Viroids

This Page Intentionally Left Blank Editors Ahmed Hadidi Agricultural Research Service U.S. Department of Agriculture ...

Author: A. Hadidi | Ricardo Flores | John W. Randles | Joseph S. Semancik

98 downloads 564 Views 8MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

This Page Intentionally Left Blank

Editors Ahmed Hadidi Agricultural Research Service U.S. Department of Agriculture Beltsville, Maryland United States of America

Ricardo Flores Instituto de Biología Molecular y Celular de Plantas Universidad Politécnica de Valencia Consejo Superior de Investigaciones Científicas Valencia Spain

John W. Randles Department of Applied and Molecular Ecology University of Adelaide Glen Osmond Australia

Joseph S. Semancik Department of Plant Pathology University of California Riverside, California United States of America

III

© 2003 CSIRO All rights reserved. Except under the conditions described in the Australian Copyright Act 1968 and subsequent amendments, no part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, duplicating or otherwise, without the prior permission of the copyright owner. Contact CSIRO PUBLISHING for all permission requests. National Library of Australia Cataloguing-in-Publication entry Viroids. Bibliography. Includes index. ISBN 0 643 06789 2 (hardback). ISBN 0 643 06985 2 (eBook). 1. Viroids. I. Hadidi, Ahmed. 579.29 Available from CSIRO Publishing 150 Oxford Street (PO Box 1139) Collingwood VIC 3066 Australia Telephone: Freecall: Fax: Email: Web site:

+61 3 9662 7666 1800 645 051 (Australia only) +61 3 9662 7555 [email protected] www.publish.csiro.au

This work has been originally published by CSIRO Publishing, Australia. Available with exclusive marketing rights for North and South America from Science Publishers, Inc. 234 May Street, Enfield New Hampshire 03748 United States of America Telephone: 603 632 7377 Fax: 603 632 5611 Email: [email protected] Website: www.scipub.net Library of Congress CIP Data will be provided on request ISBN 1-57808-272-2

Set in Adobe Garamond and Adobe Gill Sans Cover design by James Kelly Typeset by Desktop Concepts P/L, Melbourne Printed by Ligare

CONTENTS

Contributors Preface

ix xiii

A. Hadidi

Part 1

Introduction

1 Economic impact of viroid diseases

3

J.W. Randles

Part II

Properties of viroids

2 Molecular characteristics

15

G. Steger and D. Riesner

3 Biology

30

R.P. Singh, K.F.M. Ready, and X. Nie

4 Movement

49

B. Ding and R.A. Owens

5 Replication

55

R. Flores, J.A. Daròs, and J.A. Navarro

6 Pathogenesis

61

J.S. Semancik

7 Viroids and gene silencing

67

V. Conejero

8 Classification

71

R. Flores, J.W. Randles, and R.A. Owens

9 Viroid-like satellite RNAs

76

L. Rubino, F. Di Serio, and G.P. Martelli

Part III

Detection of viroids

10 Host considerations

87

J.S. Semancik

11 Biological indexing

89

R.P. Singh and K.F.M. Ready

V

CONTENTS

12 Polyacrylamide gel electrophoresis

95

D. Hanold, J.S. Semancik, and R.A. Owens

13 Molecular hybridization

103

H.-P. Mühlbach, U. Weber, G. Gómez, V. Pallás, N. Duran-Vila, and A. Hadidi

14 Polymerase chain reaction

115

A. Hadidi and T. Candresse

Part IV

Diseases and viroids associated with plant species

Viroids of solanaceous species 15 Viroids of solanaceous species

125


Cucumber viroids 16 Hop stunt viroid in cucumber

134

T. Sano

Pome fruit viroids 17 Apple scar skin viroid in apple

137

H. Koganezawa, X. Yang, S.F. Zhu, J. Hashimoto, and A. Hadidi

18 Apple scar skin viroid in pear

142

P. E. Kyriakopoulou, H. Osaki, S.F. Zhu, and A. Hadidi

19 Apple dimple fruit viroid

146

F. Di Serio, M. Malfitano, D. Alioto, A. Ragozzino, and R. Flores

20 Apple fruit crinkle viroid

150

H. Koganezawa and T. Ito

21 Pear blister canker viroid

153

R. Flores, S. Ambrós, G. Llácer, and C. Hernández

Stone fruit viroids 22 Peach latent mosaic viroid in peach

156

R. Flores, C. Hernández, G. Llácer, A.M. Shamloul, L. Giunchedi, and A. Hadidi

23 Peach latent mosaic viroid in temperate fruit hosts

161

A. Hadidi, L. Giunchedi, H. Osaki, A.M. Shamloul, A. Crescenzi, P. Gentit, L. Nemchinov, P. Piazzolla, and P.E. Kyriakopoulou

24 Hop stunt viroid in plum and peach

165

T. Sano

25 Hop stunt viroid in apricot and almond

168

V. Pallás, G. Gómez, K. Amari, M.C. Cañizares, and T. Candresse

Avocado viroids 26 Avocado sunblotch viroid

171

J.S. Semancik

Citrus viroids 27 Citrus viroids N. Duran-Vila and J.S. Semancik VI

178

CONTENTS

Grapevine viroids 28 Grapevine viroids

195

A. Little and M.A. Rezaian

Hop viroids 29 Hop stunt viroid

207

T. Sano

30 Hop latent viroid

213

D.J. Barbara and A.N. Adams

Viroids of ornamentals 31 Chrysanthemum stunt viroid

218

I. Bouwen and A. van Zaayen

32 Chrysanthemum chlorotic mottle viroid

224

R. Flores, M. De la Peña, and B. Navarro

33 Coleus blumei viroid

228

R.P. Singh, M.E.N.F. Boiteux, K.F.M. Ready, and X. Nie

34 Columnea latent viroid

231

R.W. Hammond

Palm tree viroids 35 Coconut cadang-cadang viroid

233

J.W. Randles and M.J.B. Rodriguez

36 Coconut tinangaja viroid

242

G.C. Wall and J.W. Randles

Natural history of viroids 37 Natural history of viroids — horticultural aspects

246

M. Bar-Joseph

Part V

Mapping of geographical distribution and epidemiology of viroids

38 Viroids in North America and global distribution of viroid diseases

255

R.P. Singh, K.F.M. Ready, and A. Hadidi

39 Viroids in South America

265

I. Bartolini and L.F. Salazar

40 Viroids in Europe

268

V. Pallás, G. Gómez, and N. Duran-Vila

41 Viroids in the Middle East

275

A. Hadidi, H.M. Mazyad, M.A. Madkour, and M. Bar-Joseph

42 Viroids in Australasia

279

J.W. Randles, M.A. Rezaian, D. Hanold, R.M. Harding, L. Skrzeczkowski, and M. Whattam

43 Viroids in China

283

L. Han, G. Wang, X. Yang, S.F. Zhu, and A. Hadidi

44 Viroids in Japan

286

T. Sano VII

CONTENTS

45 Viroids in Africa

290

J.V. da Graça and S.P. van Vuuren

Part VI

Control measures

46 Strategies for the control of viroid diseases

295

R.P. Singh, J.W. Randles, and A. Hadidi

47 Quarantine of imported germplasm

303

M. Barba, D.J. Gumpf, and A. Hadidi

48 Availability of viroid-tested propagation materials

312

D.J. Gumpf and L. Navarro

49 Viroid elimination by thermotherapy and tissue culture

318

M. Barba, E. Ragozzino, and L. Navarro

Part VII

Diseases of possible viroid etiology

50 Pear fruit crinkle

327

A.M. Shamloul, X. Yang, L. Han, and A. Hadidi

51 Citrus gummy bark

330

N. Önelge and J.S. Semancik

52 Eggplant latent

333

C. Fagoaga and N. Duran-Vila

53 Nicotiana glutinosa stunt

334


54 CCCVd-related molecules in oil palms, coconut palms and other monocotyledons outside the Philippines

336

D. Hanold and J.W. Randles

Part VIII

Considerations for future applications of viroids

55 Biotechnological approaches for controlling viroid diseases

343

T. Sano, R.W. Hammond, and R.A. Owens

56 Ribozyme reactions of viroids

350

F. Côté, M. De la Peña, R. Flores, and J.P. Perreault

57 Considerations for the introduction of viroids for economic advantage

357

J.S. Semancik

Index

VIII

363

CONTRIBUTORS

Adams, A.N., Horticulture Research International, East Malling, West Malling, Kent, ME19 6BJ, UK. Alioto, D., Dipartimento Arboricoltura, Botanica e Patologia Vegetale, Università di Napoli, 80055 Portici, Italy. Amari, K., CEBAS-CSIC, Campus Universitiario de Espinardo, 30100 Murcia, Spain. Ambrós, S., Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia; and Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada 46113, Valencia, Spain. Barba, M., Istituto Sperimentale per la Patologia Vegetale, Via C. G. Bertero, 22, 00156 Rome, Italy.

Crescenzi, A., Università degli Studi della Basilicata, Dipartimento di Biologia, Difesa e Biotecnologie AgroForestali, Macchia Romana Campus, 85100 Potenza, Italy. da Graça, J.V., Texas A & M University-Kingsville Citrus Center, Weslaco, TX 78596, USA. Daròs, J.A., Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia, Spain. De la Peña, M., Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia, Spain.

Barbara, D.J., Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK.

Ding, B., Department of Plant Biology and Plant Biotechnology Center, Ohio State University, Columbus, OH 43210, USA.

Bar-Joseph, M., The S. Tolkowsky Laboratory, Department of Virology, Agriculture Research Organization, The Volcani Center, Bet Dagan 50250, Israel.

Di Serio, F., Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Sezione di Bari, 70126 Bari, Italy.

Bartolini, I., The International Potato Center (CIP). P.O. Box 1558, Lima, Peru.

Duran-Vila, N., Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Invstigaciones Agrarias (IVIA), 46113 Moncada, Valencia, Spain.

Boiteux, M.E.N.F., CENARGEN/EMBRAPA, C.P. 102372, 70849 Brasilia, Brazil. Bouwen, I., Plant Research International B.V., Business unit Bio-interactions and Plant Health, Droevendaalsesteeg, P.O. Box 16, 6700 AA Wageningen, The Netherlands. Candresse, T., Equipe de Virologie, UMR GD2P, IBVM, INRA, BP 81, 33883 Villenave d’Ornon Cedex, France. Cañizares, M.C., CEBAS-CSIC, Campus Universitiario de Espinardo, 30100 Murcia, Spain. Conejero, V., Instituto de Biología Molecular y Cellular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022, Valencia, Spain. Côté, F., Département de biochimie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.

Fagoaga, C., Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada, Valencia, Spain. Flores, R., Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia, Spain. Gentit, P., Ctifl, Lanxade BP21, 24130 Prigonrieux, France. Giunchedi, L., Istituto di Patologia Vegetale, Università di Bologna, 40126 Bologna, Italy. Gómez, G., Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia, Spain. Gumpf, D.J., Department of Plant Pathology, University of California, Riverside, CA 92521, USA.

IX

CONTRIBUTORS

Hadidi, A., Lead Scientist and Research Plant Pathologist Emeritus, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705, USA. Hammond, R.W., Molecular Plant Pathology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, BARC-West, Beltsville, MD 20705, USA. Han, L., Plant Molecular Virus Laboratory, Plant Quarantine Section, CIQ-Beijing Entry & Exit Inspection and Quarantine Bureau, People’s Republic of China, Beijing 100029, P.R. China.

Mazyad, H.M., Plant Pathology Research Institute, Agricultural Research Center, Giza 12619, Egypt. Mühlbach, H.-P., Department of Genetics, Institut für Allgemeine Botanik und Botanischer Garten, University of Hamburg, 22609 Hamburg, Germany. Navarro, B., Dipartamento di Protezione delle Piante dalle Malattie, Università degli Studi di Bari, 70126, Bari, Italy. Navarro, J.A., Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022, Valencia, Spain.

Hanold, D., Department of Applied & Molecular Ecology, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia.

Navarro, L., Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada, Valencia, Spain.

Harding, R.M., Centre for Molecular Biotechnology, Queensland University of Technology, Brisbane, Queensland 4001, Australia.

Nemchinov, L., Molecular Plant Pathology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, BARC-West, Beltsville, MD 20705, USA.

Hashimoto, J., Department of Genetic Resources II, National Institutes of Agrobiological Resources, 2-1-2- Kannondai, Tsukuba, Ibaraki 305-0856, Japan.

Nie, X., Agriculture and Agri-Food Canada, Potato Research Centre, P.O. Box 20280, Fredericton, N.B., E3B 4Z7, Canada.

Hernández, C., Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia, Spain. Ito, T., Department of Citrus Research, National Institute of Fruit Tree Science, NARO, Kuchinotsu, Nagasaki 859-2501, Japan. Koganezawa, H., National Agricultural Research Center for Western Region, NARO, Nishifukatsu, Fukuyama 721-8514, Japan. Kyriakopoulou, P.E., Department of Plant Pathology, Agricultural University of Athens, Votanikos 11855, Athens, Greece.

Önelge, N., Subtropical Fruits Research and Experimental Centre, University of Cukurova, 01130 Adana, Turkey. Osaki, H., Laboratory of Plant Pathology, Department of Plant Protection, National Institute of Fruit Tree Science, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 3058605, Japan. Owens, R.A., Molecular Plant Pathology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, BARC-West, Beltsville, MD 20705, USA. Pallás, V., Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia, Spain.

Little, A., CSIRO Plant Industry, Adelaide Laboratory, PO Box 350, Glen Osmond, SA 5064, Australia.

Perreault, J.-P., Département de biochimie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.

Llácer, G., Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada 46113, Valencia, Spain.

Piazzolla, P., Università degli Studi della Basilicata, Dipartimento di Biologia, Difesa e Biotecnologie AgroForestali, Macchia Romana Campus, 85100 Potenza, Italy.

