH O

HO

OGlc COMe

H 56

Commelinids Poales Cyperaceae. A study of the hexane extracts from 17 species of Cyperus showed that they all had antifeedant activity against 3rd instar larvae of Spodoptera litura when applied to discs made from sweet potato (Morimoto et al., 1999). Cyperaquinone (57) (ED50 1.7 10–7 mol cm2) and its precursor remirol (58) (ED50 1.3 10–7 mol cm2) were isolated from C. nipponicus and were as active as rotenone (48) (ED50 1.5 10–7 mol cm2), whereas scabequinone (59) (ED50 2.6 10–9 mol cm2), isolated from C. distans, was a more potent antifeedant. It is unclear whether the antifeedant activity of extracts from species in this family could always be attributed to quinine type compounds. O

O

O

O

OH

O

H3CO

O 57

O

O

O O

58

59

Poaceae. The species of Avena and Arrhenatherum elatius are unusual within the family as they contain saponins. These compounds could contribute to the resistance of some species of Avena to fungi and it would be interesting to find out whether they play a role in resistance to attack by insects. Within Avena the triterpenoid avenacins accumulate in the roots and the steroidal avenacosides in the leaves (Osbourn, 2003). Zingiberales Cannaceae. Species of Canna are resistant to many insects and extracts from leaves of C. indica have been shown to stimulate deterrent neurones in the taste sensilla of Manduca sexta (Hanson, 1983). Eudicots: core eudicots Caryophyllales Caryophyllaceae. Many species in this family, including representatives from the genera Beta and Chenopodium, have been screened for the presence of phytoecdysteroids (Dinan et al., 1998) that can have antifeedant activity against insects (Blackford and Dinan, 1997). These compounds could contribute to or explain the early reports of antifeedant activity in extracts of these species when tested against Leptinotarsa decemlineata and Pieris brassicae (Grainge and Ahmed, 1988).


308

Rosids Geraniales Geraniaceae. Tannins can have antifeedant activity and the main tannin in Geranium is geraniin. This is an unusual ellagitannin as it lacks the astringency usually associated with this group of compounds. It is present in species of Geranium but absent from species of Pelargonium (Harborne and Williams, 2002). Its role in insect–plant interactions is not known. The zonal species of Pelargonium contain anacardic acids (60) that can deter insects from feeding (Hesk et al., 1992). These compounds also occur in representatives of the Anacardiaceae and Ginkgoaceae. Geraniums also contain indole alkaloids. Two indole alkaloids, elaeocarpidine 1 (61) and epielaeocarpidine 2 (62), were isolated from non-zonal varieties of Pelargonium. The compounds decreased the egg laying behaviour of the glasshouse whitefly Trialeurodes vaporariorum when applied at 1000 ppm to leaves from non-zonal varieties of Pelargonium but there is no data about their activity on other insects (Simmonds, 2002). OH CO2H N H H

N

R N

61 R = α-H 62 R = β-H

60

Myrtales Combretaceae. The compounds in species of Combretum, reported to deter Locusta migratoria from feeding, have not yet been characterised (Grainge and Ahmed, 1988). Myrtaceae. To date most research on the anti-insect activity of Myrtaceae-derived compounds has been on the essential oils. A C-methylated flavone, sideroxylin (63), isolated from Eucalyptus saligna (Sm.), was found to have mild antifeedant activity when tested in a glass-fibre choice bioassay at 100 ppm against Spodoptera exigua and Locusta migratoria (Sarker et al., 2001). Some species contain polyhdroxyalkaloids, such as DMDP (55) that deter feeding (Simmonds et al., 1990) but there is no published data reporting that the extracts of species of Myrtaceae that contain these compounds have antifeedant activity (Porter et al., 2000). OH

CH3 H3CO

O

H3C OH O 63

Eurosids I Celastraceae. This family contains species with dihydroagarofuran sesquiterpene polyol esters and pyridine alkaloids such as euoverrine B (64) show 50% knock


309

down activity at 21.6 mg/g against the Lepidoptera, Mythimna separate (Jinbo et al., 2002). In China many species of Celastrus are used in traditional Chinese medicine as well as insecticides. The active compounds include mytansine (65), triptolide (66), maytoline (67) and evonine (68) (Chiu, 1989). These compounds act on the nervous system and disrupt taste neurones. Alkaloids are associated with the antifeedant activity of species of Maytenus and Tripterygium. The alkaloid wilforine (69) is as active an antifeedant as the limonoid, azadirachtin (5), against some species (Delle Monache et al., 1984). Despite the early interest in these alkaloids, there is very little recent research on the antifeedant activity of alkaloids from this family. OAc R1O

OAc

R2O

R3 R4

O

CH3

O

HO

OAc

CH3

O

O

H OH N

O O H H3C

H OCH3

H CH3 O

N OCH3 CH3 Cl H CH3 O

H O H O

O

N

CH3

O

N CH3 CH3

65

64 R1 = Bz, R2 = Ac, R3 = H, R4 = OAc 69 R1 = Ac, R2 = Bz, R3 = OAc, R4 = H

O OAc AcO

O

OAc

O

AcO

O

O

CH3

O

HO

O O

H 66

OAc

O

O

OAc AcO

O

N

OH

OOC

AcO N

68

HO HO

O OAc 67

Malpighiales Euphorbiaceae. The genera in this family contain a rich diversity of terpenoids, including clerodane diterpenoids, limonoids and alkaloids. However, despite the large number of compounds isolated, only a few have been studied for their activity against insects. For example, the diterpenoid (–)-hardwickiic acid (70) isolated from Croton aromaticus, had insecticidal activity (Bandara et al., 1987). The a-seco limonoid, zumsin (71), isolated from Croton jatrophoides had antifeedant activity


310

against the pink bollworm, Pectinophora gossypiella and the fall armyworm Spodoptera frugiperda (Nihei et al., 2002). Extracts from species within the genera Euphorbia, Alchornea, Croton and Aleurites have been reported to have antifeedant activity (Grainge and Ahmed, 1988) and justify further research. O

O O AcO

OAc

H H O CO2H 70

O O

H

71

Fabales Fabaceae. There are about 18,000 different species of legumes and they contain a wide diversity of compounds known to influence insect behaviour, including alkaloids, non-protein amino acids, amines and flavonoids, including the isoflavonoids. When the distribution of the alkaloids in different genera of plants is superimposed onto a molecular phylogeny of the family then there are some trends that justify further study. For example, the quinolizidine alkaloids are known to be defence compounds. These compounds are mostly found in the genistoid alliance s.l. of the subfamily Papilionoideae. One exception within this clade are the species of Crotalaria that do not contain the quinolizidine alkaloids, but they contain pyrrolizidine alkaloids and non-protein amino acids (Wink, 2003). Many of the non-protein amino acids such as albizziine (72) accumulate in the Mimosoideae, whereas canavanine (73) occurs in tribes within the Papilionoideae that do not usually accumulate quinolizidine alkaloids. The majority of isoflavonoids are restricted to the Papilionoideae and some have been tested for antifeedant activity. For example, the isoflavonoids isolated from wild relatives of the chickpea, Cicer arietinum L., could confer resistance to attack by the polyphagous noctuid pest, Helicoverpa armigera (Simmonds and Stevenson, 2001). Of the four compounds (74–77) tested at 1–100 ppm in a choice test using glass-fibre discs, judaicin (74) and maackiain (75) were the most potent antifeedants. The antifeedant activity of 2-methoxy-judaicin (76), maackiain (75) and judaicin (74), when tested at 50 ppm, was enhanced by 20%, 19% and 10%, respectively, when tested in combination with 50 ppm chlorogenic acid (78), a compound found in many plant families. Maackiain (75) and judaicin (74) were also active at 100 ppm against Spodoptera frugiperda and Spodoptera littoralis, respectively. Judaicin 7-O-glucoside (77) was not active in these bioassays. The results of these bioassays suggest that the isoflavonoids in the wild relatives of chickpea could play an important role in modulating the responses of insects to chickpea and increasing the levels of these compounds in commercially grown chickpeas could enhance natural resistance to economically important pests. Isoflavonoids isolated from Trifolium subterraneum were also shown to have antifeedant activity (Wang et al., 1998). Flavanones, such as emoroidenone (79) isolated


311

from Tephrosia emoroides deterred Chilo partellus from feeding (Machocho et al., 1995). Some non-protein amino acids from species of legumes have antifeedant activity. For example, when tested in a choice bioassay using glass-fibre discs the non-protein amino acids, such as O-oxalyhomoserine (80) (FI of 3677.4 at 10 ppm, 2-amino-4-oxalylaminobutanoic acid (81) (FI 3576.4 at ppm) and 2,4diaminobutanoic acid (82) (FI 2674.2 ppm), isolated from Lathyrus latifolius were antifeedant against S. littoralis (Bell et al., 1996). In contrast, 2-amino-3-oxalylamino-propanoic acid (83) and 2-amino-4-hydroxybutanoic acid (homoserine) (84) stimulated feeding at 10 ppm. When combinations of these non-protein amino acids were tested in combinations that represented those present in different parts of L. latifolius they usually deterred feeding and the observed response was not a simple additive response to the non-amino acids present in the mixtures. Recent research on analogues of 2,4-methanoproline (85) isolated from Ateleia herbert-smithii have shown that although when tested on treated leaves the methanoproline was not active against S. littoralis, the synthetic derivatives had antifeedant activity that was comparable to the synthetic repellent N,N-diethy-M-toluamide (DEET) (Stevens et al., 2005). The early research on the rotenoids (e.g. rotenone (48)) in species of Lonchocarpus and Derris highlighted the importance of this family as a source of anti-insect compounds and this status is still justified. O

O H2N

H2N

OH H NH2

NH CH2 O

NH O CH2 CH2 HN

72

73

O O HO

O

O

HO

82

81

HN CH2

O OH H NH2

HO CH2 CH2

OH H NH2

O 84

83

RO

H2N CH2 CH2

O

O

HO

OH H NH2

HN CH2 CH2

80

O

O

O

OH H NH2

O CH2 CH2

OH H NH2

7

O

2

R1

HO

O

74 R = R1 = H 76 R = H, R1 = OCH3 77 R = β−Glc, R1 = H

COOH 85

O

O H3CO

N H

O O 75

O

OH H NH2


312

HO CO2H

O

O

O HO

OH

O OH

H3CO

OH

O 79

78

Fagales Fagaceae. Several triterpenes and flavonoids were isolated from species of Nothofagus (Thoison et al., 2004). Four compounds, the triterpenes 12-hydroxyoleanolic lactone (86) (FI ¼ 26) and 3-O-acetylcabraleadiol (87) (FI ¼ 26), the flavone pectolinarigenin (88) (FI ¼ 35) and the flavanone dihydrooroxylin A (89) (FI ¼ 36), when tested at a 1% concentration in a two-way choice bioassay using diet squares, deterred 5th instar larvae of Ctenopsteustis obliquane from feeding. Other genera within the family contain triterpenes that could have antifeedant activity. HO H OH O

O H

CO

H AcO

HO

H 87

86 OCH3 HO

O

HO

H3CO

O

H3CO OH O

OH O

88

89

Juglandaceae. This family is a rich source of naphthoquinones, such as juglone (90) and plumbagin (91), which deter many insects from feeding (Norris, 1986). However, there is very little data on the antifeedant activity of extracts from species in this family and activity might not always be associated with these compounds. O

O CH3

OH O 90

OH O 91


313

Cucurbitales Cucurbitaceae. The cucurbitacins are highly oxygenated triterpenoid compounds that are often cited as being the active compounds in modulating the feeding behaviour of Cucurbitaceae feeding and non-feeding species. For example, experiments with cucurbitaceous feeding beetles have shown that these compounds influence their host selection behaviour (Abe and Matsuda, 2005). Cucurbitacin B (7) stimulate the beetles Aulacophora indica and A. lewisii to feed, whereas extracts from a species Diplocyclos palmatus, even when treated with the cucurbitans, was rejected. Extracts from the bitter gourd, Momordica charantia, were antifeedant against Spodoptera litura (Yasui, 2002). The active compound was a triterpene monoglucoside momordicine II (92). Momordicines I (93) and II (92) also deterred the Cucurbitaceae feeding beetle Aulacophora nigripennis and when the compounds were tested together they also deterred Epilachna admirabilis and E. boisduvali from feeding. OR OHC

HO

H

OH 92 R = β-Glc 93 R = H

Eurosids II: Brassicales Brassicaceae. Glucosinolates, such as sinigrin (94), are often associated with the host selection behaviour of most Brassicaeae feeding insects and these compounds also have deterrent activity against non-Brassicaeae feeding insects. A sulphur containing indole alkaloid, dithyreanitrile (95), was isolated from the seeds of Dithyrea wislizenii and shown to deter Spodoptera frugiperda and Ostrinia nubilalis from feeding (Powell et al., 1991). However, very little work has been undertaken on the biological activity of this unusual type of alkaloid. O O S O N O CH2OH CH2 CH CH2 O HO S HO OH 94

H3CS

SCH3 CN

N H

OCH3 95

Sapindales Meliaceae. The family Meliaceae contains many species with very potent antifeedant compounds, such as the limonoids isolated from Azadirachta indica, Trichila, Cedrela, Toona, Khaya and Swietenia (Champagne et al., 1989). Aglaia is another genus with anti-insect compounds including the rocaglamide derivatives that have insecticidal activity (Schneider et al., 2000). They were tested in artificial medium diet using neonate larvae and the recorded mortality could be because they stopped the insects


314

from feeding. When tested against larvae of Spodoptera littoralis these rocaglamide compounds are as active as azadirachtin (5). For example, methylrocaglate (96) and C30 hydroxy methylrocaglate (97) had LC50 values of 1.3 and 1.1 ppm, respectively, compared to 0.7 ppm for azadirachtin (5) (Schneider et al., 2000). However, more recent bioefficacy studies showed that rocaglamide was not an antifeedant (Koul et al., 2004). In contrast, aglaroxin A (98), isolated from Aglaia elaegnoidea, is both an antifeedant and chronin toxin against Helicoverpa armigera and Spodoptera litura. The action of this compound differs from that of azadirachtin (5) (Koul et al., 2005).

H3CO

A

OCH3 OH OH

OCH3 OH OH

O O CO2CH3

CON(CH3)2

O

O

C

B R OCH3

OCH3

96 R = H 97 R = OH

98

Asterids: Ericales Balsaminaceae. The compounds associated with the antifeedant activity of extracts from Impatiens against Pieris brassicae and Plutella xylostella (Grainge and Ahmed, 1988) have still to be characterised. Euasterids I Boraginaceae. Extracts from Heliotropium deterred Locusta migratoria from feeding (Grainge and Ahmed, 1988). Lamiales Bignoniaceae. The presence of iridoid glycosides could contribute to the antifeedant activity of some species. For example, Catalpa contains the iridoid, specionin (99), which deters the eastern spruce budworm Choristoneura fumiferana from feeding at 50–100 ppm (Chan et al., 1990). Another iridoid, catalpol (100), present in many species deters Spodoptera exempta from feeding at 100 ppm (Simmonds et al., 1992). However, there are only a few iridoids that have been shown to deter feeding and the antifeedant activity of extracts could be associated with diterpenoids, but only a few of these compounds from this family have been tested against insects. HO O O

H

OEt

H O

O HOH2C H OEt 99

HO H H O O H HOH2C OGlc 100


315

Lamiaceae. Many of the 220 genera in the family Lamiaceae contain diterpenoids with antifeedant activity. The ent-kaurane diterpenoid sideroxol (101) isolated from Sideritis akmanii and S. rubriflora were antifeedant against Spodoptera frugiperda at 100 ppm (Bondi et al., 2000). Chemical modifications of the functional groups at C-4, C-7 and C-18 on the linearol (102) molecule modulates the activity of the compounds (Bruno et al., 2001). For example, when tested at 100 ppm linearol (102) is inactive as an antifeedant against Spodoptera littoralis, whereas the derivative of linearol (103) (FI 74%) is active. Of the 21 compounds tested, compound 103 was the only compound not to have a hydroxyl or acetyl group at C-18. This compound is more active than the neo-clerodane antifeedant ajugarin 1 (4) (FI 43%; Simmonds et al., 1989), but it is not as active as jodrellin B (3) (FI 100%; Cole et al., 1990) isolated from Scutellaria galericulata. Jodrellin B (3) is one of the most active antifeedant isolated from species of Scutellaria (Bruno et al., 2002). New neo-clerodanes with antifeedant activity against S. littoralis continue to be isolated from species of Teucrium (Bruno et al., 1999; Kumari et al., 2003; Coll and Tandron, 2004).

O

O HO

OH

OH

OH

OAc

101

102

AcO

O O

103

O

O

Iridoid glycosides are distributed within genera in the Lamioideae and are often thought to be associated with the medicinal activity of the species. Some iridoids are sequestered as defence compounds by insects and as indicated a few have antifeedant activity but very few have stimulated an antifeedant response from species such as Spodoptera littoralis. Solanales Convolvulaceae. Species in this family contain a range of alkaloids including pyrrolizidine alkaloids (Jenett-Siems et al., 2005) that could influence the feeding behaviour of a range of insects. A survey of 129 species showed that many contain from one to six polyhydroxyalkaloids (Schimming et al., 2005) compounds with known antifeedant activity against locusts and Lepidoptera larvae at concentrations between 1 and 1000 ppm (Simmonds et al., 1990). The genus Ipomoea contained calystegines in eight of the ten taxonomic sections (Schimming et al., 2005). The activity of this group of tropane alkaloids have not been fully studied against insects.


316

Earlier studies have shown that extracts of Ipomoea purpurea and Calystegia sepium deter Locusta migratoria from feeding, whereas extracts from Convolvulus arvensis deter Leptinotarsa decemlineata and Pieris brassicae (Grainge and Ahmed, 1988). Solanaceae. The Solanaceae family comprises about 96 genera and 3000 species that contain a diversity of antifeedant compounds including tropane, pyridine and steroid alkaloids and diterpenoids. The tropane alkaloids are distributed in unrelated genera within the family, whereas the steroidal alkaloids are more restricted, especially within the Solanum and very closely related genera (Wink, 2003). The genus Solanum contains a range of active antifeedant compounds including steroidal saponins and glycoalkaloids. Luciamin (104) isolated from Solanum laxum Steud. was the first steroidal glycoside to be shown to be an antifeedant against the aphid Schizaphis graminum. This is an important finding as other saponins could also deter aphids from feeding and thus decrease the spread of viral infections (Soule´ et al., 2002). This study has highlighted the importance of studying the role these compounds could play in plant–insect interactions. O OH

O O

HO HO O HO HO OH

O

OH

OH O O

O

HO OH O H3C O HO HO OH

104

Gentianales Apocynaceae. Species contain cardenolides and alkaloids with antifeedant activity. For example, the steroidal alkaloid conessine (105) from Hollarrhena antidysenterica is a potent antifeedant (Thappa et al., 1989). It is active at low concentrations (0.5–10 ppm) and can be compared favourably to azadirachtin (5). The antifeedant compounds in species of Acokanthera, Apocynum, Calotropis, Mandvilla and Nerium have still to be characterised (Grainge and Ahmed, 1988). H3C H N CH3 H H H3C

H N CH3 105

H


317

Euasterids II: Asterales Asteraceae. This family has attracted attention since the isolation of the pyrethrins (46) from Chrysanthemum cinerariaefolium (Elliot, 1977). A range of silphinene sesquiterpenes (106–108) have been isolated from species of Asteraceae including Senecio palmensis (Gonzalez-Coloma et al., 2002) and Artemisia chamaemelifolia (Marco et al., 1996). These compounds show potent antifeedant activity against the CPB but very little activity against Spodoptera littoralis. Some of these compounds are known GABA antagonists which supports the hypothesis that the antifeedant action on CPB could, like the Western Corn rootworm, be mediated by stimulation of GABA receptors on the taste neurons (Mullen et al., 1997). The difference in the responsiveness between the beetles and the Lepidoptera is most likely due to differences in the pharmacology of their GABA receptors. Species of Asteraceae contain sesquiterpene lactones, such as encelin (109), from species of Encelia that have antifeedant activity against Spodoptera littoralis (Srivastava et al., 1990). Tenulin (110) is another sesquiterpene lactone from Helenium amarum that deters Ostrinia nubilalis (Arnason et al., 1987) and lactupicrin (111) and 8-deoxylactucin (112) from Cichorium intybus deters Schistocerca gregaria (Rees and Harborne, 1985). The spiroketal enol ethers isolated from species of Artemisia have potent antifeedant against Pieris brassicae (Chen et al., 2005b). The antifeedant activity of Gnaphalium affine against the common cutworm, Spodoptera litura, was associated in part with four flavonoids: 5-hydroxy-3,6,7,8,40 -pentamethoxyflavone (113), 5-hydroxy-3,6,7,8-tetramethoxyflavone (114), 5,6-dihydroxy-3,7-dimethoxyflavone (115) and 4,40 ,60 -trihydroxy-20 -methoxychalcone (116) (Morimoto et al., 2000). Clerodane diterpenoids have been isolated from species of Baccharis and tested for antifeedant activity against Tenebrio molitor (Cifuente et al., 2002). The activity of compounds such as marrubiagenine (117), bacrispine (118) and the derivative bacchotricuneatin (119), appears to be associated with the presence of a b-substituted furan ring or a b-substituted butenolide function on the side chain. H CH3 H

OAc H

H

O

H

O O

H

R

O

H

O CH3 H3C OH

O

O

110

109

106 R = O

H O H

O OH

O

O

O

107 R = O

H O H

H

O HOH2C

H

HOH2C

O

108 R = O O O

H

111

H

112

H O O


318

O HO

O

H

H

H

O

O

H

O

OH CO2H

O

117

118

O

O

119

R2

R1 H3CO

O

O

HO

O

RO

OCH3

OH

OCH3 OH O

113 R = CH3, R1 = R2 = OCH3 114 R = R1 = OCH3, R2 = H 115 R = R1 = R2 = H

OH O 116

Conclusions This short review has shown that there are many thousands of plants that justify further study in order to further our understanding of the role secondary metabolites play in insect–plant interactions, especially their role in the modulation of insect host selection and feeding behaviour. Currently there is an increased interest in alternatives to insecticides to control insects and compounds that modify insect behaviour could have a part in an integrated pest management strategy. However, we need to further our understanding of how antifeedants work and increase our knowledge about the diversity of compounds that have this activity. However, it is very difficult to fund this type of research and very few commercial companies screen plants as part of their new molecule discovery programmes. Currently, there are many papers being published on natural products that make reference to the ecological importance of the compounds being isolated but very rarely report results of experiments to support the claims. There is a clear case for encouraging entomologist and natural product chemists to work together to increase our understanding of the role these compounds play in different aspects of plant–insect interactions. At a meeting on pest control in Kuala Lumpur in 1991, it had been hoped that by 2000 there would be an increase in the diversity of plant-derived pest control agents being commercially developed (Simmonds et al., 1992). This has not occurred. Whether this happens in the next 10 years is in part dependent on the emphasis placed in this area of research by academic scientists working on natural products.


319

Acknowledgments I thank Dr N.C. Veitch for drawing the structures of compounds and to Prof. W.M. Blaney for his encouragement and also to all those staff and students at the Royal Botanic Gardens, Kew, that have provided me with extracts and compounds to test.

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CHAPTER 14

An overview of the antimicrobial properties of Mexican medicinal plants DIANA JASSO DE RODRI´GUEZ, JOSE´ LUIS ANGULO-SAŃCHEZ, FRANCISCO DANIEL HERNAŃDEZ-CASTILLO

Introduction The use of different plants for medicinal purposes has been a common practice in Mexico well before the arrival of the Spanish in the XVI century but its knowledge was subdued, although it is being re-established nowadays. However, the scientific knowledge of the active compounds as well as the microorganisms that may be controlled by the use of those compounds has evolved at a slow pace. There are many chemical structures within the plants that protect them from the attack of different bacteria and fungi. Related to flora, Mexico is one of the countries with the greatest biological diversity due to the number of species and the biological variability levels; it possesses around 10–12% of the world total species, between 23,000 and 30,000 species, with a high endemic level due to the topography, climatic variety, and biogeographic convergence (neartic and neotropical). In this work, 12 species from different families are reviewed in terms of their characteristics, distribution in Mexico, and bioactive compounds identified for controlling, fungi, microorganisms, and insects. The plants considered here are presented in order of importance in terms of their use and knowledge of their bioactive compounds. The following list includes the scientific and most common name of the plants in Mexico: i. ii. iii. iv. v. vi. vii. viii.

Larrea tridentata (Gobernadora) Flourensia cernua (Hojase´) Chenopodium ambrosioides (Epazote) Tagetes erecta (Cempasućhitl) Metopium brownei (Checheń negro) Agave lechuguilla (Lechuguilla) Capsicum spp. (Chile) Lophophora williamsii (Peyote)


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ix. x. xi. xii.

Yucca spp. (Palma del desierto) Bidens pilosa (Chilca) Byrsonima crassifolia (Nanche) Bursera simaruba (Almacigo blanco)

All of the species are presently used as ‘‘multipurpose’’ materials ranging from shamanism to medicinal or building materials; most of them are known for their curative properties against different diseases. The information about chemical composition and bioactive compounds reviewed includes reports in the open literature and that generated at the Institutions where the authors are working.

Larrea tridentata (Gobernadora) Taxonomic classification Family: Zygophyllaceae Genus: Larrea Species: tridentate

Common names Falsa alcaparra (Sonora and San Luis Potosı´ ), Gobernadora (Northern states in Mexico); Guamis (San Luis Potosı´ and Chihuahua), Hediondilla, Huamis, Haáxat, Haájat (Seri indians in Sonora), Jarilla (Mexico) (Martı´ nez, 1994). General description Gobernadora (Figure 1) is a shrub 1–3 m in height, leaves with 3–15 leaflets 5–10 mm in length, covered with a resin of astringent odoor; small yellow flowers with 5 petals and 10 stamens; and hairy fruits with five carpels. Larrea is found in the arid Northern and Central parts and Southern USA (Martı´ nez, 1979 cited by Gamboa, 1997). Larrea tridentata Cav. is found only in North America where it occurs as a diploid (n ¼ 13) in the Chihuahuan desert, tetraploid (n ¼ 26) in the more arid Sonoran desert, and hexaploid (n ¼ 39) in the still drier Mojave desert. The chemical analysis shows that it was derived from South America similar to L. divaricata of Argentina (Mabry and Bohnstedt, 1981). Habitat The plant is resistant to drought conditions and survives where other species do not, because it collects good amounts of water even in light rain and maintains a net photosynthesis in dry lands. It grows in sandy calcareous soils with low phosphorus content. Larrea has few natural pests. It is interesting to note that plants whose aerial parts were burnt by atomic detonations were still able to sprout.

Antimicrobial properties of Mexican medicinal plants

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Fig. 1. Wild Larrea tridentata in Coahuila southeast.

Fig. 2. Distribution of Larrea tridentata in Mexico.

Distribution In Mexico (Figure 2) it is found in the states of Baja California, Sonora, Chihuahua, Coahuila, Nuevo Leoń, Tamaulipas, Durango, Zacatecas, Aguascalientes, San Luis Potosı´ , Quere´taro, and Hidalgo. Phytochemical analysis Gobernadora has been used as a source of nordihydroguayaretic acid (NDGA) (Figure 3), a commercially isolated phenol from the resin on the leaves. The resin has


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H3CO HO

OH

OH

HO

O OH

OH OCH3 OH

Nordihydroguayaretic acid

O OH Flavonoid aglycone

Fig. 3. Chemical structures of two outstanding compounds in Larrea tridentata.

been studied for its phytochemical properties and 18 flavonoids besides NDGA. Other chemicals present in lower concentration in the resin are volatile oils, waxes, and terpenes. Despite the complex phytochemical analysis there are few studies about the biological activity of the resin. The microbiological tests proved the resin to possess fungicide action against Rhizoctonia solani, Fusarium oxysporum, Pythium spp. and other phytopathogenic fungi. Some studies on the fungicide activity as a function of the season were carried out as well (Hurtado et al., 1981). Chemical analysis by GC/MS identified 67 volatile constituents which represent 90% of the volatile oil obtained by steam distillation of the resin. There are 19 aglycon-flavonoids besides different lignans which include NDGA. Some glycosides, flavonoids sapogenins, and waxes have also been isolated. Properties and documented actions This plant has been widely used in folkloric medicine by native Americans for treatment of urinary diseases such as kidney stones. The whole plant or branches are boiled in water and the tea is drunk as water. For other problems such as kidney pain or bladder inflammation the branches, cortex, or roots are used in the form of a tea and drunk before breakfast. In gynecologic disorders, vaginal douches with tea leaves are used. This infusion is also used for rheumatism, dermatitis, hepatitis, mycosis, and as an antiseptic. It purportedly cures venereal diseases and tuberculosis. Anti-amoeba activity was reported by Timmermann (1981) and Martı´ nez (1993). Larrea has been traditionally used for medicinal purposes by native Americans (Brinker, 1993). The folkloric medicinal uses are mainly based on a tea prepared from its leaves (Timmermann, 1981). Twigs and leaves contain large amounts of protein and other nutrients such that the whole plant may serve as a livestock feed. NDGA, the main phenolic constituent of Larrea and a potent antioxidant, is potentially useful in pharmaceutical products, lubricants, and rubber (Belmares et al., 1981); it also has antimicrobial properties (Vera´stegui et al., 1996). Antifungal activity The antifungal activity in vitro of Larrea extracts, has been studied carrying out inhibitory bioassays for fungi at doses of 0, 500, 1000, 2000, 4000, and 8000 ml/l. The


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resin from leaves and twigs was extracted with methanol, ethanol, or chloroform. The results showed that the extracts, despite the solvent, totally inhibited the mycelial development of Pythium spp. (Lira et al., 2003a, 2003d). Fungicide activity against Rhizoctonia solani, Fusarium oxysporum, Pythium aphanidermatum, and other phytopathogens was reported by Zavaleta (1990), against Puccinia cacabata (Marcos, 1996) at high concentrations, and fungistatic activity at low concentrations. The resin shows low efficiency in the control of the cotton smallpox caused by Puccinia cacabata (Jimeńez and Valle, 1981). At 2000 ml/l mycelial inhibition of growth was observed on Cytospora spp. and on Eutypa armeniacae (Vela´zquez, 1981). Resin extracts with dichloromethanol and methanol (500 ml/l) inhibited growth of Pythium spp. (Hurtado et al., 1981), Aspergillus flavus, and Aspergillus parasiticus (Vargas et al., 1997); however, more tests are required for a better evaluation. Activity of Larrea extracts (alone and in mixtures with chitosan) was tested against Botrytis cinerea, Colletotrichum coccodes, Colletotrichum gloeosporioides, and Fusarium oxysporum (Lira et al., 2003c); a synergic effect in the mixture was found. Solarization plus application of Larrea extract powder to the soil reduced root damage to pepper (cv. Anaheim) plants by soilborne pathogens (Lira et al., 2003b). Inhibition of Rhizoctonia solani and Phytophthora infestans was reported by Gamboa et al. (2003). The in vitro studies of different phytopathogens tested with methanol, ethanol, chloroform, or sodium hydroxide extracts showed excellent inhibition of the mycelial growth in Stramenopila (Pythium spp. and Phytophthora infestans) and fungi Rhizoctonia solani, Alternaria solani, Alternaria alternata, and Fusarium oxysporum. Methanol extracts prevented mycelial growth of Pythium spp. and P. infestans at 500 ml/l whereas at 800 ml/l it inhibited growth of all others except F. oxysporum (Lira et al., 2003c). Gordon (1987) reported that acetone extracts from twigs and leaves were fractionated into 16 different components, nine of which inhibited the growth of Trychophython mentagrophytes, T. rubrum, Microsporum canis, Bacillus subtilis, Escherichia coli, and Cladosporium cucumerinum.

Antimicrobial activity The antimicrobial activity of ethanol extracts on the development of pathogenic yeasts, molds, and bacteria was effective against 50% of the tested microorganisms (Verastegui et al., 1996). Effects on Bacillus subtilus and Escherichia coli have been reported.

Insecticide and nematicide activity Larrea extracts have shown insecticide activity against the bean weevil (Acanthoscelides obtectus, Coleoptera: Bruchidae) and the grain borer (Prostephanus truncatus, Coleoptera: Bostrichidae), and action on the nematode Meloidogyne incognita. Field test proved nematicide action on Tylenchus, Ditylenchus, and Rabditis at 100 ml/l (Marcos, 1996). Aphid control was reported by Zavaleta (1990).

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Table 1 Volatile compounds distribution in Larrea tridentata (Mabry and Bohnstedt, 1981) Chemicals Monoterpenes hydrocarbons a-Pinene b-Pinene a-Fenchene b-Ocimene Limonene Camphene Oxygeneted monoterpenes Borneol Camphor Bornyl acetate Linalool Sesquiterpenes Edulane Calamenene a-Curcumene b-Santalene Cuparene a-Bergamotene Cupaene 2-Rossalene g-eudesmol b-eudesmol a-agarofuran Farnesol Aromatics Acetphenone Ethylhydrocinnamate Benzaldehyde p-Cymene Benzylacetate o-Methylanisate Ethyl palmitate Benzylbutanoate Ethylbenzoate 1-Methyl naphthalene 1,2-dihydro-1,5,8-tri-methyl naphthalene Ethyl pentadecanoate 3-Hexenylacetate Volatile miscellaneous 2-Nonone 2-Undecanone 2-Dodecanone 2-Tridecanone 2-Tetradecanone 2-Pentadecanone 1-Hexen-3-one 1-Hepten-3-one 1-Octen-3-one n-Tridecane

Chromosomic number 2n

4n

6n

*** * * * *** **

*** * * * *** **

*** * * * *** **

*** *** ** *

*** *** ** *

*** *** ** *

* * * * * * * * *** * ** *

* * * * * * – – *** * ** *

* * * * * * – – *** – ** *

* * ** ** * * * * ** * * * *

* * ** ** * * * * ** * * * *

* * ** ** * * * * ** * * * *

* *** * *** * *** * ** **

* *** * *** * *** * ** **

* *** * *** * *** * ** **


331

Table 1 (continued ) Chemicals n-Tetradecane 3-Methyl butanal 3-Hexanol Hexanal

Chromosomic number * * * *

* * * *

* * * *

Note:* Less than 1%. ** 1–3%. *** greater than 3%.

Table 2 Flavonoids in Larrea Flavonoids Nordihydroguayaretic acid Guayaretic acid Norisoguayacin 30 -Demetoxyisoguayacin Dihydroguayaretic acid Partially demethylated dihydroguayaretic acid Flavonoid aglycones Kaemferol Kaemferol-3 methyl ether Quercetin-isorhamnetin Quercetin 3-methyl ether Gossypetin 3,7,30 -trimethyl ether Gossypetin 3,7-dimethyl ether Herbacetin 3,7-dimethyl ether Flavonols Quercetin 3,7,30 -trimethyl ether Quercetin 7,30 , 40 -trimethyl ether Flavonoid glycosides Kaemferol 3-rhamno glucoside Quercetin 3-o-glucoside Quercetin 3-o-rhamno glucoside Apigenin 6,8-di-C-glucoside (vicenin-2) Chrysoeriol 6,8-di-C-glucoside

Active chemicals The resin is constituted by complex waxes, hundreds of volatile compounds, saponins and other triterpenes, and large quantities of phenolics, including many flavonoid aglycons and glycosides (Mabry and Bohnstedt, 1981). In Tables 1 and 2, some chemicals identified by gas chromatography–mass spectrometry (Bohnstedt, 1977) are reported. Market At present, application of Larrea extracts against potato pests is being carried out.


332

Flourensia cernua (Hojase´) Taxonomic classification Family: Asteraceae Genus: Flourensia Species: cernua Common names Tarbush, blackbrush, varnish-brush, arbusto de alquitrań, escobilla negra, hojaseń, hojase´ (Valde´s, 1988). General description Flourensia cernua (Figure 4) is a perennial, single-stem erect shrub 1–2 m in height, heavily branched, tan or gray in color, having dense foliage that exudes a resin with tar fragrance (Valde´s, 1988; Za´rate, 1989). Leaves are elliptical to oblong, simple, 17–25 mm in length, 6.5–11.5 mm in width, and dark green in color. The plant flowers from September to December depending on the rainy season. The flowers are pale-yellow and grouped in panicles 1 cm in diameter (Figure 4). Fruits are achenes (Vines, 1960; Correll and Johnston, 1970; Benson and Darrow, 1981; Za´rate, 1989). Flourensia cernua may be propagated vegetatively or by seeds (Scifres, 1980). Habitat Flourensia is abundant in arid and semiarid lands between 1600 and 1900 m asl.

Fig. 4. Flourensia cernua.


333

Distribution In Mexico (Figure 5), Flourensia is found in the Chihuahuan and Sonoran deserts, as well as in states of Coahuila, Chihuahua, Durango, Hidalgo, Nuevo Leoń, San Luis Potosı´ , Sonora, and Zacatecas (Valde´s, 1988; Martı´ nez, 1993f). Phytochemical analysis The Flourensia genus is important due to the great amount of secondary metabolites it posseses; these are widely used for biological and ecological applications. Nine species of Flourensia have been reported, Flourensia cernua being the one with the highest number of chemicals (Aregullı´ n and Rodrı´ guez, 1983) having economic potential. The authors correlated the presence of benzofurans and benzopyrans with biological activity. The fact that these secondary metabolites are not present in other species led to a correlation between the ecographic distribution and a possible chemical adaptation to the environment. Properties and documented actions Several medicinal properties have been reported for the tea obtained with the leaves or flowers for indigestion and gastrointestinal problems (Arredondo, 1981). The green fruits are innocuous for cattle. However, dry fruits are toxic and when consumed at approximately 1% of the animal weight they cause death within the first 24 h (Sperry et al., 1968). Antifungal activity In vitro fungicide activity of leaf extract at a solution concentration of 1000 mg/l on Rhizoctonia solani, Pythium spp., and Fusarium oxysporum was reported (SaeediGhomi and Maldonado, 1982). The crude fractions of hexane, diethyl ether, and

Fig. 5. Distribution of F. cernua shrubs in Mexico.

334


ethanol were active against Colletotrichum fragariae Brooks, C. gloesporioides Penz., and Sacc. The essential oils from the hexane crude extracts were active at 1 mg doses, whereas the ether and ethanol fractions were active at 10 mg doses The ethanol crude extract was active against C. accutatum Simmons only at 400 mg (Tellez et al., 2001). Antimicrobial activity The mixtures of benzofurans and benzopyrans were tested against Gram-positive and Gram-negative bacteria, fungi, and Saccharomyces under two experimental conditions: One where the inoculated media was kept in darkness and the other where the inoculated media was UV irradiated (280–400 nm) for 15 min prior to incubation in darkness. Bioactivity was greatly increased by the UV irradiation (Towers et al., 1975; Aregullin and Rodrı´ guez, 1983). Insecticide and nematicide activity The insecticide activity of the benzofuran 7-methoxy-2-isopropenil-5-acetil-2,3-dihidrobenzofuran-3-ol-cinnamate proved its activity as a juvenile hormone causing anatomic malformation, juvenile characteristic retention, and sterility in the insects treated in their second to fourth stages of development (Towers et al., 1975). The results were similar to those with precosene reported by Bowers (1971). Termiticidal activity of hexane, ether, and ethanol crude extracts was found by Tellez et al. (2001). Cytotoxic activity Pure benzofurans and benzopyrans (see Figures 6e and 6f) have been studied for cytotoxic activity using red blood cells and measuring the hemoglobin released on cell destruction. The benzopyrans were more active than benzofurans, although no clear correlation between activity and structure has been obtained. The UVirradiated compounds showed higher cytotoxic activity than the non-irradiated ones; that is, they are phototoxic (Towers et al., 1980). Benzofurans and benzopyrans react with L-cystein (Towers et al., 1979), and the microbicidal and cytotoxic activities may be associated with the alkyl formation capacity. Significant inhibition of radicle growth of Amaranthus hypochondriacus was reported by Mata et al. (2003). Active chemicals Fractionation of a CHCl3–methanol (1:1) extract showed three compounds: dehyfluorensic acid (Figure 6a), fluorensadiol (Figure 6b), and methyl orsellinate (Figure 6c) and lactones (Figure 6d), besides a previously known flavonoid (Ermanin, Dominguez et al., 1973) tetracosane-4-olide, pentacosane-4-olide, hexacosane-4olide, heptacosane-4-olide, octacosane-4-olide, nonacosane-4-olide, and triacontane4-olide. Besides there are benzofurans (Figure 6e) and benzopyrans (Figure 6f).


335

HO H

O

Me COOMe COOH

H

H

OH

OH CH3-(CH2)n-CH2

HO

Dehyfluorensic acid Mata et al. (2003)

Fluorensadiol Kingston et al. (1975)

(a)

Methyl orsellinate Witiak et al. (1967)

(b)

O

Lactones Mata et al. (2003)

(c)

H

O

(d) O

H3COC

H

O

Benzopyran (e)

O

HO H

H

Benzofuran

(f) Aregullin and Rodriguez (1983)

Fig. 6. Chemical of six active compounds from F. cernua.

Chenopodium ambrosioides L. (Epazote) Taxonomic classification Family: Chenopodiaceae Genus: Chenopodium Species: ambrosioides Synonyms Ambrina ambrosioides, A. parvula, A. spathulata, Atriplex ambrosioides, Blitum ambrosioides, Chenopodium anthelminticum, C. integrifolium, C. spathulatum, C. Suffruticosum (Johnson, 1984). Common names Epazote, erva-de-santa maria, wormseed, apasote, chenopode, feuilles a vers, herbe a vers, meksika cayi, paico, pazote, semen contra, semin contra, simon contegras, mexican tea, american wormseed, jesuit’s tea, payco, paiku, paico, amush, camatai, cashua, amasamas, anserina, mastruco, mastruz, sie-sie, jerusalem tea, spanish tea, ambroisie du mexique, wurmsamen, hierba hormiguera, ipazote, lukum-xiu. General description Epazote is an annual herb that grows to about 1 m in height. It has multi-branched, reddish stems covered with small, sharply toothed leaves; flowers are numerous, small, yellow, and in clusters along stems (Figure 7). Following flowering, it produces thousands of tiny black seeds in small fruit clusters. It is easily spread and

336


Fig. 7. Panicle of C. ambrosioides.

regrown from the numerous seeds it produces which is why some consider it an invasive weed. The whole plant gives off a strong and distinctive odor (Boelcke, 1989; Amorı´ n, 1988). Habitat Epazote is native to Mexico and the tropical regions of Central and South America where it is commonly found as a garden herb (PDR, 2000). Distribution C. ambrosioides (Figure 8) is found in Mexico in Valley of Me´xico, Chihuahua, Durango, Hidalgo, State of Me´xico (Martı´ nez, 1993a).


337

Fig. 8. Distribution of C. ambrosioides herbs in Mexico.

