Bacteria in Agrobiology: Crop Ecosystems

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Bacteria in Agrobiology: Crop Ecosystems

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Dinesh K. Maheshwari Editor

Bacteria in Agrobiology: Crop Ecosystems

Editor Prof.(Dr.) Dinesh K. Maheshwari Gurukul Kangri University Deptt. of Botany and Microbiology 249404 Haridwar (Uttarakhand) India [email protected]

ISBN 978-3-642-18356-0 e-ISBN 978-3-642-18357-7 DOI 10.1007/978-3-642-18357-7 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2011926231 # Springer-Verlag Berlin Heidelberg 2011 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover illustration: Optical micrograph showing cross sections of intercellular colonization rice calli and regenerated plantlets by A. caulinodans: CS view of root uninoculated control; magnified cross section view of leaf colonized by A. caulinodans in regenerated rice plant; possible sites of infection and colonization of rice root (from left to right); see also Fig. 3.1 in “Endophytic Bacteria – Perspectives and Applications in Agricultural Crop Production”, Senthilkumar M, R. Anandham, M. Madhaiyan, V. Venkateswaran, Tong Min Sa, in “Bacteria in Agrobiology: Crop Ecosystems, Dinesh K. Maheshwari (Ed.)” Background: Positive immunofluorescence micrograph showing reaction between cells of the rhizobial biofertilizer strain E11 and specific anti-E11 antiserum prepared for autecological biogeography studies; see also Fig. 10.6 in “Beneficial Endophytic Rhizobia as Biofertilizer Inoculants for Rice and the Spatial Ecology of this Bacteria-Plant Association”, Youssef Garas Yanni, Frank B. Dazzo, Mohamed I. Zidan. in “Bacteria in Agrobiology: Crop Ecosystems, Dinesh K. Maheshwari (Ed.)” Cover design: deblik, Berlin Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Bacteria are among the most adaptable organisms. Their evolutional passage across the long timescale, extremely short generation time, and aptitude to adapt to diverse and often hostile environments, combined with the remarkable power of natural selection have made these microorganisms the most resilient of life forms on this planet. As such, bacteria and fungi abound in the soil are the essential contributors in maintaining the ecological balance. One of the most remarkable developments of the twentieth century vis-a`-vis microorganisms is the discovery of the plant growth promoting bacteria (PGPB) that offers a vast array of beneficial attributes to plants, and thereby facilitating enhancement of crop productivity in a sustainable manner. More than 97% of our food requirements are realized from terrestrial ecosystems through agricultural productivity. Diversified populations of bacterial species occur in agricultural fields and contribute to crop productivity directly or indirectly. Plants provide a substantial ecological niche for microorganisms and below ground (roots) portions of plants and soil are constantly associated with a larger number of microorganisms reaping several benefits from such associations. This volume is accordingly conceived to provide consolidated information on the subject. The book entitled Bacteria in Agrobiology: Crop Ecosystems has chapters that cover studies on various aspects of bacteria–plant interactions. Better understandings of the challenges in development of PGPB as efficient commercial bioinoculant have met in enhancing crop production. A large number of bacterial genera interplay with rhizosphere communities in different crops ecosystems, in particular, the oil-yielding crops, cereals, fruits and vegetables, forest trees, etc. Keeping in fitness with such important crops, the developmental challenges faced in the management of growth and soil and seed borne diseases associated with food crops such as rice, sesame, peanut, along with horticultural, sericultural plant ecosystems as well as in forestry are aptly covered in this volume. Detection of PGPR and biocontrol of postharvest pathogens as suitable alternatives to agrochemicals for sustainable crop production and protection, and restoration of degraded soils has also been duly addressed. I believe that this book will be useful not

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only for researchers, teachers, and students, but also for those who are interested in the subjects of applied microbiology, plant protection, ecology, environmental science, and agronomy. I would like to express my gratitude to all the authors for their scholarly contributions. I recognize with credit the continuous support that I received from my research students Mr. Abhinav Aeron, Mr. Rajat Khillon, Mr. Pankaj Kumar, and Dr. Sandeep Kumar in the preparation of this volume. I am also thankful to Council of Scientific and Industrial Research (CSIR), New Delhi; and Director, Uttarakhand Council of Science and Technology (UCOST), Dehradun, India for their support in implementation of my research projects on PGPB that served as a prolog to arrange base for compilation of this book. I extend my earnest appreciation to Dr. Jutta Lindenborn of Springer for her valuable support to facilitate completion of the task. Haridwar, Uttarakhand, India

Dinesh K. Maheshwari

Contents

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Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Abhinav Aeron, Sandeep Kumar, Piyush Pandey, and D.K. Maheshwari

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Bacillus as PGPR in Crop Ecosystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Ankit Kumar, Anil Prakash, and B.N. Johri

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Endophytic Bacteria: Perspectives and Applications in Agricultural Crop Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 M. Senthilkumar, R. Anandham, M. Madhaiyan V. Venkateswaran, and Tongmin Sa

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PGPR Interplay with Rhizosphere Communities and Effect on Plant Growth and Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Gabriele Berg and Christin Zachow

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Impact of Spatial Heterogeneity within Spermosphere and Rhizosphere Environments on Performance of Bacterial Biological Control Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Daniel P. Roberts and Donald Y. Kobayashi

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Biocontrol Mechanisms Employed by PGPR and Strategies of Microbial Antagonists in Disease Control on the Postharvest Environment of Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Anjani M. Karunaratne

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Plant Growth-Promoting Bacteria Associated with Sugarcane . . . . . 165 Samina Mehnaz

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Contents

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Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Ahmet Esitken

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Commercial Potential of Microbial Inoculants for Sheath Blight Management and Yield Enhancement of Rice . . . . . . . . . . . . . . . . . . . . . . . . 237 K. Vijay Krishna Kumar, M.S. Reddy, J.W. Kloepper, K.S. Lawrence X.G. Zhou, D.E. Groth, S. Zhang, R. Sudhakara Rao, Qi Wang M.R.B. Raju, S. Krishnam Raju, W.G. Dilantha Fernando, H. Sudini B. Du, and M.E. Miller

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Beneficial Endophytic Rhizobia as Biofertilizer Inoculants for Rice and the Spatial Ecology of This Bacteria–Plant Association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Y.G. Yanni, F.B. Dazzo, and M.I. Zidan

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Plant Growth-Promoting Bacteria: Fundamentals and Exploitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Clara Pliego, Faina Kamilova, and Ben Lugtenberg

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PGPR in Coniferous Trees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Elke Jurandy Bran Nogueira Cardoso, Rafael Leandro de Figueiredo Vasconcellos, Carlos Marcelo Ribeiro, and Marina Yumi Horta Miyauchi

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Perspectives of PGPR in Agri-Ecosystems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 Meenu Saraf, Shalini Rajkumar, and Tithi Saha

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Ecofriendly Management of Charcoal Rot and Fusarium Wilt Diseases in Sesame (Sesamum indicum L.) . . . . . . . . . . . . . . . . . . . . . . 387 Sandeep Kumar, Abhinav Aeron, Piyush Pandey, and Dinesh Kumar Maheshwari

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Crop Health Improvement with Groundnut Associated Bacteria . . 407 Swarnalee Dutta, Manjeet Kaur, and Appa Rao Podile

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431

Contributors

Abhinav Aeron Department of Botany and Microbiology, Faculty of Life Sciences, Gurukul Kangri University, Haridwar 249404, Uttarakhand, India, abhinavaeron@ gmail.com Rangasamy Anandham Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai 625104, Tamil Nadu, India, [email protected] Gabriele Berg Environmental Biotechnology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria, [email protected] Elke Jurandy Bran Nogueira Cardoso Soil Microbiology Laboratory, Department of Soil Science, Luiz de Queiroz College of Agriculture, Saõ Paulo State University, Piracicaba Saõ Paulo, Brazil, [email protected] Frank B. Dazzo Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA, [email protected] B. Du Department of Microbiology, Shandong Agricultural University, Taian Shandong Province, China Swarnalee Dutta Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India Ahmet Esitken Department of Horticulture, Faculty of Agriculture, Ataturk University, 25240 Erzurum, Turkey, [email protected] W.G. Dilantha Fernando Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada, [email protected]

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D.E. Groth LSU AgCenter, Rice Research Station, Baton Rouge, LA, USA Bhavdish N. Johri Department of Biotechnology and Bioinformatics Centre, Barkatullah University, Bhopal 462026, Madhya Pradesh, India, bhavdishnjohri@ rediffmail.com Faina Kamilova Koppert Biological Systems, Veilingweg 14, PO Box 155, 2650 AD Berkel en Rodenrijs, The Netherlands, [email protected] Anjani M. Karunaratne Department of Botany, Faculty of Science, University of Peradeniya, Peradeniya, Sri Lanka, [email protected] Manjeet Kaur Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India Joseph W. Kloepper Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA Donald Y. Kobayashi Sustainable Agricultural Systems Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, USDA-ARS, Beltsville, MD 20701, USA; Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA Sandeep Kumar Department of Botany and Microbiology, Faculty of Life Sciences, Gurukul Kangri University, Haridwar 249404, Uttarakhand, India, [email protected] K. Vijay Krishna Kumar Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA; Acharya N G Ranga Agricultural University, Hyderabad, India Ankit Kumar Department of Biotechnology and Bioinformatics Centre, Barkatullah University, Bhopal 462026, Madhya Pradesh, India, [email protected] K.S. Lawrence Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA Ben J.J. Lugtenberg Sylvius Laboratory, Institute of Biology, Leiden University, Sylviusweg 72, PO Box 9505, 2300 RA Leiden, The Netherlands, Ben.Lugtenberg @gmail.com Munuswamy Madhaiyan Department of Agricultural Chemistry, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea

Contributors

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Samina Mehnaz Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan; Institute of Pharmaceutical Biology, Bonn University, Bonn 53115, Germany, [email protected]. pk, [email protected] M.E. Miller Department of Biological Sciences, Auburn University, Auburn, AL, USA Tongmin Sa Department of Agricultural Chemistry, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea, [email protected] Marina Yumi Horta Miyauchi Soil Microbiology Laboratory, Department of Soil Science, Luiz de Queiroz College of Agriculture, Saõ Paulo State University, Piracicaba, Saõ Paulo, Brazil Senthilkumar Murugesan Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore 641003, Tamil Nadu, India, senthiltnj@ rediffmail.com Piyush Pandey Department of Biotechnology, S. B. S. P. G. Institute of Biomedical Sciences and Research, Balawala, Dehradun 248161, Uttarakhand, India Clara Pliego Instituto de Hortofruticultura Subtropical y Mediterrnea “La Mayora”, Universidad de Mlaga – Consejo Superior de Investigaciones Cientı´ficas (IHSM´ rea de Gene´tica, Universidad de Mlaga, Campus de Teatinos s/n, UMA-CSIC), A 29071 Mlaga, Spain; Division of Biology, Department of Life Science, Imperial College London, Imperial College Road, SW7 2AZ London, UK, [email protected], [email protected] Appa Rao Podile Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India, [email protected], apparaopodile@ yahoo.com Anil Prakash Department of Biotechnology and Bioinformatics Centre, Barkatullah University, Bhopal 462026, Madhya Pradesh, India, [email protected] Shalini Rajkumar Institute of Science, Nirma University, S. G. Highway, Ahmedabad 382481, Gujarat, India M.R.B. Raju Andhra Pradesh Rice Research Institute, Maruteru, India S. Krishnam Raju Andhra Pradesh Rice Research Institute, Maruteru, India

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Contributors

R. Sudhakara Rao Acharya N G Ranga Agricultural University, Hyderabad, India M. Sudhakara Reddy Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA, [email protected] Carlos Marcelo Ribeiro Soil Microbiology Laboratory, Department of Soil Science, Luiz de Queiroz College of Agriculture, Saõ Paulo State University, Piracicaba, Saõ Paulo, Brazil Daniel P. Roberts Sustainable Agricultural Systems Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, USDA-ARS, Beltsville MD 20701, USA; Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA, [email protected] Tithi Saha Institute of Science, Nirma University, S. G. Highway, Ahmedabad 382481, Gujarat, India Meenu Saraf Department of Microbiology, Gujarat University, Ahmedabad 380009, Gujarat, India, [email protected] H. Sudini International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India Rafael Leandro de Figueiredo Vasconcellos Soil Microbiology Laboratory, Department of Soil Science, Luiz de Queiroz College of Agriculture, Saõ Paulo State University, Piracicaba, Saõ Paulo, Brazil V. Venkateswaran Ministry of Food Processing Industries, New Delhi 110049, India Qi Wang China Agricultural University, Beijing, China Youssef Garas Yanni Department of Microbiology, Sakha Agricultural Research Station, Kafr El-Sheikh 33717, Egypt, [email protected] Christin Zachow Environmental Biotechnology, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria, [email protected] Shouan Zhang Tropical REC, University of Florida, Homestead FL, USA, [email protected] Mohamed I. Zidan Department of Plant Nutrition, Sakha Agricultural Research Station, Kafr El-Sheikh 33717, Egypt

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Chapter 1

Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology Abhinav Aeron, Sandeep Kumar, Piyush Pandey, and D.K. Maheshwari

1.1

Introduction

Declining crop productivity due to unsuitable agricultural practices over the years and a galloping rate of population growth have both put up a severe strain on the food supply situation in the world. To meet the food requirements of the growing population, a second green revolution has become imperative due to “loss of dynamism” in agriculture as pointed out in global economic survey during the year 2007–2008. This has two obvious objectives, firstly to rejuvenate the agricultural sector and secondly to improve the income of those dependent on it. Pertaining to massive population pressure, increase in food grain production is an uphill task in today’s world. The need of the day is sustainable agriculture without harming the delicate balance of soil ecology as well as unlocking the mystery of biota influencing plant growth by using plant growth promoting rhizobacteria (PGPR). PGPR are nowadays applied in a wide array of agro and allied industries in the form of inoculants in a range of agro-economically important plants including leguminous and nonleguminous crops, trees and plants of forest, horticulture, sericulture, medicinal, fodder, oilseed, and cash crops for enhancing their growth and productivity. Green revolution was achieved as it resulted in increased yield due to extensive use of chemical based components. The indiscriminate use of these components imparted pesticide resistance in pests and made presence in plant produce. Presence of residual pesticides cause disruption and degradation of agro-ecosystem resulting in decreased soil fertility. Excessive application of fertilizer for obtaining higher production was not only undesirable from the economic point of view, but also

A. Aeron, S. Kumar, and D.K. Maheshwari (*) Department of Botany and Microbiology, Gurukul Kangri University, Haridwar 249 404, Uttarakhand, India e-mail: [email protected] P. Pandey Department of Biotechnology, S. B. S. P. G. Institute of Biomedical Sciences and Research, Balawala, Dehradun 248 161, Uttarakhand, India

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, DOI 10.1007/978-3-642-18357-7_1, # Springer-Verlag Berlin Heidelberg 2011

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exerted an adverse effect on the environment and crop quality (Kenny 1982). It led to nutrient imbalance, whereas inefficient and overuse of chemical fertilizers resulted in considerable economic loss to the farmers (Ayala and Rao 2002). It is widely believed that agrochemicals including chemical fertilizers reduce the population of beneficial microorganisms thus having an all-embracing effect (Smiley 1981). Reduction in the population of desirable beneficial microbes alters the topology of top soil and reduces the productivity of fertile soils. Thus, an important factor in this respect is to maintain the enhancement of soil fertility through appropriate sustainable technology, which should be achieved to replenish the nutrients so as to build up the nutrient status of soils (Hera 1996). The challenges of meeting the food requirements of the burgeoning population and plateauing productivity of agricultural lands can only be met by a second green revolution or ever green revolution. Some of the strategies that can be channeled to second green revolution include micro-irrigation system, organic farming, precision farming, green agriculture, eco-agriculture, white agriculture, straw revolution, and use of PGPR and their combinations. The aim of this chapter is to elaborate the need of PGPR applications in agriculture-based industries for economic development in an eco-friendly manner.

1.2

Soil and Rhizosphere in Sustainable Agriculture

Agricultural industries are mainly soil based because they extract nutrients from the soil. Effective and efficient approaches to slowing the removal and returning nutrients to the soil is required in order to maintain and increase crop productivity apart from efforts to sustain agriculture for the long term. The overall strategy for increasing crop yields and sustaining them at high level required natural or artificial inputs. The soil is managed by both biological and nonbiological factors known to have a major impact on plant growth, soil fertility, and agricultural sustainability. The physical, biological, and chemical characteristics of soil, such as organic matter content, pH, texture, depth, and water-retention capacity, are factors that influence soil fertility. A soil’s potential for producing crops is largely determined by the environment that soil provides for root growth, such as nutrients and the surrounding microflora that may be beneficial or deleterious. Roots need air, water, nutrients, and adequate space to develop. Soil quality is defined by capacity to store water, acidity, depth, and density that determine how well roots developed. Changes in soil quality affect the health and productivity of the plants and can lead to lower yields and/or higher costs of production. Organic matter content is important for the proper management of soil fertility and helps growth by improving water-holding capacity and drought resistance. Moreover, it permits better aeration, enhances the absorption and release of nutrients, and makes the soil less susceptible to leaching and erosion. The higher plant root system significantly contributes to the establishment of the microbial population in the rhizosphere. The rhizosphere has attracted much interest

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as it is a habitat of several biologically important processes and their interactions. Acidification of the rhizosphere as a result of exudation of organic acids from root plays a pivotal role in determining the surrounding population (Dakora and Philips 2002). The rhizosphere is populated by diverse microorganisms including bacteria, fungi, actinomycetes, protozoa, algae, etc.; therefore, modifying plant root systems is considered as a means of crop improvement targeted toward low-resource environments, particularly low nutrient and drought-prone agriculture. Microbial processes and properties in the rhizosphere are crucial to support functional agriculture.

1.3

Beneficial Bacteria

The microbe–plant interaction in the rhizosphere is dynamic and complicated. Some microbes contribute to plant health by mobilizing nutrients, while some are detrimental to plant health as they compete with the plant for nutrients or cause disease and some stimulate plant growth by producing hormones or by suppressing pathogens. The bacteria useful to plants are characterized into two general types: bacteria forming a symbiotic relationship with the plant and the free-living ones found in the soil but are often found near, on, or even within the plant tissues (Kloepper et al. 1988a; Frommel et al. 1991). Different authors have found different origins with the classification and definition of beneficial rhizobacteria. Beneficial free-living soil bacteria that enhance plant growth are usually referred to as “plant growth promoting rhizobacteria” (Kloepper et al. 1989) or yield increasing bacteria (YIB) (Tang 1994). PGPR originally defined (Kloepper and Schorth 1978) as root-colonizing bacteria (rhizobacteria) cause either growth promotion or biological control of plant diseases. Bashan and Holguin (1998) proposed that the PGPR can be categorized as biocontrol-plant growth promoting bacteria (PGPB) and phytostimulating PGPB. Root-associated bacteria have a great influence on organic matter decomposition which in turn is reflected in soil nutrient availability for plant growth (Glick et al. 1994). The phosphorus- and potassium-solubilizing bacteria (PSB) may enhance plant nutrient availability by dissolving insoluble phosphorus and releasing potassium from silicate minerals (Goldstein and Liu 1987). PGPB often help increase root surface area to increase nutrient uptake and in turn enhance plant production (Mantelin and Touraine 2004). The premier example of PGPR agents occur in many genera including Actinoplanes, Agrobacterium, Alcaligens, Amorphosporangium, Arthrobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Cellulomonas, Enterobacter, Erwinia, Flavobacterium, Gluconacetobacter, Microbacterium, Micromonospora, Pseudomonas, Rhizobia, Serratia, Streptomyces, Xanthomonas, etc., as stated by several workers (Kloepper et al. 1989; Tang 1994; Okon and Labandera-Gonzalez 1994; Glick et al. 1999; Mayak et al. 2001; Lucy et al. 2004; Tahmatsidou et al. 2006; Aslantas et al. 2007; Lee et al. 2008; Pedraza et al. 2010).

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One of the dominant genera among PGPR is Pseudomonas spp. reported in the biological control of different phytopathogenic fungal species such as Rhizoctonia, Fusarium, Sclerotonia, Pythium, Erwinia, Macrophomina, etc. (Defago et al. 1990; Gupta et al. 2001a; Garbeva et al. 2004; Validov et al. 2005). Interestingly, certain rhizobia have also been noticed for the biological control of M. phaseolina (Arora et al. 2001; Deshwal et al. 2003), F. oxysporum, F. solani, R. solani, Pythium spp., etc. (Chao 1990). Rhizobia had been reported to produce several secondary metabolites for biocontrol activity, similar to fluorescent pseudomonads in their mode of action. Pseudomonads confer active role in biocontrol and yield promotion of plants, and therefore are the most widely used genera among PGPR (Gupta et al. 2001a; Kumar et al. 2005a, b, c). Among free living bacteria, various species of Azotobacter have been reported for the biological control of different phytopathogens such as Alternaria, Helminthosporium, Fusarium, etc., under in vitro conditions (Laxmikumari et al. 1975; Joshi et al. 2006a). Free nitrogen-fixing bacteria were probably the first rhizobacteria used to promote plant growth. Other bacterial genera capable of nitrogen fixation that may be responsible for growth promotion effect are Azoarcus sp., Burkholderia sp., Gluconacetobacter diazotrophicus, Herbaspirillum sp., Azotobacter sp., and Paenibacillus polymyxa. These genera have been isolated from a number of plant species such as rice, sugarcane, corn, sorghum, other cereals, pineapple, and coffee bean (Vessey 2003). Azoarcus has recently gained attention due to its great genetic and metabolic diversity. Because of their competitive advantages in a carbonrich, nitrogen-poor environment, diazotrophs become selectively enriched in the rhizosphere (Reinhold-Hurek and Hurek 2000). Azotobacter spp. is also being applied as bioinoculant due to its several direct PGP activities including asymbiotic nitrogen fixation, phosphate solubilization, growth hormones production, and vitamins production (Shende et al. 1977). The first reports on Azotobacter appeared in 1902 and it was widely used in Eastern Europe during the middle decades of the last century (Gonza´lez and Lluch 1992). As previously suggested, the effect of Azotobacter and Azospirillum is attributed not only to the amounts of fixed nitrogen but also to the production of plant growth regulators such as indole acetic acid (IAA), gibberellic acid, cytokinins, and vitamins (Rodelas et al. 1999). Azotobacter is also known to produce antifungal compound that inhibits the production of conidia of Botrytis cinerea (Doneche and Marcantoni 1992). Similarly, Azospirillum is also known to secrete phytohormones, induce root cell differentiation, and increase water uptake. Azospirillum associates with polysaccharide degrading bacteria (PDB) in rhizosphere, establishing a metabolic association (Bashan and Holguin 1997). The sugar-degrading bacteria produce degradation and fermentation products that are used by Azospirillum as a carbon source that in turn provides PDB with nitrogen. In fact, here the symbiosis is extended to multiple prokaryotic interactions. Other example includes the association between Azospirillum and Bacillus that degrades pectin, Azospirillum and Cellulomonas degrade cellulose, and Azospirillum and Enterobacter cloacae that ferment glucose (Kaiser 1995; Khammas and Kaiser 1992; Halsall 1993). Production and release of plant growth regulators by bacteria causes an alteration in the endogenous levels of the plant growth regulator. Other growth regulators such as cytokinins are less common


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among PGPR, while gibberellin production in high concentrations has only been described from the genus Bacillus. Different genera of bacteria, such as Proteus mirabilus, P. vulgaris, Klebsiella pneumoniae, B. cereus, Escherichia coli, etc., produce auxins, cytokinins, gibberellins, and abscisic acid (Griffith and Ewart 1995). Symbiotic bacteria generally termed as rhizobia for a broad group of noduleinhabiting symbionts have been used as inoculants for well over a century. These organisms were used to enhance nodulation and N-fixation among legumes. Their roles were limited earlier, but have been extended to an extent as major solubilizers of inorganic phosphate, making it available for the plants. Further their biocontrol credentials were proved when the genera Bradyrhizobium and Rhizobium reported to produce antibiotics effectively controlling fungal pathogens (Chakraborty and Purkayastha 1984; Briel et al. 1996). Sinorhizobium meliloti showed antagonism toward F. oxysporum and M. phaseolina regardless of their symbiotic effectiveness in presence of pathogen and increased overall growth of groundnut (Arora et al. 2001). Deshwal et al. (2003) isolated bradyrhizobia from the root nodules that antagonized M. phaseolina in vitro which increased under iron-limited conditions. One of the mechanisms of biocontrol by rhizobia and bradyrhizobia was established due to the production of siderophores resulting in increased growth of Arachis hypogaea. The adaptability of introduced strains to achieve equilibrium within an aboriginal niche is limited, but identification, screening, and application of local strain have been advocated (Bashan 1998; Aeron et al. 2010). Various strains of species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus are known as potential elicitors of induced systemic resistance (ISR) and exhibit significant reduction in the incidence or severity of various diseases on diverse hosts (Kloepper et al. 2004). Certain volatile compounds, especially 3-hydroxy-2-butanone (acetoin) and 2, 3butanediol, released by the B. subtilis and B. amyloliquefaciens in rhizosphere play a crucial role in the elicitation of ISR (Ryu et al. 2003). More recently, Choudhary and Johri (2008) have reviewed the significance of ISR by Bacillus spp. in relation to the biological control of pathogenic organisms. Bacillus species are believed to enhance the plant growth through synthesis of plant growth regulators such as auxins (indole-3-acetic acid) and gibberellic acid (Wipat and Harwood 1999). However, more recently representatives of B. subtilis/B. amyloliquefaciens group have been shown to produce substances with IAA-like activity; reasonable amount of IAA was produced by B. amyloliquefaciens FZB42 when fed with tryptophan (Idris et al. 2004). Based on studies of wheat rhizosphere colonization by Bacillus species, it seems that rhizosphere competent genotypes occur in this bacterium (Milus and Rothrock 1993; Mavingui et al. 1992; Maplestone and Campbell 1989; Juhnke et al. 1987). Enhancement of plant growth by root-colonizing species of Bacillus and Paenibacillus is well documented and PGPR members of the genus Bacillus can provide a solution to the formulation problem encountered during the development of biocontrol agents to be used as commercial products, due in part to their ability to form heat- and desiccation-resistant spores (Kloepper et al. 2004; Emmert and Handelsman 1999).

6

A. Aeron et al.

The organic forms of phosphorous are estimated to comprise between 30 and 50% of total soil phosphorous. This reservoir can be mineralized by microorganisms, making it available to the plant as soluble phosphates. Different PGPR genera are capable of solubilizing phosphate and include Pseudomonas, Bacillus, Rhizobium, Burkholderia, Achromobacter, Agrobacterium, Micrococcus, Aerobacter, Flavobacterium, Chryseobacterium, and Erwinia. The solubilization of phosphate occurs due to the involvement of organic acids and/or by releasing phosphatases responsible for releasing phosphate groups bound to organic matter. Most of these bacteria are able to solubilize the Ca–P complex, and there are others that operate on the Fe–P, Mn–P, and Al–P complexes. Results with PGPR capable of solubilizing phosphate are sometimes erratic, probably due to soil composition. While the inorganic forms occur in minerals as insoluble calcium, iron, or aluminum phosphates, organic phosphates are derived from the decaying plants, animals, and microorganisms. Organic matter is an important reservoir of immobilized phosphate that accounts for 20–80% of soil phosphorus (Goldstein 1986) and only a small portion (0.1%) is available to plants. Phosphatases are known to play a key role in transforming organic forms of phosphorous into plant available inorganic forms. Conversion of the insoluble forms of phosphorous to a form accessible by plants such as orthophosphates is an important criterion. Plant may poorly/not possess an innate ability to acquire phosphorus directly from soil phytate which is a major phosphorous source. The production of enzyme phytase leads to an increase in the availability of phosphorus to plants and in turn the plant uptake (Gyaneshwar et al. 2002). It is known to be secreted by many microorganisms and is involved in the stepwise degradation of phytate to lower phosphate esters. Although plants are known to produce phytase, they display poor activity in roots and other plant organs (Greiner and Alminger 2001). As zinc is a limiting factor in crop production, study on zinc solubilization by bacteria has an immense application in zinc nutrition to plants. Zinc-solubilizing potential of few bacterial genera has been studied along with mobilization of potassium (Sarvanan et al. 2003; Sperberg 1958). Potassium (K) is an essential soil nutrient that performs a multitude of important biological functions to maintain plant growth and quality. Although silicon (Si) is still not recognized as an essential element for plant growth, the beneficial effects of this element on the growth, development, yield, and disease resistance have been observed in a wide variety of plant species. However, plants cannot directly use mineralic K and Si unless they are released by weathering or dissolved in soil water. Studies have documented the release of K and Si during the degradation of silicate minerals by bacteria (Barker et al. 1998; Welch and Vandevivere 1994; Sheng and He 2006). Iron in the Earth’s crust is present in a highly insoluble form of ferric hydroxide (Fe3+), and thus unavailable to microorganisms and plants. Some bacteria have developed iron uptake systems (Neilands and Nakamura 1991). These systems involved a siderophore – an iron binding legend – and an uptake protein needed to transport iron into the cell. Siderophores are low molecular weight (~400–1,000 Da) iron-chelating compounds that bind Fe3+ and transport it back to the cell and make it available for the microbial cells (Briat 1992). The secreted siderophore molecules


7

have a very high affinity (kd ¼ 1020 to 1050) for iron and bind most of the Fe3+ that is available in the rhizosphere and prevent the pathogens present in immediate vicinity from proliferation because of lack of iron (O’Sullivan and O’Gara 1992). Antagonists can prevent the proliferation of fungal phytopathogens by producing siderophores that bind most of the Fe3+ in the rhizosphere. The resulting lack of the iron prevents any fungal pathogen from proliferating in this immediate vicinity. Kloepper et al. (1980) have supported this mechanism and stated that the production of siderophores that chelate and thereby scavenge the ferric iron in the rhizosphere may result in growth inhibition of other microorganisms whose affinity for iron is lower. It has been suggested that the ability to produce specific siderophores and/or to utilize a broad spectrum of siderophores may contribute to the root-colonization ability of biocontrol strains. In addition, siderophores also mediated the iron uptake by plant roots in ironlimiting conditions (Wang et al. 1993). Root colonization is an important first step in interaction of PGPR group of bacteria with host plant (Kloepper and Beauchamp 1992). Effective colonization of plant roots by PGPR plays an important role in growth promotion irrespective of the mechanism of action, i.e., production of metabolites, antibiotics against pathogens, or ISR or even nutrient uptake (Bolwerk et al. 2003). It is now common knowledge that bacteria in natural environments persist by forming biofilms (Davey and O’Toole 2000). The use of microorganism for biological control is a nonhazardous strategy to reduce crop damage caused by plant pathogens. The antagonistic microorganisms are ideal biocontrol agents, as the rhizosphere provides the frontline defense for roots against infection by the pathogens (Lumsden et al. 1987). Biocontrol research has gained considerable attention and appears promising as a viable alternative to chemical control strategies (Rebafka et al. 1993). The protection of root from the attack of the pathogen was due to the production of diverse metabolites like siderophore (Arora et al. 2001) and antifungal metabolites such as rhizobitoxine (Chakraborty and Purkayastha 1984). One of the most effective mechanisms, which antagonists employ to prevent proliferation of phytopathogens, is the synthesis of antibiotics. A large number of antibiotics have been reported from different fluorescent pseudomonads including agrocin-84, agrocin-434, 2, 4diacetyl phloroglucinol, herbicolin, oomycin, phenazine, pyoluteorin, pyrrolnitrin, pyrroles, etc., and they have a role to play in inhibition of pathogens (Colyer and Mount 1984; Gutterson et al. 1986; James and Gutterson 1986). Many Bacillus strains are considered as natural factories of cyclic lipopeptides, including iturins, fengycins, and surfactins, and their involvement in control of plant microbial diseases has been proved (Li et al. 2007; Romero et al. 2007; Yoshida et al. 2001; Asaka and Shoda 1996). Recently, the hydrolytic enzymes have received considerable attention because they play a role in controlling diseases due to plant pathogens. Microorganisms capable of lysing other organisms are widespread in natural ecosystems. The enzymatic digestion or deformation of cell wall components of phytopathogenic fungi by the enzymes chitinase, b-1, 3-glucanase produced by antagonistic bacteria and the hyperparasitism, lysis of phytopathogen propagules present in soil is one of the few logical methods of biological control of soil-borne plant pathogens

8

A. Aeron et al.

(Vaidya et al. 2001). Hyperparasitism occurs when a fungus exists in intimate association with another fungus which derives some nutrients while conferring no benefit in return. Hyperparasitism and lysis of propagules in soil is a logically satisfying method of biological control of soil-borne plant pathogens by microbial antagonists. The production of cell wall degrading or lytic enzymes, such as chitinase, chitosanase, b-1, 3-glucanase, b-1, 4-glucanase, b-1, 6-glucanase, proteases, and lipase (Fridlender et al. 1993; Lim and Kim 1995; Dunne et al. 1997; Vaidya et al. 2001; Vivekanathan et al. 2004), degrades fungal cell walls, resulting in lysis of wall material, leading to cell death. Induction of the systematic resistance against many pathogens has also been reported inducing long-lasting and broad-spectrum systemic resistance against disease-causing agents (Zehnder et al. 2001). Plants do not have an immune system but have evolved a variety of potent defense mechanisms, including the synthesis of low-molecular-weight compounds such as proteins and peptides that have antifungal activity.

1.4

Crop Ecosystem

PGPR can influence plant growth directly but may differ from species to species and even at strain level. Symbiotic plant colonizers and certain free-living bacteria contribute to plant growth by nitrogen fixation. The symbiotic bacteria form a hostspecific symbiosis with legumes. Molecular signal molecules (lipo-oligosaccharide) secreted by these bacteria play a critical role in this process (Lange 2000; Spaink 2000; Perrot et al. 2000). Species of Bacillus are common inhabitants among the resident microflora of inner tissues of various species of plants, including cotton, grape, peas, spruce, and sweet corn, where they play an important role in plant protection and growth promotion (Berg et al. 2005; Shishido et al. 1999; Bell et al. 1995; McInroy and Kloepper 1995; Huang et al. 1993; Hallaksela et al. 1991; Misaghi and Donndelinger 1990). Little work has been done to date concerning the beneficial relationship of Rhizobium and nonleguminous plants, although Wiehe and Hoflich (1995) demonstrated that rhizobia can multiply and survive under field conditions as well as in the rhizosphere of nonhost legumes. The attachment of bacteria with maize, wheat, rice, oat, sunflower, mustard, and asparagus has been reported along with improved growth of certain nonlegumes when inoculated with rhizobia (Planziski et al. 1985; Terouchi and Syono 1990; Yanni et al. 1995; Biswas et al. 2000a, b; Peng et al. 2002). Recently, Chandra et al. (2007) reported that a successful rhizospheric competent Mesorhizobium loti MP6 induced root hair curling, inhibited Sclerotinia sclerotiorum, and enhanced growth of mustard. Specific rhizobacteria have the ability to improve plant growth and/or root health (Kloepper et al. 1980; Suslow and Schrolh 1982; Schippers et al. 1987; Sikora 1988; Weller 1988). A key factor of all PGPR is that they colonize seed and root, or behave as endophytes. Such traits are desirable for considering them suitable for biocontrol activity (Lugtenberg and Bloemberg 2004; Compant et al. 2005).


9

Further, the phenomenon of chemotaxis, flagellar mobility, lipopolysaccharides (LPS) structure, the outer membrane protein OprF and to a lesser extent, presence of pili, all are important for competitive root colonization (Lugtenberg and Bloemberg 2004). However, in field soil, environmental conditions and competition or displacement by the myriad of microorganisms present in the rhizosphere limit colonization. Certainly, use of mutants and promoter probe techniques are the beginning to identify genes in bacteria that are important to root colonization and these are often related to nutrient uptake (Roberts et al. 1997; Rediers et al. 2005). Variation for interaction with PGPR is often dependent on environmental conditions. For example, phosphorus deficiency provokes a differential response to Rhizobium inoculation among common bean cultivars (Vadez et al. 1999; Christiansen and Graham 2002). Such phenotypic variation among cultivars may be, in part, the result of genetic variation and suggests genetic improvement of the host as an approach for development of superior plant growth promoting (PGP) strategies in conjunction with rhizosphere microbial inoculants.

1.5

PGPR in Agrobiology

PGPR are most commonly used in agriculture, and their application in various crops resulted in an average approximate increase of 20–40% in yield across multiple crops all over the world when various reports were combined over last decade. In general, PGPR-carried plant growth benefit owing to increase in seed germination rates, root growth, leaf area, chlorophyll, proteins, and hydraulic activity, fluid movement within the plant, tolerance to drought, low temperature, delayed leaf senescence, disease resistance, and finally enhanced grain size and crop yield of crop, as elaborated for some of the crops in this chapter.

1.5.1

Cereals

In recent years, crop roots association with bacteria and their proliferation in the rhizosphere has been found to be beneficial in most of the cereals (Terouchi and Syono 1990; Biswas et al. 2000a, b; Peng et al. 2002). However, the selection of effective strains is of prime importance for the growth promotion of cereals (Westcott and Kluepfel 1993; Siddiqui and Ehteshamul-Haque 2000). In rice, endophytic strains of Rhizobium leguminosarum br. trifoli E11 and E12 increased grain yield of rice in field inoculation experiment (Yanni et al. 1997). Biswas et al. (2000a, b) reported that rhizobial inoculation increased rice grain yield at different N rates. The benefit of early seedling development could carry over to a significant increase in grain yield at maturity. Earlier, Datta et al. (1982) reported that a P-solubilizing and IAA-producing strain of B. firmus increased the grain yield

10

A. Aeron et al.

and P-uptake of rice in a P-deficient soil amended with rock phosphate. Similarly, increased yield was obtained in wheat, sorghum, and barley due to application of A. brasilense (Okon and Labandera-Gonzalez 1994; Dobbelaere et al. 2001; Saubidet et al. 2002) and with Beijerinikia mobilis and Clostridium sp. in wheat and barley, respectively (Polyanskaya et al. 2000). It was interesting to note that Pseudomonas spp. increased yield of winter wheat by 27% (de Frietas and Germida 1990). Application of P. cepacia, P. fluorescens, and P. putida in winter wheat exhibited antagonism against Rhizotonia solani and Leptosphaera maculans and enhanced yield indirectly in soil reported to be relatively infertile (De Frietas and Germida 1990; 1992). Inoculation of P. cepacia R55, R85 and P. putida R104 increased root and shoot dry weight of winter wheat in R. solani infested soil (de Freitas and Germida 1991). On the other hand, P. chlororaphis 2E3 and 06 when applied on spring wheat showed increased emergence of seedlings. Both strains could also inhibit a dreaded pathogen F. culmorum (Kropp et al. 1996). In another study on winter wheat, application of P. fluorescens enhanced length of seedling and significant increase in plant height and grain yield in Pythium infested soil (Weller and Cook 1986). Iswandi et al. (1987) observed an increased yield in wheat along with maize and barley by Pseudomonas spp. 7NSK2. Rice, wheat, corn, millet, sweet potato, cotton, etc., also showed average yield increase by 10–22.5% after application of YIB (Mei et al. 1990). Chabot et al. (1993) demonstrated R. trifolii inoculation on increased yield in maize by reducing dose of phosphorous fertilizers. Seed treatment with rhizobacteria or their formulations increased the growth of maize (Jacoud et al. 1999), wheat (Khalid et al. 2004), rice (Yanni et al. 1997), and several other crops (Vidhyasekaran and Muthamilan 1995; Rabindran and Vidhyasekaran 1996; Vidhyasekaran et al. 1997a, b; Podile and Dube 1988; Kloepper et al. 1991). Recently, Ashrafuzzaman et al. (2009) isolated bacterial strains with successful root colonizer and increased plant height, root length, and dry matter production rice seedlings. Some novel efforts were made to elucidate the molecular responses of rice to P. fluorescens treatment through protein profiling (Kandasamy et al. 2009). However, the mechanism underlying such promotional activity is not yet fully understood clearly. Application of several genera, such as B. licheniformis RC02, Rhodobacter capsulatus RC04, P. polymyxa RC05, P. putida RC06, Bacillus OSU-142, B. megaterium RC01, and Bacillus M-13, showed increased root and shoot weight along with nutrient uptake in barley (Cakmacki et al. 1999). Similarly, Bacillus observed increase in yield of rice (Sudha et al. 1999), barley (Sahin et al. 2004), wheat (de Freitas 2000), canola (de Freitas et al. 1997), and maize (Pal 1998; Pal et al. 2001). Lalande et al. (1989) observed increased yield in maize by using Serratia liquifaciens, Pseudomonas spp., and Bacillus sp., but B. megaterium induced yield in rice and barley (Cakmacki et al. 1999; Khan et al. 2003). Gholami et al. (2009) reported maize seeds inoculated with bacterial strains significantly increased plant height, seed weight, number of seed per ear and leaf area along with significant increase in ear and shoot dry weight of maize. Recently, more efforts have focused on beneficial rhizobacteria in cereals that are endophytic in nature especially in the regions where legume crop season is


11

followed by cereals (Ashrafuzzaman et al. 2009). Nodule-inhabiting bacteria are now known to colonize the cereals such as rice, wheat, and sorghum. Several field inoculation trials have been conducted to assess the agronomic potential of rhizobial group in nonlegumes (Chandra et al. 2007). More efforts are required to focus on Rhizobium–cereal associations under field conditions, with the long-term goal of identifying, developing, and implementing superior PGPR inoculants for the growth promotion of rice and wheat productivity in real-world cropping systems while reducing their dependence on nitrogen fertilizer inputs.