Madkour, M.A., Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza 12619, Egypt.

Ragozzino, A., Dipartimento Arboricoltura, Botanica e Patologia Vegetale, Università di Napoli, 80055 Portici, Italy.

Malfitano, M., Dipartimento Arboricoltura, Botanica e Patologia Vegetale, Università di Napoli, 80055 Portici, Italy.

Ragozzino, E., Istituto Sperimentale per la Patologia Vegetale, Via C. G. Bertero, 22, 00156 Roma, Italy.

Martelli, G.P., Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Sezione di Bari, 70126 Bari, Italy.

Randles, J.W., Department of Applied and Molecular Ecology, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia.

X

CONTRIBUTORS

Ready, K.F.M., Agriculture and Agri-Food Canada, Potato Research Centre, P.O. Box 20280, Fredericton, N.B., E3B 4Z7, Canada.

Skrzeczkowski, L., Department of Plant Pathology, Washington State University, Prosser, Washington 99350, USA.

Rezaian, M.A., CSIRO Plant Industry, Adelaide Laboratory, PO Box 350, Glen Osmond, SA 5064, Australia.

Steger, G., Institut für Physikalische Biologie, Geb. 26.12.U1, Heinrich Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany.

Riesner, D., Institut für Physikalische Biologie, Geb. 26.12.U1, Heinrich Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany.

van Vuuren, S.P., Institute for Tropical and Subtropical Crops, Nelspruit 1200, South Africa.

Rodriguez, M.J.B., Agricultural Research and Development Branch, Philippine Coconut Authority, Albay Research Center, Guinobatan, Albay 4503, Philippines. Rubino, L., Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Sezione di Bari, 70126 Bari, Italy. Salazar, L.F., The International Potato Center (CIP), P.O. Box 1558, Lima, Peru.

van Zaayen, A., Naktuinbouw (The Inspection Service for Horticulture), Sotaweg 22, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands. Wall, G.C., Unibetsedat Guahan, College of Agriculture and Life Sciences, Agriculture Experiment Station, Mangilao, Guam 96923, USA. Wang, G., Department of Plant Protection, Huazhong Agricultural University, Wuhan, Hebei 430070, P.R. China.

Sano, T., Laboratory of Phytopathology, Faculty of Agriculuture and Life Science, Hirosaki University, Hirosakishi, Aomori-ken 036-8561, Japan.

Weber, U., Department of Genetics, Institut für Allgemeine Botanik und Botanischer Garten, University of Hamburg, 22609 Hamburg, Germany.

Semancik, J.S., Department of Plant Pathology, University of California, Riverside, CA 92521, USA.

Whattam, M., Australian Quarantine and Inspection Service, PB19, Ferntree Gully Delivery Centre, Victoria 3156, Australia.

Shamloul, A.M., Genetics Laboratory, Botany Department, Faculty of Science, Sohag, South Valley University, Sohag 82524, Egypt. Singh, R.P., Agriculture and Agri-Food Canada, Potato Research Centre, P.O. Box 20280, Fredericton, N.B., E3B 4Z7, Canada.

Yang, X., Institute of Microbiology, Chinese Academy of Sciences, P.O. Box 2714, Beijing 100080, P.R. China. Zhu, S.F., Institute of Animal and Plant Quarantine, State General Administration for Quality Supervision and Inspection and Quarantine (AQSIQ), Beijing 100029, P. R. China.

XI


PREFACE

In the early 1950s, James Watson and Francis Crick at the Cavendish Laboratory, Cambridge University, revealed the double helical structure of cellular DNA. At about the same time, Heinz Fraenkel-Conrat and Robley Williams at the Virus Laboratory, University of California, Berkeley demonstrated that the RNA, not the coat protein, of Tobacco mosaic virus is the genetic and hereditary component of the virus. These remarkable discoveries and other similar ones in the 1950s marked the birth of the modern era of molecular biology and molecular virology. Potato spindle tuber disease was identified in North America in the early 1920s. Its effects were the appearance of stunted potato plants and a harvest of spindly pointed tubers, which became a serious problem for potato certification programs. Despite the seriousness of the disease, nothing was known about the causal agent. After eliminating all the other possibilities, plant pathologists concluded that the disease might be caused by a virus. In the early 1960s a systematic study of this disease began. In 1962, the causal agent of potato spindle tuber disease was mechanically transmitted to tomato seedlings, a non-potato indicator plant species for the disease. This was the key element in enhancing research on the etiological agent of the disease because the time span for testing for the infectious agent was greatly reduced from two years in potato to produce spindled tubers to two weeks in tomato to show stunting and epinasty symptoms. By the late 1960s, research had shown that the agent causing spindle tuber disease was evidently not a conventional ‘virus’, but something entirely new. Between 1966 and 1968, it was recognized that the causal agent of potato spindle tuber disease, as well as that of citrus exocortis disease, was not a conventional viral nucleoprotein but was most likely a small RNA without coat protein. By 1971, evidence had been presented by two independent laboratories, one in the US and the other in Canada, that a low-molecular weight RNA was the causal agent of potato spindle tuber disease. In 1972, similar findings were reported in the US by a third laboratory for the causal agent of citrus exocortis disease. For both potato spindle tuber and citrus exocortis diseases, the causal RNA agent existed in an unencapsidated form in plants, unlike conventional viruses, and replicated without the help of a virus, thus differing from viral satellite RNAs. Moreover, no message function could

be demonstrated. For such an unconventional entity, various terms such as ‘viroid’ (because it is like a virus), ‘metavirus’, and ‘pathogene’ were proposed. The term ‘viroid’ was eventually accepted. In order to differentiate the abbreviation of viroid from that of virus, for viroids a lower case ‘d’ is added to the V designation of viruses. Thus Potato spindle tuber viroid becomes PSTVd and Citrus exocortis viroid becomes CEVd. Cadang-cadang disease, which causes premature decline and death of coconut palm trees, was first recognized early in the twentieth century in the Philippines. It was not until 1975 that it was determined in Australia that the disease was caused by a viroid, Coconut cadang-cadang viroid. It was the first viroid of a monocotyledonous plant species. Since the mid-1970s viroid research has emphasized conventional aspects such as viroid characterization, viroid structure and replication. However, it has also begun to explore the investigation of viroids as possible causal agents for diseases of uncertain etiology, as well as more practical aspects such as the development of rapid and accurate diagnostic tests to keep viroid diseases out of horticultural, ornamental, and field crops. Since the demonstrations that potato spindle disease of potato, exocortis disease of citrus, and cadang-cadang disease of coconut are caused by viroids, many plant diseases of once uncertain etiology were identified to have been caused by viroids. Currently, there are approximately 30 known viroid species. They belong in two families, Pospiviroidae with five genera and Avsunviroidae with two genera. Some of the viroid diseases cause significant damage to the host crop, others are latent in their primary host, but can do considerable damage to other susceptible crops located near the infected latent host species. Such latent hosts may be significant in plant quarantine and germplasm introductions, both in determining the epidemiology of viroid diseases and in formulating control strategies. In the 10-year period of 1977–1987 three books on viroids were published. They mainly covered advances in viroid research during the first and second decades after the initial viroid discovery. The purpose of the present volume is to serve as an exhaustive reference work about viroids. The editors therefore considered it appropriate that a book presenting such a comprehensive coverage of viroids, in the absence of an overview of the topic for 16

XIII

PREFACE

years, would bear the all-embracing title Viroids. The book is thus intended to provide a watershed for the next phase of viroid research, which is urgently needed to shed more light on viroid origin, structure and function, pathogenicity, epidemiology, and control. The contributing authors of this volume are an international group of scientists who have substantial experience working with viroids and viroid diseases. Viroids presents indispensable, comprehensive and up-to-date information pertinent to viroids, viroid diseases, and their control. It provides a single source of information on the economic impact of viroid diseases, properties of viroids, methods for viroid detection and control, diseases of viroids in different plant species, mapping of the geographical distribution and epidemiology of viroids, diseases of possible viroid etiology, and considerations for future applications of viroids. This book also covers plant quarantine and certification programs for viroid diseases. This information will help anyone concerned with the safe movement of plant material across international boundaries or within a single country. For the benefit of readers, chapters have been grouped into eight parts. A number of chapters focus on the current state of knowledge of the molecular characteristics of viroids, biology, localization and movement, replication, pathogenesis, viroids and gene silencing, classification, viroid-like satellite RNAs; detection of viroids using bioamplification hosts, biological indexing, polyacrylamide gel electrophoresis, molecular hybridization, polymerase chain reaction; mapping of geographical distribution and epidemiology of viroids in North

XIV

America, Australasia, China, Japan, Europe, the Middle East, Africa, South America, and at the global level. Control of viroids includes quarantine of imported germplasm, availability of viroid-tested propagation materials, thermotherapy, tissue culture, and other conventional strategies as well as biotechnological control approaches. Special topics such as ribozyme reactions of viroids and economic advantages of viroid infection are also included. Chapters that summarize the current state of knowledge concerning viroid diseases of the crop in question and aspects of the natural history of viroids in horticulture are also presented. Among the crops covered are potato, tomato, tobacco, cucumber, pome fruits, stone fruits, avocado, citrus, grapevines, hop, chrysanthemum, coleus, columnea, and coconut palm. The main aim of the editors has been to produce a cohesive, comprehensive, and up-to-date volume that can be used by students, researchers, extension agents, and regulators. It also may be of great value to science managers, policy makers, and industries in formulating policies and products to obtain viroid-free plants and control viroid diseases. On behalf of the editors, I would like to thank all contributors, whom I got to know personally over the years, for their willing participation in this project, their patience, and understanding. I would also like to express my gratitude to Mrs Marie Tousignant for her valuable expertise in preparing this book for publication. A. Hadidi

............................................................................................................................................................................................................................................................................................

PART I

INTRODUCTION

...........................................................................................................................................................................................................................................................................


PART I

CHAPTER 1

ECONOMIC IMPACT OF VIROID DISEASES ....................................................................................................

J.W. Randles

.................................................................................................................................................................................................................................................................

Viroids are pathogens of food, industrial and ornamental plants. An evaluation of their economic impact faces the same difficulties as encountered for other obligate intracellular biotrophs, such as viruses, phytoplasmas and fastidious bacteria. Viroid diseases are inconspicuous compared with diseases caused by fungi, bacteria and nematodes, and losses identified in comparative trials on their effects do not necessarily translate to global estimates of loss. Their effects also extend beyond direct effects on yield and quality. The range of direct and indirect damage associated with plant virus infection (Waterworth and Hadidi 1998) largely applies also to viroids. This chapter attempts to consider viroids as one of the many factors that affect world agriculture. It therefore commences with an outline of the history of agricultural development, world food needs and the availability of land. It discusses the role of crop protection. The deployment of new technologies in crop protection and their role in discovering viroids is a precursor to discussing the economic impact of viroids by referring to specific case studies. Crop damage caused by viroids, potential benefits of viroids, and their mode of spread are considered before considering the need for, and the design of appropriate control measures.

FORECAST OF AGRICULTURE, FOOD AND LAND REQUIREMENTS UNTIL 2150 The epoch of science and industrialization is considered to be the 300-year period from 1850 to 2150, in which the world population will show a logistic increase (Weber 1994). Economic divisions between countries with rural and industrial economies can be expected to remain for most of this era. Patterns of population growth for developing countries will be logistic, with an associated demand for greater quantities of food. In contrast, industrialized countries will show a minor increase in population and an associated demand for improved quality of food and lifestyle. It is estimated that the current world population will double to a peak of about 12 x 109 in the next 120–150 years, a limit imposed by the consumption of more resources and pollution by industrial and agricultural wastes rather than an inability to expand food production. The individual mean energy requirement of food is about 3000 cal per day, and the projected doubling of the world population, together with an increased demand for quality, will require research and development of new products and processes, as well as world peace and international support for poorer countries (Weber 1994). Up to 3 × 109 ha of land could be cultivated

3

J.W. Randles

Level of production

Characteristic of crop

Theoretical

full genetic potential expressed

Attainable

completely effective crop protection

Economic

highest net return on input costs

Actual

normal management practices

Primitive

no agronomic improvements

Impact of crop protection

gain in yield/quality

Figure l.1 The impact of crop protection on crop production. The ‘actual’ level achievable at a site where normal practices are used is compared with the level ‘attainable’ when all pests are absent. The impact of crop protection falls within the range indicated by the arrow (based on Oerke et al. 1994).

worldwide and, with improvements in agriculture, should be adequate for the expected limit of world population. However, the cost will be further clearance of natural vegetation with associated deleterious effects on ecosystems and loss of biodiversity. Land-saving advances in technology contributing to improvements in food production will continue to be the use of chemical fertilizers, plant breeding and crop protection. Of these, crop protection is the most complex because of the range of crops, range of pests, pathogens and weeds, and the different life cycles of each of these. Also, the need for crop protection increases with increases in crop yield because of the higher plant density, shortened intercrop periods, monocultures, and the increased use of fertilizers and irrigation. An essential component of crop protection in the future will be the generation of information through research and the use of this information for control of crop losses.

CROP LOSSES AND CROP PROTECTION Theoretical levels of crop production are summarized in Figure 1.1. Crop loss can be defined as the difference between actual yield under normal optimum agronomic conditions and the yield attainable for the same crop with completely effective crop protection. The effect of crop protection is a gain in yield or quality. Economic yield is the level at which incremental costs of crop protection reach but do not exceed the incremental increase in value of the crop. Ideally, estimates of loss would be a prerequisite for the rational development of plant protection programs, but crop loss assessment is complex and inexact (Oerke 1994). An estimated global figure for preharvest crop loss from all sources is considered to be about 40%, while postharvest losses are estimated to be in the range of 10–50%. Thus, more than 50% of produce grown is lost before reaching the consumer. 4

In plant pathology, research has provided better diagnostic methods, more accurate information on epidemiology, disease forecasting, cultural control measures and novel forms of resistance. Integrated crop management combines these with other strategies as a basis for sustainable agriculture, including concern for natural resources and represents a logical way forward between the extremes of ultra-intensive agrosystems and low output organic farming (Dehne and Schonbeck 1994). Because of high costs and the need for research infrastructure, these measures are first adopted by developed countries. Developing countries may only invest in selected techniques for use in crops destined for export to countries which impose strict tolerances on food quality or plant health.