Phytochemical analysis Chenopodium is rich in monoterpenes; the seed and fruit contain an essential oil called ascaridole (Bawer and Brasil e Silva, 1973). Properties and documented actions It has anthelmintic, analgesic, antitrypanosomal, amebicide, antifungal, antimicrobial, antimycobacterial, antiulcer, cytotoxic, diaphoretic, diuretic, emmenagogue, lactogogue, molluscidal, nervine, parasiticide, pectoral, poison, purgative, sedative, stimulant, stomachic, tonic, vermifuge, vulnerary, and allelopathic properties (Font, 1980; Toursarkissian, 1980; Chandra and Ghosh, 1987; Okuyama et al., 1993; Giove, 1996; Gadano et al., 2002). In one study an extract of the entire plant of Chenopodium showed the ability to kill human liver cancer cells in the test tube. Another study reported that the essential oil of Chenopodium and ascaridole showed strong antitumoral action against numerous cancerous tumor cells in the test tube (Effert, 2002; Ruffa et al., 2002). Antifungal activity Salazar et al. cited by Marcos (1996) mixed dry Larrea residues and Chenopodium extracts to test seed germination and plant development in beans grown in soil infested with Pythium aphanidermatum and Rhizoctonia solani. The results showed that germination increased 72 to 76% whereas dry matter was raised from 81.3 to 86.7%. Antifungal effects have also been documented for ascaridole (Pare et al., 1993). Activity against Phytophthora spp. and Pseudoperenospore cubensis is seen in cucumber. Inhibited mycelial growth in vitro of Alternaria solani isolated from tomato (Lycopersicon esculentum) as reported by Garcia and Montes (1992). Aqueous extracts (6 and 9% w/v) were active against Fusarium oxysporum, f. spp. radicis lycopersici in tomato plants under greenhouse conditions (Gamboa, 1997). In bean

338


plants under greenhouse condition Chenopodium extracts were active against Uromyces phaseoli, Colletotrichum lindemuthianum, and Erysiphe polygoni. Kishore (1993) reported that the essential oils are effective against dermatophytes. Antimicrobial activity Giove (1996) reported entheroparasitosic activity. Toxic effects have been found against snails, and in vitro action against drug-resistant strains of Mycobacterium tuberculosis was also reported by Lall and Meyer (1999). Finally, Pollack et al. (1990) reported the effect of ascaridole in vitro development of Plasmodium falciparum. Insecticide and nematicide activity In 2002, a U.S. patent (Zhang et al., 2003) was filed on a Chinese herbal combination containing Chenopodium for the treatment of peptic ulcers. Chenopodium essential oil was reported to inhibit various chemical and bacteria-induced ulcer formation. Activity of ascaridole against a tropical parasite called Trypanosoma cruzi as well as strong anti-malarial and insecticide actions (Kiuchi et al., 2002). Topical applications of the oil effectively treated ringworm within 7–12 days in a clinical study with guinea pigs. The leaf extract was 100% effective against the common intestinal parasites, Ancylostoma, Trichuris, and human tapeworm (Hymenolepsis nana) and 50% effective against Ascaris (Giove, 1996; Lopez De Guimaraes et al., 2001). Hmamouchi et al. (2000) reported moluscide action. Morsy et al. (1998) reported that volatile oils are active against the larvae of Lucilia sericata. Active chemicals Compounds in the C. ambrosioides oil from the leaves oil, includes: Allo-aromadendrene (0.02%), allyl levulinate (0.04%), alpha-guaiene (0.24%), alpha-pinene (0.09%), alpha-phellandrene (0.01%), alpha-terpinene (0.83%), alpha terpinyl acetate (0.05%), alpha-thujene (0.26%), amyl levulinate (0.07%), b-caryophyllene (2.09%), bcopaene (0U04%), b-pinene (0.06%), butyric-acid, carvacrol (0.08%), carvone (2.07%), carvone oxide (0.16%), cis-carveol (4.91%), cis-linalool oxide (furanoid) (0.21%), cis-p-mentha-2,8-diene-1-ol (4.00%), d-cadinene (0.08%), D-camphor (0.84%), dihydrocarveol (0.71%), geranial (5.00%), geranic acid (0.04%), geraniol (4.30%), isoborneol (0.22%), isobornyl acetate (0.20%), isobornyl propionate (0.06%), isobutyl benzoate (1.15%), isopulegyl acetate (0.08%), isopulegol (3.54%), lavandulyl acetate (0.02%), limonene (32.5%), linalool (0.07%), methyl-hexanoate (0.06%), methyl undecanal (0.03%), myrcene (0.03%), menthol (1.64%), nerol (0.41%), neryl acetae (0.01%), neryl formate (0.02%), norbornyl acetate (0.02%), p-cymene (0.03%), perillyl alcohol (0.15%), trans-carveol (2.28%), trans-pinocarveol (26.7%), trans-verbenol (1.21%), trans-sabinene hydrate (0.06%), g-terpinene (0.03%), undecanal (0.14%), (Sagrero-Nieves, 1995). Bogacheva et al. (1972) reported two glycosides named chenopodosides A and B (C52H82O22), acid hydrolysis yielded echinocystic acid, glucuronic acid, rhamnose, xylose, and arabinose. Partial hydrolysis yielded in addition echinocystic acid glucuroniside (Agarwal and Rastrogi, 1974; Pare et al., 1993) (see Figure 9a). Other chemicals found in the Chenopodium leaves are: Aritasone,


339 Me

Me

CO2 Me

Me

CH2

OH

O

OH

O

O

Me

OH

OH

Me

OH O

OH

O

CH 2

Me O

OH

OH

Me

O

O

O

OH

O

Me

OH

OH

OH

OH

OH

(a) Echinocystic acid glucuronoside (Bogacheva et al., 1972)

O O

(b) Ascaridole

Fig. 9. Chemical structure of two active compounds from C. ambrosioides.

ascaridole (C10H16O2) (Bawer and Brasil e Silva, 1973; Sagrero-Nieves et al., 1994) (Figure 9b), D-terpineol, dimethyl sulfoxide, ferulic-acid, L-pinocarvone, malic-acid, menthadiene, menthadiene hydroperoxides, methyl-salicylate, n-docosane, n-hentriacontane, n-heptacosane, p-cymol, safrole, saponins, spinasterol, tartaric-acid, terpinylacetate, terpinyl-salicylate, triacontyl-alcohol, trimethylamine, urease, vanillic acid.

Tagetes erecta (Cempasućhitl) Taxonomic classification Family: Asteraceae Genus: Tagetes Species: erecta

340


Common names Cempasuchil, Cempoalxoćhitl, Flor de muerto, Flor de tapayola, Flor de veinte pe´talos, Inditas, K-Pu-Huk (Maya), Rosa de muerto, Sempual, Tiscoque, Yita cuaá (mixteco), Zampual. Zempoalxoćhitl. General description Tagetes erecta is an annual herbaceous plant up to 60 cm height (Figure 10) grooved pubescent stem, aromatic divided leaves measuring up to 20 cm long; big orange, yellow, or reddish flowers with pungent smell; flowering in October and November. It has peduncles up to 15 cm long, with pinnate bracts, the lobes with teeth bristle ended, involucre campanulate 13–20 mm high; 9–25 mm wide, bract 5–11, glabrous with triangular apex, ligulate flowers 5–8 to numerous, the blades ablanceolate to aborate 1–2 cm long, and tubular flowers at the capitulum. Habitat The genus Tagetes is native American; approximately 20 species are distributed from Mexico to Argentina (Aranda, 1996), although its origin has been subject to speculations and confusion possibly because it spreads as a garden flower. The plant grows in mountain mesic forest with tan soil; near rivers and tropical caducifolious forest; in open areas of low caducifolious jungle; medium-height caducifolious jungle, and perennifolious high jungle; black stony soil. It is found between 800 and 2590 m above sea level. It is terrestrial, arvensis, and ruderal growing mainly in forest of oak, pine, and oak-pine. It is cultivated mainly in Argentina, Italy, Mexico, Peru, Spain, USA, and Venezuela. Distribution T. erecta is found in states of Mexico (Figure 11) in Chiapas, Estado de Me´xico, Puebla, Tlaxcala, Veracruz, Tabasco, Oaxaca, Durango, Jalisco, Michoacań, Guerrero, and Yucatań. Phytochemical analysis Tagetes possesses chemicals containing sulfur in their structure with nematicidal properties, tannins (Martı´ nez, 1993f), oleorseı´ na and xantofila. In addition, it has been reported that the plants emit ethylene which inhibits the growth of other plants in their vicinity. Properties and documented actions It is commonly used as an infusion of the flowers and leaves for curing stomach disorders (colic) (Martı´ nez, 1993f) and against parasites of the intestine. The leaves


341

Fig. 10. Tagetes erecta herb with flowers.

Fig. 11. States of Mexico with T. erecta herbs.

have been used to cure wounds, pimples, fever, and hepatic diseases. It is used for poultry forage and Ritual-religious mainly for the November 2nd celebrations. A yellow pigment is obtained from the flowers, which is used for wool, leather, fabrics, and fibers. It has veterinary use against disorders of the digestive system and

342


infections. Towers et al. (1975) reported ultraviolet-mediated antibiotic activity of thiophene. Antifungal activity Extracts of the plant showed control of the black spot of cucumber caused by Phytophthora spp. and on Pseudoperenospora cubensis (Frayre et al., 1996). It has in vitro activity against the mycelial development of Alternaria solani (Garcı´ a and Montes, 1992). It prevents damage against Uromyces phaseoli, C. Lindemuthianum, and Erysiphe poligoni under greenhouse conditions (Montes and Martı´ nez, 1989). Antimicrobial activity Bio-fumigation with Tagetes acts indirectly on the virus due to the elimination of fungi, nematodes, and insects that act as vectors. Jacobs et al. (1994) also reported the nematicide, antiviral, antibacterial, and antifungal activity of T. patula and T. erecta. Insecticide and nematicide activity Exudates from T. erecta protected tomato and capsicum against aphids (Zavaleta, 1990). There are hundreds of papers on the nematicidal activity of polythienyls from Tagetes spp. and other Asteraceae. The rotation of T. erecta or T. patula can provide economic control of P. penetrans in tobacco (Nicotiana tabacum) for two successive years (Reynolds et al., 2000). Other nematodes susceptible to Tagetes are Pratylenchus sp., M. incognita, and M. javanica (Ploeg and Maris, 1999), M. hapla and M. arenaria. The control of the nematodes has been achieved by growing Tagetes prior to tomato (Lycopersicun esculentum) plants (Ploeg, 2000). The suppression of M. incognita by Tagetes is dependent on temperature; at 30 1C galling occurred in two cultivars whereas no problem was presented at a temperature of 15 1C or lower (Ploeg and Maris, 1999). The thiophene a-terthienyl was nematicidal in vitro against the potato cyst nematode Globodera rostochiensis at 0.1–0.2 mg/ml (Uhlenbroek and Bijloo, 1958); Anguina tritici at 0.5 mg/ml; Ditylenchus dipsaci at 5 mg/ml; Caenorhabditis elegans and Steinernema glaseri at concentrations of 20 and 40 mg/ml. Besides, effect on an insect of citrus fruit (Phyllocnistis citrella) was also tested. Active chemicals The plant contains essential oils, resin, yellow pigment, tannins, and other chemicals (Martı´ nez, 1993f) in the flowers and leaves. Thiophene a-terthienyl (see Figure 12), polythienyl, 5-(3-butene-1-ynyl)-2,20 , bithieny,l and hydrogenated 5-butyl-2,20 -bithienyl (Uhlenbroek and Bijloo, 1958); oleoresin and xantofil (Esparza-Mantilla, 2002). Chemical structure of selected biotoxic phytochemicals (Figure 12)


S

S

343

S

Fig. 12. Chemical structure of Thiophene a-terthienyl, an active compound of T. erecta.

Market This plant is commonly found in the market by the end of October.

Metopium brownei (Checheń negro) Taxonomic classification Family: Anacardiaceae Genus: Metopium Species: brownei

Synonyms Brosimum conzatti Standl.; Brosimum gentlei Lundell; Brosimum terrabanum Pittier; Helicostylis ojoche K. Schum. ex Pittier; Piratinera terrabana (Pittier) Lundell; Rhus metopium L.; Terebintus brownei Jacq. Common names Checheń negro (Mexico); Boxcheche´m, Kabal-chechem (Maya, Yucatan); Checheń, Palo de rosa (Yucatan), and Madera negra venenosa (Smith and Vankat, 1992). General description Metopium brownei is a tree 12–25 m tall (Figure 13) with stem diameter of 60 cm approximately at a height of 1 m from the soil, irregularly branched, leaves in spiral 20–30 cm length including petiole, formed by 5–7 opposite leaflets, 6–11 cm in length and 3.8–8 cm width, widely elliptic; external cortex, scaly in rectangular motifs, gray to dark brown: internal cortex pink, in fibers that exudates a highly caustic fluid which darkens when in contact with air; flowers, male and female panicles axilar up to 20 cm to long glabrous; the flowers are actinomorfic, the calyx green-yellowish and the petals yellow; fruits berries in bunches, 1 cm long, oval, yellow or dark orange; the fruit has one seed 7–8 mm in length. Habitat The plant grows in coastal tropical forest along the gulf of Mexico and in the Yucatań peninsula, and sometimes form pure populations favored by fire. This tree

344


Fig. 13. Metopium brownei tree.

has economic importance in the region as its wood is utilized in the fabrication of wood floors and staves (Pennington and Sarukhań, 1968; Herrera, 1980; CabreraCano et al., 1982; Gome´z Pompa et al., 1991).

Distribution Metopium brownei is found in Mexico (Figure 14) in the states of: Campeche, Quintana Roo, Tabasco, Veracruz, and Yucatań.

Phytochemical analysis In a previous study (Rivero-Cruz et al., 1997), the composition of Metopium brownei urushiol was established. There 3-n-pentadecenylchatechols (urushiols) with antifungal properties and toxicity to brine shrimp (LC50 ¼ 89.13 mg/ml) were identified. Rivero-Cruz et al. (1997) suggested that urushiols could be of importance in the defense mechanism of the plant by preventing the attack of some fungi and bacteria.


345

Fig. 14. States of Mexico with M. brownei trees.

Alkycatechols were isolated from an acetone extract of the bark. Dihydroquercetin and eriodictyol were isolated from the chloroform method extract of the wood. In addition, masticadienoic acid was isolated from the leaves. Properties and documented actions This medicinal species produces strongly irritant, toxic exudates; these exudates are used against measles, smallpox, erysipelas, and rheumatism (Martı´ nez, 1993e). They are a poison for fishes (‘‘embarbascar’’). They are used medicinally as an antiviral, cathartic, diaphoretic, anti-inflammatory, and sedative (Amo, 1979; Mendieta and Amo, 1981) (Figure 15a to 15d). Antimicrobial activity Eriodictyol possesses antibacterial activity against larvae of Heliotis zea (Harborne, 1991). It has an inhibitory effect on the radial growth of Pythium sp. and Alternaria sp. (Anaya et al., 1999). The saps from Anacardiaceae have antimicrobial effects possibly due to the catechols present (Vogl et al., 1995). Insecticide and nematicide activity It has insecticidal action against maize worms Spodoptera frugiperda (Gonza´lezGaona and Lagunes-Tejeda, 1986). Active chemicals The active chemicals are urushiols and flavonoids, alkylcatechols such as 3(100 Z, 130 E-pentadecadienyl)-catechol (Figure 15a), dihydroquercetin (Figure 15b), eriodictyol (Figure 15c), and masticadienoic acid (Figure 15d) (Harborne and Baxter, 1993).


346 OH HO

(a) 3(10’Z, 13’ E-pentadecadienyl)catechol OH

OH

OH

OH O

HO

O

HO

OH OH

O

OH

(b) Dihydroquercetin

O

(c) Eriodyctiol

COOH

O

(d) Masticadienoic acid

Fig. 15. Chemical structure of four active compounds from M. brownei tree.

Chemical structure of selected biotoxic phytochemicals (Anaya et al., 1999) (Figures 15a to 15d)

Agave lechuguilla Torrey (lechuguilla) Taxonomic classification Family: Agavaceae Genus: Agave Species: lechuguilla Synonyms Agave poselgeri Salm-Dyck; Agave lophantha Schiede var. poselgeri (Salm-Dyck) A. Berger; Agave multilineata Baker ; Agave heteracantha hort. ; Agave lophantha Schiede var. tamaulipasana A. Berger.


347

Common names Lechuguilla, ixtle (fibra), guishe (Espejo and Lo´pez-Ferrari, 1992). General description It forms small bunches (Figure 16) with new plants emerging from a plant, generally below the soil; yellow-greenish; 0.25–0.5 m height; leaves 0.3–0.5 m in length and 3 cm in width, straight or curved at the top, 8–12 ‘‘false thorns’’ on the edge, 4–7 mm length, located approximately at 2–4 cm distance, grayish strong terminal thorn 2–3 cm long, spikes 2–3 cm height; flowers integrated by 1–3 bunches, 2.5–4 cm length, yellow greenish fusiform ovary, 12 to 14 cm long, shallow tube with 2–4 mm aperture, tepals 12–18 mm long, yellow to red or purple, 2.5–4 cm length inserted in the tube, yellow anthers 11–16 mm length, oblong capsules 2–3 cm in length (Correl and Johnston, 1970; Rzedowski and Equihua, 1987; Alanı´ s and Favela, 1997). Habitat The plant is integrated in the scrub desert with temperatures typical for the arid and semiarid lands and 200–250 mm annual rainfall, associated with Euphorbia antisyphilitica, Hechtia glomerata, Bouteloua breviseta, Larrea tridentata, Bouteloua curtipendula, Heteropogon contortus, Acacia constricta, Fouquieria splendens, Parthenium incanum, Opuntia sp., and Dasylirion sp. The plant generally grows between 200 and 2700 m above sea level; it flowers once in its life time at 6–15 years between May and August and afterwards it dies (Borja, 1962; Beltrań, 1964; Marroquı´ n et al., 1981; Lo´pez, 1990).

Fig. 16. Agave lechuguilla in the field.


348

Distribution Lechuguilla is essentially restricted to, and considered an indicator species of the Chihuahuan Desert (Gentry, 1982); it is found in the semiarid zone in Mexico (Figure 17) in the states of Chihuahua, Coahuila, Durango, Nuevo Leoń, San Luis Potosı´ , and Zacatecas (dark area in the map), and in lower proportion (light area in the map) in the states of Hidalgo, Oaxaca, and Me´xico (Freeman, 1973; Villa, 1981; Gentry, 1982; Nieto, 1983). Phytochemical analysis Marker and Lo´pez (1947a) and Marker et al. (1947b) reported that this plant contains 0.1% of smillagenin (Figure 18a), but later Wall et al. (1962) reported an average content of 1%. It also contains steroids similar to barbasco. The leaves and roots contain saponins. Properties and documented actions The native Americans used it for the production of soaps. In agriculture it is known as guishe and used for the construction of protective live fences and as forage for rabbits. The fibers are mixed with polyester to produce building materials for barns and silos, as well as ropes (Castetter et al., 1938; Maldonado, 1979; Ninõ and Patinõ, 1981). Antifungal activity Aqueous extracts from the plant bulb were active against radicular damage in tomato caused by Fusarium oxysporum f. sp. radicis lycopersici at 6% and 9% concentration (Gamboa, 1997).

Fig. 17. States of Mexico with A. lechuguilla shrubs.

Antimicrobial properties of Mexican medicinal plants H O

349

O

CH3

O

O CH3

O

CH3

O HO

HO

HO H

(a) Smillagenin (Roman, 1980)

HO H

(b) Hecogenin (Roman, 1980)

H

(c) Manogenin (Romo de Vivar, 1985)

Fig. 18. Chemical structure of three active compounds of A. lechuguilla.

Antimicrobial activity The effect of ethanolic extracts of A. lechuguilla was tested for the control of growth of yeasts, molds, and bacteria. The extracts showed good antimicrobial activity against 50% of the tested pathogens (Verastegui et al., 1996). Insecticide and nematicide activity The genus Agave contains saponins and steroidal sapogenins which are lipid-soluble steroids found in nature combined with different sugars in water-soluble form. The combination is called steroidal saponins. In lechuguilla three sapogenins have been identified (Wall, 1980): Smillagenin (Figure 18a), Hecogenin (Figure 18b), and Manogenin (Figure 18c). Active chemicals Chemical structure of selected biotoxic phytochemicals (Figures 18a to 18c) Market The present market for A. lechuguilla is related basically to the fiber production and use in textile applications.

Capsicum spp. (Chile) Taxonomic classification Family: Solanaceae Genus: Capsicum Species: spp.

Common names Ajı´ (several Amazon countries), Chile (Mexico), Katupı´ (Colombia: Tukano); Jimia (Ecuador: Shuar); Uchu, Ajı´ chinchano, Rocoto (Peru´—Bolivia); Chyoots, Iki,


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Jashfiilla, Jima (Peru´: Amuesha, Cocama, Ocaima, Aguaruna). C. annuum L. subsp. baccatum: Ajı´ chivato, Ajı´ pimiento, Chonguito, Ajı´ chirel (Colombia), C. annuum var. annuum: Chirel, Pimiento de Cayena (Venezuela). C. frutescens L.: Pimenta malagueta (Brasil); Ajı´ pique, Ajı´ huevo de araguana (Colombia). C. pubescens: Rocoto (Bolivia, Ecuador, Peru´). General description Most species are small shrubs that transform into herbaceous plants when cropped in temperate climates. It is a yearly plant, height 50 cm to 1 m (Figure 19); petiolated leaves: flowers white, small; fruit a hollow berry, cylindrical, red or yellow; seeds, white. Habitat The origin of the capsicum genus is America (Pickersgill, 1969; IBPGR, 1983). Three centers of origin are reported for the domesticated species: a) Mexico and Guatemala, C. annuum; b) the Amazons basin, C. chinense and C. frutescens and; c) the highland regions of Bolivia and Peru, C. baccatum and C. pubescens. All of them were domesticated in the last eight thousand years. In Mexico there are approximately 40 species of Capsicum native to Central America and South America. Most species are small shrubs that may transform to herbaceous plants when cultivated in temperate zones.

Fig. 19. Capsicum annuum, type baccatum bell, plant with fruits.


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Distribution In Mexico, Capsicum grows almost in all parts of the country (Figure 20). However commercial production is mainly obtained from the states of Baja California Sur, Chiapas, Chihuahua, Colima, Guanajuato, Hidalgo, Michoacan, Nayarit, Queretaro, Sinaloa, San Luis Potosi, Sonora, Tabasco, Tamaulipas, Veracruz, and Zacatecas (Pickersgill, 1969; IBPGR, 1983). Phytochemical analyses The pungent principle identified in Capsicum is capsaicin besides reports of flavonoids and steroidal alkaloids in C. annuum. There are also reports of glucosides in C. annuum var. annuum, capsaicin (Figure 21), and red capsantin (carotenoid) (Cichewicz and Thorpe, 1996). Properties and documented actions The fruits have been used as food and medicine since pre-Columbian times. It is used in the treatment of snake bites by the Jivaros (Shuar), and to ease tooth pain by Maynas (Peru); mixed with Urtica for labor problems. Chu et al. (2002) reported

Fig. 20. States of Mexico with C. annuum plants.

OMe

O

OH NH

Fig. 21. Chemical structure of capsaicin, an active compound from C. annuum.

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that red pepper consumption prevents chronic diseases related to oxidative stress in the human body. Anti-carcinogenic activity of Capsicum has been reported by Pandey and Shukla (2002). Powder of red pepper is a potential therapy for functional dyspepsia (Bortolotti et al., 2002).

Antifungal activity In an experiment conducted to study the presence of endophytic antifungal bacteria (He et al., 2002) antifungal activity was reported against Fusarium oxysporum, f. sp. Cubense, F. oxysporum, f. sp. cucumerinum, Colletotrichum gloeosporioides, and Bacillus subtilis.

Antimicrobial activity Using a filter disk assay, plain and heated aqueous extracts from fresh Capsicum annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens varieties were tested for their antimicrobial effects with 15 bacterial species and one yeast species. Two pungent compounds found in Capsicum species, capsaicin (Figure 21) and dihydrocapsaicin, were also tested for their antimicrobial effects. The plain and heated extracts were found to exhibit varying degrees of inhibition against Bacillus cereus, B. subtilis, Clostridium sporogenes, C. tetani, and Streptococcus pyogenes (Chichewicz and Thorpe, 1996). Capsaicin has been tested against Eschericha coli, Pseudomonas solanacearum, Bacillus subtilis, and Saccharomyces cerevisiae (MolinaTorres et al., 1999). Anti-yeast activity was reported by Iorizzi et al. (2002).

Insecticide and nematicide activity Not reported.

Active chemicals The chemical structures of amides, alkamides, and capsaicinoids differ mainly in the chain insaturation and the amine moiety (Christensen and Lam, 1991; Christensen, 1992). Capsaicin (Figure 21) is the pungent active compound found in the genus Capsicum, besides flavoids and steroidal alkaloids. Three furostanol saponins named capsicoside E, capsicoside F, and capsicoside G, from the seeds of Capsicum annuum L. var. acuminatum (Iorizzi et al., 2002), were active against yeast and common fungi. A steroidal saponine, CAY-I with a molecular weight of 1243.35 Da isolated from Capsicum frutescens is a potent fungicide for the germinating conidia of Aspergillus flavus, A. fumigatus, A. parasiticus, and A. niger. In vitro assays, CAY-I was effective against Pneumocystis carinii and Candida albicans (De Lucca et al., 2002). Hot peppers contain flavonoids phenolic acids, carotenoids, vitamin A, ascorbic acid, tocopherols (Rosa et al., 2002).


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Lophophora williamsii (Lem.) Coult. (Peyote) Taxonomic classification Family: Cactaceae Genus: Lophophora Species: williamsii

Synonyms Echinocactus williamsii Lem. Echinocactus williamsii Lem. ex Salm. Common names Peyote, peyotl, peiotl, jiculi. Peyotl (na´huatl), peyote (in Central Mexico); kamaba (tepehuanes), hicore o jiculi (huicholes); huaname (tarahumara), hikori (tarahumara), wokow (comanches), la Rosita. General description The plant is a small globelike green-bluish cactus, without thorns (Figure 22), flat in the apex, 2–6 cm in height, 4–11 cm in width; ribs 4–11 well defined forming tubers more or less tall; areolas separated 0.9–1.5 cm, round 2–4 mm in diameter; flowers 1–2.4 cm in length, 1–2.2 cm in width, pink; pollen 14.9–63 mm, spherical grains. In natural stands rarely exceeds 3 cm height above the soil level. The root is the higher part of the plant and may be up to 25 cm in length.

Fig. 22. Lophophora williamsii cactus with flowers, in the field.


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Habitat The plant is found in desert thickets around 1360 m asl in stone yellow soil; there are many juvenile forms that led to the great number of synonyms. Distribution In Mexico L. williamsii is found (Figure 23) in the states of Quere´taro, San Luis Potosı´ , Sonora, Zacatecas, Nayarit, and Coahuila (Martı´ nez, 1993c). Phytochemical analysis The main component is mescaline (Figure 24a); the first study dates back to 1888 when Lewin isolated analonine (Figure 24b), a crystalline tetrahydro-isoquinoleic alkaloid. Till 1973, 56 alkaloids had been characterized and classified as phenyletalamines mono-, di-, and tri- oxygenated; alkaloids from tetrahydro isoquinoleine and their amides; phenyl etalamines conjugated with acids of the Krebs cycle; and derivates from pyrrol (Trease and Evans, 1987a, 1987b). Properties and documented actions The use of peyote in religious rites by many Indian tribes is common knowledge. In addition, curative properties for such varied ailments as toothache, pain in childbirth, fever, chest pain, skin diseases, rheumatism, diabetes, colds, and blindness, among other things, have been claimed for this plant. The U.S. dispensatory lists peyote under the name Anhalonium and indicates its use to some extent in various forms for neurasthenia and hysteria and also in cases of asthma. Most of the alkaloids are located in the button.

Fig. 23. States of Mexico with L. williamsii cacti.


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H3CO

H3CO

NH

NH2 H3CO

H3CO OCH3

OCH3

(a) Mescaline

CH3

(b) Analonine

H3CO

N

COOH

H3CO OCH3

(c) Peyonine

Fig. 24. Chemical structure of three active compounds from L. williamsii.

Antifungal activity The antifungal activity of organic extracts has been tested with the following fungi: Rhizoctonia solani, Pythium sp., and Fusarium oxysporum. The extracts from the root showed 55% inhibition of the mycelial growth in Rhizoctonia solani, 90% in Pythium sp., and 54% in Fusarium oxysporum (Saeedi-Ghomi and Maldonado, 1982).

Antimicrobial activity Extracts of whole peyote plants prepared in various solvents showed positive microbial inhibition. A water-soluble, crystalline substance termed peyocactin, separated from the ethanolic extract of Lophophora williamsii exhibited antibiotic activity (Rao, 1970; Cruse, 1973). This substance was tested against fatal staphylococcal infection in mice; the protected animals survived while those in the control group succumbed within 60 h after infection with S. aureus. Peyocatin was active against 18 strains of penicillin-resistant Staphylococcus aureus (McCleary et al., 1960; McCleary and Walkington, 1964). Hordenine and tyramine are antibacterial substances (Gottlieb, 1977).

Insecticide and nematicide activity Not reported.

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Active chemicals Several alkaloids have been found among which the main are mescaline (Figure 24a), 1–6% of the button dry weight, analoine. Anhalonidine. Lophophorine, anhalamine, and peyotine besides colorants, mucilage materials, sugar, and calcium oxalate (Martı´ nez, 1993c). The plant contains 56 chemicals derived from tiroxine, 20 derived from titamine, B-fenetilamines: mescalina; tetrahydroisoquinolines: hordenine (Nacetyl-3-methoxy-4,5-dimethoxy-phenetilamine), alamine, anhalamine, anhalidine, anhalinine, anhalonidine (14% of the alkaloids), anhalonine, anhalotine, 3,4-dihydroxy-5-methoxyphenetilamine, epinine, dopamine, 3,4-dimethoxyphenetilamine, N-acetylanhalamine, N-acetylanhalonine, N-formylanhalamine, N-formylanhalinine, N-formylanhalonidine, N-formylanhalonine, N-formyl-O-methylanhalonidine, N-formyl-3-methoxy-4,5-dimethoxyphenethylamine, glycine (8% of the alkaloids), hordenine, 3-hydroxy-4,5-dimethoxyphenetilamine (1–5% of the alkaloids), isoanhalamine, isoanhalonidine, 3-hydroxy-4,5-dimethoxy-N-methylphenetilamine, isoanhalidine, isopellotine, lophophorine (5% of the alkaloids), 3-hydroxy-4,5dimethoxy-N,N-dimethoxyphenetilamine, lophorine, lophotina iodide, mescalina (30% of the alkaloids), mescaline citrimide, mescaline malaimide, mescalina maleimide, mescaline succinimide, isocitrimide lactone mescalina, N-acetylmescaline, N-formylmescaline, N-methylmescaline, mescalotam, 3-methoxytiramine, 3-methoxy-N-methyltyramine, 3-methoxy-N,N-dimethyltyramine, O-methylanhalonidine, O-methyl-peyoxylic acid, O-methylpeyoruvic acid, N-methyltyramine, peyotine (17% of the alkaloids), peyoglunal, peyoglutam, peyonine (Figure 24c), peyophorine, peyoruvico acid, peyotine iodide, peyoxilic acid, and tyramine. Chemical structure of selected biotoxic phytochemicals (Trease and Evans, 1987a) (Figures 24a to 24c). Market Peyote is not commercially exploited and its use is prohibited by law.

Yucca spp. (Palma del desierto) Taxonomic classification Family: Agavaceae Genus: Yucca Specie: spp. Common names Palma del desierto, palma samandoca. General description Yucca is a tree-like plant (Figure 25), with symmetric neighboring stems from the base and dispersing upwards, commonly 1–2 of the same or different height and


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Fig. 25. Yucca carnerosana tree in the field.

rarely more than 8, the oldest stem is 5 m long and 35 cm in diameter, occasionally divided into two at the end; leaves, 50 cm–1 m long, 5–7.5 cm wide, rigid, disperse: flowers, white sepals 7–9.5 cm length, petals 6.5–9 cm long, 2–2.8 cm wide; fruit, oblong at least 10 cm in length and 4 cm in diameter. Habitat Found from the sea level to 1850 m asl, in extense populations forming thickets with Larrea and with Pinus cembroides, generally associated with Agave lechuguilla, Euphorbia antisyphillitica; Hechtia glomerata, Dasylirion wheleri, Dasylirion spp., Parthenium argentatum, Larrea divaricata, Parthenium incanum, Opuntia leptocaulis, Echinocereus sp., and other cactaceae on limos and limestone. The plant flowers from February to April. Among the 47 species of the Yucca genus 29 grow in Mexico, the largest number being found in the Baja California peninsula (mainly Y. valida) and from the center to North-east (mainly Y. filifera) (Wall, 1980). Distribution Yucca distribution in Mexico (Figure 26) includes the states of Baja California, Coahuila, Chihuahua, Durango, Guanajuato, Hidalgo, Mexico, Michoacań, Nuevo Leon, Queretaro, San Luis Potosi, and Zacatecas. Phytochemical analysis The genus Yucca comprises 47 species native to North and Central America, the main chemicals are steroidal sapogenins (sarsapogenine, Figure 27a and hecogenine,


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Fig. 26. States of Mexico with Yucca spp. CH 3 H H O O

CH3

O O

HO

H

(a) Sarsapogenin

O

HO

H

(b) Hecogenin

Fig. 27. Chemical structure of two active compounds from Yucca spp.

Figure 27b), carbohydrates, flavonoids, tannins, terpenoids, fatty and hydrocarbon acids (Aregullin et al., 1980). Properties and documented actions As a consequence of their surface-active or detergent properties, saponins are excellent foaming agents, forming very stable foams. Yucca extracts are used in beverages in which a stable foam is desirable. Because of their surfactant properties, they are used industrially in mining and ore separation, preparation of emulsions for photographic films, and in cosmetics such as lipstick and shampoo. Their antifungal and antibacterial properties are also important in cosmetic applications, in addition to their emollient effects (Roman, 1980a, 1980b). Quillaja saponins have even been used in bioremediation of PCB-contaminated soil. The extract of the Yucca plant has been used by Native American Indians in the South West United States and Mexico for hundreds of years in medicine.


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It is reported that Yucca schidigera increases nitrogen retention to around 35% by blocking the urease enzyme, which is responsible for converting the nitrogen from protein to urea. This basically means that more lean muscle can be built from the available protein. Antifungal activity Not reported. Antimicrobial activity The protein extracted from the leaves of Yucca recurvifolia Salisb showed action against herpes simplex virus type 1. The concentrations effective at 50% (ED50) were 3, 19, and 95 mg/ml when the protein exposure begun 3 h before virus infection, 0 and 3 h after infection. The protein also inhibited herpes simples virus type 2, and human cytomegalovirus and affected only virus-infected cells (Hayashi et al., 1992). Insecticide and nematicide activity A Yucca leaf infusion was found to exert inhibitory action in vitro against cercariae (larva) of Schistosoma mansoni at dilutions of 1:200,000 to 1:20,000,000 (Cushing, 1957). Active chemicals Chemical structure of selected biotoxic phytochemicals (Figures 27a and 27b)

Bidens pilosa (Chilca) Taxonomic classification Family: Asteraceae Genus: Bidens Species: pilosa

Synonyms Bidens adhaerescens, B. alausensis, B. chilensis, B. hirsuta, B. leucantha, B. montaubani, B. reflexa, B. scandicina, B. sundaica, Coreopsis leucantha, Kerneria pilosa. Common names Alfiler, acahual, acahua, aceitilla, amor seco, beggar’s tick bident herisse, bidente piloso, cadillo, chilca, clavelito de monte, cuambu, erva-picaõ, herbe d’aiguille, jarongan, ketul, mozote, pacunga, pau-pau pasir, pirca, romerillo, saltillo, saetilla, spanish needles, yema de huevo, z’aiguille, zweizahn.


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General description Bidens pilosa is a small, erect annual herb that grows to 1 m (Figure 28). It has bright green leaves with serrated, prickly edges, small yellow flowers, and black fruit. Its root has a distinctive aroma similar to that of a carrot. It is often considered a weed in many places. It is related to Bidens tripartita, the European bur marigold, traditionally used in European herbal medicine. Habitat It is indigenous to the Amazon rainforest and other tropical areas of South America, Africa, the Caribbean, and the Philippines. It is often considered a weed in many places. It is a southern cousin to Bidens tripartita, the European bur marigold, which has an ancient history in European herbal medicine.

Fig. 28. Bidens pilosa herb with flowers, in the field.


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Distribution In Mexico B. pilosa is found (Figure 29) in the Valley of Mexico, Jalisco, Oaxaca, and Veracruz.

Phytochemical analysis There are reports (Gessberger and Seguin, 1991) that Bidens pilosa contains flavonoids, terpenes, phenylpropanoids, lipids, and benzenoids, as well as a group of chemicals called polyacetylenes including phenylheptatriyne (Figure 30a). The flavonoids possess antimalarial activity (Brandao et al., 1997).

Properties and documented actions The plant has antiseptic properties and is used for the treatment of wounds, against inflammation, and against bacterial infection of the gastrointestinal tract. Bioactive phytochemicals showed activity against transformed human cell lines (Alvarez et al., 1996). The plant has hypoglycemic properties (Dimo et al., 1999), prevents hypertension (Dimo et al., 1998, 2001, 2002), has liver-protective activity, and antiinflammatory activities, reduces gastric acid secretion (Alvarez et al., 1984; Tan et al, 2000; Ubillas, 2000). Chang (2001) reported antileukemic activity.

Antifungal activity The chemicals in Bidens pilosa showed antimycobacterial activity towards Mycobacterium tuberculosis and M. smegmatis. A water extract of the leaves has shown significant anti-yeast activity towards Candida albicans.

Fig. 29. States of Mexico with B. pilosa plants.


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(a) Polyacetylene phenylhepta-1,3,5-triyne

OR1

H

OR 2 H

R1

R2

b

H

β-D-Glucopyranose

c

Ac

β-D-Glucopyranose(I+OAc)4

d

β-D-Glucopyranose

H

Fig. 30. Chemical structure of four active compounds from B. pilosa.

Antimicrobial activity In vitro studies have demonstrated its antibacterial activity against a wide range of bacteria including Klebsiella pneumonia, Bacillus, Neisseria gonorrhea, Pseudomonas, Staphylococcus, and Salmonella. Research by different authors (Arnason et al., 1980; Khan et al., 2001; Krettli et al., 2001a, 2001b) reported that a crude ethanol extract of the plant evidenced broad-spectrum antibiotic activity against numerous microbial pathogens. Extracts of the leaves also have been documented to have this activity. Insecticide and nematicide activity Not reported. Active chemicals Aesculetin, behenic acid, beta-sitosterol, borneol, butanedioic acid, butoxylinoleates, cadinols, caffeine, caffeoylic acids, capric acid, daucosterol, elaidic acid, erythronic acids, friedelans, friedelins, germacrene D, glucopyranoses (Figure 30b–d), glucopyranosides, inositol, isoquercitrin, lauric acid, limonene, linoleic acids, lupeol, luteolin, muurolol, myristic acid, okanin-glucosides, palmiticacid, palmitoleic acid, paracoumaric acids, phenylheptatriynes, phytenoic acid, phytol, pilosola A,


363

polyacetylenes, precocene I, pyranoses, quercetin, sandaracopimaradiols, squalene, stigmasterols, tannic acid, tetrahydroxyaurones, tocopherolquinones, tridecapentaynenes, tridecatetrayndienes, and vanillic acid.

Byrsonima crassifolia (Nanche) Taxonomic classification Family: Malpighiaceae Genus: Byrsonima Species: crassifolia

Synonyms Byrsonima cumingana Juss; Byrsonima fendleri Turcz; Byrsonima panamensis Beurl; Byrsonima pulchra Sesse´ & Moc. ex DC.; Malpighia crassifolia L; Malpighia pulchra Sesse´ & Moc. (Martı´ nez, 1979). Common names Changunga, Changungo, Chengua (Michoacan state); Chi (Maya, Yucatan); Huizaa (Zapoteca, Oaxaca state; Mami-hnã (Chinanteca, Oaxaca state); Nance, Nanche, Nanchi, Nanantze (Guerrero state); Nance agrio (Guerrero, Tabasco); Nancis; Nanche amarillo (Puebla); Nanche dulce (Oax.); Nandzin (l. zoque, Chis.); Nantzincua´huitl, Nanzinxoćotl (Na´huatl). General description The plant is a crooked tree, 3–7 m in height (Figure 31) with stem diameter up to 30 cm: leaves long, simple 5–15 cm length, 2–7.5 cm width, dark green; frequently branched from soil level; cortex, scaly, gray to tan, 12–25 mm thick; flower in bunches 5–15 cm length, pubescent, yellow-reddish, 1.5 cm in diameter; fruits, globular, yellow to orange, one seed per fruit. The plant flowers from November to July (Pennington and Sarukan, 1968; Bultman and Southwell, 1976). Habitat The plant grows in open stony slopes of the tropical forest and also in flat terrains, 150–2200 m asl in temperate climate. It is found in well-degraded soils, and may support rapid or deficient drainage conditions in terrains flooded in rainy season and dry in drought. It is native, in natural stands or cultivated (Pereira and Montoya, 1991; Von Carlowitz et al., 1991; Garcı´ a and Chacon, 1994). Distribution The tree is native to MesoAmerica, widely distributed in the tropical zone of Mexico. It extends to Peru, Bolivia, Paraguay, and Brazil. In Mexico (Figure 32) it is found in

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Fig. 31. Byrsonima crassifolia branch and fruits.

Fig. 32. States of Mexico with B. crassifolia trees.


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the states of Campeche, Chiapas, Guerrero, Jalisco, Mexico, Michoacań, Hidalgo, Morelos, Nayarit, Oaxaca, Puebla, Quintana Roo, San Luis Potosı´ , Sinaloa, Tamaulipas, Veracruz, Yucatan (Croat, 1978). Phytochemical analysis The presence of tannins in the bark has been suggested since 1876 by Laso de la Vega, although only the triterpenes b-amyrin (Figure 33) and an acetate have been isolated (Dejarais et al., 1956; Mathur and Lara, 1984). The volatile composition of the fruit have been described by gas chromatography by Alves and Jennings (1979). Properties and documented actions The fruit peel produces a colorant used for cotton in Guatemala; the tree cortex contains lignans used for tanning. The cortex is used in folkloric medicine based on its astringent properties against diarrhea and other digestive disorders, used also for curing infections in the womb and ovary swelling. The use of the cortex for skin affections is well known (Sievers et al., 1949), insecticidal capacity was reported by Delaveau et al. (1980), and antimicrobial activity was reported by Mendieta and del Amo (1981). Antifungal activity Antimycotic activity was reported by Caseres et al. (1991). Antimicrobial activity Boiled stem and roots are active against Klebsiella pneumoniae, Staphylococcus aureus, S. epidermidis, S. pneumoniae, Micrococcus luteus, Escherichia colli, Salmonella

H

H HO H β amyrin (Trease and Evans, 1987)

Fig. 33. Chemical structure of an active compound from B. crassifolia.

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typhi, Pseudomonas aeruginosa, Shigella flexneri, and Bacillus subtilis (Caseres et al., 1990). Insecticide and nematicide activity Insecticide activity was reported by Ocampo (1994). Active chemicals b-Amyrin (Figure 33) and an acetate (Mathur and Lara, 1984; Bejar and Malone, 1993). Chemical structure of selected biotoxic phytochemicals (Figure 33) Market The fruit is sold in markets almost at every place where the species develops.

Bursera simaruba (Almaćigo blanco) Taxonomic classification Family: Burseraceae Genus: Bursera Species: simaruba, graveolens

Common names Almacigo Blanco, Almacigo Colorado, Almacigo, Bois D’Encens, Chique, Fragon Caranne, Gommier Blanc, Gommier Rouge, Indio Desnudo, Jobo, Gumbolimbo. General description Gumbolimbo is a large tree up to 25 m in height with red shaggy bark that peels off in paper thin strips. Its leaves are 5–12 cm long (Figure 34), and the tree produces small round fruits about 8 mm long (Beckstrom-Sternberg and Duke, 1994). Habitat The plant is abundant in high and medium rainforests, perennifolious, sub-perennifolious and caducifolious, and sometimes it becomes the dominant species in low rainforests in San Luis Potosi and Tamaulipas. It is found in a broad range of ecological conditions between 100 and 1300 m asl. Distribution It is indigenous to the Amazon, Belize, Haiti, and tropical parts of Central America. In Mexico (Figure 35) it is found in San Luis Potosi, Tamaulipas, and Yucatan.