1.5.2

Oilseeds

The importance of PGPR applications in oil seed crops production was demonstrated by several workers. The growth promotion and health of canola, sesame, and peanut were supported by using different genera of PGPR (Kumar et al. 2005a; Chandra et al. 2007; Bhatia et al. 2008; Kumar et al. 2009). Pseudomonas putida, P. fluorescens, Arthrobactro citreus, Azospirillum spp., and Serratia liquefaciens demonstrated growth promotion of canola (Brassica campestris and B. napus) in field conditions (Kloepper et al. 1988a, b; 1989). Kloepper et al. (1989) observed an increase in the yield of mustard with the application of Azospirillum spp. Selected bacterial strains showed increased seedling emergence, vigor, and yield. Non-nitrogen fixing mutants provide greater root elongation effects and greater phosphate uptake in canola (Lifshitz et al. 1987), while P. putida inoculation increased yield of canola. Van Peer and Schippers (1998) found that inoculation of Pseudomonas spp. increased root and shoot weight in canola under hydroponic growth chamber. Belimov et al. (2001) observed that inoculation of B. napus seeds with Alcaligenes sp., B. pumilus, Pseudomonas sp., and Variovorax paradoxus showed vigorous growth. Bertrand et al. (2001) observed significant increase in root dry weight due to aggressive effect of Phyllobacterium sp. apart from Variovorax sp. and Agrobacterium sp. It was demonstrated that Methylobacterium fujisawaense promoted root elongation in canola (Madhaiyan et al. 2006). Earlier, Ghosh et al. (2003) observed that B. circulans DUC1, B. wrmus DUC2, and B. globisporus DUC3 enhanced root and shoot elongation in B. campestris. Differential response of sesame under influence of indigenous and nonindigenous rhizosphere competent fluorescent pseudomonads were observed recently by Aeron et al. (2010). The results of root colonization stated the difference of using indigenous and a nonindigenous strain and the successful colonization by fluorescent pseudomonads in sesame rhizosphere promoted growth which proved efficacy of indigenous microflora over nonindigenous microflora (Table 1.1). Integrated use of organic and inorganic biofertilizers has been reported to sustain productivity of sesame by improving soil physical conditions and also reduce the costly inorganic fertilizer needs (Duhoon et al. 2001;

+

na

+ (36)

+ (30)

+ (42)

+ (40)

+ (41)

na

Groundnut

Sunflower

Tomato

Velvet Bean

Sesame

–

–

–

–

+ (31)

–

PGP attributes IAA ACC (mg/ml) deaminase

Potato

Rhizospheric origin

na

+ (76)

+ (68)

+ (75)

+ (71)

+ (55)

+ (67)

P (mg of P/ml)

na

+ (19)

+ (15)

+ (22)

+ (12)

+ (17)

+ (29)

S U/ml/h

na

–

–

–

+ (0.05)

+ (0.09)

–

HCN (OD at 625 nm)

na

+ (70)

+ (78)

+ (72)

–

+ (61)

na

+ (63.3)

–

+ (68)

–

–

Antagonism (%) 1 2 + (55) –

61

83.5**

79.1**

80.3**

77.9**

76.3**

78.8**

G (%)

10.1**

17.3**

12.4**

14.7**

14.1**

13.5**

16.2**

18.6**

45.6**

39.8**

44.3**

42.5**

40.7**

43.2**

21.6

27.1*

22.8*

26.2*

25.6*

24.7*

26.2*

Plant growth parameters RDW SDW RL (gm) (gm) (cm)

141.3

185.9**

165.3**

180.7**

173.8**

168.9**

170.3**

SL (cm)

26.3

47.3**

40.8**

41.3**

39.2**

38.8**

42.6**

G/P

PGP attributes are mean of three independent experiments; Field data is a mean of two year trials; Values are mean of 15 randomly selected plants G Germination, RDW Root dry weight, SDW Seedling dry weight, RL Root Length, SL Shoot Length, G/P seed yield per plant; + attribute positive; attribute negative; IAA Indole acetic acid; P Phosphate solubilization; Sid Siderophore; ACC 1-aminocyclopropane-3-carboxylic acid; 1 Macrophomina phaseolina; 2 Fusarium oxysporum; (%) pathogen inhibition percentage (control – treatment/control 100) [Adapted from Aeron et al. (2010)] *Significant at P > 0.01 level of ANOVA **Significant at 0.01 level of LSD as compared to control

P. aeruginosa GRC1rif+ tet+ P. aeruginosa PS2str+ P. aeruginosa PS (II) neo+ P. aeruginosa LES4tet+ P. aeruginosa PRS4gen+ P. aeruginosa PSI5azi+ kan+ Control

Treatments Isolate

Table 1.1 Plant growth promoting attributes of fluorescent pseudomonads and their effect on plant growth parameters of sesame

12 A. Aeron et al.


13

Kumar et al. 2009). Siddiqui et al. (2001) reported inoculation of P. fluorescens along with chemical fertilizers is an effective way to reduce the infestation of Meloidogyne spp. in sesame. Groundnut (Arachis hypogaea L.) is a major oilseed and food crop of the semiarid tropics. The late leaf spot disease of groundnut caused by the fungus Cercosporidium personatum almost co-exists with the crop and contributes to significant loss in yield throughout the world. Leaf spots can cause up to 53% loss in pod yield and 27% loss in seed yield (Patel and Vaishnav 1987). Smith (1992) reported pod loss of 10–50% by late leaf spot disease. Control of this disease mainly depends on fungicides, although considerable effort has been invested in developing biocontrol methods (Meena et al. 2002). On the other hand, Jadhav et al. (1994) reported a Rhizobium isolate that increased plant growth and chlorophyll content in groundnut. Earlier, Howell (1987) explained in part the rhizobiaenhanced mineral uptake in groundnut tissues. Pal et al. (2000) reported increased pod yield following seed treatment with Pseudomonas sp. Gupta et al. (2002) found reduced disease incident, better vegetative growth parameters, and ultimately enhanced grain yield in peanut by the addition of P. aeruginosa GRC2 in M. phaseolina-infested field soil. Recently, Bhatia et al. (2008) reported increased seed germination, growth promotion, and suppression of charcoal rot due to M. phaseolina with fluorescent pseudomonads. Earlier, Arora et al. (2001) observed enhanced seed germination, seedling biomass, and nodule weight with reduced disease incidence in groundnut. Similarly, Meena et al. (2006) applied P. fluorescens for plant growth and in biocontrol of late leaf spot caused by C. personatum in groundnut. Seed treated with P. fluorescens strain Pf1 recorded the highest seed germination percentage and the maximum plant height with significantly controlled late leaf spot disease of groundnut resulting in increased pod yield. In another study, B. subtilis strain AF1, isolated from soils suppressive to pigeon pea (C. cajan) wilt caused by F. udum, was presumed to induce resistance against Aspergillus niger on peanut. Strong experimental evidence that AF1 elicited ISR came from the findings of Sailaja et al. (1997) who reported a noteworthy reduction in the incidence of crown rot of peanut caused by A. niger corresponding to the increase in lipoxygenase activity, a phenomenon associated with ISR.

1.5.3

Fruits, Vegetables, and Cash Crops

Several workers have reported successful management of plant disease and increased yield in various horticultural crops such as strawberry (Tahmatsidou et al. 2006; Pedraza et al. 2010), chillies (Bharathi et al. 2004), mango (Vivekanathan et al. 2004), tobacco (Pan et al. 1991), pea (Chang et al. 1992), potato (Geels et al. 1986), red pepper (Lee et al. 2008), banana (Gunasinghe and Karunaratne 2009), and apple (Karlidag et al. 2007; Aslantas et al. 2007) with the application of PGPR. Increased seedling growth was observed in sugar beet with the application of

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Pseudomonas spp. (Williams and Asher 1996). Reddy et al. (2001) reported foliar application of PGPR bioformulation which promoted plant growth besides effectively controlling tomato bacterial spot, cucumber angular leaf spot, tobacco blue mold, and wild fire. Sarvanankumar et al. (2007) used Pseudomonas and Bacillus bioformulation against blister blight disease of tea caused by Exobasidium vexans while Chakraborthy et al. (2009) studied talc-based bioformulation of Ochrobacterium anthropi TRS-2 for plant growth promotion and management of brown root rot disease of tea. Recently, Karakurt and Aslantas (2010) investigated the effects of Agrobacterium rubi A-18, B. subtilis OSU-142, B. gladioli OSU-7, and P. putida BA-8 on growth and leaf nutrient content of apple cultivars and found interesting variations that support the application of PGPR. The role of PGPR in vegetative crops production has got less attention in comparison to that of other crops. Raupach and Kloepper (2000) reported that seed treatment of cucumber with B. amyloliquefaciens IN937a, B. subtilis GB03, and a mixture of the two strains resulted in significant increases in plant growth and reductions in disease severity. Han and Lee (2005) reported PSB B. megaterium and potassium solubilizing bacteria (KSB) B. mucilaginosus inoculated in nutrientlimited soil planted with eggplant. Inoculation of these bacteria in conjunction with amendment of its respective rock P or K materials increased the availability of P and K in soil and enhanced N, P, and K uptake, and growth of eggplant. The early seedling emergence and significant increase in yield of potato was observed with the application of PGPR. Increase in yield of potato was reported by several workers after the application of different species of Pseudomonas (Howie and Echandi 1993; Geels et al. 1986; Kloepper et al. 1989). Kloepper et al. (1980) demonstrated larger root system and significant increase in yield in different soil types. Frommel et al. (1993) found the application of Pseudomonas strain Ps JN increased whole plant dry weight. Suppression of Erwinia caratovora causing soft rot in potato was seen after the inoculation with P. putida W4P3 (Xu and Gross 1986). Raupach and Kloepper (2000) demonstrated the effect of B. amyloliquefaciens In937a and B. subtilis GB03 individually as well as in combination for plant growth promotion and reduction of disease severity on seeds of cucumber treated with these antagonists. A nonfluorescent Pseudomonas of onion rhizosphere showed significant increases in root dry weight, stem length, and lignin and enhanced stem hair formation (Frommel et al. 1991). A disease complex by Meloidogyne incognita and F. oxysporum was suppressed by the application of fluorescent pseudomonads in tomato (Santhi and Sivakumar 1995; Kumar et al. 2005a) (Table 1.2). Ekin et al. (2009) applied Bacillus sp. OSU-142 as compared to three different levels of N fertilization. The beneficial effect of Bacillus sp. OSU142 on tuber yield of potato was reported in two successive years over fertilizers. Several strains of P. fluorescens, P. cepacia, and P. aeruginosa have been used for the biological control of several plant diseases in a wide range of horticultural crops (Weller 1988; Chandel et al. 2010). Yusran et al. (2009) reported biological control of F. oxysporum f. sp. radicis-lycopersici that causes crown and root rot in tomato. The inoculation also increased the N yield and fixed N in association with banana roots subsequently increased the yield, improved the physical attributes of fruit


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Table 1.2 Effect of Pseudomonas EP10 on root disease complex and growth of tomato after 60 days Treatment Plant length Plant fresh Shoot dry Root Knot Infection (%) of (cm) weight (g) weight (g) Index F. oxysporum F. oxysporum 26.5 18.0 4.5 6.0 100 Pseudomonas 55.0** 51.0** 15.0** 3.0 – EP10 58.5** 55.0** 17.5** 2.5 4 Pseudomonas EP10 + F. oxysporum Control 32.8 27.5 6.5 7.0 – **P < 0.01, Values average of ten replicates during three trials (Modified and adapted from Kumar et al. 2005b)

quality, and initiated early flowering. More recently, PGPR proved effective as a bioenhancer and biofertilizer for banana cultivation (Mia et al. 2005). B. subtilis S499 is involved in suppression of gray mold disease caused by Botrytis cinerea on wounded apple fruits (Ongena and Jacques 2007; Jacques et al. 1999). Recently, Romero et al. (2007) showed the involvement of iturin and fengycin antibiotics from four B. subtilis strains UMAF6614, UMAF6616, UMAF6639, and UMAF8561 in suppression of powdery mildew of cucurbits caused by Podosphaera fusca. Arrebola et al. (2010) reported the production of iturin from B. amyloliquefaciens PPCB004 which inhibited seven different postharvest pathogens of citrus, avocado, and mango fruits. Recently, Choudhary and Johri (2008) implicated the mechanisms and role of Bacillus species as inducers of systemic resistance in relation to plant–microbe interactions and explicated the pathways involved in their regulation.

1.5.4

Legumes

A unique relationship was observed between two bacterial isolates Burkholderia sp. MSSP and Sinorhizobium meliloti PP3, where commensalisms between them resulted in increased IAA production in mixed-species culture and significant increase in seedling length and weight of pigeon pea (Cajanus cajan) (Pandey and Maheshwari 2007a). When wheat-bran-based bioformulation comprising consortium of PGPR was applied in field trials, significant improvement in growth and yield of pigeon pea was obtained (Pandey and Maheshwari 2007b). Kumar et al. (2010) obtained wilt disease management and enhancement of growth and yield of Cajanus cajan (L) var. Manak by bacterial combinations using root nodulating Sinorhizobium fredii KCC5 and P. fluorescens LPK2 isolated from nodules of host plant and disease suppressive soil of tomato rhizosphere, respectively (Table 1.3). Mishra et al. (2009) studied application of several potential PGPR strains on Cicer arietinum. All isolates showed significant increase in shoot length, root length, and dry matter of seedlings. Even the application of P. cepacia caused an early soybean

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Table 1.3 Effect of S. fredii KCC5, P. fluorescens LPK2, bacterial consortium (KCC5 þ LPK2) on post harvest parameters of C. cajan var. Manak, after 120 days of sowing Treatments KCC5 LPK2 Consortium (KCC5 þ LPK2) Control

*Pods plant1 115.4** 116.3** 118.6** 51.2

Grain yield (Kg ha1) 962.1** 955.2** 988.3**

A soluble protein (mg g1) 211.1* 197.2* 212.3*

Stover yield (Kg ha1) 4,230* 4,200* 4,280*

Harvest index (%) 18.53 18.52 18.76

710.0

179.1

3,150

18.03

Average value of ten plants from each treatment; a soluble protein content of seed (g1) *Significant at 5% (ANOVA) **Significant at 1% as compared to control (ANOVA) [Modified and adapted from Kumar et al. (2010)]

growth and enhanced seed germination (Cattelan et al. 1999). Ma et al. (2003) reported R. leguminosarum bv. viciae 128C53K enhanced nodulation in pea (Pisum sativum L.). Moreover, Penã-Cabriales and Alexander (1983) found that strains of rhizobia and bradyrhizobia grew readily in the presence of germinating seeds and developing root systems of soybean (Glycine max (L.) Merr.), kidney bean (Phaseolus vulgaris L.), red clover (Trifolium pretense L.), and cowpea (Vigna unguiculata L.), but Pseudomonas sp. and Bradyrhizobium sp. increased growth and promoted nodulation in mung bean (Shaharoona et al. 2006). Wiehe and Hoflich (1995) demonstrated that R. leguminosarum bv. trifolii can multiply and survive under field conditions in the rhizosphere of nonhost legumes (Lupinus albus L. and Pisum sativum L.) and nonlegumes such as corn, rape, canola, and wheat; some strains of B. subtilis that have been integrated into pest management strategies, such as biocontrol strain GB03 of B. subtilis, could inhibit the fusarial wilt caused by Fusarium species more effectively on semiresistant cultivar of chick pea than on susceptible variety (Jacobson et al. 2004; Hervas et al. 1998). There are several reports which reveal that efficacy of rhizobia could be enhanced by co-inoculation with PGPB. Co-inoculation with symbiotic and rhizosphere bacteria may improve nodulation by a number of mechanisms. Different mechanisms for such activity by Gram-positive and Gram-negative bacteria include siderophore chelating insoluble cations, LPS, flavonoids, phytoalexins, antibiotics, and colonization of root surfaces by outcompeting pathogenic organisms, and thus increase nodulation and growth (Garcia Lucas et al. 2004; Parmar and Dadarwal 2000). A common attribute, although, is efficient colonization of roots by PGPR strain to reduce the ethylene concentration inside the plant. That is so if it is able to utilize ACC as a sole nitrogen source, thereby increasing the root surface in contact with soil. Therefore, it is highly expected that presence of PGPR containing ACC deaminase on the roots of legume could suppress accelerated endogenous synthesis of ethylene during the rhizobial infection and thus may facilitate nodulation. So, co-inoculation of legumes with competitive rhizobia and PGPR-containing ACC deaminase could be an effective and novel approach to achieve successful and dense nodulation in legumes. It is highly expected that inoculation with rhizobacteria containing ACC-deaminase hydrolyzed endogenous ACC into ammonia and alpha-ketobutyrate instead of ethylene. Consequently, root and shoot growth of the


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legume plant as well as nodulation can be promoted (Garcia Lucas et al. 2004; Remans et al. 2007). Earlier, the use of specific PGPR mutant strains has indicated that bacterial indole-3-acetic-acid production and 1-aminocyclopropane-1-carboxylate deaminase activity play an important role in the host nodulation response. Tittabutr et al. (2008) conducted such a study to evaluate effect of ACC deaminase on nodulation and growth of Leucaena leucocephala. Further, Remans et al. (2007) examined the potential of ACC deaminase producing PGPR to enhance nodulation of common bean (Phaseolus vulgaris). Shaharoona et al. (2006) observed that co-inoculation with Pseudomonas and Bradyrhizobium species significantly improved root length, total biomass, and nodulation in mung bean. Belimov et al. (2009) evaluated the effect of root-associated bacterium containing ACC deaminase on pea (Pisum sativum) plants grown in dry soil. Huang and Erickson (2007) tested the effectiveness of R. leguminosarum for improving growth and yield of pea and lentil. They found improved seedling growth, nodule biomass, and shoot and root biomass in peas as we observe in velvet bean. Similarly, the effect of different methods of rhizobial inoculation on yield, root nodulation, and seed protein contents of two lentil varieties and improvement in nodulation was observed in peanut by inoculation with Rhizobium species (Ahmad et al. 2008; Dey et al. 2004). Rhizobia and other microorganisms employ various mechanisms to acquire essential nutrients such as iron, which includes production of iron-chelating molecules known as siderophores. Despite their efficient nitrogen-fixing potential, most of the times they fail to increase plant yield under field trials in agricultural soils. This has been attributed to their inefficiency to successfully colonize the rhizosphere. Iron availability is one of the limiting factors for poor rhizospheric colonization. The successful performance of rhizobial inoculant strain depends upon their capability to outcompete the indigenous soil bacteria, survive, propagate, and enter into effective symbiosis with host plant. Many studies have indicated that efficient utilization of siderophores by rhizobia is a positive fitness factor with respect to its soil survival (Carson et al. 2000). Further, Joshi et al. (2009) observed increase in nodule occupancy and higher rhizospheric colonization by pigeon pea-nodulating rhizobia expressing engineered siderophore cross-utilizing abilities. Since survival under iron limitations in soils is an important quality which every biofertilizer strain must possess, the iron sufficiency of any organism therefore largely depends on its ability to utilize siderophores present in large and small concentrations in its vicinity that may be of plant, microbial, or soil origin (Carson et al. 2000; Joshi et al. 2009; Joshi et al. 2008; Joshi et al. 2006b; Khan et al. 2006). Thus, iron availability is one of the major factors determining rhizospheric colonization. This fact is further evidenced by work of Mahmoud and Abd-Alla (2001). They showed that co-inoculation of siderophore-producing PGPR significantly enhanced nodulation and nitrogen fixation of mung bean compared to plants infected with rhizobial strain alone. Thus, siderophore plays an important role in the competition between microorganisms and may act as growth promoters as the rhizosphere is heavily populated with siderophore-producing microorganisms. There are more reports that specific siderophore producing microorganisms stimulated the nodulation, nitrogen fixation,

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and plant growth of leguminous plants (Grimes and Mount 1987; Omar and AbdAlla 1994; Shenker et al. 1999). A nodulated legume has an increased need for iron compared to non-nodulated plant since this metal is a constituent of key proteins involved in nitrogen fixation such as nitrogenase and leghemoglobin. Although scientists have reported both direct and indirect ways of growth stimulation by PGPR, there is no clear separation between these two mechanisms. A bacterium influencing plant growth by regulating synthesis of plant hormones can also play a role in controlling plant pathogens and diseases and vice versa. The presence of PGPR in the root vicinity may also improve ability of rhizobia to compete with indigenous populations for nodulation. Parmar and Dadarwal (2000) reported that increase in root growth provides more number of active sites and access to nodulation for rhizobia in chickpea. Co-inoculation of Bradyrhizobium with P. striata has also been observed to enhance biological nitrogen fixation in soybean (Dubey 1996). Rosas et al. (2006) studied the promising action of two phosphate solubilizing Pseudomonas strains on the symbiosis of rhizobial strains (S. meliloti and B. japonicum) with alfalfa and soybean. Further, differential effects on chick pea plant growth were also observed under co-inoculation with a PSB (Pseudomonas) strain and rhizobia alone (Valverde et al. 2006). There is a great advantage of using PSB in co-inoculation with rhizobia. This is because increased P mobilization in soil alleviates P deficiency. Deficit P severely limits plant growth and productivity particularly with legumes, where both plants and their symbiotic bacteria are affected. This may have a deleterious effect on nodule formation, development, and function (Robson et al. 1981). Similarly, dual inoculation of rhizobia with PGPR promoted nodulation, plant growth, and N2 fixation in Vigna radiate (Gulati et al. 2001; Gupta et al. 2003).

1.5.5

Forestry

Worldwide efforts to increase green cover and reforestation of abandoned, barren, and wasteland can benefit from a wider application of PGPR in both angiosperm and gymnosperm. There are currently very few reports on forestry-PGPR research. As a consequence, there is currently no field data for deciduous trees and still comparatively little field data for gymnosperms (Chanway 1997). Hindrance in successful application of PGPR in forestry includes aspects such as low pH conditions of forest soil, perennial nature of trees, soil type, forest environment, and survival of PGPR with trees in colder regions. In contrast to agricultural crops, the inoculation of PGPR on tree species and with special reference to their effect on seedling emergence, reduction in seedling transplant injury during the transfer from nursery to field, biomass increase due to inoculation apart from raising disease free plantlets in nursery, and increased strength of plantlets to withstand storm of antagonists in the form of pathogens have scope for investigation.


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Earlier, Pokojska-Burdziej (1982) and Beall and Tipping (1989) demonstrated increase in height and biomass in black spruce, jack pine, and white spruce by using Arthrobacter citreus, P. fluorescens, and P. putida under greenhouse conditions, while Chanway and Holl (1994) used A. oxydans and P. aureofaciens in Douglas Fir and recorded improved height and biomass. On the other hand, Arthrobacter sp. increased the shoot length of pine. Leyval and Berthelin (1989) found that a strain of Agrobacterium radiobacter increased biomass of beech and pine. According to one of the reports from authors group, Pinus roxburghii was found to show luxuriant growth due to application of Bacillus subtilis BN1. Seed treatment resulted in significant increase in seed germination, early seedling emergence, increase in biomass besides reduction in charcoal root rot in chir-pine seedlings (Singh et al. 2008; Singh et al. 2010) (Table 1.4), and reduction in the total, pre- and postemergency mortality of the P. radiata seedlings in nursery trial (Valiente et al. 2008). Inoculation of A. brasilense Cd increased root growth while inoculation of A. chroococcum increased biomass in oak (Akhromeiko and Shestakova 1958; Zaady et al. 1993; Zaady and Perevoltsky 1995), A. brasilense in river oak (Rodriguez-Barrueco et al. 1991), A. chroococcum in ash (Akhromeiko and Shestakova 1958), Quercus serrata (Pandey et al. 1986), and Eucalyptus (Mohammad and Prasad 1988). Enebak et al. (1998) studied inoculation of B. polymyxa and P. fluorescens in loblolly pine and slash pine under green house conditions. A significant increase in seedling emergence was observed along with total biomass. The postemergence damping-off was reduced in loblolly pine. B. polymyxa and Staphylococcus hominis significantly increased growth of hybrid spruce (O’Neill et al. 1992). Chanway et al. (2000) observed that B. polymyxa and P. fluorescens overwinter on the roots of field-planted trees such as spruce. Shishido and Chanway (2000) observed a significant increase in plant biomass of hybrid spruce when inoculated with Pseudomonas strain at all sites apart from reduction in seedling injury after transplant. B. licheniformis CECT5105 and B. pumilis CECT5106 led to increase in the plant growth and nitrogen content in silver spruce (Porbanza et al. 2002). Mafia et al. (2009) reported B. subtilis and Pseudomonas sp. in controlling mini-cutting rot of eucalyptus caused by Cylindrocladium candelabrum and R. solani. In fact, trees with mycorrhizal associates, with associative, or symbiotic N2-fixers, or with Table 1.4 Effect of P. aeruginosa strain PN1 on the growth of chir-pine (90DAS/Pot assay) Treatments Germination Shoot length Root length Fresh weight (g) (%) (cm) (cm) Shoot Root 10.1ns 0.92ns 0.266ns P. aeruginosa PN1 84* 7.2ns P. aeruginosa 72* 6.5ns 9.1ns 0.727ns 0.236ns PN1 þ M. phaseolina M. phaseolina 54ns 5.9ns 8.1ns 0.52ns 0.182ns Control 66 6.2 8.8 0.625 0.212 Values are the mean of triplicates; ns non-significant *Significant at 5% LSD Modified and adapted from Singh et al. 2010

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rock-weathering capacities have significant impacts on biogeochemical processes, affecting recovery of degraded ecosystems and forest sustainability.

1.5.6

Mulberry (Sericulture)

For sericulture industry, mulberry is food plant of silkworm (Bombyx mori) grown in 1, 70,000 ha in India under different agro-climatic conditions. The sustainable leaf production, silkworm rearing, and cocoon production are dependent on soil fertility of mulberry gardens maintained through periodical application of either organic manures or chemical fertilizers in required quantity. Former is an approach wherein crop can be raised without imparting any adverse effect on soil and other beneficial microbial ecology. Therefore, a shift toward nonchemical strategies has to be evolved. The biofertilizers enriched with bacteria and fungi have proven to be great importance in improving the yield and quality of mulberry (Morus alba L.) More than three decades ago, Vasantharajan and Bhat (1967) studied the interactions of beneficial microorganisms and mulberry and reported an increase in shoot length and root length of seedlings and saplings due to the application of different genera of PGPR such as Pseudomonas spp., Acetobacter, Flavobacterium, Achromatobacterium, Micrococus, Bacillus, Arthrobacterium, etc. Later, Kasiviswanathan et al. (1977) observed inoculation of Azotobacter to soil proved beneficial to increase the growth and yield of mulberry. Vijayan et al. (2007) evaluated the effect of biofertilizers Azotobacter and Azospirillum on establishment of mulberry and revealed that inoculants improved the mulberry growth and development over control in saline conditions. Even the P-solubilizing Bacillus megaterium, Bacillus sp., Aspergillus awamuri, and A. niger enhanced the growth and yield parameters of mulberry (Nagendra Kumar and Sukumar 2001). Gangwar and Thangavelu (1992) isolated the nitrogen-fixing bacteria Azotobacter and Beijerinckia from phyllosphere and rhizosphere of mulberry. The first pair of leaf was inoculated with both the bacterial isolates and showed the airborne nature of the nitrogen fixers. In rhizosphere, the population of both the genera increased corresponding to increase in the age of the plant. Yadav and Nagendra Kumar (1989) observed reduction of nitrogen fertilizer to half or one-third dose along with Azospirillum, which improved the mulberry plant growth and leaf yield at par with full dose of nitrogen fertilizer. Umakant and Bagyaraj (1998) reported improvement of plant growth in mulberry nursery with dual inoculation of Azotobacter chroococcum and Glomus fasciculatum. During several studies, Das et al. (1990, 1994) reported that biological nitrogen fixation mediated through Azotobacter is considered to be the potential system for mulberry cultivation for economizing up to half dose of N fertilizer in different mulberry cultivars without any reduction in leaf yield and quality. Rangarajan and Santhanakrishnan (1995) demonstrated the combined effect of P. fluorescens and Azospirillum more superior than that of single inoculation or uninoculated controls. This enhanced the quality of mulberry leaf and consequently improved


21

the silkworm growth and silk production. Chandrashekar et al. (1996) studied the effect of co-inoculation in mulberry with Acaulospora sevis, B. megaterrium var. phosphaticum, and A. brasillense using two sources of phosphorous and attained improvement of leaf growth, yield, and quality. Gupta et al. (2008) reported the sustainability of mulberry leaf production by reducing the application of chemical fertilizers and biofertilizers, such as Azotobacter, Azospirillum, PSB, and mycorrhizae. Baqual and Das (2006) demonstrated that dual inoculation of mulberry with Azotobacter and VAM could curtail use of fertilizers by 50% besides improving the leaf yield, cocoon production, and quality. Azotobacter inoculation to the roots caused better increase in root and shoot weight in comparison to that of leaf inoculation. The proliferation of Azotobacter in nutrient solutions was highly stimulated in the presence of mulberry plants, but the similar stimulation in the natural condition was not observed. Sudhakar et al. (2000a, b) studied the role of biotic and abiotic (seasonal variation) factors in contributing toward population buildup of diazotrophs and other microorganisms both on phylloplane and rhizosphere of mulberry. It was concluded that rainy season and the shoot age of 30–40 days after pruning appear to be ideal for the increase of diazotrophs both in phylloplane and rhizosphere by foliar application of Azotobacter and Beijerinckia. Similarly, PGPR proved to be an effective tool for increase in biomass production in som (Machilus bomycina), which in turn has an impact on the growth of silkworms to produce more silk fiber of good quality. Unni et al. (2008) isolated and exploited PGPR from rhizosphere of som plants. Muga silkworm larvae fed on some leaves of the plant treated with PGPR showed growth-promoting activities in plants. The shell weight of the cocoons formed from the larvae fed with treated som leaves was significantly higher than that of the control. Such cocoons used for fiber estimation showed considerable increase in fiber content which were not only longer but had higher nonbreakable filament length (Unni et al. 2008). B. subtilis strain Lu144 was isolated as an endophyte from the surface sterilized leaves of mulberry (Ji et al. 2008). Strain Lu144 exhibited strong in vitro antagonistic activity against Ralstonia solanacearum which causes bacterial wilt on mulberry plants and displayed reducing the disease incidence.

1.6

Limitations Associated with PGPR

In fact, the inconsistent response of field-grown crops to PGPR has limited commercial development. In natural ecosystems, the behavior of introduced bacterial inoculants (e.g., PGPR) and the subsequent expression of PGP represent a complex set of multiple interactions between introduced bacteria, associated crops, and indigenous soil microflora. These interactions are, in turn, influenced by multiple environmental variables such as soil type, nutrition, moisture, and temperature (Kloepper et al. 1989; Glick 1995). Thus, the ability of a bacterial inoculant to promote plant growth can only be fully evaluated when they are tested in

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association with all of the components of the rhizosphere (Schroth and Weinhold 1986). Inconsistent responses to beneficial bacteria are frequently reported (Brown 1974; Broadbent et al. 1977; Schroth and Hancock 1982; Howie and Echandi 1983; Schroth and Weinhold 1986; Schippers et al. 1987; Kloepper et al. 1988a, b; de Frietas and Germida 1990, 1991, 1992a, b). Moreover, in a compilation of reports of crop yield responses to bacterial inocula in field studies after 1974, Kloepper et al. (1989) noted that in 13 of 26 studies bacterial inocula previously identified as PGPR caused significant yield reductions as compared to the control. It has been suggested that inconsistencies associated with microbial inoculants for field applications are not surprising because physical and chemical factors, such as soil texture, pH, nutrient status, moisture, temperature, organic matter content, and biological interactions in the rhizosphere, may affect the establishment, survival, and activity of certain organisms whereas other organisms may remain unaffected (Schroth and Weinhold 1986; Kloepper et al. 1989). Thus, apparent discrepancies in experimental results likely reflect differences in experimental conditions including soil and associated indigenous soil microorganisms. Therefore, workers have devised new strategies to overcome these inconsistencies. Recent studies showed a promising trend in the field of inoculation technology, which is the use of mixed inoculants or application of consortia (combinations of microorganisms) that interact synergistically are currently being devised. Microbial interaction studies performed without plants indicate that some bacterial genera allow each other to interact synergistically providing nutrients, removing inhibitory products, and stimulating each other through physical or biochemical activities that may enhance some beneficial aspects of their physiology such as nitrogen fixation (Pandey and Maheshwari 2007b; Arora et al. 2008). Plant studies have shown that these beneficial effects of Azospirillum on plants can be enhanced by co-inoculation with other microorganisms (Alagawadi and Gaur 1992; Belimov et al. 1995). Co-inoculation frequently increased growth and yield compared to single inoculation, which provided the plants with more balanced nutrition and improved absorption of nitrogen, phosphorus, and mineral nutrients (Kumar et al. 2009). Application of PGPR could not only produce significant benefits that require minimal or reduced levels of fertilizers but also consequently produce a synergistic effect on root growth and development (Kumar et al. 2009).

1.7

Conclusion and Future Prospects

The application of PGPR, in recent times, is gaining attention mainly due to the environment friendly nature of bioinoculants and increased acceptability of natural “organic” plant products globally. In practice, alien strains suitable to different crops grown in various climatic conditions and their use are limited due to the unavailability of suitable climate-based microbial inoculants. In view of the knowledge of ecological specificity associated with naturally occurring microorganisms, consistent efforts from research laboratories are required for selecting and


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developing microbial performance always in field suited in the specific set of climatic conditions. Prior to registration and commercialization of PGPR products, a number of hurdles must be overcome. These include scale up and production of the organism under commercial fermentation conditions while maintaining quality, stability, and efficacy of the product. Formulation development must consider factors such as shelf life, compatibility with current application practices, cost, and ease of application. Health and safety testing are also required to address such issues as nontarget effects on other organisms including toxigenicity, allergenicity and pathogenicity, persistence in the environment, and potential for horizontal gene transfer. Capitalization costs and potential markets must be considered in the decision to commercialize the product for their application in agro and allied industries. Acknowledgement Thanks are due to UCOST (Dehradun), UGC, and CSIR (New Delhi) for providing financial support in the form of research project to DKM.

References Aeron A, Pandey P, Maheshwari DK (2010) Differential response of sesame under influence of indigenous and non-indigenous rhizosphere competent fluorescent pseudomonads. Curr Sci 99(2):166–168 Ahmad ZI, Ansar M, Tariq M, Anjum MS (2008) Effect of different rhizobium inoculation methods on performance of lentil in Pothowar region. Int J Agric Biol 10:81–84 Akhromeiko AI, Shestakova VA (1958) The influence of rhizospheric microorganisms on the uptake and secretion of us and sulphur by the roots of arboreal. In: Proceedings of second UN Internal Conf Peace: Uses Atomic Energy seedlings. pp. 193–199 Alagawadi AR, Gaur AC (1992) Inoculation of Azospirillum brasilense and phosphate solubilizing bacteria on yield of sorghum (Sorghum bicolor L. moench) in dry land. Trop Agric 69:347–350 Arora NK, Kang SC, Maheshwari DK (2001) Isolation of siderophore producing strains of Rhizobium meliloti and their biocontrol potential against Macrophomina phaseolina that causes charcoal rot of groundnut. Curr Sci 81:673–677 Arora NK, Khare E, Oh JH, Kang SC, Maheshwari DK (2008) Diverse mechanisms adopted by fluorescent Pseudomonas PGC2 during the inhibition of Rhizoctonia solani and Phytophthora capsici. World J Microbiol Biotechnol 24:581–585 Arrebola E, Jacobs R, Korsten L (2010) Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens. J Appl Microbiol 108(2):386–395 Asaka O, Shoda M (1996) Biocontrol of Rhizoctonia solani damping of tomato with Bacillus subtilis RB14. Appl Environ Microbiol 62(11):4081–4085 Ashrafuzzaman M et al. (2009) Efficiency of plant growth-promoting rhizobacteria (PGPR) for the enhancement of rice growth. Afr J Biotechnol 8(7):1247–1252 Aslantas R, Cakmacki R, Sahin F (2007) Effect of plant growth promoting rhizobacteria on young apple tree growth and fruit yield under orchard conditions. Sci Hortic 111:371–377 Ayala S, Rao EVSP (2002) Perspective of soil fertility management with a focus on fertilizer use for crop productivity. Curr Sci 82:797–807 Baqual MF, Das PK (2006) Influence of biofertilizers on macronutrient uptake by the Mulberry plant and its impact on silkworm bioassay. Caspian J Env Sci 4(2):98–109

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Chapter 2

Bacillus as PGPR in Crop Ecosystem Ankit Kumar, Anil Prakash, and B.N. Johri

2.1

Introduction

Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria which have the ability to colonize the roots and either promote plant growth through direct action or via biological control of plant diseases (Kloepper and Schroth 1978). They are associated with many plant species and are commonly present in varied environments. Strains with PGPR activity, belonging to genera Azoarcus, Azospirillum, Azotobacter, Arthrobacter, Bacillus, Clostridium, Enterobacter, Gluconacetobacter, Pseudomonas, and Serratia, have been reported (Hurek and Reinhold-Hurek 2003). Among these, species of Pseudomonas and Bacillus are the most extensively studied. These bacteria competitively colonize the roots of plant and can act as biofertilizers and/or antagonists (biopesticides) or simultaneously both. Diversified populations of aerobic endospore forming bacteria (AEFB), viz., species of Bacillus, occur in agricultural fields and contribute to crop productivity directly or indirectly. Physiological traits, such as multilayered cell wall, stress resistant endospore formation, and secretion of peptide antibiotics, peptide signal molecules, and extracellular enzymes, are ubiquitous to these bacilli and contribute to their survival under adverse environmental conditions for extended periods of time. Multiple species of Bacillus and Paenibacillus are known to promote plant growth. The principal mechanisms of growth promotion include production of growth stimulating phytohormones, solubilization and mobilization of phosphate, siderophore production, antibiosis, i.e., production of antibiotics, inhibition of plant ethylene synthesis, and induction of plant systemic resistance to pathogens (Richardson et al. 2009; Idris et al. 2007; Gutierrez-Manero et al. 2001;

A. Kumar (*), A. Prakash, and B.N. Johri Department of Biotechnology and Bioinformatics Centre, Barkatullah University, Bhopal 462026, Madhya Pradesh, India e-mail: [email protected]


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Whipps 2001). It is very likely that plant growth promotion by rhizosphere bacilli may be a result of combined action of two or more of these mechanisms (Fig. 2.1). Pathogenic microorganisms affecting plant health are a major threat to food production, and traditional methods, viz., crop rotation, breeding for resistant plant cultivars, and application of chemical pesticides, seem to be insufficient to control root diseases of important crop plants (Johri et al. 2003). Further, it appears inevitable that fewer pesticides will be used in future and that greater reliance will be laid on biotechnological applications including use of microorganisms as antagonists. Therefore, interest in biological control has been increased in the past few years partly due to change in the public concern over the use of chemicals and the need to find alternatives of chemicals used for disease control. Both Bacillus and Paenibacillus species express antagonistic activities by suppressing the pathogens and numerous reports covering this aspect both under in vitro and in vivo conditions are available (Arrebola et al. 2010; Chen et al. 2009; Joshi and McSpadden Gardener 2006). Enhancement of plant growth by root-colonizing species of Bacillus and Paenibacillus is well documented and PGPR members of the genus Bacillus can provide a solution to the formulation problem encountered during the development of BCAs to be used as commercial products, due in part to their ability to form heatand desiccation-resistant spores (Kloepper et al. 2004; Emmert and Handelsman 1999). In the past few years, research has been directed more toward the induced systemic resistance (ISR), a process by which PGPR stimulate the defense

Fig. 2.1 Schematic illustration of important mechanisms known for plant growth promotion by PGPR. Different mechanisms can be broadly studied under (1) Biofertilization, and (2) Biocontrol of pathogens. Biofertilization encompasses: (a) N2 Fixation, (b) Siderophore production, (c) Pinorganic solubilization by rhizobacteria. Biocontrol involves: (a) Antibiosis, (b) Secretion of lytic enzymes, and (c) Induction of Systemic Resistance (ISR) of host plant by PGPR

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Bacillus as PGPR in Crop Ecosystem

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mechanisms of host plants without causing apparent harm to the host. More recently, Choudhary and Johri (2008) have reviewed ISR by Bacillus spp. in relation to crop plants and emphasized on the mechanisms and possible applications of ISR in the biological control of pathogenic microbes. Various strains of species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus are known as potential elicitors of ISR and exhibit significant reduction in the incidence or severity of various diseases on diverse hosts (Choudhary and Johri 2008; Kloepper et al. 2004). It is believed that plants have the ability to acquire enhanced level of resistance to pathogens after getting exposed to biotic stimuli provided by many PGPRs and this is known as rhizobacteriamediated ISR (Choudhary et al. 2007). The aim of this chapter is to perpetuate the ecological perspectives and role of Bacillus species studied in the past few years, pertaining to its plant growth promotory activities with emphasis on the biocontrol mechanisms and possible implications in crop ecosystem. Published and some previously unpublished work have been summarized in this chapter, showing that strains of Bacillus and Paenibacillus species, including B. subtilis, B. cereus, B. amyloliquefaciens, B. pumilus, B. pasteurii, B. mycoides, B. sphaericus, P. polymyxa, P. azotofixans, and some other newly discovered species (B. endophyticus), influence the growth, development, and yield of crops under controlled and varied natural conditions either directly or indirectly following various mechanisms.