THE DEPLOYMENT OF NEW TECHNOLOGIES IN PLANT PATHOLOGY

The range of techniques available does not provide an adequate solution to all disease losses. Where therapeutic means of control are not available, as for the intracellular biotrophic pathogens, indirect control measures are needed, but they can only be applied if the disease cycle is known. Such solutions are often temporary and require continued intellectual input to remain relevant to the requirement for sustainability. Biotechnology and genetic engineering are the most recent contributors to new control strategies. Direct incorporation of resistance genes against many plant viruses by transformation of plants with pathogen-derived nucleotide sequences is now well tested and the phenomenon of gene silencing is now under intensive study (Hull 2002). Moreover, the development of highly sensitive and specific nucleic acid based diagnostic tests promises to revolutionize quarantine practice and pathogen-testing schemes for exclusion of infected plant material. The cost effectiveness of this technology is now widely recognized and it only remains for it to be deployed where it is likely to be most effective. Research on viroids over the last 30 years has paralleled and contributed to the development of biotechnology. The discovery of the first viroid, Potato spindle tuber viroid (PSTVd), exemplifies the ground-breaking consequences of identifying a new paradigm in plant biochemistry and pathology. The positive economic impact of the original discoveries that previously enigmatic diseases were due to viroids has been greatly multiplied not only by the ability of workers in quarantine and pathogen-testing schemes to identify, evaluate and control viroids, but by their ongoing contribution to understanding cellular processes in molecular biology and particularly the puzzle of how they produce disease. This positive economic impact is highly significant but impossible to quantify because many of the long-term consequences are not obvious.

LOSSES DUE TO VIROIDS Viroids vary in the hosts they infect, the type and severity of disease that they cause, their mode of spread and their epidemiol-

ECONOMIC IMPACT OF VIROID DISEASES

ogy. They also vary in pathogenicity, time to induce disease, interactions with other pathogens and response to the environment. Control measures may be available for some, while the lack of knowledge of the epidemiology may prevent the formulation of reliable control strategies for others. Many of the known viroids were discovered because they induced serious damage to their host crops. Following their characterization, attempts were made to monitor their incidence and effects. This was intended to lead to quantification of the dynamics of incidence, spread and distribution, followed by the prediction of outbreaks and the deployment of control treatments.

Meristems, at both apex and root tips, may show reduced cell division, altered patterns of differentiation leading to hypoplasia or hyperplasia in plant tissues. Any reduction in cell division will be a precursor of reduced growth rate in both roots and tops of plants. This may be reflected in shortened internodes or loss of apical dominance in dicotyledonous plants, or ‘pencilling’ of the trunks of monocotyledons such as palms. These reduced outputs by the plant occur despite normal inputs of labor, nutrients and water.

As each viroid has a unique disease cycle, each disease cycle needs to be defined so that a specific system of assessment, loss prediction and control can be established. The measurement of losses due to viroids has two aspects. The first is the severity of the disease induced by infection with the viroid and its variants. The second is the prevalence of the viroid and its ability to spread and produce an epidemic. Direct losses can be measured either as a depression in yield or as a cash loss. These losses will vary with the viroid, the crop, time and the environment. The types of loss that have been reported from viroid infection include losses of whole plants, damage to parts of plants, damage to plant products, damage to subsequent generations, and the effects of coinfection with other endoparasitic pathogens.

If the economic product of the plant is a tuber, nut or fruit, downgrading of quality, predisposition to damage at harvesting or in storage, and reduced yield at harvest, may be components of loss. Seed viability, strength of the plant’s framework and of timber harvested from the plant may be reduced by viroid infection and incur a cost. Variation in growth rates of infected compared with viroid-free plants may incur labor costs in plantation management, as for hops with reduced extension of bines when infected with Hop stunt viroid (HSVd).

Whole plant losses

The effect of lethal viroid diseases of coconut palm such as cadang-cadang and tinangaja, as well as those leading to the removal of unproductive annual or perennial plants, can be evaluated using a descriptive formula: Loss = value of plant + cost of replacement + time lost from interrupted production + cost of diagnosis This implies that where viroid control is achieved by eradication and replanting, the effect on loss will be the same as natural death of the plant. This situation allows individual losses to be calculated with reasonable accuracy. Damage to parts of plants

Specific tissues and organs of plants may be directly affected, or a general reduction in growth rate may cause stunting and reduced yield. For example, foliage may be distorted, chlorotic or damaged through increased susceptibility to sun or wind. Plant structure may be affected by reduced growth, lesions on the stem or reduced translocation of water and nutrients. Reproductive structures may be affected. For example, flower production may cease, or flowers may show necrosis, reduced size or change in pigmentation. Fruit may be altered in size, shape, quality, color or markings. Seed may be small or shrivelled, with changed composition or viability.

Damage to plant products

Effects on subsequent generations

If a viroid is transferred to the next generation of the host species, in true seed or vegetative propagules, the rate of transmission and the cost of an epidemic resulting from secondary spread from these primary sources of infection will determine the extent of future losses. Mixed infections

A range of consequences may arise from mixed infection either with different strains of viroids, with different viroids or with viroids and viruses (Garnsey and Randles 1987). Cross-protection occurs between mild and severe isolates of several viroids. It has not been used as a control measure for viroids, but has been used to identify mild strains of PSTVd in biological indexing programs. Molecular analysis has indicated that for viroid-infected perennial crops such as grapevine, stonefruit and citrus, a most important consequence of mixed infection is the appearance of recombinant ‘new’ viroids, with the potential for genetic properties to be derived from several co-infecting viroids. Synergism has been reported in potato between PSTVd and Potato virus Y (Singh 1982). The report (Francki et al. 1986) that PSTVd could be encapsidated in Velvet tobacco mottle virus, which is mirid transmitted in nature, indicated that co-infection may have major consequences for viroid epidemiology by providing an unexpected mode of spread. Salazar et al. (1995) reported very high rates of transmission of PSTVd by Myzus persicae when source plants were doubly infected with Potato leafroll virus (PLRV) and PSTVd. The epidemiological consequences of this report are very important because of the widespread distribution of PLRV. 5

J.W. Randles

Losses due to spread

Epidemics of viroid diseases will depend on the ability of the causal viroid to spread. Epidemics can be classed broadly as either monocyclic (showing a linear increase with time) or polycyclic (a logistic pattern of increase). The type of epidemic is generally closely linked to the mode of spread of the viroid, and analysis of the progress of an epidemic may help to identify the mode of spread and possibly identify the type of vector if one is involved. Spread can be described in terms such as time, rate, range and pattern of spread. All of these descriptors are important in measuring, predicting and controlling losses from spread. Where vegetative propagules are the only known means of spread, the use of ‘clean’ or ‘pathogen-tested’ propagules can be recommended. Where seed or pollen transmission occur, eradication of infected trees together with replanting of pathogentested plants is recommended. If mechanical transmission occurs, either eradication and replanting, or avoidance of mechanical processes, can be recommended. Known arthropodborne viroids are uncommon, but if the pattern of spread could be explained by the activity of a vector it needs to be identified and cultural control measures similar to those used for controlling arthropod-borne virus diseases need to be considered. Where the mode of spread is unknown or uncertain, as for the palm viroids, an analysis of epidemiology may provide clues for testing a range of control options. For example, because the risk of cadang-cadang disease occurring in a replanted palm is no greater than that occurring in other palms in a plantation, a replanting strategy can be recommended for minimizing the effects of cadang-cadang disease. When considering the selection of control strategies for viroids, the following points need to be considered: • accurate and sensitive diagnostics are needed for the implementation of control schemes; • each viroid has a unique disease cycle and epidemiology; • all mode(s) of spread need(s) to be known for each viroid/ host/environment combination; • control strategies need to interrupt spread.

CASE STUDIES Table 1.1 is a summary of viroids listed by their economic host group. For each host group either single or multiple viroid infections are considered for their economic impact. The known distribution of the viroid in relation to the distribution of the host species is described as either widespread or limited, and is included as a guide to whether exclusion strategies such as quarantine may be relevant to control. The severity of the viroid diseases in the economic host is listed, together with the main

6

source of loss from the viroid disease. Known mode of spread is listed as a guide to the control strategy either suggested or practised. Specific points about the economic impacts of the viroids on the crop groups are also provided in Table 1.1. Avocado sunblotch viroid (ASBVd)

As described by Desjardins (1987) the sunblotch disease of avocado has been known for over 70 years and has been reported in most countries where avocados are grown. Symptomatic trees show stem streaks, bright chlorotic leaf blotches and variegations, leaf distortion, discolored depressions and distortion of the fruit, and a sprawling stunted growth habit of the tree. On ‘symptomless’ carrier trees economic effects include markedly reduced production and downgrading of a high proportion of fruit. Where a symptomless shoot develops on a symptomatic tree (a phenomenon described as recovery) there are a number of unusual consequences. Symptoms cannot be induced in the symptomless carrier tree, seed transmission of the viroid increases dramatically (to 90–100%) compared with symptomatic trees, progeny seedlings of symptomless carriers are themselves symptomless carriers and show a high rate of seed transmission of ASBVd to the next generation and they can act as a reservoir for mechanical and pollen transmission of ASBVd. The direct economic impact of this viroid is therefore lost production, the cost of selecting viroid-free rootstocks and scions for propagation, and the cost associated with eradication of infected trees. The low rate of pollen transmission, apparently to the fertilized ovule and seed but not to the pollinated tree, indicates the need for isolation of elite germplasm source trees from orchards, and the regular molecular indexing of these trees for infection. The existence of many variants of ASBVd (Rakowski and Symons 1989) needs to be considered when devising RTPCR diagnostics for indexing to ensure that all variants are detected. Coconut viroids

Coconut cadang cadang viroid (CCCVd) and Coconut tinangaja viroid (CTiVd) are of concern not only because of their lethality, but because diseased palms cease production of nuts many years before they die. The diseases are frequently not recognized by growers so that non-producing palms may be kept for years in the hope that they will become productive again or simply because there is a reluctance to cut down any living palm whether it is bearing or not. Both diseases were first reported about 85 years ago, and have had a similar effect on the coconut industries of the Philippines and Guam, respectively. The distribution of cadang-cadang disease in the Philippines has been mapped since the 1950s and estimates of incidence since that time have indicated that the annual loss of palms from the disease is in the range of 200 000 to 500 000 with a total loss of over 40 x 106 palms since the disease was first recognized. As


Table 1.1 Viroid(s)

Example of the economic effects of some viroids on the basis of crops affected, distribution of the viroid in relation to the distribution of the crop species, mode of spread and control strategy. Economic host

Locality

Damage

Cost

Mode of Spread

Control strategy

Vegetative propagules

Seed/pollen

Insect

Mechanical

+

+

–

–

ASBVd

avocado

widespread

severe

reduced yield, discarded fruit

CCCVd & CTiVd

coconut palm

limited

lethal

replacement

not applicable

low rate

not known

not known

CSVd

chrysanthemum

widespread

severe

yield, quality

+

–

–

+

eradication, pathogen tested propagules

Citrus viroids

citrus

widespread

variable

reduced yield & longevity

+

–

–

+


Grapevine viroids

grapevine

widespread

minor

impact on vine improvement schemes

+

+

–

–


HSVd

hops

limited

moderate

yield & quality

+

–

–

+

eradicate and replant

HSVd

cucumber

limited

pale fruit

yield & quality

–

–

–

+

eradication

Pome fruit viroids

apple & pear

widespread

mild

quality

+

–

–

–


PSTVd

potato

limited

moderate

yield & quality

+

–

–

+

eradication, pathogen tested propagules and quarantine

PSTVd

tomoto

limited

stunting

yield & quality

not applicable

+

+

+

eradication

Stone fruit viroids

prunus spp.

widespread

mild

quality

+

–

–

–


TASVd

tomato

limited

mild

minor

not applicable

not known

not known

not known

TPMVd

tomato

limited

severe

yield

not applicable

not known

+

+

eradication, pathogen tested propagules replant, quarantine

not applicable eradication

7

J.W. Randles

described by Randles and Rodriguez later in this volume, as palms cease production an average of five years before death and replacement palms do not reach full bearing for 5–8 years, each infected palm may interrupt production from that site for 10–13 years. An average loss in production of copra of about US$ 100 per infected site has been calculated (Randles 1987). This allows the annual loss to be calculated in the range US$ 20–50 million. This is a minimum value as new alternative uses for coconuts are lifting the value of individual nuts in the local retail market. If palms could be removed immediately after early symptoms are recognized and replaced with precocious hybrids, the interruption would be reduced to 5–8 years, and this would be a major saving. There is little opportunity to replace coconuts with other crops in the Philippines, as the coconut palm is still a major industrial crop and export earner, and the coconut palm as the ‘tree of life’ is an intrinsic part of local culture. Another cost to the global coconut industry is the embargo, due to cadang-cadang, on the collection of breeders’ germplasm from the Philippines. Export markets for unprocessed coconut products are also generally unavailable because of the disease. When evaluating economic impact, the loss of nut production should be combined with the cost of researching and understanding the cause of cadang-cadang. For example, the loss of 40 x 106 palms at a cost of US$ 100 per site provides a total loss value of US$ 4000 million at current prices. The cost of research on the disease can be calculated simplistically on the basis of employing two full-time international scientist equivalents per annum, starting from the mid-1950s when international experts were first supported to work on cadang-cadang disease in the Philippines. To the completion of international involvement in 1993, costs of research may be calculated as follows:

be a suitable strategy to minimize the impact of the disease. The epidemiology of CCCVd needs to be investigated in detail using the molecular probes that have been developed. Losses from tinangaja disease on Guam result from premature cessation of nut production and death of the palm. The loss can be calculated from the value of individual trees and nuts. Mature palms used as ornamental trees for new tourist hotel developments have had a reported value of up to US$ 700–1000, and a drink based on a single coconut at a hotel bar can cost over US$ 5 (G. Wall, personal communication). Chrysanthemum stunt viroid (CSVd)

CSVd and the coleus viroids are the only viroids known to be important in ornamentals. While the effect of the coleus viroids is not widely reported, the economic impact of CSVd on chrysanthemums is well described. Chrysanthemum stunt disease was first recognized in 1945 at the time of rapid expansion of the cultivated chrysanthemum industry in the US, and was soon reported in other countries following international movement of cuttings (Lawson 1987). The affected plants are unproductive and the causal viroid is readily transmitted by foliar contact, cultivation practices and cutting knives. The bio-assay and elimination procedures by meristem culture after thermotherapy are inefficient and expensive. An outbreak of CSVd causing a loss of about $3 million was reported in Australia in 1987, although the incidence of infection is now negligible with indexing by molecular hybridization detecting only a few infections in some greenhouses (Hill et al. 1996). Growers are warned of the consequences of allowing CSVd into their propagating material and diagnostic services are generally available to identify new outbreaks. Awareness of the effect of other virus infections has led to good management practices for both viruses and viroids.