Fig. 34. Bursera simaruba branch with leaves.

Fig. 35. States of Mexico with B. simaruba trees.

367


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Phytochemical analyses The Bursera genus has triterpenes (Jolad, 1977a; Parsons et al., 1991; Symasundar, 1991), bilignans (Hernandez et al., 1983a), podophyllotoxin-like lignans (Bianchi et al., 1968; Mc Doniel and Cole, 1972; Jolad et al., 1977b) and flavonoids (Soyuza et al., 1989). In the resin of Bursera simaruba are elemicine and amyrenol (Pernet, 1972); besides, the resin was fractionated and the bioactive substance identified as picropolygamain (Figure 36), a L-aryltetralin lignan having a 2,3-cis-lactone ring and two methylenedioxy moieties in its structure (Peraza Sańchez and Penã Rodriguez, 1992). Properties and documented actions The plant has the following actions: analgesic, anti-inflammatory, aphrodisiac, asthma, bite (snake), cytostatic, depurative, diaphoretic, diuretic, dropsy, dysentery, enterorrhagia, expectorant, febrifuge insecticide, purgative, stomach, swelling, venereal, yellow fever, and topical remedy for skin infections (Roig and Mesa, 1945; Logan, 1973; Ramirez, 1988; Arvigo and Balick, 1993). Antifungal activity Antimitotic activity reported by Peraza-Sanchez and Penã-Rodriguez (1992). Antimicrobial activity Antiviral and antitumor activity reported by Peraza Sanchez and Penã Rodriguez (1992).

O O

O O

O O Picropolygamain

Fig. 36. Chemical structure of an active compound from B. simaruba.


369

Insecticide and nematicide activity Not reported. Active chemicals Studies of the chemical constituents of different species from the genus Bursera reported triterpenes, bilignans, podophyllotoxin-like lignans and flavonoids. Besides, there are reports related to the chemical structure of metabolites produced by Bursera sinaruba, with elemicine, amyrenol, and picropolygamain (Peraza Sanchez and Penã Rodriguez, 1992), see Figure 36. Chemical structure of selected biotoxic phytochemicals (Figure 36)

Acknowledgements The authors thank Dr. Jose Angel Villarreal-Quintanilla for access to the material at the Herbarium of the University (UAAAN) used for some pictures presented in this chapter.

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CHAPTER 15

Promissory botanical repellents/deterrents for managing two key tropical insect pests, the whitefly Bemisia tabaci and the mahogany shootborer Hypsipyla grandella LUKO HILJE, GERARDO A MORA

Introduction In the past 60 years, conventional chemical pesticides have been the predominant method for controlling pests worldwide. Nonetheless, the recognition and documentation of many unwanted agroecological, environmental, social, and economic problems resulting from pesticide overuse, has led scientists to look for alternatives, among which integrated pest management (IPM) has stood out. IPM tactics include plant breeding and cultural practices, as well as physical, biological, and selective chemical control. In fact, in recognition of the concept of co-evolution between herbivorous insects and their host plants, as well as its practical implications, IPM also promotes the search and utilization of natural active principles (insecticides, repellents, attractants, etc.) which could be helpful in dealing with insect pests of crops and forest plantations. Historically, some of the insecticides first used in agriculture and forestry were derived from plants (Stoll, 2000), such as nicotine from tobacco (Nicotiana tabacum, Solanaceae) leaves; rotenone, from the roots of ‘‘timbo’’ (Derris spp.), ‘‘chaperno’’ (Lonchocarpus spp.), yam bean (Pachyrhizus spp.) and other leguminous plants; quassinoids, from bitterwood (Quassia amara, Simaroubaceae) wood; azadirachtin, from neem (Azadirachta indica, Meliaceae) seeds; and pyrethrum, from Chrysanthemum cinerariifolium (Asteraceae) flowers; other plants well known for having substances with insecticidal properties include ryania (Ryania speciosa, Flacourtiaceae) and ‘‘sabadilla’’ (Schoenocaulon officinale, Lilliaceae). However, their use in agriculture, and even in traditional tropical systems, vanished in the 1950s, as a result of the appearance and widespread use of synthetic insecticides, as their rather simple molecules lend themselves for these materials to be manufactured at an industrial scale and a relatively low cost.

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Today, as a consequence of the wide promotion and acceptance of the sustainable development paradigm among conservation and farmer organizations, government policy- and decision-makers, and international donor agencies, environmentfriendly and highly profitable production schemes and practices, such as organic agriculture, have gained a lot of adoption and support in many countries. This new scenario opens better opportunities and possibilities for agrichemical companies involved in manufacturing non-conventional insecticides (bioinsecticides or biorationals) and other types of pesticides (Rodgers, 1993; Hall and Menn, 1999) harmless to non-target organisms, generally perceived as environmentally benign, and best suited for IPM programs within those production schemes. Such bioinsecticides can be obtained by extracting the natural active principle per se, as it currently occurs with neem, or by using them as leads to synthesize their analogue molecules (Pillmoor et al., 1993), as it happens with pyrethroid insecticides.

Tropical biodiversity as a source of bioactive substances against insects The issue of biodiversity has received a lot of attention in recent years, because of the worldwide concern about the high rates of destruction of some of the last major forest masses on Earth, which are mainly located in the neotropics (Wilson, 1988). These forests contain many organisms not yet described, some of them potentially useful for humankind. Nevertheless, few resources have been allocated to search for biodiversity applications to agriculture, including IPM programs, despite biodiversity and IPM being closely interrelated (Hilje and Hanson, 1998); new products (genes, natural pesticides, and beneficial organisms) for IPM programs can be obtained from tropical species and, conversely, implementation of IPM programs can have beneficial effects on both terrestrial and aquatic biodiversity. One of the best ways to take advantage of the remarkably high tropical biodiversity is to explore, identify, and utilize plant-derived substances in IPM programs aimed at key insect pests of crops and forest plantations (Hilje and Hanson, 1998). For instance, secondary metabolites with defensive properties against insects are rather common in plants, including alkaloids, non-protein amino acids, steroids, phenols, flavonoids, glycosides, glucosinolates, quinones, tannins, and terpenoids (Harborne, 1977; Panda and Khush, 1995). An excellent example along these lines is the highly successful and widespread use of neem-seed derivatives, a very well-known tree from India, Pakistan, Indonesia, and Thailand (Schmutterer et al., 1982; Walter, 1999). But it would also be possible to exploit a large number of tropical plants with an untapped potential as sources of active principles against insect pests (Grainge and Ahmed, 1988; Stoll, 2000), some of which are present in pristine ecosystems (Wilson, 1988) and even in man-made environments (such as agroforestry systems) and in other disturbed habitats.

Insecticides or repellents/deterrents? In addition to insecticides, plants may contain a wide array of substances acting against insects, including several types of allomones (those providing an adaptive

Promissory botanical repellents/deterrents for managing two key tropical insect pests

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advantage to the emitter), such as repellents, suppressants, deterrents, antibiotics, and anorexigenics (Warthen and Morgan, 1990). Substances belonging to the first three categories mentioned above act by interfering with the ability of insects in locating, feeding or ovipositing on their host plants. Even though there are long lists of plant species claimed as having compounds with such properties (Grainge and Ahmed, 1988; Warthen and Morgan, 1990; Stoll, 2000), many references are rather anecdotal, thus requiring scientific confirmation. For those substances commonly named as repellents, there are several possible levels of response from an insect to a plant, which are not always easy to differentiate. A true repellent is a substance which acts at a distance, causing oriented movements away from the source, i.e. a plant (Matthews and Matthews, 1978), whereas other substances cause an effect once an insect has got in contact with the plant structure emitting such substances. Thus, for instance a suppressant inhibits the initiation of feeding or oviposition, a deterrent impedes the continuation of such processes, and an anorexigenic causes a loss of appetite (Warthen and Morgan, 1990). It is very difficult to exactly differentiate and recognize such effects, unless highly sensitive and sophisticated equipment and tools are available, such as electronic feeding monitor, which provides electrical penetration waveforms or graphs (EPG) when a wired insect feeds or oviposits (Walker and Perring, 1994). Therefore, for the purpose of this chapter, the terms repellent and deterrent will be the only ones used here, defining repellents as those substances that keep an insect away before it lands on a plant, and deterrents as those substances inhibiting feeding or oviposition once an insect has landed and got in contact with the plant structure emitting such substances. The acceptance and spread of IPM as a sound strategy to deal with pest problems has made a growing number of agrichemical companies to develop commercial bioinsecticides (Hall and Menn, 1999), for which there has been a renewed interest in revisiting ethnobotanical knowledge to formally pursue screening of crude extracts for biological activity. But, unfortunately, the discovery, characterization, and exploitation of natural products against insects have proceeded in a biased trend towards bioinsecticides, disregarding repellent and deterrent substances. Perhaps this is because their production process is as complex as that for an insecticide, whereas their effects under field conditions do not eliminate a pest problem, but rather could transfer it to neighboring farmers (Schoonhoven, 1982). Nevertheless, for key insect pests showing a very low damage threshold, as it will be discussed in the next section, semiochemicals (repellents and deterrents) could play an important role as a component of IPM programs based upon a preventive approach. This is the case of the whitefly Bemisia tabaci and the mahogany shootborer Hypsipyla grandella, on which the authors have conducted research in recent years.

The target pests: why these ones? Currently, B. tabaci (Gennadius) (Homoptera: Aleyrodidae) and H. grandella Zeller (Lepidoptera: Pyralidae) can be considered as the main agricultural and forest pests

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in Latin America, respectively, so that their management is relevant in both economic and social grounds for agricultural and forest producers in the continent. On one hand, B. tabaci is a cosmopolitan insect and a key pest in many tropical and subtropical cropping systems (Brown and Bird, 1992; Brown, 1994). Currently, it is causing serious problems all the way from the U.S. throughout Argentina, including Caribbean countries, as well as in many African, Asian and European countries, and in Australia. Estimated economic losses amount to several hundreds or even thousands of millions of dollars a year, worldwide (Oliveira et al., 2001). Crop damage occurs directly through excessive sap removal, or indirectly by promoting the growth of sooty mold, inducing systemic disorders (syndromes) through feeding, or by vectoring plant viruses. It is a highly polyphagous pest, which can develop or reproduce on over 500 different plant species, belonging to 74 families (Greathead, 1986). It can attack some 30 crops worldwide, including both cash and staple crops (tomato, peppers, melon, watermelon, soybean, cotton, beans, and cassava). However, in Mesoamerica and the Caribbean, as well as in paleotropical areas, it acts mainly as a virus vector in a number of such crops. Moreover, B. tabaci has at least 19 well-documented races or biotypes (Perring, 2001) which may vary in their degree of association with particular host plants and the induction of specific syndromes, as well as their reproductive potential and response to climatic changes. On the other hand, H. grandella is one of the two shootborer species (Lepidoptera: Pyralidae) which attack precious wood plants of the Meliaceae family (Schabel et al., 1999) worldwide, the other being H. robusta, restricted to paleotropical areas. H. grandella (Zeller) is distributed throughout the neotropics, where it damages some 17 species of mahoganies (Swietenia spp.), cedars (Cedrela spp.) and related species. Its larva mainly bores the terminal shoots of trees, which causes forking of the stems, which has prevented attempts to establish commercial plantations of such species in Latin America and the Caribbean. Even though B. tabaci and H. grandella are very different insects both taxonomically and biologically, they are treated together here, as they have some commonalities in regards to their damage and possible management approaches. Considerable research aimed at managing each pest species has been conducted and summarized in Naranjo and Ellsworth (2001) and Newton et al. (1993), respectively. Nevertheless, management tactics, including chemical control by novel and effective insecticides, have not been successful enough due to a number of reasons, the main one being that both H. grandella and B. tabaci as a virus vector have a extremely low damage threshold. For H. grandella, damage caused by a single larva normally results in severe economic losses, as forking of the main stem since early stages of tree development renders them unmarketable. For B. tabaci, in the case of geminiviruses affecting tomato – which is probably valid to other viruses and crops – viral disease incidence can reach 100% with an average vector density as low as 0.3 adults/plant (Hilje, 2001). Then, since in both cases damage caused by even a single insect can result in irreversible losses, it would be worthless to kill either H. grandella larvae or B. tabaci adults once damage is done. Therefore, a preventive management scheme would be well in place, with semiochemicals (repellents and deterrents) precluding these pests


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from causing serious damage. Hopefully, such a scheme should also be environmentfriendly, cost-effective, and fully compatible with other IPM tactics.

Methodological approaches In order to detect the biological activity of plant extracts on B. tabaci and H. grandella herewith reported, a number of methodological approaches were used, some of them so far unpublished. Hydroalcoholic extracts were prepared at CIPRONA (Research Center on Natural Products), and laboratory and greenhouse bioassays carried out at CATIE. Extract preparation Each type of plant material was collected from a single location and at the same time, in order to avoid undesirable variability due to geographic or seasonal differences. Crude extracts For extracts prepared from leaves, corresponding to the majority of the species tested (Table 1), as well as those from seeds, bulbs, flower buds, and fruits (excepting wild ‘‘tacaco’’), plant material was dried in an oven at 40 1C, ground and placed in 70% methanol in a suitable flask for 24 h; the solvent was drained and the residue was treated again with methanol for 24 h. The pooled extracts were filtered through a Whatman No. 4 filter paper, and concentrated at 40 1C using a rotary evaporator. The final residue was freeze-dried to eliminate any water remaining in the crude extract. The same procedures were followed for preparing extracts from woody tissue, such as bitterwood (Quassia amara), which started by drying wood chips. In the case of leaves from mother-of-cocoa (Gliricidia sepium), the crude extract was defatted with hexane before freeze-drying it. Extract fractions Fractions were obtained only for the most promising of all plant extracts studied. A column 31 cm high and 4.5 cm diameter was prepared with 100 g of the synthetic resin Diaion HP-20 (Mitsubishi Chemical Industry, Yokohama, Japan). The resin was washed with water, water/methanol (1:1), methanol and diethyl ether. A maximum of 10 g of the crude freeze-dried extract was placed on the column and eluted with 1 l each of the solvents, starting with water and finishing with diethyl ether. The column was used as many times as necessary to completely process one batch of crude extract. The solvents were evaporated and freeze-dried, if necessary, to have the weight of each fraction in order to establish the proper dose to be used in the bioassay. In the case of extracts prepared from fruits, such as wild ‘‘tacaco’’ (Sechium pittieri), which was prepared for other kind of experiments (Castro et al., 1997), fresh fruits were extracted with methanol and the extract was concentrated in vacuo to give an aqueous suspension which was passed through the Diaion HP-20 column. The


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Table 1 Approximate degree of feeding deterrence caused by crude plant extracts to Bemisia tabaci adults (Bt) and Hypsipyla grandella larvae (Hg) Common name

Scientific name

Family

Structure tested

Deterrencea Bt

Allspice Balsam pear Bitterwood Bitterwood Cardamomo ‘‘Chile muelo’’ ‘‘Chile muelo’’ Clove Common rue Coriander Eucalyptus Fish bean Garlic Gumbo-Limbo Hot pepper Jackass bitters Lemongrass Marigold Marigold Mexican oregano Mother-of-cocoa Onion Peppermint Portugal cedar Spiked pepper Spiny coriander Sweet lime

Pimenta dioica Momordica charantia Quassia amara Quassia amara Elettaria cardamomum Drimys granadensis Drimys granadensis Syzygium aromaticum Ruta chalepensis Coriandrum sativum Eucalyptus deglupta Tephrosia vogelii Allium sativum Bursera simaruba Capsicum frutescens Neurolaena lobata Cymbopogon citratus Tagetes jalisciensis Tagetes microglossa Lippia graveolens Gliricidia sepium Allium cepa Satureja viminea Cupressus lusitanica Piper aduncum Eryngium foetidum Citrus limetta

Myrtaceae Cucurbitaceae Simaroubaceae Simaroubaceae Zingiberaceae Winteraceae Winteraceae Myrtaceae Rutaceae Apiaceae Myrtaceae Fabaceae Alliaceae Burseraceae Solanaceae Asteraceae Poaceae Asteraceae Asteraceae Lamiaceae Fabaceae Alliaceae Labiatae Cupressaceae Piperaceae Umbelliferae Rutaceae

Sword bean Sword bean Wild ‘‘tacaco’’ Wild sunflower Worm-seed

Canavalia ensiformis Canavalia ensiformis Sechium pittieri Tithonia diversifolia Chenopodium ambrosioides

Fabaceae Fabaceae Cucurbitaceae Asteraceae Chenopodiaceae

Essential oil (leaves) Leaves Leaves Wood Essential oil (seed) Leaves Essential oil (leaves) Flower buds Leaves Leaves Leaves Leaves Bulb Essential oil (fruit) Fruits Leaves Leaves Leaves Leaves Leaves Leaves Bulb Leaves Essential oil (leaves) Spikes (essential oil) Leaves Essential oil (fruit skin) Leaves Seeds Fruits Leaves Leaves

Hg

+ +++ + +++ + +++ ++ ++ + ++ ++ +++ 0 0 + ++ 0 ++ ++ + +++ + ++ + ++ + ++

0 ++ +++ 0 0 +++ 0 0 0 0 0 0 0 0 0 0 0 0

+++ ++ +++ +++ +++

T 0

Sources: Cubillo et al. (1994, 1997, 1999); Go´mez et al. (1997); Mancebo et al. (2000a, 2000b, 2001); Soto (2000); Hilje and Stansly (2001), Aguiar et al. (2003); Flores (2003). Note: The word ‘‘approximate’’ is used because it is not possible to compare results from different sources and even different methodologies. a Response: Non-tested (), nil (0), weak (+), mild (++), strong (+++) and toxic (T).

column was washed with water, water: methanol (1:1), methanol, and ethyl acetate or diethyl ether. Essential oils These were obtained by hydro-distillation (Ciccio´, 1996). The plant material, normally 500—800 g, was placed on a round bottomed flask and water was added up to a volume of 3 l. The essential oil was collected by means of a Clevenger-type


385

apparatus in the lapse of 2.5 h. The oil obtained was dried on anhydrous sodium sulfate and then filtered through a cotton plug. The sample was kept under refrigeration at 10 1C until it was used. Laboratory and greenhouse tests B. tabaci adults and H. grandella larvae for the experiments were taken from colonies kept at the Entomology Laboratory at CATIE. Whiteflies So far, 32 candidate crude plant extracts have been tested, including samples from leaves, seeds, bulbs, flower buds, fruits, and essential oils (Cubillo et al., 1994, 1997, 1999; Go´mez et al., 1997; Hilje and Stansly, 2001; Aguiar et al., 2003). They were selected based on ethnobotanical references, as well as on their low or nil taxonomic affinity with the most common hosts of B. tabaci (Greathead, 1986). Each plant extract was tested individually, at the following doses: 1, 5, 10, and 15 ml/l water (0.1%, 0.5%, 1.0%, and 1.5%, v/v). They were compared with an insecticide (endosulfan), a control treatment (distilled water), of either Volck 100 Neutral or Sunspray 9E, which are agricultural oils that strongly deter whitefly adults (Hilje and Stansly, 2001), and the emulsifier Citowett. Endosulfan (Thiodan 35% CE; Hoechst, Germany) (350 g a.i./l) was used at its commercial dose (2.5 ml/l water), and Volck (Chevron Chemical Co., CA) or Sunspray (Sun Co., Philadelphia) at 1.5% v/v. Citowett (BASF, Germany) (0.25 ml/l) was applied in all treatments, at its commercial dose (0.025%). Treatments were applied to tomato plants (var. Hayslip) with two true-leaves. This was done with a hand-sprayer DeVilbiss 15, with an adjustable tip (The DeVilbiss, Somerset, PA), which was connected to an air pump, under a constant pressure (10 kg/cm2). Plants from each treatment were separately sprayed with each substance in an isolated room, for which they were placed on a table and thoroughly sprayed to run-off. Treated plants were introduced into sleeve cages (30 30 45 cm, with walls made of wood, a fine net, and glass) 30 min after being sprayed. A randomized complete block design with four replicates was used for the three experiments. For these restricted choice experiments, two pots with a tomato plant in each one were placed in a sleeve cage. One of them has been sprayed with a given substance (either an extract or the control treatments), whereas the other plant was treated with distilled water. For the absolute control treatments, one of the potted plants was sprayed with Citowett and the other with water. The experimental unit was represented by each potted plant receiving a given treatment. Fifty B. tabaci adults were collected with a hand aspirator from a greenhouse colony reared on tomato, and released into each cage. Release took place between 8:30 and 10:30 h; 2 min later, the aspirator flask was checked, in order to count and release additional adults for replacing those which had died because of handling. In addition to these experiments, unrestricted choice experiments were performed, by exposing potted plants to flying whiteflies inside a greenhouse where their colony is maintained. Plants were placed on a bench and arranged in a randomized complete block design. Treatments included the best doses from the previous experiments, and were compared to the same controls, except endosulfan, in order not to disturb

386


colony development. Also, the experimental unit was represented by each potted plant receiving a given treatment. Moreover, in order to gain insight into more specific groups of substances responsible for causing phagodeterrence, testing of fractions (water, methanol: water, methanol, and ether) of some promising extracts was carried out by means of both types of experiments just described, at the same doses tested for the crude extracts (0.1%, 0.5%, 1.0%, and 1.5%, v/v). For all experiments described above, counts were made on the foliage of the whole plant. The criterion to appraise feeding deterrence was the number of landed adults at 48 h, in combination with the number of those surviving within such interval. Oviposition response was appraised by counting the number of eggs laid up to 48 h. Also, mortality was determined by counting the total number of living adults in each cage (in both plants) at 48 h, and subtracting the product from 50; in this case, the experimental unit was represented by each cage containing two potted plants. Eggs were counted under a stereo-microscope.

Mahogany shootborer Thus far, 20 out of the 32 crude plant extracts prepared for the experiments on whiteflies have been tested (Table 1) on this pest (Mancebo et al., 2000a). H. grandella larvae were taken from colonies, where they were initially reared on tender foliage of Spanish cedar (Cedrela odorata) and later transferred to an artificial diet (Vargas et al., 2001). Third-instar larvae, which had been fed exclusively on cedar foliage, were selected because their size allowed easy handling. Experiments were carried out in environmental chambers (Percival I-35L) set at 22 1C, 80%–90% RH, and 12:12 (L:D) photoperiod. Bioassays included treatments with both wood and leaf extracts of bitterwood, as well as leaf extracts of mother-of-cocoa and wild ‘‘tacaco,’’ at five increasing concentrations of each extract (0.10%, 0.312%, 1.00%, 3.16%, and 10.00%) mixed with a surfactant (Nu film 17, at 0.03%). They were compared to two relative controls (70% methanol, and Nu film 17 at 0.03%), and an absolute control treatment (distilled water). All dissolutions were prepared just before the experiment was set up, with distilled water as a carrier. Disks of cedar tender foliage (2.3 cm in diameter) were cut with a cork-borer, dipped in the selected treatment for 10 s, and allowed to dry for 30 min. Treated disks were placed individually in 30 ml glass flasks, along with a third-instar H. grandella larva which had been deprived of food for 3 h. A piece of paper towel was fastened with the lid of each flask and was moistened periodically, in order to retain leaf turgor. A randomized complete block design, with four replications, was used. The experimental unit consisted of seven larvae, except in the control (14 larvae). Blocks were represented by plastic trays, and flasks representing each treatment were randomized within each tray. After being exposed to the treatment for 24 h, each larva was transferred to a flask containing about 6 ml of artificial diet (Vargas et al., 2001), where it was allowed to complete its development; larvae were transferred to other flasks in cases where it was judged that the diet was not suitable for their development.


387

Such procedures were also used to test fractions (methanol, water, methanol: water, and ether) of the wood of Q. amara. To prepare the bitterwood test solutions, the equivalent amount for each fraction, in accordance to the yield of the fractionation process, was weighted and dissolved into 100 ml of the respective solvent. So, because 10% w/v was the highest concentration of crude extract tested at which feeding deterrence was observed (Mancebo et al., 2000b), treatments corresponded to the following concentrations (weight/volume) for each fraction: 2.3% (water), 0.625% (methanol), and 0.14% (ether). The absolute control treatment corresponded to a 10% bitterwood crude extract (in water), obtained from a 21.2% solution, which was the concentration at which it was obtained in the process of extraction. Relative control treatments corresponded to each one of the solvents (water, methanol, and diethyl ether). In addition, greenhouse experiments were carried out, where first-instar larvae were carefully placed on the main shoot of potted cedar plants previously treated with each one of such fractions. Three types of variables were measured in response to the bitterwood, motherof-cocoa, and wild ‘‘tacaco’’ extracts: food consumption, mortality, and developmental effects. Food consumption was assessed for each disk, by recording the percentage of leaf area which was consumed in 24 h. This was done by means of a visual scale of the program Distrain 1.0 (Tomerlin and Howell, 1988). Mortality was determined for each larva every 24 h, and the instar at which mortality occurred was recorded; cessation of movement and color change to black were the criteria used for judging mortality. Developmental effects included developmental time for each larval instar and the pupa, as well as pupal weight on the day after pupation; dates for larval moulting, conversion into pupae and adult emergence were recorded. For the greenhouse experiments with Q. amara fractions, variables included the number of orifices, sawdust mounds, and tunnels made by larvae, as well as the number of wilt and fallen shoots. Moreover, since bitterwood and common rue showed promise as deterrents, and even wild ‘‘tacaco’’ showed biological (insecticidal) activity to the mahogany shootborer (Mancebo et al., 2000a, 2000b, 2001), they were assessed for their systemic activity. Therefore, they were tested at a high enough concentration (10%), in order to clearly detect any possible systemic effects, and were compared to carbofuran (Furadan, FMC Corp. and Mobay Chem. Corp.) and Azatin EC (AgriDyne Technologies Inc., Salt Lake City, UT). The former is a very effective systemic insecticide against H. grandella larvae when it is applied as a granular material under field conditions (Wilkins et al., 1976), whereas the latter was shown to act as a strong toxicant to H. grandella (Mancebo et al., 2002). Plant extracts and the control treatments were dissolved into distilled water and mixed with a basic tissue culture media (Murashige and Skoog, 1962), supplemented with sucrose and solidified with agar. Two-month old plantlets grown from microcuttings or apical shoots of 45-day old cedar plants, were individually transplanted into 250 ml glass flasks containing the culture media. Systemic activity was assessed 3 days, a week and 2 weeks later by excising folioles from any of the upper leaves from each plant and exposing them to first or second-instar larvae which had been deprived of food for 3 h, inside 30 ml glass flasks (Soto, 2000). Variables measured

388


included leaf area consumed as well as mortality, according to the procedures previously described.

Achievements Whiteflies The majority (29) of the crude extracts tested thus far have induced some degree of response by B. tabaci adults (Table 1), meaning that they have substances acting as feeding deterrents on them (Cubillo et al., 1994, 1997, 1999; Go´mez et al., 1997; Hilje and Stansly, 2001; Aguiar et al., 2003). The following nine species have stood out for their ability to deter adult whiteflies: bitterwood, ‘‘chile muelo,’’ fish bean, mother-of-cocoa, ‘‘sorosı´ ,’’ sword bean, wild sunflower, wild ‘‘tacaco,’’ and worm-seed. Such an effect has been detected at doses as low as 0.1% v/v (1 ml/l water) for bitterwood, 0.1% (wild ‘‘tacaco’’), 0.5% (‘‘sorosı´ ’’), 1% (‘‘chile muelo’’), 1% (mother-of-cocoa), 1% (fish bean), 1% (sword bean), 1% (wild sunflower), and 1% (worm-seed). Phagodeterrence is revealed by the reluctance of whitefly adults to remain on the tomato plant treated with a given extract once they have landed on it and, presumably, made contact with the deterrent substances present in the extract, so that over time they tend to accumulate in the untreated plant. This is illustrated here with their responses to the mother-of-cocoa extract (Figure 1A). In general, the same pattern holds for oviposition response (Figure 1B), as the latter is a direct expression of the number of females present on the tomato plants. In all cases there is a clear-cut deterrence effect by the mineral oil (Figures 1A and 1B), as well as a toxic effect by endosulfan (Figure 1C). Mortality is revealed by the total number of living adults inside the cage at the end of each experiment, regard less of the plant where they are located. In fact, sometimes deterrence itself is so strong that it causes a high degree of mortality (Figure 1C), statistically similar (p>0.05) to that of the insecticide control, endosulfan. Spoiled by these findings, the authors purposely decided to concentrate research efforts on three promising extracts: bitterwood, mother-of-cocoa, and wild ‘‘tacaco’’. This is so because they represent three ‘‘prototypes,’’ considering a number of factors such as plant habit (tree, shrub, vine, etc.) and life cycle (perennial or annual); temporal availability and operational difficulties in harvesting the specific plant structures from which they are obtained (wood, foliage, and fruits, respectively); degree of harmfulness of their chemical components to farmers, consumers, and wildlife; and easiness to establish rather large-scale plantations of these species, either in open areas, within common tropical agroforestry systems (associated with coffee and cacao) or in enriched forests, for the industry to count on a permanent supply of raw material for preparing such extracts. Therefore, in order to gain insight into more specific groups of substances responsible for causing phagodeterrence, the next step was to test fractions of these promising extracts. Results are encouraging (Table 2), showing that methanol and methanol: water fractions provoked a stronger response by whitefly adults, except for wild ‘‘tacaco,’’ for which the response to the methanol fraction was weak,


30

N° adults b

A

25 b

20 15

389

a

a

b

a a

a 10

a a a a

5

a

a a

a

0 Water Citowett

Endos

Oil

0.1%

0.5%

1.0%

1.5%

N° eggs 60

a

50 a 40

b

B a

b a a

a

30 a a

10

a

b a

0

Water Citowett Endos

a Oil Treated

35 30 25

a

b

20

0.1%

0.5%

1.0%

1.5%

Untreated

N° adults

C

a

a ab

ab

a

ab

20 ab

15 10 5

b

0 Water Citowett Endos

Oil

0.1%

0.5%

1.0%

1.5%

Fig. 1. Average number of landed B. tabaci adults (A) and deposited eggs (B) at 48 h after the mother-of-cocoa (G. sepium) extract was applied to tomato plants, as well as the average number of surviving adults (C) in that interval. Means followed by the same letter in each pair of bars for A and B, and between individual bars for C, are not significantly different (p ¼ 0.05) (Hilje and Stansly, 2001). Abbreviations: Endos (endosulfan), Oil (Volck 100 Neutral).

390


Table 2 Minimum concentration of either the crude extracts of three plant species or their fractions at which feeding deterrence to Bemisia tabaci adults was detected, using the assays described in the text Plant species and fractions

Deterrence

Bitterwood (Q. amara) Crude Methanol Water Methanol: water Ether

0.1% 0.1% 1.0% 0.5% No

Mother-of-cocoa (G. sepium) Crude Methanol Water Methanol: water Ether

1.0% 0.1% No 0.5% 1.5%

Wild ‘‘tacaco’’ (S. pittieri) Crude Methanol Water Methanol: water Ether

0.1% No 0.5% 0.5% 0.1%

Source: Flores (2003).

whereas the ether one showed the strongest response. This could be explained considering that the more polar components of this extract are glycosides of bayogenin (saponins) (Castro et al., 1997) and the ether fraction could contain some of the aglycone bayogenin as a product of partial decomposition of the saponins. So, eventually, a further experiment has to be performed to obtain some of the bayogenin itself and test the pure compound on whiteflies. Results are illustrated here with adult responses to the bitterwood methanol fraction, showing strong deterrence at a dose as low as 0.1% (Figure 2A), the same pattern holding for oviposition (Figure 2B). Also, deterrence is so strong that it causes a high degree of mortality (Figure 2C), statistically similar (p>0.05) to that of endosulfan. Likewise, the methanol fraction mother-of-cocoa, as well as the aqueous fraction of wild ‘‘tacaco’’ caused deterrence at doses as low as 0.1 and 0.5%, respectively. Deterrence by bitterwood could be explained by the presence of quassinoids, such as quassin and neoquassin, which are common in this species (Polonsky, 1973). For example, Leskinen et al. (1984) found that a type of quassin from Q. amara deters feeding by Epilachna varivestis (Coleoptera: Coccinellidae). In the case of motherof-cocoa, its foliage contains a wide array of compounds, including terpenoids, flavonoids, anilpropanoids, and isoflavonoids (Lo´pez, 1995), some of which may have deterrent activity.


60

391

N° adults

A

50 40 b

30

a a

b

b

b

b

a

20

b b

10

a

0

a

a

a

a

a

Oil

0.1%

0.5%

1.0%

1.5%

Water Methanol Endos N° eggs 160

b

140 a

120 100 80

b

b

b

B b

a b

a

60

b

40 20 a

0

Water Methanol Endos

a Oil Treated

60 50 40

a 0.1%

a

a

a

0.5%

1.0%

1.5%

Untreated

N° adults a

C ab

ab

a

ab

ab

0.1%

0.5%

30 20

ab

b

10 0 Water Methanol Endos

Oil

1.0%

1.5%

Fig. 2. Average number of landed B. tabaci adults (A) and deposited eggs (B) at 48 h after the methanol fraction of bitterwood (Q. amara) was applied to tomato plants, as well as the average number of surviving adults (C) in that interval. Means followed by the same letter in each pair of bars for A and B, and between individual bars for C, are not significantly different (p ¼ 0.05) (Flores, 2003). Abbreviations: Endos (endosulfan), Oil (Sunspray).

In regards to the wild ‘‘tacaco’’ extract, chemicals causing the observed effects remain unknown, although probably they are a series of glycosides known as tacacosides, which are very bitter and irritating. Six of these bayogenin saponins have been isolated from fruits and aerial parts of S. pittieri and S. talamancense

392


(Castro et al., 1997), in an effort to look for antiproliferative principles in neotropical plants. Cucurbitacins, which have several kinds of activities, including toxicity and feeding deterrence (Mabry and Gill, 1979), are not found in this plant. When tomato plants treated with all bitterwood fractions were exposed to flying whiteflies, the methanol fraction performed better than the rest, closely followed by the ether one, but none of them did better as well as the mineral oil (Figure 3A), a trend that lasted for only 48 h (Figure 3B). Within a week (Figures 3C and 3D), none of these fractions performed better (p>0.05) than the absolute control (water), which suggests that the deterrent principles decompose under the experimental conditions. Quassinoids, which are possibly responsible for such effects, are not volatile, but probably are decomposed by the air and/or light. Other components of Q. amara which could have a similar activity are some indole alkaloids of the canthin-6-one type, mainly present in the leaves (Saénz and Nassar, 1970) but which can be found in the wood (Barbetti et al., 1990; Coe and Anderson, 1996), especially if the preparation of the sample included some bark. These compounds can also be decomposed by air and/or light. Mahogany shootborer Some 20 plant extracts, from the 32 ones also tested for B. tabaci, have been assessed (Table 1), and three of them have shown some type of biological activity against larvae: bitterwood, common rue, and wild ‘‘tacaco,’’ the first two acting as feeding deterrents and the latter as a powerful insecticide (Mancebo et al., 2000a, 2000b, 2001). Mortality by the wild ‘‘tacaco’’ extract, which kills larvae within a few hours or days, depending on its concentration, is probably due to tacacosides (Castro et al., 1997). Both foliage and wood methanol extracts of bitterwood, as well as that of common rue have substances acting as feeding deterrents, so that larvae refuse to eat treated leaf disks but, once they are placed into flasks containing an artificial diet they continue feeding and complete their development, normally reaching the adult stage. In terms of leaf disk consumption, there were very large differences between wood and leaf extracts of Q. amara (po0.0001), the former showing far higher antifeedant activity than leaf extracts (Figure 4). Such activity was detected at a concentration as low as 0.32% for the wood extract and as high as 3.16% for the leaf extract, with the response curve for the extract concentrations and leaf disk consumption being best fitted by a potential model in both cases. Such differences are probably explained by the specific chemical constituents in each plant structure, and especially by the concentration of quassinoids. For instance, bitterwood foliage contains substances toxic to insects such as larvae of the mosquito Culex quinquefasciatus (Diptera: Culicidae) (Evans and Raj, 1988), some of which could be quassinoids, which appear in low concentrations (Robins and Rhodes, 1984). Also, quassin and neoquassin concentrations vary within the tree branches, and increase with branch diameter (Villalobos, 1995). In addition, the wood contains some beta-carboline related indole alkaloids (Barbetti et al., 1987). In the case of the common rue extract, the lower consumption averages were attained at the 3.16%, followed by 0.32%, 1.0%, and 10% concentrations, which did

N° adults

350

A

250

150

ab

a a

100 d

0 MF

MWF

EF

Oil

C

d

100 a

a

MF

MWF

EF

100 c

Water

D a

a a

80 ab

60 40

a

Oil

N° adults

120

a

200

50

WF

Water 140

a

c

0

N° adults

150

b

50

c WF

a

150 bc

50

250

250 200

a

100

300

B

300 a

200

N° adults

b

b

Oil

Water

20 0

0 WF

MF

MWF

EF

Oil

Water

WF

MF

MWF

EF

Fig. 3. Average number of B. tabaci adults on tomato plants at intervals of 1 (A), 2 (B), 8 (C), and 15 days (D), in response to four fractions (water, methanol: water, methanol, and ether) of bitterwood (Q. amara) and two control treatments (Sunspray oil and water). Means followed by the same letter are not significantly different (p ¼ 0.05) (Flores, 2003). Abbreviations: WF (water), MF (methanol), MWF (methanol: water), and EF (ether) fractions, and Oil (Sunspray).


300

393


394 60 50

y = 1.9126 x

40

A

-1.0395

2

R = 0.93

30

Consumption (%)

20 10 0 0

1

2

3

4

5

6

7

8

9

10

60

B

50

y = 13.993 x

40

-0.6381

2

R = 0.90

30 20 10 0 0

1

2

3

4

5

6

7

8

9

10

Concentration (%)

Fig. 4. Average cedar leaf disk consumption (% area) by third-instar H. grandella larvae at 24 h, in response to increasing concentrations of wood (A) and leaf extracts (B) of bitterwood (Q. amara). The continuous line depicts the predicted response curve (Mancebo et al., 2000b).

not differ among themselves (p>0.05), the response curve also being best fitted by a potential model (Figure 5). Common rue extracts can deter Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Coccinellidae) larvae and adults (HoughGoldstein, 1990), and repel the cat flea, Ctenocephalides canis (Siphonaptera: Pulicidae) (Cox, 1980). Common rue foliage contains a number of chemicals, such as benzenoids (moskachans), quinoline alkaloids (arborinine and several acridone derivatives), terpenoids (elemol and beta-eudesmol from the essential oil), flavonoids (rutin), and coumarins derivatives (coumarin, bergapten) (Torres, 1950; Vasudevan and Lukner, 1968; Kong et al., 1984), but it remains unknown if any of them are responsible for causing either antifeedant or repellent activities. Results were encouraging regarding systemic activity of the extracts, which would be an asset for using them or their analogs in IPM programs. Foliole consumption by H. grandella larva was nil for those plantlets grown on culture media treated with carbofuran, as expected (Soto, 2000), being followed by Azatin, bitterwood, common rue, and wild ‘‘tacaco’’ all of them differing from the control (po0.05) (Figure 6). Therefore, substances causing either deterrence or toxicity can be translocated within the plant, reaching the leaves. Afterwards, once the crude extract showed this valuable asset, bitterwood fractions were analyzed to gain insight into more specific groups of substances responsible for causing phagodeterrence, as previously described.


395

Consumption (%)

30 25

y = 9.0044 x -0.3615 R2 = 0.75

20 15 10 5 0 0

1

2

3

4

5

6

7

8

10

9

Concentration (%)

Consumption (%)

Fig. 5. Average cedar leaf disk consumption (% area) by third-instar H. grandella larvae at 24 h, in response to increasing concentrations of a leaf extract of common rue (R. chalepensis). The continuous line depicts the predicted response curve (Mancebo et al., 2001).

20 18 16 14 12 10 8 6 4 2 0

a

b bc c cd d Water

Carbofuran Bitterwood

Azatin

Common Wild tacaco rue

Fig. 6. Average foliole consumption (% area) by first-instar H. grandella larvae at 24 h, in response to cedar plantlets grown in vitro in culture media treated with different crude plant extracts, neem (Azatin) and two control treatments (carbofuran and water). Means followed by the same letter are not significantly different (p ¼ 0.05) (Soto, 2000).

Methanol and ether fractions stood out and performed as well as the crude bitterwood extract in precluding larvae from feeding on leaf disks (Figure 7A). This finding was corroborated when larvae were exposed to shoots treated with such fractions under greenhouse conditions (Figure 7B); in both cases the water fraction performed very poorly, probably because the water solubility of the quassinoids at room temperature is scarce and the effect of the very small amount of these compounds present in the water fraction could be arrested by the presence of other components. Results with the bitterwood methanol extract are used here to illustrate the concentrations at which phagodeterrence to H. grandella occurs (Figure 8). When exposed to disks impregnated with increasing concentrations of that fraction, leaf disk consumption by larvae was significantly lower (po0.05) at doses as low as 0.02%.


396 Consumption (%) 25

A

a

20

ab 15 10

bc bcd

5 0

d Crude

WF

cd

d

MF

EF

Water

Methanol

a

ab

Ether

N° holes 2.5

ab

2.0

B

1.5

b 1.0 0.5 0.0

c

c Crude

WF

MF

c EF

Water

Methanol

Ether

Fig. 7. Response of H. grandella larvae to four fractions (water, methanol: water, methanol, and ether) of bitterwood (Q. amara) and the respective solvents, expressed as: the average consumption (% area) by third-instar larvae in 24 h to leaf disks treated with them (A), and the average number of holes in cedar plants treated with them, two days after their exposure to first-instar larvae I (B). Means followed by the same letter are not significantly different (p ¼ 0.05) (Soto, 2000). Abbreviations: WF (water), MF (methanol), and EF (ether) fractions.

Concluding remarks Even though findings herewith reported are still preliminary, they clearly show that a rather wide range of tropical plant crude extracts – some of them not reported in the literature yet – contain substances that can act as strong feeding deterrents of B. tabaci and H. grandella. It has been shown that, prior to insert their stylet or sucking tube into plant tissue B. tabaci adults rub or tap the apex of their labium on the plant surface, where they have several pairs of sensilla whose ultrastructure suggests that they can act either as chemoreceptors or mechano-chemoreceptors (Walker and Gordh, 1989). Likewise, H. grandella larvae possess deterrent receptors in the medial and/or lateral sensilla styloconica located on the maxillae (Schoonhoven, 1980).


397

Consumption (%) 12 10

a a

8

ab

6

bc

4

c

2

c

c

0.2%

0.625%

0 Water

Methanol 0.00625%

0.02%

0.0625%

Fig. 8. Average cedar leaf disk consumption (% area) by third-instar H. grandella larvae, in 24 h, in response to increasing concentrations of the methanol fraction of bitterwood (Q. amara). Means followed by the same letter are not significantly different (p ¼ 0.05) (Soto, 2000).