2.2

Ecology of Bacillus and Paenibacillus Species

Most species of Bacillus and Paenibacillus are distributed globally and the widespread occurrence of subspecies of B. subtilis and B. cereus with their ability to suppress the plant pathogens has been widely recognized.

2.2.1

Distribution, Diversity, and Population Dynamics

Plant growth promoting strains of Bacillus and Paenibacillus have been widely studied for enhancement of plant growth (Choudhary and Johri 2008; Kloepper et al. 2004). Cultivation-dependent approaches have revealed the occurrence of multiple isolates of phylogenetically and phenotypically similar species of B. subtilis and B. cereus ranging from log 3 to log 6 counts (CFU) per gram fresh weight (Vargas-Ayala et al. 2000). While culture-independent studies of soil confirmed the uncultured diversity of both Bacillus and Paenibacillus rRNA lineages, there are contradictions about the relative abundance of culturable and unculturable representatives of these genera in different soils (McSpadden Gardener 2004; Smalla et al. 2001). Though multiple species of Bacillus and Paenibacillus are frequently found in the soil and rhizosphere, only limited

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information is available about the most commonly isolated species of this genus. In some cases, B. megaterium has been found as the most abundant species, but it is improbable that a single species will dominate numerically in most soils (Liu and Sinclair 1992). Species of B. polymyxa group, recently renamed Paenibacillus, are autotrophs, commonly associated with rotting plant materials, composts, and the rhizosphere. Some of them are able to fix nitrogen and thus contribute significantly to the acquisition of nitrogen by crops such as Canadian wheat (Priest 1993). Members of B. brevis group, renamed Brevibacillus, are found in both soil and water habitats. The species B. sphaericus is most noted as an insect pathogen and is found in the sediments of pools, lakes, and drainage ditches where insect larvae thrive. Limited attempts have been made to study the diversity of bacterial populations in and around the rhizosphere, probably due to lack of appropriate techniques required to isolate sufficient number of strains belonging to the same species. Due in part to the unavailability of suitable methods to explore the community dynamics, our understanding of the variation in microbial community dynamics in response to soil type, plant type, or stage of plant development is limited as yet (McSpadden Gardener 2004; Duineveld et al. 1998). In fact, bacterial communities residing in the rhizosphere respond, in particular, with respect to density, composition, and activity, to the plethora and diversity of organic root exudates, resulting in plant species-specific microflora which may eventually vary with the stage of plant growth (Wieland et al. 2001 and references therein). To come to an improved understanding of factors affecting the ability of bacteria to colonize the rhizosphere, the plant has to be taken into account. Rhizospheric competence is a necessary prerequisite for PGPR. It comprises of effective root colonization combined with the ability to survive and proliferate along the growing plant roots in the presence of indigenous microbiota over a period of time. Given the importance of rhizospheric competence as a prerequisite, understanding the plant–microbe communication as affected by genetic and environmental factors in the context of their ecological niche can contribute significantly toward understanding the mechanisms of action (Bais et al. 2004; Whipps 2001). Bacillus species are believed to be less rhizosphere competent than Pseudomonas species. As a consequence, most research even today is aimed at the development of BCAs based on Pseudomonas species (Weller 1988). However, studies on the genetic diversity of Bacillus from soil as well as from the wheat rhizosphere implied that rhizosphere competence is a characteristic of the strain (genotype) not exclusive to the genus or species. Based on studies of wheat rhizosphere colonization by Bacillus species, it seems that rhizosphere competent genotypes occur in this bacterium (Milus and Rothrock 1993; Maplestone and Campbell 1989). Experiments with different wheat varieties conducted by Juhnke et al. (1987) and Milus and Rothrock (1993) have revealed that seeds bacterized with selected strains of Bacillus could successfully establish in the rhizosphere. But, whether the colonization attained by introduced strains was on the entire root or only the top few centimeters of root below the seed could not be confirmed. However, in another

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study, high populations of B. mycoides and B. pumilus in the rhizosphere of wheat at a depth of 20–30 cm below the site of bacterial inoculation (200 ml/seed) at the time of planting have been reported; the bacterial population was believed to be carried downward either in conjunction with water infiltration or along with elongating tips of growing roots (Maplestone and Campbell 1989).

2.2.2

Spatiotemporal Aspects

Variations are known to exist in the genetic microdiversity within the species of Bacillus and Paenibacillus (McSpadden Gardener 2004). Wieland et al. (2001) studied the spatiotemporal variation among the microbial communities from soil, rhizosphere, and rhizoplane with respect to crop species (clover, bean, and alfalfa), soil type, and crop development following a comparative study of 16S rRNA sequences employing temperature gradient gel electrophoresis (TGGE). According to their study, the type of plant species had profound effects on microbial community dynamics, with the effect of soil type typically exceeding that of plant type. Plant development had only minor habitat-dependent effect and insignificant variations were observed in time-dependent shifts among the microbial communities compared to the soil type or plant type in all the habitats under study. Systematic community shifts could not be recognized in samples from bulk soil; however, some variations in the TGGE patterns could be correlated to time of development in the rhizosphere and rhizoplane. Nearly, similar findings were reported by Mahaffee and Kloepper (1997) who used fatty acid methyl ester analysis (FAME) to determine the community shifts in the rhizosphere of cucumber. However, only an altered window of observations generated by the use of specific primers could possibly reveal a stronger time-dependent stimulation of certain bacterial groups. McSpadden Gardener (2004) studied the population structure of these two groups by terminal restriction fragment length polymorphism (TRFLP) using group specific primers Ba1F and Ba2R and characterized the plant growth promoting population of PGPR; only minor differences were observed in the number and relative abundance of Bacillus-like ribotypes from different sites all the way through Ohio (USA). Despite environmental constraints and interactions with other microorganisms, some bacteria are able to colonize the phylloplane with higher frequency than others. Arias et al. (1999) evaluated the diversity and distribution of Bacillus spp. from soybean phylloplane wherein a decline was observed in the population of Bacillus spp. from 80% of total bacterial isolates in early stages to 0% at the time of harvesting. In addition, the diversity of Bacillus spp. decreased from nine species at 45 days to just one species at 133 days, shortly before harvesting. B. pumilus was reported as the most prominent species from soybean phylloplane at all sampling times till the end of cropping season, followed by B. subtilis as second most abundant species from 15 to 108 days after sowing. Several other Bacillus spp., such as B. subtilis, B. brevis, B. firmus, and B. circulans, were found as regular or as dominant microflora at an early stage of plant growth, but were no longer detected

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after 85 days from the phylloplane of trifoliate leaves. The cause of apparent reduction in Bacillus spp. populations at the end of soybean cropping season, however, remained unclear. The genus Paenibacillus encompasses several species described as nitrogenfixing bacilli, including P. polymyxa, P. azotofixans, and P. macerans (Ash et al. 1993). In contrast to other species of this genus, strains belonging to P. azotofixans are efficient nitrogen fixers and are prevalent in the rhizosphere of maize, sorghum, sugarcane, wheat, banana, and forage grasses (Rosado et al. 1998a; Seldin 1992). Rosado et al. (1998b) showed that bacterial diversity of P. azotofixans was high in bulk soil compared to the rhizosphere. Seldin et al. (1998) determined the diversity of P. azotofixans strains isolated from the rhizoplane, rhizosphere, and nonroot associated soil of maize grown in two different field soils of Brazil (Cerrado and Varzea). On the basis of phenotypic traits, 60 strains from Varzea soil and 46 strains from Cerrado were identified as P. azotofixans and they could be categorized into six groups for each soil. Fifteen different hybridization patterns were obtained in 60 P. azotofixans strains from Varzea while only two patterns were obtained from 46 strains of Cerrado when specific plasmids for nifH genes were used as probes. Data from the phenotypic and hybridization studies were used to construct a dendrogram; all strains could be distributed into 29 groups. Strains isolated from Varzea soil were more heterogeneous than those obtained from Cerrado soil. This heterogeneity is believed to be a result of difference in soil type but it remained unclear whether the difference in soil type could account for differences demonstrated by the heterogeneity between Varzea and Cerrado soil populations. These observations were in agreement with the findings of Berge et al (1991) who also reported variations in the population structure of B. circulans from the rhizosphere of maize with the soil type.

2.2.3

Rhizospheric Effect and Host Specificity

It is not certain if plants actively select beneficial soil microbial communities in their rhizosphere through rhizodeposition, though earlier studies showed that plants select for taxonomic functional groups in the rhizosphere (Grayston et al. 2001; 1998). Although some field studies with mixed plant communities did not find such selections in the rhizosphere, there are reports that suggest a strong correlation between plant and soil microbial communities (Duineveld et al. 2001; Smalla et al. 2001). The root exudation is believed to be plant specific and this specificity may reflect the evolution or specific physiological adaptation to conditions of a particular soil habitat (Crowley and Rengel 1999). The type of root exudates is crucial for the ecosystem distribution and niche specificity of certain plants. Composition of root exudates was shown to vary with plant species and stage of plant growth (Jaeger et al. 1999). Concomitantly, the plant is supposed to influence the population structure of indigenous rhizobacteria as well as the population dynamics of introduced BCAs. Under certain conditions, many compounds present in the root

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exudates (sugar, amino acids, or organic acids) stimulate a positive chemotactic response in bacteria (Somers et al. 2004). Being a major driving force for microbial root colonization, plant root exudation could be engineered precisely to stimulate specific microbial colonization on the roots. Oger et al. (1997) demonstrated that genetically engineered plants producing opines have an altered rhizosphere community. In fact due to high diversity of chemical influences in the rhizosphere of different plants, roots drive specific selections of microbes out of indefinite pool of soil microbial diversity. Nevertheless, the cultivation practices being followed have also been recognized as an important determinant of rhizospheric microbiota (Mittal and Johri 2007). Agriculture management strategies can induce clear shifts in the structures of plantassociated microbial communities (Garbeva et al. 2004). For example, plant genotype can exert strong effects on the bacterial communities associated with the plants (Gu and Mazzola 2003; Adams and Kloepper 2002). Growth stage of plant is another important factor that provides shape to the rhizobacterial community structure and as reported in case of potato rhizosphere it could be the strongest one affecting the bacterial communities (van Overbeek and van Elsas 2008). Besides, land use, soil history, cultivation practices, and plant growth stage are some of the other factors which govern the structure of plant-associated microbial communities (van Overbeek and van Elsas 2008 and references therein, Mittal and Johri 2007). Among the existing practices, use of biofertilizer is of utmost importance in crop ecosystem pertaining to agriculture production. A study was carried out to evaluate the effect of cultivation practices (traditional and modern), on the community structure of culturable bacteria antagonistic toward soilborne pathogenic fungus Sclerotinia sclerotiorum, associated with the soybean (Glycine max L.) rhizoplane and rhizosphere/endorhizosphere and bulk soil (Kumar et al. 2009). The cultivation parameters for both kinds of practices were otherwise similar except that the traditional system of cultivation involved use of farmyard manure (FYM) as fertilizer input while modern cultivation system was based on application of commercially available inorganic chemical fertilizers. The community structure of bacterial antagonists isolated following traditional system of cultivation was structurally more diverse than modern system. Further, traditional system of cultivation was found to support higher population density of the antagonists. The bacterial diversity was found to increase with the stages of plant growth gradually from seedling up to maturation stage and then eventually followed a decline with only transient changes. Little variation was observed in bulk soil for community structure, implying that the bulk soil was highly stable while the gradual shifts observed in bacterial diversity may be a consequence of change in composition of root exudates excreted from the plant roots which are known to change the chemistry and biology of root microenvironments (Hartmann et al. 2009). The nature of organic amendments used in traditional system of cultivation may account for the occurrence of high bacterial diversity of antagonists in the traditional system. As a matter of fact, these organic substrates can act as ideal source of nutrients for the antagonists in soils and offer an opportunity to introduce

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and establish specific BCAs into soils, which in turn leads to sustainable disease control based on activities of microbial communities. Smalla et al. (2001) demonstrated for the first time that roots of each model plant species are colonized by its own bacterial communities using cultivation-independent methods on three phylogenetically different and economically important crops – strawberry (Fragaria ananassa Duch.), potato (Solanum tuberosum L.), and oilseed rape (Brassica napus L.). It was possible to differentiate the plant species on the basis of the rhizosphere communities using DGGE in a randomized field trial (Smalla et al. 2001). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere. All rhizobacteria showed some bands in common, and also specific bands intriguingly, e.g., Nocardia populations were identified as strawberry-specific bands.

2.2.4

Endophytic Colonization and Plant Growth Promotion

Bacteria residing in the rhizosphere of plants may gain access into the root interior and establish endophytic populations. Several bacteria can transcend the endodermis barrier, reach the vascular system by crossing through the root cortex, and subsequently thrive as endophytes in plant tissues, viz., stem, leaves, tubers, etc. (Compant et al. 2005). The endophytic colonization of host plant by bacteria reflects on their ability to selectively adapt themselves to these specific ecological niches resulting in an intimate association without any apparent harm to the plant (Compant et al. 2005 and references therein). Bacterial endophytic communities are presumed to be a product of colonization process initiated in the root zone but they may originate from other sources, viz., phyllosphere, anthosphere, or spermosphere (Sturz et al. 2000). Species of Bacillus are common inhabitants among the resident microflora of inner tissues of various species of plants, including cotton, grape, peas, spruce, and sweet corn, where they play an important role in plant protection and growth promotion (Berg et al. 2005; Shishido et al. 1999; Bell et al. 1995). Almost all the endophytic, aerobic, spore forming bacteria described so far belong to the species generally recognized as free-living soil organisms, such as B. cereus, B. insolitus, B. megaterium, B. pumilus, B. subtilis, and P. polymyxa, though in some cases the bacteria have not been identified beyond the genus level (Shishido et al. 1999; Benhamou et al. 1996; Sturz et al. 1997; Bell et al. 1995). Reva et al. (2002) studied the diversity of endophytic AEFB in the inner tissues of healthy cotton plants (Gossypium sp. Dushanbe, Tajikistan). A total of 76 strains were characterized phenotypically and majority of them were identified as B. amyloliquefaciens, B. licheniformis, B. megaterium, B. pumilus, and B. subtilis; four strains could not be assigned to any known species. Among the isolates, B. subtilis was most abundant (43 strains) followed by B. licheniformis (15 strains), B. megaterium (eight strains), and B. pumilus (six strains). Phenotypically all the four unusual strains appeared similar and showed some resemblance to B. insolitus, another well-known colonizer of plants but differed from the latter in some

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physiological properties (Sturz et al. 1997; Bell et al. 1995). Molecular typing of these four strains revealed similar RAPD patterns that were different from those of the reference strains of common plant-associated species such as B. licheniformis, B. megaterium, B. pumilus, and B. subtilis. Based on similarity level of RAPD profiling, the four strains were grouped into single distinct taxon but two different amplification profiles were obtained when the hypervariable spacer regions between 16S and 23S rRNA genes were targeted, suggesting that these four bacteria encompass two lineages within the same taxon. Complete 16S rRNA sequencing of the two representatives unravelled the distinction between them; one of these was characterized as a new species, B. endophyticus.

2.3

Phtyostimulation and Biofertilization Effects

The physiology of plant and signaling are affected by bacterial hormones in different ways depending upon the physiological role played by hormone or recalcitrance of the plant tissues to change in hormonal level and the concentration of the hormone being produced. Biofertilizing PGPR, in particular, refers to the rhizobacteria that are able to promote plant growth by increasing nutrient uptake by plants.

2.3.1

Phytostimulation

Enhancement of plant growth by root colonizing species of Bacillus and Paenibacillus is well known (Idris et al. 2007; Kloepper et al. 2004). It is also very likely that growth promoting effects of various PGPRs are due to bacterial production of plant growth regulators such as indole-3-acetic acid (IAA), gibberellins, and cytokinins (Bottini et al. 2004; Bloemberg and Lugtenberg 2001). A large proportion (80%) of bacteria colonizing the rhizosphere have been reported positive for IAA production, but reports depicting IAA production by Gram-positive soil-living bacteria are only few (Loper and Schroth 1986). However, Idris et al. (2004) showed production of substances with auxin (IAA)-like bioactivity from strains of B. subtilis/B. amyloliquefaciens including strain FZB42. Further, gibberellin production was confirmed from B. pumilus and B. licheniformis (Gutierrez-Manero et al. 2001). Tryptophan has been identified as main precursor molecule for biosynthesis of IAA in bacteria. IAA controls a diverse array of functions in plant growth and development and acts as a key component in shaping plant root architecture such as root vascular tissue differentiation, regulation of lateral root initiation, polar root hair positioning, and root gravitropism (Aloni et al. 2006). Idris et al. (2007) first demonstrated the production of reasonable quantities of IAA from Gram-positive bacterium B. amyloliquefaciens FZB42 and IAA production was enhanced when the bacterium was fed with tryptophan. Production of IAA was dramatically reduced in the mutants deficient in trp gene responsible for biosynthesis

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of IAA, suggesting that main route of IAA biosynthesis in this bacterium was dependent on tryptophan. Spaepen et al. (2007) reviewed different pathways involved in the biosynthesis of IAA based on the chemical nature of intermediate molecules produced using tryptophan as precursor. The plant beneficial Gram-negative bacteria synthesize IAA following different pathways that involves indole-3-pyruvic acid (IPA), indole-3-acetamide (IAM), or indole-3-acetonitrile (IAN) as important intermediates (Patten and Glick 1996; Kobayashi et al. 1995). However, in Gram-positive bacteria the main route for biosynthesis of IAA involves IPA (Vandeputte et al. 2005). Plant hormones affect the spatial and temporal expression of various phenotypes such as cell elongation, division, and differentiation. Besides they are believed to play an important role in plant’s response to biotic and abiotic stresses. Many bacteria are capable of producing more than one type of plant hormone; however, some of them can produce and degrade the same hormone, produce one, and degrade the precursor of another, thus affecting the physiology of plant in several ways (Boiero et al. 2007; Leveau and Lindow 2005). Further, bacterial production of IAA may be beneficial or detrimental to the plant health. For example, IAA production by P. putida GR12-2 has been found to improve the root proliferation of Azospirillum brasilense resulting in increased root surface area which helps in augmentation of nutrient and water uptake from soil (Patten and Glick 2002). On the other hand, in some reports IAA production has been found necessary for pathogenesis (Yang et al. 2007; Vandeputte et al. 2005). There is a growing body of literature showing that IAA can act as a signal molecule, indicating that use of hormones as signaling molecules is not confined only to the plants but also takes part in communication between bacteria and other microorganisms (Spaepen et al. 2007).

2.3.2

Biofertilization

PGPR stimulate the plant growth directly through increase in nutrition acquisition, such as phosphate solubilization, or more generally by rendering the inaccessible nutrients available to the plants (Persello-Cartieaux et al. 2003). After nitrogen, perhaps the essential mineral element that most frequently limits the growth of plants is P, which is taken up from soil solution as phosphate (Pi, H2PO4 ). Although soils generally contain a large amount of total P but only a small proportion is available for uptake by the plants. On an average, most of mineral nutrients in soil are present in millimolar amounts but P is present in micromolar or even lesser quantities (Khan et al. 2006). However, plants are well adapted to uptake of P from low concentration soil solution (Jungk 2001). Therefore, it is presumed that the supply and availability of P to the root surface is influenced by the root and microbial processes. Phosphate-solubilizing microorganisms (PSM) include a wide range of symbiotic and nonsymbiotic organisms, such as Pseudomonas, Bacillus, and Rhizobium

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species; actinomycetes; and various fungi-like Aspergillus and Penicillium species (Richardson et al. 2009 and references therein). Phosphate-solubilizing bacteria have already been applied in the agronomic practices as potential bioinoculants to increase the productivity. For example, in Soviet Union, a biofertilizer product under the trade name “phosphobacterin” was prepared and commercialized for agricultural applications. Phosphobacterin contained Bacillus megaterium var. phosphaticum and later on it was also introduced to other countries, like Eastern Europe and India (Khan et al. 2006). Similarly, in India, a consortium, termed as Indian Agricultural Research Institute (IARI) microphos culture, has been developed containing two very efficient phosphate-solubilizing bacteria (Pseudomonas striata and Bacillus polymyxa) and three phosphate-solubilizing fungi (Aspergillus awamori, A. niger, and Penicillium digitatum) (Gaur 1990). Application of phosphate solubilizers alone or in combination with nitrogen fixers has been found beneficial for cotton and wheat fields (Zaidi and Khan 2005; Kundu and Gaur 1980). A study had been carried out under green house conditions to explore the effects of combined inoculation of Rhizobium and phosphatesolubilizing P. striata or B. polymyxa with or without added fertilizers on chickpea yield and nutritional contents (Algawadi and Gaur 1988). Whereas, inoculation with Rhizobium alone was found to increase nodulation, addition of phosphate solubilizers increased the phosphorus content of the soil. Combined inoculation increased the nodulation and available phosphorus of the soil coupled with improved grain yield and phosphorus and nitrogen uptake by the plants. Natarajan and Subramainan (1995) suggested that following a combined inoculation of Rhizobium (strain Tt 9) with B. megaterium var. phosphaticum could meet with about 50% of the phosphatic fertilizer requirement of the groundnut. This consortium was found very effective for groundnut, resulting in increased nodulation, increased root and shoot length, as well as increased pod yield. Tomar et al. (1993) reported that inoculation with the phosphate-solubilizing bacterium B. firmus resulted in significant increase in seed yield in field trials on lentil (Lens esculentus) and black gram (Vigna mungo). Similarly, Bethlenfalvay (1994) demonstrated the impact of a consortium comprising Glomus mosseae, Bacillus sp., and Rhizobium sp. on plant growth and soil aggregation upon Pisum sativum cultivation and observed a dramatic increase in plant growth and soil aggregation. While in case of P. sativum, inoculation of Rhizobium, B. polymyxa, and Glomus faciculatum resulted in enhanced dry matter production and PO43 uptake, no significant response of soybean to dual inoculation was observed (Kloepper et al. 1980).

2.4

Biological Control: Gram-Positive Perspectives

Biological control, using microorganisms to suppress plant disease, offers a powerful alternative to the use of synthetic chemicals. The rich diversity of the microbial world provides a seemingly endless resource for this purpose. While a diverse array

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of microorganisms contribute toward the biological control of plant pathogens, most research has utilized species of Bacillus, Trichoderma, and Pseudomonas (McSpadden Gardener and Driks 2004). There are eight species of microorganisms registered by U.S. Environmental Protection Agency for commercial use against soilborne plant pathogens in the United States (Cook et al. 1996). These include two fungi (Gliocladium virens G-21 and Trichoderma harzianum KRL-AG2), three Gram-negative bacteria (Agrobacterium radiobacter K84, Pseudomonas fluorescens EG1053, and Burkholderia cepacia type Wisconsin), and three Gram-positive bacteria (Bacillus subtilis GB03, B. subtilis MBI 600, and Streptomyces griseoviridis K61). Other than A. radiobacter K84, all others are used to control damping-off diseases and improve stand establishment and seedling vigour. There is a growing body of literature which describes different mechanisms for biocontrol ability of Bacillus, viz., siderophore production, secretion of hydrolytic enzymes, antibiosis, ISR, etc. However, discussion on all these aspects of biocontrol is beyond the scope of this chapter, hence antibiosis, quorum quenching (QQ), and ISR, the mechanisms of major importance being emphasized in current scenario involved in biocontrol, will be discussed in detail. Moreover, numerous reports on in vitro antimicrobial activity of Bacillus species are available, but here we emphasize on the selective studies that combine the successful in situ demonstration of antagonism in addition to in vitro studies, i.e., success stories of Bacillus species used as BCAs in the field.

2.4.1

Success Stories of Bacillus Species as Biocontrol Agents

Extensive research including the field testing of different Bacillus strains has led to the development of a number of products widely used as commercial BCAs (McSpadden Gardener and Fravel 2002). There is a list of biopesticides (available online: http://www.oardc.ohio-state.edu/apsbcc) registered for pests and disease control in the United States, approved by the U.S. Environmental Protection Agency (EPA), wherein the commercial formulations of different Bacillus strains used as BCAs are specified. The products are available as different formulations, viz., liquid or suspension in a liquid, wettable powder, or dry cakes depending upon the compatibility of the biocontrol strain with the carrier molecule. Products like Companion, Kodiak, Serenade, Subtilex, and Taegro are based on exploitation of different strains of B. subtilis as BCAs. Although Companion and Kodiak manufactured by Growth Products Ltd, NY, and Gustafson Inc., TX, of the United Staes, respectively, use the same strain B. subtilis GB03, the formulations used differ; the former is used as liquid while the latter as dry flakes. While Kodiak is labeled for the control of root pathogens of cotton and legumes (soybean) such as Rhizoctonia solani, Fusarium spp., Aspergillus spp., and Alternaria spp., Companion is known to control the diseases caused by species of Rhizoctonia, Phytophthora, Pythium, and Fusarium. The principal component of Subtilex (Becker Underwood, Ames, IA) is B. subtilis MBI600 and is marketed for control of root- and

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seed-borne infections of ornamental and vegetable crops, such as root rot of soybean and Botrytis species, infection of vines, strawberry and cucumber, and brown rust of cereals. Likewise, Serenade (AgraQuest, Davis, CA, USA) containing B. subtiis strain QST713 has been proposed to mitigate the downy mildew, Cercospora leaf spot, and early blight and late blight diseases associated with various crop plants. However, until today the genetic basis of biocontrol ability of B. subtilis strains is not clearly understood and much has been emphasized on the antibiotic production (Joshi and McSpadden Gardener 2006).

2.4.1.1

Antibiosis

Bais et al. (2004) demonstrated the protective action of surfactin produced by B. subtilis against infection caused by Pseudomonas syringae in Arabidopsis thaliana and suggested that surfactin was necessary not only for root colonization but also provided protection against the pathogen. The disease suppression was correlated with inhibitory concentrations of surfactin produced by the organism on roots. Moyne et al (2001) identified B. subtilis strain AU195 capable of producing antifungal peptides showing similarity with bacillomycin (group iturin A). The strain AU195 exhibited strong antagonistic activity against Aspergillus flavus and a broad range of other plant pathogenic fungi. In another study, B. amyloliquefaciens strain A1Z isolated from soybean rhizosphere was found to produce iturin-like compounds, which successfully inhibited three taxonomically diverse fungal pathogens, Sclerotinia sclerotiorum, Macrophomina phaseolina, and Fusarium oxysporum, the causal agents of sclerotinia stem rot, charcoal rot, and fusarial wilt of soybean plants, under controlled conditions. Chromatographic analysis and mass spectrometric studies showed that the principal antifungal components show similarity with iturin-like compounds (Kumar et al. unpublished). However, the efficacy of antifungal compounds has not been evaluated in the field as yet. Romero et al. (2007) showed the involvement of iturin and fengycin antibiotics from four B. subtilis strains UMAF6614, UMAF6616, UMAF6639, and UMAF8561 in the suppression of powdery mildew of cucurbits caused by Podosphaera fusca. The culture supernatant could successively inhibit the powdery mildew at levels previously reported for vegetative cells (Romero et al. 2004). The chemical analysis of culture filtrate together with the recovery of inhibitory components (surfactin, fengycin, and iturin A or bacillomycin) from the melon leaves treated with two strains (UMAF6614 and UMAF6639) strongly supported the evidence of in situ production of these antimicrobials.

2.4.1.2

Quorum Quenching and Biological Control

Bacteria sense their population density and coordinate the expression of target genes, including the virulence factors in Gram-negative bacteria, by N-acylhomoserine lactones (AHLs) dependent mechanism known as quorum sensing (QS). While

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AHLs and other substituted g-butyrolactones are synthesized by Gram-negative bacteria, certain oligopeptides and substituted g-butyrolactones are the primary signal molecules found in Gram-positive bacteria (Faure et al. 2009). The most widely studied signal molecules involved in quorum sensing are the AHLs (Whitehead et al. 2001). In Gram-positive bacteria, QS signaling molecules are generally peptides, except for the universal pheromone LuxS found in both Gram-positive and Gramnegative bacteria (Schauder et al. 2001). QS is believed to play a crucial role in bacterial physiology including regulation of rhizospheric competence factors such as antibiotic production, horizontal gene transfer, and control of those functions that are directly or indirectly related to plant–microbe interactions (Whitehead et al. 2001). However, several soil bacteria are able to interfere with the QS by enzymatic degradation of AHLs, a process known as QQ. AHL inactivation has been reported in a-proteobacteria (e.g., Agrobacterium, Bosea, and Ochrobactrum), b-proteobacteria (e.g., Variovorax, Ralstonia, Comamomonas, and Delftia), and g-proteobacteria (e.g., Pseudomonas and Acinetobacter) (Faure et al. 2009). In case of Gram-positive bacteria, AHL degradation occurs in both low G + C% strains, i.e., Firmicutes, such as Bacillus, and in high G + C% strains or actinobacteria, such as Rhodococcus and Arthrobacter. Acylhomoserine lactonase activity (AiiA) that hydrolyzes the lactone ring of AHLs was first observed in a Bacillus isolate from soil (Dong et al. 2001, 2000). Until now, two types of enzymes that inactivate AHLs have been identified in several species/genera of bacteria: the AHL lactonases that cause lactonolysis (opening of the gamma-butyrolactone ring) resulting in acyl-homoserine with reduced biological activity and AHL acylases that break the amide linkage of AHLs to produce homoserine lactone and fatty acids with no biological activity (Uroz et al. 2008; Zhang and Dong 2004). QQ covers various phenomena that lead to perturbation of expression of QSregulated functions. Dong et al. (2007) evaluated the mechanisms and functions of QQ in vivo and threw light on the possible applications of this phenomenon in control of plant diseases and promotion of plant health. It has been suggested by many researchers to take advantage of QQ to develop novel biocontrol strategies for plant pathogens (Dong et al. 2007). For example, Park et al. (2008) identified a potential AHL-degrading enzyme, AiiA, from B. thuringiensis which could effectively attenuate the virulence of Gram-negative bacterium Erwinia carotovora in the root system of pepper plant by QQ. Recent studies on B. thuringiensis show that many subspecies of this organism produce AiiA homolog enzymes to degrade AHLs (Dong et al. 2004, 2000). In another case, genetically modified plants which expressed AHL lactonase, AiiA of Bacillus, were found to be more resistant to Pectobacterium carotovorum infection than their parental, wild-type plants (Dong et al. 2001). Moreover, studies carried out by Molina et al. (2003) clearly demonstrated the role of AHL-lactonase enzyme in biocontrol of phytopathogens. A significant reduction was observed in the severity of soft rot of potato caused by P. carotovorum and crown gall of tomato caused by A. tumefaciens when applied with soil bacterium Bacillus sp. A24 or P. fluorescens P3 modified with lactonase gene AiiA, suggesting that disease inhibition was a result of QQ.

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Thus, QQ, in a way, can be used under antivirulence/antidisease strategies to develop novel medical/animal therapies or novel biological control strategies for phytopathogens (Dong et al. 2007). These studies elegantly suggest that QQ can be used as a potential weapon for biological control of pathogenic microorganisms targeting the QS pathway, however, little is known toward ecological aspects of QQ enzymes under in situ conditions. All QQ strategies have so far been developed under in vitro or under the green house conditions and their efficacy under field conditions remains to be evaluated. Assessment of interconnections in the signal molecules is a future challenge that needs the help of advanced analytical tools and techniques including transcriptomics, proteomics, and metabolomics to account for the intra- and inter-species communications in the rhizosphere and their ecological impact on the rhizospheric microbiota.

2.4.1.3

Induced Systemic Resistance: Ecological Significance and Applicability

Induced resistance may be defined as a physiological “state of enhanced defensive capacity” elicited in response to specific environmental stimuli and consequently the plant’s innate defenses are potentiated against subsequent biotic challenges (van Loon 2000). In addition, there is another defined form of induced resistance, popularly known as systemic acquired resistance (SAR) which is different from ISR in context to the nature of elicitor and regulatory pathways involved. While ISR relies on pathways regulated by jasmonic acid (JA) and ethylene (ET), SAR involves accumulation of salicylic acid (SA) and pathogenesis related (PR) proteins – chitinase and cellulase. PGPRs are among the various groups of plant-associated microorganisms that can elicit the plant defense systems resulting in reduction of disease severity or incidence of diseases caused by pathogens which are spatially different from the inducing agent (van Loon and Glick 2004). Recently, Choudhary and Johri (2008) explicated the mechanisms and role of Bacillus species as inducers of systemic resistance in relation to plant–microbe interactions and demarketed the pathways involved in their regulation. Available reports suggest that specific strains of the species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus elicit significant reductions in the incidence or severity of various diseases on a diversity of hosts including greenhouse studies or field trials on tomato, bell pepper, muskmelon, watermelon, sugarbeet, tobacco, Arabidopsis species, cucumber, loblolly pine, and tropical crops (Kloepper et al. 2004).

2.4.1.4

Greenhouse Studies on Induction of Plant Resistance Systems

A greenhouse test was performed for ISR study of B. mycoides strain Bac J isolated from sugarbeet leaves infected with Cercospora beticola, the causal agent of Cercospora leaf spot on sugarbeet. The strain was sprayed (1.0 log 8 CFU/ml)

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onto one leaf of test plant and bagged. After 3 days of treatment with Bac J, plants were challenge inoculated with the spore suspension of pathogen. There was a significant reduction in disease severity in plants treated with Bac J on a highly susceptible and a moderately susceptible variety of sugarbeet (Bargabus et al. 2002). In another study performed by Krause et al. (2003), bacterial strains isolated from compost were screened for their capacity to elicit systemic protection against Xanthomonas campestris py. armoraciae. A total of eleven isolates were found to elicit significant reduction in the disease severity in two of the three repeated experiments: four of the top performing strains were characterized as members of Bacillus species. A comparative study of the results obtained by microtiter-based bioassays to assess elicitation of ISR and pot experiments was conducted in greenhouse against blue mold of tobacco caused by Peronospora tabacina Adam (Zhang et al. 2002). The disease incidence was significantly reduced in terms of mean percentage of leaf area under infection from P. tabacina Adam when strains of B. pasteurii C-9 and B. pumilus SE34 and T4 were applied as soil drenches on three tobacco cultivars. Also, the sporulation of the pathogen was significantly decreased when compared with the treated strains and nonbacterized control. To explore the relationship between elicitation of plant growth promotion and ISR, the three strains were further evaluated and applied separately as seed treatment. Tobacco growth was significantly increased by strains SE34 and C-9 but not by T4. It was found to be induced by C-9, not by SE34 and T4. However, application of bacteria by seed treatment following soil drenches resulted in elicitation of ISR by all three strains, in addition to the enhancement of plant growth. In another study, B. subtilis strain AF1, isolated from soils suppressive to pigeon pea (Cajanus cajan) wilt caused by Fusarium udum, was presumed to induce resistance against Aspergillus niger on peanut (Arachis hypogea) (Podile and Dube 1988). Further, it was found that strain AF1 stimulated production of phenylalanine ammonia lyase (PAL) and peroxidase activity, indicating that AF1 elicited ISR (Podile et al. 1995). Strong experimental evidence that AF1 elicited ISR came from the findings of Sailaja et al. (1997) who reported a notable reduction in the incidence of crown rot of peanut caused by A. niger corresponding to increase in lipoxygenase activity, a phenomenon associated with ISR.

2.4.1.5

Field Experiments for Protection Against Systemic Disease

It is not surprising that many biological control agents showing promising results under the controlled environmental conditions of greenhouse fail to exhibit same results in the field under natural environments where competition is more severe. Therefore, shifting from greenhouse to field trials is an important step to evaluate the efficacy of PGPR eliciting ISR and Bacillus. Species were found effective in reduction of disease incidence or plant growth promotion have been examined under field conditions.