2 scientists at US$ 50 000 for 40 years = US$ 4 million For on-costs to cover travel, infrastructure and support from international agencies, a conservative multiplier value of 2.7 can be used, to give an overall cost of research at current dollar values of US$ 10.8 million. Thus, the cost of research using these figures comes to 0.27% of the cost of the disease, and relative to the cost of the disease is trivial. This illustrates that the benefits of research far outweigh the cost of research. The research has provided an understanding of the etiology and epidemiology of cadang-cadang, has produced a platform for assessing the risks presented by the disease, and diagnostic tools needed for further studies are now available. It has also been used in the unsuccessful search for resistance to CCCVd. A low rate of seed transmission and a report of pollen transmission indicate that it is unlikely that cadang-cadang can be eradicated from the Philippines. Thus, calculations of infection rates and spatial patterns of spread (Zelazny et al. 1982) may be used to recommend that replanting of diseased coconut palms would 8

Citrus viroids

Exocortis and cachexia (xyloporosis) are the economically important viroid diseases of citrus. The variable expression of viroids in different citrus species, cultivars and stock-scion combinations, and the recognition that a number of viroids infect citrus has presented a confusing picture to horticulturists. Exocortis disease has received most attention since it was first reported in 1948, and Citrus exocortis viroid (CEVd) as well as the other viroids are expected to be present in all citrus growing countries. As the modes of transmission seem to be limited to propagation and mechanical transmission (Garnsey and Randles 1987), control strategies are based on the use of propagating material from mother trees certified to be free of the citrus viroids. The beneficial effect of dwarfing of citrus and the use of viroids to develop a predictable range of tree size in intensive citrus plantings is reviewed by Hutton et al. (2000). Increasing the planting density of citrus trees increases the yield per unit area, but the relationship is asymptotic in that yield rapidly rises to near maximum levels but then levels out at very high


planting densities. Also, as trees age, cropping moves higher into the tree canopy and spraying and harvesting becomes very difficult. Viroid dwarfing controls vegetative vigor, and compared to uninoculated trees, citrus viroid dwarfed ‘Valencia’ trees on Poncirus trifoliata stocks grown at Dareton, Australia, remained highly productive throughout their mature bearing life when grown at high density. During the first eight years after planting, yields per hectare were greater for viroid inoculated trees planted at high density than for uninoculated trees planted at medium density. An economic model indicated that the break-even point for orchards was reached earlier for the highest density plantings. The economic factors favoring high density plantings of viroid dwarfed trees are described as the early income on investment and the containment of fixed costs by increasing production efficiency. Fungicide and insecticide applications are more efficient. Disadvantages were the higher costs for planting and an increased need for optimum orchard management. Grapevine viroids

The grapevine viroids have not been reported to cause noticeable economic effects on winegrape production. Grapevine yellow speckle viroid (GYSVd) 1 and 2 produce yellow speckling and biological indexing programs have been used to exclude infected germplasm from improved selections. The other three grapevine viroids seem to be asymptomatic. As reviewed by Krake et al. (1999), there is no published evidence of a significant adverse effect of yellow speckle disease although a mixed infection of GYSVd with HSVd may alter grapejuice pH and reduce vegetative growth. Vein banding disease has been described as an interaction of GYSVd with Grapevine fanleaf virus (Szychowski et al. 1995). Because of the possibility that viroids could also interact with any of the many known viruses, and the phytoplasmas, coinfecting grapevines, there is good reason to consider excluding viroids from elite vine germplasm by investing in schemes to remove all viroids, viruses and phytoplasmas. The main economic impact of the grapevine viroids is therefore the cost of setting up elite pathogen tested grapevine germplasm schemes using molecular diagnostic methods. It can be foreseen that the free exchange of grapevine germplasm of known pathogen status will follow the international adoption of such a scheme. The epidemiology of the grapevine viroids needs to be further investigated following the report of transmission of GYSVd 1 and Hop stunt viroid (HSVd) in the seed of grapevines (WanChow-Wah and Symons 1999). Seed transmission would influence breeding practices and may account for survival of viroids in seedling reservoirs. Hop stunt viroid (HSVd)

Hop stunt disease appears to have originated in Honshu Island, Japan. Although the commercial hop was introduced to Japan in the late 19th century, the disease has not been reported out-

side Japan (Shikata 1987). Affected plants are stunted in height by about 40% with shortening of the internodes of the main and lateral bines, bine and leaf weight is reduced by about 30%, and cone weight is reduced by about 50%. Of the bitter resins contained in the cones, the alpha acid content is reduced whereas the beta acid content is not. Lupulin glands on bracteoles are severely shrivelled. The recommended method for control of the disease is the eradication of plants with the causal HSVd, taking special care to kill or remove deep roots. Cucumber pale fruit disease (van Dorst and Peters 1974) was first reported around 1963 in greenhouse cucumbers in the western Netherlands. It has not been reported elsewhere. The incidence was low in affected greenhouses, mostly less than 0.1%. Surveys provided no evidence that the disease was soilborne, seedborne or nematode-borne, but it is mechanically transmitted during pruning. The incubation period was about two months, and high temperatures favored disease expression. A variant of HSVd (HSVd-c) with about 95% nucleotide sequence homology to HSVd has been isolated from pale fruit affected cucumber, and the variants are biologically indistinguishable on cucumber (Diener 1987b; Shikata 1987). HSVdc shows less sequence similarity to HSVd than does the grapevine isolate, HSVd-g. Pome fruit viroids

All of the characterized viroids isolated from pome fruit trees, Apple dimple fruit viroid (ADFVd), Apple scar skin viroid (ASSVd), Pear blister canker viroid (PBCVd), have been assigned to the genus Apscaviroid (Flores et al. 2000). They are associated with the apple dimple fruit (ADFVd), apple scar skin, dapple apple, pear rusty skin, Japanese pear fruit dimple (ASSVd) and pear blister canker (PBCVd) diseases, respectively. The ADFVd has not yet been assigned officially to a genus. All diseases are graft transmitted, without other known means of spread. The significance of these diseases on tree health, and fruit appearance and quality, has resulted in the imposition of germplasm indexing protocols for both international and local quarantine. The availability of nucleic acid probes and RT-PCR protocols for these viroids will reduce the costs and delays previously associated with exclusion of infected lines from pome fruit germplasm. Potato spindle tuber viroid (PSTVd)

The spindle tuber disease of potato originated in the USA before 1917, and was at first ascribed to a degeneration associated with prolonged asexual propagation, before its infectious nature was recognized (Diener 1987a). The economic damage arises from effects on yield and quality as well as threats to seed production and germplasm collections. It has been calculated on the basis of incidence and loss per infected plant, that overall yield loss is about 1% of the crop in North America. Although this loss is 9

J.W. Randles

apparently small, because of the worldwide importance of the crop as a staple food, a 1% loss is very large in absolute terms. For example, the loss due to PSTVd in potatoes can be calculated to have been 197,850 tons in North America in the period 1988–90 on the production figures provided by Oerke (1994). Costs due to down-grading on appearance of tubers for retail markets would be an additional loss. Outbreaks of PSTVd have been occasionally reported in tomato crops in countries which exclude PSTVd in potatoes by quarantine practice (Elliott et al. 2001). Of greater concern to these countries is the potential for establishment of PSTVd in potato germplasm because viroid indexing would then need to be included in seed certification schemes. Stone fruit viroids

Peach dapple viroid, a variant of HSVd, and Peach latent mosaic viroid (PLMVd), are the only viroids reported in stone fruit trees. Peach and nectarine are the only reported economically important hosts of these viroids, with effects on fruit quality the main economic effect of infection. Either delayed (the most general situation) or premature ripening (Di Serio et al. 1999) of fruit have been reported in trees containing PLMVd. In South Australia, the premature ripening of one line of nectarines in which PLMVd was detected has provided an economic benefit to the grower because of an opportunity for the fruit to be marketed in advance of the normal variety. These trees are therefore retained for their economic advantage. Unwanted effects such as premature ageing of trees, and decreased resistance to cold and various diseases warrant control by eradication and planting with viroid tested trees. The epidemiology of these viroids needs to be determined to evaluate whether natural spread by mechanical damage is a risk factor in orchards (Hadidi et al. 1997). Molecular indexing would be the main cost of control. Tomato apical stunt viroid (TASVd) and Tomato planta macho viroid (TPMVd)

The limited distribution of these tomato pathogens, their low incidence, and the annual nature of the crop are presumably responsible for the low economic impact of TASVd and TPMVd (Walter 1987; Galindo 1987).

CONCLUSIONS An assessment of the economic impact of viroids first needs to take account of quantitative factors such as monetary losses in capital and the cost of labor. Crop loss assessments use inputs such as incidence of disease, the area affected and the effect of infection on the yield or quality of individual plants or plant products. Losses will vary with the value of the commodity. The cost of labor will vary between that of a farmer in a developing country, at a few dollars per day, and that of a scientist or administrator in a developed country, at over $100 per day. However, the actual cost of labor should not necessarily be measured in dollar terms, but in terms of productivity. This is more difficult 10

to evaluate because productivity may be realized some years later for a scientist who makes a basic discovery, or a farmer who develops a valuable landrace or maintains genetic diversity through practising sustainable agriculture. Table 1.1 is an example of a semiquantitative summary of the comparative impacts of the viroids known to affect different crop groups. It lists factors such as distribution, severity of damage, and the mode of spread as components which lead to determining the economic importance of a given viroid group. These are then available for decision making, considering first the economic impact, and second whether a control strategy is appropriate. Qualitative factors include education, time required for basic research, intellectual input associated with discovery, information exchange, technology transfer, change of management structures, infrastructure for ongoing research and development, and commercialization. Less tangible effects are the necessity to provide food for the community and products for trade, and aspects of agricultural economics such as production efficiency, marketing, depreciation and politics at micro- and macro-economic levels. A major difference in impact is evident between developing and industrialized countries. The rural economies of developing countries, where food needs are paramount, require imported technologies for disease control. For example, international collaboration led to the molecular characterization of CCCVd and CTiVd and provided the means for determining epidemiology and possible control strategies in the countries where they occur and control of international movement of the viroids. The industrialized countries have the means to research viroid problems where they occur, and highly skilled input is available for control. Moreover, alternative crops or land uses can replace affected crop areas to minimize losses. Thus, rural economies are more vulnerable to the effects of viroid infection. While it may appear that in some cases individual viroids are not significant causes of loss in particular crops, viroids are known to mutate or occur in mixed infections with other pathogens. Because of the need to allow plants to perform as close as possible to their genetic potential, the exclusion of viroids from crops should be considered necessary. Now that the nucleotide sequences of all assigned viroids are available, molecular diagnostic methods for viroids and other intracellular biotrophs of known sequence can be combined to achieve viroid freedom from vegetatively propagated germplasm. However, viroids which spread naturally present a continuing challenge for attempts to elucidate their epidemiology and minimize their economic impact. References Dehne, H.W., and Schonbeck, F. (1994). Pages 45-71 in: Crop production and crop protection. E.C. Oerke, H.W. Dehne, F. Schonbeck, and A. Weber, eds. Elsevier: Amsterdam.