In fact, 20 out of 32 as well as three out of 20 plant extracts tested on B. tabaci and H. grandella, respectively, showed deterrence or toxicity. This general trend is kind of unexpected, since B. tabaci is quite polyphagous, whereas H. grandella is rather oligophagous, as its host range is restricted to members of the Meliaceae family. At any rate, in order for the agrichemical industry to get involved into developing commercial deterrents based upon plant extracts, as it currently occurs with neem derivatives (Walter, 1999), a number of questions and concerns ought to be addressed. First of all, there should be a demand strong enough as to justify their involvement in such a business. However, nowadays market considerations are closely tied to other issues related to the concepts and practices of sustainability, aimed at reconciling both agricultural production and economic development with environmental conservation. On one hand, since IPM practitioners are continuously striving for developing such sustainable production systems, commercial deterrents would fit very well into these systems, provided that they are compatible with the conservation of water, soil, and wildlife, as well as with farmer and consumers’ health. On the other hand, since the only discrepancy between IPM and organic farming is that the former approach allows a rational use of some synthetic inputs (fertilizers and pesticides), safe and botanical-based deterrents would also fit well into organic systems, as well as in certified forest systems and products. Since both systems allow producers to take advantage of some economic benefits, such as particular niches in international markets associated with a premium value compared to the price paid for conventional products, they would be willing to adopt environment-friendly semiochemicals, provided that they are cost-effective. This situation represents a unique opportunity for local small and medium-size companies in developing countries. For instance, in Mesoamerica, both microbial and botanical pesticides have been widely accepted, which explains the ever increasing number of companies involved in biopesticide production, such as neem

398


products, entomopathogens, botanical products, and pheromones (Hilje et al., 2003). Local companies could not only have a rather easy access to tropical biodiversity resources, but also add value to their products, favoring local economies and communities. Secondly, toxicity to wildlife and people is a major concern not only for conventional insecticides, but also for natural products, including botanical ones. Of course, toxicological aspects for the majority of plant materials, as well as for those reported here, remain unknown. Nevertheless, some of the latter seem not to pose risks to humans and other mammals. For instance, common rue and bitterwood are commonly used as natural medicines in some neotropical countries and elsewhere. Likewise, leaves and shoots of mother-of-cocoa are normally used as fodder for ruminants (CATIE, 1991). As for wild ‘‘tacaco,’’ most of what is known about it responds to ethnobotanical knowledge but, being very bitter, this is normally a plant which people regard as a weed. In the case of bitterwood, it was one of the botanical insecticides widely used before synthetic insecticides were developed (Metcalf et al., 1951). Also, Q-assia is the brand of a new pharmaceutical product for digestive problems recently released into the market by Lisanatura, a local pharmaceutical company in Costa Rica. It has been shown that, on rats, the aqueous extract of the dry wood of Q. amara was capable of preventing the formation of ulcers as induced by indomethacin, ethanol, or stress (Badilla et al., 1998). In a previous study, no sign of acute toxicity was observed at any oral dose of an aqueous extract of the wood; however, the intraperitoneal administration of 500 mg/kg, presented acute toxicity signs with a 24 h recovery, but the 1000 mg/kg dose was lethal to a 100% within 24 h (Garcı´ a et al., 1996). The crude methanol extract of the stem wood significantly caused a reduction in the weight of the testis, epididymis, and seminal vesicle, but an increase in that of the anterior pituitary gland. Quassin produced similar biological actions as the crude extract while the effects of 2-methoxycanthin-6-one did not seem to differ from those of the control (Raji and Bolarinwa, 1997). Quassin was also shown to inhibit the synthesis of testosterone in rat Leydig cells in a dose-dependent fashion (Njar et al., 1995). Thirdly, the desired degree of industrialization for plant semiochemicals is a highly relevant matter in practical terms. In fact, some of these would lend themselves to be applied as crude extracts, as it normally occurs with many plant extracts in tropical rural communities (Stoll, 2000). Moreover, some of them could be further processed as to obtain semi-rustic products based on the most active fractions against B. tabaci and H. grandella, in which farmer organizations may participate, as it has happened with IPM projects aimed at other pests, such as the small-scale processing plants for the production of Beauveria bassiana, an entomopathogenic fungus attacking the coffee berry borer (Hypothenemus hampei), in Colombia and elsewhere. In such cases, farmers could require periodical support from governmental or academic entities to guarantee adequate standards of quality control for their products. Otherwise, farmer organizations could participate in the initial steps of industrial processes leading to the production of well-elaborated materials by the agrichemical industry, which has shown a real interest in developing bioinsecticides (Hall and Menn, 1999). For instance, there is a growing interest in promoting the utilization of the bitterwood tree as an economic resource for indigenous communities in


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Mesoamerica, so that several of its ecological, silvicultural, and marketing aspects have been researched in recent years (Ocampo, 1995). Currently, Bougainvillea S.A., which is a new Costa Rican company, is starting an effort to produce an industrial extract of bitterwood for insecticidal purposes (Rafael Ocampo 2005, pers. comm.). This initiative begins with the domestication of the plant. Until the present day the plant has been managed – not cultivated – by the native population of Talamanca. The domestication of the plant includes a study of the best conditions for cultivation and observation and control of its pests. Harvesting practices compatible with the conservation of the species and assurance of a high content of quassinoids have been contemplated, as well as the optimization of the extraction conditions. Also, now that it has been determined the most active fractions of bitterwood, mother-of-cocoa, and wild ‘‘tacaco’’ against B. tabaci and H. grandella, agrichemical companies interested in developing commercial products based upon either natural substances or use them as leads to obtain their synthetic analogues (Hall and Menn, 1999) could proceed in identifying and characterizing the specific substances responsible for the phagodeterrent effects and eventually manufacture them in formulations well fitted to meet farmer’s needs. And, last but not least, there is the concern about the availability of raw material in large enough amounts to supply industry on a continuous and reliable basis, which could be a hindrance when dealing with tree species. Fortunately, in the case of bitterwood, mother-of-cocoa, and wild ‘‘tacaco’’ this is not a serious problem, as all lend themselves to be planted in different types of schemes for commercial purposes, outside natural forests. The bitterwood tree, Q. amara L. ex Blom (Simaroubaceae) is a native tropical shrub, whose range extends from Mexico to Ecuador, including the Caribbean (Ocampo, 1995). It normally grows in the forest understory, where it reaches up to 9 m in height and 10 cm in diameter. However, it can also grow easily in disturbed areas and can be reproduced readily by vegetative cuttings. Due to the interest in utilizing it for pharmaceutical and agricultural purposes, silvicultural aspects have been researched in recent years, including pruning regimes and resprout responses, as to guarantee a continuous harvest of wood for quassinoid extraction (Brown, 1995). Mother of cocoa, G. sepium (Jacquin) Kunth ex Walpers (Fabaceae) is a perennial shrub that occurs in tropical seasonal lowlands, from Mexico to Panama. It reaches up to 15 m in height and 40 cm in diameter, and is commonly used in agroforestry systems to provide shade to cacao and coffee plantations. The trees are also used as living supports for growing black pepper and vanilla, as living fences, in alley cropping systems, and as fodder (CATIE, 1991). Wild tacaco, Sechium pittieri (Cogn.) C. Jeffrey (Cucurbitaceae) is a perennial vine that occurs at a very wide altitudinal range (100–2500 m) from Nicaragua to Panama, where it grows on both wild and disturbed habitats, usually near rivers or creeks, and even in flooding areas (Lira, 1995). Its fruits, 4–6 cm long and 3–4 cm wide, are green, kind of ovoid or fusiform, very bitter, and can appear all year round; the fruits of its congeneric species S. tacaco are edible. In case of eventually counting upon formulated deterrents to use against B. tabaci and H. grandella in the field, their use could be optimized by deploying them only during certain times in the crop life (critical period), aimed at minimizing contact

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between the insect and the host plant. For instance, since the impact of viral diseases on yields is higher at earlier stages of plant development (Hilje, 2001), any management scheme for B. tabaci should focus on this critical period (60 days after germination), by spraying the product on the plant. For H. grandella, the critical period corresponds to the first 5–8 years of tree development, depending on the region (Cibriań et al., 1995), and the product could be delivered into trees as a slow-release formulation through an implant or a microinjection. However, in both cases, during such a period the deterrents could be complemented with other IPM tactics, as semiochemical by themselves very seldom provide robust pest control (Pickett et al., 1997). Tactics like plant breeding, cultural practices, and biological control, along with semiochemicals, should be aimed at reconciling production with environmental conservation, in accordance to the paradigm of sustainable development, for the economic benefit of farmers and of society as a whole.

Acknowledgments The authors thank their students Fernando Mancebo, Francisco Soto, Guillermo Flores, Alana Aguiar, and Paul Go´mez, as well as their assistants Douglas Cubillo, Manuel Carballo, Guido Sanabria, and Arturo Ramı´ rez, who have supported these efforts over the years at CATIE. To Juan Carlos Brenes (CIPRONA), for preparing the extracts for the experiments described here, and to Vı´ ctor Castro and Jose´ Francisco Ciccio´ (CIPRONA) for providing samples of wild ‘‘tacaco’’ and essential oils of some plants, respectively. To Bernal Valverde, Phillip J. Shannon, and Phillip A. Stansly, for their valuable contributions at early stages of our research development. To Francisco Soto, for his valuable help in preparing the illustrations.

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CHAPTER 16

Naturally occurring anti-insect proteins: current status and future aspects TZI BUN NG

Introduction Insects, fungi, viruses, and bacteria may cause devastating damage to agricultural crops that incur enormous losses in revenue. Research has thus been undertaken to devise strategies that can protect plants from invasion by the aforementioned predators and microorganisms and thereby minimize the reduction in crop yield and deterioration of crop quality. The present chapter focuses on proteins that are toxic to predatory insects.

Thiol methyltransferases Attieh et al. (2002) reported the presence of five isoforms of plant thiol methyltransferase (TMT) that catalyze the methylation of the hydrolytic products of glucosinolates to volatile sulfur compounds with putative anti-insect and anti-pathogenic activities. Two cDNAs encoding these enzymes (cTMT1 and cTMT2) were isolated by screening a cabbage cDNA library with an Arabidopsis EST that displayed marked sequence similarity to one of the TMT isoforms. Both cDNAs encoded 25kDa proteins with 227 amino acid residues. The cDNAs were distinct from conventional known N-, O-, or S-methyltransferases although they contained the typical methyltransferase signatures. The only gene with an assigned function and significant similarity to the TMT cDNAs was a chloride methyltransferase gene. The recombinant proteins obtained by expressing the two cDNAs in Escherichia coli had properties similar to those of the native enzymes. The expression pattern of these enzymes in cabbage tissues was consistent with their association to glucosinolates. Elucidation of the defensive role of the TMTs against biotic stress is facilitated by cloning of these enzymes.

Lectins Griffonia simplicifolia lectin delayed development of the cowpea bruchid Callosobruchus maculates (Coleoptera: Bruchidae). Site-directed mutagenesis studies

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revealed that carbohydrate binding and resistance to proteases in the insect gut were essential to the anti-insect activity of G. simplicifolia lectin (Zhu et al., 1996). The resistance of the lectin to digestion by two cathepsin L-like proteases from third and fourth instar C. maculatus larvae and carbohydrate binding of the lectin to a target ligand in the insect gut were independent although both contributed to its defense function (Zhu-Salzman and Salzman, 2001) and were Ca2+-dependent (Zhu-Salzman et al., 2002). The recombinant lectin displayed insecticidal activity indicating that glycosylation was not required for the activity (Zhu et al., 1996). The large subunit of the lectin accounted for much of the insecticidal activity of the lectin (Zhu-Salzman et al., 1998). Talisia esculenta lectin induced approximately 90% mortality in the larvae of the bruchid insects C. maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) when it was included in an artificial diet at a level of 2% (w/w). The lectin was not digested by crude midgut preparations of the two insects. The transformation of the genes encoding this lectin may be useful in conferring insect resistance to agricultural crops (Macedo et al., 2002). Macedo et al. (2004) noted that T. esculenta lectin recognized glycoproteins in the midgut of C. maculatus. Digestive proteases from the larvae of C. maculatus were incapable of digesting the lectin. The insecticidal activity was abolished by mannose. Phaseolus vulgaris erythro- and leucoagglutinating isolectins included in the diet or present in transgenic Arabidopsis plants expressing the lectins did not adversely affect the survival, growth, and development of tomato moth (Lacanobia oleracea) (Lepidoptera: Noctuidae) larvae. The lack of lectin binding to L. oleracea gut glycoproteins probably accounts for the lack of insecticidal activity of the lectins (Fitches et al., 2001a). Cratylea argentea lectin exerted insecticidal activity against the cowpea bruchid C. maculatus and adversely affected the growth rate of the digestive tract of rats that prey on the plant. This constituted a mechanism against herbivory (Oliveira et al., 2004). The N-acetylglucosamine-binding lectin from Koelreuteria paniculata seeds displayed anti-insect activity against the larvae of the bruchid C. maculatus and Anagasta kuehniella (Lepidoptera: Pyralidae) (Macedo et al., 2003). Some chitin-binding lectins like the stinging nettle lectin (Urtica dioica agglutinin) exhibited insecticidal activity (Lerner and Raikhel, 1992). The entomotoxic activity of Lathyrus ochrus lectin was related to its glucose-binding activity. Inclusion of glucose in the medium used to raise larvae led to a reduction in the lectin toxicity (Trigueros et al., 2000). Snowdrop lectin (Galanthus nivalis agglutinin, GNA) was toxic to the rice brown planthopper (Nilaparvata lugens) (Delphacidae: Delphacinae). Expression of GNA in transgenic rice plants conferred resistance to the rice planthopper (Rao et al., 1998; Sudhakar et al., 1998). Liang (2004) observed that transgenic wheat plants expressing GNA were resistant to the aphids Rhopalosiphum padi and Maerosiphum avenae (Hemiptera: Aphiidae). The insecticidal lectins snow drop lectin and Con A induced a 50–60% weight reduction and significant decline in midgut a-glucosidase activity in tomato moth (L. oleracea) larvae chronically exposed to the lectins (Fitches and Gatehouse, 1998). In potato, the presence of the gregarious hymenopteran ectoparasitoid Eulophus pennicornis brought about a reduction in the damage caused by the phytophagous

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lepidopteran pest, the tomato moth L. oleracea. The level of damage was further reduced by expressing the GNA gene in transgenic potato plants (Bell et al., 2001). Both Con A (jackbean lectin) and GNA exerted systemic effects in tomato moth larvae via transport from the gut contents to the hemolymph across the gut epithelium. Con A was more toxic than GNA and persisted in the fat body and gut tissues (Fitches et al., 2001). Larvae exposed to Con A and GNA exhibited weight reductions (Fitches and Gatehouse, 1998). Con A binds more than GNA to larval tissues in vitro (Fitches et al., 2001). When the tritrophic interactions between GNA-expressing transgenic potato plants, Myzus persicae (an aphid pest) and the two-spot ladybird Adalia bipunctata (a beneficial predator) (Coleoptera) were studied, no deleterious effects of GNA on development and survival of ladybird larvae fed on aphids from these transgenic plants were seen (Down et al., 2003). Fitches et al. (2004) reported that whereas neither GNA nor spider (Segestria florentina) (Arachinda: Segestridae) venom toxin SFII alone demonstrated acute toxicity when fed to tomato moth (L. oleracea) (Lepidoptera: Noctuidae) larvae, the fusion protein formed from both proteins had entomotoxic and insecticidal activities. Zephyranthes candida lectin possessed a three-dimensional structure markedly similar to that of GNA, suggesting that the former lectin might resemble GNA in insecticidal activity (Pang et al., 2003). Narcissus pseudonarcissus lectin displayed insecticidal activity and might be able to increase resistance of transgenic crops to pests (Summers et al., 2002). Other mannose-binding monocotyledonous lectins, including those from the garlic Allium sativum, the taro Colocasia esculenta, and Diffenbachia sequina, adversely affected the growth and development of a homopteran insect, the red cotton bug, with A. sativum lectin exhibiting the most potent insecticidal effect (Roy et al., 2002). The Chinese daffodil Narcissus tazetta produces lectins highly homologous to GNA (Ooi et al., 1998, 2000a, 2000b). They possessed insecticidal activity (Ooi et al., unpublished data). The insecticidal activity of ground ivy (Glechoma hederacea) leaf lectin was linked to its carbohydrate-binding specificity (Wang et al., 2003). The black bean (P. vulgaris) phytohemagglutinin exerted insecticidal action against the beetle C. maculatus (Janzen et al., 1976). A lectin from the edible mushroom Xerocomus chrysenteron had a more potent insecticidal activity than GNA and L. ochrus lectin. It was toxic to insects such as Acyrthosiphon pisum (Hemiptera: Aphiidae) and Drosophila melanogaster (Diptera: Drosophilidae) (Trigueros et al., 2003).

Arcelins Arcelins are lectin-like proteins with insecticidal activities, e.g., against the larvae of the Mexican bean weevil Z. subfasciatus. Arcelin-1 from kidney bean (P. vulgaris L. cv. ‘‘RAZ-2’’) seeds was a 60-kDa homodimeric glycoprotein that was resistant to proteases and chaotropic agents (Fabre et al., 1998). Arcelin-1 did not bind monosaccharides. Its crystal structure has been studied (Mourey et al., 1997, 1998). Five variants of arcelin have been identified.

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Ribosome inactivating proteins An extract of Ricinus communis, which contained the ribosome inactivating protein ricin, exerted toxicity on larvae and adults of the housefly Musca domesticus. Pupal development was also adversely affected (Alvarez Montes de Oca et al., 1996).

Ureases Jack bean urease (JBU) and embryo-specific soybean urease (SBU), but not Bacillus pasteurii urease, manifested potent insecticidal activity on the cotton sucker hemipteran bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) (Follmer et al., 2004). However, the ureases were not lethal in mice. The insecticidal activity was not inhibited by p-hydroxymercuribenzoate that irreversibly inhibited the ureolytic activity. The ureases exhibited different susceptibilities to inhibitors like acetohydroxamic acid (a chelator of nickel and zinc ions) and p-benzoquinone (a chemical modification reagent for cysteine residues).

Avidin Transgenic tobacco plants expressing avidin demonstrated insecticidal activity toward the noctuid lepidopterans, Helicoverpa armigera and Spodoptera litura (Lepidoptera: Noctuidae) (Burgess et al., 2002b).

Chitinases Chitin is a component of insect article. The synthesis and degradation of chitin are targets for pesticide action (Cohen, 1993). Both the chitinases cloned from tomato moth larvae, and a fusion protein containing chitinase joined to the N-terminus of GNA, retarded growth, reduced food consumption, and induced mortality in the larvae (Fitches et al., 2004).

a-Amylase inhibitors A dimeric a-amylase inhibitor (aAI) from the wheat kernel, designated as inhibitor 0.19, inhibited midgut a-amylase of Tenebrio molitor (Coleoptera: Tenebrionidae) (Petrucci et al., 1976). As shown by circular dichroism measurements in the farultraviolet, the inhibitor possessed about 50% ordered structure. Ionizable tyrosine groups contributed to the ellipticity bands in the near-ultraviolet. A monomeric aAI, designated as inhibitor 0.28, was also isolated (Silano and Zahnley, 1978; Buonocore et al., 1980). Another dimeric aAI designated as WDAI-3 inhibits T. molitor a-amylase but not porcine a-amylase or trypsin. Its N-terminal sequence resembles the dimeric inhibitor 0.19 more than the monomeric inhibitor 0.28. WDAI-3 is encoded by a duplicated gene in the short arm of chromosome 3B (Sanchez-Monge et al., 1989). A wheat germ protein that simultaneously inhibited insect a-amylase and proteinase K has been crystallized, and so has its complex with proteinase


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K (Pal et al., 1986). Four 14-kDa aAIs have been isolated from wheat flour. They differed in their inhibitory potencies toward human salivary a-amylase and a-amylases from the yellow mealworm, red flour beetle, and rice weevil (Feng et al., 1996). Franco et al. (2000) purified five wheat kernel aAIs of the structural 0.19 family and demonstrated their inhibition of a-amylase from the bean weevil Acanthoscelides obtectus (Coleoptera). The inhibitors exhibited differences in inhibition profiles despite substantial sequence identity. Residue size and charge loop lengths, and the conformational effects of a mutation from Cys to Pro, were among the factors contributing to the observed differences in specificity. The knowledge gained in this study may facilitate future rational design of inhibitors with modified inhibition characteristics. The BIII aAI from rye kernels was inhibitory to a-amylase from the cotton boll weevil (Anthonomus grandis) (Coleoptera: Curculionidae) while several other plant aAIs were ineffective (Oliveira-Neto et al., 2003). A 14-kDa protein from corn seeds, referred to as corn inhibitor of activated Hageman factor (CHFI) or as the popcorn inhibitor, had multiple activities including inhibitory activities toward human b-factor XIIa (Hageman factor), porcine trypsin, and insect a-amylase. The recombinant protein was similar to the native protein in activities. However, a recombinant form with a deletion of the 11 N-terminal residues was devoid of insect a-amylase inhibitory activity, suggesting that the N-terminal region was involved in interaction with insect a-amylase (Hazegh-Azam et al., 1998). The Ragi bifunctional aAI trypsin inhibitor, a member of the cereal inhibitor superfamily that inhibited both trypsin and a-amylase, may be used to protect crop plants from predatory insects (Strobl et al., 1998b). Wheat aAI inhibited a-amylases in the salivary glands of Lygus hesperus (Hemiptera: Miridae) and L. lineolaris (Zeng and Cohen, 2000). Wheat aAI and common bean aAI incompletely inhibited the a-amylase isozymes from Western corn rootworm (Diabrotica virgifera virgifera) (Coleoptera: Chrysomelidae) larvae (Titarenko and Chrispeels, 2000). A 13.75-kDa monomeric aAI has been isolated from rye (Secale cereale). It was more active against insect a-amylases than against mammalian a-amylases (Iulek et al., 2000). A dimeric protein composed of about 120 amino acid residues, and designated as BDAI-1, was isolated from mature barley endosperm. It inhibited T. molitor a-amylase but not salivary or pancreatic a-amylases. It exhibited sequence homology to wheat aAIs (Lazaro et al., 1988). Rasmussen and Johansson (1992) showed that the primary structure of barley aAI deduced from the cDNA was 60–85% identical to wheat aAIs but had less than 50% identity to barley and wheat trypsin inhibitors. However, the 10 conserved Cys residues and Pro-x-Cys motif were present. A 14.5-kDa insect aAI from barley has been identified as a major IgE-binding component of sera from patients with baker’s asthma (Barber et al., 1989). A 26-kDa dimeric aAI with endochitinase activity was purified from seeds of Job’s tear (Coix lachryma-jobi). Its function might be related to protection from fungal infection and insect predation (Ary et al., 1989). aAI in P. vulgaris showed insecticidal activity against cowpea weevil (C. maculatus) and azuki bean weevil (C. chinensis). In addition, an aAI-like protein with activity against C. maculatus was isolated. It was a putative precursor of aAI (Ishimoto et al.,

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1999). Grossi de Sa et al. (1997) isolated from P. vulgaris aAI-1 and aAI-2. aAI-1 inhibits aAIs from certain seed weevils and porcine pancreas but not aAI from Z. subfasciatus; aAI-2 inhibits aAI from Z. subfasciaturs but not that from porcine pancreas. aAI-2, composed of 240 amino acid residues, was a seed storage protein isolated from a bruchid-resistant wild common bean (P. vulgaris). It inhibited the growth of bruchid pests and was structurally homologous to aAI-1, arcelin-1, and phytohemagglutinin of the common bean, suggesting an evolutionary relationship among these proteins (Suzuki et al., 1994). The insecticidal activities of aAI from the common bean (P. vulgaris), aAI-Pa from tepary bean (P. acutifolius) seeds, and aAI-Pc from scarlet runner bean (P. coccineus) seeds were studied. It was found that they differed in their activities against the a-amylases of the azuki bean weevil Callosobruchus chinensis and the Mexican bean weevil Z. subfasciatus (Ishimoto and Chrispeels, 1996). Morton et al. (2000) showed that bean aAI-1 in transgenic pea (Pisum sativum) offered total protection from pea weevil (Bruchus weevil) under field conditions. High temperatures reduced the protection (Sousa-Majer et al., 2004). A 32-residue aAI with 3 S–S bridges has been isolated from the seeds of a Mexican crop plant, Amaranthus hypocondriacus. It inhibited the activity of a-amylases from the larvae of Tribolium castaneum (Coleoptera: Tenebrionidae) and Prostephanus truncatus (Colerptera: Bostrickidae) but not mammalian a-amylases (ChagollaLopez et al., 1994). The inhibitor was composed of a short triplet-stranded b-sheet stabilized by 3 S–S bonds. When its first intercystine segment with the sequence IPKWNR was inserted into a homologous position of the spider toxin Huwen toxin I, the resulting chimera exhibited aAI activity, indicating the involvement of the segment in enzyme binding (Lu et al., 1999). The crystal structure of yellow mealworm a-amylase in complex with amaranth a-amylase inhibitor was determined at 2.0 A˚ resolution. The remarkable structural similarity between amaranth aAI and knottins would prompt engineering of novel activities onto the small scaffold of this group of proteins (Pereira et al., 1999). Molecular cloning of Z. subfasciatus a-amylase cDNA and expression of the enzyme with a baculovirus vector in cultured insect cells have been achieved. The protein, composed of 466 amino acids, showed 50–60% amino acid identity with five other insect a-amylases. Three residues important for catalysis and three histidine residues for substrate binding were conserved in Z. subfasciatus a-amylase. The aamylase was inhibited by aAI-2 but not by AI-1 (Grossi de Sa and Chrispeels, 1997). The crystal structure of a-amylase from the yellow mealworm T. molitor has been reported. Its structure showed, as reflected in the substrate and inhibitor binding region, a marked variation from its mammalian counterparts. It was lacking in a highly flexible, glycine-rich loop involved in a ‘‘trap-release’’ mechanism of substrate hydrolysis by mammalian a-amylases. The structural differences may account for the specificity of inhibitors against insect a-amylases (Strobl et al., 1998a). The structure of a-amylase from Mexican bean weevil (Z. subfasciatus) was modeled based on the crystal structure of yellow mealworm a-amylase (Da Silva et al., 2000). The a-amylase from T. molitor larvae showed large deviations from mammalian a-amylase models that occurred in the loops. Nevertheless, aAI from the bean P. vulgaris inhibited both insect and mammalian a-amylases (Nahoum et al., 1999).


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The a-amylase of the coffee berry borer (Hypothenemus hamepei) (Coleoptera: Scolytidae) was potently inhibited by P. vulgaris aAI-I but much less so by Amaranthus aAI (Valencia et al., 2000). Structural comparison of the solution structure of amaranth aAI with the X-ray structure of the inhibitor bound to T. molitor revealed that the backbone conformation was only slightly modified on complex formation while that of side chains involved in protein–protein contacts was similar to those present in solution. Thus, the overall conformation of aAI seemed to be predisposed to binding to its target a-amylase, corroborating that it acted as a lid covering the active site of a-amylase (Martins et al., 2001). An aAI from the bean Lablab purpureus inhibited fungal but not animal or plant a-amylase. Its amino acid sequence resembled lectin members of a lectin–arcelin– a-amylase inhibitor family described in common bean (Fakhoury and Woloshuk, 2001).

Protease inhibitors Corn cystatin inhibited proteinases in the gut of the insect pest Sitophilus zeamais (Coleoptera: Curculionidae) (Irie et al., 1996). One of the soybean cystatins, soyacystatin N, inhibited insect gut proteinases much more potently than the other cystatin, soyacystatin L. Soyacystatin N retarded the growth and development of cowpea weevil while soyacystatin L was inactive (Koiwa et al., 1998). Introduction of potato proteinase inhibitor II gene into rice plants conferred on the plants augmented resistance to a major rice insect pest, the pink stem borer Sesamia inferens (Lepidoptera: Noctuidae) (Duan et al., 1996). Aprotinin, a mammalian serine protease inhibitor, exerted insecticidal action (Burgess et al., 2002a). A genomic clone encoding a serine proteinase inhibitor II was isolated from tomato seedlings. The proteinase inhibitor could be expressed in tobacco cells and the protein products demonstrated insecticidal activity. Its expression in roots, stems, and leaves could be activated by mechanical injury, jasmonic acid and a-linolenic acid, but not by abscisic acid (Zhang et al., 2004). Pal et al. (1986) showed that a 21-kDa proteinase K inhibitor from wheat germ was bifunctional in that it also inhibited insect a-amylase. The protein was crystallized. Foissac et al. (2002) demonstrated cathepsin B-like and trypsin-like proteases in gut of rice brown planthopper (N. lugens) (Delphacidae: Delphacinae). Transgenic rice plants expressing soybean trypsin inhibitor were partially resistant to the insect. The genes of protease inhibitors can be used to engineer resistance to insects and other pests (Haq et al., 2004). However, there were isolated cases in which this strategy did not work well (Winterer and Bergelson, 2001). Although diamond moths have a lower growth rate caused by consumption of plants transformed with potato protease inhibitor, the moths eat more plant tissue to mitigate the effect.

Glycoprotein toxin of Bacillus thuringiensis The 135-kDa protoxin from B. thuringiensis could be cleaved using trypsin or its own neutral metalloproteases to yield the entomocidal 68-kDa toxin (Andrews et al.,

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1985). Proteolytic activatin of the protoxin could also be brought about by the 25kDa trypsin-like gut enzyme from the spruce budworm Choristoneura fumiferana (Milne and Kaplan, 1993). The toxin bound to a lectin-like gut receptor in the mosquito (Aedes aegypti) larvae (Diptera: Culicidae) (Muthukumar and Nickerson, 1987). Two preparations of the delta-endotoxin were used. The dipteran-specific preparation (protoxin treated with trypsin and then with mosquito gut proteases) bound to the dipteran cells but not to lepidopteran or fruitfly cells. The lepidopteran-specific preparation (trypsintreated protoxin) bound to lepidopteran cells but not to dipteran cells. The toxicity of trypsin-activated delta-endotoxin was abolished by preincubation with D-glucose and reduced by osmotic protectants, indicating binding of toxin with a unique receptor determined toxin specificity. Cell death caused by colloid osmotic lysis then followed (Haider and Ellar, 1987). N-acetylglucosamine-containing oligosaccharides on the delta endotoxin of B. thuringiensis were required for its toxicity toward A. aegypti larvae but not toward Culex quinquefasciatus larvae (Diptera: Culicidae) (Pfannenstiel et al., 1990). The sugar N-acetylgalactosamine diminished the binding of the delta-endotoxin to brush-border membrane vesicles of Manduca sexta (Lepidoptera: Sphingidae) and Heliothis virescens (Lepidoptera: Noctuidae) but not to those of Pieris brassicae (Lepidoptera: Pieridae), indicating that N-acetylgalactosamine might be a component of the toxin receptor (Knowles et al., 1991). The receptor for the endotoxin in the brush-border membrane of M. sexta was the metalloprotease aminopeptidase N (Knight et al., 2004). The O-glycosylated N-acetylgalactosamine-rich C-terminal stalk region of aminopeptidase N was the most likely binding site for the toxin (Knight et al., 2004). A receptor for the delta-endotoxin was identified in the midgut of Bombyx mori (Lepidoptera: Bombycidae). However, binding proteins for the toxin were also identified in gut membrane of T. molitor larvae (Coleoptera: Tenebrionidae), a coleopteran not sensitive to the toxin. This indicates that the specificity of the toxin was not only due to existence of the binding protein (Nagamatsu et al., 1998). A 170-kDa endotoxin-binding aminopeptidase from the tobacco budworm H. virescens was partially purified and its cDNA cloned. Another glycoprotein, 130 kDa in molecular mass and possessing N-terminal sequence and immunological characteristics identical to the 170-kDa aminopeptidase, was also isolated. The 130kDa protein had a higher binding affinity. The results indicated post-translational modification that affected toxin interaction with specific insect midgut proteins (Oltean et al., 1999). Jenkins et al. (2000) showed that the delta-endotoxin bound to the aminopeptidase N receptor in gypsy moth (Lymantria dispar) (Lepidoptera: Lymantriidae). Banks et al. (2001) demonstrated that the 110-kDa aminopeptidase in H. virescens was distinctive in that it, unlike other endotoxin-binding aminopeptidases, did not contain N-acetylgalactosamine. Burton et al. (1999) showed that B. thuringiensis toxin recognizes N-acetylgalactosamine on the putative insect receptor aminopeptidase N. A possible structural basis for the participation of domain III of the toxin in carbohydrate-mediated receptor recognition lies in the similarity between the domain III fold of a related toxin and a carbohydrated-binding domain in Cellulomonas fimi glucanase. Mutagenesis of residues N506, Q509, or Y513 leads to decreased binding and reduced pore


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formation in M. sexta midgut membrane vesicles, and diminished binding to the putative receptor aminopeptidase N. Transgenic tobacco plants simultaneously expressing two kinds of insect-resistant genes, the delta-endotoxin and snowdrop lectin, demonstrated resistance to the peach aphid M. persicae (Hemiptera: Aphiidae) and the cotton bollworm Heliothis armigera (Lepidoptera: Noctuidae) Hubner (Zhao et al., 2001). Fan et al. (1999) demonstrated that transgenic tobacco plants expressing both B. thuringiensis deltaendotoxin and cowpea trypsin inhibitor had higher insecticidal efficacy against the cotton bollworm Helieoverpa armigera (Lepidoptera: Noctuidae) than transgenic tobacco plants expressing the delta-endotoxin gene only. Similarly, Wei et al. (2000) reported that transgenic rice plants expressing both delta endotoxin and soybean trypsin inhibitor were more resistant to the leaf folder Cnaphalocrosis medinalis (Lepidoptera: Pyralidae) than transgenic rice plants expressing only soybean trypsin inhibitor. Synergism of insecticidal activities of delta-endotoxin and soybean trypsin inhibitor was also noted in H. armigera larvae (Zhang et al., 2000). Crude proteinase inhibitor extracts of black gram chickpea, chickling vetch, French bean pea, and soybean, but not those of kidney bean, horse gram, and finger millet, potentiated the insecticidal activity of B. thuringiensis subsp. kurstaki HD-1 on H. armigera larvae (Gujar et al., 2004).

Peptide toxins from venom of the Chinese bird spider Selenocosmia huwena Wang (also known as Ornithoctonus huwena Wang) (Theraphosidae) The majority of these peptide toxins, designated as huwentoxins (HWTX), had 30–40 amino acids and 3 S–S bonds. With the exception of HWTX-II that adopted a novel scaffold, most of them adopted the ‘‘inhibitor cystine-knot’’ motif. The HWTX exhibited a variety of activities that included insecticidal activity, inhibition of voltage-gated sodium and calcium channels, trypsin inhibition, and lectin-like agglutination (Liang, 2004).

Insect-sensitive scorpion toxins Toxin from the venom of Leiurus quinquestriatus guinquestriatus (Buthidae) induced flaccid paralysis in insects but lacked toxicity in mice. Its primary structure, composed of 64 amino acids and 4 S–S bridges, has been determined (Kopeyan et al., 1990; Landon et al., 1996). The toxin was engineered into the Autographa californica Nuclear Polyhedrosis Virus genome. Larvae of Spodoptera littoralis and H. armigera injected with the recombination budded virus exhibited characteristic intoxication symptoms (Chejanovsky et al., 1995). Aromatic and non-polar amino acids were found in patches on the surface of the toxin, alternating with patches of charged, non-polar residues. This topology facilitated the interactions of the toxin with membrane sodium channels. Two weakly constrained loops conferred flexibility to the structure that could be related to the activity of the toxin. Cysteine-stabilized alpha beta motif occurred in the central core, in common with defensins and thionins

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(Landon et al., 1996). Its solution structure was similar to those of other scorpion toxins (Landon et al., 1997). Another toxin Lqh III has been isolated from the scorpion Leiurus quinquestriatus hebraeus. It inhibited sodium current inactivation in the cockroach axon but induced in addition a resting depolarization due to a slowly decaying tail current atypical of the action of other toxins (Krimm et al., 1999). A toxin Lqh beta l from L. quinquestriatus hebraeus recognized both insect and mammalian sodium channels (Gordon et al., 2003). Another neurotoxin, AaH IT4, was isolated from the venom of the North African scorpion Androctonus australis Hector. It had 65 amino acids and was deficient in proline that played a role in the folded structure of other scorpion neurotoxins. Its amino acid sequence did not resemble those of other scorpion toxins. It competed with anti-insect scorpion toxins for binding to the insect sodium channel, modulated the binding of anti-mammal scorpion toxins to the mammalian sodium channel, and was toxic to insects and mammals (Loret et al., 1991). Sequence-specific nuclear magnetic resonance assignments for the polypeptide backbone and for most of the amino acid side-chain protons, and the general folding of the neurotoxin, have been described (Darbon et al., 1991). A fraction (fraction II) of the venom of the scorpion Buthus martensii (Karsch) contained anti-insect toxins (Bauer et al., 1992). Two 66-amino acid peptides, designated as BmK AS and BmK AS-1, have been purified from the venom. They exhibited 86% sequence identity to each other and marked sequence homology to AaH IT4 (Ji et al., 1999). The crystal structure of an anti-insect toxin from the scorpion Buthotus judaicus, Bj-xtrIT, has been determined. Substitution of the last seven amino acids with a single glycine residue abolished its activity, indicating the involvement of the last seven residues in interaction with the sodium channel (Oren et al., 1998). Toxin TbIT-1 from the South American scorpion Tityus bahiensis was lethal to houseflies but virtually inactive in vertebrates. Another toxin Tb2-II from the same scorpion was lethal to both houseflies and mice (Pimenta et al., 2001). A neurotoxic protein CsE-v5 was purified from the venom of the scorpion Centruroides sculpturatus Ewing. It was highly specific for insect sodium channels, unlike CsE-V that had both anti-insect and anti-mammal activities. The differences in primary structure and electrostatic potential surface around areas with residues 8, 9, 17, 18, 32, 43, and 57 might account for the differences in activity between CsE-v5 and CsEV (Jablonsky et al., 2001). Two 70-amino acid peptides with 4 S–S bridges have been isolated and sequenced from the venom of the scorpion Isometrus vittatus. Their electrophysiological actions were similar to other anti-insect scorpions (Coronas et al., 2003). Anti-insect scorpion toxins include alpha toxins without strict selectivity for insects, excitatory insect-selective toxins that produce immediate fast paralysis in blowfly larvae, and depressant insect-selective toxins that induce slow progressive flaccid paralysis in larvae (Pelhate et al., 1998). The higher insecticidal efficacy of the baculovirus expressing the depressant toxin relative to the excitatory potency suggests that pharmacokinetic factors and/or promoter efficiency may play a role during infection of insect pest larvae by recombinant baculoviruses (Gershburg et al., 1998). A dense core of secondary structure, 2½ turns of alpha-helix, and a short segment


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of anti-parallel b-sheet exist in all known structures of scorpion toxins, regardless of their size, sequence, and function (Legros and Martin-Eauclaire, 1997). Gordon et al. (1996) suggested the existence of a cluster of receptor sites for scorpion toxins inhibiting sodium current inactivation that was very much alike on insect and rat brain sodium channels despite their structural and pharmacological differences.

Conclusion and future perspectives It can be seen from the foregoing account that a variety of structurally different proteins display anti-insect activity. They include thiol methyltransferses, lectins, arcelins, ureases, avidin, chitinases, a-amylase inhibitors, protease inhibitors, B. thuringiensis toxin, spider toxins, and scorpion toxins. They have different entomotoxic mechanisms. Lectins of fungal, monocot, legume, and other dicot origins have been shown to possess insecticidal activity. In some lectins it has been demonstrated that carbohydrate binding and resistance to proteases in the insect gut are important to their anti-insect activity. Some arcelins, which are lectin-like proteins, may also exhibit protease resistance and anti-insect activities. Some ribosome inactivating proteins, which manifest toxic effects on tumor cells and arrest protein synthesis by virtue of their N-glycosidase activity, are also insecticidal. Some chitinases may exert their anti-insect activity by their enzymatic activity on chitin that is a constituent of the insect cuticle. aAIs from monocots and dicots, some being monomeric and others being dimeric, have been reported to have insecticidal activity. Some inhibit insect but not mammalian a-amylase, due to structural differences between the enzymes. aAIs and protease inhibitors including cystatins and serine protease inhibitors produce their toxic actions on insects by inhibiting carbohydrate and protein digestion, respectively. The glycoprotein toxin from B. thuringiensis exerts its anti-insect activity by binding to a lectin receptor in the insect gut. N-acetylgalactosamine is probably a component of the toxin receptor that is the metalloprotease aminopeptidase. Neurotoxic peptides from venoms of different species of scorpions inhibit sodium current inactivation in insect axons. Transgenic plants expressing one or two of the genes of the following insecticidal proteins: protease inhibitors, a-amylase inhibitors, lectins, arcelins, B. thuringiensis delta-endotoxin, spider toxins, and scorpion toxins, usually acquire a stronger resistance against predatory insects. A comparative investigation of neurotoxin receptor sites on mammalian and invertebrate sodium channels may reveal new targets and ways to develop selective pesticides (Gordon, 1997). The safety of these transgenic plants for human consumption is a cause of concern (Carlini and Grossi-de-Sa, 2002) although the use of cooking that denatures the anti-insect proteins should help dispel some of the skepticism. However, the problem remains with livestock ingesting feeds containing these transgenic plants. More research should be conducted to check the safety of these transgenic products to human and livestock.

Acknowledgments We thank Miss Fion Yung for excellent secretarial assistance.

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CHAPTER 17

Antifungal natural products: assays and applications DORIS ENGELMEIER, FRANZ HADACEK

Introduction Natural products or plant secondary metabolites comprise low-molecular-weight compounds that are regarded as dispensable for sustaining life, but as indispensable for the survival of the producing organism (Hartmann, 1996; Hadacek, 2002). For ages, plants have provided mankind with medicines and food-conserving additives. Still, plant- or microbial-derived compounds are regarded as a substantial source for novel lead structures to develop medicines and biocides. Recently, especially huge expectations were directed to those from hitherto uncharacterized microorganisms that live as endophytes in plants or occur in deep-sea habitats (Clardy and Walsh, 2004). Furthermore, the application of combinatorial chemistry in generating structural diversity has somehow affected the significance of natural products in biological activity screening. However, especially concerning the identification of novel mode of actions, natural products are still regarded as a valuable pool for lead structures. Further, for the release of novel antimycotic drugs and fungicides to the market, costs have increased dramatically during the last decades due to additionally imposed conditions to elucidate mode of actions and side effects; an increasingly chemistry-critical public has also fuelled this process. As a result, cost estimates for the development of a new drug or biocide as well as the formulation of their application amount to around 150 millions US Dollar today. These facts may somehow impose a constraint on the development, all the more as some representatives from the industry regard the quality of existing biocides more or less as sufficient to combat the recognized pest organisms (Stetter and Lieb, 2000). Contrary to this view, other authors voice increasing concern regarding the rapid emergence of resistance phenomena in pathogens toward specific applied fungicides (Knight et al., 1997; Henningsen, 2003). In antimycotic therapy, resistance also constitutes a recognized problem (Baddly and Moser, 2004). The tremendous pathotype diversity of some pathogenic fungi may additionally complicate the development of efficient control mechanisms as, e.g., shown for the rice blast fungus Pyricularia ( ¼ teleomorph Magnaporthe) grisea (Kareiva, 1999). Further, a broad


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successful introduction of transgenic disease resistant crop cultivars is not expected to take place before 2015 (Henningsen, 2003). In a concomitant development of increased alertness toward the application of synthetic chemicals in agricultural practice, the implementation of integrated pest management programs has increased (Yang and del Rio, 2002). This approach generally favors fungicides that affect non-target mechanisms less than their synthetic analogues, and here natural products certainly constitute potential candidates. Consequently, there still exists interest in screening low-molecular-weight compounds for antifungal activities within various research approaches that reflect themselves in the choice of assays. So far, key areas for the application of antifungal bioassays include control of crop pathogens in phytopathology and human pathogenic fungi in antimycotic chemotherapy. Further, the authors opine that resource utilization and susceptibility to secondary metabolites will attract more attention in future to address basic ecological questions, such as the functioning and structuring of plant communities. In this context, the impact of microbial symbionts and pathogens will attract more attention as was hitherto been paid to. Antifungal modes of actions have been primarily elucidated for antimycotic drugs. The available antimycotic drugs show various modes of actions and, as a result of the dire consequences, resistance phenomena are much more attended to than in previous times, for a review see Baddly and Moser (2004). Polyenes, such as amphotericin B (1 in Figure 1), are in use since the 1950s and bind to ergosterol units of the fungal cell membrane (Figure 1). Changes in the sterol content decrease binding of the drug and contribute to resistance. Further, an alteration in cell wall 1,3-b-D-glucan restricts the ability of this drug to reach its target site.