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In a field trial conducted on sugarbeet for six consecutive growing seasons, the disease severity due to Cercospora leaf spot was reduced significantly when sprayed with B. mycoides strain Bac J (log 7.0 CFU/ml). About 38–91% reduction in disease severity was found in comparison to the nontreated control. However, in 2 of the 6 years, reduction in disease severity achieved by treatment with Bac J was not significantly different from that attained by using triphenyltin hydroxide, the most commonly used fungicide for Cercospora leaf spot. It has been suggested previously that ISR was presumed to be the mechanism of disease control in greenhouse test that provided spatial separation of pathogen and PGPR but spatial separation was not maintained in the field experiments (Bargabus et al. 2002). In addition to the bacterial and fungal diseases, reduction in the incidence or severity of viral diseases has also been studied in the field employing selected strains of ISR-eliciting Bacillus species. Zehnder et al. (2000) assessed three strains, B. subtilis IN937b, B. pumilus SE34, and B. amyloliquefaciens IN937a for ISR activity against CMV on tomato plants under field conditions for two consecutive cropping seasons. The PGPR strains were applied as seed treatments at the time of transplanting to the pots prior to their transplantation in the field, while CMV inoculation was done on plants 1 week before transplantation to the field. Treatment with all three Bacillus strains resulted in significant reduction of disease compared to the nonbacterized control. Resistance-inducing rhizobacteria offer an attractive alternative, providing a natural, safe, effective, persistent, and durable type of protection. But protection based on biological agents is not always trustworthy and is seldom as effective as chemical treatments. However, different treatments may be combined and combinations of BCAs that suppress diseases by complementary mechanisms may further reduce the incidence or severity of disease. Rhizobacteria-mediated ISR thus may be a valuable addition to the alternatives available for environmentally friendly plant disease control.

2.5

Conclusions

Considerable efforts toward understanding the ecology and management of PGPR have been directed, yet their development as inoculants remains a considerable challenge. The rhizospheric community is highly complex, comprises of a myriad of organisms interacting in various ways, acting upon each other and reacting to the external environment. Several isolates of Bacillus spp. have been developed as BCAs of plant pests and pathogens. However, to be used as successful BCAs a greater understanding of their ecology is desired. In this context, greater knowledge of the diversity, distribution, and physiology of Gram-positive species will be helpful for identification of new strains compatible with the cropping systems. Paramount to success of PGPR is a need to better understand the ecology of rhizobacteria either indigenous or introduced within the rhizosphere. Exploration and identification of traits involved in the ability of certain bacteria to establish

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themselves into the rhizosphere at levels sufficient to exert effects on plant growth, effectively compete with the indigenous microflora, cooperatively interact with other beneficial members of rhizospheric biota, and understand the mechanisms (signaling, growth promotory actions, disease suppression etc.) that occur between plants and bacteria are also required. Clearly, the taxonomic and physiological diversity of Bacillus spp. appears capable of reducing the disease incidence or severity but also indicates that much remains to be done on the mechanisms by which these bacteria promote plant growth. The molecular mechanisms involved in the root colonization are under study nowadays and advancement in the molecular and genomic tools offers new possibilities for improving the selection, characterization, and management of biological control. Development of proteomics and functional genomics will be helpful to determine and follow expression of crucial genes of BCAs during mass production, formulation, and application. Transformation of BCAs by inserting genes that improve the tolerance of antagonists to abiotic stresses, such as increased tolerance or resistance to cold, heat, drought, high salinity, heavy metal rich soils, or acidic soils, etc., could be another exciting and challenging task and may provide with better opportunities to implement the concept of biocontrol in the field under the dynamic natural environments. Acknowledgements This work was supported in part by grant received in the form of Silver Jubilee Fellowship to BNJ from Madhya Pradesh Council of Science and Technology, Bhopal. The authors are thankful to Dr. Shipra Singh, DST Young Scientist for critical reading of the manuscript and Mr. Sandeep Saini, Research Fellow, Department of Biotechnology and Bioinformatics Centre, Barkatullah University, Bhopal for help in preparation of the manuscript.

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Tomar SS, Pathan MA, Gupta KP, Khandkar UR (1993) Effect of phosphate solubilizing bacteria at different levels of phosphate on black gram (Phaseolus mungo). Ind J Agron 38:131–133 Uroz S, Oger PM, Chapelle E, Adeline MT, Faure D, Dessaux Y (2008) A Rhodococcus qsdAencoded enzyme defines a novel class of large-spectrum quorum-quenching lactonases. Appl Environ Microbiol 74:1357–1366 van Loon LC (2000) Systemic induced resistance. In: Slusarenko AJ, Fraser RSS, van Loon LC (eds) Mechanism of resistance to plant diseases. Kluwer, Dordrecht, pp 521–574 van Loon LC, Glick BR (2004) Increased plant fitness by rhizobacteria. In: Sandermann H (ed) Molecular ecotoxicology of plants, vol 170. Springer, Berlin, pp 177–205 van Overbeek L, van Elsas JD (2008) Effect of plant genotype and growth stage on the structure of bacterial communities associated with potato (Solanum tuberosum L.). FEMS Microbiol Ecol 64:283–296 Vandeputte O, Oden S, Mol A, Vereecke D, Goethals K, El Jaziri M, Prinsen E (2005) Biosynthesis of auxin by the Gram positive phytopathogen Rhodococcans fascians is controlled by compounds specific to infect plant tissues. Appl Environ Microbiol 71:1169–1170 Vargas-Ayala R, Rodriguez-Kaban R, Morgan-Jones G, McInroy JA, Kloepper JW (2000) Shifts in soil microflora induced by velvetbean (Mucuna deeringiana) in cropping systems to control root-knot nematodes. Biol Control 17:11–22 Weller DM (1988) Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu Rev Phytopathol 26:379–407 Whipps JM (2001) Microbial interactions and biocontrol in the rhizosphere. J Exp Bot 52:487–512 Whitehead NA, Barnard AML, Slater HLSNJ, Salmond GPC (2001) Quorum sensing in Gram negative bacteria. FEMS Microbiol Rev 25:365–404 Wieland G, Neumann R, Backhaus H (2001) Variation of microbial communities in soil, rhizosphere and rhizoplane in response to crop species, soil type and crop development. Appl Environ Microbiol 67:5849–5854 Yang SH, Zhang Q, Guo JH, Charkowski AO, Glick BR, Ibekwe AM (2007) Global effect of indole-acetic acid biosynthesis on multiple virulence factors of Erwinia chrysanthemi 3937. Appl Environ Microbiol 73:1079–1088 Zaidi A, Khan MS (2005) Interactive effect of rhizospheric microorganisms on growth, yield and nutrient uptake of wheat. J Plant Nutr 28:2079–2092 Zehnder GW, Yao C, Murphy JF, Sikora EJ, Kloepper JW (2000) Induction of resistance in tomato against cucumber mosaic cucumovirus by plant growth-promoting rhizobacteria. Biol Control 45:127–137 Zhang LH, Dong YH (2004) Quorum sensing and signal interference: diverse implications. Mol Microbiol 53:1563–1571 Zhang S, Moyne A-L, Reddy MS, Kloepper JW (2002) Development of assays for assessing induced systemic resistance by plant growth promoting rhizobacteria against blue mold of tobacco. Biol Control 25:288–296

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Chapter 3

Endophytic Bacteria: Perspectives and Applications in Agricultural Crop Production M. Senthilkumar, R. Anandham, M. Madhaiyan, V. Venkateswaran, and Tongmin Sa

3.1

Introduction

Both aerial and subterranean plant organs are constantly exposed to intimate contacts with a plethora of microorganisms, including members of phyla as diverse as viruses, bacteria, oomycetes, fungi, and eukaryotic protozoans. The outcome of interactions between plants and microbes can be neutral, detrimental, or even beneficial for the photoautotrophic organisms. Disadvantageous encounters typically manifest themselves as diseases, which in extreme cases can result in full collapse of plant tissues (Volker and Panstruga 2005). By contrast, benign contacts usually give rise to symbiotic relationships that typically support the plant’s nitrogen metabolism and mineral uptake. Surprisingly, although many microbes have a principal phytopathogenic potential, the majority of interactions between plants and microbes remain macroscopically symptomless. Beneficial plant–microbe interactions that promote plant health and development have been the subject of considerable study. Endophytes, microorganisms that reside in the tissues of living plants, are potential sources of novel natural products for exploitation in medicine, agriculture, and industry but these are relatively unstudied. It is noteworthy that, of the nearly 300,000 plant species that exist on earth, each individual plant is host to one or more endophytes. Only a few of these plants have been completely

M. Senthilkumar Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India R. Anandham Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai 625 104, Tamil Nadu, India M. Madhaiyan and T. Sa (*) Department of Agricultural Chemistry, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea e-mail: [email protected] V. Venkateswaran Ministry of Food Processing Industries, Government of India, New Delhi 110 049, India


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studied relative to their endophytic biology. Consequently, the opportunity to find new and interesting endophytic microorganisms among myriads of plants in different settings and ecosystems is great.

3.2

“Endophytes” What It Means?

For more than 50 years, bacteria have been observed to exist inside plants without causing apparent disease symptoms (Tervet and Hollis 1948). Various reports indicate that such bacteria exist in a variety of tissue types within numerous plant species, suggesting a ubiquitous existence in most plants. Taken literally, the word endophyte means “in the plant” (endon Greek, within; phyton, plant). Since the discovery of endophytes in Darnel, Germany, in 1904 (Tan and Zou 2001), various investigators have defined endophytes in different ways, which is usually dependent on the perspective from which the endophytes were being isolated and subsequently examined. Bacon and White (2000) gave an inclusive and widely accepted definition of endophytes as “microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects.” While the symptomless nature of endophyte occupation in plant tissue has prompted focus on symbiotic or mutualistic relationships between endophytes and their hosts, the observed biodiversity of endophytes suggests that they can also be aggressive saprophytes or opportunistic pathogens. The usage of the term is as broad as its literal definition and spectrum of potential plant hosts and inhabitants includes bacteria (Kobayashi and Palumbo 2000), fungi (Stone et al. 2000), algae (Peters 1991), and insects (Feller 1995). Any organ of the host can be colonized. The endophytic partners and their relationships to each other vary. There are pathogenic endophytic algae (Bouarab et al. 1999), parasitic endophytic plants (Marler et al. 1999), mutualistic endophytic bacteria (Chanway 1996), ectomycorrhizal helper bacteria (Founoune et al. 2002), as well as endophytic bacteria in pathogenic and commensalistic symbioses (Sturz and Nowak 2000). Both fungi and bacteria are the most common microbes existing as endophytes (Strobel and Daisy 2003). It seems that other microbial forms, e.g., mycoplasmas and archaebacteria, exist in plants as endophytes, but no evidence for them has yet been reported. Kloepper et al. (1992) called bacteria found within tissues internal to the epidermis as endophytes. However, quiescent endophytic bacteria can become pathogenic under certain conditions and within different host genotypes (Misaghi and Donndelinger 1990). James and Olivares (1997) adjusted the definition and stated that all bacteria that colonize the interior of plants, including active and latent pathogens, can be considered to be endophytes. Considering all bacteria that colonize the interior of plants, one should also take into account those bacteria that reside within living plant tissues without doing substantive harm or gaining benefit other than securing residency (Kado and Kado 1992), as well as those bacteria that establish endosymbiosis with the plant, whereby the plant receives an ecological benefit from the presence of the symbiont (Quispel 1992).

3 Endophytic Bacteria: Perspectives and Applications

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The definition of endophytic bacteria should, in accordance with the definition of endomycorrhizal fungi, also include bacteria that reside in the cortex of the root. These subdefinitions may provide an operational overview of what is considered to be an “endophyte,” and consequently this might be regarded as the most general definition for which the term “endophyte” stands.

3.3

Sources of Endophytic Bacteria

Two of the most frequently raised questions in connection with endophytic bacteria are what is the origin of endophytes and how do they enter plant tissues in nature? The sources of endophytes are various. These can appear to originate from seeds (McInory and Kloepper 1995), vegetative planting material (Sturz 1995), rhizosphere soil (Patriquin et al. 1983), and the phylloplane (Beattie and Lindow 1995). The importance of seeds as a source of endophytic bacteria is still controversial. The role of the spermosphere as a source of bacterial endophytes is also evident by observations that bacterial endophytes introduced as seed treatments colonized the internal tissues of root radical newly emerged from the seed coat (Musan et al. 1995). In detail, endophytic colonization appeared to begin with the migration of bacteria through the germination slit and into the starchy endosperm, from which these bacteria colonized the radicle and coleoptile and finally spread systematically through the plant. Besides seeds and spermosphere, several observations favor the rhizosphere soil as the primary source for endophytic colonization. Axenic potatoes planted into field soil mainly harbored genera of commonly found soil saprophytes (De Boer and Copeman 1974). Comparing the internal and external bacterial communities of cucumber, cotton, and potato, almost all endophytic bacteria were found also in the rhizosphere, thus supporting the hypothesis that there is a continuum of root-associated microorganisms from the rhizosphere to rhizoplane to epidermis and cortex (Kloepper et al. 1992). Statistical analysis of the bacterial diversity from rhizosphere (zone outside roots) and endorhiza (root interior) indicated that the initial composition of the endorhiza was dependent on the rhizosphere community. However, the endorhiza community quickly differentiated from that of the rhizosphere with fewer genera present, suggesting that the endorhiza is a distinct habitat from the rhizosphere. Rosenblueth and Martıńez-Romero (2006) published a list of endophytic bacteria reported in various plant species (Table 3.1) but the list continues.

3.4

Modes of Entry of Endophytes

With the exception of seed transmitted bacteria, which are already present in the plant, potential endophytes must first colonize the root surface prior to entering the plant. Potential internal colonists find their host by chemotaxis, electrotaxis, or

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Table 3.1 Reported putative endophytes in cultivated plants (Rosenblueth and Martıńez-Romero 2006) Endophytes Plant species References a-Proteobacteria Azorhizobium Rice Engelhard et al. (2000) caulinodans Azospirillum brasilense Banana Weber et al. (1999) Azospirillum Banana, pineapple Weber et al. (1999) amazonense Bradyrhizobium Rice Chantreuil et al. (2000) japonicum Gluconacetobacter Sugarcane, coffee Cavalcante and D€obereiner (1988); diazotrophicus Jimeńez-Salgado et al. (1997) Methylobacterium Citrus plants Araujo et al. (2002) mesophilicuma Methylobacterium Scots pine, citrus plants Araujo et al. (2002); Pirttil€a et al. (2004) extorquens Rhizobium Rice Yanni et al. (1997) leguminosarum Rhizobium Carrot, rice Surette et al. (2003) (Agrobacterium) radiobacter Sinorhizobium meliloti Sweet potato Reiter et al. (2003) Rice Engelhard et al. (2000) Sphingomonas paucimobilisa b-Proteobacteria Azoarcus sp.

Kallar grass, rice

Burkholderia pickettiia Burkholderia cepaciab Burkholderia sp.

Maize Yellow lupine, citrus plant Banana, pineapple, rice

Chromobacterium violaceuma Herbaspirillum seropedicae Herbaspirillum rubrisubalbicans

Rice

g-Proteobacteria Citrobacter sp. Enterobacter spp. Enterobacter sakazakiia Enterobacter cloacaea Enterobacter agglomeransa Enterobacter asburiae Erwinia sp. Escherichia colib Klebsiella sp.

Engelhard et al. (2000); Reinhold-Hurek et al. (1993) McInory and Kloepper (1995) Araujo et al. (2001); Barac et al. (2004) Weber et al. (1999); Engelhard et al. (2000) Phillips et al. (2000)

Sugarcane, rice, maize, sorghum, banana Sugarcane

Olivares et al. (1996); Weber et al. (1999)

Banana Maize Soybean Citrus plants, maize

Martıńez et al. (2003) McInory and Kloepper (1995) Kuklinsky-Sobral et al. (2004) Araujo et al. (2002); Hinton and Bacon (1995) Kuklinsky-Sobral et al. (2004)

Soybean Sweet potato Soybean Lettuce Wheat, sweet potato, rice

Klebsiella pneumoniaeb Soybean

Olivares et al. (1996)

Asis and Adachi (2003) Kuklinsky-Sobral et al. (2004) Ingham et al. (2005) Engelhard et al. (2000); Iniguez et al. (2004); Reiter et al. (2003) Kuklinsky-Sobral et al. (2004) (continued)

3 Endophytic Bacteria: Perspectives and Applications Table 3.1 (continued) Endophytes Klebsiella variicolab

65

Klebsiella terrigenaa Klebsiella oxytocab Pantoea sp.

Plant species Banana, rice, maize, sugarcane Carrot Soybean Rice, soybean

Pantoea agglomerans

Citrus plants, sweet potato

Pseudomonas chlororaphis Pseudomonas putidaa Pseudomonas fluorescens Pseudomonas citronellolis Pseudomonas synxantha Salmonella entericab

Marigold (Tagetes spp.), carrot Carrot Carrot

Surette et al. (2003) Kuklinsky-Sobral et al. (2004) Kuklinsky-Sobral et al. (2004); Verma et al. (2004) Araujo et al. (2001, 2002); Asis and Adachi (2003) Sturz and Kimpinski (2004); Surette et al. (2003) Surette et al. (2003) Surette et al. (2003)

Soybean

Kuklinsky-Sobral et al. (2004)

Scots pine Alfalfa, carrot, radish, tomato Rice Rice Dune grasses (Ammophila arenaria and Elymus mollis)

Pirttil€a et al. (2004) Cooley et al. (2003); Guo et al. (2002); Islam et al. (2004) Sandhiya et al. (2005) Gyaneshwar et al. (2001) Dalton et al. (2004)

Citrus plants Maize, carrot, citrus plants Grass Miscanthus sinensis Sweet potato Carrot

Araujo et al. (2001, 2002) Araujo et al. (2001)McInory and Kloepper (1995); Surette et al. (2003) Miyamoto et al. (2004) Reiter et al. (2003) Surette et al. (2003)

Rice

Phillips et al. (2000)

Maize

Chelius and Triplett (2000)

Citrus plants

Araujo et al. (2002)

Marigold Marigold

Sturz and Kimpinski (2004) Sturz and Kimpinski (2004)

Maize

Zinniel et al. (2002)

Wheat, Scots pine

Conn and Franco (2004); Pirttil€a et al. (2004) Araujo et al. (2002) Coombs and Franco (2003)

Serratia sp. Serratia marcescensa Stenotrophomonasa

Firmicutes Bacillus spp. Bacillus megaterium Clostridium Paenibacillus odorifer Staphylococcus saprophyticusb Bacteroidetes Sphingobacterium sp.a Actinobacteria Arthrobacter globiformis Curtobacterium flaccumfaciens Kocuria varians Microbacterium esteraromaticum Microbacterium testaceum Mycobacterium sp.b

Nocardia sp.b Citrus plants Streptomyces Wheat a Opportunistic human pathogenic bacteria b Common human pathogenic bacteria

References Rosenblueth et al. (2004)

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accidental encounter. Motility of beneficial associative rhizosphere bacteria has been described for several bacteria such as Alcaligenes faecalis, Azospirillum brasilense, and Pseudomonas fluorescens (Bashan 1986; You et al. 1995). In general, entry into a plant tissue can be via the stomata, lenticels, wounds (including broken trichomes), areas of emergence of lateral roots and germinating radicles. However, the main entry for endophytic bacteria appears to be through wounds that naturally occur as a result of plant growth or through root hairs and at epidermal conjunctions. Several authors have reported extensive colonization of the secondary root emergence zone (site of root branches) by bacterial endophytes. Because of breaks in the endodermis at these points, bacteria colonizing the cortex can be observed to extend to and across the epidermis into the vascular tissue. Plant wounds, in general, induced either by biotic or abiotic factors, are ubiquitous in any agroecosystem and are probably a major factor for bacterial entrance. Besides providing entry avenues, wounds also create favorable conditions for the approaching bacteria by allowing leakage of plant exudates, which serve as a food source for the bacteria (Hallmann et al. 1997). Wounds and lateral roots are not, however, absolutely required for entrance of endophytic bacteria. Several workers have proposed that the enzymatic degradation (cellulolytic and pectinolytic enzymes) of plant cell walls by these bacteria was only observed when they colonized the root epidermis but never after colonizing intercellular spaces of root cortex. These results suggested that endophyte induced production of cellulose and pectinase was only for penetration into the host plant. Although these observations demonstrate the possibility of active penetration mechanisms for some endophytic bacteria, very little is known about the origin and regulation of these enzymes. Nevertheless, active penetration is still controversial and the fact that soil bacteria show a higher frequency of hydrolytic enzymes than xyleminhabiting bacteria suggests that it is unlikely for systemic endophytic bacteria to gain plant entry primarily via production of hydrolytic enzymes (Bell et al. 1995). Benhamou et al. (1996) concluded that hydrolytic enzymes might only be produced by endophytes during the early invasion phase and not after residing in plant tissues. In addition, one must differentiate between bacterial enzyme production in vitro and in planta. However, constitutive release of plant cell degrading enzymes by endophytic bacteria is undesirable as this would confer plant pathogenicity (Collmer et al. 1982). Therefore, endophytic bacteria must have some regulatory mechanisms to specifically regulate their enzyme production in terms of quantity and time of expression, a fact well known for Xanthomonas campestris where virulent and avirulent strains differ in their cellulose activity (Knosel and Garber 1967). Although the root zone offers the most obvious site of entry for many endophytes, entry may also occur at sites on aerial portions of plants. Sharrock et al. (1991) suggested that, in some cases, endophytic populations within fruit may arise by entry through flowers. Endophyte penetration is also believed to occur through natural openings on the leaves (e.g., stomata) or through stem lenticels (Kluepfel 1993). A completely different way of penetration is described by Ashbolt and Inkerman (1990) for sugarcane, via the mealybug, and by Kluepfel (1993), via a range of different insects.


3.5

67

Endophytic Movement Inside the Plant

Once inside the plant tissue, endophytes either remain localized in a specific plant tissue like the root cortex or colonize the plant tissues systematically by transport through the conducting elements or apoplast (Hurek et al. 1994). The prevailing thought that systematic colonization of the vascular system from cortical tissues is limited appears to be due to the belief that the endodermis represents a physical barrier to the apoplastic movement of bacterial endophytes into the vascular tissue (Kloepper et al. 1992). This theory is based upon the research demonstrating that the root endodermis functions physiologically to regulate chemical flow into the vascular region and that casparian strip, when intact, limits apoplastic movement of solutes from the cortex to the stele. Therefore, it was believed that endophytes present in the root cortex could not traverse the endodermis to enter the vascular system. However, as discussed previously, the endodermis is not an unbroken barrier, as epidermal disruption occurs at the sites of secondary root formation, providing an apoplastic route to the root stele, which can be visualized by fluorescent dyes (Peterson et al. 1981). Bacterial endophytes may also follow this same path to colonize the vascular tissue. The endodermal barrier can also be surpassed bacterial entrance via the undifferentiated cells of the root tissues (Mahaffee and Kloepper 1994). Once inside the plant tissue, endophytic bacteria remain localized in a specific plant tissue, such as the root cortex, or colonize the plant systematically by transport or active migration through the conducting elements or the apoplast (Hurek et al. 1994; James et al. 1994; Hallmann et al. 1997; Patriquin and D€obereiner 1978). The different mechanisms of distribution might be due to interactions with other bacteria or to the different requirements of each microorganism that allows them to inhabit different niches, represented by tissue and, more specifically, by the intercellular spaces inside each tissue.

3.6

Endophytic Colonization in Plant Tissues

The high population density of endophytes in carrot crowns indicates the preferential colonization of these tissues by bacterial endophytes. Surette et al. (2003) speculated that the greater abundance of colony-forming units (CFU) in the crown tissues may perhaps be due to the higher sugar content of crowns which can vary from 6 to 8% compared to the slender slicer carrot which varies from 3 to 4%. Fisher et al. (1992) found similar accumulations in the base of corn stems. Preferential colonization of crown tissues may also be a function of proximity to the soil surface. In this instance, bacterial colonization may be influenced by environmental factors such as oxygen concentration (Sitnikov et al. 1995). Higher oxygen concentrations for the multiplication and survival of the more aerobic bacterial species would be found in host tissues that were located nearer the soil surface.

68


The fact that colonization is especially abundant in root tissue may reflect the fact that the root is the primary site where endophytes gain entry into plants. As mentioned earlier, except for bacteria transmitted through seeds other potential endophytes must first colonize the root surface prior to entering the plant. This might explain the close relationship between endophytic and rhizosphere colonizing bacteria, that is, many facultative endophytic bacteria can also survive as rhizosphere bacteria. Potential internal colonists find their host by chemotaxis, electrotaxis, or accidental encounter. The fact that bacteria seem to be capable of colonizing the internal tissues of plants could confer an ecological advantage over bacteria that can only colonize plants epiphytically. The internal tissues of plants are thought to provide a more uniform and protective environment for microorganisms than plant surfaces, where exposure to extreme environmental conditions, such as temperature, osmotic potentials, and ultraviolet radiation, is a major factor limiting long-term bacterial survival. The plant interior could also be a hideout from grazing by soil protozoa. However, there are probably other limiting factors that must be overcome when establishing populations in the internal tissues of plants. Endophytic colonization of the shoot and root seem to differ. For most of the endophytes that have been investigated to date, colonization of the shoot is either intracellular and then confined to individual cells or intercellular but localized. Colonization of roots by endophytes, on the other hand, is usually extensive but may also be inter- or intracellular. A criterion for some endophytes to colonize the plant is these must find their way through cracks formed at the emergence of lateral roots or at the zone of elongation and differentiation of the root. The evidence for the penetration and root colonization by the rhizosphere bacteria came from the recovery of rhizosphere bacterial populations from the endodermis and root cortex of plants (Quadt-Hallman et al. 1997). The means of infection by the endophyte into the host plants in most cases is not clearly understood, where no specialized structures such as root nodules are formed as in legume–rhizobia symbioses. According to the model proposed by Darbyshire and Greaves (1973) and supported by Old and Nicolson (1978), the root cortex becomes an integral part of the soil–root microbial environment, resulting in a continuous apoplastic pathway from the root epidermis to the shoot which favors the movement of microorganisms into the xylem (Peterson et al. 1981). Thus, a continuum of root-associated microorganisms, which are able to inhabit the rhizosphere, the root cortex, and other plant organs, exists (Kloepper et al. 1992). The major reason for better root colonization in contrast to the above-ground organs of the plants may be due to the fact that roots are in intimate contact with an environment harboring many different mainly degradatively active microorganisms that can potentially provide the plants with water and essential minerals. Hence, a mutualistic interaction has been developed between microorganisms and the roots, because the roots as a natural carbon sink of the plants can supply dual and multiorganism symbioses with nutrients. In return the host can be supplied with minerals and water by the microorganisms (Schulz and Christine 2005). Another important fact is that the organisms occupying the endosphere are not accidentally there but most probably have been selected for this niche by the plant, because of the beneficial effects they offer their host and their


69

abilities to resist the effects of plant defense products. The energy lost by the plant in the production of endophyte biomass is in all likelihood adequately compensated for by the improvements in plant health derived from the presence of mutualistic microorganisms. It has also been proposed that cell wall-degrading enzymes, such as endogluconase, polygalacturonase, pectate lyase, cellulose, and pectinolytic enzymes, produced by endophytes are involved in the infection process (Hallmann et al. 1997; Kovtunovych et al. 1999; Compant et al. 2005b). Some endophytic plant growth promoting rhizobacteria (PGPR) may utilize other organisms as vectors to gain access to apoplastic spaces in their host. For example, both the pink sugarcane mealybug (Saccharicoccus sacchari) (Franke et al. 2000) and arbuscular mycorrhizae (Isopi et al. 1995) have been implicated in the infection of host plants by the endophytic diazotroph, Gluconacetobacter diazotrophicus. Endophyte colonization has also been visualized with the use of the b-glucuronidase (GUS) reporter system. A GUS marked strain of Herbaspirillum seropedicae Z67 was inoculated onto rice seedlings. GUS staining was most intense on coleoptiles, lateral roots, and also at some of the junctions of the main and lateral roots (James et al. 2002). This study by James et al. (2002) showed that endophytes entered the roots through cracks at the point of lateral root emergence. H. seropedicae subsequently colonized the root intercellular spaces, aerenchyma, and cortical cells, with a few penetrating the stele to enter the vascular tissue. The xylem vessels in leaves and stems were also colonized. Currently, one can only speculate on the reasons for these different colonization patterns, because many factors may be involved, e.g., anatomical differences, source–sink relationships, and differences in permeability or nutrients supplied by the micropartner or by the host (Schulz and Christine 2005).

3.7

Ecology of Endophytic Bacteria

Abundant microorganisms live both within and on crop plants. They possess functional diversity – the diversity of metabolism and interspecific relationships. The crop plant and its microorganisms constitute a holistic system. The complex relationships between plant and microorganisms and among the microorganisms themselves constitute the key to plant health and growth. Moreover, selective utilization of the functional diversity of microbial species provides us with several opportunities to improve agricultural production. Roots, stems, leaves, flowers, and fruits of plants all provide suitable habitats for microbial populations (Campbell 1989; Atlas and Barthar 1993). Plants can be considered to be complex microecosystems where different habitats are exploited by a wide variety of bacteria (McInroy and Kloepper 1994). These habitats are not only represented by plant external surfaces, where epiphytic bacteria predominate, but also by internal tissues, where many microorganisms penetrate and survive. Inside the plant microecosystem, different microbial species, both bacterial and fungal (Fisher et al. 1992)

70


are able to interact and establish an equilibrium. Some of these microorganisms can be considered to be dominant species (Van Peer et al. 1990) and may be represented by those that are most frequently, and in large numbers, isolated from the host plant. In addition to the dominants, there is a large variety of species that cannot be isolated easily because of their low numerical consistency. These are considered to be rare species. Endophytic bacteria have been isolated from both monocotyledonous and dicotyledonous plants, ranging from woody tree species, such as oak (Brooks et al. 1994) and pear (Whitesides and Spotts 1991), to herbaceous crop plants, such as sugar beets (Jacobs et al. 1985) and maize (Fisher et al. 1992; Gutierrez-Zamora and Martinez-Romero 2001). Diversity associated with bacterial endophytes exists, not only in the plant species colonized but also in the colonizing bacterial taxa. Plants can be colonized simultaneously by a large variety of endophytic bacteria. The variation in bacteria that has been reported as endophytes spans a significant range of Gram-positive and Gram-negative bacteria and include genera such as Acidovorax, Acinetobacter Actinomyces, Aeromonas, Afipia, Agrobacterium, Agromonas, Alcaligenes, Alcanivorax, Allorhizobium, Alteromonas, Aminobacter, Aquaspirillum, Arthrobacter, Aureobacterium, Azoarcus, Azomonas, Azorhizobium, Azotobacter, Azospirillum, Bacillus, Beijerinckia, Blastobacter, Blastomonas, Brachymonas, Bradyrhizobium, Brenneria, Brevundimonas, Burkholderia, Chelatobacter, Chromobacterium, Chryseomonas, Comamonas, Corynebacterium, Delftia, Derxia, Devosia, Enterobacter, Flavimonas, Flavobacterium, Flexibacter, Frankia, Halomonas, Herbaspirillum, Matsuebacter, Mesorhizobium, Moraxella, Nevskia, Nocardia, Ochrobactrum, Pantoea, Pectobacterium, Phenylbacterium, Phyllobacterium, Photobacterium, Porphyrobacter, Pseudoalteromonas, Pseudomonas, Psychrobacter, Ralstonia, Renibacterium, Rhizobacter, Rhizobium, Rhizomonas, Rhodanobacter, Rhodococcus, Shewanella, Sinorhizobium, Sphingobacterium, Sphingomonas, Spirillum, Stenotrophomonas, Streptomyces, Thauera, Variovorax, Vibrio, Xanthomonas, Xylella, Zoogloea, Zymobacter, Zymomonas, or members of the group of the pink-pigmented facultatively methylotrophic bacteria, such as Methylobacterium. Even though it remains difficult to compare earlier and more recent studies that identify bacteria, certain trends are apparent with predominant bacterial types isolated as endophytes (Kobayashi and Palumbo 2000).

3.8

Endophytes and Their Role in Plants

From literatures available, endophytic bacteria were shown to have beneficial effects on plant growth and health, and the main modes of action described are diazotrophy, production of plant growth hormones and antifungal compounds, and induced systemic resistance (Benhamou et al. 1996; Hallmann et al. 1997). Interactions of endophytic bacteria with their host plants are not only beneficial for the host, but provide enough nutrients for the endophytes to extensively colonize the


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host’s roots and potentially for growth in the rhizosphere, which in turn could improve the host’s mineral and nutrient supply as in the case of mycorrhizal fungi. Mutualistic endophytic associations have been reported more frequently in associations with roots than with the aerial plant organs, may be due to the fact that colonization of the aboveground organs is frequently localized, whereas that of the roots is more often extensive and sometimes systemic (Stone et al. 2000). Most of the endophytes that have been investigated to date revealed colonization of the shoot is either intracellular and then confined to individual cells or intercellular but localized. On the other hand, colonization of roots by endophytes is usually extensive, but may also be inter- or intracellular. Specialized structures that are presumed to improve the exchange of metabolites have been observed in both shoots and roots. An endophyte cannot improve the nutrient status of the photosynthetic organs directly. Thus, in general, a mutualistic systemic interaction with the roots of a putative host is more probable than with the aboveground organs. And, recently a molecular basis for mutualistic interactions of roots with microorganisms was found (Imaizumi-Anraku et al. 2005). The aboveground organs – leaf community is colonized by endophytes in the internal tissues and epiphytes on the surface. These colonizers are of bacterial or fungal community and may be of three types: (1) pathogens of another host that are nonpathogenic in their endophytic relationship, (2) nonpathogenic microbes, and (3) pathogens that have been rendered nonpathogenic but still capable of colonization by selection methods or genetic alteration (Imaizumi-Anraku et al. 2005). Endophytic bacteria in a single plant host are not restricted to a single species but comprise several genera and species. So far, no studies have indicated endophytic communities’ interaction inside the plants, and it has been speculated that beneficial effects are the combined effect of their activities. The population density of endophytes is highly variable, depending mainly on the bacterial species and host genotypes and also in the host growth stage, inoculum density, and environmental conditions. Generally, bacterial populations are larger in roots and decrease in the stems and leaves (Lamb et al. 1996). Natural endophyte concentrations can vary between 2.0 and 6.0 log10 CFU g 1 for alfalfa, sweet corn, sugar beet, squash, cotton, and potato, as described by Kobayashi and Palumbo (2000). Similar results were obtained for endophytic bacteria inoculated by root or seed drenching, with the population levels reaching between 3.0 and 5.0 log10 CFU g 1 of plant tissue for tomato and potato (Kobayashi and Palumbo 2000). Population densities of bacterial endophytes have been shown to be greatest in plant roots (McInory and Kloepper 1995) with densities ranging from 104 to 106 CFU g 1 fresh weights in cotton and sweet corn roots. In potato, the average bacterial densities over two seasons were 5.6 107 in the rhizosphere, 2.2 106 in the endorhiza, and 5.2 105 phyllosphere and were lowest in the endosphere 3.9 104 CFU g 1 fresh weight basis (Berg et al. 2005). The total number of endophytes present at a particular time is being controlled by the plant and environment (Hallmann et al. 1997). Recent papers indicate that the phyllosphere is much more diverse than previously thought. Using molecular techniques, studies by Yang et al. (2001) evaluating cultivated citrus, and Lambais et al. (2006) evaluating nine tree species in an ancient

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subtropical forest, determined a high level of diversity with many unculturable and often unidentifiable species. To learn more about the functions of endophytes inside plants and the conditions to which they are exposed there, it will be crucial to develop appropriate methods to localize bacterial gene expression in plants. To study endophytic gene expression in situ in uninoculated natural systems, immunolocalization of specific enzymes (such as nitrogenase) or in situ hybridization studies (Hurek et al. 1994) have recently been introduced. The interrelationship and the ecological role of these organisms and how these functions are related to the metabolic capabilities of microbe and plant are just now beginning to be studied.

3.9

Beneficial Effects on Plant

There have been vast studies describing potential advantage of plant-associated bacteria as agents inducing plant growth and maintaining soil and plant health. Because of various factors such as small size, diversity, and culturable nature, the endophytes are unnoticed in the plants. This makes plant physiologists to consider the plants as a single organism. As a result, the latter’s crucial roles were sometimes overlooked or unnoticed. Endophytic bacteria ubiquitously inhabit most plant species, and have been isolated from a variety of plants. Among the plant-associated microorganisms, endophytic bacteria are regarded as a largely unexplored potential resource for the discovery of isolates with novel antibiotic substances and PGP traits (Lodewyckx et al. 2002; Rosenblueth and Martıńez-Romero 2006).

3.10

Endophytic Diazotrophs

Much progress has been made in the area of biological nitrogen fixation (BNF) with nonleguminous plants over the last 10 years. Several new species of nitrogen-fixing bacteria have been identified but special attention has been given to endophytic diazotrophs. The concept of diazotrophic endophytes was introduced to the area of BNF by D€oobereiner (1992) although the term endophyte was coined more than 150 years ago by Leveille (1846) to define a special class of fungi living inside plant tissues. The term was later extended to bacteria by Chanway (1996) who also observed that some bacteria colonize the interior of plant tissues without causing disease symptoms. Although there are several definitions of bacterial endophytes, the role of the endophytic diazotrophs in association with graminaceous plants is still not yet well understood. Splitting the term diazotrophic endophytes into facultative and obligate was suggested to distinguish, respectively, strains that are able to colonize both the surface and root interior and to survive well in soil from those that do not survive well in soil but colonize the root interior and aerial parts.