Desjardins, P.R. (1987). Avocado sunblotch. Pages 299-313 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Di Serio, F., Malfitano, M., Flores, R., and Randles, J.W. (1999). Detection of peach latent mosaic viroid in Australia. Austr. Plant Pathol. 28, 80-81. Diener, T.O. (1987a). Potato spindle tuber. Pages 221-233 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Diener, T.O. (1987b). Cucumber pale fruit. Pages 261-263 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Elliott, D.R., Alexander, B.J.R., Smales, T.E., Tang, Z., and Clover, G.R.G. (2001). Infection of glasshouse tomatoes by Potato spindle tuber viroid in New Zealand. Page 224 in: Proceedings, 13th Biennial Conference, Australian Plant Pathology Society, Cairns, Australia. Flores, R., Randles, J.W., Bar-Joseph, M., and Diener, T.O. (2000). Subviral agents: viroids. Pages 1009-1024 in: Virus taxonomy. M.H.V. van Regenmortel, C.M. Fauquet, D.H.L. Bishop, E.B. Carstens, M.K. Estes, S.M. Lemon, J. Maniloff, M.A. Mayo, D.J. McGeoch, C.R. Pringle, and R.B. Wickner, eds. Academic Press: San Diego. Francki, R.I.B., Zaitlin, M., and Palukaitis, P. (1986). In vitro encapsidation of potato spindle tuber viroid by velvet tobacco mottle virus particles. Virology 155, 469-473. Galindo, J.A. (1987). Tomato planta macho. Pages 315-320 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Garnsey, S.M., and Randles, J.W. (1987). Biological interactions and agricultural implications of viroids. Pages 127-160 in: Viroids and viroid-like pathogens. J.S. Semancik, ed. CRC Press: Boca Raton, FL. Hadidi, A., Giunchedi, L., Shamloul, A.M., Poggi Pollini, C., and Amer, A.M. (1997). Occurrence of peach latent mosaic viroid in stone fruits and its transmission with contaminated blades. Plant Dis. 81, 154-158. Hill, M.F., Giles, R.J. , Moran, J.R., and Hepworth, G. (1996). The incidence of chrysanthemum stunt viroid, chrysanthemum B carlavirus, tomato aspermy cucumovirus and tomato spotted wilt tospovirus in Australian chrysanthemum crops. Aust. Plant Pathol. 25, 174-178. Hull, R. (2002). Matthew’s plant virology (4th edition). Academic Press: San Diego. Hutton, R.J., Broadbent, P., and Bevington, K.B. (2000). Viroid dwarfing for high density citrus plantings. Horticultural Reviews 24, 277-317. Krake, L.R., Steele Scott, N., Rezaian, M.A., and Taylor, R.H. (1999). Graft-transmitted diseases of grapevines. CSIRO Publishing: Collingwood, Australia.

Lawson, R.H. (1987). Chrysanthemum stunt. Pages 247-263 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Oerke, E.C. (1994). Estimated crop losses due to pathogens, animal pests and weeds. Pages 72–741 in: Crop production and crop protection. E.C. Oerke, H.W. Dehne, F. Schonbeck, and A. Weber, eds. Elsevier: Amsterdam. Rakowski, A.G., and Symons, R.H. (1989). Comparative sequence studies of variants of avocado sunblotch viroid. Virology 173, 352-356. Randles, J.W. (1987). Coconut cadang-cadang. Pages 265-277 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Salazar L.F., Querci, M., Bartolini, I., and Lazarte, V. (1995). Aphid transmission of potato spindle tuber viroid assisted by potato leafroll virus. Fitopatologia 30, 56-58. Shikata, E. (1987). Hop stunt. Pages 279-290 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Singh, R.P. (1982). A unique interaction of spindle tuber viroid and virus Y in potatoes. Phytopathology 72, 962. Szychowki, J.A., McKenry, M.V., Walker, M.A., Wolpert, J.A., Credi, R., and Semancik, J.S. (1995). The vein-banding disease syndrome: A synergistic reaction between grapevine viroids and fanleaf virus. Vitis 34, 229-232. Van Dorst, H.J.M., and Peters, D. (1974). Some biological observations on pale fruit, a viroid-incited disease of cucumber. Neth. J. Plant Pathol. 80, 85-96. Walter, B. (1987). Tomato apical stunt. Pages 321-327 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Wan-Chow-Wah, Y.F., and Symons, R.H. (1999). Transmission of viroids via grape seeds. J. Phytopathol. 147, 285-291. Waterworth, H.E., and Hadidi, A. (1998). Economic losses due to plant viruses. Pages 1-13 in: Plant virus disease control. A. Hadidi, R.K. Khetarpal, and H. Koganezawa, eds. APS Press: St. Paul. Weber, A. (1994). Population growth, agricultural production and food supplies. Pages 1-44 in: Crop production and crop protection. E.C. Oerke, H.W. Dehne, F. Schonbeck, and A. Weber, eds. Elsevier: Amsterdam. Zelazny, B., Randles, J.W., Boccardo, G., and Imperial, J.S. (1982). The viroid nature of the cadang-cadang disease of coconut palm. Scientia Filipinas 2, 46-63.

11


............................................................................................................................................................................................................................................................................................

PART II

PROPERTIES OF VIROIDS

...........................................................................................................................................................................................................................................................................


PART II

CHAPTER 2

MOLECULAR CHARACTERISTICS ....................................................................................................


.................................................................................................................................................................................................................................................................

Mature viroids consist of a covalently closed circular RNA that ranges in size from 246 to 399 bases (see Table 2.1). Because viroids do not code for any peptide or protein, they have to utilize proteins of the host for most biological functions like replication, processing or transport. Therefore, viroids can be regarded as minimal parasites of the host machinery. To act as parasites, viroids have to present to the host machinery the appropriate signals that have to be based either on the sequence or the structure of viroid RNA. The thermodynamically optimal, stable structure of most viroids consists of an unbranched series of short helices and small loops; that is their sequence is an imperfect palindrome (see structural model of PSTVd in Figure 2.3). In this rod-like structure several structural domains may be defined. Most viroids have a ‘central conserved region’ (CCR, marked by ‘C’ in Figure 2.3), which is located in the centre of the rod-like structure and is formed by two conserved nucleotide stretches. The upper stretch is flanked by an inverted repeat (Figures 2.5 and 2.6). In addition to the rod-like structure, viroids are able to form further structures, which may be favorable either in thermodynamic equilibrium or trapped kinetically in a metastable state. Several structural elements, present either in stable or in metastable structures, are linked to biological functions.

Viroids replicate by a rolling circle mechanism (see Chapter 5 ‘Replication’), using either an asymmetric or a symmetric pathway (see Figure 2.9). Different enzymes are involved in the different types of replication cycle. In either case replication includes a processing step of oligomeric replication intermediates to molecules of unit length. This step proceeds either by a viroid-internal ribozyme or by proteinaceous RNase(s) of the host. According to these features viroids are classified (Flores et al. 2000) into two families (see Table 2.1 and Chapter 8 on ‘Classification’): Pospiviroidae possess a thermodynamically stable rod-like secondary structure with a CCR and do not self-cleave. According to sequence and structural homology, Pospiviroidae may be divided into five genera, which are named according to the respective type member: Pospiviroid, Potato spindle tuber (PSTVd); Hostuviroid, Hop stunt (HSVd); Cocadviroid, Coconut cadang cadang (CCCVd); Apscaviroid, Apple scar skin (ASSVd); and Coleviroid, Coleus blumei-1 (CbVd-1). Avsunviroidae do not possess a CCR and self-cleave via a hammerhead ribozyme. This family consists of three members, Avocado sunblotch (ASBVd), Peach latent mosaic (PLMVd), and Chrysanthemum chlorotic mottle (CChMVd). ASBVd is the

15


Table 2.1 Genus

Compilation of viroids. Viroid

# variantsa

Length/nt

Family Pospiviroidae Pospiviroid

Potato spindle tuber Chrysanthemum stunt Citrus exocortis Columnea latent Iresine Mexican papita Tomato apical stunt Tomato chlorotic dwarf Tomato planta macho

PSTVd CSVd CEVd CLVd IrVd MPVd TASVd TCDVd TPMVd

45 10 33 3 1 9 3 1 1

341b, 356–361 354–356 368–374, 463c 370, 372, 373 370 359, 360 360, 363 360 360

Hostuviroid

Hop stuntd

HSVd

82

294–303, 318

Cocadviroid

Coconut cadang-cadang Citrus IV Coconut tinangaja Hop latent

CCCVd CVd-IV CTiVd HLVd

7 1 2 10

246, 247, 287, 296, 297, 301e 284 254 255, 256

Apscaviroid

Apple scar skin Apple dimple fruit Australian grapevine Citrus viroid III Citrus bent leaf Grapevine yellow speckle 1 Grapevine yellow speckle 2 Pear blister canker

ASSVd ADFVd AGVd CVd-III CBLVd GYSVd-1 GYSVd-2 PBCVd

4 1 1 22 7 35 1 11

329–331 306 369 293, 294, 297 315, 318, 326, 327, 328, 329 366–369 363 315, 316

Coleviroid

Coleus blumei-1 Coleus blumei-2 Coleus blumei-3

CbVd-1 CbVd-2 CbVd-3

4 2 9

248, 250, 251 295, 301 361, 362, 364

Avsunviroid

Avocado sunblotch

ASBVd

33

245–251

Pelamoviroid

Peach latent mosaic Chrysanthemum chlorotic mottle

PLMVd CChMVd

98 13

335–339, 342 397–401

Family Avsunviroidae

a b c d e

Number of variants present in “The subviral RNA database” (Pelchat et al. 2000). An infectious PSTVd evolved from an in vitro-generated deletion mutant (Wassenegger et al. 1994). A CEVd variant produced by terminal repeats (Semancik et al. 1993, 1994). Variants from different host plants and/or diseases have different names: hop, HSVd-h; grapevine, HSVd-g; citrus, HSVd-cit; plum and peach, HSVd-p; Prunus, HSVd-apr; cucumber, HSVd-c (Cucumber pale fruit, CPFVd); almond, HSVd-alm. Circular dimeric forms of these RNAs are present in a late stage of the disease (Mohamed et al. 1982).

single member of the genus Avsunviroid, and PLMVd and CChMVd belong to the genus Pelamoviroid.

BIOPHYSICAL PROPERTIES Biophysical properties of some viroids had been determined even before the nucleotide sequences became known (for review see Riesner and Steger 1990). For example the molecular weights were measured by analytical ultracentrifugation, the nature of a covalently closed circular single stranded RNA was deduced from electron-microscopy studies under denaturing

16

conditions, the rod-like native structure from hydrodynamic and thermodynamic studies, and the secondary structure of a series of short double helices and small internal loops was concluded originally from the extraordinary cooperativity in thermal denaturation experiments. Some physical parameters can presently be derived directly from the nucleotide sequence and the secondary structure (see ‘Structure of Pospiviroidae’ below) like the molecular weight, the circular structure, and so on; those features will not be discussed here. Other parameters, however, cannot be derived from the sequence with sufficient accuracy and therefore will be described briefly. These are

MOLECULAR CHARACTERISTICS

Table 2.2

Sedimentation coefficients of viroids.

s 20,w 0

Viroid

∆∆Α/∆Τ

∆Α

Length [nt]

[S]

260 nm

200

PSTVd

6.7±0.1

359

CEVd

6.7±.1

371

CCCVd-1s

5.9

247

CCCVd-1l

6.3±.1

302

CCCVd-2s

7.6±.1

494

CCCVd-2l

8.2

604

20 0.6

150 280 nm

Data are taken from Sänger et al. (1976) and Randles et al. (1982).

hydrodynamic, thermodynamic and kinetic properties of general interest.

25 0.8

15

100

0.4 10

50

0.2 5

0

0 0.0 40

50

60

70

80

T / °C Hydrodynamic properties

For PSTVd, CEVd, and for molecular species of CCCVd, sedimentation coefficients s020,w have been determined under native conditions (0.01 M Na-cacodylate, pH 6.8, 0.1 M NaCl, 5 mM MgCl, 1 mM EDTA) and extrapolated to standard conditions (zero concentration, 20, water). The values are listed in Table 2.2.

Figure 2.1 Optical denaturation curve of circular PSTVd. Buffer conditions are 500 mM NaCl, 0.1 mM EDTA, 1 mM Na-cacodylate, pH 6.8. In addition to the differentiated curve at 260 nm (∆∆A/∆T, thin line), the hypochromicities (∆A, thick lines) at 260 and 280 nm are depicted. The ordinates are given in milliabsorption units, the absorption of the sample at 40°C and 260 nm was 1.

When the hydrodynamic theory of a rigid prolate rotational ellipsoid has been applied to calculate the axial ratio a/b (large diameter/small diameter), for PSTVd the following value was obtained:

length the spatial orientation is lost due to flexibility. In other words, the molecule of length P is bent statistically to a quarter of a circle. The dependence of the S-values of viroids upon the molecular weights could be fitted by a persistence length P of 300 Å and a hydrodynamic diameter, i. e. including bound water, of 29 Å (Riesner et al. 1982).

a/b = 20. The partial specific volume v¯2 of viroids was evaluated as 0.53 cm3/g. Because the hydrodynamic behavior of viroids deviates from the model of a rigid rod, the data of the sedimentation coefficients have been interpreted in terms of the ‘worm-like chain’ model for a stiff polymer. The stiffness was described by the persistence length P according to Kratky and Porod, meaning that over this Table 2.3

Midpoint-temperatures Tm and halfpointtemperature ∆T1/2 of the thermal denaturation of viroids. Tm [°C]

∆T1/2 [°C]

Length [nt]

PSTVd

49.5

0.9±0.1

359

CEVd

50.5

1.0±0.1

371

CSV

48.5

1.1±0.1

354

CCCVd-1l

48.1

1.2±0.1

302

CCCVd-1s

48.8

1.4±0.1

247

ASBVd

37.5

1.5±0.1

247

Viroid

Buffer conditions were 10 mM NaCl, 0.1 mM EDTA, 1 mM Nacacodylate, pH 6.8. Data are taken from Henco et al. (1979) and Randles et al. (1982).