OH OH OH

O

HO HOOC HO

OH OH O

O

OH O

O

HO

1

OH NH2 N O

O

OH N N

Cl

O Cl

N O

N

F

F

N N N

N O

N

2

3

Fig. 1. Antimycotics active at the cytoplasmic membrane. 1, amphotericin B; 2, the azole ketoconazole; 3, the triazole fluconazole.

Antifungal natural products: assays and applications

425

Azoles constitute less toxic and effective alternatives to the former antimycotic. These compounds do not react with but inhibit the biosynthesis of ergosterol. Ketoconazole (2 in Figure 1) contains two nitrogen atoms in the five-membered azole ring whereas fluconazole (3 in Figure 1) contains three. Resistance to the azoles may be caused by several mechanisms: (1) enhanced efflux by up-regulation of multidrug efflux reporter genes (ABC transporter genes); (2) amino acid substitutions in the 14-a-demethylase (catalyzing the demethylation of 24-methylendihydrolanosterin); (3) up-regulation of the 14-a-demethylase gene ERG11; or (4) alterations in the ergosterol biosynthetic pathway (Figure 2). Echinocandins are semi-synthetic lipopeptide antimycotics developed from naturally occurring polypeptides that were originally isolated from Aspergillus nidulans var. echinulatus (Nyfeler and Keller-Schierlein, 1974). They act on the fungal cell wall by inhibiting the synthesis of 1,3-b-D-glucan, which leads to osmotic instability and finally to lysis of the cell wall. Caspofungin is the only commercially available derivative (Figure 3). Resistance can result from mutations of FKS1 and FKS2, genes that encode subunits of the 1,3-b-D-glucan synthase. The mode of action of flucytosine (1 in Figure 4) is based on antimetabolite properties. After uptake by fungi, it is converted by intercellular deamination into 5fluorouracil (2 in Figure 4). The converted 5-fluorouridine triphosphate (3 in Figure 4) incorporates into fungal RNA and inhibits protein synthesis. 5-Fluorouracil is also converted into fluorodeoyxuridine monophosphate (4 in Figure 4), which interferes with DNA synthesis by inhibiting thymidylate synthetase. Various mechanisms of resistance exist: (1) mutation of the enzymes resulting in decreased uptake or conversion of the drug; (2) loss of activity of uracil phosphoribosyltransferase; and (3) increased synthesis of pyrimidines that compete with the fluorinated antimicrobials (Figure 3). However, not the whole world population benefits from antimycotic drugs. Especially in developing countries, people rely and still have to rely on traditional medicines from plant sources. This fact, the emerging resistance phenomena, the increased occurrence of fungal strains with multiple antibiotic resistance, and new emerging fungal diseases still fuel interest in screening studies for antimycotic natural products (Ficker et al., 2004). Consequently, plants from countries, such as India (Vonshak et al., 2003), Latin America (Freixa et al., 1998), and Africa (Cos et al., 2002), or even Canada (Jones et al., 2000), are still under investigation, in most cases guided by an ethnopharmacological background.

Fungicides in agriculture A compendium of pesticide common names can be found under the following hyperlink: http://www.hclrss.demon.co.uk/class_fungicides.html. Anderson et al. (2004) have listed various emerging fungal diseases in agricultural ecosystems: Phytophthora infestans, potato blight in south America; Pyricularia grisea, rice blast in all rice-producing areas including USA; Tilletia indica, carnal bunt on gramineous crop plants originally in India, in the last decade in South Africa and USA; and Puccinia kuehnli, sugar cane orange rust which reduced sugar cane production in Australia by 25%. Compared to our knowledge of crop plant emerging infectious


426

X

A HO

HO

HO 3

2

1

X

B

X

C

HO

HO

HO

6

B

5

4

X

HO

HO

HO 9

8

7

A

B

C

N N

Cl

N Cl

O O O N F

HO

H N O

Cl epiconazole

spiroxamines

fenhexamide

Fig. 2. Steroid biosynthesis and exemplary structures of inhibitors (A, B, and C); inhibited reactions are marked with X. 1, lanosterol; 2, 2,4-methylendihydrolanosterin; 3, 4,4-dimethylergosta-8,14,24-triene-3b-ol; 4, 4,4-dimethylzymosterol; 5, 4-methylzymosterol; 6, zymosterol; 7, fecosterol; 8, episteriol; 9, ergosterol.

diseases, we know definitely less about those of wild plants. Examples of studied cases include Ophiostoma ulmi, the Dutch elm disease, Cryphonectria parasitica, the chestnut blight in America, Phytophthora cinnamoni, an emerging root rot disease in Proteaceae, Fabaceae, Mimosaceae, and Epacridaceae in Australia, Pestalotiopsis microspora, Floradia torreya mycosis, Discula destructiva, dogwood anthracnose on


427

H 2N NH

OH

O

O

HO

NH

NH O

N

H 2N

HN

O HO

O

NH H N

O

OH

N OH

O

HO

OH

OH

Fig. 3. Caspofungin, an echinocandin polypeptide antimycotic acting on fungal cell walls.

H N

O N

NH2

O

H N

HN

F

F O

1

2

H N

O

O HO P HO O

O

N

F O

OH

HO

3

H N

O

O HO

O HO P O P O P O O HO OH

O

N

F O

OH

HO

4

Fig. 4. Mode of action of flucytosine (1): uptake and desamination to 5-fluorouracil (2), conversion to fluorouridine triphosphate (3) that inhibits protein synthesis and to fluorodeoxyuridine monophosphate (4) that inhibits DNA synthesis.

Cornus florida in USA, and Phytophthora ramorum, sudden oak death syndrome in England and Poland. Fungicides do not exclusively target true fungi. Although Phytophthora is classified as a fungus in many instances, it is not treated as a true fungus by modern classifications systems. True fungi are characterized by a number of specific traits, many of which they have in common with insects, with whom they are classified as

428


ophistokonts, whereas oomycetes belong to the heterokonts including diatoms and brown algae and share more characters with plants (Table 1). However, both true fungi and oomycetes produce highly similar disease symptoms. Their pathogenicity is often caused or tremendously enhanced by the production of huge numbers of asexual conidia, another reason for their often-to-be observed joint treatment. As many classic fungicides target the biosynthesis of ergosterol, they fail to affect oomycete fungal pathogens because their cell walls are made of cellulose, such as those of higher plants (Deacon, 1997). One of the first pathogenic organisms that caused substantial effects on human crops was potato blight, Phytophthora infestans. Potatoes were introduced to Europe during the 16th century. About 300 years later, in 1845, the first severe outbreak of potato blight in Ireland caused severe famine in the following years and induced many people to leave the country forever. This incident is often used as marketing argument for the application of fungicides by the agrochemical industry. Large (1940) published a most recommendable survey on the development of phytopathology as a scientific discipline and recapitulates how we became aware of many fungal pathogens and devised control mechanisms, the development of which was not always as straightforward as it seems today. According to up-to-date estimates, about 10–20% of today’s production of staple foods and cash crops are destroyed by plant pathogens, always depending on the crop and the region. Despite the existence of disease-tolerant cultivars, crop rotation, and sanitation practices, the use of fungicides is still regarded as indispensable to maximize yields (Knight et al., 1997; Hewitt, 2000; Henningsen, 2003). In attempts to lower biocide input, attention has shifted from discovering novel fungicides with known mode of actions to the identification of novel modes of fungicidal activities. In this context, we have also to consider the meaning of the term ‘‘fungicide.’’ The designation of fungicide to a specific compound defines the ability of this compound to kill fungal hyphae or its propagules. Usually, most so-called antifungal compounds are fungistatic, i.e., they inhibit or delay conidia and spore germination or hyphal growth to a certain extent. Even if some compounds can be identified as fungicides in in vitro and in vivo glasshouse experiments, the thus determined effects may be totally different when the candidate fungicide is actually applied in the field. The more complex the environment of the application becomes, the more difficult it is to predict the actual nature and strength of a biocide’s efficiency. And nature is very complex. In order to become a pathogen, conidia or spores have to germinate on leaf or root surfaces. Especially on leaves, in most cases the development of specific infection structures, so-called appressoria, is required that allow the fungus to establish itself in the leaf’s tissues (Dean, 1997). Once this is accomplished, it starts attempting to penetrate cell walls. Plant cells contain specific receptors to recognize cell wall fragments caused by fungal invasions, glucans from oomycetes, chitosans from true fungi, polygalacturonids from their own cell walls, or extracellular fungal proteins. Efficient recognition of these so-called ellicitors triggers a signal cascade initiating the hypersensitive reaction process (HR) that includes the expression of pathogenesis-related proteins (PR-proteins) and genes for phytoalexin biosynthesis, and ultimately may lead to local cell death in the affected tissue (Knogge, 1996; Baker et al., 1997; Morel and Dangl, 1997; Dixon, 2001; Lam et al., 2001).

True fungi

Animals

Oomycota

Plants

Growth habit

Hyphal, tip growth

Not hyphal

Hyphal, tip growth

Not hyphal

Nutrition

Heterotrophic, absorptive

Heterotrophic, ingestive

Heterotrophic, absorptive

Autotrophic

Cell wall

Chitin

Chitin in exoskeleton cellulose

Cellulose

Cellulose

Nuclei

Haploid, membrane persists during division; spindle pole bodies do not have a centriolar arrangement

Typically diploid; typical centrioles

Diploid; typical centrioles

Typically diploid; typical centrioles

Microtubules

Sensitive to benzimidazoles and griseofulvin

Sensitive to colchicine



Golgi cisternae

Unstacked, tubular

Stacked

Stacked

Stacked

Mitochondria

Plate- or disc-like cisternae

Plate- or disc-like cisternae

Tubular

Tubular

Lysine

Synthesized by aaminoadipic acid pathway

Not synthesized

Synthesized by diaminopimelic acid pathway

Synthesized by diaminopimelic acid pathway

Translocable carbohydrates

Polyols, trehalose

Trehalose in insects

Glucose, sucrose, etc.

Glucose, sucrose, etc.

Storage compounds

Glycogen, lipids, trehalose

Glycogen, lipids, trehalose in some

Mycolaminarin

Starch, lipids, sucrose

Mitochondrial UGA codon usage

Tryptophan

Tryptophan

Chain termination

Chain termination

Sterols

Ergosterol

Cholesterol

Sitosterol

Sitosterol

Taxonomy

Ophistokonts

Ophistokonts

Heterokonts

Plants

429

Character


Table 1 Some major characteristics of the chitin-walled (true) fungi compared to animals, oomycetes, and plants, after Deacon (1997), modified


430

Pathogenic fungi may show manifold mechanisms of disease development (Agrios, 1997), and thus the successful use of a fungicide usually also requires the dissemination of its correct application procedure. The development of germ tubes from conidia and spores represents on the most sensitive developmental stages in the life of a pathogenic fungus and thus offers itself as target for a preventive fungicide. Once the fungus has entered the plant’s tissues, a curative systemic fungicide is required. However, the increased insights into plant resistance mechanisms, the hypersensitive response (see previous paragraph), has stimulated the exploitation of respective signal molecules, such as salicylic and jasmonic acid and their mimics, as inductors of plant resistance in disease control (Feys and Parker, 2000; Hewitt, 2000; Conrath et al., 2001; Terry and Joyce, 2004). Consequently, recent formulations of fungicides comprise various compounds with preventive, curative, or resistance-eliciting and potentiating effects (e.g., Labourdette and Latorse, 2003). The quest to discover novel modes of actions has significantly spurred and is still the major driving force in the search for natural, semi-natural, and synthetic compounds (Lyr, 1995; Hewitt, 2000; Henningsen, 2003). One of the classical modes of actions of fungicides that affect true fungi is the inhibition of the steroid biosynthesis leading to ergosterol, an important building block of the fungal cell wall, such as shown by diazoles and triazoles, spiroxamines and morpholins (Figure 2). As a matter of fact, numerous cases of resistance are known in the literature. Strobilurins and oudemansins (Figure 5) have been discovered in culture filtrates of basidiomycetes to colonize dead wood and produce these antifungal compounds to gain advantages toward other fungi competing for the same nutrient source. These

Lead structures OCH3

OCH3

O

OCH3

O O

O

1

2

Stabilization with aromatic rings N

N

O N

O

OCH3

O

CN

O

O 3

OCH3

O

4

Fig. 5. Strobilurin and oudemansin-based fungicides inhibit enzymes of the respiratory chain. 1, Strobilurin A; 2, oudemansin A; 3, kresoxym-methyl; 4, azoxystrobin.


431

compounds inhibit mitochondria by blocking an ubichinone receptor in the respiratory chain, an hitherto unknown mechanism of inhibition (Kraiczy et al., 1996). As a consequence, these compounds inhibit both oomycetes and true chitin fungi and thus offered themselves as promising lead structures for broadband fungicides; today, azoxystrobin is commercialized by Zeneca and kresoxim-methyl by BASF, both being less photolabile than the original natural products (Figure 5; Sauter et al., 1999; Henningsen, 2003). However, resistant strains surfaced within Erysiphe graminis (Chin et al., 2001; Hollomon, 2001) and Podosphaera fusca as well as Pseudoperonospora cubensis (Ishii et al., 2001). Monsanto has introduced a fungicide, with silthiopham as active compound (Figure 6), against the take-all fungus, Gaeumannomyces graminis, which specifically inhibits ATP transporter molecules. In case of the rice blast fungus, Pyricularia grisea, various compounds were found that inhibit the melanin biosynthesis that is required for the development of efficient appressoria facilitating the penetration of the leaf epidermis by the hyphal tip (Figure 7). Flumorph, dimethomorph, iprovalicarb, and benthiavalicarb also inhibit cell wall biosynthesis of various oomycetes and true fungi; however their mode of action has not been elucidated yet (Figure 8). Though the introduction of these fungicides occurred only recently, tolerance was already noted; Phytophthora capsici to flumorph (Huang et al., 2004), Phytophthora infestans to dimethomorph (Stein and Kirk, 2003), and Plasmopara viticola as well as Phytophthora infestans to iprovalicarb (Suty and Stenzel, 1999; Kast, 2004). Other fungicides with a rather broad activity against true fungi and oomycetes include various inhibitors of mitosis, such as zoxamides (Figure 9); quinoxifens affects the G proteins of the fungal hyphae, which are essential for successful signal transduction (Figure 9). After treatment, the hyphae of Plasmopara viticola failed to penetrate the leaf epidermis of vine. However, recently resistant strains have surfaced (Anonymous, 2004). Despite of the preponderance of semi-synthetic and synthetic compounds in the control of postharvest diseases, natural products are still viewed as potential alternatives. The application of volatile compounds, such as monoterpenes and glucosinolates, as fumes may gain importance in the future. Besides, the application of resistance-inducing elicitors, such as plant and fungal cell wall components, and signal compounds, such as salicylic and jasmonic acid, also shows potential. As a consequence, consumers will be less subjected to the cancerogenic and teratogenic properties besides the high and acute residual toxicity known for most non-volatile synthetic fungicides (Tripathi and Dubey, 2004).

S

Si H N O

silthiopham

Fig. 6. Silthiopham, an inhibitor of ATP transporter molecules.


432

Co

a

leaf epidermis

App Hy

OH OH acetate

pentaketide HO

OH 1

b OH OH

OH O

A, B, C X

HO

HO

OH

3

O

2

OH

O

OH

A, B, C melanin

X HO 5

4

A

B

C

O N H Cl

O Cl

N H

Cl Cl

capropamid

Cl

CN

diclocymet

O N H

Cl

CN

fenoxanil

Fig. 7. (a) Appressorium exemplified by the rice blast fungus Pyricularia grisea; App, appressorium; Co, conidium; Hy, hyphae. (b) Melanin biosynthesis in the appressorium and structures of inhibitors; inhibited catalytic reactions are marked with X; 1, 1,3,6,8,-tetrahydronaphthalene; 2, scylatone; 3, 1,3,8-trihydronaphthalene; 4,vermelone; 5, 1,8-dihydronaphthalene.

Antifungal molecules in biotic interactions The majority of authors consent that fungal communities are defined by bottom-up factors, i.e., by competition rather than by predators (top-down control, Wardle, 2002). This notion agrees with the presence of numerous antibiotic natural products that are produced by fungi. The strobilurins, one of the most successful discoveries of


433

F

Cl

O

O N

N O

H3CO

O

H3CO

OCH3

OCH3 2

1

O

O O

N H

N

O

N H S

O

O

F 4

3

Fig. 8. Inhibitors of fungal cell wall biosynthesis with yet unknown mode of action. 1, flumorph; 2, dimethomorph; 3, iprovalicarb; 4, benthiavalicarb. F

O Cl

Cl

Cl

O

O Cl

Cl 1

N 2

Fig. 9. Zoxamide (1) inhibits mitosis in true fungi and oomycetes; Quinoxyfen (2) affects G proteins in fungal hyphae and thus inhibits signal transduction.

antifungal natural products within the last decade, originate from a wood-decaying basidiomycete (Sauter et al., 1999). However, the production of antibiotic natural products is restricted to the true fungi, the oomycetes being less talented producers. Especially those fungi that are classified as necrotrophic pathogens rely on the phytotoxicity of their secondary metabolites (Prell and Day, 2001). This ability to produce secondary metabolites is usually lost when isolates are cultured on axenic media for longer periods. From efforts to optimize culture conditions in biofermenters we know today that fungi require nutrient shortage to produce these toxic secondary metabolites (Demain, 1996). Such situations are also caused by a competitive environment and thus these traits concur with the hypothesized bottom-up definition of their communities. Many mycotoxin-producing fungi are thought to originate from lichen-associated fungi (Lutzoni et al., 2001). In this form of symbiosis, the ability to produce toxic

434


secondary metabolites in a competitive environment may have contributed to defense against herbivore predators. Today, several fungi that colonize crop plants may often severely affect human health by production of mycotoxins, e.g., the trichothecenes from Fusarium on wheat. Mycotoxins constitute an additional reason why fungal control on crop plants is mandatory (Cardwell et al., 2001). A fungus may not only be confronted by secondary metabolites from competitors. As a plant saprophyte and pathogen it has to tolerate the constitutive defenses of a plant, i.e., diverse secondary metabolites stored in adapted tissue compartments, such as resin ducts and idioblasts. Further, during the colonization process, induced secondary metabolites, also called phytoalexins in many instances, may suppress the infection process (see Hadacek, 2002). Ecologists have attempted to develop models that try to explain species diversity and agreement exists that microbial biodiversity is closely linked to plant biodiversity. A mathematical exploration of a feedback model between plants, their competitor plants, and the microbial communities of both concludes that negative feedbacks promote the coexistence of species (Bever, 2003). In this context, the evaluation of antifungal properties of natural products may constitute an important tool to test the proposed hypotheses in exploring extant interactions of plant and fungi in a community scenario. As a consequence, bioassays determining susceptibility of fungal isolates to specific secondary metabolites may become a key technology in this field, all the more as belowground plant defense may affect spatiotemporal processes in plant communities (van der Putten, 2003). As disease outbreaks also occur in natural plant communities (Burdon, 1993), a parallel exploration of agricultural and natural ecosystems has to be carried out to develop new and optimize existing sustainable control mechanisms. In recent years, reports emerged suggesting that complex symbiotic communities, such as the fungal species composition of fungus gardens of leaf cutting ants, may be determined by antifungal compounds produced by ant-associated bacteria belonging to genera Streptomyces (Currie et al., 1999) and Burkholderia (Santos et al., 2004). These reports somehow illustrate the complexity of the issue. These examples also suggest that antifungal susceptibility testing constitutes an essential methodology in dissecting scenarios of biotic interactions. Properly set-up bioassays combined with adequate statistics will probably help to obtain better insights into complex biological phenomena. For secondary metabolites, the more or less anthropocentrically coined function of pure chemical defense metabolites will have to be revised, and, most certainly, in this process, antifungal susceptibility assays will constitute an important tool.

Assay procedures Going through the numerous publications dealing with the assessment of susceptibility effects of specific fungi to specific antifungal natural products or synthetic compounds, the interested reader becomes confronted with numerous methodologies, always depending on the specific aim of the published investigation and, of course, the type of fungus the focus is set on. Consequently, in many instances, the choice of assay constitutes the first arising difficulty. One of the most inherent


435

problems is that we have to be aware that the single methodologies do not really produce comparable results (Hadacek and Greger, 2000). Further, when assayed against the identical chemical compound, results may vary using various fungal strains of the same species and the identical isolate cultivated under different regimes in the same assay. Researchers involved in antimycotic chemotherapy are especially aware of this problem of interlaboratory result comparability (e.g., Calhoun et al., 1986; Rex et al., 1993; Cormican and Pfaller, 1996). This concern has led to the development of guidelines as how to perform antifungal assays for clinical susceptibility assays with yeasts and filamentous fungi (Pfaller et al., 1997, 2002a, 2002b; Sheehan et al., 2004), and for recommendations to use specific strains from culture collections. To all interested in performing clinical-related assays we recommend to consult these guidelines. Besides, there exist various publications that aim to aid with this issue (Rios et al., 1988; Paxton, 1991; Chand et al., 1994; Cole, 1994; Kerwin and Semon, 1999; Hadacek and Greger, 2000).

Culture of fungi in the laboratory Fungi can colonize a broad range of substrates of living and dead tissues of organisms. Their propagules can be found in the air and on surfaces of nearly everything. The variety of substrate sources for fungi is too diverse to be reviewed. In the ongoing text we will predominantly focus on filamentous fungi and, in selected cases, also on yeasts from filamentous fungi. As our research concentrates on fungi associated with plants we will only superficially treat fungi that are associated with human diseases. Fungi that attack humans have specific requirements that have to be taken into consideration. Isolates may be recovered from nails, hairs, sputum, pus, cerebrospinal fluids, blood, urine, etc. For those interested in introductory information about culture techniques media, microscopy techniques, and stains as well as specimen collection and processing, we recommend to visit the Mycology Online homepage (http://www.mycology.adelaide.edu.au/). However, many principles of what we state is also valid for those fungi. Further, the experimental procedures are much more standardized in this area of antifungal activity research and we recommend consulting the published NCCLS guidelines (Pfaller et al., 1997, 2002a, 2002b; Sheehan et al., 2004). One fundamental requirement in obtaining and maintaining a monoxenic culture of a fungus is to exclude bacteria. For this purpose, a broad range of antibiotics is available that may be included into the culture medium of the Petri dishes. As the media have to be autoclaved, the thermal stability of the antibiotic has to be considered. In case of thermally labile additives, a sterile filtered solution of the respective compound may also be added after autoclaving. Other annoying contaminants are mites that may be introduced with samples or with impure cultures obtained from elsewhere (Figure 10). In this case, all strains have to be destroyed and further culture is only possible on agar media with insecticides incorporated; Bills (1996) recommends dieldrin that has no side effects on fungal growth and, furthermore, does not decompose during autoclaving. We have also observed that tobacco taken from cigarettes serves as an efficient repellent to mites when deposited at the place of storage.


436

Fig. 10. Mite in a fungal Petri dish culture (140 ).

Virtually, there exists no limitation in modifying the media composition. The largest biodiversity of fungi occurs in soil, and the majority of fungi is considered as unculturable (Bridge and Spooner, 2001). In attempts to find the appropriate recipe, we have to consider that structures of fungal communities are usually regulated by bottom-up effects, i.e., by the quality of resource. As a conclusion, to successfully establish a culture of a particular fungal strain, a specific set of available nutrients that more or less resembles those present in its natural environment has to be offered. These circumstances reflect themselves in numerous recipes for selective media for phytopathological and endophytic fungi from plants (e.g., Singleton et al., 1992; Dhingra and Sinclair, 1995; Bills, 1996). As natural carbon sources usually contain mixtures of carbohydrates, they have to be also offered in respective artificial media. Figure 11 illustrates the extent how plant root carbohydrate mixtures may stimulate the growth of rhizosphere microfungi compared to offered single sugars. Obtaining conidia or spores With the exception of biotrophic plant pathogenic fungi, either conidia or spores may be obtained from quite a large number of fungi grown in axenic cultures, in Petri dishes, within manageable efforts. In some instances, however, certain isolates may be reluctant to conidiate or sporulate. The literature is full with various recommendations to overcome this problem. However, we opine that modification of the culture medium yields the best results anyhow. Conidia are directly produced on conidiophores, and it is comparatively simple to collect them by overlaying the Petri dish culture by 0.9% aqueous NaCl with an addition of 5% DMSO (to reduce

Antifungal natural products: assays and applications Penicillium citrinum

Doratomyces stemonitis

Relative growth to control (%)

437

100

Cylindrocarpum destructans

Glucose

90 80 70 60 50 40 30 20 10 0 5000

2500

1250

625

313

156

78

39

20

10


Conc (µg/mL)

Sucrose

100 90 80 70 60 50 40 30 20 10 0 5000

2500

1250

625

313

156

78

39

20

10

39

20

10


Conc (µg/mL)

Host plant carbohydrates

100 90 80 70 60 50 40 30 20 10 0 5000

2500

1250

625

313

156

78

Conc (µg/mL)

Fig. 11. Utilization of carbohydrates by rhizosphere soil fungi of a calcareous grassland in central Europe. Doratomyces stemonitis and Penicillium citrinum occur in the rhizosphere of the umbellifer Peucedanum alsaticum and Cylindrocarpum destructans in the rhizosphere of Peucedanum cervaria. Glucose and sucrose were obtained commercially whereas the watersoluble fraction of the methanolic root extract represented the host plant carbohydrates. GC–MS analysis revealed that besides glucose and sucrose, fructose and mannitol were accumulated as major carbohydrates apart from various trace compounds (Hadacek and Kraus, 2002). Growth rates considerably vary among isolates, and thus the relative growth was determined as percentage of the maximum growth that a fungus could achieve within the assayed period in an offered substrate in the dilution series. The assay was performed in microwell plates. The duration varied until growth was sufficient for scoring.


438 Conidia attached to hyphae x 400

Filter using a sieve large enough not to retain the conida or spores

Add liquid and loose attached conidia by scraping with a Drigalski spatula, and ...

Determine CFU number and store until further use at low temperatures

Concentrate suspension by centrifugation at 3000 g

Fig. 12. Preparation of a stock suspension for the conidia or spore inoculum. Petri dish cultures containing spore- or conidia-producing mycelia are overlaid with 0.9% aqueous NaCl and scraped off with a Drigalski spatula. The suspension is filtered and centrifuged for concentration.

surface tension of the liquid) and scraping off the conidia with a sterilized Drigalski spatula (Figure 12). For storage, the suspension has to be centrifuged to concentrate the liquid. Small cryovials are ideal for storage at –201C. The quality of the propagule suspension has to be assessed and the ideal storage temperature has to be determined for each isolate. The described conditions may suffice in most instances, but in case of failure the proper set of modifications has to be found. A number of issues have to be considered: for successful storage, the suspension medium has to penetrate the propagules, and this takes time. Thus, immediate transfer to the low temperature may compromise the conservation process. It is advisable to store the vials at 41C and then to transfer them to lower temperatures. However, if the described set of condition fails altogether, you may change the regime and use an aqueous solution of sucrose (140 g/L) and peptone (10 g/L) as medium instead (Hadacek and Kraus, 2002). In some cases, storage may only be possible at temperatures above zero. The viability of the spore or conidia suspension can be determined by the method as illustrated in Figure 13. As a matter of fact, to obtain reliable CFU (colony forming units) numbers, a series of replicates has to be performed. Compared to just counting the number of spores or conidia, determining the CFU number is better suited for adjusting the right inoculum’s size, which is around 105 CFU/mL for the majority of applications in assaying fungi. In


439

8

1

Agar plate 180 µL aqueous NaCl (12 g/L)

7

2

6

3

....

100 µL spore suspension

5 ....

4

CFU = 50 n X 10 x After 2 days:

20 µL

20 µL

20 µL

....

8

20 µL

1

n = germinated conidia x = order of dilution

Agar plate

x= 1

2

3

4

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Microwell plate

5

4

7

1.5 x 10 CFU/mL

Fig. 13. Determination of colony forming units (CFU) of conidia and spore suspensions, 10fold dilutions of the suspension are performed as illustrated. Twenty microliters of each dilution are plated on the agar medium and the number of germinated propagules is assessed. The number of CFUs is calculated using the given equation.

certain instances, automation by using calibrated optical density measurements in microwell plates is also applicable (Espinel-Ingroff et al., 1991). Gehrt et al. (1995) demonstrated the species-dependent propensity of some filamentous fungi to show lower susceptibility above CFUs of 105. Similar effects were also reported for yeasts (Rex et al., 1993). The authors interpret these phenomena with the fact that an increasing amount of microbial targets may exceed the number of available molecules and, as a consequence, advance the development of comparatively more resistant genotypes among the inoculum population. However, in some situations, CFU numbers lower than 105 will have to be utilized due to various difficulties obtaining enough propagules, which are either culture-related or fungal species-related, or caused by low tendency of conidia to free themselves from the conidiophores. Conidia may be obtained from both oomycetes and true fungi, and are especially characteristic for the majority of pathogenic fungi because they facilitate a quick and efficient asexual propagation of the pathogenic genotypes. Especially in the case of phytopathogenic fungi, direct isolates from the infected plant organs are advantageous in every respect: they represent the proper pathotype and usually copiously produce conidia, definitely much more readily than the majority of isolates available from culture collections. The latter, in many cases, at least in our experience, seems

440


to have been re-inoculated too often on axenic media, and thus usually fail to compare to freshly isolated strains. Concerning biotrophic fungi, such as arbuscular mycorrhizal fungi and powdery mildews, spores and conidia may be obtained by recovering the propagules from soil (mycorrhiza, Daniels and Skipper, 1982) or culturing the fungus on sterilized plant tissue, respectively (e.g., powdery mildews). In the latter case difficulties may arise to obtain suspensions that are completely free of bacterial contaminants. If the focus is on the discovery of compounds that are to be applied in antifungal chemotherapy, culture collections such as ATCC (American Type Culture Collection; http://www.lgcpromochem.com/atcc/), BCCM (Belgium Coordinated Collections of Microorganisms; http://www.belspo.be/bccm/), CBS (Centraalbureau voor Schimmelcultures; http://www.cbs.knaw.nl/), or JCM (Japan Collection of Microorganisms; http://www.jcm.riken.go.jp/) constitute recommendable sources. Conversely, they may be also used to deposit strains for documentary purposes. What to do when there are no conidia or spores available Spores and conidia are characterized by the inherent advantage that they present a defined starting point for the development of fungal growth. Hyphae are characterized by apical tip growth (Deacon, 1997), which means that they are not growing uniformly. The antifungal assays focus on the inhibition of the germ tube that is usually most sensitive to the presence of chemicals (Guarro et al., 1997). Germ tubes are easily obtained from conidia and spores, but what to do if neither is available. This will be the case with those isolates that fail to produce spores or conidia or yeasts that propagate by budding (Figure 14). Reliable germination of the inoculum represents a fundamental requirement to obtain reproducible results. This presents no problem with conidia or spores, but affects all assays that have to be performed with developed mycelia or yeasts. The budding yeasts resemble bacteria, and growth curves have to be assessed to determine the optimal time for inoculation, which usually occurs during the acceleration phase and first stage of exponential growth phase (Figure 15). Conventionally, if filamentous fungi fail to produce conidia, agar plugs taken from the actively growing border of the colony may be used for inoculation. Here, control of the quality of the inoculum usually depends more on the care and expertise of the experimenter than on objectively assessable parameters. Recovery of strains and preservation Figure 16 illustrates a simple isolation procedure for the recovery of culturable fungi colonizing leaf tissues including surface sterilization, incubation of the source tissue in a moist chamber, and transfer of conidia formed on the colonized tissue to a Petri dish medium. Underground organs, such as stolons and roots also contain endophytic fungi, symptomless colonizers of plant tissues. Surface sterilization methods have to be adjusted to the type of plant tissue; structured surfaces have to be treated differently than smooth leaf cuticles; instead of the commonly used hydrogen peroxide, 70% ethanol and subsequent flaming proves more efficient. Sieber (2002) represents an excellent source for suggestions on how to proceed. If roots represent


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Fig. 14. Budding yeast (Taphrina sp.) (H.J. Prillinger, with permission).

Inoculation Stationary phase

Declining phase

Log cell number

Deceleration phase

Exponential phase

Acceleration phase

Lag phase 0

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6 7 Time

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(a)

(b)

2 min H20

2 min H202

2 min H20

Surface sterilization

Infected plant organs

(c)

Development in moist chamber

(d)

Transfer to agar plate

Fig. 16. Recovery of a filamentous fungus (Drechslera sp.) from a plant leaf. (a) Dark spots or lesions often indicate fungal infections on plant tissues, aerial parts, or roots; (b) to isolate the pathogen or endophyte of interest the plant tissue has to surface sterilized, e.g., with hydrogen peroxide; (c) incubation of the plant tissue in moist chambers (Petri dishes with moistened filter paper); (d) Petri dish culture with conidia.

the tissue under investigation, another fact has to be considered: plant roots usually accumulate distinctly larger amounts of secondary metabolites than aerial tissues. In attempts to isolate a fungus, the tissue has to be damaged and this action also affects the compartmented secondary metabolites. Many plant roots may contain a sticky resin or latex that quickly polymerizes into a callous layer representing an impenetrable barrier for the endophyte. In this case, covering the plant tissue with agar medium is advantageous because (1) the secondary metabolites diffuse into the agar and dilute themselves and (2) the formation of a callous crust on the surface of the plant tissue is avoided. The largest biodiversity of fungi occurs in soil. Gams and Domsch (1967) have devised a combination of sieves that is used to wash soil samples. The advantage is that fungi occurring as dormant spores or conidia can be differentiated from those that have developed a mycelium. The latter are retained by the sieves whereas the


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former can be found in the water used for the washing procedure. As interest of ecologists is increasingly focused on rhizosphere biology, this classical regime to assess the biodiversity may re-attract attention as complementary direct accession methodology to the currently favored indirect molecular methods, such as DGGE and T-RFLP (Anderson and Cairney, 2004). If special care is taken with the choice of the sieve pore sizes, the direct approach also yields rewarding results. We found a combination of sieves with a pore size of 0.8, 0.4, 0.2, and 0.05 mm as efficient. Particles retained by the 0.2 and 0.05 mm sieve were collected and transferred to agar media (Hadacek and Kraus, 2002). More likely, small particles contain only mycelial fragments of a single fungal strain. The obtained suspensions can be diluted accordingly and, upon transfer on agar media, develop into cultures containing a single strain. If the washed particles contain more than one fungus, the more vigorous strain may suppress the other, which, as a consequence, may not be recovered. The problem of antagonism reducing the diversity of strain recovery is well known and some researches even attack vigorous isolates, which emerged in the Petri dishes, with a soldering iron (Dreyfuss, 1986). Once a fungal strain is isolated, it has to be preserved with its given characteristics. Culture collections usually cryopreserve their strains at –801C, or even better at –1301C (American Type Culture Collection, 1991; Espinel-Ingroff et al., 2004); for a review of the actual standard methodologies see Smith and Onions (1994). The primary objective of culture collections is to ensure that the specific characteristics of a given strain remain stable throughout storage. However, Ryan et al. (2001) showed that secondary metabolite profiles, extracellular enzyme production, and DNA polymorphisms may be affected by non-optimal cooling and thawing regimes. If regeneration rates decline, improvements of the regime are mandatory. Insights from preserving fungal cultures do also apply to the preservation of inoculum suspensions and suggestions for its improvement of the viability may also be gleaned from published efforts to improve strain preservation. Still, the regime has to be adapted to the fungus of interest. This can be quite laborious sometimes. However, there exist alternatives to the cryopreservation, such as the filter paper technique (Dhingra and Sinclair, 1995; Fong et al., 2000). Sterile filter paper disks of 5–10 mm diameter – paper disks as used for antibiotic disk diffusion assays offer themselves – are placed into actively growing fungal cultures. After a couple of days, when they are completely covered by fungal mycelia, the disks are removed, dried, and preserved at –201C. In our laboratory, thus preserved strains could still be regenerated after 6 years. Figure 17 illustrates this technique. Preparation of microscopic slides The shape of spores, sporangia, conidia, and condiophores represent relevant characters for the proper identification of filamentous fungi. Figure 18 illustrates the ways to obtain permanent slides adapted from Kreisel and Schauer (1987). As mounting medium, LPCB (lactophenol cotton blue) or methylene blue in lactic acid meet most of the requirements. Potassium hydroxide (KOH) can be used to improve the visibility of fungi in pigmented structures. If the above-described procedures do not yield satisfactory results we recommend searching species-specific literature for suggestions. For fixation, the coverslip is usually sealed with nail polish.

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Fig. 17. Filter paper technique to preserve fungal strains, a simple regime that may work in many cases: (a) add filter paper discs to freshly inoculated fungal cultures; (b) wait until fungal hyphae penetrate the discs; (c) remove discs; (d) store them in glass vials.

The various types of antifungal assays The choice of bioassays we introduce here is biased by those techniques that are established in our laboratory, and the focus will be more on filamentous fungi colonizing living or dead plant tissues. However, many of the stated principles and exemplified techniques are also valid when focusing on fungi from other sources or yeasts. Most of the assay methodologies will be described in detail to allow even those, which are less familiar with microbiological protocols, to follow instructions. However, several issues are valid for all techniques: (1) the test organisms should grow exponentially, (2) positive controls are to be presented to facilitate more or less a comparison with published data, and (3) negative controls are to be set up to assess any affects caused by added organic solvents and surfactants. Bioautography on thin-layer plates This technique was introduced by Homans and Fuchs (1970) and is preferably carried out on thin-layer plates (TLC), but is also applicable on polyacrylamide gels (De Bolle et al., 1991). TLC has an enormous potential for separating mixtures of low-molecularweight compounds. Egon Stahl (1967), one of the pioneers in this field, reviews numerous methods to be applied for a various compound classes. However, advances in LC–DAD and LC–MS technologies have ousted TLC as analytical tool in the majority of applications. Still, TLC has merits for bioautography as it constitutes the only directly


445

Fig. 18. Permanent slide technique.

combined application of an analytical chemical method with an in situ bioassay that allows a rapid identification of the active compound or compounds in a complex mixture. Accordingly, it is very popular among those researches that are also experienced in the application of chromatography techniques, such as organic chemists and pharmacognosists. As only few journals nowadays accept reports that focus only on

446


novel structures alone, this rather easy-to-perform assay is very popular among articles dealing with natural product chemistry (e.g., Hostettmann et al., 2000). Autobiography on TLC plates facilitates the submission of a wide range of filamentous fungi to antifungal testing. Preference is given to those fungi that are characterized by pigmented hyphae, spores, or conidia. If contrast is poor, it can be enhanced by treatments with iodine vapor (Gerlach, 1977). Similarly, this visualization method is also applicable for yeasts (Hadacek, unpublished). Figure 19 illustrates the procedure. Total evaporation of the organic solvent is mandatory to obtain a mycelial layer on the surface of the plate. In some applications, the choice of solvents will have to be constrained to more volatile ones, such as diethyl ether and hexane. We successfully employ an air brush to spray the inoculum suspension onto the thin-layer plate. The suspension usually consists of malt extract broth or glucose medium with mineral salts added (Homans and Fuchs, 1970). However, the nutrient medium composition may have to be adjusted to the specific requirements of each test fungus. The application procedure of the inoculum is difficult to standardize and this may affect the comparability of results with other laboratories, besides a wide range of other factors. Figure 20 illustrates the testing of extract fractions obtained from column chromatography of a crude extract of underground organs of tarragon, Artemisia dracunculus, which contains antifungal polyacetylenes and isocoumarins (Engelmeier et al., 2004). Apart from the advantages of rapidly detecting active compounds in mixtures, the depicted bioautography also points to a potential disadvantage of this diffusion assay. The more lipophilic the compounds are, the more they show diffusion effects. These diffusion effects may significantly hamper a comparison of activities between different compounds with differing chemical properties. Another factor that may also affect results is the stability of the compound on the TLC plate as the duration of the assay may last for several days and exact quantization of the amounts of the compound that survived on the TLC plate are rarely performed due to the amount of effort required. The usually applied method of evaluation is assessing the MIC (minimum inhibitory concentration) that designates the lowest concentration in a dilution series that still yields a detectable effect. Undoubtedly, this assay has its merits for identifying active compounds in complex mixtures. Wedge and Nagle (2000) published the application of 2D-TLC as efficient approach to obtain improved separation of compounds with a concomitant gain in sensitivity of the assay. For exploring the efficacy of specific compounds in comparison to positive controls, we rate this assay as less apt due to previously discussed limitations. Diffusion assays are – despite their quick and versatile application – generally less suitable to assess the quality of the antifungal activity in comparison to positive controls. Disk diffusion This technique belongs to one of the most widely employed antifungal screening methodologies, and it is primarily used to determine if a compound or a compound mixture, such as crude extracts, possesses any activity at all. One of the major shortcomings is that, as for all diffusion assays, the concentration of the test compound or test compound mixture is unknown. The simplicity of the procedure (Cole, 1994) and


447

Prepare the appropiate diluten of the conidia suspension

Spray homogenously on the developed TLC plate, e.g. as illustrated here with an air brush, and transfer into a bioassay chamber; incubatel until fungal growth becomes visible

Bioassay chamber (25 x 25 cm)

Moistened filter paper

U-shaped glass rod

Fig. 19. Bioautography on TLC plates.

the relative good comparability to another widely employed method, broth microdilutions (Trancassini et al., 1986), have considerably contributed to its wide distribution; a guideline has even been published recently for antimicrobial chemotherapy purposes (Sheehan et al., 2004). Figure 21 illustrates this procedure affording a minimum of equipment. Antibiotic paper disks and stars that facilitate the application of the test compound or mixture (Figure 22) are commercially available. Conventionally, diameters of inhibition zones are presented to document the observed antifungal activity. In

448


Fig. 20. TLC bioautography of the fractionated lipophilic extract of underground organs of Artemisia dracunculus, the tarragon with Cladosporium herbarum as tested fungus. White zones in the layer of mycelium indicate active compounds, polyacetylenes and isocoumarins (Engelmeier et al., 2004), that inhibit the development of dark pigmented hyphae and conidia. Note that diffusion of compounds increases with lipophilicity. The TLC plate was developed in a mixture of diethyl ether and hexane (4:6, v/v).

interpreting these diameters, the fact has to be considered that variable diffusion properties of the test compound may affect the outcome, especially if results from this assay are used to compare MIC values of different compounds. There exist modifications of this method, such as the agar well diffusion, including the hole-plate (diffusion of the aqueous test compound solution into the agar medium from a vertical hole in the agar layer) and the cylinder method (stainless-steel or ceramic cylinders placed on top of the agar medium) (Rios et al., 1988). These two modifications have their merits when the test compound shows good solubility in aqueous solvents. However, as the majority of active compounds are better soluble in organic solvents, the addition of a specific portion of organic solvent to obtain an aqueous suspension of the test compound is required – in all instances, avoid adding more than 5% of organic solvent (v/v). This modification has the evident advantage that pure organic solvent can be used for the stock solution, which gets completely lost during the preparation of the disks after efficient drying. Figure 23 exemplifies the effects of an active compound, the naphtochinone juglone. Applying the compound


449

Fig. 21. Disk diffusion test.

with a pipette dissolved in a micro-volume of solvent allows the incorporation of the compound into the medium in high concentrations even in situations when test compound amounts are limited. The inhibition zones are usually distorted as this application procedure does not guarantee the test compound to be evenly distributed across the disk. However, if the solvent has not been removed properly and causes inhibition effects by itself, the zones are highly concentric to the disk. The peculiarity of this phenomenon facilitates the experienced researcher to become aware of the deficiency in his work. Microdilution For sure, diffusion assays have their merits as preliminary screening methods, but subsistence with minimal test compound amounts and concomitant provision of a wide range of concentrations together with the hydrophobic nature of the majority of the candidate compounds turns broth dilutions assays, especially those that are carried out in microwell plates, into one of the most attractive and concomitantly efficient methodologies to characterize antifungal activity of defined compounds (Wedge and Kuhajek, 1998; Kerwin and Semon, 1999; Hadacek and Greger, 2000). Recommendations of NCCLS for antifungal susceptibility testing focus also on this technique (Pflaller et al., 1997, 2002a, 2002b). Figure 24 illustrates the procedure required to test a potential antifungal hydrophobic compound – such as is the nature of the majority of the candidate compounds. In this case, the use of organic solvents and surfactants is mandatory to obtain a viable suspension for the assay. The concentration of organic solvents should not exceed 5% of

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Fig. 22. Antibiotic test disks and stars. Copyright and provided courtesy of MACHEREYNAGEL. All rights reserved.

the volume of the stock solution. Organic solvent (250 mL) is used to dissolve 2 mg of test compound; this solution is added to 4750 mL of medium – the quotient of organic solvent is 5%. Usually, to obtain a practicable emulsion a surfactant also needs to be added. The amount of the latter should be kept to a manageable minimum. The viscosity of the surfactant complicates the application of its dosage. Variable combinations of organic solvents and surfactants may cause different effects of the growth of the test fungus. In some cases, even significant inhibition effects may occur. Thus, all of these additives have to be present in the stock solutions of the control as well. During the dilution procedure, concentrations of the additives become lower, which usually improves the analysis of the results obtained from the diluted concentrations of the stock solution. Partially, the dilution attenuates this unwanted effect as it proceeds. Depending on the fungus, concentrations of organic solvents, such as DMSO, acetone, or ethanol, not exceeding 5% per well could also be useful if test compounds exhibit very low solubility in water and therefore precipitate when added to the aqueous culture medium. This phenomenon may seem to be idiosyncratic but, once its merit is recognized, it proves as helpful. However, the optimum relation of applied concentrations, combinations of organic solvents, and surfactants has to be individually determined for each test scenario.