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In nature, legumes benefit directly from biologically fixed nitrogen, provided that they are in symbiotic association with root-nodulating bacteria, such as Rhizobia. However, nonleguminous plants, most of which belong to the Gramineae, do not have this symbiosis (Hurek and Reinhold-Hurek 2003). Studies of plant bacteria that might make reliable contributions to the growth of nonlegumes such as cereals revealed that there are groups of bacteria which intimately associate with nonleguminous crops called diazotrophic endophytes. In general, the term “endophyte” includes all the microorganisms that are capable of colonizing the inner tissues of plants. The term endophyte was first introduced to the area of nitrogen fixation research associated with graminaceous plants by D€oobereiner (1992). Among nonleguminous plants, several diazotrophic endophytes have been isolated and characterized as nitrogen fixing endophytes including Acetobacter (Sevilla et al. 2001), Azoarcus spp. (Reinhold-Hurek and Hurek 1998), Serratia spp. (Gyaneshwar et al. 2001), Burkholderia spp. (Baldani et al. 2000), and Herbaspirillum spp. (Elbeltagy et al. 2001; Gyaneshwar et al. 2002). The interaction of endophytic diazotrophic bacteria with plants has been extensively studied through the inoculation of sugarcane and rice plants grown under sterile conditions followed by microscopic analysis. These bacteria enter the plant tissues primarily through the root zone. They colonize the spaces at the junctions of the lateral roots and the intercellular spaces of the root epidermis (Roncato-Maccari et al. 2003) and penetrate deeply to enter the internal tissues of the roots and basal stem (James et al. 2000; Zakria et al. 2007) and colonize the aerial parts by entering in the xylem tissues of the roots and stem (Hurek et al. 1994; James et al. 2000). The nitrogen-fixing bacteria are reported to provide biologically fixed nitrogen to their hosts, but the amounts of nitrogen that they supply are highly variable, for example, in rice it ranged from 0 to 36% (Malarvizhi and Ladha 1999; Shrestha and Ladha 1996) and in sugarcane from 4 to 70% of the host plant’s nitrogen requirement (Yoneyama et al. 1997). The variation in the amount of fixed nitrogen is believed to depend on variety, plant stage, endophyte strain, inoculation method, and environmental conditions. Several diazotrophic endophytes have been isolated from rice and they can provide fixed N (Gyaneshwar et al. 2001, 2002; Verma et al. 2001). Many endophytes appear to have a broad host range. For example, H. seropedicae has been found in a variety of crops, including maize, sorghum, sugarcane, and other Gramineae plants (Baldani et al. 1986; Olivares et al. 1996). This indicates that an endophyte isolated from one host family member can colonize other nonhost members of the same family, which is suggestive of host nonspecificity in Gramineae plants. Endophytes also show host nonspecificity among families. Burkholderia sp. isolated from the onion can colonize grapes (Compant et al. 2005b), potatoes, and vegetables (Nowak et al. 1995). However, interaction studies involving rice and endophytes isolated from other host families have not been done in an efficient way, and the issue needs to be addressed. The nitrogen supplied by indigenous endophytic N2-fixing bacteria is inadequate for crop growth (Nishiguchi et al. 2005). Asis et al. (2000) isolated several putative strains of G. diazotrophicus and Herbaspirillum from sugarcane, and reported that only 40% of these strains had acetylene reduction activity. Thus, there is potential

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to introduce nonindigenous endophytes that can contribute to plant growth. Therefore, studies of the interactions among the host, endophyte, and environmental conditions are very important in defining the nitrogen-fixing associations between hosts and endophytes. Researchers are interested in finding bacterial strains with enhanced PGP capabilities. As new beneficial bacterial strains are identified, the delivery of these strains to specific plant tissues becomes an issue. To facilitate the use of endophytic bacteria in practical agronomic production, reliable and practical methods of inoculum delivery must be developed. The ability of an endophyte to fix atmospheric nitrogen within a host has been proved using different approaches: acetylene reduction assay, 15N isotope dilution experiments, 15N2 reduction assays, or 15N natural abundance assays. These experiments have conclusively shown that an increase in the host–plant N content as high as 30–45 mg of N per plant (6-week-old seedlings) in rice and to 170 kg of N per hectare per year in sugarcane was a result of BNF (Iniguez et al. 2004). Due to their ability to colonize the root surface and interior of many cereals and forage grasses, the first report showing the presence of Azospirillum in cells of the cortex, in intercellular spaces between the cortex and endodermis, and in the xylem cells of maize roots by applying the tetrazolium reduction staining technique (Patriquin and D€obereiner 1978). Many inoculation experiments with Azospirillum spp. have shown a positive effect of the bacteria on crops (Boddey and D€obereiner 1995) but still a debate remains about the exact mode of action by which endophytic diazotrophs contribute to the nitrogen accumulated in the plants. Effects of PGP substances (Zimmer et al. 1988), nitrogen fixation per se, or the ability of the bacterial nitrate reductase to help in the incorporation of the nitrogen assimilated from soil by the plant have been demonstrated (Ferreira et al. 1987). Despite the different mechanisms exerted by Azospirillum in association with graminaceous plants, increases in the range of 5–30% in yield have been observed in several inoculation experiments (Okon and Labandera-Gonzalez 1994). Systematic studies by various workers in Brazil over the years led to the observation that some sugarcane varieties grown for decades or even a century do not show any decline in the soil N reserve or yield despite the supply deficit of N (Boddey et al. 1995). In the wild rice variety Oryza officinalis, acetylene reduction and 15N2 gas incorporation were deployed to determine the in planta nitrogen fixation after inoculation with endophytic Herbaspirillum sp. strain B501 was 381% as compared to 0.4% of the uninoculated plant, which proved the role of nitrogen fixation by Herbaspirillum sp. strain B5 (Elbeltagy et al. 2001). Nitrogen-fixing Burkholderia vietnamiensis (Gillis et al. 1995) has been isolated from the rhizosphere of rice in Vietnam. It was the first species of Burkholderia reported to fix nitrogen. More recent reports suggest that many species of this genus actually contain diazotroph strains. A phytohormone-producing diazotroph Enterobacter of sugarcane inoculated to roots of micropropagated sugarcane assimilated 29% of nitrogen by atmospheric fixation (Mirza et al. 2001). In all the above cases, the bacteria that colonized and invaded the plant upon inoculation contributed the fixed nitrogen (Boddey et al. 1995; Oliveira et al. 2002). The crucial for the understanding of functions of diazotrophic endophytes is the long-standing question, whether the


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host plants profit from nitrogen fixation. This has been successfully demonstrated for Azoarcus sp. BH72 and its host Kallar grass (Leptochloa fusca L. Kunth). The comparison of wild type and a nif mutant showed a significant gain of plant nitrogen after inoculation with the wild type; total N balance and natural 15N abundance corroborated that fixed nitrogen was contributed, moreover nifH-mRNA of strain BH72 was found to be predominant in plant roots (Hurek et al. 2002). Thus, grass endophytes are able to supply fixed nitrogen to the plant, as has also been demonstrated for G. diazotrophicus in sugar cane (Sevilla et al. 2001). This makes the Azoarcus sp. – grass system a highly interesting model system for a novel type of plant–microbe interaction. If several diazotrophs are found on one plant, it becomes difficult to judge their relative significance. In the case of Kallar grass, different nitrogen-fixing bacteria associated with distinct root zones (Reinhold et al. 1986). This result suggests that different diazotrophs might be adapted to colonize different root zones of their host plants and to contribute there to the association. It may be considered that the dominant diazotrophs, i.e., those occurring in highest numbers, to be the most important ones for an association, because they may be able to make a significant contribution to the nitrogen supply of the plant.

3.11

Endophyte’s Physiological Role

Plants infected with endophytes are often healthier than endophyte-free ones (Waller et al. 2005). This effect may be partly due to the endophytes’ production of phytohormones (such as indole-3-acetic acid (IAA) (Lee et al. 2004), cytokinins, and other PGP substances) and/or partly owing to the fact that endophytes can enhance the hosts’ absorption of nutritional elements such as nitrogen (Reis et al. 2000) and phosphorus (Guo et al. 2000) and that they regulate nutritional qualities such as carbon–nitrogen ratio. Moreover, a number of other beneficial effects on plant growth have been attributed to endophytes and include siderophore production (Costa and Loper 1994), supply of essential vitamins to plants (Pirttil€a et al. 2004), osmotic adjustment, stomatal regulation, modification of root morphology, enhanced uptake of minerals, and alteration of nitrogen accumulation and metabolism (Compant et al. 2005a, b). Protective effects on endophyte-infected host plants greatly enhance their resistance to unfavorable challenges. The evidence suggests that plants infected with endophytes often have a distinct advantage against biotic and abiotic stress over their endophyte-free counterparts. Beneficial features have been offered in infected plants, including drought acclimatization, improved resistance to insect pests and herbivores, increased competitiveness, enhanced tolerance to stressful factors such as heavy metal presence, low pH, high salinity, microbial infections, biocontrol of phytopathogens in the root zone (through production of antifungal or antibacterial agents, siderophore production, nutrient competition and induction of systematic acquired host resistance, or immunity), or enhanced availability of minerals (Sturz et al. 2000). Endophyte infected plants also gain protection from herbivores and pathogens due to the bioactive secondary metabolites

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secreted by the endophytes in plant tissue. An increasing number of antimicrobial metabolites biosynthesized by endophytic microorganisms, such as alkaloidal mycotoxins and antibiotics, have been detected and isolated (Strobel 2003; Strobel and Daisy 2003). But only a few studies have been published describing the molecular basis of the interactions between endophytic bacteria and plants. Adapting strategies that have been used to study bacterial gene expression in the rhizosphere and phyllosphere such as in vivo expression technology (IVET) and recombination IVET (Leveau and Lindow 2001; Zhang et al. 2006) may provide an insight into genes that are required by bacteria to enter, compete, colonize the plant, suppress pathogens, and generally survive within the plant.

3.12

Biotization

Plant propagation technology via tissue culture has been developed over the last 30–40 years as a spinoff of in vitro studies on differentiation. Typically, aseptic explants are grown under low light intensity in small containers, on artificial culture media containing sucrose, mineral salts, vitamins, and growth regulators, in concentrations exceeding levels recorded under natural environments. During the last decade, some researchers have searched for natural inhabitants of plants, epiphytes, and endophytes to enhance adaptation of tissue culture propagules to environmental stresses (Herman 1996). In nature, microorganisms inhabit the interior and exterior of plant organs (McInroy and Kloepper 1994). Some of these microorganisms, plant-beneficial bacteria, and vesicular-arbuscular mycorrhizae in particular can improve plant performance under stress environments and consequently enhance yield. The tissue culture approach is one way of method to introduce the selected endophytes into the host plant (Nowak 1998). Biotization, in the current context, may be defined as the metabolic response of in vitro grown plant material to microbial inoculants which promote developmental and physiological changes that enhance biotic and abiotic stress resistance in subsequent plant progeny. Such systems allow for mutual adaptation between the host plant and the introduced bacteria. Induction of stress resistance in plant propagules produced in vitro prior to transplanting is a primary target of several research groups attempting utilization of microbial inoculants in micropropagation (Nowak et al. 1995). Such responses under in vitro conditions are referred to as “biotization” (Herman 1996). Biotization of potato plantlets enhanced the transplant stress tolerance and eliminate an expensive greenhouse hardening step (Herman 1987). Greenhouse experiments also demonstrated that plants derived from dual cultures of potato and pseudomonad bacterium had larger root system, set stolons, tuberized earlier, and gave better tuber yield than nonbacterized control. Both in vitro and ex vitro benefits of biotization depended on plant species, cultivar, and growth conditions (Nowak et al. 1997; Pillay and Nowak 1997). In vitro cocultivation of soybean cotyledon explants with two strains of Pseudomonas maltophilia stimulated development of


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nodular callus with high regeneration potential. Improvement of somatic embryogenesis by selected bacterial inoculants in genotypes recalcitrant to regeneration has further been reinforced by Visser-Tenyenhuis et al. (1994). The in vitro growth responses depend on the degree of endophytic colonization, and a certain threshold of the bacteria concentration is required to trigger beneficial responses (Pillay and Nowak 1997). Sharma and Nowak (1998a) postulated similar induction patterns for the resistance to pathogens. A study by Senthilkumar et al. (2008) aimed at developing biotized rice investigated the effects of Azorhizobium strain isolated from the stem nodule of Aeschynomene aspera on the rice calli colonization, dispersion, colonization in roots, leaves, and leaf sheath, growth physiology, and yield under greenhouse condition. In this study, a natural endophytic association between A. caulinodans and tissue culture rice plants was observed with migration of rhizobia to roots, stem, and leaves with nitrogen fixation, plant growth promotion, and increased yield (Fig. 3.1). There appears to be some variation in the colonization pattern of rice calli and plantlets by the A. caulinodans strains studied. Several factors like host specificity, geographical distribution, plant age and tissue type may explain these differences. These strains also confirmed the Koch’s postulate when they were able to recolonize the original plant host. Our findings have shown that the qualitative parameters of rice calli such as protein and total nitrogen content can be modulated by microbial co-bionts when imbibed with calli. Although the mechanism for the growth promoting capacity has not been established, evidence gained so far suggests that the growth and development stimulatory properties of the isolated strains were mediated through microbial-derived compounds. Qualitative evidence based on callus induction, tissue subculture, and plantlet regeneration experiments suggests that the strains-induced growth and cell proliferation are mediated by microbial-derived cytokinin-like or auxin-like substances. Endophytic bacterial associations with rice are generally nonspecific (Reddy et al. 2002), and the size of the bacterial population density in rice tissues is too low to support adequate N2 fixation. Hence, it is important to develop strategies that enable enhanced diazotrophic bacterial colonization for significant endophytic BNF in rice. N2 fixation by inoculated diazotrophs per se seems to have minimal but significant effect on the nitrogen content of the biotized plantlets, as it was clear from the data of percent N content of inoculated samples (Muthukumarasamy et al. 2007; Senthilkumar et al. 2008). Bacterized plantlets did not only grow faster than unbacterized plantlets (Chanway 1997; Bensalim et al. 1998) but are also sturdier and have a better developed root system (Nowak 1998) and a significantly greater capacity to withstand adverse biotic stresses (i.e., drought) and low level disease pressures (Sharma and Nowak 1998b). Frommel et al. (1991) reported that the endophyte bacteria can be translocated to successive generations of potato plants during multiplication through stem explants (Fig. 3.1). If the culture conditions are refined, artificial associations between callus and individual microorganisms or their groups can be created. Such microorganisms not only would act as inducers of the stress resistance responses but also could occupy microsites on host plants, making them unavailable to pathogens. An example of successful artificial association between plants and a nitrogen-fixing bacterium

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Fig. 3.1 Optical micrograph showing cross section of intercellular colonization rice calli and regenerated plantlets by A. caulinodans. (a) Embryogenic calli (b) Cross section of rice calli colonized by A. caulinodans 15 days after inoculation (c) CS view of colonized rice leaf (d) Magnified cross section view of leaf colonized by A. caulinodans in regenerated rice plant (e) CS view of root uninoculated control (f) Possible sites of infection and colonization of rice root (g) Shoot and (h) Root development (i) Regenerated plantlets


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has been described by Varga et al. (1994). The authors created a symbiotic culture system, callus-bacterium, between Daucus carota L. and Azotobacter zettuovii (CRS-H6) which could grow for 4 years on a nitrogen-free medium with lactose as a carbon source. The bacterium, located in intracellular spaces, could also be transmitted to and fix nitrogen in newly regenerated plantlets (Varga et al. 1994; Preininger et al. 1997). The authors were also able to establish similar associations in tomato, potato, wheat, sugarcane, and poplar, using 11 strains of 8 Azotobacter species and in strawberries with Azomonas insignis (Preininger et al. 1997). The fact that we can culture probably only a few percent or even less of naturally occurring microorganisms is one of the challenges of the utilization of microbial inoculants in plant production. Development of new culture methods will allow establishment of stable association between plants and beneficial organisms in vitro and ex vitro and understanding of mechanisms of signal recognition and transduction in plant–microbial associations under different environments are probably the most critical elements of this challenge.

3.13

Biological Control

The intimate association of bacterial endophytes with plants offers a unique opportunity for their potential application in plant protection and biological control. Although biocontrol activity of microorgansims involving synthesis of allelochemicals has been studied extensively with free-living rhizobacteria, similar mechanisms apply to endophytic bacteria (Lodewyckx et al. 2002), as they can also synthesize metabolites with antagonistic activity toward plant pathogens. Certain endophytic bacterial isolates may play a significant role in plant protection against soilborne pathogens and in the overall productivity of an agricultural ecosystem (Hallmann et al. 1997; Sturz et al. 2000). Establishing a stable microbial endoplant communities may induce disease resistance through de novo synthesis of structural compounds and inhibition of fungal penetration (Benhamou and Nicole 1999), the induction and expression of general molecular-based plant immunity (Benhamou and Nicole 1999), or a simple exclusion of other organisms (phytopathogens and colonists) by niche competition (Sturz et al. 2000). The widely recognized mechanisms of biocontrol mediated by both organisms are competition for an ecological niche or a substrate, production of inhibitory allelochemicals, and induction of systemic resistance (ISR) in host plants to a broad spectrum of pathogens and/or abiotic stresses. But the effectiveness of endophytes as biological control agents is dependent on many factors, which include the host specificity, the population dynamics and pattern of host colonization, the ability to move within host tissues, and the ability to induce systemic resistance (Backman et al. 1997). For example, Chen et al. (1995) found five endophytic species, Aureobacterium saperdae, Bacillus pumilus, Phyllobacterium rubiacearum, Pseudomonas putida, and Burkholderia solanacearum, which could significantly reduce vascular wilt in cotton caused by Fusarium oxysporum f. sp. vasinfectum. Pseudomonas sp. strain PsJN, an onion

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endophyte, inhibited Botrytis cinerea Pers. and promoted vine growth in colonized grapevines, demonstrating that divergent hosts could be colonized (Barka et al. 2002). Colonization of multiple hosts has been observed with other species of endophytes and plants. For example, P. putida 89B-27 and Serratia marcescens 90-166 reduced Cucumber Mosaic Virus in tomatoes and cucumbers (Raupach et al. 1996) as well as anthracnose and Fusarium wilt in cucumber (Liu et al. 1995). Mahaffee and Kloepper (1994) have shown that biological control by endophytic bacteria is possible and can involve induced resistance to soilborne pathogens. Inoculation with some bacterial endophytes has also been demonstrated to reduce disease incidence and symptoms of F. oxysporum in cotton (Chen et al. 1995). Castillo et al. (2002) demonstrated that munumbicins antibiotics produced by the endophytic bacterium Streptomyces sp. strain NRRL 30562 isolated from Kennedia nigriscans can inhibit in vitro growth of phytopathogenic fungi, P. ultimum and F. oxysporum. Subsequently, it has been reported that certain endophytic bacteria isolated from field-grown potato plants can reduce the in vitro growth of Streptomyces scabies and X. campestris through production of siderophore and antibiotic compounds (Sessitsch et al. 2004). Interestingly, the ability to inhibit pathogen growth by endophytic bacteria, isolated from potato tubers decreases as the bacteria colonize the host plant interior, suggesting that bacterial adaptation to this habitat occurs within their host and may be tissue-type and tissue-site specific (Sturz et al. 1999). Aino et al. (1997) have also reported that the endophytic P. fluorescens strain FPT 9601 can synthesize 2, 4-diacetylphloroglucinol (DAPG) and deposit DAPG crystals around and in the roots of tomato, thus demonstrating that endophyte can produce antibiotics in planta. Most reports of plant growth promoting bacteria (PGPB)-mediated ISR involve free-living rhizobacterial strains, but endophytic bacteria have also been observed to have ISR activity. For example, ISR was triggered by P. fluorescens EP1 against red rot caused by Colletotrichum falcatum on sugarcane (Viswanathan and Samiyappan 1999), Verticllium dahliae on tomato (Sharma and Nowak 1998b), P. fluorescens against F. oxysporum f. sp. radicislycopersici on tomato (M’Piga et al. 1997), B. pumilus SE34 against F. oxysporum f. sp. pisi on pea roots (Benhamou et al. 1996), and F. oxysporum f. sp. vasinfectum on cotton roots (Conn et al. 1997). In all the reports that exist, the exact mode of action of disease suppression by particular endophytes has not been studied. Additional research is needed in order to better understand the parameters of this biocontrol system. The ISR in plants following treatment with endophytic fungi or bacteria and the mechanism of action opens a new line of research on the biochemical and genetic nature of signaling and gene induction.

3.14

Rice Endophytes

Diazotrophs, in rice cultivation, can be broadly grouped into two existing BNF systems, with the possibility of an additional third system. The existing systems include (1) indigenous (autochthonous) BNF systems comprising of heterotrophic


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and phototrophic bacteria as well as cyanobacteria native to soil–plant–floodwaters and (2) exogenous (allochthonous) BNF systems include Azolla that harbor symbiotic N2-fixing cyanobacteria, and aquatic legumes like Sesbania and Aeschynomene species that form symbioses with heterotrophic and phototrophic rhizobia. These exogenous BNF systems are not ubiquitous and hence need to be applied/inoculated to rice fields. The third system, endogenous (in planta) BNF, would come about from transforming rice to an autonomous N2-fixing plant. D€obereiner et al. (1993) speculated that endophytic diazotrophs in certain rice genotypes may, in fact, be responsible for the substantial contributions of BNF to rice. Studies have shown that rice indeed harbors a wide range of endophytic diazotrophs (Barraquio et al. 1997; Gyaneshwar et al. 2001; James et al. 2000). Some or all of them may be responsible for supplying plants with fixed N. Inoculation experiments with the endophytes S. marcescens and Herbaspirillum sp. in nonsterilized soil under greenhouse conditions have shown that they can be readily introduced into the rice plant by applying bacterial cultures on seeds or roots prior to planting. Endophytic associations with rice are nonspecific and the size of the bacterial populations in rice tissues is low. To overcome some of these problems, de Bruijn and his associates examined the possibility of creating “biased rhizospheres” to selectively encourage the growth of introduced microorganisms (Rossbach et al. 1994). Rice has been intensively studied in the last decade because of their potential to associate with endophytic diazotrophs and contribute to nitrogen nutrition of the plant through BNF. The discovery of endophytic diazotrophs such as H. seropedicae (Baldani et al. 1986) and Herbaspirillum rubrisubalbicans (Baldani et al. 1986) and the recent isolation of a new N2-fixing bacterium within the genus Burkholderia, provisionally named Burkholderia brasiliensis (Baldani et al. 1997b) based on morphological and physiological characteristics (Baldani et al. 1997a) and 23S rDNA oligonucleotide probes (Kirchhof et al. 1997), could explain the BNF observed in certain cereal genotypes. Azoarcus originally isolated from Kallar grass (Leptochloa fusa Kunth) growing in saline-sodic soils of Pakistan, a strain of Gram-negative nitrogen-fixing bacterium Azoarcus sp BH72 has been described by Reinhold-Hurek et al. (1993) that also colonizes rice in laboratory experiments (Hurek et al. 1994). These diazotrophs colonize roots, stems, and leaves of cereals endophytically (Olivares et al. 1996; Baldani et al. 1997a) and therefore probably suffer much less competition from other microorganisms for C substrates than rhizosphere bacteria, and possibly excrete part of their fixed N directly into the plant (Stoltzfus et al. 1997). The finding of a beneficial PGP association of Rhizobium leguminosarum bv. trifolii with rice roots (Yanni et al. 2001) is particularly relevant to assessments of whether rhizobia can fix N2 endosymbiotically in cereals. It was concluded that the benefits of this association leading to greater production of vegetative and reproductive biomass more likely involved rhizobial modulation of the root architecture for more efficient acquisition of certain soil nutrients rather than biological N2 fixation. By inhabiting the interior of the plants, these bacteria are thought to (1) avoid competition with bacteria of the rhizosphere and (2) derive nutrients directly from host plants (Baldani et al. 1997a; Boddey et al. 1995; James and Olivares 1997). In return, the plant interior (which is low in O2 and relatively

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high in carbon) provides an environment conducive to N2 fixation allowing the bacteria to efficiently transfer fixed N products to the host (James and Olivares 1997). D€obereiner et al. (1993) speculated that endophytic diazotrophs in certain rice genotypes may, in fact, be responsible for the substantial contributions of BNF to rice as reported earlier by App et al. (1984). Studies have shown that rice indeed harbors a wide range of endophytic diazotrophs (Barraquio et al. 1997; Gyaneshwar et al. 2001; James et al. 2002; Stoltzfus et al. 1997). Mano and Morisaki (2008) reported in their recent review the occurrence of diverse endophytic bacteria throughout the rice plant irrespective of cultivar (Tables 3.2 and 3.3).

3.15

Sugarcane Endophyte

A variety of diazotrophic bacteria have been isolated from rhizosphere (Beijerinckia) and roots (Azospirillum, Bacillus, Klebsiella, Enterobacter, Erwinia) of sugarcane plant. Diazotrophs such as G. diazotrophicus and Herbaspirillum spp. grow endophytically in the stems and leaves of sugarcane. In addition, Azospirillum, Burkholdaria, and perhaps other diazotrophs also inhabit sugarcane. Even though it is still not clear which of these endophytes is the main contributor to the observed BNF in sugarcane, evidence, nevertheless, suggests that the total endophytic BNF, according to N-balance studies, can be as high as 150 kg N ha 1 year 1 in sugarcane (Boddey et al. 1995). In 1988, Cavalcante and D€obereiner (1998) reported an acid-tolerant N-fixing bacterium, Acetobacter diazotrophicus, associated with sugarcane which contributed abundant N to sugarcane crops, with a capability to excrete almost half of the fixed N in a form that is potentially available to plants. A. diazotrophicus has also been isolated from other plants, viz., Cameroon grass (Pennisetum purpureum), sweet potato (Ipomoea batatas), coffee (Coffea arabica), tea, banana, ragi, rice, and pineapple (Table 3.4) and even from insects that infest sugarcane. The close association between a plant and an endophyte may provide suitable conditions for nutrient transfer between the bacteria and their host, than the association between predominantly rhizosphere bacteria and plants. It was reported that up to 80% of the plant nitrogen in certain sugarcane varieties has been derived as a result of BNF (Boddey et al. 1995). Application of G. diazotrophicus to sugarcane has been proved beneficial where the plant height, nitrogenase activity, and yield of the inoculated plants were higher than the control. Plant inoculation studies revealed the abundant population of G. diazotrophicus in the artificially inoculated sugarcane plantlets, reflecting through enhanced ARA, supporting the suggestion of James et al. (1994) that direct inoculation of G. diazotrophicus is possible. Inoculation of G. diazotrophicus was reported to enhance leaf N, biomass, and yield. Field trials conducted in sugarcane system revealed the usefulness of G. diazotrophicus with other diazotrophs, which have contributed to the yield equal to that of control (275 kg N ha–1). Mixed inoculation of VAM spores and G. dizaotrophicus also proved beneficial in improving the yield of different sugarcane varieties. The yield


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Table 3.2 The endophytic bacteria isolated from various parts of the rice plant (Mano and Morisaki 2008) Rice part Rice species Bacterial taxa Reference Seed Oryza alta Pantoea ananatis Elbeltagy et al. (2000) Seed Oryza Herbaspirillum seropedicae, Elbeltagy et al. (2000) meridionalis Methylobacterium sp. Seed Oryza sativa Klebsiella oxytoca Elbeltagy et al. (2000) Seed Oryza sativa Acidovorax sp., Bacillus pumilus, Mano et al. (2006) B. subtilis, Curtobactrium sp., Methylobacterium aquaticum, Micrococcus luteus, Panibacillus amylolyticus, Pantoea ananatis, Sphingomonas melons, S. yabuuchiae, Xanthomonas translucens, B. cereus, Azosprillum amazonense, Flavobacterium gleum Seed Oryza sativa Sphingomous echinoides, Okunishi et al. (2005) S. parapaucimobilis Seed Oryza sativa Ochrobactrum anthropi, Pantoea Verma et al. (2001) agglomerans, Pseudomonas boreopolis, P. fulva Leaf Oryza sativa Aurantimonas altamirensis, Bacillus Mano et al. (2007) gibsonii, B. pumilis, Curtobacterium sp., Diaphorobacter nitroreducens, Methylobacterium aquaticum, Methylobacterium sp., Pantoea ananatis, Sphingomonas echinoides, S. melonis, S. yabuuchiae, Stenotrophomonas maltophili, Streptomyces sp. Leaf Oryza sativa Methylobacterium sp. Elbeltagy et al. (2000) sheath Stem Oryza alta Methylobacterium sp. Elbeltagy et al. (2000) Stem Oryza barthii Methylobacterium sp. Elbeltagy et al. (2000) Stem Oryza Methylobacterium sp. Elbeltagy et al. (2000) brachyantha Stem Oryza Methylobacterium sp. Elbeltagy et al. (2000) glandiglumis Stem Oryza latifolia Methylobacterium sp. Elbeltagy et al. (2000) Stem Oryza longiglumis Herbaspirillum seropedicae Elbeltagy et al. (2000) Stem Oryza Rhodopseudomonas palustris Elbeltagy et al. (2000) meridionalis Stem Oryza minuta Methylobacterium sp., Sphingomonas Elbeltagy et al. (2001) adheasiva Stem Oryza officinalis Azosprillum brasilense, Enterobacter Elbeltagy et al. (2000) cancerogenus, Herbasprillum seropedicae Stem Oryza ridleyi Cytoohagales str. MBIC4147, Elbeltagy et al. (2001) Methylobacterium sp. Stem Oryza rufipogon Azosprillum lipoferum, Ideonella Elbeltagy et al. (2001) dechloratans (continued)

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Table 3.2 (continued) Rice part Rice species Stem Oryza rufipogon

Stem Stem Stem

Oryza sativa Oryza sativa Oryza sativa

Bacterial taxa Agrobacterium vitis, Azorhizobium caulinodans, Azosprillum sp., Bacillus megaterium, B. subtilis, Pseudomonas cepacia Herbaspirilium seropedicae Serratia marcescens Klebsiella sp.

Reference Elbeltagy et al. (2000)

Leaf Stem, Root Stem, Root Root Root

Oryza sativa Oryza sativa

Azoarcus sp. Gallionella sp.

Stoltzfus et al. (1997) Olivares et al. (1996) Gyaneshwar et al. (2001) Engelhard et al. (2000) Engelhard et al. (2000)

Oryza sativa

Azocarcus sp.

Engelhard et al. (2000)

Oryza granulate Oryza minuta

Engelhard et al. (2000) Engelhard et al. (2000)

Root Root

Oryza nivara Oryza officinalis

Root

Oryza sativa

Root

Oryza sativa

Sphingomonas paucimobilis Azoarcus sp., Azoarcus indigens, Azorhizobium caulinodans, Azosprillum brasilense, A. lipoferum, Burkholderia sp., Hespaspirillum sp. Klebsiella pneumoniae Ochrobactrum sp., Sphingomonas paucimobilis Azosprillum irakense, Bacillus luciferensis, B. megaterium, Bradyrhizobium elkanii, B. japonicum, Brevibacillus agri, Burkholderia kururiensis, Caulobactor crescentus, Chryseobacterium taichungense, Enterobactor cloacae, E. ludwigii, Hyhomicrobium sulfonivorans, Methylocapsa acidiphila, Micrococcus luteus, Mycobacterium petroleophilum, Paenibacillus alvei, Rhizobium loti, Roseateles depolymerans Burkholderia cepacia, Rhizobium leguminosarum, R. leguminosarum

Engelhard et al. (2000) Mano et al. (2007) Singh et al. (2006)

Yanni et al. (1997)


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Table 3.3 Bacterial diversity in rice plants as revealed by culture independent method (Mano and Morisaki 2008) Rice part Rice species Bacterial group Bacterial taxa Surface and Oryza sativa g-Proteobacteria Erwinia amylovora inside of Pseudomonas fluorescens milled rice Firmicutes Xanthomonas sacchari Actinobacteria Staphlococcus sp. a-Proteobacteria Nocardia globerula Brevundimonas diminuta, Caulobacter sp. Kaistiana koreensis, Methylobacterium sp., Novosphingobium tardaugens, Sinorhizobium terangae Inside root Oryza sativa b-Proteobacteria Achromobacter xylosoxidans Acidovorax facilis Burkholderia fungorum Burkholderia sp. Comamonas testerone Curvibacter gracilis Delftia acidovorans D. tsuruhatensis Duganella violaceinigra Gallionella ferruginea, Herbaspirillum frisingense Hydrogenophaga taeniospiralis Methyloversatilis universalis Sterolibacterium denitrificans Variovirax sp. Acinetobacter baumannii Alkanindiges illinoisensi g-Proteobacteria Enterobacter sp. Methylophaga marina Pantoea sp. Plesiomonas shigelloides Pseudomonas stutzeri Stenotrophomonas maltophilia Stenotrophomonas sp. Bdellovibrio bacteriovorus Geobacter sp. Sulfurospirillum multivorans d-Proteobacteria Flavobacterium frigoris e-Proteobacteria F. psychrophilum Sphingobacterium sp. Bacteroidetes Acidaminobacter hydrogenoformans Clostridium sp. Firmicutes Lachnospiraceae bacterium Planomicrobium okeanokoites DeinococcusThermus, P. mcmeekinii Deinococcus indicus Acidobacteria Holophaga foetida

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Table 3.4 Sources of G. diazotrophicus (Muthukumarasamy et al. 2002)

Source Sugarcane Cameroon grass Sweet potato Coffee Ragi Tea Pineapple Mango Banana Others: mealy bugs, VAM spores

Part Root, root hair, stem, leaf Root, stem Root, stem tuber Root, rhizosphere, stem Root, rhizosphere, stem Root Fruit Fruit Rhizosphere Internal environment

was also not reduced even under 50–100% reduction from the recommended dose of chemical N compared to the control, attributing the role of inoculated G. diazotrophicus in N contribution (Muthukumarasamy et al. 1999). It has been reported that inoculation of micropropagated sugarcane seedlings would make the plants not only grow faster, but also ensure efficient N-fixing plants in fields (Reis et al. 2000). The 15N incorporation experiments, using sterile sugarcane plants, have also demonstrated the potential for N fixation in G. diazotrophicus– sugarcane interaction (Sevilla et al. 1998). Use of mutant strains (carrying nif D::kan interposan mutation that prevents N fixation entirely) in plant experiments proved the participation of G. diazotrophicus in N fixation. It is an established fact that the growth hormones, viz., auxins, cytokinins, and gibberellins, play a role in enhancing the growth of grasses associated with diazotrophs. Apart from N fixation, G. diazotrophicus is also reported to benefit sugarcane through production of PGP factors (Fuentes-Ramirez et al. 2001).

3.16

Conclusion

Currently, endophytes are viewed as an outstanding source for undescribed microbes because there are so many of them occupying literally millions of unique biological niches (higher plants) growing in so many unusual environments. Exploitation of endophyte–plant interactions can result in the promotion of plant health and can play a significant role in low-input sustainable agriculture applications for both food and nonfood crops. An understanding of the mechanisms enabling these endophytic bacteria to interact with plants will be essential to fully achieve the biotechnological potential of efficient plant–bacterial partnerships for a range of applications. Acknowledgment This study was supported by Rural Development Administration (RDA), Republic of Korea and Tamil Nadu Agricultural University, Coimbatore, India.


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Chapter 4

PGPR Interplay with Rhizosphere Communities and Effect on Plant Growth and Health Gabriele Berg and Christin Zachow

4.1

Introduction

The interface between soil and plant roots – the rhizosphere – is, due to root exudates and the resulting high nutrient content, a unique microenvironment in terrestrial ecosystems (Sørensen 1997; Raaijmakers et al. 2009). Although ubiquitous and cosmopolitan rhizosphere-associated bacterial genera are known, e.g. Pseudomonas, Bacillus and Methylobacterium, specific populations have been detected for each plant species (Berg and Smalla 2009). The function of rhizosphere-associated bacteria is only partly understood. Firstly, bacteria play a role for plant growth. They can supply macro- and micro-nutrients. The most prominent example is bacterial nitrogen-fixation. The symbiosis between rhizobia and its legume host plants is an important example for plant growth-promoting rhizobacteria (PGPR). Bacteria of this group metabolise root exudates (carbohydrates) and in turn provide nitrogen to the plant for amino acid synthesis. The ability to fix nitrogen also occurs in free-living bacteria like Azospirillum, Burkholderia and Stenotrophomonas (Dobbelare et al. 2003). Another nutrient is sulphate, which can be provided to the plant via oxidation by bacteria (Banerjee and Yesmin 2002). Bacteria may contribute to plant nutrition by liberating phosphorous from organic compounds such as phytates and thus indirectly promote plant growth (Unno et al. 2005). Mineral supply is also involved in plant growth promotion and includes synthesis of siderophores and siderophore uptake systems. Poorly soluble inorganic nutrients can be made available through the solubilisation of bacterial siderophores and the secretion of organic acids. Recently, de Werra et al. (2009) showed that the ability of Pseudomonas fluorescens CHA0 to acidify its environment and to solubilise mineral phosphate is strongly dependent on its ability to produce gluconic acid. In the processes of plant growth, phytohormones, e.g. indole-3-acetic acid (IAA), ethylene, cytokinins, and gibberellins, play an important role. Furthermore, plant-associated bacteria can

G. Berg (*) and C. Zachow Graz University of Technology, Environmental Biotechnology, Petersgasse 12, A-8010 Graz, Austria e-mail: [email protected]


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influence the hormonal balance of the plant (Costacurta and Vanderleyden 1995). An interesting phenomenon is the enhancement of stress tolerance by lowering the ethylene level (Glick et al. 1998). Another important function is the involvement of rhizosphere-associated bacteria in pathogen defence. Whereas resistance against leaf pathogens is often encoded in the plant genome, it is difficult to find resistance genes against soil-borne pathogens. Cook et al. (1995) suggest that antagonistic rhizobacteria fulfil this function. Interestingly, besides direct antagonism, plantassociated bacteria can induce a systemic response in the plant, resulting in the activation of plant defence mechanisms (Pieterse et al. 2003). However, several studies suggest that there are many more plant–microbe interactions and resulting functions. A fascinating example is endophytic methylobacteria, which use C1 compounds from the plant for their energy production (Zabetakis 1997). The (intermediate) catabolic product hydroxypropanol is given back to the plant and works as precursor of the flavour compounds mesifuran and 2,5-dimethyl-4hydroxy-2H-furan (DMHF). The latter posses additional antifungal activity and can be responsible for pathogen defence. Methylobacterium treatment resulted in both a statistically significantly higher content of flavour compounds and a better taste of strawberries (Verginer et al. 2010). Interestingly, an earlier report provided evidence that hormone-producing methylobacteria are essential for germination and development of protonema of the moss Funaria hygrometrica (Hornschuh et al. 2002). Another function of rhizobacteria can be the degradation of root exudates with allelopathic or even autotoxic functions (Bais et al. 2006). To study plant-associated bacteria and their structure and functions is important not only for understanding their ecological role and the interaction with plants and plant pathogens, but also for any biotechnological application. In biotechnology, rhizosphere-associated bacteria can be applied directly for biological control of plant pathogens as biological control agents (BCAs), for growth promotion as PGPR or as biofertilisers and rhizoremediators (Whipps 2001; Lugtenberg et al. 2002). During the last years, there are an increasing number of products based on microbial inoculants on the market (rev. in Berg 2009). However, for many promising candidates the translation into practical approaches failed due to technical problems but also due to (1) their human pathogenic potential or (2) due to negative interactions with the environment (Fig. 4.1). Risk assessment studies at an early stage of the product development can avoid these problems.

4.2

Interplay with Eukaryotic Hosts: The Human Pathogenic Potential of PGPR

PGPR interact intensively not only with their host plant but also with other eukaryotic hosts living in the rhizosphere. The interaction will be analysed not only to understand the human pathogenic potential of PGPR but also to develop assays, which allowed assessing the latter.

4 PGPR Interplay with Rhizosphere Communities

99

Disease Plant

Soil-borne pathogens Induced resistance PGPR INTERPLAY Biocontrol Competition Change of composition

Root exudation Plant growth promotion Hormomal stimulation

Plant Growth Promoting Rhizobacteria PGPR

Humans ct?

pa

Im

i) Human health ii) Impact on environment

ct?

pa

Im

Microbial communities

Fig. 4.1 Interplay of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere: key players, modes of action and impact on (i) human health and (ii) on the environment

4.3

The Rhizosphere as Reservoir for Potential Human Pathogenic Bacteria

During the last few years, it has been shown that plants, especially in the rhizosphere, can harbour not only beneficial bacteria, but also those that potentially can cause diseases in humans (Berg et al. 2005; Opelt et al. 2007). These pathogens are called opportunistic or facultative human pathogens and they cause diseases only in patients with a strong predisposition to illness, particularly in those who are severely debilitated, immuno-compromised or suffering from cystic fibrosis or HIV infections (Parke and Gurian-Sherman 2001; Steinkamp et al. 2005). This group of bacteria cause the majority of bacterial infections associated with significant case/fatality ratios in susceptible patients in Europe and Northern America. A special group are those bacteria responsible for hospital-acquired diseases which are called nosocomial infections. For example, in intensive care units in Europe, 45% of the patients were infected by opportunistic pathogens (Vincent et al. 1995). In the last two decades, the impact of opportunistic infections on human health has increased dramatically. Many plant-associated genera, including Burkholderia, Enterobacter, Herbaspirillum, Ochrobactrum, Pseudomonas, Ralstonia, Serratia, Staphylococcus and Stenotrophomonas contain root-associated bacteria that enter bivalent interactions with plant and human hosts (Fig. 4.1). Several members of these genera show plant growth-promoting as well as excellent antagonistic properties against plant

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pathogens and therefore were utilised as PGPR and for the development of biological control products (Whipps 2001). However, many strains also successfully colonise human organs and tissues and thus cause diseases. The mechanisms behind these interactions are similar; they include recognition, adherence, colonisation as well as survival (Rahme et al. 1995; Cao et al. 2001). Furthermore, the rhizosphere is a tool for naturally occurring antibiotics produced by bacteria and their resistance genes (Martinez 2009). The problems with biofungicides based on strains of the genus Burkholderia underlines the importance of thorough risk assessment studies prior to registration (Govan et al. 2000; Parke and GurianSherman 2001). Another example is given by Stenotrophomonas maltophilia, a PGPR and emerging pathogen in humans (Ryan et al. 2009). Interestingly, for Stenotrophomonas strains a study indicate that clinical environments select bacterial populations with high mutation frequencies from rhizosphere populations (Turrientes et al. 2010). In a study published by Alonso et al. (1999) it was shown that clinical and environmental isolates of P. aeruginosa, which is the major cause for morbidity and mortality in cystic fibrosis patients, share several phenotypic traits with respect to both virulence and environmental properties. Several studies support the view that the environmental Pseudomonas strains are indistinguishable from those from clinical sources in terms of genotypic, taxonomic or metabolic properties (Kiewitz and T€ ummler 2000; Wolfgang et al. 2003). But there is also an impact of the clinical environment: a study showed that P. putida as well as P. stutzeri strains acquired the antibiotic resistance genes under selective pressure of antibiotic exposure in the hospital environment (Carvalho-Assef et al. 2010). Rhizosphere-associated bacteria with a high capacity for biocontrol and plant growth promotion can be potentially dangerous for human health. Therefore, it is important to understand the mode of action and specific properties of the PGPR. It is well known that antagonistic properties and underlying mechanisms are highly strain-specific (Berg et al. 2002, 2006) but identification of bacteria is based mainly on 16 S rDNA sequencing. Thus, from sequencing information it is difficult to draw conclusions about potential pathogenicity: neutral bacterial strains can be dangerous due to pathogenicity islands or pathogenic bacteria can be harmless because of the absence of any pathogenicity factor. Therefore, models to assess the pathogenicity of individual BCAs are important as risk assessment studies.