Structure formation and thermodynamic properties

Thermal denaturation of viroids was studied by optical melting curves (UV-absorption vs. temperature) and temperature-gradient gel electrophoresis (TGGE, migration velocity in polyacrylamide gel electrophoresis vs. temperature). Structure formation after synthesis by T7 polymerase from a DNA template was analyzed by TGGE. All viroids studied experimentally show a highly cooperative thermal transition (as shown in Fig. 2.1). The data for the midpoint-temperature Tm and the halfwidth ∆T1/2 (width of the transition in differentiated form ∆A/∆T vs. T at half height) are listed in Table 2.3. In most viroids a second transition with about 10% hypochromicity of the main transition could be detected 5 to 10°C above the main transition. The influence of ionic strength and of several organic solvents on the transition of viroids are useful to know for comparison of the results from different methods, in which different solvent conditions have to be used. The Tm- values of viroids depend upon the logarithm of ionic strength linearly between 5 mM and 1 M NaCl with a slope of 13.2°C/logcNa+. The cooperativity decreases slightly with increasing ionic strength, except CCCVd

17


35 oC

75 oC

-

cPSTVd PSTVd (5 S)2

IPSTVd 7S

+

Figure 2.2 Temperature-gradient gel-electrophoresis of a crude RNA extract from tomato plants infected with PSTVd. cPSTVd, lPSTVd: circular and linear PSTVd, respectively; (5 S)2: dimeric complex of 5 S RNA. Buffer conditions are 8 mM Tris-HCl, 20 mM NaOAc, 0.2 mM EDTA, pH 8.4; 5% acrylamide, 40:1 acrylamide/bisacrylamide. The stained material near the top of the gel is double-stranded DNA.

for which one transition at low ionic strength is split into three transitions at high ionic strength. Regarding the influence of Mg2+, as little as 10 M MgCl2 raises the Tm- value by about 30°C. Increasing urea or formamide concentrations decrease the Tm- value by about 4°C/1 M urea in the range between 0 and 8 M or by about 5°C/10% formamide in the range between 0% and 60%, respectively (Henco et al. 1979). In TGGE circular viroids show a highly cooperative transition at a temperature equivalent to that observed in optical denaturation curves (Figures 2.1 and 2.2). In samples also containing linear viroid molecules, several additional transitions may be seen at temperatures a few degrees below the main transition. Several well defined transition curves indicate that circular viroids were nicked, not randomly but at preferential sites. These data confirm the formation of linear viroid molecules in vivo (Hadidi and Diener 1977). A significant advantage of TGGE over optical transition curves is the potential to analyze simultaneously the transition curves of different viroid species or different RNAs in the same solution (see Figure 2.2). Thermal transition curves have been simulated using the sequence information, temperature-dependent thermodynamic stability parameters, and values for the hypochromicity of G:C, A:U and G:U base pairs. Experiment and theory can be compared best at 1 M ionic strength. For PSTVd, CEVd, and CSVd calculated and experimentally determined Tm- values agreed within 2°C or better. For CCCVd the split of one transition at low ionic strength into three transitions at high ionic strength was predicted with a fair accuracy of the single Tm- values (Randles et al. 1982; Steger et al. 1984). Together with kinetic studies on the thermal denaturation a detailed mechanism of the reversible denaturation-renaturation was derived and described in most detail for PSTVd (Riesner et al. 1979). Two characteristic regions — the pathogenicity region (see below), and a region from nt 74–92 in the upper strand with nt 281–267 in

18

the lower strand — show lowest stability and denature 5–10°C below the main transition. During the main transition all base pairs are disrupted and 1–3 (depending on the viroid species) particularly stable hairpins are newly formed. Hairpin II is of particular functional interest (see below). At higher temperatures the stable hairpins denature according to their individual stabilities. Thermodynamically metastable structures containing functionally relevant motifs are gene-rated during (-)- and (+)-strand replication intermediate synthesis. Those motifs will be discussed in a later section. Formation and dissolution of metastable structures were studied in vitro when PSTVd (-)-strands were synthesized by T7-RNA-polymerase and folded sequentially during synthesis into a variety of metastable structures that rearrange only slowly into equilibrium structures. The rate of synthesis was adapted to the in vivo rate of Pol II by altering the concentration of nucleotide triphosphates, and the structure distribution was analyzed by TGGE. It was concluded that viroids are able to form and use metastable structures on a realistic time scale for their biological functions (Repsilber et al. 1999).

STRUCTURE OF POSPIVIROIDAE In the following, secondary structures are calculated with RNAFold (Hofacker et al. 1994) version 1.31 and ConStrict (Lück et al. 1999) for a temperature of 25°C and drawn with RNAviz (Rijk and Wachter 1997). Sequences are aligned with help of ClustTalx (Jeanmougin et al. 1998) using standard parameters. Sequences are taken from ‘The subviral RNA database’ (Pelchat et al. 2000). The native structure of mature, circular forms of Pospiviroidae is generally described as rod-like without any bifurcations. This was established by biophysical means (electron microscopy, sedimentation analysis, dye binding, gel-electrophoresis, etc.), by chemical and enzymatic mapping, and by theoretical analysis for several viroids (for review see Riesner and Gross 1985); a standard example for PSTVd is shown in Figure 2.3. For CCCVd, partial duplications of the right structural end (see Section on Terminal right (TR) domain) as well as dimeric forms have been found (Mohamed et al. 1982). All these duplications would allow for branched, cruciform-like structures. The rod-like structure, however, could be confirmed also for CCCVd (Riesner et al. 1982). At least for PSTVd the presence of a tertiary structure (in the sense of tRNA-L-conformation) could be safely excluded. Several loops, however, may contain unusual base pairs; for an example see the description of the C domain (see below). As mentioned above the rod-like structure denatures with high cooperativity at unphysiologically high temperatures into branched conformations. The cooperativity is based on forma-


P

TL 40

20

1

C

60

80

V

100

I

TR

Pol II

I’

120

140

GCbox

160

C AAA A C AC UA C A -A U CC C UGGC A A AGG GGUGGGG GUG CC GCGGCCG AGGAG A U CCCG GA A A A G G G UU U U U U U C C C CGC U C C C A C G G C G C C G G C U C C U U U G G G G C C U U U U C C C AC U C C U U G G U U G A C G C C A A G G UUC C C G AUUU G U GGGAGGGGG U U C G U U C - AU U C U A U C - U C U U U U U C G C C G A G C C C U C G A A G U CA ACAAA G G G G C C C A U CAU C G G C U U C G C U G U C G C A A U UU C C C G CC U CC A A C C CCU G A G A C U GA U C G G A A C U A A C U G U G G U U C C U G G G U U C A C A C C U C C C C U G A G C A G A - AA A G A A A A A - A G A A G G C G G C U C G G G A

359

GCbox

II’

340

320

A A C GCUUCAGGG A U C C C C G G G G A A C CUGGAGCGA

280

300

240

260

Pol II

II

220

200

180

loop E

- UU A A G U A U 80 A U 40 C G 60 20 100 A U A AC U U U A G UUU C U U G U U U AG A A 240 C A A G U G A G G A U A U G A U U A A A C U U U G UU UG A CG AA C C A G GU C UGU U C C G A C U U U C GC CU U G A G A CG A G U G A G A G A G A G G A C G G G U G A A C U A U U U A U U A C U U C U A U A C U A U U U G G A A C G C U G C A G A A G G G U G A A A G G GA CUC G C U C A U U C U C U C U U C U G C U C A C U U G A U A A U G C U G U C A A CU A A U U U A A A A AG A C C C G U CU U C A U U U A A U 200 U 220 180 140 U U 160

HHrz (+)

NEP

HHrz ()

1

80

60

C G A A A U G A G G G U G U G U U C G G

C C A GU U U C U U C U C A A AG C C U A G G A C A

40

U

U U C -

G C U C U C C A C A G C C

A G A U C C A U G

U C GA G A U G C G A U G C C G

120

100 U C A U C AG G

A A A C C C A C U U C A G G U C U

AGU AGU C C

U G U G G C U G C A G U C C A G G C C

20

G U

AA

C A

1 U G

C C C

360

U

380

240

220

320

A G A A U G G A G G A G GC A AU U A C C U C GGG C A G - U C C A A C C C C G

180

160

200

340

300

260

100

20

140 A A CC U U A C U U C C U C UA

G C A A G A G G G UC U G U U U A C G A C A G G G U U G G G GA G AC G A A G G G C G C AG U C G C CU AG C A C G UC CC C U G U U A GU G AC U A AG G C CU UG A CA G A GG G U A C G C G CG G G U C C C A G AC A U A U A C G G A U A G A U U G G C A U G G G C A C G G C U A U A C U G C G C G A U GU C C GC U U C G G C G U G C C A U A U A U C C C U A G U U G C A U U C G U C U

C U G A C C G GA A U G U C U G C C C U C AG C A U U G C U U A A A C A C U G C A U U C U U A A C G C C G A C G U C G G G C C G C A G A A C U C G A C G G C G UG G A A U A A G C G C U U A G U C A U A A GU U U C U C A U C AG U G G G C U U A G C C C A G A C U U A A U C U U C U C A A AG G C A C C AGC AGU C A C A C G A A U C G U G U C UGA G A U U C GC A A C G U G U G G A G U A A U UG G G A G C A C A G C G A U C G U G G C C C A U U U C U C C A G G U UU G AGA U - U A U A A C C GUG C C G G U C U U G G C G G AA G A U A G G C A C G U A C U U C U G C G A C A U A G A G A G U U C GG A U G U C C U C G U A U C A C C G C G G U A A G GG C U C G C C C G A U A A A G

120

1

337

40

300

60

140

280

320

260

160

200

180

240

220

Figure 2.3 Structure of viroids. Consensus structures are shown but with sequence and numbering of individual variants. In structures of PSTVd (45 sequences) and ASBVd (33 sequences), the size of dots connecting base pairs is proportional to the pairing probability. For further details see text.

19


C A A

C U

C A GG A A C U A A C U G

CC U U G G U U

U GGU U C C U

G

U

A G U G C U A U A C G CU 3’ G U G G U U C A C A C C U G C

20

20 1

UC

1

3’ U G G G U U C A C A C C U

U

U G U G G G C C C G A G A C G C C A A G G UU U U

U G U G G G C C G A U U

U

C U C 359 UC GG U A G A 340 G C U C

5’

359

PSTVd

340

U G

5’

G AC

IRVd

20

A A U

1 370

G UG GU U C C C UCC C A A G G A A A

G G U G C A C C C

A U

U C G G G U G G G U C

350 Figure 2.4 Structures of TL region. The bold characters should help to identify the repeat regions in PSTVd; this bold sequence is, however, not conserved in all Pospiviroidae. In the sequence of IrVd only one of the repeat regions is present. Size of dots connecting base pairs is proportional to the pairing probability.

tion of stable hairpins that are not part of the rod-like structure. Common to all Pospiviroidae is Hairpin I formed from regions located in the upper center of the rod-like conformation (see Section on Central (C) domain). Hairpin II, unique to Pospiviroid, is a GC-rich hairpin of up to 12 base pairs (see Section on Hairpin II). Keese and Symons (1985) proposed a scheme for division of the rod-like structure into five domains. These domains comprise the left and right terminus (TL and TR, respectively), the central C domain including the CCR, and the P and V domains located in between the termini and the C domain (see labeling of PSTVd in Figure 2.3). This scheme relies mainly on sequence homology of viroid sequences available at that time. Because these domains seem to be correlated also with biological functions (see Section on ‘Structural motifs and their biological function’ below) we will follow this scheme for a more detailed description of the structure of Pospiviroidae. Take note that several viroids seem to be the result of recombination or polymerase template swapping between different parental viroids. For detailed information on this topic refer to the literature (Koltunow and Rezaian 1989; Hammond et al. 1989; Rezaian 1990; Spieker 1996; Kofalvi et al. 1997; Sano and Ishiguro 1998; Owens et al. 2000). In addition the terminal right domain seems to be a preferred site for sequence duplications; to a lesser extent this is also true for the terminal left domain which is discussed next.

20

Terminal left (TL) domain

The left terminal end of most Pospi- and Apscaviroid contains an imperfect repeat; in PSTVd the respective sequences are from nt 341 to 358 and from nt 2 to 21 (see Figure 2.4). Each repeat is an imperfect palindrome (PSTVd: nt 341-347/352-358 and 28/15-21) that allows for formation of a rod-like (top left in Figure 2.4) or a Y-shaped (top right) structure. According to calculations the rod-like structure is thermodynamically preferred for most Pospiviroidae; the only exception is a single CEVd variant (EMBL-ID S67437). The experimental analysis of both structures is described in a following section (Elongated left terminal domain of PSTVd). IrVd does not possess the above mentioned repeat sequence in the TL region (see bottom right in Figure 2.4). Thus, Spieker (1996) proposed that IrVd’s sequence is the result of a deletion of one of the repeats in the TL region of an ancestral Pospiviroid. Pathogenic (P) domain

The P domain, located in between TL and C domain, contains an oligopurine stretch in the top strand and the corresponding oligopyrimidine stretch in the bottom strand of most Pospiviroid. The resulting oligopurine/oligopyrimidine pairings result in a structural region that has a relatively low thermodynamic stability; hence the name ‘premelting’ (PM) region (Steger et al. 1984). Furthermore, after sequencing of the first pathogenicity variants of PSTVd, it was realized that slight sequence variations in this region tend to influence the pathogenicity of the viroid; hence the name ‘pathogenicity modulating’ (PM) region


(Schnölzer et al. 1985). It should be noted that pathogenicitymodulating mutations and/or regions are not restricted to this domain in other viroids (for examples see Visvader and Symons 1985; Sano et al. 1992).

would allow for Y-shaped or even cross-like cruciform structures that were excluded, however, by sedimentation analysis. A similar sequence duplication of the right terminal end was found with CEVd (Semancik et al. 1994).

Central (C) domain

Hairpin II

The central domain of Pospiviroidae is the most highly conserved region among viroids. In addition to the possibility for pairing between the top strand (upper central conserved region, UCCR) and the bottom strand (lower central conserved region, LCCR) both strands have the possibility for pairing with itself. In Pospi-, Hostu- and Cocadviroid the top strand is able to form a thermodynamically stable hairpin of nine base pairs, called hairpin I, during thermal denaturation (Henco et al. 1979; Riesner et al. 1979); the nucleotides of hairpin I in PSTVd are shown in bold in Figure 2.3. The hairpin I loop with 14 to 15 nt contains a palindrome 5’KCSYCGRSGM3’ (K = G∨U; S = C∨G; Y = U∨C; R = A∨G; M = A∨C; see Figure 2.5).