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Fig. 23. Disk diffusion test of the naphtochinone juglone. Test fungus Cladosporium herbarum; 100 mg (upper left), 50 mg (upper right), 25 mg (lower right), control (lower left, treated with the same amount of organic solvent). Incubation for 3 days at room temperature.

Growth rates of fungi may vary depending on the strain and the quality of the medium used and the optimal point of time to evaluate the growth has to be determined for each test strain. The dilution assays represent typical quantitative bioassays, which will be discussed later in more detail. Characteristically, they allow to estimate effective concentrations (EC50, Finney, 1972; Roberts and Boyce, 1972). The inclusion of a wide range of concentrations to obtain values that range between 10% and 90% is conditional to obtain a sound estimate. Roughly, there exist three types of evaluation methodologies that are characterized by both advantages and disadvantages: in case of yeasts, turbidity of the culture broth indicates growth and this phenomenon can be easily scored with the naked eye, especially when the assay is targeted at assessing the MIC of the tested compound. This procedure is widespread in the evaluation of efficacy of various antimycotic drugs (Cormican and Pfaller, 1996). This approach is also applicable for yeasts. However, the filamentous growth of the majority of fungi prevents its broad application because their growth cannot be recognized by the naked eye unambiguously. A certain amount of magnification is required to determine if germ-tube formation has occurred or not and to what extent it differs from the control. The advent of digitized micrographs enormously contributed to increased throughput rates in determining germ-tube growth (Figure 25), similarly as microwell plates facilitated microdilutions that considerably reduced the amounts of required test compounds (Figure 24, Hadacek and Greger, 2000).

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Fig. 24. Broth dilution in microwell plates, the method of choice to perform assays with minimal amounts of test compounds in a wide range of test concentrations to facilitate an estimate of the EC50.


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For comparative analyses, one approach recommends the usage of indices that represent different degrees of growth inhibition (Kobayashi et al., 1996). The evaluation of micrographs has a distinctive advantage: morphological deformations that are caused by the test compound, such as curling (see Figure 26), swelling, hyperdivergency, and beads shape can be detected (Kobayashi et al., 1996), even in case that no significant growth reduction occurs. Although means for automation have been suggested (e.g., Hilber and Schuëpp, 1992; Oh et al., 1996), its broad application for various groups of fungi has never been established. Appressorium formation (Figure 27) constitutes another important morphological character that is characteristic for many plant pathogenic fungi infecting leaves (Dean, 1997). Thines and co-workers (Thines et al., 1997) developed an assay system that not only allowed assessing inhibitory effects of chemicals but also pointed to the affected signal pathways. The authors used either Parafilm ‘M’ (American National Can) or GelBond (Sigma-Aldrich Chemical Company, St Louis, MO)-coated slides as hydrophobic surfaces and various chemicals, among the cAMP (Sigma-Aldrich Chemical Company) and 1,16-hexadecanediol (Sigma-Aldrich Chemical Company) to induce appressorium formation on hydrophilic surfaces (microwell plates).

Fig. 25. Principle procedure in applying image analysis as tool to assess germ-tube size (magnification 140 ).

Fig. 26. Morphological deformations, such as curling (depicted here, fungus Pyricularia grisea, magnification 140 ), may be missed in microplate reader-based evaluations. Consequently, in certain instances, preference may be given to morphological analysis. (A) control; (B) curling effect.

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Fig. 27. Appressorium formation of germ tubes (magnification 140 ). Certain fungi, such as the rice blast fungus Pyricularia grisea, may develop infection structures of leaf surfaces that are decisive for phytopathogenicity. (A) control; (B) appressoria (a).

For automation, other methodologies, such as those developed for the assessment of cell proliferation, have been applied to fungi: in metabolically active cells, mitochondrial dehydrogenases reduce the soluble tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; BD Biosciences) and turns it into a blue formazan, which can be measured photometrically (Clancy and Nguyen, 1997; Adam et al., 1998); redox-based indicator dyes, such as Alamar Blue (Sigma BD Biosciences) or similar products, indicate cell growth by a colorimetric change and fluorescent signal (e.g., Espinel-Ingroff et al., 1997; Pelloux-Prayer et al., 1998). Other reagents that are turned into fluorescent products include FungiqualTM (Coleman et al., 1989) and fluorescein diacetate (Chand et al., 1994; Hadacek and Greger, 2000). In a comparative study of assessing fungal biomass by weighing the dried mycelia and turbidity around 600 nm, Broekaert et al. (1990) demonstrated the applicability of the latter method. However, for all these microwell plate-based assays the following issues should be taken into consideration: positive controls are to be included in any case to allow extrapolation of the chosen test methodology, and also to facilitate practicable comparisons between different assay methodologies. The positive controls should consist of efficient drugs and fungicides and share one trait: ready availability from a commercial source to facilitate comparison of assays systems. Further, matrix effects from the diluted test compound or mixture of compounds may affect the readings of the microwell plates. Thus, a blank of the test compound is mandatory to assess the actual strength of the inhibitory effect. Inhibition of radial growth Dilution assays can be performed in liquids or in agar medium. The main difference is that in liquids growth is submersed, and on agar media superficial. As a result, susceptibilities of the same fungus may vary considerably if tested by microdilution or radial surface growth methodologies (Hadacek and Greger, 2000). However, the latter assay may prove useful in certain questions, such as in screening fungicides to preserve surfaces. Figure 28 illustrates the procedure of this assay. As alternative to inoculations with micro-volumes of conidia or spore suspensions, plugs from the actively growing zones of Petri dish cultures may also be used. Here, the advantage lies in the fact that fungi that fail to produce conidia or spores may be used as tested


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Fig. 28. Inhibition of radial growth on media supplemented with the test compound.

organism. Conversely, the inoculation procedure may be disadvantageous in case of profusely sporulating fungi with the unpleasant consequence that emerging stray colonies may contaminate the assay. Disk diffusion and radial growth have one characteristic in common: they are easy to perform and also require minimal laboratory equipment. Thus, radial growth assays are often employed in ecological studies (more lipophilic compounds) similarly as disk diffusion assays (more hydrophilic compounds) are used in clinical studies. The inclusion of positive controls is highly recommended since this test

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system is less sensitive than others. The fungal mycelium that develops on the surface of the agar is less confronted to the test compound’s effects compared to the submersed growth in the microdilution assay (Hadacek and Greger, 2000).

Statistics In this final chapter we recommend some statistical procedures that we think to be useful for the discussed antifungal bioassays. It is limited to those assays where the concentration of the test compound in the medium is known. Accordingly, this excludes all diffusion bioassays. In this case, the report of the MIC or, in case of disk diffusion assays, the diameter of the inhibition zone is sufficient. We have to bear in mind that the development of an endpoint is specific for each compound and does not inform about when the effect starts. Consequently, this value is biased as it favors compounds with an inhibition effect that (1) is limited to few dilution steps and (2) is clearly shown. The inherent problems associated with ‘‘trailing endpoints’’ are extensively discussed by Rex et al. (1997). A good example for this is the inhibition of the rice blast fungus Pyricularia grisea (Figure 29) by the commercially available fungicide BenlateTM (50% benomyl). All assays that operate with known concentrations in the medium can be evaluated with statistical procedures recommended for a quantitative biological assay (Roberts and Boyce, 1972). In those cases where the dose–response relation is not sigmoid, no estimates are probable, and this applies more often to quantitative than to quantal assays. We chose the naphtochinone juglone and the polyacetylene falcarindiol (Figure 30) to illustrate the statistical procedures on specific examples; for juglone, the dose–response relationship follows a sigmoid curve and for falcarindiol it does not. Moreover, we compared two scoring methodologies: germ-tube size assessment on one hand and turbidity at 620 nm on the other hand. Table 2 lists the germ-tube sizes in pixels obtained while assaying juglone and falcarindiol against Botrytis cinerea. Usually, two rows were prepared for each test compound and the control. Ten representative germ tubes were selected for measurements. The means were used for calculation of the percentages of control growth for each dilution step. This procedure helps to minimize potential effects of the organic solvent and added surfactants, such as Tween 80. The inhibitory effects of the latter can be noted in the reduced growth of the control at higher dilutions. Table 3 presents the readings for juglone obtained from the microplate reader at 620 nm. A blank is measured about 1 h postinoculation after the conidia suspension has settled. Turbidity that may be caused by higher concentrations of the test compounds is thus taken into account. In our experience, the assessment of the difference in turbidity increase between 24 and 48 h yields practicable results. Both readings were previously corrected by deducting the blank. In higher concentrations of juglone, precipitation of the originally dissolved test compound occurred which affected the readings. As juglone is a highly active compound and inhibition already occurs at lower concentrations, the readings at higher concentrations can more or less be omitted. Alternatively, the dilution series can be performed in molten agar medium that adds temperature stress to, but prevents precipitation of the test compound or mixture.

Antifungal natural products: assays and applications 1008

457

BenlateTM Control

1006 1004

pixels

1002 1000 800 600 400 200 endpoint

0 100

50

25

13

6

3 µg/mL

1.5

0.8

0.4

0.2

0.1

Fig. 29. Inhibition of BenlateTM to the rice blast fungus Pyricularia grisea (microdilution in U-bottomed microplates; CFU 104, medium 4% malt extract; germ-tube size assessed after 16 h growth in darkness at 251C, magnification 120 ; pixel counts by Scion Image 4.02).

OH

O

OH O 1

OH 2

Fig. 30. Juglone (1) and falcarindiol (2) serve as examples for the statistical evaluation of microdilution assay scores by germ-tube image analysis and turbidimetry at 620 nm.

Analogously, Table 4 lists the readings for falcarindiol. Test compound and control are run in four replicates each in the identical microwell plate. Figure 31 compares the results from both assays. In both assay methodologies, the inhibitions of juglone are comparable; the test compound caused a sigmoidal dose–response relation. The 95% fiducial limits do not differ significantly, the EC50 estimates differ by 20%, but this is negligible in a two-fold dilution series. Both EC50 values fall within the fiducial limits of the other assay. Consequently, the assays can be regarded as highly reproducible and their results as comparable. Some authors report the mean estimates of several replicates and present the variation as standard variation or error. Such a procedure treats the estimates as measurements and ignores the asymmetry of the fiducial limits. These values contain valuable additional information about the accuracy of the measurement and the inherent raw data and should thus not be ignored.


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Table 2 Microdilution of juglone and falcarindiol in U-bottomed microwell plates, test fungus Botrytis cinerea, CFU 104, medium 4% malt extract, assessment of germ-tube size after 16 h growth at 251C by digitizing micrographs at 140 magnification; number of pixels determined by Scion Image 4.02 Dilution Concentration (mg/mL)

1 200 100

2 50

3 4 5 25 12.5 6.3

6 3.1

7 1.6

8 0.8

9 0.4

10 0.2

11 0.1

622 1289 1091 1007 860 721 1475 1921 907 913 1081 388 0.6 0.87

1078 852 1169 623 505 646 699 526 900 793 779 223 0.44 0.99

590 1257 706 804 607 509 525 683 495 912 709 235 0.65 0.8

835 933 806 686 435 774 818 594 1085 909 788 183 0.54 0.93

471 488 508 694 383 715 768 619 829 559 603 145 0.46 0.98

Control

Mean Standard deviation Kolmogorov-Smirnov Z Asymptotic significance Juglone

370 682 1143 803 281 663 331 546 733 409 513 1113 529 660 568 640 757 388 581 782 967 523 657 268 259 529 1064 461 345 442 491 575 735 160 166 321 0.33 0.6 0.52 1 0.87 0.95

Mean Standard deviation Kolmogorov-Smirnov Z Asymptotic significance % of control p (Mann– Whitney U-test) Falcarindiol 150 111 253 208 127 155 330 109 204 80

1517 1026 770 1020 727 765 816 413 623 512 819 313 0.64 0.8

662 1203 683 913 1024 713 712 776 598 432 1077 814 813 816 601 756 896 484 525 1020 725 871 846 1143 923 424 408 1243 749 360 785 883 653 228 217 225 0.55 0.53 0.55 0.93 0.94 0.92

-

-

-

-

-

196 339 264 95 125 92 274 173 182 338

393 218 361 274 411 621 103 127 118 184

512 558 410 160 355 285 261 216 947 414

89 99 459 511 522 185 365 573 611 235

226 693 228 339 772 440 310 705 483 644

-

173 171 210 644 904 205 317 505 774 1133 165 228 453 391 1090 208 174 768 237 720 309 268 464 421 1248 131 197 573 419 612 183 182 713 656 199 162 523 812 586 729 90 178 499 589 344 80 210 254 326 435 171 245 525 504 741 65 108 200 170 353 0.58 0.83 0.51 0.6 0.44 0.89 0.5 0.96 0.87 0.99 16 31 74 64 123 0 0 0.089 0.001 0.393

938 465 670 768 953 682 808 777 403 207 536 335 281 595 639 245 648 531 600 1183 650 866 474 554 976 514 483 311 484 736

342 728 373 563 945 581 839 670 504 802

875 480 472 863 618 380 842 609 501 366 660 640 282 358 552 1109 376 818 503 675


459

Table 2 (continued ) Dilution Concentration (mg/mL)

1 200 100

2 50

3 4 5 25 12.5 6.3

Mean 173 208 281 412 Standard deviation 77 92 166 226 Kolmogorov-Smirnov Z 0.61 0.48 0.47 0.62 Asymptotic significance 0.85 0.98 0.98 0.84 % of control 35 36 38 50 p (Mann– Whitney U-test) 0 0 0.002 0.002

365 199 0.58 0.89 46 0

484 207 0.57 0.9 55 0

6 3.1

7 1.6

8 0.8

9 0.4

10 0.2

11 0.1

600 663 568 635 568 630 310 240 131 199 188 247 0.71 0.71 0.65 0.34 0.43 0.46 0.69 0.69 0.79 1 0.99 0.99 92 61 73 90 72 104 0.853 0.007 0.052 0.631 0.035 0.912

Table 3 Microdilution of juglone in U-bottomed microwell plates, test fungus Botrytis cinerea, CFU 104, medium 4% malt extract, measurement of turbidity at 620 nm at the beginning, after 24 and 48 h Juglone Dilution Concentration (mg/mL) Blank Juglone

Control

Absorbance_24 h Juglone

Control

Absorbance_48 h Juglone

Control

(abs_48 h-blank)–(abs_24 h-blank) Juglone

200

1 100

0.14 0.17 0.19 0.17 0.04 0.05 0.04 0.04

0.15 0.15 0.08 0.07 0.04 0.04 0.05 0.05

0.27 0.31 0.19 0.21 0.23 0.24 0.23 0.23

2 50.0

3 4 5 25.0 12.5 6.3

6 3.1

7 1.6

8 0.8

9 0.4

10 0.2

11 0.1

0.08 0.08 0.08 0.07 0.04 0.05 0.04 0.04

0.06 0.06 0.06 0.06 0.05 0.05 0.05 0.04

0.06 0.06 0.06 0.06 0.04 0.04 0.04 0.04

0.06 0.06 0.05 0.06 0.05 0.05 0.05 0.05

0.06 0.06 0.05 0.06 0.05 0.05 0.05 0.05

0.05 0.05 0.05 0.06 0.04 0.05 0.05 0.05

0.05 0.05 0.05 0.05 0.05 0.05 0.04 0.04

0.05 0.06 0.05 0.06 0.05 0.05 0.05 0.05

0.05 0.06 0.05 0.06 0.05 0.05 0.05 0.05

0.06 0.06 0.05 0.06 0.05 0.05 0.05 0.05

0.22 0.29 0.20 0.19 0.21 0.22 0.22 0.23

0.26 0.22 0.22 0.25 0.21 0.20 0.21 0.21

0.11 0.11 0.10 0.12 0.19 0.19 0.19 0.20

0.10 0.09 0.09 0.11 0.17 0.17 0.17 0.19

0.10 0.09 0.08 0.09 0.17 0.17 0.17 0.18

0.08 0.07 0.07 0.08 0.17 0.17 0.17 0.17

0.07 0.06 0.07 0.07 0.14 0.16 0.15 0.16

0.07 0.09 0.11 0.11 0.15 0.15 0.15 0.15

0.13 0.13 0.16 0.17 0.13 0.13 0.13 0.14

0.18 0.18 0.20 0.22 0.11 0.12 0.12 0.14

0.21 0.20 0.21 0.21 0.12 0.06 0.07 0.13

0.35 0.46 0.37 0.36 0.62 0.66 0.73 0.75

0.26 0.41 0.32 0.31 0.58 0.58 0.56 0.65

0.26 0.20 0.21 0.23 0.56 0.58 0.53 0.58

0.12 0.11 0.10 0.12 0.48 0.46 0.45 0.50

0.10 0.10 0.10 0.11 0.46 0.42 0.52 0.56

0.10 0.09 0.09 0.10 0.43 0.60 0.58 0.54

0.09 0.08 0.08 0.09 0.41 0.41 0.42 0.56

0.08 0.07 0.11 0.09 0.41 0.45 0.41 0.56

0.17 0.23 0.32 0.28 0.39 0.43 0.42 0.50

0.32 0.29 0.34 0.36 0.39 0.44 0.44 0.59

0.45 0.38 0.45 0.42 0.44 0.42 0.44 0.50

0.59 0.50 0.51 0.53 0.47 0.47 0.47 0.55

0.08 0.16 0.18 0.15

0.04 0.00 0.01 0.00 0.13 0.01 0.00 0.00 0.12 0.01 0.00 0.00 0.12 0.02 0.00 0.00

0.00 0.00 0.00 0.01

0.01 0.01 0.01 0.01

0.01 0.01 0.04 0.02

0.10 0.14 0.21 0.17

0.20 0.17 0.17 0.19

0.27 0.20 0.25 0.20

0.38 0.30 0.31 0.32


460 Table 3 (continued ) Dilution Concentration (mg/mL) Mean Standard deviation Kolmogorov-Smirnov Z Asymptotic significance % of control p (Mann– Whitney U-test) Control

Mean Standard deviation Kolmogorov-Smirnov Z Asymptotic significance

200

1 100

0.14 0.10 0.04 0.04 0.64 0.82 0.81 0.51 29 27 0.02 0.02 0.39 0.37 0.42 0.36 0.50 0.35 0.52 0.42 0.48 0.38 0.06 0.03 0.50 0.63 0.96 0.83

2 50.0 0.00 0.01 0.50 0.96 0 0.02 0.35 0.37 0.32 0.38 0.36 0.03 0.43 0.99

3 4 5 25.0 12.5 6.3

6 3.1

7 1.6

0.00 0 0.88 0.42 1 0.02 0.28 0.27 0.26 0.29 0.28 0.01 0.30 1.00

0.01 0 3 0.01 0.25 0.24 0.25 0.40 0.30 0.08 0.85 0.46

0.02 0.16 0.18 0.23 0.32 0.01 0.05 0.01 0.03 0.04 0.52 0.26 0.60 0.60 0.67 0.95 1.00 0.87 0.86 0.77 6 52 51 71 79 0.02 0.02 0.02 0.02 0.04 0.27 0.24 0.26 0.33 0.35 0.30 0.28 0.30 0.29 0.41 0.25 0.27 0.32 0.32 0.40 0.41 0.35 0.45 0.36 0.42 0.32 0.30 0.36 0.32 0.41 0.07 0.05 0.08 0.03 0.03 0.58 0.59 0.62 0.36 0.63 0.89 0.89 0.84 0.99 0.83

0.00 0 0 0.01 0.29 0.25 0.35 0.37 0.32 0.05 0.48 0.98

0.00 0 0.88 0.42 1 0.02 0.26 0.43 0.41 0.37 0.40 0.08 0.53 0.95

8 0.8

9 0.4

10 0.2

11 0.1

Test compound precipitated as crystals that influence the readings of the microplate reader.

Falcarindiol (Figure 31) is inhibited in both assays, but the effect does not follow a sigmoidal dose–response effect. In our experience, such results are generally more the rule than the exception. The development of the visible inhibition in both methodologies is comparable, but the EC50 values differ more, and moreover the fiducial limits differ significantly. The endpoint in the turbidity scoring is much lower than in the germ-tube size assessment. One explanation might be that the inhibitory effect of the test compound reduces the number of germinating conidia, an effect that is more accurately assessed by the turbidity measurements than by the selected germ-tube measurements. In this context, one may point out that counting germinated versus non-germinated spores or conidia might facilitate an approach to solve this problem. Such a scoring procedure is possible but cumbersome in case of small size of the propagules and often complicated by incomplete germination of the control inoculum. In this aspect, turbidity certainly holds advantages compared to germ-tube size assessment. However, in the latter we can directly monitor the actual inhibition whereas in the former we have to trust the readings of the microplate reader. In specific cases one might be tempted to explore synergism in the activities of compound mixtures. Here we recommend to consult Tallarida (2000).

Conclusions The research outset and goals ultimately determine the assay to be actually used. On one hand different kinds of researchers prefer different assays, and on the other hand lacking facilities may exclude the application of some specific assays. Generally we would welcome to see the standardizations of procedures, as it is done for screenings in antifungal chemotherapy, to be extended to other fields. However, the incorporation of positive controls may help to facilitate comparison of results to some extent. Antifungal assays will continue to be applied in a wide range of basic and applied research problems, from antifungal chemotherapy to agrochemistry in applied sciences. Biotic interactions are


461

Table 4 Microdilution of falcarindiol in U-bottomed microwell plates, test fungus Botrytis cinerea, CFU 104, medium 4% malt extract, measurement of turbidity at 620 nm at the beginning, after 24 and 48 h Falcarindiol Dilution Concentration (mg/mL) Blank Falcarindiol

Control

Absorbance_24 h Falcarindiol

Control

Absorbance_48 h Falcarindiol

Control

200

1 100

0.11 0.11 0.11 0.11 0.04 0.05 0.04 0.05

0.08 0.07 0.07 0.07 0.05 0.05 0.05 0.04

0.06 0.06 0.06 0.06 0.04 0.05 0.05 0.04

0.05 0.06 0.06 0.06 0.04 0.05 0.05 0.05

0.20 0.20 0.21 0.22 0.23 0.24 0.23 0.23

0.19 0.20 0.19 0.21 0.21 0.22 0.22 0.23

0.21 0.20 0.19 0.20 0.21 0.20 0.21 0.21

0.13 0.14 0.14 0.17 0.47 0.47 0.47 0.55

0.13 0.12 0.13 0.18 0.44 0.42 0.44 0.50 0.00 0.00 0.00 0.00 0.00 0.00 0 0.014 0.23 0.2 0.22 0.27 0.228 0.03 0.52 0.95

(abs_48 h–blank)–(abs_24 h–blank) Falcarindiol 0.00 0.00 0.00 0.00 Mean 0.00 Standard deviation 0.00 Kolmogorov-Smirnov Z Asymptotic significance % of control 0 p (Mann– Whitney U-test) 0.014 Control 0.24 0.23 0.24 0.32 Mean 0.264 Standard deviation 0.04 Kolmogorov-Smirnov Z 0.76 Asymptotic significance 0.61

2 3 4 50.0 25.0 12.5

5 6.3

6 3.1

7 1.6

8 0.8

9 0.4

10 0.2

11 0.1

0.06 0.06 0.06 0.06 0.05 0.05 0.05 0.07

0.05 0.06 0.05 0.06 0.04 0.05 0.05 0.05

0.05 0.05 0.05 0.06 0.05 0.05 0.05 0.06

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.07

0.05 0.05 0.06 0.05 0.05 0.05 0.05 0.05

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

0.06 0.05 0.06 0.05 0.05 0.04 0.05 0.05

0.05 0.06 0.06 0.06 0.04 0.05 0.05 0.05

0.21 0.20 0.20 0.22 0.19 0.19 0.19 0.20

0.22 0.20 0.22 0.20 0.17 0.17 0.17 0.19

0.21 0.19 0.20 0.22 0.17 0.17 0.17 0.18

0.22 0.21 0.21 0.23 0.17 0.17 0.17 0.17

0.21 0.19 0.22 0.23 0.14 0.16 0.15 0.16

0.13 0.16 0.16 0.18 0.15 0.15 0.15 0.15

0.10 0.13 0.11 0.11 0.13 0.13 0.13 0.14

0.10 0.10 0.11 0.11 0.11 0.12 0.12 0.14

0.10 0.12 0.12 0.13 0.12 0.06 0.07 0.13

0.18 0.22 0.19 0.19 0.39 0.44 0.44 0.59

0.26 0.30 0.31 0.33 0.39 0.43 0.42 0.50

0.36 0.35 0.38 0.39 0.41 0.45 0.41 0.56

0.45 0.37 0.36 0.40 0.41 0.41 0.42 0.56

0.40 0.38 0.37 0.41 0.43 0.60 0.58 0.54

0.48 0.39 0.40 0.39 0.46 0.42 0.52 0.56

0.50 0.37 0.43 0.42 0.48 0.46 0.45 0.50

0.54 0.40 0.41 0.41 0.56 0.58 0.53 0.58

0.47 0.37 0.43 0.49 0.58 0.58 0.56 0.65

0.59 0.57 0.55 0.58 0.62 0.66 0.73 0.75

0.00 0.02 0.00 0.00 0.006 0.01 0.88 0.42 0 0.018 0.18 0.23 0.23 0.38 0.282 0.09 0.71 0.69

0.05 0.10 0.11 0.11 0.093 0.03 0.71 0.70 37 0.020 0.2 0.24 0.23 0.3 0.255 0.04 0.57 0.91

0.14 0.15 0.16 0.19 0.159 0.02 0.50 0.96 54 0.012 0.24 0.28 0.23 0.38 0.297 0.07 0.52 0.95

0.23 0.18 0.16 0.18 0.189 0.03 0.70 0.71 64 0.020 0.24 0.24 0.25 0.39 0.292 0.07 0.84 0.48

0.18 0.17 0.15 0.17 0.167 0.01 0.66 0.78 41 0.020 0.26 0.43 0.41 0.38 0.405 0.08 0.59 0.88

0.27 0.20 0.18 0.15 0.201 0.05 0.50 0.96 58 0.043 0.32 0.27 0.37 0.4 0.344 0.06 0.38 0.99

0.37 0.21 0.27 0.24 0.272 0.07 0.53 0.94 84 0.248 0.33 0.32 0.31 0.34 0.323 0.01 0.37 0.99

0.44 0.27 0.30 0.30 0.329 0.07 0.78 0.58 76 0.080 0.43 0.44 0.41 0.44 0.43 0.02 0.53 0.95

0.37 0.27 0.33 0.38 0.334 0.05 0.49 0.97 71 0.021 0.47 0.45 0.44 0.52 0.471 0.03 0.52 0.95

0.48 0.45 0.43 0.45 0.452 0.02 0.60 0.87 72 0.020 0.49 0.60 0.66 0.62 0.624 0.07 0.59 0.88


462

control juglone falcarindiol

Assessment of germ-tube size, n=10 1600

EC50 = 0.5 (0.2–1) EC50 = 21 (6–141)

1400 1200

a

pixels

1000 800

a

a a

a

600 400

a

a

c

0.4

0.2

a

a a a

c

c

c

c

c

a

c

c

200

a

a

a a

a

a

a

c

0 200

100

50

25

13

6

3

1.6

0.8

0.1

µg/mL control juglone falcarindiol

Difference of turbidity (48h–24h), n=4

0.8

EC50 = 0.4 (0.2–0.7) EC50 = 4 (1–39)

0.7

a

absorbance

0.6 0.5

a

a

0.4

a

a a

0.3

a

a

a

a

0

b

b b b 200

100

50

a

b 25

13

6

a ab

a

b

a b b

b b

b

b

b

a

a

b

b

b 0.1

a

a

a

a

a

a

0.2

a

a

b

a

3

1.6

0.8

0.4

0.2

0.1

µg/mL juglone (germ-tube size)/juglone (turbidity):p = 0.268 (Mann-Whitney U test ) falcarindiol (germ-tube size)/falcaridiol (turbidity):p < 0.001 (Mann-Whitney U test)

Fig. 31. Result comparison of two scoring methodologies: germ-tube size (10 germ tubes, control in the same microwell) and turbidity at 620 nm (four replicates; controls in each microwell), for juglone and falcarindiol; test fungus Botrytis cinerea, microdilutions in 4% malt extract, CFU 105; probit-log estimates by SPSS 10.0.7; bars indicate mean values+standard deviation; significance levels: a, p>0.05; b, po0.05; and c, po0.005.


463

gaining more attention in community ecology. Natural products, as pharmacognosists call them, or allelochemicals or secondary metabolites, as biologists call them, will thus be evaluated for their antifungal activities by many researchers from different disciplines in the future. In many cases, optimization of high-throughput procedures will be a major requirement. This can be achieved by using robot pipetting and automated scoring systems. Despite the enthusiasm we might develop for the gain in results, we have to be aware that we lose awareness of what is actually going on in the single well of the microtiter plate, and thus we might fail to interpret our assays correctly. Further, another methodology is offering itself to be applied for antifungal testing: metabolomics, sometimes also called metabonomics or metabolic profiling (Hall, 2006). Here, NMR- or MS-based methods may allow not only to determine the antifungal effect alone but also to obtain insights into its mode of action. An example how such an approach might work is given by Aliferis and Chrysati-Tokousbalides (2006), who studied the effects of naturally occurring phytotoxins and synthetic herbicides on oat seedlings.

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468

469

Contributors Deepak Acharya – Department of Biotechnology, SGB Amravati University, Amravati 444 602, Maharashtra, India. Young-Joon Ahn – School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea. Jose´ Luis Angulo-Sańchez – Centro de Investigacioń en Quı´ mica Aplicada, Blvd. Enrique Reyna Hermosillo #140, Saltillo, Coahuila 25100, Me´xico. Eduardo Aranda – Biological Control Lab, Center of Biotechnology, UAEM, Cuernavaca, Morelos State, Me´xico. J. Guillermo Avila – Phytochemistry Lab, UBIPRO-FES-Iztacala, UNAM, Av. de Los Barrios s/n, Los Reyes Iztacala, Tlalnepantla, CP 54090, Edo. de Mexico. Me´xico. Wendy J Bryan – Pesticides Research Group, Biological Sciences, University of Paisley, Paisley, PA1 2BE, UK. Marı´ a C Carpinella – Laboratorio de Quı´ mica Fina y Productos Naturales, Fac. Ciencias Quı´ micas, Universidad Cato´lica de Co´rdoba, Camino a Alta Gracia Km 10 (5000), Co´rdoba, Argentina. Carlos L Ce´spedes A – Phytochemistry Lab, UBIPRO-FES-Iztacala, UNAM, Av. de Los Barrios s/n, Los Reyes Iztacala, Tlalnepantla, CP 54090, Edo. de Mexico. Me´xico. Marı´ a T Defago´ – Centro de Investigaciones Entomolo´gicas de Co´rdoba, Fac. Ciencias Exactas, Fı´ sicas y Naturales, Universidad Nacional de Co´rdoba, Av. Velez Sa´rsfield 299 (5000), Co´rdoba, Argentina. Diana Jasso de Rodrı´ guez – Universidad Autońoma Agraria Antonio Narro, Buenavista, Saltillo, Coahuila 25315, Me´xico. Mariana Domı´ nguez L – Phytochemistry Lab, UBIPRO-FES-Iztacala, UNAM, Av. de Los Barrios s/n, Los Reyes Iztacala, Tlalnepantla, CP 54090, Edo. de Mexico. Me´xico. Doris Engelmeier – Department of Chemical Ecology and Ecosystem Research, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. Aniket Gade – Department of Biotechnology, SGB Amravati University, Amravati 444 602, Maharashtra, India.

470

Contributors

Franz Hadacek – Department of Chemical Ecology and Ecosystem Research, Faculty of Life Sciences, University of Vienna, Athanstrasse 14, A-1090 Vienna, Austria. Stuart Heritage – Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK. Francisco Daniel Hernańdez-Castillo – Universidad Autońoma Agraria Antonio Narro, Buenavista, Saltillo, Coahuila 25315, Me´xico. Luko Hilje – Department of Agriculture and Agroforestry, Tropical Agricultural Research and Higher Education Center (CATIE), Turrialba, Costa Rica. Murray B Isman – Faculty of Land and Food Systems, University of British Columbia, Vancouver, BC, Canada V6T 1Z4. Hyun-Hyung Kim – School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea. Soon-Il Kim – School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea. Elisabeth H Koschier – Department of Applied Plant Sciences and Plant Biotechnology, University of Natural Resources and Applied Life Sciences (BOKU), Institute of Plant Protection, Peter-Jordan-Strasse 82, 1190 Vienna, Austria. Isao Kubo – Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA. Tong-Xian Liu – Vegetable IPM Laboratory, Texas Agricultural Experiment Station, Texas A&M University, 2415 E, Highway 83, Weslaco, TX 78596-8399, USA. Wan-Chun Luo – Department of Pesticide Science, College of Plant Protection, Shandong Agricultural University, Taian, Shandong 280001, China. Cristina M Machial – Faculty of Land and Food Systems, University of British Columbia, Vancouver, BC, Canada V6T 1Z4. J. Camilo Marin – Phytochemistry Lab, UBIPRO-FES-Iztacala, UNAM, Av. de Los Barrios s/n, Los Reyes Iztacala, Tlalnepantla, CP 54090, Edo. de Mexico. Me´xico. Olı´ via C Matos – Estac- aõ Agrono´mica Nacional, Departamento de Fisiologia Vegetal, Quinta do Marqueˆs, Av. Da Repu´blica, 2784-505 Oeiras, Portugal. Gerardo A Mora – Natural Products Research Center (CIPRONA), Universidad de Costa Rica, San Jose´, Costa Rica.

Contributors

471

Tzi Bun Ng – Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. Tetsuya Ogura – Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA. Sara M Palacios – Laboratorio de Quı´ mica Fina y Productos Naturales, Fac. Ciencias Quı´ micas, Universidad Cato´lica de Co´rdoba, Camino a Alta Gracia Km 10 (5000), Co´rdoba, Argentina. Mahendra Rai – Department of Biotechnology, SGB Amravati University, Amravati 444 602, Maharashtra, India. Caˆndido P Ricardo – Instituto de Tecnologia Quı´ mica e Biolo´gica, Av. da Repu´blica (EAN) 2781-901 Oeiras, Portugal. Monique SJ Simmonds – Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK. Robin HC Strang – Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK. Jun-Hyung Tak – School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea. George P Tegos – Wellman Laboratories of Photomedicine Massachusettes General Hospital and Department of Dermatology, Harvard School, 50 Blossom Street, Boston, MA 02114, USA. Jonathan RM Thacker – Pesticides Research Group, Biological Sciences, University of Paisley, Paisley, PA1 2BE, UK. Patricio Torres – Department of Botany, Faculty of Natural Sciences and Oceanography, University of Concepcion, Chile. Graciela Valladares – Centro de Investigaciones Entomolo´gicas de Co´rdoba, Fac. Ciencias Exactas, Fı´ sicas y Naturales, Universidad Nacional de Co´rdoba, Av. Velez Sa´rsfield 299 (5000), Co´rdoba, Argentina. Michael Wink – Institute of Pharmacy and Molecular Biotechnology, Heidelberg University INF 364, 69120 Heidelberg, Germany. Han-Hong Xu – Laboratory of Insect Toxicology, College of Natural Resources, South China Agricultural University, Guangzhou, Guangdong 510642, China.