4.4

Caenorhabditis elegans: A Model to Assess Potential Human Pathogenic Bacteria

To assess the pathogenic potential is particularly difficult in many opportunistic human pathogens as well as BCAs because of the lack of adequate animal models. Until now, this procedure for BCAs is based on rules originally developed for


101

synthetic pesticides (EU Council Directive 91/414/EEC, see also http://www. rebeca-net.de). Methods adopted from standardised tests for chemical-based agents, including elaborate animal tests are not only time-consuming and expensive but also their results are difficult to interpret. Pathogenicity and the mode of action of facultative pathogens such as Burkholderia and Stenotrophomonas could not be analysed in traditional animal models. Therefore, alternative models using the slime mould Dictyostelium discoideum (Alonso et al. 2004), duckweed (Lemna minor L.; Radic´ et al. 2010), the zebrafish Danio rerio (van der Sar et al. 2003) or the nematode C. elegans (Tan et al. 1999) were developed. The model organism C. elegans has valuable advantages, enabling it to be used in many bacteria–pathogen interaction analyses to evaluate the pathogenic potential of these bacteria (Aballay and Ausubel 2002; Cardona et al. 2005). C. elegans is a free-living terrestrial nematode that feeds on bacteria in its environment (Beale et al. 2006). Extensive information about C. elegans research is available in well-resourced internet databases (http:// www.wormbook.org, http://www.wormbase.org). In an extensive study, we applied a broad range of BCAs, pathogens and plantassociated bacteria to a rapid and inexpensive bioassay, using C. elegans to estimate the risk of bacteria to harm human health (Zachow et al. 2009). The nematode killing assay described as slow killing assay by K€othe et al. (2003) was used. Movement and reproduction behaviour of C. elegans with BCAs were compared with those fed with the human pathogen P. aeruginosa QC14-3-8 (positive control) and Escherichia coli OP50 (negative control). In Fig. 4.2, the kinetics of killing C. elegans under slow killing conditions is shown (a) for different Pseudomonas strains: P. fluorescens L13-6-12, P. trivialis RE*1-1-14 and Pseudomonas sp. ¼ Proradix# [product produced by Sourcon-Padena GmbH & Co.KG] and by (b) different enterics: Pantoaea agglomerans L24-6-12, Rhanella aquatilis G3SM41, Serratia liquefaciens N1SM25, S. grimesii N1SM34, S. marcescens and S. plymuthica HRO C48 ¼ RhizoStar# [product produced by e ~ nema Gesellschaft f€ur Biotechnologie und biologischen Pflanzenschutz mbH]. All strains were isolated from the rhizosphere but some of them are selected according to their identification and grouping into risk group 2 (P. aeruginosa, P. agglomerans, R. aquatilis, S. grimesii, S. liquefaciens and S. marcescens). Indeed, these bacteria from risk group 2 showed a significantly higher rate of killing. In contrast, results obtained from other pseudomonads and S. plymuthica gave hints to a low risk. Altogether, results indicate that C. elegans provides a reliable model system to assess the human pathogenic potential of BCAs prior implementation of extensive studies using animal test systems. The C. elegans assay can be integrated into initial screens for BCAs and is useful to exclude pathogens in a very early stage of the product development. There are some restrictions for the C. elegans assay. The model of pathogenicity is limited by the amount of bacteria infecting the worm, which was shown for P. aeruginosa, P. fluorescens, S. marcescens, Burkholderia cepacia, B. pseudomallei, B. thailandensis, Salmonella spp. and Bacillus megaterium (Tan and Ausubel 2000). Therefore, in this study we used an overnight bacterial culture with approximately 107 cells/ml, which provides an appropriate thin cell layer to evaluate the

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a

100

Pseudomonas aeruginosa QC14-3-8

90

Escherichia coli OP50 Pseudomonas fluorescens L13-6-12

80

Pseudomonas trivialis RE*1-1-14 Pseudomonas sp.

Dead worms (%)

70 60 50 40 30 20 10 0 0h

24 h

48 h

72 h

Time (hour)

b

100 90 80

Dead worms (%)

70

Pseudomonas aeruginosa QC14-3-8 Escherichia coli OP50 Pantoea agglomerans L24-6-12 Rahnella aquatilis G3SM41 Serratia liquefaciens N1SM25 Serratia grimseii N1SM34 Serratia marcescens Serratia plymuthica C48

60 50 40 30 20 10 0 0h

24 h

48 h

72 h

Time (hour)

Fig. 4.2 Kinetics of killing of Caenorhabditis elegans by different PGPR under slow killing conditions. Worms grown on NGMII and feeding on Pseudomonas aeruginosa QC14-3-8 (positive control, black circle), Escherichia coli OP50 (negative control, black square). The PGPR were represented by (a) different Pseudomonas strains: Pseudomonas fluorescens L13-6-12 (white square), Pseudomonas trivialis RE*1-1-14 (white circle) and Pseudomonas sp. ¼ Proradix# (white triangle), and by (b) different enterics: Pantoaea agglomerans L24-6-12 (black cross), Rhanella aquatilis G3SM41 (white circle), Serratia liquefaciens N1SM25 (white square), Serratia grimesii N1SM34 (grey square), Serratia marcescens (white triangle), and Serratia plymuthica HRO C48 ¼ RhizoStar# (grey triangle). Data points represent means standard errors of at least five independent experiments


103

behaviour of the transparent worm on the Petri dishes. In a pilot study, this concentration of cells was found to permit detection of differences in survival among worm strains after 24 h (Schulenburg and Ewbank 2004). Furthermore, the developmental stage of the applied worms influenced the slow killing. Adult worms were more sensitive and died faster than fourth-stage larvae. Therefore, in this study we used second-stage larvae in the assay, exactly 48 h after egg preparation. Another restriction is associated with B. thuringiensis, a well-known BCA. Although B. thuringiensis is used world-wide against insect pests without reports that it has caused harm to humans, the bioactive toxin does act against C. elegans (Devidas and Rehberger 1992).

4.5

Interplay with Rhizosphere Communities: The Impact on the Environment

Although originating from plant-associated microenvironments themselves, beneficial bacteria, if applied to plant roots in adequate numbers, may perturb indigenous microbial populations and their associated important ecological functions (Fig. 4.1). Therefore, unwanted, unspecific actions of the introduced beneficial microbes against nontarget organisms have to be assessed. To this end, sufficient knowledge about the microbial ecology of the target habitats is necessary for reasonable risk assessment studies concerning the release of beneficial microorganisms. As only a small proportion of the microorganisms can be analysed by common cultivation techniques, several DNA-based, cultivation-independent methods, have been developed to overcome the limitations of cultivation (Smalla 2004). The use of such molecular methods is urgently needed in order to include the highest possible number of total microorganisms in risk assessment studies to determine non-target effects of introduced beneficial bacteria (Winding et al. 2004). Several studies using cultivation-independent methods exist. They focus mainly on the effects of genetically modified microorganisms (GMOs) such as Pseudomonas (Viebahn et al. 2003; Glandorf et al. 2001) and Sinorhizobium (Schwieger and Tebbe 2000) on non-target microorganisms. Examples of studies, which analysed the fate and ecosystem effects of introduced PGPR and antagonistic bacteria, are given in Table 4.1. Generally it can be concluded from these studies that the impact of bacterial inoculants is either negligible or small compared with effects of general agricultural practises, and more or less all effects are transient. However, for strains with a strong production of antifungal antibiotics or genetically modified strains with additional genes to synthesise antibiotics, effects were observed (Viebahn et al. 2003; Walsh et al. 2003; Blouin-Bankhead et al. 2004). Interestingly, also pathogenic bacteria are able to persist in plant-associated microenvironments, especially in the rhizosphere (Table 4.1). As their non-pathogenic counterparts they caused only transient effects on microbial communities.

Table 4.1 Examples for risk assessment studies for PGPR and facultative human pathogenic bacteria Strains Plant/pathosystem Results PGPR Pseudomonas putida QC14-3-8 Potato No differences between the inoculated Serratia grimesii L16-3-3 and non-inoculated communities Pseudomonas fluorescens CHA0 Cucumber Differences in the composition and/or and GMO relative abundance of species in the fungal community, no effect on species diversity indices, and species abundance Impact of treatments was smaller than the effect of growing cucumber repeatedly in the same soil Pseudomonas putida WCS358r Wheat Transient change in the composition of and GMOs the rhizosphere microflora No influence on soil microbial activities Pseudomonas fluorescens F113Rif Clover No influence on the structure of the Rhizobium community Small influence on the proportion of Phl-sensitive isolates Wheat Treatment with mixture disrupted the Microbispora sp. strain EN2 natural actinobacterial endophyte Streptomyces sp. strain EN27 population, reducing diversity and Nocardioides albus EN46 colonisation levels Single isolates; mixture Treatment with single isolates – population was not adversely affected Wheat Inoculation with Z30-97 resulted in Pseudomonas fluorescens strains several shifts in rhizosphere bacterial 2-79, Q8rl-96, and a community structure recombinant strain, Z30-97 Serratia plymuthica HRO-C48 Strawberry and potato – Only negligible, short-term effects due Streptomyces sp. HRO-71 Verticillium dahliae to the bacterial treatments Lettuce – Rhizoctonia Only negligible, short-term effects due Serratia plymuthica 3Re4-18 solani to the bacterial treatments Pseudomonas trivialis 3Re2-7 Pseudomonas fluorescens L13-6-12 Scherwinski et al. (2007, 2008)

Scherwinski et al. (2006)

Blouin-Bankhead et al. (2004)

Conn and Franco (2004)

Walsh et al. (2003)

Glandorf et al. (2001) Viebahn et al. (2003)

Girlanda et al. (2001)

Lottmann et al. (2000)

Reference

104 G. Berg and C. Zachow

Lettuce – Rhizoctonia solani

Maize

Pseudomonas jessenii RU47

Pseudomonas fluorescens strains F113, CHA0 and Pf153

Lettuce

Spinach

Escherichia coli O157:H7

Escherichia coli O157:H7

Hydroponically grown wheat

Soil

Collimonas spp.

Facultative human pathogenic bacteria Burkholderia cepacia, E. coli, Pseudomonas aeruginosa, Streptococcus pyogenes

Wheat

Pseudomonas fluorescens SBW25

While B. cepacia and P. aeruginosa strains showed considerable growth in the rhizosphere, E. coli and Staphylococcus aureus survived without substantial growth and Streptococcus pyogenes cells died The time for pathogens to reach detection limits (real-time PCR) on the leaf surface by plate counts was 7 days after planting in comparison with 21 days in the rhizosphere Escherichia coli O157:H7 persisted in soil for at least 28 days, not on the plant

Only minor impacts were found on native microflora due to bacterial (GMM or wild-type) inoculation The introduction of collimonads altered the composition of all fungal communities studied but had no effects on fungal biomass increase, cellulose degrading activity or plant performance RU47 established as the dominant Pseudomonas population in the rhizosphere but reduced the impact of R. solani on fungal communities A persistence study of the three strains indicated that the strains persisted differently over a period of 5 weeks

Patel et al. (2010)

Mark Ibekwe et al. (2009)

Morales et al. (1996)

Von Felten et al. (2010)

Adesina et al. (2009)

H€ oppener-Ogawa et al. (2009)

J€aderlund (2008)

4 PGPR Interplay with Rhizosphere Communities 105

106

4.6


Conclusions

Rhizosphere-associated bacteria are as PGPR or BCAs an interesting bio-resource for biotechnological applications. On the other hand, problems with opportunistic infections, which are originally from rhizosphere, will become even more severe due to the increasing numbers of at-risk individuals in the human population. Therefore, it is important to understand the ecological behaviour of rhizosphereassociated bacteria. Further, it is essential to exclude potential pathogenic bacteria at an early stage of product development. Criteria are growth at 37 C, grouping in risk groups (http://www.dsmz.de or Dir. 2000/54 EC) or any toxic effect in the C. elegans assay. Otherwise, more research and toxicological data are necessary for risk assessment. In all studies assessing the risk for the environment, mainly transient short-term effects were reported. Due to the fact that a broad spectrum of microorganisms was already investigated, it is not necessary to perform such studies with each individual strain. Acknowledgements The work has been financed in part by the Austrian Science Fund (FWF) and by KWS SAAT AG.

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Chapter 5

Impact of Spatial Heterogeneity Within Spermosphere and Rhizosphere Environments on Performance of Bacterial Biological Control Agents Daniel P. Roberts and Donald Y. Kobayashi

5.1

Introduction

There has been considerable effort directed at finding alternatives to chemical pesticides for suppression of soilborne pathogens due to environmental and human health risks associated with their use (Larkin et al. 1998; Raupach and Kloepper 1998). Interest in developing these alternative disease control measures has increased as there is no guarantee that registered chemicals may remain available for commercial growers as exemplified by the phase-out in use of the broad-spectrum chemical fumigant methyl bromide (Martin 2003). There is added concern since nonregistered chemical compounds currently in development will not be made available for commercial use (Duniway 2002; Noling 2002). Biological control, applied alone or as a component of an integrated pest management strategy, is one tactic being investigated as a means to reduce reliance on chemical pesticides for control of soilborne plant pathogens. The use of biological controls instead of more traditional chemical methods has great appeal (Pielach et al. 2008) due to the view that these biologicals are more environmentally benign and of less risk to human health than synthetic chemical compounds. Numerous small-scale experiments have demonstrated the potential of bacterial biological control agents for control of soilborne plant pathogens (Compant et al. 2005; Kloepper et al. 1989; Larkin et al. 1998; Lugtenberg et al. 1991; Weller 1988). Unfortunately, there is a great deal of inconsistency in performance by biocontrol agents when used in larger scale field trials that more accurately mimic production agriculture (Compant et al. 2005; Handelsman and Stabb 1996). While many factors are attributed to inconsistent biocontrol performance, one major factor receiving increased attention is the effect of spatial heterogeneity occurring among the

D.P. Roberts (*) USDA-ARS, Sustainable Agricultural Systems Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Beltsville, MD 20705, USA e-mail: [email protected] D.Y. Kobayashi Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA


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physical, chemical, and biological components of the spermosphere and rhizosphere environments (Pielach et al. 2008). The spermosphere and rhizosphere are the regions of soil directly under the influence of seeds and roots, respectively, and are the critical interfaces between plants and microbes where beneficial and detrimental interactions that lead to disease and disease suppression occur (Rudrappa et al. 2008). It will be a challenge to develop effective strategies to decrease inconsistencies in performance of bacterial biocontrol agents in field settings without a better understanding of how these individual physical, chemical, and biological components of the spermosphere and rhizosphere influence these bacterial agents. Without adequate or consistent performance, the use of bacterial and other biocontrol agents in commercial agriculture will most likely remain limited (Pielach et al. 2008). As a result of extensive work on a range of experimental systems, significant advances have been made with regard to our understanding of mechanisms and traits required for suppression of soilborne plant pathogens by biocontrol agents (Haas and De´fago 2005; Pielach et al. 2008). Standard mechanisms of biocontrol include exclusion of the pathogen through competition for limited resources, production of antibiotics or other inhibitory molecules, predation and parasitism, and induction of plant host defense pathways (Pielach et al. 2008). It is widely held that in most applications bacterial biocontrol agents must also colonize the developing rhizosphere to effectively express these mechanisms (Pielach et al. 2008; Haas and De´fago 2005; Compant et al. 2005; Gamalero et al. 2004; Lugtenberg et al. 2001; Weller 1988) and that biocontrol agents are capable of colonizing the rhizosphere only if they have the appropriate traits to defend against harmful chemicals, utilize available nutrients, and contend with the indigenous microflora (Hartman et al. 2009). However, our knowledge of biological control at the whole systems level remains limited. Despite shortfalls in our knowledge, there is solid evidence demonstrating the impact of various rhizosphere environmental factors on expression of genes contributing to biocontrol activity (Haas and De´fago 2005; Kiely et al. 2006). In this chapter, we discuss the heterogeneity of the physical, chemical, and biological components within the spermosphere and rhizosphere as they impact (1) the expression of traits and mechanisms associated with biocontrol, and (2) the performance of bacterial biocontrol agents.

5.2

The Spermosphere and Rhizosphere Influence Behavior of Bacterial Biological Control Agents

In Vitro Expression Technology (IVET)- and microarray-based experiments provide snapshots at the genetic and genomic levels of the impact that various soil and plant influences have on bacterial biocontrol agents during colonization of seeds and roots (Matilla et al. 2007; Rainey 1999; Silby and Levy 2004). These studies suggest a highly dynamic environment that necessitates the adaptation of bacteria

5 Impact of Spatial Heterogeneity Within Spermosphere and Rhizosphere

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to detrimental environmental stresses and a varied nutritional environment (Matilla et al. 2007; Rainey 1999). IVET experiments with Pseudomonas fluorescens in soil show differential upregulation of genes functioning in nutrient transport and utilization, detoxification, and regulation (Silby and Levy 2004). Pseudomonas genes differentially expressed during colonization of the rhizospheres of sugar beet and corn include those functioning in nutrient utilization and stress responses and reflect the effects of the rhizosphere environment on general metabolism and the need for protection against detrimental factors encountered within this environment (Matilla et al. 2007; Rainey 1999). Expression of genes functioning in type III secretion in the plant rhizosphere suggests an intimate association of the biocontrol bacterium with the plant (Jackson et al. 2005; Mark et al. 2005; Rainey 1999).

5.3

Soil Factors Influence Biological Control Agents in the Spermosphere and Rhizosphere

Soils consist of aggregates that vary spatially in size, composition (clay, silt, sand, organic matter), and chemistry, as well as in the voids between and within these aggregates. The voids can be occupied by gases, differing aqueous solutions, and a variety of soil microbial communities (Buyer et al. 1999; Foster 1988; Garbeva et al. 2004; Hattori and Hattori 1976; Wieland et al. 2001) thereby creating vastly diverse microenvironments that can profoundly affect the distribution, persistence, and physiology of biocontrol agents. As an example, in the absence of percolating water, which in itself can greatly influence downward movement of bacteria on roots, soil matric potential becomes a major factor controlling the downward movement of bacteria (Liddell and Parke 1989; Parke et al. 1986; Scott et al. 1995). Voids of smaller sizes represent microenvironments that retain water longer during dry periods, decreasing exposure of inhabiting microbes to desiccation stress (Foster 1988). Void size can also influence predation of bacteria by protozoans (Postma and van Veen 1990; van Veen et al. 1997). Abiotic soil factors, such as pH, temperature, high osmotic conditions, or matric tension, are also known to impose stresses on microorganisms that influence their survival (van Elsas and van Overbeek 1993; van Veen et al. 1997). Chemical compositions of soil appear to influence survival of microorganisms in the soil. Bashan et al. (1995) demonstrated that concentrations of nitrogen, potassium, and phosphate in soil are correlated with survival of Azospirillum brasilense. In addition to influencing survival, abiotic soil factors have demonstrated their impact on the expression of biocontrol genes in bacterial agents (Haas and De´fago 2005). For example, a number of soil factors influence secondary metabolite production in P. fluorescens. Excess iron concentrations repress biosynthesis of the iron siderophores pyoverdine and pyochelin, while low oxygen tensions and low iron availability are required for production of volatiles like hydrogen cyanide in certain pseudomonads (Haas and De´fago 2005). Some other factors such as oxygen concentration, pH, and temperature were shown to impact expression of the

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antibiotic phenazine-1-carboximide in P. chlororaphis (van Rij et al. 2004). These in vitro studies on gene expression were corroborated by studies where certain physical and chemical factors in soil were shown to influence disease suppression by bacterial biocontrol agents (Duffy and De´fago 1997; Duffy et al. 1997; Haas and De´fago 2005; Ownley et al. 2003). It is clearly evident from the examples described earlier that variation in numerous soil factors, including aggregation, voids, chemistry, and resident microflora, can impose heterogenous conditions in a relatively small volume of the field such as a single plant rhizosphere. The end result is a spatially diverse collection of unique and localized microenvironments (Smiles 1988; van Elsas and van Overbeek 1993), each with the potential to influence persistence, distribution, gene expression, and ultimately the performance of bacterial biocontrol agents in different ways.

5.3.1

Plants Influence the Complexity and Heterogeneity of the Spermosphere and Rhizosphere

As a consequence of the growth and development of the root system, an extremely diverse range of organic and inorganic compounds can be taken up or released by seeds and roots into the soil. The uptake and release of these compounds has a profound effect on the physical and chemical properties of the soil and on the indigenous microflora (Badri and Vivanco 2009; Rougier 1981), and add to the complexity and spatial heterogeneity of microenvironments encountered by bacterial biological control agents. For example, protons and electrons are secreted within carbon compounds as undissociated acids or compounds with reducing capabilities. Acidification of the surrounding soil can occur with the release of protons and organic acids from the seed and root, and uptake of nutrient ions by the plant (Hartman et al. 2009). While pH levels represent one example of how plants can significantly alter soil properties, there are other examples of how plants influence soil changes. Oxygen consumption, due to respiration by the root and associated microflora, can result in steep redox gradients in the rhizosphere (Hartman et al. 2009). Organic material released into the soil by the plant in the form of rhizodeposits significantly influences the nutritional environment encountered by bacterial biocontrol agents. Estimates regarding the percentage of photosynthate released vary with the methods employed in the analysis (Farrar et al. 2003) and have been reported to be as high as 30–40% in young seedlings (Lugtenberg et al. 1999; Meharg 1994; Whipps 1990). Root products and rhizodeposits entering the soil probably consist of almost every type of plant compound as sloughed cells from the root cap and root hairs decay or the rhizodermis degrades in older portions of roots. Root products with specific biological activity can also be actively secreted (De-la Penã et al. 2008; Hartman et al. 2009; Wen et al. 2007). Reduced carbon compounds can be divided into low- and high-molecular-weight compounds with the low-molecular-weight compounds consisting of a diverse mixture of


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carbohydrates, amino acids, organic acids, phenolics, and secondary metabolites. High-molecular-weight components such as proteins and mucilage have been reported (Badri and Vivanco 2009; Wen et al. 2007). The quantity and quality of root exudates are determined by plant species, age of the plant, soil nutritional environment, and biotic agents and abiotic soil factors that cause plant stress (Badri and Vivanco 2009). For example, plants under iron stress release phytosiderophores or protons and chelators (phenolics, carboxylates) to acquire iron while phosphorous deficiency in many plants enhances the production and release of phenolic and carboxylate compounds (Hartman et al. 2009). Organic compounds released by seeds and roots can impact bacterial biological control agents in many ways, serving as important nutrients, attractants, and deterrents (Badri and Vivanco 2009; Pielach et al. 2008). Arguably, one of the most significant influences plants impose on bacterial biocontrol agents residing in the spermosphere and rhizosphere, however, is on nutritional content, which is discussed in detail in the following section.

5.3.1.1

Plant Inputs Serve as Nutrients and Influence the Behavior of Biological Control Bacteria

Soluble reduced carbon compounds within rhizodeposits are thought to have the greatest stimulatory impact on growth and metabolic activity of microbes in the spermosphere and rhizosphere (Kraffczyk et al. 1984; Lynch and Whipps 1990). Sugars, amino acids, and organic acids are typically the dominant soluble reduced carbon compounds in rhizodeposits (Farrar et al. 2003; Lynch and Whipps 1990) and their availability affects growth and metabolism of biocontrol agents in the soil. The importance of reduced carbon inputs from plants into soil is perhaps most easily seen in studies determining the significance of these nutrients on bacterial colonization. Roberts et al. (1999a, 2000) studied the impact of nutrients released from cucumber and pea seeds on spermosphere populations of Enterobacter cloacae. These two seed types differ dramatically in the quantity, and therefore availability, of reduced carbon nutrients within their exudates to colonizing bacterial biocontrol agents. Pea seeds release two to three orders of magnitude more carbohydrate and amino acid during the first 24 h after imbibition than cucumber seeds. Consequently, pea seeds support substantially greater growth and population sizes of E. cloacae than cucumber seeds (Roberts et al. 1999a, 2000). Further evidence that differences in populations of E. cloacae carried by pea compared with cucumber seeds are directly related to the quantity of reduced carbon nutrients in exudate comes from studies using E. cloacae mutant strains that are impaired in catabolic capabilities. Mutants disrupted in key enzymatic steps of catabolic pathways are no longer capable of using certain compounds in exudate as nutrient sources. The net result is one that mimics a reduction in exudate nutrients available for use during growth in the spermosphere or rhizosphere (Liu et al. 2007; Roberts et al. 1999a, 2000, 2007). E. cloacae A-11 contains a mutation in pfkA, which encodes the key glycolytic enzyme phosphofructokinase. With the exception of fructose, this mutant cannot catabolize hexose sugars found in seed exudate resulting

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in a reduction in sugar compounds in the spermosphere available to the mutant compared with the wild-type strain 501R3. Indeed, seed colonization experiments demonstrated that strain A-11 is significantly reduced in population levels in the cucumber spermosphere relative to the wild-type strain (Fig. 5.1b). When exogenous fructose is supplied to cucumber spermosphere to compensate for the nutritional deficit, colonization ability by strain A-11 can be restored to levels comparable to the wild type (Roberts et al. 1999a, 2000). In addition to the glycolytic mutant A-11, colonization effects have been observed with E. cloacae catabolic mutant strains containing mutations in sdhA and aceF, which encode subunits for succinate dehydrogenase and the pyruvate dehydrogenase complex, respectively (Liu et al. 2007; Roberts et al. 2007). The sdhA mutant strain M2 is severely impaired in in vitro growth on almost all amino acids and organic acids detected in seed exudates but relatively unaffected in growth on the carbohydrates. The aceF mutant strain M43 is more severely affected, with only limited in vitro growth in almost all components of seed exudate. The resulting reduction in nutrient utilization capabilities of these strains is reflected in their colonizing populations in cucumber spermosphere. The sdhA mutant strain M2 is significantly reduced in colonization relative to wild-type strain 501R3, while the aceF mutant strain M43 is completely deficient in colonization (Roberts et al. 1996, 2007). Along with cell population levels, metabolic activity (as measured by metabolic energy status) of colonizing bacteria has been found to correlate with plant exudate content. Using a bioluminescence-based reporter system, it was shown that E. cloacae displayed significantly higher metabolic energy status per cell on pea seed than on cucumber seed (Roberts et al. 2009), corresponding directly with differences in quantities of exudate between the two seed types. In vitro experiments using oxygen consumption to measure metabolic activity by E. cloacae corroborated the role of pea seed exudate in supporting higher metabolic activity in pea spermosphere. Additionally, the pfkA mutant strain A-11 had lower average metabolic energy status per cell than the wild-type strain in both cucumber and pea spermosphere due to its inability to use the full range of sugars available to the wild type (Fig. 5.1a, c). These results indicate that greater exudation by pea seed not only supports higher populations of cells, but that the average bacterial cell colonizing a pea seed is more active metabolically than that colonizing a cucumber seed due to this greater exudation. These observations suggest profoundly different influential effects of microenvironments that vary in nutrient levels on expression of traits by biocontrol bacteria. When analyzed further, the example described previously illustrates the complexity of the influence that plant exudation imposes on the behavior of bacterial biocontrol agents. In addition to quantity of exudate, individual components of the exudate have been shown to differentially influence colonization and metabolic activity by biocontrol agents. Roberts et al. (2000, 2009) demonstrated that qualitative differences in seed exudate not only influenced growth rate during seed colonization by E. cloacae, but also the average metabolic energy status per cell. In this particular study, hexose sugars had the greatest stimulatory effect on the


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Fig. 5.1 Colonization and metabolic energy status of Enterobacter cloacae strains 501R3 and A-11 in cucumber spermosphere in the presence and absence of exogenous fructose. Both strains

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enteric bacterium, E. cloacae. However, the metabolic influence by both the type and concentration of exudate components may be completely different in taxonomically unrelated bacteria, such as fluorescent pseudomonads, which favor organic acids over simple sugars as preferred carbon sources for growth. While greater exudation or nutrient availability may prolong metabolic activity, extend colonization persistence, and enhance expression of certain traits; its influence on overall biological control performance is directly dependent on the pathosystem, the biocontrol agent, and the mechanism by which disease suppression occurs (Pielach et al. 2008). An excellent example of the confounding influence of exudation on performance by bacterial biocontrol agents comes from a series of studies investigating the mechanism by which E. cloacae suppresses damping-off caused by Pythium ultimum. van Dijk and Nelson (2000) demonstrated that degradation of certain fatty acids by E. cloacae was responsible for suppression of P. ultimum damping-off. These plant-derived fatty acids serve as signals for spore germination by P. ultimum and therefore are important for infection and disease. A subsequent study by Kageyama and Nelson (2003) demonstrated that variation in nutrient availability between spermospheres of different plant species had a profound effect on biocontrol of P. ultimum damping-off. Biological control activity by E. cloacae was much less effective in nutrient-rich spermospheres, such as pea, compared with nutrient poor spermospheres, such as cucumber. Winstam and Nelson (2008) demonstrated that concentrations of sugars in the nutrient-rich spermospheres were sufficient for catabolic repression of fatty acid degrading enzymes in E. cloacae, resulting in loss of biocontrol activity. This variable disease suppression behavior by E. cloacae underscores the impact of differential plant inputs and/or availability of certain nutrients in plant-associated environments on biocontrol activity. Hence, differential plant inputs of certain nutrients may have an effect on the pathogen as well and thus indirectly impact performance of the biocontrol agent. It has been posed that the metabolically active portion of colonizing populations of plant-beneficial bacteria is most important for success in their biotechnological applications (Heijnen et al. 1995; Ramos et al. 2000; Unge et al. 1999) as metabolic energy is necessary for expression of required traits (Crowley et al. 1996; Sørenson et al. 2001). Clearly, metabolic activity is essential for biological control to occur successfully; however, the above studies with E. cloacae represent a caveat regarding how one addresses the importance of bacterial metabolic activity to biocontrol performance. The relevance of metabolic activity is system dependent, further emphasizing the need to better understand the mechanisms by which biocontrol bacteria suppress disease.

Fig. 5.1 (continued) contained the bioluminescence reporter plasmid pGL3.1 (Roberts et al. 2009). (a) Relative luminescence units (RLU) from total populations of E. cloacae strains. (b) Colony-forming units (CFU) of E. cloacae strains. (c) Average metabolic energy status per cell of E. cloacae strains. Experiments were performed as described in Roberts et al. (2009) with the exception that strains were not starved prior to application to cucumber seed


5.3.1.2

119

The Distribution and Bioavailability of Nutrient Compounds to Bacterial Biological Control Agents Are Nonuniform in the Spermosphere and Rhizosphere

The previous section emphasizes the impact of the variability in types and concentrations of plant-derived nutrients from different plant host species on the populations and physiology of bacterial biocontrol agents. Nutrient availability also varies spatially within the spermosphere and rhizosphere of an individual plant leading to impacts on the biocontrol agent situated in different locations in the spermosphere or along the root. The spermosphere and rhizosphere can be thought of as gradient systems where diffusible compounds released from seeds and roots influence microbes in regions of the soil that extend for millimeter distances radially from the plant (Helal and Sauerbeck 1983, 1986; Toal et al. 2000). As distance from the plant increases, the concentration of plant-released compounds decreases. There are many factors that contribute to the formation of this gradient, including binding of these compounds by soil particles and their degradation or uptake by the associated microflora (Farrar et al. 2003; Hartman et al. 2009). In contrast, concentrations of soil-derived nutrients such as nitrogen or phosphorus tend to increase with increasing distance from the root due to their uptake by roots. Exudation of organic compounds into the soil varies along the root longitudinally with the maturation phase of the root system (Badri and Vivanco 2009; Walker et al. 2003). Most roots can be divided into four classes ranging from the tip to the base of the root: (1) the root tip, consisting of the root cap and meristematic region; (2) the elongation zone; (3) the maturation zone; and (4) the mature zone (Gilroy and Jones 2000). Experiments involving 14C-labeling and the use of reagents such as ninhydrin to detect organic compounds released from roots have identified exudation sites along the root system (McDougall and Rovira 1970; Van Egeraat 1975). In general, the zone immediately behind the root tip is considered a major site of exudation (Badri and Vivanco 2009). This is not surprising as the xylem and phloem are not yet mature necessitating the flow of nutrients through the apoplast to the root tip. These nutrients are readily released into the adjoining soil due to diffusion, as this region of the root is not yet suberized (Cardon and Gage 2006). Regions of older roots, such as points of secondary root emergence, release exudate as well (Van Egeraat 1975). Additionally, the root cap, border cells, and root hair cells have been reported to be involved in the release of compounds into the rhizosphere (Czarnota et al. 2003; Farrar et al. 2003; Nguyen 2003; Pineros et al. 2002; Wen et al. 2007). Therefore, the distribution of reduced carbon compounds and other nutrients in the rhizosphere vary both radially away from the root as well as longitudinally along the root. Distribution of nutrients in the spermosphere and rhizosphere at a given point in space will also vary over time due to the growth and maturation of the root and to the development of attendant soil microbial communities. This is evidenced by wave-like patterns of indigenous bacterial populations developing while root tips move through soil; pulsing the localized soil environment with nutrients (Semenov et al. 1999; van Bruggen et al. 2008).

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Molecular tools, based on whole-cell bioreporter systems, are being constructed to better determine the availability of spermosphere and rhizosphere nutrients to microbes on micrometer scales, which is representative of the scale that individual microbial cells sense their environment (Leveau and Lindow 2001). Although current biosensors have been constructed to detect only a small portion of compounds in the spermosphere and rhizosphere (Farrar et al. 2003), work with these biosensors is providing insight into the spatial distribution of nutrients that are available for uptake by bacterial biocontrol agents. Working with an ice-nucleating-reporter-based system, Jaeger et al. (1999) demonstrated that availability of sucrose was maximal near the root tip and decreased dramatically with increasing distance along the root away from the root tip. On the other hand, bioavailability of the amino acid tryptophan was minimal at the root tip and increased along the root until 12–16 cm back from the tip. Bringhurst et al. (2001) detected bioavailable galactosides around root hairs of legumes and in regions where lateral roots emerged from main roots, but not at root tips. Joyner and Lindow (2000) demonstrated substantial heterogeneity in iron bioavailability to Pseudomonas syringae in the rhizosphere. The emerging picture is that the spermosphere and rhizosphere are spatially and temporally heterogeneous on micro and macro scales with regard to the concentration of individual nutrients and their availability to biocontrol agents. The impact of this spatially heterogeneous distribution of nutrients on bacterial biocontrol agents is readily seen when analyzing the distribution of these bacteria in the rhizosphere and their metabolic activity. Several studies, including those using fluorescent pseudomonads and E. cloacae on roots of different crop species, indicate that these bacteria have a wide, but nonuniform distribution in the rhizosphere (Bahme and Schroth 1987; Bull et al. 1991; Dandurand et al. 1997; Lohrke et al. 2002; Loper et al. 1984; Roberts et al. 1999a, b, 2003; Weller 1988). Metabolically active cells of bacterial biocontrol agents are nonuniformly distributed in the rhizosphere and form a subset of the colonizing population. Microbes are known to have greater activity in association with plants than in bulk soil (Kroer et al. 1998; Rattray et al. 1995). However, only a small portion of the bacterial population maintains high levels of metabolic activity in the rhizosphere; the majority of microbial cells having metabolic characteristics similar to those of starved cells (Marschner and Crowley 1996; Normander et al. 1999; Ramos et al. 2000). It is thought that variations in nutrient availability as well as differences in the compositions of plant-derived compounds result in this spatial heterogeneity of metabolic activity (Brennerova and Crowley 1994; Crowley et al. 1996; Heijnen et al. 1995; Kragelund et al. 1997; Ramos et al. 2000; Rattray et al. 1995; Sørenson et al. 2001). Specific studies have indicated that regions along roots where root exudation is greatest, such as behind root tips and near lateral roots, support the highest metabolic activity by bacterial cells (Boldt et al. 2004; Brennerova and Crowley 1994). At the single-cell level, only small portions of the colonizing population within these regions are metabolically active (Ramos et al. 2000).


5.3.1.3

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Molecules Released by Plants into the Spermosphere and Rhizosphere Can Have Detrimental Effects on Residing Microorganisms, Including Biological Control Bacteria

Roots produce a number of organic compounds with antibacterial and/or antifungal properties that can be released into the rhizosphere, and some of these compounds have broad-spectrum activity (Bais et al. 2004b). These compounds most likely function in protecting the plant from infection by pathogens but also may be detrimental to bacterial biocontrol agents. In particular, border cells sloughed from the root tip appear to have a fundamental role in protecting the plant from pathogen attack (Wen et al. 2007). Border cells release a number of compounds that attract, repel, and influence gene expression in soilborne microbes (Farrar et al. 2003). The border cell secretome of pea was shown to contain a complex mixture of proteins that potentially function in protection of the vulnerable root cap from pathogen infection (Wen et al. 2007). The root cap is surprisingly devoid of infection by pathogens (Olivain and Alabouvette 1999; Turlier et al. 1994; Wen et al. 2007) or colonization by beneficial microbes (Assmus et al. 1997; Gamalero et al. 2005) despite being a major site of organic carbon input into the soil. In hydroponic culture, 98% of organic material released into the medium is thought to be derived from the root apex (Farrar et al. 2003). Rudrappa et al. (2007) present findings that demonstrate the impact of rootproduced detrimental compounds on biofilm formation and disease suppression by the biocontrol agent Bacillus subtilis FB17. Strain FB17 did not form biofilms on the surface of roots of the NahG transgenic line of Arabadopsis. Inhibition of biofilm formation was shown to be a response by strain FB17 to the presence of catechol on the surface of roots; catechol leading to the production of reactive oxygen species. The presence of this compound led to the downregulation of the yqx -sipW-tasA and epsA-O operons which are important for biofilm formation by this bacterium. Biofilm formation was previously demonstrated to be of importance in disease suppression by B. subtilis (Bais et al. 2004a). Evidence suggests that plants can selectively detect a pathogen and respond through the release of antimicrobial compounds (Bais et al. 2004b). Earlier, Bais et al. (2002) demonstrated that release of rosmarinic acid by roots was elicited by cell walls from the fungal plant pathogen Phytophthora cinnamoni. This compound is inhibitory to a number of fungal and soilborne microorganisms, indicating that the ability of biocontrol agents to escape such inhibitory activity may be of primary importance for successful plant associations to occur.