From optical melting curves and kinetic studies it was concluded that viroids denature in a highly cooperative transition due to dissociation of the base pairs of the native conformation and formation of stable hairpins that are not part of the native conformation (Henco et al. 1979; Riesner et al. 1979; Randles et al. 1982; Steger et al. 1984). In case of Pospiviroid (PSTVd, CEVd, CSVd) the stable hairpins are hairpin I (see Section on ‘Central (C) domain’) and hairpin II, which is located in the bottom strand of V and TL domain (nt 227–236/319–328 in PSTVd; see Figure 2.3). The hairpin II helix has a length of 11 to 12 nt and is rich in GC content (see Figure 2.8).

In the centre of the C domain of many Pospiviroid is located a particular internal loop (nt 98-102 and 256-261 in PSTVd; see Figure 2.3); it shows homology to loop E of eukaryotic 5 S RNA. In the eukaryotic loop E all nucleotides are involved in non-WC pairings (Wimberly et al. 1993) that results in the possibility for UV-crosslinking. UV-irradiation of native PSTVd RNA crosslinks G98 with U260 (Branch et al. 1985; Paul et al. 1992). A variant of PSTVd, able to infect Nicotiana tabacum, differs from a PSTVd, which is not able to infect tobacco but tomato, by a single nucleotide substitution of C259 to U located in the lower part of the loop E-like element (Wassenegger et al. 1996). In Apscaviroid the hairpin I consists of two helices separated by a mismatch; the hairpin I loop contains again a palindrome 5’ UYGUCGRCGA3’ (see Figure 2.6). In all Pospiviroidae the C domain sequences allow for alternative structures, especially when a true 5' end is located in the palindrome region (for an example see Figure 2.10). Variable (V) domain

This domain is the most variable region showing low sequence homology between otherwise closely related viroids. The boundaries of the V domain have been defined by a change from low sequence homology to the adjacent C and TR domains (Keese and Symons 1985). Terminal right (TR) domain

By sequence duplications of the right terminal end, the smallest CCCVd, termed CCCVd-1fast or -1small (246/247 nt), gives rise to longer molecules, termed CCCVd-1slow or -1long (287–301 nt). These molecules, depicted in Figure 2.7, do exist also in dimeric, circular forms, termed CCCVd-2fast/small or 2slow/long (492/494 nt and 574–602 nt). All the duplications

In PSTVd an additional hairpin III (nt 127–134/160–168) takes part in the cooperative transition. In CCCVd only hairpin I is present.

STRUCTURE OF AVSUNVIROIDAE Avsunviroid (ASBVd) possesses a rod-like native structure except a Y-shaped conformation at the left terminal end as predicted by calculations (see Figure 2.3). Members of Pelamoviroid do not share a remarkable sequence similarity with ASBVd, apart from the short sequence regions necessary for their hammerhead ribozymes (see the following Section). The structure of a Pelamoviroid deviates more drastically from a rod-like conformation (see Figure 2.3). Furthermore, ASBVd is an unique viroid with a GC-content of only 37.8% whereas the mean GCcontent of the other viroids is near or even above 50%.

STRUCTURAL MOTIFS AND THEIR BIOLOGICAL FUNCTION

Viroid structure can be regarded as a collection of structural motifs which play their specific functional role in viroid replication, processing, transport, and pathogenesis. As discussed previously, viroids assume a stable secondary structure as (+)-stranded mature circles. However, during (-)-strand- or (+)-strand synthesis they can be trapped kinetically in metastable structures, in which local structures or structural motifs can be stable for some time and might carry out specific functions. In the following paragraphs the structural motifs are discussed together with their functional roles; for an overview see Figure 2.9. Elongated left terminal domain of PSTVd

As mentioned earlier, secondary structure modeling predicted for the left terminal domain of PSTVd an elongated stem-loop structure and a less stable branched structure (Riesner et al. 1979), whereas Gast et al. (1996) favored a branched native

21


| A C A G AUCC CGG GAAA CU GA | GA -

A

A

G

ACC U

U

-

G

-

C

U

CA

UG

U

GCU U

A GC

C

U

-

UC

G

A

U

U

C

AG

C

U

bits

MMAMM U AG U AACG

M

C GC G U UG CCA

CGU

G

M

A

G CG

UC CAA A

G

M

A

G A

M

M

UGGCG

MMA M G U

AGU G UC MC CU CU G UA A G

M

U

0

M

M

A

1

MM

G C C G

U

M

GUG

M M

C

M

UC

2

C C U A

G G A A G

G G A C U U C G 5’ A G GA G C

: : : : : : : : :

A C C U G G A G C 3’ GA A C U GG C AA A

PSTVd Figure 2.5 Structural alignment of the UCCR of Pospi-, Hostu- and Cocadviroid. Top: A structure logo (Schneider & Stephens 1990; Gorodkin et al. 1997) with height of characters proportional to the frequencies of the nucleotide in this position. When a nucleotide character appears less than expected the character is displayed upside-down; the expected frequencies are based on full sequences of all Pospi-, Hostu- and Cocadviroid available from ‘The subviral RNA database’ (Pelchat et al. 2000). The mutual information, marked by character ‘M’, is calculated as the relative entropy between the fractions of complementary bases at indicated base-paired positions in the alignment and the number of base pairs one would expect by chance from the distribution of nucleotides at the involved positions; i.e. the size of the character M is proportional to the statistical significance for base-pairing of this position. Middle: Consensus secondary structure in bracket-dot notation. Note that the central base pairs are not statistically supported because of their absolute conservation. Furthermore, in monomeric sequences the central base pairs are not possible due to a minimum hairpin size of three nucleotides. Bottom: Alignment of the UCCR of selected Pospiviroidae. The EMBL-IDs of the sequences are given in brackets. Right: Secondary structure model of PSTVd's UCCR; dotted lines show complementary nucleotides in the hairpin I loop.

structure. The structure of the left terminal stem-loop was investigated by site-directed mutagenesis and Agrobacterium-mediated inoculation. It was concluded that disruption or destabilisation of this structure primarily prevented transcription (Hammond 1994; Hu et al. 1997). Recently the structure was investigated in detail by NMR and TGGE (Dingley et al. 2002) with the PSTVd-fragment (nt 326-359/1-35; see Figure 2.4). In addition two mutant fragments were studied, one (U18 to C, A344 to G) strongly favoring the elongated structure (G:C pair instead of A:U in the elongated structure and A.C in the branched structure), and the second (U18 to C, A5 to G) favoring the branched structure (G:C pair instead of A:U in the branched and A–C in the elongated structure). TGGE analysis showed that the wt fragment behaved like the elongated mutant, and quite different from the branched mutant. In the analysis by NMR all signals could be attributed to the base pairs of the secondary structure, and the base pairs of the wt fragment were identical to the elongated mutant but different from the branched mutant. In the presence or absence of Mg2+ the same

22

conclusions could be drawn, from TGGE as well as from NMR. It should be noted that the study of this viroid fragment led to a breakthrough in NMR methodology. Dingley and Grzesiek (1998) discovered the effect of J-coupling through hydrogen bonds, by which method they could clearly identify the individual nucleotides of opposite strands involved in the particular base pair. The three-dimensional structure of the left terminal domain with atomic resolution should be available in the near future. Motifs involved in (-)- and (+)-strand synthesis

Start sites and structural motifs involved in complementary strand synthesis were studied in PSTVd and ASBVd. PSTVd is transcribed by the host-encoded DNA-dependent RNA Pol II. In an in vitro transcription extract prepared from a non-infected cell culture with circular PSTVd added as template, two specific start sites could be identified. In both cases the nucleotides transcribed first are adenosines, A111 and A325, and the first 7 nucleotides are identical with one exception,


U

C

-

AC

G C

G

U

G

U C

A

A C U C C G

: : : : :

G G G G U

G U G A G 5’ GA GG G G

: : : : : :

G C A C U C 3’ C G A GU GC C UG A

G

A

UC A GU G

C G

C G A

U

G AG

AAGAUCAUC

C

G

U

GU A

C

UGAUC

A AG

C

U

C

G

U G C U

M

C

U C

0

C CU

M MM A GMUMGUC CAMMUCG GUG CGU GA UG C C C AAU

A

M

M

G

bits

| AGGG UCG CGUCGACG AG A C | M M MA M MAM GAUUA GMC GACG GC G M

GUA

1

C

2

A

GYSVd1 Figure 2.6

Structural alignment of the UCCR of Apscaviroid. For description see legend to Figure 2.5.

GGAGCGA325 and 319GGGGCGA325. In a distance of 15 or 16 nts upstream, respectively, GC-boxes are located in the native viroid structure which might act as promoters (Fels et al. 2001). In Figure 2.3 the sections of the native structure containing the start sites and the GC boxes are depicted.

105

From site-directed mutagenesis of PSTVd it was concluded that the formation of a thermodynamically metastable structure including a GC-rich hairpin (Hairpin II, Figure 2.9) is critical for infectivity and in particular acts as a functional element of the (-)-strand replication intermediate (Loss et al. 1991; Qu et al. 1993). Its presence in vivo in the (-)-strand could be demonstrated recently by a combination of oligonucleotide probing and TGGE (Schröder and Riesner 2002). Navarro and Flores (2000) characterized initiation sites of both polarities in ASBVd. ASBVd is quite different from PSTVd in the sense that it is located in the chloroplast and is replicated by a nuclear encoded polymerase (NEP). The replication intermediates were extracted from infected cells and analyzed by an in vitro capping assay. Linear (+)- and (-)-strands begin with a UAAAA sequence that maps to the corresponding right terminal loops in the (+)- (nt 121) and (-)-strand (nt 119 with numbering of (+)-strand) structures (cf. Figure 2.3). Motifs involved in processing oligomeric intermediates to circles

In Pospiviroidae cleavage and ligation of replication intermediates is catalyzed by host encoded nucleases and ligases. Both reactions were analyzed in a potato nuclear extract using a longer-than-unit-length (+)-strand-transcript of PSTVd that

contained the UCCR twice, i. e. at the 5'- and the 3'-terminus. As depicted in Figure 2. 10 both termini together with the lower central conserved region fold into a multi-helix junction containing at least one GNRA tetraloop-hairpin. This local structure is metastable, converts slowly into the extended rod-like structure and thereby loses its template activity for the processing reaction. By a combination of biochemical, biophysical and phylogenetic analysis the mechanism was elucidated: the first cleavage occurs between G95 and G96 within the stem of the GNRA tetraloop; a local conformational change switches the tetraloop motif into a loop E motif which is present also in the circular rod-like structure. After the second cleavage between G95 and G96, this time close to the 3'-terminus, both terminal nucleotides are base paired in an optimal juxtaposition for ligation. The ligation to mature circles can occur autocatalytically with low efficiency, or enzymatically with high efficiency (Baumstark et al. 1997). In contrast to Pospiviroidae, Avsunviroidae (ASBVd, PLMVd, and CChMVd) can form hammerhead structures, in strands of both polarities (for review see Flores et al. 1999). Accordingly they act as ribozymes and mediate self-cleavage of the oligomeric RNA-intermediates. Ligation is catalyzed by an RNA ligase. In Figure 2.11 the hammerhead structures of ASBVd, PLMVd, and CChMVd are shown for the (+)-strand polarity. Similar hammerhead structures were given for (-)-polarity strands. The sequence regions taking part in hammerhead formation are given by bold characters in Figure 2.3. The monomeric strands of PLMVd and CChMVd can form stable hammerhead structures and self-cleave accordingly in vitro. However, the hammerhead structure of the monomeric

23


100 A

U G C G C G C

C

120 A - -

C G U A UU C U G G G C G U C G U G C G C G G G A G

G G C C C G C A G C C G U G C C C U C UU UU A A

G -

CCCVd1fast - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

140 U G C G C G C


C G C

A

U G C G

G -


- - - - - C A G C C G U G C C C U C AG UU UU A

CCCVd.6 C U A UU U G G G C G U C G U G C G C G G G A G GA G C C C G C A G C C G U G C C C U C AG UU UU A

A A C

CCCVd.5


C

-

-


A

U A UU - - - - C G U C G U G C G C G G G A G GA

- -


C

CCCVd1slow

A - -

G G C C C G C A G C C G U G C C C U C UU UU A A -

C

U G C G

C G C

A

G -

G C

C U A UU U G G G C G U C G U G C G C G G G A G GA

G C C C G C A G C C G U G C C C U C AG UU UU A G

Figure 2.7 Partial sequence duplications in CCCVd. Black bars denote the ends of partial sequences and structural elements that are duplicated in the respective variant.

2

| CC UCGC C G GCGAG GU| M

M

G

C A

C G

C

G A

G

A

C

C

U

U

A GG

U

U

0

82 nt

M

C

M

M

U

1

M

A

bits

M

M

C C C C G C U C C C

5’

: : : : : : : : : :

G G G G C G A G G G

3’

PSTVd Figure 2.8 Structural alignment of hairpin II from Pospiviroid. The numbers in between the sequence parts give the size of the hairpin loop. For further description see legend to Figure 2.5.

24


I

TL

I’ III

II’

III’

TR

II

II TR

tl

TL

TR

TL tl

tl TR

TL

Figure 2.9 The replication cycle of PSTVd is a structural cycle. The mature circular viroid assumes a rod-like secondary structure under native conditions (top). During thermal denaturation the rod-shaped structure dissociates cooperatively and stable hairpins are formed by pairing of regions I/I', II/II' and III/III' (not shown). Transcription by host-endcoded DNA-dependent RNA polymerase II results in (-)-stranded oligomers, which are sequentially folded into thermodynamically metastable conformations including hairpin II (right). This hairpin II is critical for transcription by polymerase II into (+)-stranded oligomers. The (+)-strands rearrange into structures with termini TL and TR, which are similar to those of mature RNA; their central region forms a metastable structure that contains tetraloop-hairpins tl (bottom). Host-encoded enzymes recognize this conformation and process the oligomers to mature circles (left; the cleavage and ligation point is marked by a triangle). The major part of mature circles accumulates in nucleoli of infected cells.