472

473

Subject Index 1-acetyl-2-deacetyltrichilin H in Melia azedarach L. 85 1-acetyl-3-deacetyltrichilin H in Melia azedarach L. 85 1-acetyl-3-tigloyl-11-methoxymeliacarpinin in Melia azedarach L. 85 1-acetyltrichilin H in Melia azedarach L. 85 1-cinnamoilmelianolona in Melia azedarach L. 85 1-cinnamoyl-3-11 dihydroxymeliacarpin in Melia azedarach L. 86 1-cinnamoyl-3-acetyl-11hydroxymeliacarpin in Melia azedarach L. 86 1-cinnamoyl-3-acetyl-11methoxymeliacarpinin in Melia azedarach L. 86 1-cinnamoyl-3-feruloyl-11-hydroxy-22,23dihydro-23-b-methoxymeliacarpin in Melia azedarach L. 86 1-cinnamoyl-3-feruloyl-11hydroxymeliacarpin in Melia azedarach L. 86 1-cinnamoyl-3-methacrylyl-11-hydroxymeliacarpin in Melia azedarach L. 86 1-deoxy-3-methacrylyl-11methoxymeliacarpinin in Melia azedarach L. 86 1-deoxy-3-tigloyl-11-methoxymeliacarpinin in Melia azedarach L. 87 1-desacetylnimbolinin B in Melia azedarach L. 87 1-O-detigloyl-1-O-benzoylohchinolal in Melia azedarach L. 87 1-O-detigloyl-1-O-cinnamoylohchinolal in Melia azedarach L. 87

1-methacrylyl-3-acetyl-11methoxymeliacarpinin in Melia azedarach L. 87 1-tigloil-11-methoxy-20-acetylmeliacarpin in Melia azedarach L. 87 1-tigloyl-3,20-diacetyl11methoxylmeliacarpinin in Melia azedarach L. 87 1-tigloyl-3-acetyl-11-methoxymeliacarpinin in Melia azedarach L. 87 1-O-deacetylochinolide A in Melia azedarach L. 84 1-O-deacetylochinolide B in Melia azedarach L. 84 1-O-deacetyl-1-O-benzoylochinolide B in Melia azedarach L. 84 1-O-deacetyl-1-O-tigloylochinolide A in Melia azedarach L. 84 1-O-deacetyl-1-O-tigloylochinolide B in Melia azedarach L. 84 1-(2-methylpropanoyl)-3-acetyl-11methoxymeliacarpinin in Melia azedarach L. 83 1,5-dihydroxy-8-methoxy-2methylanthraquinone-3-O-a-Lrhamnopyranoside in Melia azedarach L. 87 1,8-dihydroxy-2-methylanthraquinone-3-Ob-D-galactopyranoside in Melia azedarach L. 87 1,12-di-O-acetyltrichilin B 1,3-dicinnamoyl11-hydroxymeliacarpin in Melia azedarach L. 83 1,2,3,4,6-penta-O-galloyl-b-D-glucose 64 1,3-b-D-glucan 424, 425 1,8-cineole 30, 31, 35, 235 1-cinnamoyl-3-hydroxy-11methoxymeliacarpinin in Melia azedarach L. 83

474 11-methoxy-20-acetylmeliatinin in Melia azedarach L. 83 12-a-acetoxyfraxinellone in Melia azedarach L. 83 12-acetoxyamoorastatin see toosendanin in Melia azedarach L. 83 12-hydroxyoleanolic lactone 312 12-deacetyltrichilin I in Melia azedarach L. 84 12-hydroxyamoorastatin in Melia azedarach L. 84 12-deacetyltoosendanin see 12hydroxyamoorastatin in Melia azedarach L. 84 12-hydroxyamoorastatone in Melia azedarach L. 85 12-O-acetylazedarachin A in Melia azedarach L. 85 12-O-acetylazedarachin B in Melia azedarach L. 85 12-O-acetyltrichilin B in Melia azedarach L. 85 135-kDa protoxin 411 16,17-didehydro-16(E)-stemofoline 304 29-deacetylsendanin see toosendanin in Melia azedarach L. 83 29-isobutylsendanin in Melia azedarach L. 88 2b,3b-dihydroxy-5a-pregn-17(20)-(2)-en-16one in Melia azedarach L. 88 2,2-diphenyl-1-pycril-hydrazyl 4, 14, 17 2,4-D 151, 158 2,4-dihydroxybenzoic acid 65 2,4-methanoproline 311 20 ,40 , 60 -trihidroxy acetophenone 4 2-mercaptopyridine-1-oxide 49 2-phenethyl propionate 38 3-carene in Norway spruce 202 in Scots pine 202 3-O-[O-b-D-glucopyranosyl-(1-4)–b-Dglucopyranosyl]-hederagenin in Barbarea vulgaris 295 3-hydroxy-4-prenyl-5-methoxystilbene-2carboxylic acid 297 3-hydroxi-4,5-dimethoxy-Nmethylphenetilamine in Lophophora williamsii (Lem.) Coult. 356

Subject Index 3-hydroxi-4,5-dimethoxy-N,Ndimethoxyphenetilamine in Lophophora williamsii (Lem.) Coult. 356 3-deacetyltrichilin H in Melia azedarach L. 88 3-methoxytiramine in Lophophora williamsii (Lem.) Coult. 356 3-methoxy-N-methyltyramine in Lophophora williamsii (Lem.) Coult. 356 3-methoxy-N,N-dimethyltyramine in Lophophora williamsii (Lem.) Coult. 356 3-n-pentadec(en)ylchatechols (urushiols) in Metopium brownei 344 3-tigloyl-1,20-diacetyl-11methoxylmeliacarpinin in Melia azedarach L. 88 3,30 ,5,50 -tetrahydroxy-4-methoxystilbene in Yucca periculosa F. Baker 4 3,4-dihydroxy-5-methoxiphenetilamine in Lophophora williamsii (Lem.) Coult. 356 3,4-dimetoxiphenetilamine in Lophophora williamsii (Lem.) Coult. 356 30-hydroxy-fraxinellone in Melia azedarach L. 88 4,40 -dihydroxstilbene in Yucca periculosa F. Baker 4 4,40 ,60 -trihydroxy-20 -methoxychalcone in Gnaphalium affine 317 40 ,5,7-trihydroxyisoflavone 49 4,8-dimethoxy-1-vinyl-b-carboline in Melia azedarach L. 88 4-hydroxy-3-methoxycinnamaldehyde in Melia azedarach L. 88 4-methoxy-1-vinyl-b-carboline in Melia azedarach L. 88 5-deoxikievitol 146 5-dimethyl-2-furanene 129 5-fluorouracil 425, 427 50 -methoxyhydnocarpin-D 50, 51 5-hydroxy-3,6,7,8,40 -pentamethoxyflavone in Gnaphalium affine 317 5-hydroxy-3,6,7,8-tetramethoxyflavone in Gnaphalium affine 317 5,6-dihydroxy-3,7-dimethoxyflavone in Gnaphalium affine 317

Subject Index 6-methoxymellein 146 6-acetoxy-3b-hydroxy-7-oxo-14b,15bepoxymeliac-1,5-diene-3-O-b-Dglucuronopyranoside in Melia azedarach L. 88 6-acetoxy-7a-hydroxy-3-oxo-14b,15bepoxymeliac-1,5-diene in Melia azedarach L. 89 6-acetoxy-11a-hydroxy 7-oxo-14b,15bepoxymeliacin-1,5-diene-3-O-a-L rhamnopyranoside in Melia azedarach L. 89 6-hydroxy-7-methoxycoumarin in Melia azedarach L. 89 6-methoxy-7-acetoxycoumarin in Tagetes lucida 15 6,7-diacetoxy coumarin in Tagetes lucida 15 6b-hydroxy-4-campesten-3-one in Melia azedarach L. 89 6b-hydroxy-4-stigmasten-3-one in Melia azedarach L. 89 7-hydro-coumarin in Stellera chamaejasme L. 187 7-methoxy-2-isopropenil-5-acetil-2,3dihidrobenzofuran-3-ol-cinnamate 334 7-methoxy-6-hydroxycoumarin in Tagetes lucida 15 7a-acetoxy-14b,15b-epoxy-gedunan-1-ene-3O-b-D-glucopyranoside in Melia azedarach L. 89 7-deacetyl-7-oxogedunin in Melia azedarach L. 89 8-deoxylactucin in Cichorium intybus 317 8,20 -dihydroxygenistein 146 9a-hydroxyfraxinellone in Melia azedarach L. 89 9b-hydroxyfraxinellone in Melia azedarach L. 89 a-amylase inhibitor 408–411 a-amylase inhibitor-1 410 a-amylase inhibitor-2 410 a-chalcone 146 a-guaiene in Chenopodium ambrosioides L. 338 a-linolenic acid 411 a-phellandrene in Chenopodium ambrosioides L. 338

475 a-pinene in Chenopodium ambrosioides L. 338 in Norway spruce 202 in Scots pine 202 a-solanine 146 a-terpinene in Chenopodium ambrosioides L. 338 a-terpinyl acetate in Chenopodium ambrosioides L. 338 a-thujene in Chenopodium ambrosioides L. 338 a-tomatine in Lycopersicon esculentum 146, 225, 237 A. nidulans 130, 425 ABC 45, 47 ABC transporter genes 425 abemectin 187 Abrus precatorius L. 126 abscisic acid 411 Acanthoscelides 32 Acanthoscelides obtectus 32, 329, 409 Acarapis woodi 35, 36 acari 81, 115, 116 acaricidal 270, 272, 275, 278, 279, 283, 284 acaricidal activity 36, 270, 272, 273, 279, 281 acaricides 270, 272, 278, 279, 282–284 Acarus siro (L.) 269 acetate 6, 7, 12, 105, 106, 130, 183, 236, 275, 330, 338, 365, 432 acetone 16, 76, 103, 108, 114, 128, 130, 177, 180, 190, 450 acetylcholinesterase (AchE) 2, 17, 31, 182, 278 Achillea millefolium 208 Acokanthera 316 aconitine 256, 300 Aconitum hemsleyanum Pritz 190, 191 Acorus 304 acridon 148 active principles 82, 102, 103, 106, 108, 110, 111, 113, 128, 150, 379, 380 acute toxicity 5, 12, 13, 16, 19, 36, 182, 240, 241, 283, 398, 407 Acyrthosiphon pisum 407 Adalia bipunctata 407 adult emergence 4, 12, 103, 105, 106, 108, 109, 182, 241, 387 Aedes aegypti Linnaeus 104 Aeolothrips intermedius Bagnall 233 aescin 295

476 aesculetin (6,7-dihydroxycoumarin) in Bidens pilosa 362 in Melia azedarach L. 89 in Tagetes lucida 15 Aesculus hippocastaneum 295 aflatoxin(s) 142, 143 agar well diffusion 448 Agavaceae 2, 4–6, 305 aglaroxin A in Aglaia elaegnoidea 314 agricultural 35, 38, 115, 125, 126, 148, 171, 172, 216, 235, 271, 381, 385, 399, 406, 424, 434 Agrobacterium tumefaciens, 49, 126, 127, 130 agrochemistry 460 agronomically 130 Agrotis ipsilon (Hufn.) 106 ajmalicine 142, 148 Ajuga 68 ajugarin 1 294, 315 Alamar Blue 454 alamine in Lophophora williamsii (Lem.) Coult. 356 albizziine 310 Alchornea 310 alcoholic extracts 130 alcohols 18, 31, 68, 71, 73, 278, 279 aldehydes 31, 229, 258, 275, 279 alfalfa 102, 130, 146 alismatales 304 alkaloids in Chrysanthemum coronarium L. 224 in Sophora alopecuroids L. 186, 187 in Tripterygium wilfordii Hook. F. 189 alkanols 19, 68–73, 75–77 alkycatechols in Metopium brownei 345 allelochemicals 1, 172, 208, 221, 222, 225–227, 234, 239, 240, 242, 264, 463 allelopathic 3, 4, 8 allelopathic properties 337 allergenicity 280 allicin 253, 258, 264 Allium 305 Allium sativum 32, 384, 407 allo-aromadendrene in Chenopodium ambrosioides L. 338 allomones 221, 240, 380 allyl levulinate in Chenopodium ambrosioides L. 338

Subject Index almond bitter 265 Aloe 306 aloe-emodin 128, 131 alpha-amylases 131 alpha-cypermethrin 206, 207 Alternaria brassicae 141 Alternaria solani 129, 130, 329, 337, 342 Amaranth 410, 411 Amaranthus 411 Amaranthus hypochondriacus 410, 334 Amaryllidaceae 305 Amazon 127, 349, 360, 366 amino acids 68, 223, 251, 252, 258, 264, 310, 311, 380, 410 Ammi visnaga 141 amoorastatone in Melia azedarach L. 89 amphipathic 46–49, 51 amphiphilic 255 amphotericin B 424 amyl levulinate in Chenopodium ambrosioides L. 338 Amynothrips andersoni ONeill 223 amyrenol in Bursera simaruba 368, 369 Anaphothrips obscuratus (Mu¨ller) 233 Anastasia green 52 Anastrepha fraterculus (Wied.) 104 Ancylostoma 338 Androctonus australis Hector 414 angelicin 298 anhalamine in Lophophora williamsii (Lem.) Coult. 356 anhalidine in Lophophora williamsii (Lem.) Coult. 356 anhalinine in Lophophora williamsii (Lem.) Coult. 356 anhalonidine in Lophophora williamsii (Lem.) Coult. 356 anhalonine in Lophophora williamsii (Lem.) Coult. 356 anhalotine in Lophophora williamsii (Lem.) Coult. 356 anhydrocinnzeylanine 300 anilpropanoids 390

Subject Index anisic acid 4, 19, 182 in Parthenium argentatum Gray 182 Annona squamosa L. 300 Annonaceae 272, 273, 300 anorexigenic 381 antagonism 443 antennal receptors 225, 226 anthocyanins 296 anthothecol 2 anthracnose 141, 426 anthrones, 306 anti-amoeba 328 antibacterial 16, 82, 127, 130–132, 342, 345, 355, 358, 362 antibiotic activity 342, 355, 362 anticancer chemotherapy 45 Anticarsia gemmatalis 99 antifeedant(s) 116, 238, 242, 243, 292–294, 298–300, 302, 310, 318 antifeedant activity 4, 12, 19, 62, 97–101, 105, 177–179 antifeedant compounds 238, 299, 304, 306, 313, 316 antifeedant effect 3, 8, 15, 99, 100, 103, 110, 112, 113, 115, 172, 174, 177 antifeedant index 98 antifeedant response 315 antifungal 144–145, 328, 333, 337, 342, 348, 352, 355, 359, 361, 365, 368, 423–463 antifungal assays 155, 156, 435, 440, 444, 460 antifungal compounds 140, 144, 145, 154, 156, 158, 428, 430, 434 anti-inflammatory 8, 14, 82, 345, 368 anti-malarial 338 antimicrobial potential 126, 132 antimicrobials 45, 46, 49, 52, 54, 55, 130, 425 antimycotic drugs 423, 424, 425, 451 antioxidant 1, 3, 328 antiseptic 328, 361 antitrypanosomal 337 antitumor 368 antiulcer 337 antiviral 342, 345, 368 ants 304, 434 aphanastatin in Melia azedarach L. 89 aphid 62–64, 66, 171, 186, 238, 241, 316, 329, 413 Aphidicide 38 Aphididae 113

477 aphids antennae 302 Aphis gossypii 172 Aphis gossypii Glover 171 Apiaceae 30, 264, 272 apigenin-C-glycosides 297 Apocynaceae 150, 188, 261, 316 Apocynum 316 apolysis 20, 68 apoptosis 65, 264 appressorium formation 453, 454 apricots 52, 229 aprotinin 411 aqueous extract(s) 99, 102–104, 106, 107, 114, 115, 130, 151, 154, 337, 348, 352 Arabidopsis thaliana 141, 145 Araceae 304 arachidic acid 126, 129 Arachis hypogaea 146 Araucaria araucana (Mol.) K. Koch 15–16 Araucariaceae 15 arcelins 407, 415 arcelin-1 in Phaseolus vulgaris L. cv. ‘RAZ-2’ 407, 410 argentatins A in Parthenium argentatum 182 argentatins B in Parthenium argentatum 182 Aristolochia 303 Aristolochiaceae 303 armyworm 16, 62, 100, 171, 175, 182, 297, 305, 310 Arrhenatherum elatius 307 Artemia salina 83 Artemisia absinthium 52 Artemisia annua 52 Artemisia chamaemelifolia 317 Artemisia dracunculus 446, 448 arthropods 30, 31, 35, 146, 171, 176, 209, 239, 242, 279, 294 artificial diet 61, 63, 64, 66, 67, 100, 104–106, 111, 183, 386, 392, 406 ascaridole 337–339 Ascaris 338 ascorbic acid 52, 352 asparagales 305 Aspergillus flavus 130, 131, 141, 142, 150, 161, 329, 352 Aspergillus fumigatus 127, 141 Aspergillus niger 16, 126, 127, 130, 141 Aspergillus parasiticus 130, 329

478 Aspergillus spp. 37 Asphodelaceae 306 Asteraceae 2, 10, 13, 18, 150, 176, 182, 294, 317, 342, 379 Asterales 317 asthma 270–272, 354, 368, 409 Ateleia herbert-smithii 311 atopic dermatitis 270 ATP Binding Cassette Transporters 45 atractyloside 174, 190, 262 in Xanthium sibiricum Patrin ex Widder 190 attractants 35, 204, 221, 222, 224, 225, 233–235, 243, 379 Aulacophora indica 313 Aulacophora lewisii 313 Aulacophora nigripennis 313 Authonomus grandis Boh. 99 Avena 307 avidin 408, 415 in Nicotiana tabacum L. 408 axenic cultures 436 Aylthonia 304 aylthonic acid 304 ayurveda 127 Azadirachta excelsa 199 Azadirachta indica A. Juss 172, 173, 199 azadirachtin in Azadirachta indica A. Juss 81, 172, 275, 379 azadirachtine 2, 20, 81, 97, 111, 172, 175, 176, 181, 185, 208, 209, 275, 291, 294, 309, 314, 316, 379 azedarachin A in Melia azedarach L. 89 azedarachin C in Melia azedarach L. 90 azedarachol in Melia azedarach L. 90 azedaralide in Melia azedarach L. 90 azedaric acid in Melia azedarach L. 90 azinphosmethyl 38 azoles 425 azoxystrobin 430, 431 b-agarofurans in Maytenus spp. 10 b-amyrin in Byrsonima crassifolia 365

Subject Index b-aryophyllene in Chenopodium ambrosioides L. 338 b-carboline 146, 264, 392 b-copaene in Chenopodium ambrosioides L. 338 (E)-b-farnesene 228, 234, 240 b-hydrastine 293 b-phellandrene in Norway spruce 202 in Scots pine 202 b-pinene in Chenopodium ambrosioides L. 338 in Norway spruce 202 in Scots pine 202 b-rosorcylic acid 65 B-fenetilamines in Lophophora williamsii (Lem.) Coult. 356 bacchabolivic acid in Gutierrezia microcephala. Gray 10 Bacillus cereus 129, 352 Bacillus megaterium 132 Bacillus pasteurii urease 408 Bacillus subtilis 46, 126, 127, 329, 352, 366 Bacillus thuringiensis 108, 411 Bacillus thuringiensis delta-endotoxin 412 bacrispine 317 bacteria 16, 46, 48, 49, 52, 53, 55, 73, 127–129, 132, 146, 252, 264, 329, 344, 349, 352, 362, 405, 434, 435 bacterial blight 131 baculovirus 410, 414 Balsaminaceae 314 bark extract 97, 99 barley 144, 409 basal stem rot 141 Basidiomycetes 144, 430 basil 34, 235 bean 101, 114, 130, 131, 146, 238, 272, 337, 379, 407, 408, 410, 411 bean weevil 200, 329, 407, 409, 410 Beauveria bassiana 398 behenic acid 129, 362 in Bidens pilosa 362 Bemisia spp. 171, 172 Bemisia tabaci 114, 379, 381, 390 Bemisia tabaci (Genn.) 114 benalaxyl 144 benthiavalicarb 431, 433 benzene 130, 177, 178

Subject Index benzenoids (moskachans) in Ruta chalepensis 394 benzofurans 333, 334 benzoic acid 65, 152, 153, 182, 279 benzopyrans 333, 334 benzyl benzoate 269, 272, 275, 278, 281 berberine 47–49, 51, 52, 264 Berberis aquifolia 51 Berberis fremontii 51 Berberis repens 51 bergamot 272 bergapten 298, 394 Bersama abyssinica 67 Beta 307, 413 Beta vulgaris var. cicla L. 104 beta-monoenol 130, 131 beta-sitosterol in Bidens pilosa 362 BHA 4 Bidens pilosa 359–363 Bidens tripartita 360 bilignans in Bursera simaruba 368, 369 bioactive compounds 4, 8, 19, 126, 149, 152, 156, 160, 208, 325, 326 bioautography 444, 446, 447 biochanin 52 biochemical 1, 3, 61, 69, 71–73, 133, 145, 223, 225, 291 bioguided fractionation 150 bioinsecticides 380, 381, 398 biomembrane 254, 255, 258 biorationals 380 biosynthesis 106, 209, 252, 264, 425, 428, 430, 431 Biscutella lusitanica 150, 157, 159 bitter gourd 313 bitter orange 272 bitterwood 379, 383, 386–388, 390, 392, 394, 395, 398, 399 black bean 407 Blattella germanica 33, 34 Bombyx mori 412 Boophilus microplus (Canestrini) 115 Boraginaceae 314 borbonol-2, a hydrocarbonated lactone in Persea borbonica 145 Bordeaux mixture 144 borneol in Bidens pilosa 362

479 botanical insecticides 29, 171, 172, 182, 189–192, 398 botanicals 29, 125, 133, 192 Botrydiplodia theobromae 127 Botrytis cinerea 131, 132, 146, 150, 161, 329, 456 bottlebrush 292 Brassica juncea 279 Brassica napus L. 297 Brassicaeae 313 Brazil 103, 104, 106, 107, 112, 114, 115, 127, 363 Brevicoryne brassicae (L.) 171, 172, 178 brine shrimp 73, 344 broadbean 146 broad-range 129 broad-spectrum antibacterial 130 broth dilutions assays 449 brown planthopper 297, 406, 411 bruchids 300 bryophytes 133 bufadienolides in Crassulaceae (Kalanchoe) 264 in Hyacinthaceae (Urginea) 264 in Ranunculaceae (Helleborus). 264 Bursera simaruba 366–369 Busseola fusca (Fuller) 107 butanedioic acid in Bidens pilosa 362 Buthotus judaicus 414 Buthus martensii (Karsch) 414 butoxylinoleates in Bidens pilosa 362 butylidenephthalide 275, 278, 279 butyric-acid in Chenopodium ambrosioides L. 338 Byrsonima crassifolia 363–366 C. Lindemuthianum 132, 338, 342 C. reticulata 272 C. sinensis 272 cabbage 100, 113, 171, 174, 175, 177, 180, 181, 189, 238, 405 cabbage aphid 171 cadalenes 146 cadinols in Bidens pilosa 362 Caesalpinia sapan 130, 132 caffeic acid 4, 182 in Parthenium argentatum 182

480 caffeine 142, 276, 362 in Bidens pilosa 362 caffeoylic acids in Bidens pilosa 362 Cajanus 297 calcium hydroxide 144 Callistemon 292 Callosobruchus chinensis Lucas 103 Callosobruchus maculatus 33 callus 114, 115, 132 Calotropis 316 Calystegia sepium 316 calystegines 315 calyx 128, 343 campesterol in Melia azedarach L. 90 camphor 36, 38, 330 camptothecins 52 Canavalia ensiformis 384 canavanine 310 cancer 53, 304, 337 Candida albicans 52, 127, 129, 131, 352, 361 canker 140, 143 Canna 307 Cannaceae 307 caper spurge 52 capric acid in Bidens pilosa 362 Caprifoliceae 150 caprilic acid 152 capsaicin 351, 352 in Capsicum annuum 351 in Capsicum annuum var. annuum 351 capsicoside E in Capsicum annuum l. var. acuminatum 352 capsicoside F in Capsicum annuum l. var. acuminatum capsicoside F 352 capsicoside G in Capsicum annuum l. var. acuminatum 352 Capsicum annuum L 352 Capsicum baccatum 350, 352 Capsicum chinense 350, 352 Capsicum frutescens 350, 352 Capsicum pubescens 350, 352 capsidiol 146 capsidiol in Nicotiana tabacum L. 146

Subject Index caraway 275 carbohydrates 223, 251, 252, 358, 436 carbosulfan 206, 207 carboxyatractyloside 174, 190 in Xanthium sibiricum Patrin ex Widder 190 cardenolides 188, 261, 316 in Apocynaceae (Apocynum, Nerium, Strophanthus, Thevetia) 261 in Asclepiadaceae (Periploca, Xysmalobium) 263–264 in Brassicaceae (Erysimum, Cheiranthus), 264 in Celastraceae (Euonymus) 264 in Convallariaceae (Convallaria) 264 in Ranunculaceae (Adonis) 264 in Scrophulariaceae (Digitalis) 261 cardiac glycosides 256, 261, 264 cardiotonic heterosides 139 carnal bunt 425 carrot 30, 146, 360 Carum carvi 141, 275 carvacrol 31, 34, 36, 152, 153, 161, 242, 338 in Chenopodium ambrosioides L. 338 carvomenthenol 36 carvone in Chenopodium ambrosioides L. 338 carvone oxide in Chenopodium ambrosioides L. 338 Caryophilaceae 150 caryophyllene 235, 338 casbene 146 caspofungin 425, 427 cassia 126, 270, 281, 282 Cassia alata 126, 127 Cassia occidentalis 127 Cassia tora 127, 128, 131, 132, 141, 142 castor bean 146 Catalpa 314 catalpol 314 caterpillars 109, 110 catharantine 142, 148 catnip 279 caudoside in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 CAY-I in Capsicum frutescens 352 cedar 275, 280, 386, 387, 394–396 Cedrela 2, 6, 313 Cedrela dugessi 6, 7

Subject Index Cedrela oaxacensis 6, 7 Cedrela odorata, 6, 7, 386 Cedrela salvadorensis 6, 7, 14, 20 Cedrela spp. 2, 9, 208, 382 Cedrelone 208 in Cedrela spp. 208 Celastraceae 8–10, 189, 264, 308 Celastrus 309 celery 146 Centruroides sculpturatus Ewing 414 Ceratocystis pirifera 16 Chakramard 127 chalcones 52, 296 chamaechromone in Stellera chamaejasme L. 187 in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 Chamaecyparis obtusa 275, 280 Chamaecyparis taiwanensis 273, 275 chelerithrine 152, 153, 161 Chelidonium majus 141, 150 chemical barriers 208 chemical constituents 126, 128, 369, 392 chemoreception 209, 222, 224, 236 chemoreceptors 225, 396 chemosensory structures 225 chemotherapy 45, 52, 261, 424, 435, 440, 447, 460 chenopodosides A in Chenopodium ambrosioides L. 338 chenopodosides B in Chenopodium ambrosioides L. 338 Chenopodium 307, 337, 338 Chenopodium ambrosioides 141, 240, 335–339 chestnut blight 426 chickpea 310, 413 chiconine 142, 148 chile muelo 388 Chilo partellus 311 Chinese starwort 187 Chinese stellera 187 chitinase(s) 408, 415 chitosans 428 chloramphenicol 46 chlordimeform 278, 283 chloroform 106, 108, 115, 127, 128, 130, 131, 150, 174, 177, 186, 190, 329, 345 chlorogenic acid 182, 310 in Parthenium argentatum Gray 182 chlorothalonil 144

481 Choristoneura fumiferana 314, 412 Choristoneura rosaceana 32 chromatographic 128, 150, 152, 158, 162 Chrysodina sp. 99 chymotrypsin 64, 65 cinamodiol in Melia azedarach L. 90 cineole 38 cinnamaldehyde 30, 31, 35, 38, 229, 281, 282 cinnamates 30 cinnamic acid in Melia azedarach L. 90 Cinnamomum Cassia 270, 277 cinnamon 31, 37, 280, 285 cinnamyl alcohol, 35, 229, 275, 278, 279, 281 4-methoxy-cinnamaldehyde 35 cinnamylphenols 146 cinnzeylanone 300 cis-carveol in Chenopodium ambrosioides L. 338 cis-linalool oxide (furanoid) in Chenopodium ambrosioides L. 338 cis-p-mentha-2,8-diene-1-ol in Chenopodium ambrosioides L. 338 citral 31, 37, 259 citronella 35, 280 citronella java 272 citronellal 30, 31, 33 citronellol 152, 153, 161 Citrus 30, 101, 112, 115 Citrus aurantifolia 272 Citrus aurantium 272 Citrus bergamia 272 Citrus limonum 272 Citrus paradisi 272 Cladosporium cladosporioides 141 Cladosporium cucumerinum 131, 143, 150, 155, 156, 158, 329 Cladosporium herbarum 130, 448, 451 Cladosporium werneckii 127 clerodane in Gutierrezia microcephala. Gray 11 Clitoria ternatea 131 Clostridium sporogenes 352 Clostridium tetani 352 clove oil 31, 35, 37, 38 clubroot 142 Cnaphalocrosis medinalis 413 Cnidium officinale 279 Cocholearia armoracia 279 cockroach 31, 105, 278, 414

482 codein 148 coffee berry borer 398, 411 colchicines 256, 261, 429 in Colchicum autumnale 261 Coleoptera 98–103, 172, 175, 200, 294, 306, 329, 390, 394, 405–407, 409–412 Colias lesbias 99 Colletotrichum acutatum 143 Colletotrichum lindemuthianum 131, 338 Colletotrichum magna 143 Colorado potato beetle 200, 293, 394 Combretaceae 308 Combretum 308 common rue 384, 387, 392, 394, 398 complex 1 300 confertin in Parthenium schottii 184 conidia 352, 428, 430, 436, 438–440, 442, 443, 454, 456, 460 conidial germination 131, 132 conidiophores 436, 439 conifer 15, 200, 213 conjunctivitis 270 consumers 125, 171, 283, 388, 397, 431 convallatoxin in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 Convolvulaceae 315 Convolvulus arvensis 316 copper sulphate 144 Coprinus comatus 130 Coptotermes formosanus 32–34 Coriandrum sativum 141, 384 corn earworm 171 corn rootworm 34, 102, 293, 299, 317, 409 corn rootworm beetles 35 Cornus florida 427 cortex 328, 343, 363, 365 cosmetic, 125, 358 cotton 107, 146, 224, 365, 382 cotton aphid 171 cotton smallpox 329 cotton sucker 408 cotyledons 101, 108, 130, 147 coumaric acid 15, 49 coumarin 152, 161, 394 in Ruta chalepensis 394 coumarins derivatives in Ruta chalepensis 394 coumestrol 49, 51 covalent bonds 258

Subject Index cowpea bruchid 405, 406 Cratylea argentea 406 crocin 5, 6 cross-striped cabbageworm 171 Crotalaria 130, 310 Croton aromaticus 309 Croton jatrophoides 61, 309 Cruciferae 150 crude extracts 68, 102, 131, 149–151, 172, 179, 334, 381, 383, 386, 390, 398, 447 Cryphonectria parasitica 426 Cryptomeria japonica 275, 280 crysoplenetin 52 crysoplenol 52 Ctenocephalides canis 394 Ctenopsteustis obliquane 312 cTMT1 405 cTMT2 405 Cucumis sativus L. 104 cucurbitacin B 294, 313 cucurbitacins 265, 294, 313, 392 cucurbitans 313 Culex pipiens molestus 33 Culex pipiens pallens 303 Culex quinquefasciatus 392, 412 Culex tarsalis 68 Culiseta incidens 68 culture medium 151, 435, 436, 450 culture techniques media 435 Cuminum cyminum 141, 276 Cunninghmella sp. 130 Cupressaceae 272, 273 curative 326, 354, 430 cutworms 171 cyanin 296 cyanogenic glycosides 251, 252, 261, 265 cycloeucalenone in Melia azedarach L. 90 Cydia pomonella 32 cylobrassin 141 cymarin in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 Cymbopogon 272 Cymbopogon citratus 274, 280, 384 Cymbopogon martini 274 Cymbopogon nardus 37, 280 Cymbopogon spp., 37 Cyperaquinone 307 Cyperus distans 307 Cyperus nipponicus 307

Subject Index cystatin in corn 411 in soybean 411 cytisin in Sophora alopecuroids L. 186 cytochrome P450 297 cytotoxic activities 334 d-cadinene in Chenopodium ambrosioides L. 338 D. dumentorum 141 d-camphor in Chenopodium ambrosioides L. 338 D-glucuronic acid in Melia azedarach L. 90 D-strophanthin in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 Dacus dorsalis 35 daffodil 407 Dalbergia sissoo 130 Dalea versicolor 48, 52 damping-off 141 Daphne tangutica Maxim. 173, 177 daphnoritin in Stellera chamaejasme L. 187 daucosterol in Bidens pilosa 362 Davidia involucrate Baill. var. vilmoriniana (Dode) Hemsl. 190, 191 deacetylsalannin in Melia azedarach L. 90 decline 141, 204, 406, 443 DEET 35, 269, 311 deguelin 178 dehyfluorensic acid in Fluorensia cernua 335 delta-endotoxin 412, 413, 415 Dendroctonus micans 200, 201 deoxynivalenol 144 dermatitis 270, 328 Dermatophagoides 275 Dermatophagoides farinae 269, 275 Dermatophagoides pteronyssinus 36 Derris elliptica 2, 178 deterrence 39, 101, 172, 174, 175, 181, 209, 236, 239, 278, 386–388, 390, 397 deterrent activity 187, 313, 390 di-glycerides 63 Diabrotica speciosa (Germ.) 99, 103 Diabrotica spp. 35

483 Diabrotica undecimpunctata Barber 102 Diabrotica virgifera virgifera 293, 299, 409 diamondback moth 108, 171, 296, 304 diaphoretic 337, 345, 368 Diaphorina citri Kuwayama 112 dicaffeoylquinic acids 52 dichlorophenoxiacetic acid 151 Dicladispa armigera Olivier 103 dictamus 37 die-back 140 digestive proteases 406 digitoxigenin-based glycosides in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 dihydroactinidiolide 129 dihydrobenzofuran 129 dihydrocapsaicin 352 dihydrocarveol in Chenopodium ambrosioides L. 338 dihydrooroxylin A 312 dihydroquercetin in Metopium brownei 345 dimethomorph 431, 433 dioscin 295 Dioscorea 142, 295 Dioscorea bulbı´fera 141, 147 Dioscoreales 304 diosgenin 129, 148 Diplocyclos palmatus, 313 diptera 103, 104, 171, 172, 392, 412LBV Dipterix odorata 272, 273 Discula destructiva 426 disk diffusion 443, 446–449, 455 diterpene alkaloids 139 diterpenes 12, 19, 139, 146, 255, 261 diterpenoids 52, 275, 294, 302, 309, 315, 316 in Nicotiana glutinosa 145 Dithyrea wislizenii 313 diuretic 337, 368 divaricoside, sinlside in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 diversity 45, 133, 139, 149, 199, 291–293, 297, 299, 304, 309, 310, 316, 318, 325, 423, 434, 443 d-limonene 33, 37 DNA repair 264 DNA replication 255, 264 DNA-topoisomerase 264 DNA transcription 255, 264 docosadienoic acid 126

484 docosatrienoic acid 126 docosenoic acid 126 dodecanol tapers 69 DON 144 DPPH 3, 4, 6, 14, 16, 19 dried stem 128, 129 drimanes 293, 302 Drimys granadensis 384 Drimys lanceolata 302 Drosophila auraria 32 Drosophila melanogaster 31, 33, 407 dry rots 141 dust mite nests 269, 270, 283 Dutch elm disease 426 Dysdercus peruvianus 408 ecdysis inhibitors 2, 112 echinocandins 425 eco-friendly 125 ectoparasitoid 406 eczema 129 edible mushroom 407 efflux 45, 47, 48, 52, 425 eicosadienoic acid 126 eicosanoic acid, 126 eicosatrienoic acid 126 eicosenoic 128 eithyreanitrile 313 elaeocarpidine 1 308 elaidic acid in Bidens pilosa 362 electrodyn application principle 206 elemicine in Bursera simaruba 368, 369 elemol in Maclura pomifera 279 ellagitannin 308 embryo-specific soybean urease 408 emergency 107, 108, 261 emetine 256, 264 emmenagogue 337 Emmenopterys henryi Oliv. 190, 191 emodin, 128, 131 emoroidenone in Tephrosia emoroides 311 encelin in Encelia 317 endochitinase activity 409 endophytes 423 entandrophragmin 2

Subject Index ent-kaurane diterpenoid 315 entomopathogenic fungus 234, 398 entomotoxic activity 406 epazote 335, 336 Ephestia kuehniella 32 epielaeocarpidine 2 308 Epilachna admirabilis 313 Epilachna boisduvali 313 Epilachna paenulata Germ 99, 101 Epilachna varivestis Muls. 101, 390 Epilachna vigintioctopunctata 99 epinine, dopamine in Lophophora williamsii (Lem.) Coult. 356 Epitrix argentiniensis 99 equinofuran 148 ergosterol 46, 152, 153, 161, 424, 425, 428, 430 Ericaceae 184, 273 Erigorgus femorata Aubert 109 eriodictyol in Metopium brownei 345 eruptions 127 Erwinia chrysanthemi EC16 49 erychroside in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 erysimoside in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 Erysiphe graminis 128, 131, 431 Erysiphe poligoni 342 erythromycin 52 erythronic acids in Bidens pilosa 362 Escherichia coli 49, 126, 127, 129, 130, 329, 405 essential oils 29–44, 125, 141, 226, 227, 229, 235–237, 239, 241, 242, 253, 270, 272, 273, 275, 276, 279, 280, 334, 384–385 in Pimpinella anisum L. 273, 276, 280 ethanolic extract 115, 130, 355 ethereal oils 139 ethidium bromide 47 ethyl gallate 64, 65, 67 ethyl nicotinate 229, 232 Euasterid I 300, 301 Euasterids II 301 eucalyptol 37, 228 eucalyptus 35, 274, 278 Eucalyptus saligna (Sm.) 308

Subject Index eudesmin in Araucaria araucana (Mol.) K. Koch 15 Eugenia caryophyllata 274, 279, 280 eugenol 30, 31, 35, 37, 38, 235, 275, 278, 279 Eulophus pennicornis 406 Eumolpinae sp. 99 euonine 189 euoverrine B 308 Euphorbia 310 Euphorbia lathyris 52 Euphorbiaceae 61, 261, 273, 309 eurosids 300, 308, 313 Eutypa armeniaca 329 evaluated 6, 127, 128, 172, 182, 183 Evergestis rimosalis (Guenee) 171, 172 evonine 309 Fabaceae 2, 126, 132, 172, 178, 264, 310, 399, 426 falcarindiol 456, 460 in Lycopersicon esculentum 146 falcarinol 146 in Lycopersicon esculentum 146 farnesol 68, 71, 73, 330 fatty acids 63, 252 fecundity 82, 107, 116 feeding index 297 fenugreek 129 ferrulic acid 141 fertility 82, 107, 116 field study 187, 203 finotin 131 filamentous fungi 435, 439, 440, 443, 444, 446 Filipendula ulmaria Maxim 228 filter disk assay 352 fish bean 384, 388 flavone 152, 161, 297, 308, 312 in Gutierrezia microcephala. Gray 10 flavonoids in Bidens pilosa 361 in Bursera simaruba 368, 369 in Melia azedarach L. 82 in Ruta chalepensis 394 in Yucca spp. 358 flavonol 129, 131, 297 flies 109 Floradia torreya 426 Flourensia cernua 332–335 fluconazole 52, 424, 425 flucytosine 425, 427

485 flumorph 431, 433 fluorensadiol in Fluorensia cernua 335 fluoroquinolones 55 folkloric medicine 328 forage 126, 131, 341, 348 formazan 454 formosan cypress 275, 277 forskolin 261 Frankliniella intonsa (Trybom) 230, 233 Frankliniella occidentalis Pergande 222 Frankliniella tenuicornis (Uzel) 233 Frankliniella tritici Fitch 229 fraxinellone in Melia azedarach L. 90 fraxinellonone in Melia azedarach L. 91 friedelans in Bidens pilosa 362 fruit extract 82, 98, 102–105,107–109, 112 fruit pulp 128 fruitfly 412 Fuchsia tetradactyla 61 fumigants 35, 39, 281, 282, 284 fumigation 342 fumonisins 143, 144 functional 31, 133, 264, 352 fungicidal 125, 128, 131, 140, 428 fungicide 38, 154, 281, 328, 329, 352, 428, 430, 431 fungiqual 454 fungistatic 329, 428 fungitoxins 145, 161 furanocoumarins 146, 256, 258, 264, 297, 298 furanocromones 146 furoquinoline alkaloids 259, 263, 264 fusarins 143 Fusarium culmorum 141, 150, 151, 158, 163 Fusarium graminearum 143, 144 Fusarium moniliforme 16, 37, 131, 144 Fusarium nivale 141 Fusarium oxysporum 37, 130, 132, 141, 145, 328, 329, 333, 355 Fusarium oxysporum f.sp. cubense 143, 150, 155, 156, 158, 161, 352 Fusarium oxysporum f.sp. cumini 143 Fusarium oxysporum f.sp. dianthi 141, 143 Fusarium oxysporum f.sp. gladioli 143 Fusarium oxysporum f.sp. lentis 143 Fusarium oxysporum f.sp. lycopersici 143

486 Fusarium oxysporum f.sp. melonis 150–152, 156, 159, 161 Fusarium oxysporum f. sp. radicis lycopersici 337, 348 Fusarium solani 37, 127, 131, 132 Fusarium sp. 141, 143, 146 g-terpinene in Chenopodium ambrosioides L. 338 GABA receptors 317 Gaeumannomyces graminis 431 galanthamine 305 Galanthus nivalis agglutinin 406 Gallae Rhois 62–64, 66 gallic acid 3, 6, 62–67, 224 galls 62, 67, 142 garajonone 300 garlic, 32, 407 gastrointestinal 333, 361 gedunin, 2, 4, 11, 14, 20, 91, 305 in Cedrela dugessi 6 in Cedrela salvadorensis 6, 14, 20 in Melia azedarach L. 91 genistein 49, 52, 256 genistoid 310 Geraniaceae 273, 308 geranial 129, 338 in Chenopodium ambrosioides L. 338 geranic acid in Chenopodium ambrosioides L. 338 geraniol 35, 129, 231, 240, 338 in Chenopodium ambrosioides L. 338 Geranium 235, 308 Geranium caespitosum 52, 53 geranylacetone 35 germacrene D in Bidens pilosa 362 germination 129, 131, 132, 150, 252, 337, 400, 428, 440, 460 gitogenin 129 gitoxigenin in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 glabrous 126, 340, 343 Gliricidia sepium 383, 384 Globodera rostochiensis 342 glucans 428 glucopyranoses in Bidens pilosa 362 glucopyranosides in Bidens pilosa 362

Subject Index glucosinolates 251, 252, 265, 266, 313, 380, 405, 431 glutinosone in Nicotiana tabacum L. 146 glyceolinol 148 glyceollin 146 glycine in Lophophora williamsii (Lem.) Coult. 356 glycoalkaloid leptine 293 glycosidase inhibitors 306 glycosides 139, 145, 188, 251, 252, 261, 264, 314, 328, 331, 380, 391 GNA 406–408 Gnaphalium affine 18, 317 gobernadora 326, 327 gossypol 49, 51, 146, 224 gram-negative 46, 48, 49, 53, 73, 127, 129, 132, 334 gram-positive 46, 49, 52, 53, 55, 73, 127, 132, 334 grapefruit 272 grapevine 146 graveolone 148 green peach aphid 171 greenhouse 38, 103, 104, 227–229, 233, 234 238, 240, 337, 342, 383, 385, 387, 395 gregarization 114 Griffonia simplicifolia 405 ground ivy 407 groundnut 130 growth index (GI) 12, 13, 16 growth inhibition properties 189 guayule 12, 182 gums 126 Gutierrezia microcephala. Gray 10–12 gypsy moth 300, 412 habitat 8, 271, 326–327, 332, 336, 340, 343, 347, 350, 354, 357, 360, 363, 366 (-)-hardwickiic acid 309 Haplothrips aculeatus Fabricius 223 heartwood 132 hecogenin in Agave lechuguilla Torrey 349 hedges 126 Hedomea mandonianum 32 Helicoverpa armigera 297, 306 310, 314, 408 Helicoverpa spp. 172 Helicoverpa zea (Bodddie) 171 Heliothis subflexa 293

Subject Index Heliothis virescens 293, 306, 412 Heliothis zea (Boddie) 107, 184, 297 Heliothrips haemorrhoidalis (Bouche´) 223 Heliotropium 314 Helminthosporium sp. 130, 131 Hemigossypol 146 Hemiptera 100, 111, 113, 306, 406–408 hepatitis 328 heptacosane-4-olide in Fluorensia cernua 335 herbivores 235, 252, 261, 265, 266, 296 herpes simples virus type 2 359 hexacosane-4-olide in Fluorensia cernua 335 hexadecanol. 69, 71–74 hexadecenoic acid 126 hexane, 11, 12, 63, 106, 108, 110, 115, 130, 226, 272, 307, 333, 334, 446 hiba wood 277 hinoki 275, 280 hojase´ 332 Hollarrhena antidysenterica 316 Holy basil 32 Hompotera 172 hordenine (N-acetyl-3-metoxi-4,5dimetoxiphenetilamine) in Lophophora williamsii (Lem.) Coult. 356 horsechestnut 295 horticultural 38, 305 house dust mite allergens 270, 284 house dust mite Psoroptes cuniculi 36 HR 428 human cytomegalovirus 359 Huwen toxin I 410 huwentoxins (HWTX) 413 Hyacinthaceae 264, 306 hydrogenated 5-butyl-2,2’-bithienyl in Tagetes erecta 343 hydrophilic 12, 47, 71, 76, 251, 255, 453, 455 hydrophobic 18, 71, 75, 280, 449, 453 hydrophobic alkyl 76 hydroxiacetophenone 141 hydroxilubimin 146 Hylobius abietis (L.) 199, 200 Hymenolepsis nana 338 Hypera postica Gyllenh. 102 Hypericum perforatum 52 hypersensitive reaction process 428

487 hyphal 131, 428, 439, 431 Hypothenemus hamepei 411 Hypsipyla grandella 379, 381 ichthyotoxic 83, 179 IGR 3, 4, 6, 10, 12, 13, 15, 18, 19, 21 imparipinnate 128 imperatorin 298 indigenous 127, 129, 190, 292, 360, 366, 398 indigestion 333 indole 139, 299, 308, 313, 392 indomethacin 398 industrial 47, 67, 126, 152, 266, 379, 398 INF271 47, 48 infections 45, 125, 127, 133, 143, 144, 161, 164, 251, 266, 316, 365, 368 inhibitory activity 3–6, 12, 19, 50, 52–54, 63–65, 125, 132, 305, 409 inositol in Bidens pilosa 362 insect growth regulator 4, 185 insecticidal activity 2, 6, 12, 15, 19, 20, 177, 178, 183, 275, 291, 300, 303, 304, 309, 313, 406–408, 411, 413, 415 insecticide 329, 334, 338, 342, 345, 349, 359, 362, 366 insecticide-resistant 270 integrated pest management (IPM) 35, 39, 140, 172, 192, 216, 221, 281, 318, 379, 424 iodine vapor 446 ipomeamarone 146 Ipomoea 315 Ipomoea purpurea 316 iprovalicarb 431, 433 Iridaceae 306 iridodial 130, 131 iridoid(s) 139, 256, 314, 315 iridoid aldehydes 258 irradiation 153, 154, 161, 334 isoanhalamine in Lophophora williamsii (Lem.) Coult. 356 isoanhalidine in Lophophora williamsii (Lem.) Coult. 356 isoanhalonidine in Lophophora williamsii (Lem.) Coult. 356 isoborneol in Chenopodium ambrosioides L. 338

488 isobornyl acetate in Chenopodium ambrosioides L. 338 isobornyl propionate in Chenopodium ambrosioides L. 338 isobutyl benzoate in Chenopodium ambrosioides L. 338 isocitrimide lactone mescalina in Lophophora williamsii (Lem.) Coult. 356 isoflavones in Lupinus albus 145 isoflavonoids 146, 256, 306, 310, 390 isokinoline 139 Isometrus vittatus 414 isopellotine in Lophophora williamsii (Lem.) Coult. 356 isoprene derivatives 139 isopulegol 152, 153, 161 in Chenopodium ambrosioides L. 338 isopulegyl acetate in Chenopodium ambrosioides L. 338 isoquercitrin 297, 362 in Bidens pilosa 362 isoquinoline alkaloids, 47, 256 isosinotrodise in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 isoterpenoides in Lupinus 145 isothiocyanates 256, 258 itching 68, 127, 270 Jack bean urease (JBU) 408 Japanese beetle 35, 200 jasmonic acid 145, 252, 411, 430, 431 Jodrellin B 294, 315 judaicin 310 juglone 312, 448, 456, 457 Juncaceae 150 juvenile hormone 68, 73, 107, 209, 334 juvenile rainbow trout 38 K-strophanthoside in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 K-strophanthin-beta in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 Kakothrips pisivorus (Westwood) 223 Kakothrips robustus Uzel 238