5.3.1.4

Indigenous Microbial Communities Are Distributed Nonuniformly in the Spermosphere and Rhizosphere

Plant inputs into soil also affect the local soil environment by stimulating the development of indigenous microbial communities in the rhizosphere. Plant exudates have been shown to select bacteria from the indigenous bacterial community

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in bulk soil, resulting in microbial communities within the rhizosphere that are different from those observed in bulk soil. Such communities not only can vary between plant species, but also between locations along the root. This effect is probably linked to root exudate composition which, as discussed above, varies not only between plant species and regions of the root, but also as the result of environmental influences (Garbeva et al. 2004; Haichar et al. 2008; Wieland et al. 2001; Vandenkoornhuyse et al. 2007; Yang and Crowley 2000). The combined effect of plant and soil factors on the indigenous microflora is a spatially diverse collection of microbial communities with different capacities for nutrient competition and production of inhibitory metabolites. Green fluorescent protein (gfp)tagged cells of the biocontrol bacterium P. fluorescens introduced into the soil as a treatment on barley seeds were often physically associated with indigenous bacteria on barley roots (Normander et al. 1999). This would allow for negative interactions such as competition for nutrients and other resources, antibiosis, and disruption of regulatory networks that control expression of genes involved in disease suppression mechanisms (Haas and De´fago 2005). Thus, bacterial biocontrol agents must also contend with indigenous microflora in order to express intended suppressive effects on pathogens.

5.3.1.5

Biological Control Agents Must Colonize the Rhizosphere for Effective Disease Suppression to Occur

As discussed in the preceding sections, biocontrol agents can only colonize the rhizosphere if they have the appropriate traits to defend against harmful chemicals and other adverse environmental conditions, utilize available nutrients, and contend with the indigenous microflora (Hartman et al. 2009). If a bacterial biocontrol agent does not effectively colonize and become widely distributed throughout the rhizosphere, it is thought that effective disease suppression will not be achieved in most cases. This is because many pathogens are believed to infect plants near root tips, in the zone of root elongation, or through root hairs, which represent infection courts that can be relatively distant from the point of introduction of the biocontrol agent into the soil. Close proximity of biocontrol agents to pathogens is likely to be important in cases where they suppress pathogens through the production of secondary metabolites, such as antibiotics or siderophores, so that these inhibitory compounds are present in sufficient concentration to be effective (Gamalero et al. 2004). Occupation of the same microenvironment by the bacterial biocontrol agent and pathogen is also likely to be important in cases where they suppress pathogens through competition for resources such as nutrients or through predation/parasitism. Even with biocontrol agents that suppress disease through induction of plant defense responses, it is likely that redistribution of populations from the dead seed coat to other plant tissues capable of a physiological response is necessary. Close contact of the bacterial elicitor to the plant cell membrane is thought to be needed for induction of a plant response (Leeman et al. 1995).


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Strong evidence of a role for extensive colonization of the rhizosphere by biocontrol agents came from Lugtenberg’s group in The Netherlands working with suppression of Fusarium oxysporum f.sp. radicis-lycopersici on tomato with P. chlororaphis PCL1391 (Chin-A-Woeng et al. 2000). This pathogen approaches the root via root hairs, colonizes the root surface, and then infects at random sites throughout the root system (Lagopodi et al. 2002). For this work, three classes of colonization mutants of strain PCL1391 were constructed that were deficient in traits or loci previously demonstrated to be important for root colonization (Lugtenberg et al. 1999). All mutants produced wild-type levels of phenazine1-carboximide, an antibiotic crucial for control of F. oxysporum f.sp. radicislycopersici (Chin-A-Woeng et al. 1998). Production of other inhibitory compounds potentially involved in disease suppression (hydrogen cyanide, chitinase, protease) was shown to be unaffected by these mutations. All colonization mutants were reduced in disease suppression despite being unaffected in production of these metabolites (Chin-A-Woeng et al. 2000). In subsequent work, it was shown that autofluorescent protein-tagged PCL1391 and F. oxysporum f.sp. radicis-lycopersici strains compete for the same sites on tomato roots and that PCL1391 readily colonized the hyphae of this pathogen (Bolwerk et al. 2003). Colonization density of the pathogen was reduced when in close proximity with PCL1391. Only with damping-off pathogens has the importance of extensive spatial distribution of bacterial biocontrol agents throughout the rhizosphere been demonstrated to be of lesser importance (Roberts et al. 1997). With damping-off diseases the infection courts are the seed coat, endosperm, embryo, the emerging radicle, hypocotyl, and cotyledons (Paulitz 1992). These tissues have a limited spatial distribution in soil at the end of a brief period of high susceptibility to disease. Consequently, large populations of biocontrol bacteria can be readily delivered directly to the infection court as seed treatments (Paulitz 1992). We demonstrated that it is possible to suppress pre-emergence and post-emergence damping-off of cucumber caused by P. ultimum with E. coli S17R1 in the absence of extensive or persistent root colonization (Roberts et al. 1997). This bacterium was root-colonization deficient on cucumber plants, being limited to the seed coat and upper 1 cm of cucumber root at 7 days and nondetectable after 42 days. E. coli S17R1 and a root-colonizationproficient strain of E. cloacae provided similar and effective levels of biological control of damping-off of cucumber seeds and seedlings (Roberts et al. 1997).

5.4

Conclusion

In this chapter, it is highlighted that variations in the numerous soil- and plantderived factors within the spermosphere and rhizosphere impose heterogeneous conditions in relatively small volumes of the field. On field scales, the end result is a collection of plant rhizospheres containing myriad unique and localized microenvironments, each microenvironment with the potential to influence persistence, distribution, expression of biocontrol traits, and ultimately the performance of

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bacterial biocontrol agents in different ways. Inconsistent performance in disease suppression on field scales occurs when the influences of these microenvironments (1) preclude sufficient colonization of certain rhizospheres by the biocontrol agent necessary for effective disease suppression and/or (2) result in subpopulations of biocontrol bacteria residing in certain rhizospheres that are not in the correct physiological state for disease suppression. Overcoming inconsistent performance by bacterial and other biocontrol agents at the field-scale level is necessary if the use of these biological controls is to become widespread in production agriculture. Approaches to overcoming inconsistent performance by biocontrol agents resulting from the spatial heterogeneity in physical, chemical, and biological components of the spermosphere and rhizosphere include their integration into multitactic disease management strategies, where different biocontrol agents are combined with each other or with other biologically based disease management technologies such as cover crops. These approaches have been documented in the literature for some time (Lemanceau and Alabouvette 1991; Lemanceau et al. 1993; Pierson and Weller 1994; Raupach and Kloepper 1998). A combination of biocontrol agents is more likely to have a greater variety of traits responsible for suppression of the pathogen. Further, it is likely to have these traits expressed over a wider range of microenvironmental conditions due to the different ecological adaptations of the producing strains. Likewise, combining biocontrol agents with cover crops with disease suppressive capabilities will increase the likelihood of disease suppressive activity over a wider range of microenvironmental conditions. The current challenge is to develop a greater understanding of spermosphere and rhizosphere factors, and their impacts on biocontrol agents, so that strategic combinations of biocontrol agents or other biologically based technologies can be made; the combinations being made to overcome the known susceptibilities of each individual component to given microenvironmental conditions.

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Meharg AA (1994) A critical review of labeling techniques used to quantify rhizosphere carbon flow. Plant Soil 166:55–62 Nguyen C (2003) Rhizodeposition of organic C by plants: mechanisms and controls. Agronomoie 23:375–396 Noling JW (2002) The practical realities of alternatives to methyl bromide: concluding remarks. Phytopathology 92:1373–1375 Normander B, Hendriksen NB, Nybroe O (1999) Green fluorescent protein-marked Pseudomonas fluorescens: localization, viability, and activity in the natural barley rhizosphere. Appl Environ Microbiol 65:4646–4651 Olivain C, Alabouvette C (1999) Process of tomato root colonization by a pathogenic strain of Fusarium oxysporum f.sp. lycopersici in comparison with a non-pathogenic strain. New Phytol 141:497–510 Ownley BH, Duffy BK, Weller DM (2003) Identification and manipulation of soil properties to improve the biological control performance of phenazine-producing Pseudomonas fluorescens. Appl Environ Microbiol 69:3333–3343 Parke JL, Moen R, Rovira AD, Bowen GD (1986) Soil water flow affects the rhizosphere distribution of a seed borne biological control agent, Pseudomonas fluorescens. Soil Biol Biochem 18:583–588 Paulitz TC (1992) Biochemical and ecological aspects of competition in biological control. In: Baker RR, Dunn PE (eds) New directions in biological control: alternatives for suppressing agricultural pests and diseases. Alan R Liss, New York, pp 713–724 Pielach CA, Roberts DP, Kobayashi DY (2008) Metabolic behavior of bacterial biological control agents in soil and plant rhizospheres. In: Laskin AI, Sariaslani S, Gadd GM (eds) Applied Microbiology, vol 65. Academic, New York, pp 199–215 Pierson EA, Weller DM (1994) Use of mixtures of fluorescent pseudomonads to suppress take-all and improve the growth of wheat. Phytopathology 84:940–947 Pineros MA, Magalhaes JV, Alves VMC, Kochian LV (2002) The physiology and biophysics of an aluminum tolerance mechanism based on root citrate exudation in maize. Plant Physiol 129:1194–1206 Postma J, van Veen JA (1990) Habitable pore space and survival of Rhizobium leguminosarum biovar trifolii introduced into soil. Microb Ecol 19:149–161 Rainey PB (1999) Adaptation of Pseudomonas fluorescens to the plant rhizosphere. Environ Microbiol 1:243–257 Ramos C, Molbak L, Molin S (2000) Bacterial activity in the rhizosphere analyzed at the singlecell level by monitoring ribosome contents and synthesis rates. Appl Environ Microbiol 66:801–809 Rattray EAS, Prosser JI, Glover LA, Killham K (1995) Characterization of rhizosphere colonization by luminescent Enterobacter cloacae at the population and single-cell levels. Appl Environ Microbiol 61:2950–2957 Raupach GS, Kloepper JW (1998) Mixtures of plant growth-promoting rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology 88:1158–1164 Roberts DP, Marty AM, Dery PD, Yucel I, Hartung JS (1996) Amino acids as reduced carbon sources for Enterobacter cloacae during colonization of the spermospheres of crop plants. Soil Biol Biochem 28:1015–1020 Roberts DP, Dery PD, Hebbar KP, Mao W, Lumsden RD (1997) Biological control of damping-off of cucumber caused by Pythium ultimum with a root-colonization-deficient strain of Escherichia coli. J Phytopathol 145:383–388 Roberts DP, Dery PD, Yucel I, Buyer JS, Holtman MA, Kobayashi DY (1999a) Role of pfkA and general carbohydrate catabolism in seed colonization by Enterobacter cloacae. Appl Environ Microbiol 65:2513–2519 Roberts DP, Kobayashi DY, Dery PD, Short NM Jr (1999b) An image analysis method for determination of spatial colonization patterns of bacteria in plant rhizosphere. Appl Microbiol Biotechnol 51:653–658


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Chapter 6

Biocontrol Mechanisms Employed by PGPR and Strategies of Microbial Antagonists in Disease Control on the Postharvest Environment of Fruits Anjani M. Karunaratne

6.1

Introduction

The immediate microenvironment surrounding a plant root in the plant–soil interface, having much microbial activity (due to bacteria, yeast and fungi), is referred to as the rhizosphere. When referring to plant growth promotion, bacteria are given a special place although the rhizosphere fungi (as ectomycorrhizae and endomycorrhizae) also play a significant role in nutrient acquiring function of roots. The term “rhizobacteria” decribes the ability of certain bacteria to colonize the rhizosphere aggressively (Rao 1993). Certain strains of rhizosphere bacteria are known as plant growth promoting rhizobacteria (PGPR) because their application can stimulate growth and improve plant stand under stressful conditions (Van Loon et al. 1998). Well over a decade ago, demonstrating the attention PGPR have attracted, Bashan and Holguin (1998) have reported that close to 4,000 publications have appeared in the decade prior to their publication in the mid-1998. There is no reason to believe that the attention has declined over the years thereafter, as different aspects of PGPR have been reviewed in the recent past as well (Lucy et al. 2004; Van Loon 2007; Lugtenberg and Kamilova 2009). The term PGPR dates back to the 1970s, and this term has been used in the early days to describe the biocontrol ability of these bacteria along with their aggressive colonization and plant growth stimulation (Kloepper and Schroth 1978). Davison (1988) has mentioned that the beneficial effects of PGPR fall into two categories according to whether the bacteria benefit the plant directly or indirectly by antagonizing a phytopathogen (for which the term “biocontrol” is used) or removing a growth inhibitor. Hence a decade ago, two new terms for general scientific usage as biocontrol-PGPB (PGPB standing for “plant growth promoting bacteria”) and PGPB have been suggested (Bashan and Holguin 1998). These two authors argued that since many beneficial bacteria are not rhizosphere bacteria, replacing the term

A.M. Karunaratne Department of Botany, University of Peradeniya, Peradeniya, Sri Lanka e-mail: [email protected]


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“rhizobacteria” with “bacteria” to coin the term PGPB is more appropriate. However, not all publications thereafter have adhered to this terminology. Bashan and Holguin (1998) correctly pointed out that the term PGPR does not cover nonrhizosphere interactions like biocontrol in the phyllosphere. It is also striking that the term PGPB has been used in connection to phyllosphere, anthosphere or spermosphere (Hallman et al. 1997; Compant et al. 2005). Bashan and Holguin (1998) further pointed out that at the early stages its broad reference (meaning PGPR in biocontrol) was an advantage, but today (referring to 1998) the term was too general and nonspecific. Interestingly, chronological detection of Burkholderia phytofirmans initially on root surfaces, next in root internal tissues, then in the internode and on xylem vessels and substomatal chambers of leaf tissue has been described (Compant et al. 2006) giving weightage to the idea proposed by Bashan and Holguin. Some PGPR being established as endophytic population in stem, leaves, tubers, and other organs, after transcending the endodermis barrier, crossing from the root cortex to the vascular system has also been noted (Compant et al. 2005; Kloepper et al. 1999), further substantiating the wisdom of the statement by Bashan and Holguin, indicating the absence of clear demarcations for their location on plants. Postharvest scientists also have noted that the antagonists encountered in the fructoplane may have come from other closely related or unrelated sources, and phylloplane has been a good source of antagonists (Janisiewicz and Korsten 2002). This phenomenon has been expressed for the occurrence of pathogens too. For instance, the presence of pathogen conidia on dead floral parts as debris on the ground has been recorded for the banana fruit pathogen Colletotrichum musae (Adikaram 2005) and avocado fruit pathogen C. gloesporioides (Karunaratne et al. 1999) in twigs, leaves, flowers, and debris under the tree. Further Pinto et al. (2000) have documented that the postharvest pathogens of banana C. musae and Fusarium moniliforme live endophytically in plant tissues asymptomatically and could have pathogenic activity (in causing banana crown rot by a pathogen complex or anthracnose by C. musae) at a later stage, causing visible symptoms. Initial emphasis on biocontrol has been on soilborne plant pathogens (Cook 1990). The very first mention of postharvest disease control by a biological agent is the control of Botrytis rot of strawberry with a fungal antagonist Trichoderma (Tronsmo and Dennis 1977) which was more than 40 years ago. However, much focus on the biocontrol of postharvest pathogens of fruits has been observed in the last decade of the twentieth century and the first decade of the twenty-first century. For instance, in his text book Campbell (1989) has mentioned that there is little research on the biological control of diseases of flowers and fruit and even less practical applications. As late as 2002, Janisiewicz and Korsten have compared the long-standing interest in biological control of soilborne pathogens, with research into biological control of postharvest diseases and noted that the latter is in its infancy. A recent review by Droby et al. (2009) states that while in the early 1980s one could find 1–2 publications per year on postharvest biocontrol, now a literature search on the topic will bring up at least a hundred related publications per year. Of the postharvest environment, fruits have attracted more attention than vegetables.

6 Biocontrol Mechanisms Employed by PGPR

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In fact, Wilson and Wisniewski (1989) have reported that since 1983, an explosion of research has occurred in the area of postharvest biocontrol of mainly fruit diseases. Therefore, in retrospective it appears that the accelerated interest in biocontrol of postharvest pathogens is more recent. Unlike research on PGPR, research on biocontrol for postharvest pathogens appeared to have gained interest, as a pressing need to search for alternatives to replace agrochemicals. An impetus to look for biological control strategies to control postharvest diseases of fruits and vegetables was a report of the US National Academy of Sciences that indicated the dangerous presence of oncogenic compounds in pesticides (NAS 1987). In fact, the use of agrochemicals has been the main means of preventing postharvest pathogen attack (Eckert and Ogawa 1985) until the need to look for alternatives was felt and even thereafter due to the lack of suitable alternatives to takeover their role completely. Wilson and Wisniewski (1989) have correctly pointed out that postharvest diseases have not received the attention that the magnitude of the problem warrants. By late 1980s and early 1990s, a number of book chapters and reviews on biological control of postharvest disease (Wilson and Pusey 1985; Chalutz et al. 1988; Janisiewicz 1988; Jeffries and Jegger 1990; Janisiewicz 1991; Wilson and Wisniewski 1989; Wisniewski and Wilson 1992; Barkai-Golan 2001) indicate the attention on this agrochemical-free alternative to combat disease on harvested produce. Since then, biological control still remains as a potential area requiring further investigation for successful field application. It is reported that about 90% of the 2,000 major diseases of the 31 major crops in the USA are caused by soilborne plant pathogens (Wilson 1968; Lewis and Papaviza 1991). This gives the cue that answers may lie in the rhizosphere. The long-standing interest on PGPR appears to present a wealth of knowledge to applications on biocontrol strategies. In fact Lucy et al. (2004) have expressed similar sentiments by stating that PGPR present an alternative to the use of chemicals for plant growth enhancement in many different applications. By and large, it appears that the development of research on postharvest applications of biocontrol has taken a technological route with applied aspects taking a lead role. On the other hand, research on PGPR, having a longer history seems to have taken a fundamental approach and may have much to offer to this relatively younger area of research on biocontrol. Therefore, it may be timely to look at the development of these two areas of research in perspective for a mutual benefit for both disciplines. Some selected milestones for the present discussion in the two disciplines are summarized in Table 6.1. It appears that other than the rhizosphere bacteria, early emphasis on plant tops to look for antagonists has been on the phylloplane. Up to 1984 weightage has been on basic research with regard to rhizosphere bacteria, particularly PGPR. In the latter part of 1980s, there has been interest among postharvest scientists to experiment with biocontrol strategies to control pathogens as a result of a report indicating health issues related to use of agrochemicals (NAS 1987). While other biocontrol applications have been concentrating on molecular biological aspects, postharvest work has been on the modes of antagonist selection and application. In the decade of 1990s, there has been an obvious emphasis on

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Table 6.1 Some selected, historically important key observations and findings during different periods in the fields of rhizosphere biocontrol and postharvest biocontrol Upto 1984 1985–1990 1991–2000 2001–2010 Induced systemic Use of root exudate as Auxin production by Fungicides are a resistance of energy to degrade phylloplane fungi primary means of Pseudomonas pollutants (Kuiper (Buckley and Pugh controlling mentioned (Van et al. 2001) 1971) postharvest Peer et al. 1991) diseases (Eckert and Ogawa 1985) PGPR mixtures to Commercialized Antagonist selection Fungi showing combat multiple Pseudomonas from the resistance to pathogens syringae used in phylloplane fungicides (Lewis (Jetiyanon and the fruit industry (Andrews 1985) 1977) Kloepper 2002) (Janisiewicz and Marchi 1992) Phylloplane Bacillus Antagonists show Integrated PGPR induced spp. control greater success management with increased plant anthracnose in when applied after biocontrol for growth is related mango and harvest PH disease control in part to antibiosis avocado (Korsten (Wisniewski and suggested (Spadaro in root zones and Kotze 1992) Wilson 1992) and Gullino 2004) displacing some microorganisms (Kloepper and Schroth 1981) PH disease control by Phylloplane Erwinia Phylloplane Plant growth was inducing resistance herbicola as a Pseudomonas enhanced by by applying siderophores of biocontrol agent fluorescens from jasmonate (Yao PGPR (Kloepper (Kempf and Wolf mango leaves and Tian 2005) et al. 1980) 1989) controlled latent fungal infections (Koomen and Jeffries, 1993) PGPR produce Soilborne plant Use of UV light to Method to enrich for extracellular pathogens produce induce resistance in biocontrol strains siderophores antimicrobial PH disease control of rhizobacteria (Neilands 1981) compounds (Wilson et al. (Kamilova et al. (Bruehl 1987) 1994) 2005) Fluorescent Pseudomonas spp. is Antagonist selection Root exudates Pseudomonas best mentioned from the composition known exclusively as a phylloplane for PH reviewed (Uren microorganisms PGPR (Schippers avocado fruit 2007) with PGPR activity 1988) anthracnose (Kloepper et al. (Stirling et al. 1980) 1995) Soil pH is crucial in Strain improvement by Bacillus thuringiensis Concern on invasive disease suppression genetic engineering looked at as a microbes (Van der (Scher and Baker suggested (Davison biopesticide Putten et al. 2007) 1980) 1988) (Cannon 1996) Genetic manipulation Rhizobacteria elicited Suggestion for the of pseudomonads induced systemic need to occupy to show antibiosis resistance activates specific niches on (Thomashow and signal transduction the rhizosphere by Weller 1988) pathways involving antagonists for (continued)


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Table 6.1 (continued) Upto 1984 1985–1990

1991–2000 2001–2010 successful jasmonic acid and biocontrol (Pliego ethylene (Van et al. 2007) Loon et al. 1997) Microorganisms in the Influence of a mineral Importance of molecular tools in rhizosphere are ion on biocontrol rhizosphere ideal for use as (Duffy and Defago microbiology biocontrol agents 1997) (Sorensen et al. (Weller 1988) 2009) Several unsuccessful Use of antagonist Genetically modified attempts of mixtures in PH organisms as transferring research biocontrol agents effective biocontrol (Janisiewicz 1998) may be a way from the lab to field to achieve in PHa research commercial use of (Janisiewicz 1988) biocontrol agents for PH use (Droby et al. 2009) Need to explore Labeling rhizobacteria chemical and with fluorescent physical elicitors tags (Bloemberg among biocontrol et al. 2000) agents that could induce resistance in harvested commodities (Wilson and Wisniewski 1989) Suggestion that nonindigenous antagonists might be successful in the aerial plant surfaces (Pusey 1990) Competition for niches and nutrients as a mechanism of biocontrol rhizobacteria (Lemanceau and Alabouvette 1990)

a

PH postharvest

Pseudomonas spp. as biocontrol agents, and Bacillus thuringiensis also has been in the limelight. While postharvest scientists have concentrated on applications, rhizosphere scientists have continued on basic research and molecular biological aspects. A slight change in the emphasis by both groups is seen in the decade of 2001–2010. While work on rhizosphere biocontrol has shown an interest on

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applications, as late as 2009, postharvest scientists have critically evaluated research done and have suggested ways to go forward (Droby et al. 2009). It appears that both groups of scientists will move towards molecular biological applications in the future.

6.2

Biocontrol Strategies of PGPR of the Rhizosphere and of Antagonists in the Postharvest Environment

The term biocontrol appears to have different nuances of meaning based on where it is applied. While there is diversity among the antagonists in the postharvest environment, the modes of action of the antagonists too appear to be diverse.

6.2.1

The Philosophy of Biocontrol

Biological control has been defined by Campbell (1989) giving a broad definition including crop rotation, direct addition of microbes antagonistic to pathogens or favorable to the plant, use of chemicals to change the microflora, and plant breeding as it is known that changes in the plant genome may affect disease resistance and also the surface microflora. In a broader sense, the positive and negative factors governing the sizes of bacterial populations in a natural habitat have been identified as (1) the effect of nutrients and growth factors and the ability to invade and (2) predation and desertion, respectively (Watanabe and Baker 2000). When the above definition is applied to biocontrol of pathogens in the postharvest environment, manipulation of the microenvironment to favor the needs of the antagonists and hindering the progress of pathogens by a variety of means have been practiced over the years, for the antagonists to win the “battle” against the pathogens. Elsewhere, in unifying the terminology of biocontrol, four strategies of biological control as classical, inoculation, inundation, and conservation have been mentioned (Eilenberg et al. 2001). Of these the intentional introduction of an exotic, usually co-evolved, biological control agent for permanent establishment and long-term pest control, which is referred to as classical biological control, has been the main strategy in the past. In fact looking back, the term biological control has been initially used to describe suppression of insect pests by natural enemies (Smith 1919). In a critical look at the practices adopted over the past years, Droby et al. (2009) have noted that plant pathologists have mainly adopted the entomologists’ definition of biocontrol which involves the control of one organism with another, and they further pointed out that a plant disease is not an organism, but a process influenced at different levels: the pathogen, microenvironment, and host. Therefore, the need for a broader approach to combat plant pathogens has been highlighted. When reviewing the methods employed, selection of one antagonist to control one pathogen referred to as the “silver-bullet” approach


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(Spurr and Knudsen 1985) has been the trend in a majority of publications on biocontrol of postharvest pathogens. Here the emphasis has been only on the pathogen and antagonist (in line with controlling insect pests by natural enemies in the past). Spurr et al. (1990) have mentioned that a “non-silver-bullet” approach is important to make progress toward achieving biological control. Among the criticisms of the “silver-bullet” approach are that it omitted testing of antagonism in the natural environment (in planta) and it did not determine the antagonists impact on disease reduction (inoculum dose: disease index) (Edwards et al. 1994). The “silver-bullet” method could be considered a simplistic method without considering the interactions that occur simultaneously with the antagonist effect on the pathogen. Alternatively, making the system favorable to the biocontrol agents over the pathogen by determining the population sizes has been suggested by Edwards et al. (1994) as a more appropriate strategy. This calls for the need for a broader understanding of the microbial ecology of the environment in question to target biocontrol strategies based on the knowledge of how a mixed population multiplies and survives. Further substantiating this idea, the importance of studies of soil microorganisms at the community level in addition to research on population dynamics and metabolic activity of individual strains has been highlighted by researchers working on rhizosphere bacteria (Gilbert et al. 1993, 1994). In their review on biological control of postharvest fruit diseases, Spadaro and Gullino (2004) have stated that biological control fits in well with the concept of sustainable agriculture because it mostly exploits natural cycles with reduced environmental impact. This idea also is biased towards ecological conservation and appears to fit in with the definition of conservation biological control, which is modification of the environment or existing practices to protect and enhance specific natural enemies or other organisms to reduce the effect of pests (Eilenberg et al. 2001). Confining to the biological control methods adopted in postharvest technology, a broad definition of the term biological control encompasses constitutive or induced resistance, natural plant products, and antagonistic microorganisms to control pathogens (Baker 1987; Wilson and Wisniewski 1989). The current review will focus mainly on antagonistic microorganisms with a brief account of host resistance.

6.2.2

Antagonist Candidates in the Postharvest Environment

Although the emphasis is on antagonist bacteria, this section explores briefly other microbial antagonists with the intension of identifying the key favorable characteristics of potential antagonist. In the postharvest environment among potential antagonists, yeast has commanded a special place (Chalutz et al. 1988) as they could colonize the surface for long periods under dry conditions by rapidly using available nutrients and thereby they may restrict not only colonization sites for pathogens, but also flow of germination cues to fungal propagules (Janisiewicz 1988). Thus, yeasts have been the target of interest for postharvest biocontrol in the late 1980s and early 1990s and

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they also form the major component of flora of fruit surfaces (Janisiewicz 1991). Yeasts do not seem to have got a special mention in the rhizosphere probably because they are not the dominant flora in the rhizosphere. There is another more important reason for yeasts to have a special place in postharvest applications. The treated product (fruit) is often consumed raw, and the antagonists used to control pathogens should be absolutely safe. Although there are exceptions of pathogenic yeasts, their general inability to produce toxic metabolites (antibiotics) also has been considered as a reason for yeasts to be preferred as biocontrol agents. An overview of commercial products of yeasts is given elsewhere (Droby et al. 2009; Sharma et al. 2009). The biocontrol potential of bacteria and fungi has been investigated with much promise. Fungi, especially certain groups in the Fungi Imperfecti, are promising candidates as antagonists, and isolates of mainly Trichoderma, Gliocladium, Penicillum, and Aspergillus have been observed to be the primary colonizers of steamed, pasteurized, or fumigated soils (Cook and Baker 1983; Lewis and Papavizas 1991) which indicate their competitive edge. Among the fungal antagonists, the very first biocontrol agent tried out on a postharvest environment (Tronsmo and Dennis 1977), the Genus Trichoderma has got a special place. Trichoderma has been a well-known mycoparasite and has been commercially available from 1987 (Campbell 1989). Many investigations using Trichoderma spp. have been documented elsewhere (See references listed in Droby et al. 2009 and Sharma et al. 2009). Other than Trichoderma spp., mycotoxin nonproducing strains of fungi have been the popular topics of interest with regard to fungal antagonists. Use of nontoxigenic/nonpathogenic strains of the same species to control toxigenic/ pathogenic strains has been reported to control aflatoxin producing Aspergillus flavus (Ichielevich-Auster et al. 1985; Herr 1988; Cotty 1989). The philosophy behind this is that there is competition between nonpathogenic/nontoxic strains to occupy the same ecological niche as the toxigens/pathogens where the more aggressive antagonist could dominate. In the postharvest environment, the ability of bacteria to grow very rapidly in wounds but not on undamaged fruit surfaces, and therefore bacteria being more effective as biocontrol agents in wounds, has been highlighted (Smilanick 1994). Looking at the antagonist bacterial genera common to the postharvest environment and the rhizosphere, both Bacillus spp. (Edwards et al. 1994) and Pseudomonas spp. have been mentioned, among other genera. More recently, Compant et al. (2005) have remarked that in spite of the focus on free-living rhizobacterial strains especially on Pseudomonas and Bacillus much remains to be learned from nonsymbiotic endophytic bacteria that have unique associations and apparently a more pronounced growth enhancing effect on host plants. Of the above the most commonly mentioned are the pseudomonads. Many bacilli have been used as postharvest biocontrol agents with successful results and Bacillus spp. in the rhizosphere have been demonstrated to promote induced systemic resistance (ISR) and plant growth (Kloepper et al. 2004). Currently, the use of B. thuringiensis seems to have surpassed all other biocontrol agents as it has almost become a household name.


6.2.3

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Diverse Mechanisms of Action

The common strategies mentioned for PGPR and biocontrol of postharvest pathogens in several publications include competition for nutrients and/or space, antibiotic protection and synthesis of cell wall lysing enzymes. Additionally, siderophore production has been noted in bacteria (Rao 1993). Fungal antagonists have demonstrated the ability to attach to the pathogenic fungal hyphae (parasitism) (Campbell 1989), although information on this on subsequent publications appears to be scarce. The above overlap of strategies in the two disciplines is not surprising considering the species mentioned as PGPR and as biocontrol agents of postharvest pathogens. As there are specific conditions favorable for pathogens, the necessity of specific conditions that are favorable for antagonists should be acknowledged. Eckert and Ogawa (1985) have noted that the number of infectious propagules at a potential infection is usually a major determinant of disease severity following either field inoculation or postharvest wound inoculation. The existence of an inoculum threshold implies that a reduction in the amount of inoculum on the harvested crop will result in less postharvest disease. Similarly, the threshold for effective biocontrol by antagonists should be expected. However, information on the mechanisms of action employed by a majority of antagonists is still incomplete probably because of the difficulties encountered in planning long-term longitudinal studies, under controlled conditions incorporating the complex interactions between host, pathogen, antagonist, other associated microorganisms, as well as the environmental effects. The biocontrol strategies to be used on a pathogen would vary depending on the stage of pathogenicity, i.e., quiescent infection, wound infection, primary infection, secondary infection, etc. Even preharvest applications of biocontrol agents for postharvest pathogen control have been suggested (Ippolito and Nigro 2000). Preharvest applications may have an added advantage as it would be possible to transfer some of the findings on research on PGPR of the rhizosphere directly to the postharvest environment. The idea of manipulation of rhizosphere bacteria to combat plant pathogens is not a new concept in spite of the fact that the results have been variable (Chet and Baker 1980; Mazzola 2007). Additionally, the preharvest application of biocontrol agents seems an appropriate strategy also where postharvest handling is unacceptable because the produce may appear less appealing. Looking at the problem from an environmental approach, growth prevention of pathogens can be achieved by making the microclimate undesirable. The strategies employed could be limiting the essential requirements such as space, nutrients, and physical factors such as pH, water activity, and temperature. In fact, soil pH has been shown to be an important criterion for disease suppression (Scher and Baker 1980). Interestingly, it has been noted that the suppressiveness of some soils could be lost by a change of pH (Haas and Defago 2005). It should be acknowledged that even the presence of a single undesirable condition/compound would hinder growth, even if all other essential requirements are available.

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From the organisms’ point of view, Wilson and Wisniewski (1989) have mentioned two basic approaches of using antagonistic microorganisms to control postharvest diseases: promoting and managing natural antagonists that already exist on fruit and vegetable surfaces or artificially introducing antagonists against postharvest pathogens. Plants already harboring pathogens remain healthy most of the time, as the pathogens are suppressed by natural means. Referring to previous work on natural antagonists in the phyllosphere and rhizosphere of plants that could suppress disease development, Wilson and Wisniewski (1989) have queried the presence of natural epiphytic antagonist populations on the surfaces of fruits and vegetables that might be managed to control postharvest diseases. An interesting finding in this regard was when concentrated washings from surfaces of citrus fruit were plated on an agar media, only bacteria and yeast appeared initially, but when these washings were diluted further, rot fungi appeared on the plates indicating that the yeasts and bacteria may be suppressing the growth of the latter (Chalutz 1990). To draw an analogy, the existence of disease suppressive soils has been reported (Schroth and Hancock 1982) and pasteurizing such soils is known to lose their suppressiveness, and when disease suppressive soil is transferred to another location, conducive soil has shown to establish disease suppression (Campbell 1994; Haas and Defago 2005). The above observations show the “behind the screen” role played by the antagonists in natural systems. Extending a similar idea to pesticide applications, Ippolito and Nigro (2000) have commented that pesticide applications can have a harmful effect on nontarget, nonpathogenic phylloplane microbial populations as well. The role of these populations in natural disease suppression is largely unknown and unacknowledged. Additionally, while nonspecific agrochemicals may target both pathogens and antagonists, resistance to them too could be pathogen oriented or antagonist oriented. Combining of antagonists with different modes of action has also been suggested and is thought to have at least three main advantages: broaden the spectrum of activity, enhance efficacy, and allow combination of various mechanisms of biocontrol strategies (Janisiewicz 1998; Ippolito and Nigro 2000). Treatment of plants with a combination of rhizobacteria antagonistic to various soilborne plant pathogens could have a marked effect in reducing root disease if the rhizobacteria are not mutually inhibitory (Kloepper and Schroth 1981). Even more recently, the idea of development of antagonist mixtures consisting of complementary and noncompetitive antagonists to combat postharvest pathogens has been put forward as a promising approach (Spadaro and Gullino 2004). In our experience in combating crown rot of banana caused by a pathogen complex, two different antagonists investigated had different modes of action (Gunasinghe and Karunaratne 2009). However, there appears to be a word of caution from the scientists working on PGPR. Lugtenberg and Kamilova (2009) in their review on PGPR pointed out that although its logical to inoculate seeds with two strains that use different mechanisms of biocontrol, in their experience such mixtures (so called cocktails) never resulted in better disease control. Their explanation for this unexpected result was that the cell numbers of each of the two bacteria on the root are reduced below the threshold level required to cause control. Ippolito and Nigro (2000) have also


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mooted the idea that if populations of bacteria decline that could lead to densities insufficient to control disease. Poor rhizoplane colonization has been considered as a factor that can limit biocontrol efficacy and is reported to be required for some biocontrol mechanisms (Lugtenberg and Kamilova 2009 and references listed therein). However earlier findings have shown enhanced plant root elongation as a consequence of initial binding of bacteria to seed rather than the roots, under controlled experimental conditions (Hong et al. 1991). It has also been noted by Van Loon (2007) that in 1938, Van Luijk has reported that grass seeds germinated to a higher percentage in nonsterile than in sterilized soil. It turned out that nonpathogenic Pythium spp. took over and counteracted the actions of pathogenic Pythium spp. and other deleterious soil microorganism. Inadequate biocontrol in field experiments has often been correlated to poor root colonization (Bloemberg and Lugtenberg 2001). Chin-AWoeng et al. (2000) using colonization mutant strains showed that root colonization played a crucial role in biocontrol, disputing earlier claims that colonization is not important for biocontrol (Gilbert et al. 1993; Roberts et al. 1994). Motility, later refined to chemotaxis toward root exudates, appeared to be an important colonization characteristic (Lugtenberg and Kamilova 2009). In retrospect it appears that the motility of the organisms in question would have contributed to the above dispute. Additionally, the phenomenon discussed earlier in this section of the “behind the screen” role of antagonists also would have had a confounding effect. Unfortunately, on many postharvest biocontrol applications, the role of colonization has not been monitored and chemotaxis has not been considered as a trait of importance for potential biocontrol agents. Selection of enhanced root tip colonizers after plant growth in a gnotobiotic system has been tried out successfully using grass root tips (Kuiper et al. 2001; Kamilova et al. 2005). The other characteristics required to accomplish successful colonization by antagonists were noted as the high level of competitive capability and niche overlap (Ippolito and Nigro 2000). While concepts in biocontrol such as antagonism, antibiosis, resistance, and suppressiveness had already been established by about 1935 (Lewis and Papavizas 1991), competition for nutrients and niches as a mechanism of biocontrol bacteria in the rhizosphere has been a comparatively new finding (Lemanceau and Alabouvette 1990). More recent work has focused on biofilms proposed earlier by Casterton (1990) where colonies of microbes attached to the host exert their effects on the pathogens and host. It is now known that most bacteria grow as complex multicellular-like communities attached to surfaces and immersed in polysaccharides, known as biofilms of slime (Marques et al. 2005). Attachment to the fruit surface is probably an important trait for the antagonists to possess for successful preharvest applications because persistent attachment would contribute to a better colonization and avoiding their dislodging due to wind, rain, or water level fluctuations (Dickinson 1986; Ippolito and Nigro 2000). Furthermore, root colonizing Psedomonas bacteria have been shown to alter plant gene expression in roots and leaves to different extents, indicative of recognition of one or more bacterial determinants by specific plant receptors (Van Loon 2007). The above phenomena demonstrate the

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complexity of the events leading to biocontrol involving the plant host, microbes on the host plant and the soil in which the host plant grows. More than 2 decades ago, expressing similar sentiments as Lemanceau and Alabouvette (1990), Wilson and Wisniewski (1989) have expressed the view that other than antibiosis little information exists on other modes of action of biocontrol. Even as late as 2009, successful biocontrol by certain bacteria has been reported but the explanation of the control mechanism was inadequate (Sharma et al. 2009; Droby et al. 2009). We demonstrated that a strain of Erwinia herbicola (synonym to Pantoea agglomerans) suppressed C. musae on banana (Gunasinghe et al. 2004). Antibiotic production could not have been the mode of suppression as there was no conclusive evidence for the presence of antibiotics. It was noted that previous researchers too have mentioned the existence of nonantibiotic producing strains of P. agglomerans that could also suppress disease (Wilson et al. 1992) but their mode of action remained elusive. The plant roots are known to offer a niche for the proliferation of soil bacteria that thrive on root exudates and lysates (Van Loon 2007). Fungi too are known to utilize root exudates (Campbell 1989). The fruit surface has been described as a moist, nutrient-rich environment in which resistance to disease decreases as maturation progresses (Janisiewicz 1988). The gradual abundance of nutrients on the fruit surface (among other factors related to ripening) may help the fungal pathogens to progress. Therefore, the presence of competitors for these nutrients (and space) would help in disease control. In postharvest biocontrol, competition for nutrients and space by antagonists has been demonstrated by using radiolabeled glucose, where the deprivation of pathogenic conidial germination due to consumption of glucose by antagonists has been demonstrated (Spadaro and Gullino 2004; Filonow 1998). Another dimension to this aspect is offered by researchers on rhizosphere colonizers. It is reported that in addition to the root surface bacteria utilizing the nutrients that are released from the host for their growth, it also secretes metabolites into the rhizosphere and several of these metabolites could act as signaling compounds that are perceived by neighboring cells within the same microcolony or by cells of other bacteria or by root cells of the host (Van Loon 2007). The presence of a complex network of genetic signaling is apparent from the above findings. Perhaps related to genetic signaling, it is also interesting that different sources of information show the involvement of metal ions in pathogenesis. For instance, Duffy and Defago (1997) have reported that zinc improved biocontrol of Fusarium crown and root rot of tomato. More recently, Conrath et al. (2006) noted that almost immediately after pathogen recognition, ion fluxes (Ca2+, NO3, Cl, K+) and changes in the electrical potential differences across the plasma membrane can be detected in plant cells, which they attributed to the enzyme dependence of metal ions. There may be similar applications with benefits to the plant in question. In the postharvest environment, the effects on mineral ions (except the siderophores) have not attracted the attention of researchers. However, addition of nutrients preferably metabolized by the antagonist and not by the pathogen has been suggested (Janisiewicz 1998; Spadaro and Gullino 2004). Even the mobile signal which moves from the rhizosphere bacteria to the specific aerial organ for systemic


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resistance on the plant has remained elusive (Van Loon 2007). However, this is a cue for postharvest scientists to exploit the possibility of improving postharvest shelf-life of fruits by a preharvest mode of treatment perhaps initiating from the rhizosphere through a mobile signal.

6.3

The Role of Host Plant in Biocontrol of Pathogens

While earlier literature has concentrated on compounds formed as strategies of defense by host plants, newer information has ventured into signal transduction and genetics.

6.3.1

Strategies of Defense by Host Plants

In the postharvest environment, an intriguing question has been why ripe fruits become more susceptible to disease unlike unripe fruits. The presence of preformed antimicrobial compounds (currently referred to as phytoanticipins) in unripe mature fruits which breakdown when the fruit ripens has been demonstrated (Sivanathan and Adikaram 1989; Cojocaru et al. 1986; Adikaram et al. 1993; Terry and Joyce 2004). Phytoalexins can be elicited to protect the plant by the use of nonpathogenic microorganisms as well as by other means. One of the earliest recorded studies in this regard has been on peanuts. Being a susceptible commodity to aflatoxins, peanut is reported to produce phytoalexins in response to invasion by storage fungi (Vidhyasekeran et al. 1972). Later, Wotton and Strange (1985) determined how phytoalexin synthesis by peanuts affected invasion by A. flavus. They found that conditions which promoted invasion of peanuts by A. flavus also inhibited phytoalexin production. In addition, Fujita and Yoshizawa (1989) have recorded that mycotoxins such as T2 toxin, deoxynivalenol, and ochratoxin A, which, have the ability to induce phytoalexins in sweet potato discs. In a time course experiment, these authors have shown the metabolization of mycotoxins into other unknown compounds. However, the safety of having phytoalexins in edible commodities has also been questioned (Frank 1987). ISR (Jetiyanon and Kloepper 2002; Kloepper et al. 2004; Manonmani et al. 2007) and systemic acquired resistance (SAR) (Van Loon 2007) have been mentioned as biocontrol mechanisms of PGPR. Specific defense mechanisms may be expressed as reinforcement of plant cell walls, production of antimicrobial phytoalexins, or synthesis of pathogenesis-related (PR) proteins (Van Loon 2007). Induced or acquired resistance of plants has been long documented by Chester (1933) as reported by Terry and Joyce (2004). It is interesting to note how resistance of plants has been reported by scientists working on rhizosphere microorganisms and on postharvest produce; it is perhaps indicative of the limited sharing of information within the two disciplines. While SAR and ISR are referred to as biologically

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induced defense responses, by Pieterse and Van Loon (2004) (who have published on rhizosphere bacteria), they are referred to as not only of biological origin, but also as chemical or physical origin, by Terry and Joyce (2004). In fact SAR has been defined by Conrath et al. (2002) as the systemic resistance response activated upon infection of plants with necrotizing pathogens. Van der Ent et al. (2008) noted that phenotypically ISR resembles SAR that develops upon primary infection with a necrotizing pathogen, unlike SAR, ISR is not marked by transcriptional activation of PR genes encoding PR proteins. Additionally, while Terry and Joyce (2004) refer to local acquired resistance (LAR), Pieterse and Van Loon (2004) refer to wound induced defense typically elicited upon tissue damage, i.e., feeding insects, probably meaning LAR. Later, Van Loon (2007) has discussed the role of nonpathogenic rhizobacteria in antagonizing pathogens through many mechanisms including activating inducible defense mechanisms. Various induced resistance phenomena are reported to be associated with an enhanced capacity for the rapid and effective activation of cellular defense responses, induced only after contact with a (challenging) pathogen, and the responses include the hypersensitive reactions, cell-wall strengthening, oxidative burst, and expression of defense-related genes (Conrath et al. 2002). By analogy with a phenotypically similar phenomenon in mammalian monocytes and macrophages, the augmented capacity to mobilize cellular defense responses has been called the “primed” or “sensitized” state of the plant (Conrath et al. 2002). In their review titled “Priming: getting ready for battle”, Conrath et al. (2006) noted that plants can be primed for more efficient activation of cellular defense responses. These authors have defined “primed state” as the physiological condition in which plants are able to better or more rapidly mount defense responses, or both, to biotic or abiotic stress. Interestingly Terry and Joyce (2004) have used the word hormesis (after Luckey 1990) to describe the involvement of stimulation of a beneficial plant response by low or sub-lethal doses of an elicitor/agent, such as a chemical inducer or a physical stress. Conrath et al. (2006) hypothesized that the primed state could be based on accumulation or posttranslational modification of one or more signaling proteins that, after being expressed or modified, still remained inactive, they pointed out that molecular mechanisms underlying priming are not understood. Terry and Joyce (2004) mentioned that considerably more applied and basic research is required to fully understand the role that induction of LAR, SAR, and/or ISR could play in achieving practical suppression of postharvest diseases, and they have acknowledged the need for enhanced fundamental and applied knowledge.

6.3.2

Signal Transduction and Genetic Involvement

Many root colonization genes and traits from Pseudomonas biocontrol species have been identified (Bloemberg and Lugtenberg 2001). Genetic manipulation of pseudomonads dates back to 1988 (Thomashow and Weller 1988). Simons et al. (1996) developed a monoaxenic system to demonstrate rhizosphere colonization.


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P. fluorescens can be labeled with fluorescent tags for in situ detection (Bloemberg et al. 2000). Using the above technology, Lugtenberg and Kamilova (2009) have reported sequence of events leading to biofilm formation by P. fluorescens WCS365 and induction of systemic signaling. Aiding in signaling, the plant hormones, such as salicylic acid, jasmonic acid, and ethylene that accumulate in response to pathogen infection or herbivore damage, are noted as major players in the regulation of signaling networks involved in induced defense responses against pathogens and insects (Pieterse and Van Loon 2004). Interestingly, both salicylic acid and jasmonic acid (additionally methyl jasmonate and acibenzolar, a functional analogue of jasmonic acid) as well as other plant hormones such as cytokinins and gibberellic acid have been tried out as chemical elicitors of natural disease resistance to postharvest pathogens in horticultural produce as reported by Terry and Joyce (2004). Ethylene could induce fruit ripening which is detrimental as it shortens shelf-life, and therefore, with respect to ethylene in postharvest technology, only methods of reducing levels attract attention unless it is specifically used to induce ripening of fruits. Fuqua et al. (1994) coined the term quorum sensing (QS) to describe communications occurring between bacterial communities occupying a single niche. Among the QS bacteria of PGPR, Pseudomonas spp., Erwinia spp., and Burkholderia cepacia have also been used successfully as postharvest antagonists. Interestingly, B. cepacia isolated from banana fruit skin has been used as biocontrol agents on controlling banana fruit pathogens (De Costa and Erabadupitiya 2004). It appears that understanding the molecular basis of biocontrol mechanism has much to offer to fill in the missing gaps. For instance, a better understanding of QS would perhaps be the key to harness benefits of biocontrol strategies displayed by different biocontrol agents. True to this point, Defago and Moenne-Loccoz (2006) have mentioned that discovery of alternative mechanisms by QS interference based on signal degradation shows that not all biocontrol mechanisms are documented yet. These authors further pointed out the need to understand the social life and biogeography of biocontrol pseudomonads along with other indigenous plantbeneficial bacteria to determine how they could confer disease suppressiveness to soils and expressed the need to understand the microevolution of biocontrol pseudomonads. QS has been shown to influence biofilm development for several species (Parsek and Greenberg 2005). QS of Gram-negative PGPR has been reported to produce N-acyl-homoserine lactones (Sharma et al. 2003). Thus, QS within and between bacterial populations is a major regulatory mechanism in bacteria to adjust their metabolism to crowded conditions or other changes in the biotic and abiotic environment (Whitehead et al. 2001; van Loon 2007). Parsek and Greenberg (2005) have also noted that there is growing appreciation within the biofilm field that individual cells of a variety of bacterial species are capable of actively leaving a biofilm and in crowded conditions QS would be an ideal way to mediate exodus from a biofilm. The influence on genes of the host plant by rhizobacteria has been reported (Verhagen et al. 2004). Conversely, plants could alter root exudation and secrete compounds that interfere with QS (van Loon 2007). It could be assumed that this two-way sensing need not be restricted to only the rhizosphere.

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Challenges in the Postharvest Environment

The postharvest environment is unique as the harvested organ is independent of the parent plant and should be able to survive independently in the absence of any nutritional input. While continuing life’s processes is essential to maintain freshness, the slower and steady this process is, the longer it can stay fresh. Janisiewicz (1988) has reported that fruits have a moist, nutrient-rich environment in which resistance to disease decreases as maturation progresses. Very broadly fruits would have a lower pH compared to vegetables, resulting in their pathogens to be of mainly fungal origin, and vegetables in general would have a pH near neutral being susceptible to both bacteria and fungi. Earlier research on biocontrol in plant tops (unlike control of root pathogens) has been less attractive because of more severe and fluctuating conditions which adversely affect many biocontrol agents resulting in inconsistent control (Janisiewicz 1991). However, unlike the rest of the plant, in harvested commodities disease should be easier to control than preharvest disease because conditions encountered in storage are less variable (Spurr et al. 1990) and could be maneuvered to suite the needs. For instance, postharvest diseases of fruits and vegetables can be suppressed by low temperature storage, low oxygen atmosphere and treatment with growth regulators that delay tissue senescence, but these beneficial practices may not adequately protect the crop from microbial attack especially during prolonged storage (Eckert and Ogawa 1985). Turning to the pathogen, some pathogens not only spoil the commodity but also make it dangerous for consumption by the presence of toxins, i.e., toxins from fungi (mycotoxins) and bacteria which can cause bacterial intoxications and infections. The deleterious effects of toxigenic organisms can be overcome by preventing its growth, if not by preventing toxin production or by metabolizing the preformed toxins into harmless compounds. Of these three options, the first is obviously the ideal although it is not possible all the time. Additionally, many postharvest diseases are known to be caused by a complex of pathogens. These pathogens have distinct roles to play and therefore their control has to be carefully planned by understanding their modes of action (Gunasinghe and Karunaratne 2009). Eckert (1990) has noted three types of infections that may lead to postharvest disease, as latent (quiescent) infections (i.e. anthracnose and stem-end rots) initiated on immature fruit in the field, infection of unripe fruit at an advanced stage of development in the field, where progressive development of the disease is suppressed for a while (i.e., Botrytis rot), and infection through wounds. Based on the above information, in planning disease control strategies, the above types of infections are taken into account. To compete successfully with pathogens at the wound site, the microbial antagonist should be better adapted to various environmental and nutritional conditions than the pathogen (Barkai-Golan 2001; Sharma et al. 2009). Thus, the focus on initial attempts on biological control of postharvest diseases has been on the control of wound pathogens on a selected number of fruits (Wilson and Wisniewski 1989; Eckert 1990).


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The inability to control previously established infections, quiescent infections and incipient infections occurring through wounds by potential biocontrol agents has been of concern. Quiescent infections have been the most difficult to control, of all pathogen infections even with the use of agrochemicals. Eckert and Ogawa (1985) have noted that the development of benzimidazole fungicides (i.e., dicloran, imazalil, prochloraz, etaconazole and guazatine) in the late 1960s was a breakthrough as they could penetrate into fruit tissues to prevent development of quiescent infections and other subsurface inocula without injuring host cells. If biocontrol agents are to replace benzimidazole fungicides, they should demonstrate the same capability. For this, knowledge of the epidemiology of the target disease is crucial for choosing the right time for the application of biocontrol agents (Ippolito and Nigro 2000). It has been stated that preharvest, harvest (transition) and postharvest activities impact epiphytic microflora and in postharvest disease control, proper management of this microflora determines success (Spurr et al. 1990). Therefore, getting a biocontrol agent established before the pathogen arrives (i.e., preharvest application of biocontrol agents for controlling postharvest pathogens) has been suggested as more effective (Smilanick 1994; Ippolito and Nigro 2000). Surprisingly, in spite of the skepticism, several investigations have shown that postharvest applications of biocontrol agents can accomplish at least partial control of quiescent infections (Chuang and Yang 1993; Janisiewicz and Korsten 2002; De Costa and Erabadupitiya 2004; Gunasinghe et al. 2004; Sharma et al. 2009). Overall, Janisiewicz (1991) has mentioned that most fruit and vegetable storage diseases are caused by fewer than 30 pathogen species, giving the idea that controlling them by biological means would not be a difficult target to achieve. Lack of consistent control of pathogens by antagonists has been reported in the past and the reasons for this have been identified as lack of survival of antagonists in the environment, effects of environmental and edaphic (soil related) factors on the live organisms and interactions with other microorganisms (Campbell 1994). However, being able to maneuver storage conditions in the postharvest environment gives the opportunity to switch the host pathogen antagonist equilibrium towards the antagonist (Spadaro and Gullino 2004), provided the strategies of pathogens and antagonists are better understood.

6.5

Ecology of the Microbial Environment

At a very early stage, Janisiewicz (1988) has contributed immensely to applications of biocontrol technology to the postharvest environment. It was correctly pointed out that the outcome of biocontrol by antagonists will be subject to biotic and abiotic factors and stresses. Recently, in the control of postharvest diseases of tropical fresh produce, Korsten (2006) has noted that natural antagonistic populations could be exploited to attain natural disease control with a broader understanding of population dynamics and ecological interactions. With this regard, in spite of the complexity of planning even short-term experiments, it appears that not having

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a holistic understanding of the microbial population dynamics could be a major limitation in harnessing maximum benefits obtainable from the approach of biological control. Looking back at the biocontrol methods employed on harvested produce, the “silver-bullet” approach cannot be considered farsighted as the aspect of microbial ecology involving associations between microfloras has been neglected. Turning to research on the rhizosphere, synergistic effects of arbuscular mycorrhizal fungi, PGPR and yeast on root colonization have been documented (Linderman and Paulitz 1990; Singh et al. 1991; Bhowmik and Singh 2004). In spite of the comparatively better awareness among researchers working on the rhizosphere microflora, Van Loon (2007) noted that the ecological diversity and its consequences for metabolic activity of the rhizosphere bacteria deserve further investigation. With regard to the postharvest environment too it appears that this aspect has not received due consideration. The approach of biocontrol by introduction of antagonists has been looked at by certain earlier workers with much skepticism, as nonconductive habitats will not sustain introduced microflora (Garrett 1956; Lewis and Papavizas 1991). However, studies of the multiple interactions among saprophytes, pathogens, nutrients and fungicides have been used for the development of a simulation model to guide the introduction of biocontrol agents (Spurr et al. 1990; Knudsen et al. 1988). Therefore, the trend thereafter was that environments must be altered to accept biocontrol agents or that formulations must be developed which allow the antagonist to survive, proliferate, become active (produce toxins or lytic enzymes), and establish themselves in an alien environment (Lewis and Papavizas 1991). This idea appears to be in par with conservation biological control (Eilenberg et al. 2001) and seems to fit in with traditional cultural practices used for pest control (Kean et al. 2003). In postharvest applications of fruits and vegetables, the ecology of the microflora on exudates of the intact fruit has not received much attention while root exudates have been reviewed extensively (Uren 2007). Although addition of nutrients preferably metabolized by the antagonists and not likely to be metabolized by the pathogen has been suggested (Janisiewicz 1998; Spadaro and Gullino 2004), determining the composition of fruit exudates with this regard has not received much attention. The fruit surface is considered as a very good food base for epiphytic microorganisms as it is rich in nutrients coming from plant leakage, outside deposits of pollen, organic debris and honeydew (Janisiewicz 1988). Later works reported on fruit exudate have been to determine exudates from plum and nectarine fruit to relate to Botrytis cinerea causing rot development (Fourie and Holz 1998) and to determine exudates from cucumber slices to design a method to measure chilling injury (Cabrera and Saltveit 1992). The need for this aspect to be investigated is further intensified by the fact that exudates found on the rhizosphere are composed of organic acids such as malic acid (Rudrappa et al. 2008) which are found in abundance in the pulp of many fruits. The rhizosphere microbes are known to benefit by metabolizing nutrients that plant roots secrete (Lugtenberg and Kamilova 2009). It is also noted that plants could alter root exudation and secrete compounds that interfere with QS regulation in the bacteria


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(Van Loon 2007). The ability of plants to react to root colonization by rhizobacteria by increasing the release of exudates and the ability to change quantity and composition of root exudates with the developmental stage of the plant have been noted (Van Loon 2007). The author is not aware of any comprehensive study on fruit or vegetable exudates, which may explain the microbial ecology on their surfaces. Such information would be valuable, having acknowledged the need of beneficial bacteria to colonize the root surface to exert their effect (Lugtenberg and Kamilova 2009). In one of the few such studies done on fruits, it is mentioned that sugars in fruit exudates of plum and nectarine may contribute to susceptibility of fruits to B. cinerea infection and noted that their concentrations increased as the fruit ripened (Fourie and Holz 1998). An interesting account involving P. fluorescens in a postharvest environment is reported by Campbell (1989), which again emphasizes the need to have a holistic approach. It is on a failed attempt by Swinburne (1986) to control C. musae, which causes latent infections on the banana skin. It is said that its germination is stimulated by leachates from the fruit, especially anthranilic acid, which may act as a siderophore, sequestering iron. Germination and appressorium formation are also reported to be stimulated by the siderophores produced by P. fluorescens and other bacteria on the fruit surface. The exact reverse of the mode of action proposed for some biocontrol agents whose siderophores reduce infection by starving the pathogen of iron is reported on the banana skin as free iron, and various chelates in which iron is available to the plant, inhibit germination, possibly by stimulating phytoalexin production. Here, instead of the pathogen being starved of iron, the removal of iron by other microorganisms stimulates pathogen germination. This shows the significance of ecology of the surface microorganisms in question, as well as acknowledging the different modes of interactions of pathogens and antagonists. Wisniewski and Wilson (1992) have remarked on the difficulties involved with biological control agents tested in the laboratory to the field largely because of problems of ineffectiveness when exposed to the “uncontrolled” environment of the latter. Campbell (1994) too has cautioned against selection of organisms by known modes of action, as selection based on tests in vivo has not shown such inhibition when tested in vitro. Managing naturally occurring population of microorganism of the surfaces of fruits and vegetables to enhance resistance of harvested commodities to disease has been postulated by Wilson and Wisniewski (1989). Cook and Baker (1983) suggested that antagonists should be sought from where disease does not occur in spite of considerable inoculum pressure. Since the fruit surface supports the growth of a variety of interacting microorganisms, it has also been postulated that a form of biological control occurs in fruits in nature and that some of these may be potential biocontrol agents for fruit pathogens (Janisiewicz 1991). In retrospect, the inability to transfer technology from the laboratory to the field is likely due to the “silver-bullet” approach adopted by postharvest scientists. In fact, Halverson et al. (1993) working on rhizosphere microorganisms have shown the need to perform multiple year field experiments for successful results. In spite of the fact that the postharvest environment could be considered to be less variable and well defined, compared to the field conditions of a plant which depend on climatic

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and weather conditions, the host characteristics may be influenced by field conditions, thus having an indirect effect on the influence directed at the microbes on the surface. Considering the interactions between the rhizobacteria and the host plants, it is possible that the fruit physiology and hence the organoleptic qualities of the fruit too is influenced by the composition of the surface microflora. It is reported that microbial populations in the natural environment are much more diverse than microorganisms so far isolated in the laboratory (Watanabe and Baker 2000). The complexity of microbial interactions that occur and the importance of investigating the microbial ecology on the surface in question are apparent for successful biocontrol. To conclude this section, it appears that two decades ago some of the questions raised by Wilson and Wisniewski (1989) appear to be very pertinent currently. They are as follows: “What are the effects of antagonists on wound healing and host resistance? How important and widespread in nature are the direct effects of antagonists on pathogens? How do incidental microorganisms or mixtures of antagonists affect the pathogen/antagonist interaction? And how does the nutrient/chemical composition at the wound site affect antagonists, other microflora and the infection process and the fruit wound response?” The authors have correctly stated that a greater understanding of the microecology of fruit and vegetable surfaces would shed light to this area of research. It is timely therefore to survey the strategies of PGPR in controlling postharvest pathogens, to draw parallels between the control strategies, so that we could obtain a better understanding of the mechanisms involved in postharvest biocontrol strategies. One major attribute seen when talking of PGPR and biocontrol agents is that the emphasis on the latter is on its direct effect on the pathogen. PGPR, on the other hand, may influence indirect growth promotion of crops. In a recent review (Droby et al. 2009), the need for a more thorough understanding of the microbial ecology of fruit surface has been emphasized.

6.6

Nature and Role of Bacterial Antagonists

Wilson and Wisniewski (1994) have mentioned that an ideal antagonist should be genetically stable, should be efficient at low concentrations and against a wide range of pathogens on various fruit products, should have simple nutritional requirements and survive in adverse environmental conditions. Also, when used on edible plant matter, being 100% safe in terms of not being toxic or allergenic and not producing toxic metabolites are important considerations. The necessity to manage the epiphytic microflora to control postharvest diseases has been recognized (Spurr et al. 1990) and this management has to be at different phases of progression of disease. Therefore, it appears that an ideal antagonist should have multiple mechanisms of action such as niche and nutrient competition, enzymes to attack pathogen cells, induction of resistance, antimicrobial production and additionally help the host tissue to maintain metabolic processes. Additionally at a time


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when agrochemicals are still in use for postharvest applications, in spite of fungicide resistance of pathogens has been of concern (Campbell 1989) the resistance of biocontrol agents to fungicides may be useful in integrated disease control programs (Ippolito and Nigro 2000). It is apparent from the remarks made by different authors that the attention on Gram-positive bacteria on the rhizosphere has been studied much later (Kloepper and Schroth 1981; Gilbert et al. 1993; Halverson et al. 1993). At an early stage, the emphasis was on Gram-negative rhizobacteria (which are the more dominant population in the rhizosphere), mainly the pseudomonads. The importance of Gram-positive bacteria, mainly of Bacillus spp. is discussed much later, with the note that the rich diversity of the microbial world provides a seemingly endless resource for biocontrol (Emmert and Handelsman 1999). However, the extensive use of B. thuringiensis (a Gram-positive bacterium) that has been patented for use on control of pests of several agricultural crops, trees, and ornamental plants (Falcon 1971) is unparalleled to any other bacterial biocontrol agent. Gram-positive bacteria are known to utilize signaling molecules such as short peptide pheromones to inhibit pathogens (Sharma et al. 2003). Parsek and Greenberg (2005) mentioned two most thoroughly described QS systems of which are the acyl-homoserine lactone systems of many Gram-negative species and peptide-based signaling systems of many Gram-positive species. Also mentioned are AI-2 systems common to several Gram-positive and Gram-negative species. Among the PGPR, P. fluorescence has received much attention (Schippers 1988; Van Loon et al. 1997; Lugtenberg and Kamilova 2009), and this species has been mentioned exclusively in determining survival in soil and rhizosphere (Mazzola et al. 1992, 2001; Mazzola 2007). Interestingly, the word “pseudomonads” (referring to Pseudomonas fluorescence-putida group) has even been used synonymously with PGPR (Kloepper et al. 1980). The awareness of suppression of plant diseases by fluorescent Pseudomonas spp. has a long history dating back from 1976 and has made a steady progress over the years (Cook and Rovira 1976; Weller and Cook 1983; Baker et al. 1986; Parke et al. 1991). Antibiotics produced by Pseudomonads are known to be effective against not only bacteria but also fungi (Howell and Stipanovic 1979). Additionally, Davison (1988) has reported that many rhizosphere bacteria (such as P. fluorescens and P. putida) have been isolated and screened for inhibition of phytopathogenic fungal or bacterial growth. Much information on biocontrol strategies of microbial antagonists has been unraveled by using pseudomonads. On the postharvest environment, the studies on antagonistic activity of Pseudomonas spp. by their production of antibiotics are reviewed by Spadaro and Gullino (2004). P. cepacia has been analyzed on postharvest rot of apples (Janisiewicz and Roitman 1988) and on pome fruit (Janisiewicz et al. 1991). It is reported that poorly soluble inorganic nutrients that are rate limiting for growth could be made available through the solubilizing action of bacterial siderophores or the secretion of organic acids (Vessey 2003; Van Loon 2007). The role of siderophores which efficiently complex environmental iron, making it less available to certain native microflora has been documented (Kloepper et al. 1980).

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Postharvest fruit pathogens are described mainly as necrotrophs and depend on exogenous nutrients for germination and initiation of the pathogenic process (Janisiewicz 1991). On the fruit surface, application of fungicides is prophylactic and therefore application of biocontrol agents to a fruit surface should serve the same purpose. No mention has been made of the ability of pathogen antagonists to have a direct effect on growth of the host plant in postharvest applications. This is in spite of several reports that have appeared on the effect of phytohormones produced by antagonists in the rhizosphere. For instance, reducing ethylene production during the postharvest shelf life of a fruit is a major requirement for increased shelf-life. Ethylene is considered a menace as it hastens fruit ripening and senescence of the host tissue, and promotes growth of pathogens. 1-aminocyclopropane-1-carboxylate (ACC), the immediate precursor of ethylene can be metabolized to prevent formation of ethylene. Breakdown of ethylene by bacterial ACC deaminase has been reported (Glick et al. 1998; Lucy et al. 2004). The bacterial enzyme ACC deaminase is known to be the only nonplant enzyme that metabolizes ACC (Grichko et al. 2000). It is noted that transforming the ACC deaminase gene, which directly stimulates plant growth by cleaving the immediate precursor of plant ethylene into P. fluoresens CHAO, not only increases plant growth but can also increase biocontrol properties of PGPB (Glick et al. 1998; Wang et al. 2000; Compant et al. 2005). The use of antagonists capable of metabolizing ACC in the postharvest environment would be a very valuable tool in postharvest applications. Apart from lowering ethylene levels, production of plant hormones like auxins, cytokinins and gibberellins (Glick et al. 1998; Lucy et al. 2004) known as juvenile phytohormones may be helpful in the postharvest environment. Incidence of enhanced pathogen control when microbial antagonists are applied in the presence of certain additives, including phytohormones has been demonstrated by several investigations on postharvest applications (Sharma et al. 2009). Other stimulants of plant growth include certain volatiles and the cofactor pyrrolquinoline quinine in several enzymes involved in antifungal activity and induction of systemic resistance (Lugtenberg and Kamilova 2009). Among the other attributes listed for PGPR are increases in chlorophyll content, magnesium content, nitrogen content, protein content, hydraulic activity, tolerance to drought and delayed leaf senescence (Lucy et al. 2004). Such attributes would also have a direct positive effect in the postharvest environment of specific fruits and vegetables. With regard to mycotoxin detoxification, one of the earliest records known is the work of Ciegler et al. (1966) who screened about 1,000 microorganisms which included yeasts, fungi, actinomycetes, bacteria, and algae. These authors reported that Flavobacterium aurantiacum was capable of removing aflatoxins from test substrates. Later various scientists have determined the capability of this bacterium to detoxify aflatoxin contaminated food substrates with positive results (Lillehoj et al. 1971; Hao and Brackett 1988). Much later work concentrated on other nontoxigenic microorganisms capable of detoxifying preformed mycotoxin (Cotty 1989). It has been correctly reported by Gilbert et al. (1993) that an improved understanding of the influence of the introduced organism on microbial


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communities is required for biocontrol or growth promotion of plants to be accomplished through the introduction of microorganisms.

6.7

Application and Commercialization of Microbial Antagonists

While postharvest applications could be accomplished by drenching or dipping in an antagonist suspension, pressure infiltration of fruit with the suspension or mixing the antagonists with waxes on sorting lines is also suggested (Janisiewicz 1991). Vacuum infiltration and high pressure processing are also more complex methods which have given positive results in the laboratory, but they need to be further developed to suit field applications. Preparations of biocontrol formulations such as dusts, wettable powders, granules, pellets, prills, pastes, tablets, emulsifiable liquids, fluid-drill gels have been discussed (Vidhyasekaran 2004; Lewis and Papavizas 1991). Several microbial antagonists have been patented and evaluated for commercial use of which ASPIRE, YieldPlus and BIOSAVE-110 are used worldwide for controlling postharvest diseases of fruits and vegetables and the continuous increase in the use of BIOSAVE without failure since 1996 indicates that current biological control practices can be cost effective in large packing houses (Sharma et al. 2009). Some commercial products using free-living PGPR are listed elsewhere (Lucy et al. 2004). By 2005 more than 33 products of PGPR have been registered for commercial use in greenhouse and field in North America (Nakkeeran et al. 2006). There have been suggestions to formulate mixtures containing biocontrol agent(s) with chemical fungicide(s) to broaden the spectrum of activity (Eckert 1990). Such investigations have provided substantial evidence of success (Chand-Goyal and Spotts 1997; Sharma et al. 2009). Also suspending the conidia of antagonists in nutrients, such as malt and yeast extractions, and use of various inorganic salts as the suspension medium have given desired results (Ippolito and Nigro 2000).

6.8

Current Challenges in the Field of Biocontrol

At an early stage of investigation although there has been concerns regarding public reaction in the application of “living fungicides” to food (Wisniewski and Wilson 1992), the number of commercial products that are available now bears testimony to the fact that it has been accepted by the public. However, there are several issues to be addressed currently, if we are to make progress. It appears that many researchers are ready to move out for field trials, but the necessary wherewithal for such an attempt is not there. Except for a few countries who have commercialized products, scientists in other countries have restricted their know-how only to

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the laboratory and publications restricted to the scientific community. Droby et al. (2009) have noted that large-scale production of formulated biocontrol agents requires costly trials and no serious attempts have been made to address the large-scale production and formulation technology of biocontrol agents. It appears that the problem is deeper and wider than that. Inability to move forward after laboratory investigations can be cited as a major drawback for scientists who have found positive results in the laboratories of many countries. The absence of a central body giving guidelines on specific tests to be conducted for safety, in terms of health, and the absence of a ready repository of biocontrol agents and their information, may be cited as snags in the process. Droby et al. (2009) have noted that even with the large number of researchers all over the world working on biocontrol applications, currently the use of chemical agents remains the major method of choice for managing postharvest rots and the few postharvest biocontrol products commercially available have limited use, mostly in niche markets. It appears a battery of standard protocols to be adopted to facilitate the link research findings and field applications. Issues on concerns of safety and environmental impact should be addressed following a parallel protocol for commercializing novel agrochemicals. Linked to this is the concern posed by Van der Putten et al. (2007) on moving plants, animals, or microbes around the globe as invasive microbes (plants and animals) could be a major threat to the composition and functioning of ecosystems and can have a major impact on the abundance of individual species, affecting the diversity of native communities. Additionally, agrochemical companies do not seem to have felt the pressing need to replace their current products with biocontrol agents, in spite of the effort of the researchers. The lack of knowledge of the current issues to be addressed among law makers to facilitate such a process also could be cited as another dimension to the failure. With the concept of a global village, suitable means of sharing such information of isolated scientists, on the pretests required and registration protocol for new biocontrol agents, at a central body (perhaps in par with the Codex Alimentarius Commission of WHO/FAO), would benefit the entire world to address global food insecurity at large.

6.9

Concluding Remarks

In this chapter, of the several benefits for PGPR, biological control of plant pathogens, one of the main benefits, was reviewed by comparing biological control strategies employed in postharvest technology of fruits. Biological control of postharvest pathogens has become a popular research topic over the years, with a high demand for immediate applications, and several biocontrol formulations are now allowed in the postharvest environment. In spite of this, many of the positive findings in research on the postharvest environment cannot be explained. Besides, many of the postulations have given opposite results. Revisiting ideas mooted by two postharvest scientists Wilson and Wisniewski (1989), who have been


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publishing on the subject for the last 20 years, it is anticipated that our knowledge will increase rapidly by drawing and building upon the foundation of information developed in other areas of biocontrol and phylloplane research. According to Wilson and Wisniewski (1989) understanding the mode of action of antagonists is important for two reasons, as it will allow the development of more reliable procedures for effective application of known antagonists and it should provide a rationale for selecting more effective antagonists. It is interesting to note that the current strategies to combat pathogens by using microbial antagonists have revolved round the broader definition for biocontrol given by Campbell (1989) with the last option gaining ground with the refining of molecular biological tools. Work on PGPR has a longer and a more comprehensive development, and has ventured into molecular biological applications as seen in several reviews (Bloemberg and Lugtenberg 2001; Conrath et al. 2002; Duffy et al. 2003; Pieterse and Van Loon 2004; Haas and Defago 2005; Compant et al. 2005; Van Loon 2007; Sorensen et al. 2009). It appears that some of the fundamental research ideas put forward by scientists working on PGPR could be applied by scientists working on postharvest applications of biocontrol strategies. On the other hand, work on postharvest biocontrol, which has concentrated more on applications having many unexplained positive results, may have answers from the work on PGPR. Additionally, such applications also may give research ideas for scientists working on PGPR. Marques et al. (2005) have indicated that novel pesticides and disinfectants, and/ or decontamination procedures should be designed which could attack biofilms as current pesticides and protocols are based on killing of single-celled organisms. Applying this idea into antagonists of postharvest pathogens, it appears that a novel way of thinking is needed. It is known that conducive soil can establish disease suppression by transferring an inoculum of 0.1–10% of suppressive soil (Haas and Defago 2005). This shows the potential of the elusive ideal antagonist to be encountered by future scientists. One area that has not been addressed by researchers on postharvest application of biocontrol is the capability of the fruit surface microflora to influence gene expression of the host. Whether it happens and how it happens remains elusive. This may be an interesting area to explore, considering the numerous gene related events that could occur in relation to fruit maturation and ripening. Van Loon (2007) has cited several examples to indicate that root-colonizing Pseudomonas spp. may activate signaling pathways. The reader is referred to comprehensive reviews which discusses current applications and future opportunities for improving pseudomonad-based biological control (McSpadden Gardener 2007) and on the genetics of disease suppression by fluorescent pseudomonads (Haas and Defago 2005). The use of genetic engineering for enhancement of biological control efficacy has been suggested (Spadaro and Gullino 2004). If future scientists could consider genetically engineering an ideal antagonist with multiple mechanisms of actions with characteristics of being environmentally safe and being nontoxic, and also perhaps having some prebiotic characteristics, that would be a breakthrough. Meanwhile it is obvious that there is much to give and take from the two disciplines.

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.

Chapter 7

Plant Growth-Promoting Bacteria Associated with Sugarcane Samina Mehnaz

7.1

Introduction

Sugarcane is an important industrial and cash crop in many countries of the world. It is grown in over 110 countries, in tropical and sub-tropical regions, in a range of climates from hot dry environment near sea level to cool and moist environment at higher elevations. Besides sugar production, sugarcane produces numerous valuable by-products like ethanol, bagasse, press mud, molasses, and essential items for industries like chemicals, plastics, paints, synthetics, fiber, insecticides, and detergents (http://www.pakissan.com). This crop is perhaps the most economically competitive source of ethanol and can effectively contribute to a cleaner environment. Ways of improving its productivity are subject to investigation in several countries. Worldwide climate change due to the intense use of greenhouse gasproducing energy sources has resulted in the development of sustainable energy. Consequently, sustaining and enhancing the growth and yield of sugarcane have become a major focus of research. Sugarcane and other grasses such as rice, wheat, maize, and sorghum, currently have much of their nitrogen (N) needs supplied by costly mineral fertilizers. It has been a general practice to apply 250 kg N ha 1 year 1, or more in most of the sugarcane cultivating countries. In 2008, an estimated 1,743 million metric tons of sugarcane were produced worldwide, with about 50% of production occurring in Brazil and India. In India, sugarcane is grown over 4.2 million ha, producing about 250 million tons of canes annually and the nitrogen requirement of Indian sugarcane ranges from about 250 to 350 kg ha 1. Brazil is the largest sugarcane producer in the world, with the crop occupying more than five million hectares with a yield of 495 M tons in 2007/2008 (UNICA 2009), 16 million m3 of alcohol in 2006 (Mendes

S. Mehnaz Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan and Institute of Pharmaceutical Biology, Bonn University, Bonn 53115, Germany e-mail: [email protected]


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et al. 2007; Oliveira et al. 2006) and annual application of nitrogen fertilizer for sugarcane is around 50 kg N ha 1, with a cost near US$ 500 t 1 (http://www.udop. com.br). Researchers in Brazil are intensively working on further reducing the use of N-fertilizer application by one half (

Bacteria in Agrobiology: Crop Ecosystems

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Bacteria in Agrobiology: Plant Nutrient Management

Bacteria in Agrobiology: Plant Growth Responses

Bacteria

Variation in Bacteria

Bacteria classification

Bacteria phylogeny

Bacteria Genetics

Lacewings in the Crop Environment

Digital Ecosystems

Bee Pollination in Agricultural Ecosystems

Chemical Biomarkers in Aquatic Ecosystems

Biological Invasions in Marine Ecosystems

Carbon Sequestration in Urban Ecosystems

Bee Pollination in Agricultural Ecosystems

Carbon Sequestration in Forest Ecosystems

Maintaining Biodiversity in Forest Ecosystems

Bacteria and Cancer

Plant-Associated Bacteria

Bacteria and Cancer

Bacteria and Viruses

Magnetoreception and Magnetosomes in Bacteria (Microbiology Monographs)

Molecular genetics of bacteria

Wastewater Bacteria (Wastewater Microbiology)

Lactic Acid Bacteria

The Fecal Bacteria

Molecular Genetics of Bacteria

Bacteria and Viruses

Bacteria in Agrobiology: Crop Ecosystems

Bacteria in Agrobiology: Stress Management (Bacteria in Agrobiology)

Bacteria in Agrobiology: Plant Nutrient Management

Bacteria in Agrobiology: Plant Growth Responses

Bacteria

Variation in Bacteria

Bacteria classification

Bacteria phylogeny

Bacteria Genetics

Lacewings in the Crop Environment

Digital Ecosystems

Bee Pollination in Agricultural Ecosystems

Chemical Biomarkers in Aquatic Ecosystems

Biological Invasions in Marine Ecosystems

Carbon Sequestration in Urban Ecosystems

Bee Pollination in Agricultural Ecosystems

Carbon Sequestration in Forest Ecosystems

Maintaining Biodiversity in Forest Ecosystems

Bacteria and Cancer

Plant-Associated Bacteria

Bacteria and Cancer

Bacteria and Viruses

Magnetoreception and Magnetosomes in Bacteria (Microbiology Monographs)

Molecular genetics of bacteria

Wastewater Bacteria (Wastewater Microbiology)

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