Subject Index kanamycin 47 karanjin 129 ketoconazole 424, 425 ketones 31, 275 Khaya 313 khellin 141 kidney bean 407, 413 kievitone 148 kinoline alkaloids 139 kinolisidine alkaloids 139 knockdown 31, 189, 191, 278, 279 Koelreuteria paniculata 406 kresoxim-methyl 431 kulactone in Melia azedarach L. 91 kulinone in Melia azedarach L. 91 kulolactone in Melia azedarach L. 91 L. sativae 171, 172, 184 Lablab purpureus 131, 411 Lacanobia oleracea 406 lacinilenes, 146 lactogogue 337 lactophenol cotton blue 443 lactupicrin in Cichorium intybus 317 ladybird 407 lamdacyhalotrin 107 Lamiaceae 30, 235, 272, 294, 315 Lamioideae 315 lariciresinol in Araucaria araucana (Mol.) K. Koch 15 Larrea tridentata 326–331 larvae development 102, 106 Lathyrus latifolius 311 Lathyrus ochrus 406 Lauan 275 Lauraceae 272, 273, 300 lauric acid 126, 129, 362 in Bidens pilosa 362 lavandulyl acetate in Chenopodium ambrosioides L. 338 leaf curls 142 leaf cutting ants 434 leaf disc bioassays 225, 227, 236 leaf extract(s) 99, 102, 103, 109, 111, 155, 178, 183, 386, 392 lecithins 73

Subject Index lectins 25, 25,3 266, 267, 405–407, 415 in Colocasia esculenta 407 in Cratylea argentea 406 in Diffenbachia sequina 407 in Glechoma hederacea 407 in Griffonia simplicifolia 405 in Lathyrus ochrus 406 in Narcissus pseudonarcissus 407 in Narcissus tazetta 407 in Talisia esculenta 406 in Xerocomus chrysenteron 407 in Zephyranthes candida 407 Leguminosae 61, 68, 76, 126, 147 leguminous 126, 132, 186, 379 Leiurus quinquestriatus guinquestriatus 413 Leiurus quinquestriatus hebraeus 414 lemongrass 31, 272, 280 Lepidoptera 3, 16, 82, 99, 100, 105, 107, 109, 110, 171, 172, 209, 210, 294, 300, 302, 306, 315, 317, 381, 382, 406 Leptinotarsa decemlineata 200, 293, 294, 300, 307, 316, 394 leptospermone 292 Leptospheria maculans 143 leucine-aminopeptidase 65 levofloxacin 47 lignans in Melia azedarach L. 112 lignoceric acid 126, 127, 129 Liliales 304–305 Lilium longiflorum 304 lima bean 146 limonene 30, 31, 202, 330, 338, 362 in Bidens pilosa 362 in Chenopodium ambrosioides L. 338 in Norway spruce 202 in Scots pine 202 limonin in Citrus spp 208 limonoids in Melia azedarach L. 82 Limothrips cerealium Haliday 223 Limothrips denticornis Haliday 233 linalool 36, 228, 231, 235, 275, 278, 330, 338 in Chenopodium ambrosioides L. 338 linearol 315 linoleic acid 127, 127, 129, 152, 153 in Bidens pilosa 362 linolenic acid 126, 127, 129, 411 Lipaphis erysimi 172

489 Lipaphis erysimi (Kaltenbach) 171 Liriodendron tulipifera (L.) 300 Liriomyza huidobrensis (Blanchard) 103 Liriomyza sativae Blanchard 171 l-menthone 279 Locusta migratoria 107, 306, 308, 316 Locusta migratoria migratorioides (Reiche & Fairmaire) 113 long-chain alcohols 68 lophophorine in Lophophora williamsii (Lem.) Coult. 356 lophorine in Lophophora williamsii (Lem.) Coult. 356 lophotina iodide in Lophophora williamsii (Lem.) Coult. 356 LPCB 443 lubimin 146 in eggplant 146 in Nicotiana tabacum L. 146 lubrication 129 luciamin in Solanum laxum Steud 316 lupeol in Bidens pilosa 362 luteolin 296, 362 in Bidens pilosa 362 Luzula lactea 150, 157–159 Lycopersicon esculentum 108, 337 Lygus hesperus 409 Lygus lineolaris 409 Lymantria dispar 300, 412 Lyriomiza huidobrensis 103, 104, 108 M. arenaria 342 M. gypseum 127, 130 M. hapla 342 M. incognita 342, 329 M. javanica 342 Maackiain 310 Maerosiphum avenae 406 Magnoliaceae 300 Mahonia 52 maitencito 9 malformation 209, 334 Mamesta configurata 294 mandarine 272 Manduca sexta 307, 412 Mandvilla 316

490 manogenin in Agave lechuguilla Torrey 349 marine 133, 252 marjoram 37, 241 marrubiagenine 317 Mascarene Islands 128 masticadienoic acid in Metopium brownei 345 matrine in Sophora alopecuroids L. 186 Maytenus disticha (Hook) 9 maytoline 309 MC207110 47, 48, 50 mechanical barriers 208 medical mycology 435 medicarpin 146 Megalurothrips sjostedti (Trybom) 238 melain in Melia azedarach L. 91 melain G in Melia azedarach L. 91 Melaleuca 36 Melaleuca alternifolia 274, 278 Melaphis chinensis 62 melazolide A in Melia azedarach L. 91 meldenin in Melia azedarach L. 91 Melia azedarach L. 81, 173, 181, 199 Melia toosendan 173, 181 meliacarpinin A in Melia azedarach L. 91 meliacarpinin B in Melia azedarach L. 91 meliacarpinin C in Melia azedarach L. 92 meliacarpinin D in Melia azedarach L. 92 meliacarpinin E in Melia azedarach L. 92 Meliaceae 2, 6–7, 9, 81, 106, 172, 181, 313, 379, 382, 397 meliandiol in Melia azedarach L. 92 melianin A in Melia azedarach L. 92 melianin B in Melia azedarach L. 92 melianol in Melia azedarach L. 92

Subject Index melianolide in Melia azedarach L. 92 melianone in Melia azedarach L. 92 melianoninol in Melia azedarach L. 92 Melianthaceae 67 meliantriol in Melia azedarach L. 92 meliartenin in Melia azedarach 181 meliatoxin A1 in Melia azedarach L. 93 meliatoxin A2 in Melia azedarach L. 93 meliatoxin B1 in Melia azedarach L. 93 meliatoxin B2 in Melia azedarach L. 93 Meloidogyne incognita 329 membrane proteins 75, 256 258, 264 Mentha 272 Mentha aquatica 278 Mentha piperita 272 Mentha pulegium 272 Mentha spicata 272, 273, 276 menthol in Chenopodium ambrosioides L. 338 menthone 30, 33, 278 mescalina in Lophophora williamsii (Lem.) Coult. 356 mescalina maleimide in Lophophora williamsii (Lem.) Coult. 356 mescaline in Lophophora williamsii (Lem.) Coult. 355 mescaline citrimide in Lophophora williamsii (Lem.) Coult. 356 mescaline malaimide in Lophophora williamsii (Lem.) Coult. 356 mescaline succinimide in Lophophora williamsii (Lem.) Coult. 356 Mescalotam in Lophophora williamsii (Lem.) Coult. 356

Subject Index methanolic extracts 11, 100, 104, 106, 111, 129, 155, 159 methanoproline 311 methoxybrassinin 141 methyl eugenol 35 methyl gallate 64, 65, 67 methyl-hexanoate in Chenopodium ambrosioides L. 338 methyl kulonate in Melia azedarach L. 93 methyl orsellinate in Fluorensia cernua 335 methyl-pinoresinol in Araucaria araucana (Mol.) K. Koch 15 methyl salicylate 228, 235, 276, 339 methyl undecanal in Chenopodium ambrosioides L. 338 methylchalcone 146 methylene blue 443 methylene cycloartanone in Melia azedarach L. 93 methyleugenol 278 MexAB-OprM, 47 MexCD-OprJ, 47 MexEF-OprN 47 mexicanolide 2, 6 microbes 1, 129, 132, 252, 254, 265, 267 microbial growth 132 Micrococcus luteus 127, 365 microdilution 449–454, 456–459 Microsporum canis 126–128, 329 microtubule 256, 261 midrib 62 mildew 142 Mimosoideae 310 minimum inhibitory concentration (MIC) 74, 96, 446, 448, 456 mint 30, 34, 37, 236, 278 Minthostachys andina, 32 mites 35, 38, 269, 270, 275, 278, 279, 282, 283, 285, 435 miticide 38 mitochondrial 178, 297, 300, 454 mitochondrial glutathione 65 moderate 7, 8, 13, 35, 128 molluscidal 337 molting 1, 2, 19, 20, 68, 172 momilactones 146 Momordica charantia 313, 384 momordicine II 313 momordicines I 313

491 Monilinia fructicola 37 monophagous 109, 222, 223, 233, 293 monoterpenes 30, 31, 34, 202, 223, 231, 235, 236, 241, 337, 431 monoterpenoids 18, 31, 208, 275, 278, 279 morphine 148 mosquito 39, 61, 62, 68, 71–73, 76, 77, 104, 303, 392, 412 mother-of-cocoa 383, 384, 386, 388, 390, 398, 399 moulting 86, 105, 107, 109, 110, 112, 114, 116, 209, 241, 387 mounting medium 443 mouthparts 101, 209, 225, 302 MTT 454 Mucor miehei 16 Mucuna pruriens 128, 132 multidrug and toxic compound extrusion (MATE) 45, 47 multidrug endosomal transporter (MET) 45 multidrug resistant pumps (MDRs) 45–48 Musca domestica 32–34 mutant 47–49 muurolene 129 muurolol in Bidens pilosa 362 mycelial 129, 329, 337, 342, 355, 443, 446 mycelium 142, 152, 154, 442, 456 Mycobacterium tuberculosis 338, 361 mycorrhizal fungi 440 mycosis 328, 426 myrcene in Chenopodium ambrosioides L. 338 in Norway spruce 202 in Scots pine 202 myristic acid 93, 126, 362 in Bidens pilosa 362 in Melia azedarach L. 93 Myristica fragrans 32, 274 Myrtaceae 30, 272, 274, 308, 384 myrtle 30 mytansine 309 Mythimna separata (Walker) 100, 107, 179 Myzus persicae (Sulzer) 171, 241 N-acetylanhalamine in Lophophora williamsii (Lem.) Coult. 356 N-acetylanhalonine in Lophophora williamsii (Lem.) Coult. 356

492 N-acetylglucosamine-binding lectin in Koelreuteria paniculata 406 N-acetylmescaline, N-formylmescaline in Lophophora williamsii (Lem.) Coult. 356 N-formylanhalamine in Lophophora williamsii (Lem.) Coult. 356 N-formylanhalinine in Lophophora williamsii (Lem.) Coult. 356 N-formylanhalonidine in Lophophora williamsii (Lem.) Coult. 356 N-formylanhalonine in Lophophora williamsii (Lem.) Coult. 356 N-formyl-O-methylanhalonidine in Lophophora williamsii (Lem.) Coult. 356 N-formyl-3-methoxy-4,5dimethoxyphenethylamine in Lophophora williamsii (Lem.) Coult. 356 N-methylmescaline in Lophophora williamsii (Lem.) Coult. 356 N-methyltyramine, peyotine in Lophophora williamsii (Lem.) Coult. 356 N,N-diethyl-m-touamide 35 Na+,K+-ATPase 261 NADH dehydrogenase 55, 178 NADPH cytochrome c reductase 105 naphtochinone juglone 448, 451, 456 (7)-naringenin 4, 19, 49, 296 NCCLS 435, 449 NDGA 327, 328 necrosis 140 necrotrophic 433 neem 29, 81, 102, 111, 112, 172, 174–176, 183, 192, 199, 200, 208–216, 241, 275, 281, 294, 379, 380, 397 nematicide 329, 334, 338, 342, 345, 349, 352, 359, 366 nematode 329, 342 Neohydatothrips tiliae (Hood) 233 neoquassin in Quassia amara 390

Subject Index Nepeta catariai 279 nepetalactone in Nepeta cataria 279 Nephotettix virescens (Distant) 112 Nerium 261, 316 nerol in Chenopodium ambrosioides L. 338 neryl acetae in Chenopodium ambrosioides L. 338 neryl formate in Chenopodium ambrosioides L. 338 neurons 317 Neurospora crassa 128 neurotoxic 1, 31, 414, 415 neurotoxin 414, 415 neurotransmitter 31, 75, 261 nicotine in Nicotiana tabacum L. 172, 379 nidulans var. echinulatus 425 Nilapavarta lugens Stal. 100, 112 nimbolidin A in Melia azedarach L. 93 nimbolidin B in Melia azedarach L. 94 nimbolin A in Melia azedarach L. 94 nimbolin B in Melia azedarach L. 94 nimbolinin B in Melia azedarach L. 94 nomilin 2 non-protein amino acids (NPAA) 252, 256, 310, 311, 380 nonacosane-4-olide in Fluorensia cernua 335 NorA mutant 47, 48 NorA pump 46, 47, 52 norbornyl acetate in Chenopodium ambrosioides L. 338 nordihydroguayaretic acid in Larrea tridentate 328 Nothofagus 312 nucleic acids 254, 263 nutmeg 32, 280 O-methylanhalonidine in Lophophora williamsii (Lem.) Coult. 356 O-methylpeyoruvic acid in Lophophora williamsii (Lem.) Coult. 356

Subject Index O-methylpeyoxylic acid in Lophophora williamsii (Lem.) Coult. 356 ochinolid A in Melia azedarach L. 94 ochinolid B in Melia azedarach L. 94 Ocimum basilicum L 235 Ocimum sauve 32 o-coumaric acid 15, 141 octacosane-4-olide in Fluorensia cernua 335 octadecadienoic acid 126 octadecatrienoic acid 126 octadec-cis-9-enoic acid 128 octanoic acid 153, 161, 162 octopamine 31, 283 odoratol 2 ohchinin in Melia azedarach L. 95 ohchinolal in Melia azedarach L. 95 ointment 127 okanin-glucosides in Bidens pilosa 362 oleandrin in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 oleic acid 126, 127, 152, 153, 161, 162 oleoresin in Tagetes erecta 343 olfactory response 236 olfactory stimuli 222, 224–227, 235 Onagraceae 61, 67 Oncorhynchus mykiss 38 oocytes 114, 264 oomycete 144, 428 Ophiostoma ulmi 426 orange 126, 272, 340, 343, 363, 425 oregano 31, 34, 37 organic solvent 68, 446, 448, 450, 456 organophosphate 38 oriental fruit fly 35 Origanum marjorana L. 241 Orius laevigatus Fieber 239 ornamental 126, 129, 143, 228, 234, 239 Ornithonyssus sylviarum 36 orobol 52 Orseolia oryzae (Wood-Mason) 104 Orthoptera 113 Oryzaephilius surinamensis 32

493 oryzalexins 146 osage orange tree 279 Osmunda japonica Thunb. 190, 191 Ostrinia furnacalis (Guenee) 181 Ostrinia nubilalis 33, 313, 317 oudemansins 430 ovicidal effects 112 oxadiocyl 144 oxygen species 65 oxymatrine in Sophora alopecuroids L. 186 F-caudostroside in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 p-cymene in Chenopodium ambrosioides L. 338 pachyelaside A 68 pachyelaside B 68 pachyelaside C 68 pachyelaside D 68 Pachyelasma tessmannii 61 paclitaxel 261 Paecilomyces variotii 16 Paeonia suffruticosa 274, 276, 279 paeonol 275, 276, 279 palmarosa 37, 272 palmatine 47, 48, 51 palmitic 128 palmitic acid 126, 152, 153, 162 in Bidens pilosa 362 palmitoleic acid in Bidens pilosa 362 palmitos 4 pandanales 304 p-anisaldehyde 227, 229, 233, 234, 240, 275 p-anisic acid in Parthenium argentatum Gray 182 panicles 128, 332, 343 Panolis flammea 200 Panonychus citri (Mc Gregor) 115 Pantomorus leucoloma 98 Papaveraceae 150 Papilionaceae 128, 186 Papilionoideae 310 paracoumaric acids in Bidens pilosa 362 parahydroxy benzoic acid in Parthenium argentatum Gray 182 Paraı´ so 81, 97, 99, 103, 104, 109, 110, 113, 114, 116

494 parasitoids 104, 109, 208 parsley 146 parsnip 146 parthenin in Parthenium argentatum 182 Parthenium argentatum Gray 182 Parthenium hysterophorus L. 182 Parthenium schottii Greenman 182 Parthenium tomentosum DC 182 parthenolide 4 patayuc 67 Patchouli, 32, 280 pathogenic 36, 38, 45, 125, 127, 128, 130, 132, 140, 147, 329, 423, 424, 428, 430, 436, 439 Pogostemon cablin 32 p-coumaric acid 49 pea 130, 146 238, 297, 410, 413 peanut 130, 143, 146 Pectinophora gossypiella 61, 310 pectolinarigenin 312 Pediculus humanus 32 Pelargonium 308 Penicillium digitatum 37, 130 Penicillium italicum 127, 130 Penicillium notatum 16, 130 Penicillium species 127 pennyroyal 36, 272 pentachloronitrobenzene 144 pentacosane-4-olide in Fluorensia cernua 335 pentadecanoic acid 126 pentadecanol 69–72, 76 pepper 52, 143, 146, 223, 280, 329, 352, 399 peppermint 37, 272, 384 peptic ulcers 338 peptide 130–132, 258, 413 Pericoń 14 perilla 275 Perilla frutescens var. Japonica perillyl alcohol in Chenopodium ambrosioides L. 338 Periplaneta americana 33 permethrin 205, 207, 213, 284 Persea indica 300 Pestalotiopsis microspora 426 peyocactin in Lophophora williamsii (Lem.) Coult. 355

Subject Index peyoglunal, peyoglutam in Lophophora williamsii (Lem.) Coult. 356 peyonine in Lophophora williamsii (Lem.) Coult. 356 peyophorine in Lophophora williamsii (Lem.) Coult. 356 peyoruvico acid in Lophophora williamsii (Lem.) Coult. 356 peyotine iodide in Lophophora williamsii (Lem.) Coult. 356 peyoxilic acid in Lophophora williamsii (Lem.) Coult. 356 P-glycoprotein 45 phagodeterrent 399 phagostimulant(s) 302 Pharbitis purpurea (L.) Voigt 190, 191 pharmaceuticals 125, 271, 272 pharmacophor 265 phaseolin 146, 148, 296 Phaseolus vulgaris 103, 132, 406 Phaseolus vulgaris L. cv. ‘RAZ-2’ 407 phenolic acids 49, 182, 352 phenolics 1, 3, 19, 20, 253, 272, 296–299, 331 phenoloxidase 102 phenols 3, 30, 34, 35, 380 phenylamide fungicides 144 phenyl-heptatrine 148 in Bidens pilosa 362 phenylpropanoid methyl salicylate 235 phenylpropanoids 3, 148, 223, 229, 235, 241, 258, 275, 361 phenylpropenes 30, 34 pheromone 227, 236, 238 Philippines 128, 360 phytenoic acid in Bidens pilosa 362 phytol in Bidens pilosa 362 pinoresinol in Araucaria araucana (Mol.) K. Koch 15 Phoma lingam 143 Phorocera grandis Rondani 109 photogedunin epimers in Cedrela dugessi 6 in Cedrela salvadorensis 6

Subject Index photogedunins in Cedrela dugessi 6 in Cedrela salvadorensis 6 photosensitizers 146, 148 Phryxe caudata Rondani 109 Phthorimaea operculla Zeller 110 p-hydroxymercuribenzoate 408 Phyllocnistis citrella 342 Phyllotreta vittata 172, 175 physcion, 128, 131 Physostigma venenosum Balf. 172 Physostigmine 18, 172, 262 Phytium sp. 146 phytoalexins naringenin 49 phytoalexins 45, 49, 146, 148, 434 phytoanticipins 45 phytochemicals 61, 76, 126, 224, 228, 229, 234, 239, 242, 270, 342, 346, 349, 356, 359, 361, 366, 369 phytopathogenic 140–142, 145, 149–151, 328, 439 phytophagous 3, 36, 224, 237, 291, 406 Phytophthora infestans 128, 131, 147, 329, 425, 428, 431 Phytophthora cambivora 150, 156, 158 Phytophthora capsici 431 Phytophthora cinnamomi 132, 143, 426 Phytophthora drechsleri 141, 145 Phytophthora ramorum 427 Phytoseilus persimilis 36, 38 phytotoxic 214, 242 phytuberin 146 in Nicotiana tabacum L. 146 phytuberine 148 phytuberol 146 in Nicotiana tabacum L. 146 Picea sitchensis (Bongard) 200 Picrasma quassioides (D. Don) Benn. 190, 191 Picris spinifera 150, 157–159 picropolygamain in Bursera simaruba 368, 369 Pieris napi oleracea 188 Pieris rapae L. 92, 171 pigeon pea 297 pilosola A in Bidens pilosa 362 Pinaceae 272, 274 pine 109, 146, 202, 204, 275, 340 pine weevil 199, 200, 210, 213, 215 pink bollworm 62, 63, 65, 310

495 pink stem borer 411 pinnate 126, 129, 340 pinoresinol in Melia azedarach L. 95, 112 Pinus densiflora 275, 280 Pinus edulis, 52 Pinus sylvestris 146 Piperaceae 303, 384 piperales 303 pipercide 303 piperidine alkaloids 139 piperitone 36 pirolidone 139 pirolysine 139 pisatin 146 Pisum sativum 130, 147, 410 Plagioneda erythroptera 99 plant antimicrobials 45, 46, 49, 52–55 plant extracts 68, 125, 149, 150, 154–156, 158, 159, 181, 182, 190, 192, 208, 226, 239, 270, 272, 275, 279, 281, 294, 297, 383, 387 plant resistance 46, 145, 224, 430 Plasmodium falsiparum 338 Plasmopara viticola 431 Pleurotus ostreatus 130 plumbagin 49, 51, 312 Plutella xylostella L. 100, 108, 171, 174, 179, 296, 304, 306, 314 Pneumocystis carinii 352 Poaceae 272, 274, 307, 384 Podocarpus 68 podophyllotoxin 256, 261, 262, 368 in Linum species 261 in Podophyllum 261 podophyllotoxin-like lignans in Bursera simaruba 368, 369 Podosphaera fusca 431 Pogostemon cablin 32 polyacetylenes in Bidens pilosa 361, 362 polyacylated neohesperidosides 52 polyetilenes 146 polygalacturonids 428 polymethoxyflavones 52 polyphagous 66, 222, 224, 225, 233, 293, 310, 382, 397 polyphenol oxidase (PPO) 2 polythienyl, 5-(3-butene-1-ynyl)-2,2’bithienyl in Tagetes erecta 343

496 pongam oil 129 Pongamia pinnata 128–129, 131, 132 pongarotene 129 Popillia japonica 35 postinhibitins 145 potato 143, 144, 146, 161, 265, 307, 331, 342, 406, 407, 411 potato blight 425, 428 Pratylenchus sp. 342 precocene I in Bidens pilosa 362 precosene 334 predator 39, 113, 115 predatory mites 38 preservation 125, 140, 440, 443 preventive 381, 382, 430 Priocyphus bosqui 99 propagules 428, 435, 438–440, 460 propanol 69, 70 Propionibacterium acnes 74 Prostephanus truncates 200, 329, 410 Proteaceae 426 protease inhibitors 253, 265–267, 411, 415 protein kinase C 261 protein receptors 300 proteinases 64, 411 proteins 3, 18, 65, 75, 223, 254, 256, 258, 405, 408 protoanemonin 258 PR-proteins 428 Prunus dulcis var. amara 274, 276 Prunus sp. Pseudomonas aeruginosa 47, 127, 129, 132, 366 Pseudomonas solanacearum 352 Pseudomonas syringae pv maculicola 52 Pseudoperonospora cubensis 431 Pseudoscorporella herpotrichoides 143 Psoralea corylifolia 130, 132 psoralen 148, 152, 161, 298 pteridophytes 133 Puccinia cacabata 329 Puccinia kuehnli 425 Puccinia recondita 128, 131 pulegone 30, 31, 33, 36, 37, 152, 153, 161, 278 pupal weight 107, 109, 185, 387 pupation 4, 7, 10, 13, 20, 111, 182, 387 pure components 125 purgative 337, 368 purine 139, 299

Subject Index pyranoses in Bidens pilosa 362 pyrethroid 199, 205, 206, 241, 380 pyrethroid-resistant 304 pyrethrum in Chrysanthemum cinerariifolium [(Trevir.)Vis.] 172, 281, 379 Pyricularia (= teleomorph Magnaporthe) grisea 423 Pyricularia grisea 128, 131, 425, 431, 432, 454, 456 pyrithione 49 pyroangolensolide in Melia azedarach L. 95 pyrones 306 pyrrolizidine 256 264, 299, 310, 315 Pythium ultimum 37, 141 Pythium violae 143 QacA pump 46 quality control 281, 282, 398 Quassia amara 379, 383, 384 quassin in Quassia amara 390 quassinoids in Picrasma ailenthoides 296 in Quassia amara 379 quercetin 3, 4, 19, 297, 363 in Bidens pilosa 362 quercetin-3-methyl ether 297, 331 quercetin-3-O-rhamnosyl[1?6]glucoside see rutin Quercus robur L. 110 quinine(s) 144, 146, 256, 264, 299 quinoline alkaloids (arborinine and several acridone derivatives) in Ruta chalepensis 394 quinolizidine 262, 265, 266, 310 quinolizidine alkaloids 262, 265, 266, 310 quinones 64, 139, 256, 258, 380 racemes 128 Rachiplusia nu Gueneé 99 radial growth assays 455 ranunculales 47 raw materials 126, 241, 281 receptors 31, 75, 225, 226, 229, 258, 283, 300, 306, 317, 396 red capsantin in Capsicum annuum var. annuum 351 red thyme 37

Subject Index refinement 30, 281 registration 281 remirol 307 reniform 128 repellency gustatory 236 olfactory 236 repellent 35, 109, 172, 176, 192, 208, 224, 235, 237–239, 270, 275, 282, 381 reserpine 45 resveratrol in Yucca periculosa F. Baker 4 resistance 1, 3, 9, 45, 66, 425, 430 resistance nodulation division (RND) 45–47 respiratory chain 261, 300, 431 resveratrol 3, 4, 6, 49, 51, 146, 305 Retithrips syriacus (Mayet) 224 rhein, 49, 51, 55, 128, 131 Rheum rhapunticum 208 rheumatism 328, 345, 354 rhinitis 270, 271 Rhizobium etli 49 Rhizoctonia solani 37, 128, 131, 141, 143, 145, 328, 329, 333, 337 Rhizoctonia sp. 146, 150, 151, 155 Rhizopus nigricans 130 rhizosphere 145, 146, 443 rhizosphere microfungi 436 Rhodnius neglectus 32 Rhodnius prolixus 112 Rhododendron molle G. Don 173, 184 rhodojaponin-III in Rhododendron molle G. Don 184 rhodomolin A (R-A) in Rhododendron molle G. Don 185 rhodomolin B (R-B) in Rhododendron molle G. Don 185 rhodomolin C (R-C) in Rhododendron molle G. Don 185 Rhodotorula rubra 127 Rhopalosiphum padi 406 rhubarb 49, 208 Rhus javanica 62 Rhyzopertha dominica 32, 200 ribosome inactivating proteins 408 rice 34, 99, 103, 146, 224, 297, 406, 409, 411, 413 rice blast 145, 423, 425, 431, 432, 456 ricin 408 ringworm 127, 338

497 rishitin 146 in Lycopersicon esculentum 146 in Nicotiana tabacum L. 146 RNA 254, 264, 425 rocaglamide 313, 314 Roldana barba-johannis 10, 13–14 Roman candle tree 126 romasillo 9 root-rot 140, 143, 426 ROS 65 Roselinea necatrix 150, 156, 158 rosemary 31, 34, 36, 236, 237 rotenoids 129, 131 in Stemona collinae Craib 304 in Tephrosia vogelii (Hook f.) 179 rotenone in Amorpha fruticosa L. 172 in D. chinesis (L.)178 in Derris elliptica (Wallich) Benth 178 in Derris indica (Lam.) Bennet 172 in Derris spp 178, 379 in Lonchocarpus nicou (Aublet) 172 in Lonchocarpus spp.178, 379 in Milletia pachycarpa Baker 178 in Milletia reticulate L. 178 in Tephrosia purpurea L. 172 in Tephrosia vogelii Hook 178 rust 142, 150, 425 Ruta chalepensis 384 Rutaceae 30, 264, 384 rutin in Melia azedarach L. 95 in Ruta chalepensis 394 ryania in Ryania speciosa 379 ryanodol monoacetate 300 rye 409 sabadilla, 305, 379 in Schoenocaulon officinale 305, 379 Saccharomyces cerevisiae 52, 127, 334, 352 salannal in Melia azedarach L. 95 salannin in Melia azedarach L. 95 salicylaldehyde 229, 231, 235, 275, 278, 281 salicylic acid 145, 252, 262 Salmonella typhi 129, 130 Salmonella typhosa 126, 127

498 Sambucus nigra 150, 155 sandaracopimaradiols in Bidens pilosa 362 sanguinarine 142, 152, 153, 161, 256, 264 sanskrita 127 santamarin 4 Sapium sebiferum (L.) Roxb 190, 191 sapogenins 129, 328, 349, 357 in Yucca spp. 356 saponins 61, 68–77, 251, 252, 256, 295–296, 331, 339, 390 saprophytes 434 sargachromenol in Bidens pilosa in Roldana barba-johannis 13 sargahydroquinoic acid in Roldana barba-johannis 13, 14 sargaquinoic acid 13 sarmutodise in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 sativan 147 sativin 130 Satureja thymbra 141, 161 SBU 408 scab 141, 143, 144 scabequinone 307 scabies 127 scarification 208 schaftosides 297 Schistocerca gregaria 107, 209, 317 Schistocerca gregaria (Forsk.) 113 Schistosoma mansoni 359 Schizaphis graminum (Rondani) 113 Schoenocaulon officinale 305, 379 Sclerotinia sclerotiorum 37 Sclerotium rolfsii 131, 132, 143 sclerotization 2, 20 scoparone (6,7-dimethoxy-coumarin) in Tagetes lucida 15 scopoletin in Melia azedarach L. 95 scopoletin (6-methoxy-7-hydroxycoumarin) in Tagetes lucida 14 scorpion 414 scorpion toxins 413–415 Scutellaria 315 Scutellaria galericulata 315 SDDS 35 Secale cereale 409 Sechium pittieri 383, 399

Subject Index secoisolariciresinol in Araucaria araucana (Mol.) K. Koch 15 Secondary metabolitos 1–3, 15, 20, 45, 46, 61, 63, 66, 81, 139, 142, 145, 149, 188, 228, 251–267 sedative 337, 345 seed husk 131 seed kernel extract 82, 97 Segestria florentina 407 selective pesticides 415 selinene 129 sendanin in Melia azedarach L. 95 Senecio palmensis 317 senecionine N-oxide 299 sensilla 225, 226, 293, 300, 302, 306, 307, 396 sensilla styloconica 396 Sesamia inferens 411 Sesamia nonagrioides Lefe`bre 110 sesquiterpene lactones 139, 182–184, 258, 317 sesquiterpenes 30, 31, 232, 252, 317, 330 sesquiterpenic lactone in Chrysanthemum parthenium 145 sesquiterpenoid dialdehyde 302 Shigella flexneri 366 Shorea spp. 275 Sideritis akmanii 315 Sideritis rubriflora 315 sideroxol 315 sideroxylin 308 Silene foetida 150, 157–159 silthiopham 431 Simaroubaceae 296, 379, 399 sinigrin 293, 313 Sitophilus granarius 33 Sitophilus oryzae (L.) 32, 33, 99 Sitophilus zeamais 32, 411 sitosterol in Melia azedarach L. 95 Sitotroga cerealella 32 sloperine in Sophora alopecuroids L. 186 small multi-drug resistance (SMR) 45, 46 smillagenin 348, 349 in Agave lechuguilla Torrey 349 smut 142 sodium channel antagonists 300 soft rots 141 Sogotella furcifera (Horvath) 112

Subject Index Solanaceae 147, 299, 316, 379 Solanum 66, 115, 147, 148, 316 Solavetivone 146 in Nicotiana tabacum L. 146 sophocarpine in Sophora alopecuroids L. 186 sophoradine in Sophora alopecuroids L. 186 sophoramin in Sophora alopecuroids L. 186 sorosı´ 388 South America 127, 326, 336, 350, 360, 425 soyacystatin L in soybean 411 soyacystatin N in soybean 411 soybean(s) 49 spearmint 272 specionin 314 specks 128 spider toxins 415 Spilosoma virginica 99 Spiro enol ether 173, 176, 177 spiroketal enol ethers in Artemisia 317 Spodoptera eridania (Cramer) 241 Spodoptera exigua 308 Spodoptera exigua Hu¨bner 171 Spodoptera frugiperda (J.E. Smith) 3, 105 Spodoptera littoralis (Boisd.) 106 Spodoptera litura (Fabricius) 99, 105 Spodoptera venalba 181 Spofdoptera ornithogalli(Guen) 99 Spongospora subterranea 143 sporangia 443 spores 142, 234, 238, 428, 430, 436, 438, 440, 443, 443, 446, 454, 460 spruce 200, 202, 205, 209, 210, 213 spruce budworm 210, 314, 412 squalene in Bidens pilosa 362 St. John’s Wort 52 standardization 29, 39, 270, 281 Staphylococcus epidermidis 365 Staphylococcus pneumoniae 365 Staphylococcus aureus 46, 126, 127, 129, 130, 355, 365 stearic acid 129 Steinernema glaseri 342 Stellera chamaejasme L. 173, 187 Stemonaceae 304

499 Stenchaetothrips biformis Bagnall 224 steroid saponins 139 steroidal avenacosides 307 steroidal saponin 295 steroide alkaloids 265, 316 steroids 148, 252, 348, 349, 380 stigmasterols in Bidens pilosa 362 stilbene 4, 6, 19, 52, 146, 297 stilbenoids 3, 19 stimulodeterrent diversionary strategy 35 Stramenopila 329 Streptococcus pyogenes 352 streptomycin 47, 54 strobilurins 430, 432 strophanthidin-based glycosides in Strophanthus divaricatus (Lour.) Hooker & Arnnott 188 structural 2, 20, 52, 133, 252, 258, 411, 423 strumaroside 174, 190 in Xanthium sibiricum Patrin ex Widder 190 strychnine 293, 299 subtropics 126 sugar cane orange rust 425 sulphurated aminoacids 145 superoxide anion 65 suppressant 381 surfactant 70, 75–77, 103, 358, 386, 450 sustainable plant disease management 126 swallowtail 303 sweet potato 147, 307 Swietenia 2, 313, 382 sword bean 384, 388 symbionts 424 synthetic 1, 20, 39, 47, 55, 61, 67, 81, 104, 108, 139, 164, 171, 172, 191, 199, 205, 266, 269, 279, 294, 311, 379, 397, 399, 431, 463 T. rubrum 127, 128, 130, 329 tacaco 383, 386–388, 390–392, 394, 398, 399 Taeniothrips inconsequens (Uzel) 233 Tagetes erecta 339–343 Tagetes lucida 10, 14–15 tail 71, 76, 414 Talisia esculenta 406 Tamarindus indica 129 tamaron 104 Tanacetum vulgare 32

500 tannic acid 62, 64–66, 279, 280, 284, 363 in Bidens pilosa 362 tannin 18, 61–67, 127, 139, 224, 237, 308, 364, 380 in Melia azedarach L. 82 in Yucca spp. 358 tansy 32, 238 taro 407 tarragon 446, 448 taxol A 261 Taxols 261 Taxus baccata 261 Taxus brevifolia 261 t-cinnamic acid 49 tea tree 35, 278 techniques 128, 150, 151, 205, 208, 435 tenulin in Helenium amarum 317 Tephrosia vogelii 178, 179, 384 tephrosin 178 teracrylmelazolide A in Melia azedarach L. 96 teracrylmelazolide B in Melia azedarach L. 96 termiticidal activity 334 terpenoids in Ruta chalepensis 394 in Yucca spp. 358 terpin-4-ol 36 terpinen-4-ol 36 Tessaratoma papillosa 86 choice 98–101, 108, 227 no choice 8, 99, 101, 102, 108, 115, 227 tetracosane-4-olide in Fluorensia cernua 335 Tetradenia riparia 32 tetrahydrofuran acetogenins 300 tetrahydroxyaurones in Bidens pilosa 362 Tetrahymena pyriformis 73 tetranortriterpenoid 172 Tetranycus urticae 36 tetrazolium salt 454 Teucrium 315 Thaumatopoea pityocampa (Den. & Schiff.) 109 Thaumatopoea processionea L. 109 Thevetia peruviana 141, 150, 155 thickets 126, 354, 357 thiol methyltransferase 405 thiophenes 146

Subject Index thiophene a-terthienyl in Tagetes erecta 343 THQ 4 threo-12-octadec-trans-9-enoic acid 128 thrips 221–249 Thrips calcaratus Uzel 233 Thrips imaginis Bagnall 233 Thrips major Uzel 233 Thrips obscuratus (Crawford) 228 Thrips palmi Karny 115, 222 Thrips pillichi Priesner 233 Thrips tabaci Lindeman 227 Thrips vulgatissimus Haliday 233 triacontane-4-olide in Fluorensia cernua 335 Trichila 313 trichilin D in Melia azedarach L. 93 trichilins in Trichilia spp. 208 tridecapentaynenes in Bidens pilosa 362 tridecatetrayndienes in Bidens pilosa 362 triterpenes in Bursera simaruba 368, 369 Thuja 36 thujone 37 Thujopsis dolabrata var. hondai 273, 275, 277 Thyma spicata 141 Thymbra spicata 161 thyme 31, 34, 37, 285 Thymelaeaceae 177, 261 thymol 30, 31, 33, 34–37, 152, 153, 161, 236 Thysanoptera 115, 222, 226, 228, 230, 237, 242 thysanopteran 221, 226, 229, 234, 241, 242 tigogenin 129 Tilletia indica 425 Tithonia diversifolia 384 Tityus bahiensis 414 TMT 405 tobacco cutworm 34 tocopherol 4 tocopherolquinones in Bidens pilosa 362 tolC mutant 49 tomato 103, 107, 111, 114, 143, 146, 223, 225, 337, 342, 382, 385, 406, 408 tonka bean 272

Subject Index Toona 2, 313 toosendanin 2, 4, 9, 12, 13, 19, 20, 97, 100, 173, 181, 182, 185, 189, 294 topically 102, 108, 112, 127, 180, 240 toxicarol 178 toxicity acute 5, 12, 13, 16, 19, 36, 182, 240, 241, 283, 398, 407 topical 16, 240 toxin(s) 46, 114, 145, 182, 413–415 tracheal mite 35 Trametes versicolor 16 trans-Anethole 30, 34, 37, 278 trans-carveol in Chenopodium ambrosioides L. 338 trans-cinamic acid 141 trans-pinocarveol in Chenopodium ambrosioides L. 338 trans-sabinene hydrate in Chenopodium ambrosioides L. 338 trans-verbenol in Chenopodium ambrosioides L. 338 Trialeurodes vaporariorum 32, 308 Triatoma infestans Klug. 111 Tribolium castaneum 32, 34, 410 Tribolium confusum Duv. 99, 103 trichilin B in Melia azedarach L. 96 trichilin H in Melia azedarach L. 96 Trichloronitrobenzene 144 Trichoderma viride 130 Trichophyton mentagrophytes 16, 126–128, 130 Trichoplusia ni Hu¨bner 171 Trichothecenes 143, 434 Trichothecium roseum. 130 Trichuris, 338 Trifolium subterraneum 310 triglycerides 63 Trigonella foenum-graecum 129, 130 Tripterygium hypoglaucum (level) Hutch. 174, 189 Tripterygium wilfordii Hook. F. 174, 189 triptolide in Tripterygium wilfordii Hook. F. 189 triptonide 189 in Tripterygium wilfordii Hook. F. 189 triterpenoid saponin 295 tropane alkaloids 315, 316

501 tropical forage legume 131 tropics 53, 126, 128, 206 Trychophython mentagrophytes 329 Trypanosoma cruzi 338 Tryporyza incertulas (Walker) 181, 188 trypsin inhibitors 409 tsuga heterophylla 274, 275 tuberculosis 328, 338, 361 tulip tree 300 tumor 264, 337, 415 turnip aphid 34, 171 Tuta absoluta (Meyrick) 107 twinning plant 126 tyramina in Lophophora williamsii (Lem.) Coult. 356 Tyrophagus longior 36 Tyrophagus putrescentiae (Schrank) 269 tyrosinase 2, 3, 18–20, 66 ubichinone receptor 431 ulcers 95, 127, 338, 398 Ulmus spp. 102 undecanal in Chenopodium ambrosioides L. 338 unspecific 148 urace 144 ureases 408, 415 Uromyces phaseoli 338, 342 Urtica dioica 406 Ustilago nuda 128 Uvaria klaineana 273 Uvaria mocoli 272 Uvaria pauci-ovulata 272, 273 valepotriates 258 vanillic acid in Bidens pilosa 363 in Melia azedarach L. 182 in Parthenium argentatum 182 vanillin in Melia azedarach L. 96, 182 Varroa jacobsoni 35, 36 varroa mite 35 vegetable leaf miners 171 Velloziaceae 304 venereal diseases 328 Verbena hybrida 228, 234 vermifuge 337 vernolic acid 128

Subject Index

502 vertebrates 132, 242, 252, 283, 294, 414 Verticillium dahliae 130, 143 Verticillium albo-atrum 143 vesitol, 146 vetiver, 32, 280 Vetiveria zizanioides 32, 274, 280 viability 438, 443 Vicia faba 104 vinblastine in Catharanthus roseus 261 vine 128, 388, 399, 431 viniferin 146 viridiflorol 278 vitamins 52 Vitis vinifera 146 volatile oils 125, 235, 328, 338 V-shaped olfactometer 227 warburganal 302 Warburgia 302 warts 142 wasp 109 water mint 278 waxes 252, 328, 331 Western corn rootworm 293, 299, 317, 409 wheat 103, 114, 144, 406, 408, 409, 434 white pine 200, 210, 275 whiteflies 114, 115, 171, 385–386, 388–392 wild ‘‘tacaco’’ 383, 386–388, 390, 391, 394, 398, 399 wild sunflower 384, 388 wilfordine in Tripterygium wilfordii Hook. F. 189 wilforine 174, 189, 309 wilfortrine 174, 189 wilt 142, 161, 238, 387 winter cress 295 Winteraceae 302, 384 witches’ brooms 142 worm seed 384, 388

wormwood 36, 37 wyerone 146 xanthanodiene 174, 190 in Xanthium sibiricum Patrin ex Widder 190 xanthatin 174, 190 in Xanthium sibiricum Patrin ex Widder 190 xanthin 174, 190 in Xanthium sibiricum Patrin ex Widder 190 Xanthium sibiricum Patrin ex Widder 174, 190 xantofil in Tagetes erecta 343 Xanthogalleruca luteola (Mu¨ller) 99, 102 xanthostrumarin 174, 190 in Xanthium sibiricum Patrin ex Widder 190 xanthotoxin 148, 298 xanthunin 174, 190 in Xanthium sibiricum Patrin ex Widder 190 Xerocomus chrysenteron 407 yam(s) 295 yamogenin 129 yang jiao niu 188 yarrow 208 yeasts 46, 53, 329, 349, 435, 439, 440, 444, 446, 451 yellow mealworm 409, 410 Y-tube olfactometer 227, 233 Yucca periculosa F. Baker 4 Yucca recurvifolia Salisb 359 Yucca schidigera 359 Zabrotes subfasciatus 32, 406 zearalenone 143 zingiberales 307 Zygophyllaceae 326

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