5’

80 G C 96 3’ U U G C G A C C G C G A C G G U A 80 G A U G G C C G G A G C C C G G A G C U U C A G C C U C

G A A G U C A

280

G G

A U C G A G A A

100 A A C 110 120 C A C U A G G A G C G A U G G C A A A A

C U U C G C U C G C G C C U A U CA

G U C G C G U U

240

260 Figure 2.10 Consensus structure of the CCR in processing configuration.The sequence and numbering of PSTVd is shown, for which the model was established (Baumstark and Riesner 1995; Baumstark et al. 1997). The structure is based on a consensus model of 11 Pospiviroid sequences (PSTVd, MPVd, CEVd, CSVd, TASVd, TPMVd, CCCVd1f, HLVd, CPFVd, HSVd, and AGVd). Size of dots connecting base pairs is proportional to the consensus pairing probability. The triangle marks the processing site.

25


5’

ASBVd (simple)

5’

U UG A C

A

G

C C A A

A

PLMVd

G G U C

C

U

3’

U G U U CC

G

UC U G A 3’ G

U A G

A C A A GA C 5’ U

AA

A U

G A G

A

A

U U C U C A

3’ A A U G A A G G U d C

G

U G U G C U A . U G A G A C A A A C A C G A AGU C U C U G C A C A U U G A U A G

U

C

ASBVd (double) U 5’ C A G A A C A

G

A U G

CC U U G U

3’

C U G G A C C A A

5’ U G A CG UC U G A 3’ G

A

3’ A G U CU

G

C A GU

5’

A

A

C

3’

U G U U CC

U A G

5’

CChMVd

A C C A G G U

A C A A GA C 5’ U

U U C U C C A A

C AG G A U CG A G

U AC C U A G A A

A

3’ A A G A G G U

d

C

G C A

U G

A C C U G U G G G C U C U d d G G G C A C C

U

U

Figure 2.11 Schematic drawings of the hammerhead structures that can be formed in (+)-strand sequences of ASBVd, PLMVd, and CChMVd. Self-cleavage sites are given by a bolt arrow. Small arrows show nucleotide substitutions in variants of the respective viroid; a ‘d’ marks deletion of a nucleotide. For location of the hammerhead sequences in the native structure see Figure 2.3.

ASBVd is thermodynamically unstable, mainly because of loop III with two basepairs in the stem and three nucleotides in the loop in (+) polarity, and three base pairs and three nucleotides in (-) polarity, respectively. Thus, self-cleavage is restricted in monomeric structures. In oligomeric intermediates of ASBVd a stable double-hammerhead structure can be formed (see Figure 2.11), that very likely catalyze their selfcleavage to linear monomers. Cell-to-cell and long distance movement motif

Applying Agrobacterium-mediated inoculation of PSTVd cDNAs onto tomato it was found that some mutants could not replicate at all, some mutants did replicate but were restricted to specific tissues like gall and roots, whereas the corresponding inoculation with wildtype PSTVd led to a systemic infection. Obviously Agrobacterium-mediated inoculation could result in local replication but cell-to-cell or long distance movement was prevented. It was concluded that the structure or stability of the right terminal stem-loop is essential for cellto-cell and/or long distance movement. Possibly an essential

26

RNA movement protein interaction was disrupted (Hammond 1994). Pathogenicity (or virulence) modulating domain

As determined from sequencing PSTVd-strains of different pathogenicity (Schnölzer et al. 1985), only 40 of the 359 nucleotides represent the pathogenicity modulating (P, Figure 2.3) region. The segments nt 40–60 in the upper strand and 300–321 in the lower strand form a partially double stranded region. Several attempts to correlate the thermodynamic stability of this region with virulence finally failed (Owens et al. 1996). Another hypothesis which was derived from model building postulated a higher degree of bending for more virulent strains (Figure 2.12). This hypothesis could be confirmed experimentally by electrophoresis in polyacrylamide gels. In the absence of Mg2+ the P-regions differ only in terms of flexibility. Addition of Mg2+ induces rigid bending, and the angle of bending increases monotonically with the pathogenicity of the strain (Schmitz and Riesner 1998). Binding of the dsRNA-activated protein kinase (PKR) to the P-region was discussed earlier as a primary pathogenic event (Diener et al. 1993).


Mild

36 .. . . ..

50

40

G A C C U C C U G A G C A G A G G G G C U U C G U U A G C

325 36

C

G G G G C U U C G U U A G C

320

300

... .. .

292

.. . ...

G G G G C U U C G U U A G C

311a

320 40

G G G G C A G C

320

A

A G A A G G C G G

U

U U U U U C G C C C

300

A A

A UC

CU

CA A

G

G U U C C

... .. .

292

43

310

Severe 60

68

A A A A G A A A

A G A A G G C G G

U U C U A U C

310

A

68

A A A A G A A A

U U C U A U C A C

U

U U U U U C G C C C

309

300

.. . ...

292 310

RG1

50

G A C C U C C U G A

.. . ...

Intermediate 60

50 50a

40

325

U U U U U C G C C C

310

G A C C U C C U G A G C A G

36

U

50

G A C A C U C C U G A G C A G

325

A G A A G G C G G

U C U A U C

311

.. . ...

36

68

A A AA A G A A A

U UU

311a 310

320

40

325

60

60 A A

68

A A A A G A A A

A G A A G G C G G

U U C U A U C

U U U U U C G C C C

310

U

300

.. . ...

292

45

310

Figure 2.12 Schematic representations of the PSTVd pathogenicity-modulating region. For each natural sequence variant, the vertical arrows denote corresponding positions in the two- and three-dimensional structures. Left: Thermodynamically optimal secondary structures of selected PSTVd variants. Right: Two-dimensional representations of the corresponding three-dimensional structures. A standard A-type helix is assumed, and the helix axis (indicated by dotted lines) is bent to compensate for missing nucleotides in asymmetrical loops. Modified from Owens et al. (1996).

References Baumstark, T., and Riesner, D. (1995). Only one of four possible secondary structures of the central conserved region of potato spindle tuber viroid is a substrate for processing in a potato nuclear extract. Nucleic Acids Res. 23, 4246-4254. Baumstark, T., Schröder, A.R., and Riesner, D. (1997). Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation. EMBO J. 16, 599-610. Branch, A.D., Benenfeld, B.J., and Robertson, H.D. (1985). Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5 S RNA. Proc. Nat. Acad. Sci. USA 82, 6590-6594. Diener, T.O., Hammond, R.W., Black, T., and Katze, M.G. (1993). Mechanism of viroid pathogenesis: differential activation of the interferon-induced, double-stranded RNA-activated, M r 68,000 protein kinase by viroid strains of varying pathogenicity. Biochimie 75, 533-558. Dingley, A.J., Esters, B., Steger, G., Riesner, D., and Grzesiek, S. (2002). The structure of the TL domain of potato spindle tuber viroid. (Submitted.)

Dingley, A.J., and Grzesiek, S. (1998). Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide 2JNN couplings. J. Am. Chem. Soc. 120, 8293-8297. Fels, A., Hu, K., and Riesner, D. (2001). Transcription of potato spindle tuber viroid by RNA polymerase II starts predominantly at two sites. Nucleic Acids Res. 29, 4589-4597. Flores, R., Navarro, J.A., de la Peña, M., Navarro, B., Ambrós, S., and Vera, A. (1999). Viroids with hammerhead ribozymes: some unique structural and functional aspects with respect to other members of the group. Biol. Chem. 380, 849-854. Flores, R., Randles, J.W., Bar-Joseph, M., and Diener, T.O. (2000). Subviral agents: viroids. Pages 1009-1024 in: Virus taxonomy, 7th Report of the International Committee on Taxonomy of Viruses. M.H.V. van Regenmortel, C.M. Fauquet, D.H.I. Bishop, E.B. Carstens, M.K. Estes, S.M. Lemon, J. Maniloff, M.A. Mayo, D.J. McGeoch, C.R. Pringle, and R.B. Wickner, eds. Academic Press: San Diego, CA. Gast, F.U., Kempe, D., Spieker, R.L., and Sänger, H.L. (1996). Secondary structure probing of potato spindle tuber viroid (PSTVd) and sequence comparison with other small pathogenic RNA replicons

27


provides evidence for central non-canonical base-pairs, large Arich loops, and a terminal branch. J. Mol. Biol. 262, 652-670. Gorodkin, J., L.J., Heyer, Brunak, S., and Stormo, G.D. (1997). Displaying the information contents of structural RNA alignments: the structure logos. Comp. Appl. Biosci./Bioinformatics 13, 583-586. Hadidi, A., and Diener, T.O. (1977). De novo synthesis of potato spindle tuber viroid as measured by incorporation of 32P. Virology 78, 99-107. Hammond, R.W. (1994). Agrobacterium-mediated inoculation of PSTVd cDNAs onto tomato reveals the biological effect of apparently lethal mutations. Virology 201, 36-45. Hammond, R., Smith, D.R., and Diener, T.O. (1989). Nucleotide sequence and proposed secondary structure of Columnea latent viroid: a natural mosaic of viroid sequences. Nucleic Acids Res. 17, 10083-10094. Henco, K., Sänger, H.L., and Riesner, D. (1979). Fine structure melting of viroids as studied by kinetic methods. Nucleic Acids Res. 6, 3041-3059. Hofacker, I.L., Fontana, W., Stadler, P.F., Bonhoeffer, S., Tacker, M., and Schuster, P. (1994). Fast folding and comparsion of RNA structures. Monatsh. Chem. 125, 167-188. Hu, Y., Feldstein, P.A., Hammond, J., Hammond, R.W., Bottino, P.J., and Owens, R.A. (1997). Destabilisation of potato spindle tuber viroid by mutations in the left terminal loop. J. Gen. Virol. 78, 1199-1206. Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G., and Gibson, T.J. (1998). Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23, 403-405. Keese, P., and Symons, R.H. (1985). Domains in viroids: evidence of intermolecular RNA rearrangement and their contribution to viroid evolution. Proc. Nat. Acad. Sci. USA.82, 4582-4586. Kofalvi, S.A., Marcos, J.F., Cañizares, M.C., Pallás, V., and Candresse, T. (1997). Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. J. Gen. Virol. 78, 3177-3186. Koltunow, A.M., and Rezaian, M.A. (1989). Grapevine viroid 1B, a new member of the apple scar skin viroid group contains the left terminal region of tomato planta macho viroid. Virology 170, 575-578. Loss, P., Schmitz, M., Steger, G., and Riesner, D. (1991). Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA. EMBO J. 10, 719-727. Lück, R., Gräf, S., and Steger, G. (1999). ConStruct: A tool for thermodynamic controlled prediction of conserved secondary structure. Nucleic Acids Res. 27, 4208-4217. Mohamed, N.A., Haseloff, J., Imperial, J.S., and Symons, R.H. (1982). Characterization of the different electrophoretic forms of the cadang-cadang viroid. J. Gen. Virol. 63, 181-192. Navarro, J.A., and Flores, R. (2000). Characterization of the initiation sites of both polarity strands of a viroid RNA reveals a motif conserved in sequence and structure. EMBO J. 19, 2662-2670. Owens, R.A., Steger, G., Hu, Y., Fels, A., Hammond, R.W., and Riesner, D. (1996). RNA structural features responsible for potato spindle tuber viroid pathogenicity. Virology 222, 144-158. Owens, R.A., Yang, G., Gundersen-Rindal, D., Hammond, R.W., Candresse, T., and Bar-Joseph, M. (2000). Both point mutation and RNA recombination contribute to the sequence diversity of citrus viroid III. Virus Genes 20, 243-252.

28

Paul, C.P., Levine, B.J., Robertson, H.D., and Branch, A.D. (1992). Transcripts of the viroid central conserved region contain the local tertiary structural element found in full-length viroid. FEBS Lett. 305, 9-14. Pelchat, M., Deschenes, P., and Perreault, J.P. (2000). The database of the smallest known auto-replicable RNA species: viroids and viroid-like RNAs. Nucleic Acids Res. 28, 179-180. Qu, F., Heinrich, C., Loss, P., Steger, G., Tien, P., and Riesner, D. (1993). Multiple pathways of reversion in viroids for conservation of structural elements. EMBO J. 12, 2129-2139. Randles, J.W., Steger, G., and Riesner, D. (1982). Structural transitions in viroid-like RNAs associated with cadang-cadang disease, velvet tobacco mottle virus, and Solanum nodiflorum mottle virus. Nucleic Acids Res. 10, 5569-5586. Repsilber, D., Wiese, U., Rachen, M., Schröder, A.R., Riesner, D., and Steger, G. (1999). Formation of metastable RNA structures by sequential folding during transcription: time-resolved structural analysis of potato spindle tuber viroid (-)-stranded RNA by temperature-gradient gel electrophoresis. RNA 5, 574-584. Rezaian, M.A. (1990). Australian grapevine viroid—evidence for extensive recombination between viroids. Nucleic Acids Res. 18, 1813-1818. Riesner, D., and Gross, H.J. (1985). Viroids. Ann. Rev. Biochem. 54, 531-564. Riesner, D., Henco, K., Rokohl, U., Klotz, G., Kleinschmidt, A.K., Domdey, H., Jank, P., Gross, H.J., and Sänger, H.L. (1979). Structure and structure formation of viroids. J. Mol. Biol. 133, 85-115. Riesner, D., Kaper, J.M., and Randles, J.W. (1982). Stiffness of viroids and viroid-like RNA in solution. Nucleic Acids Res. 10, 5587-5598. Riesner, D., and Steger, G. (1990). Viroids and viroid-like RNA. Pages 194-243 in: Nucleic acids, Subvolume d, Physical Data II, theoretical investigations. W. Saenger, ed. Landolt-Börnstein, Group VII Biophysics, Vol I. Springer-Verlag: Berlin. Rijk, De, P., and Wachter, De, R. (1997). RnaViz, a program for the visualisation of RNA secondary structure. Nucleic Acids Res. 25, 4679-4684. Sänger, H.L., Klotz, G., Riesner, D., Gross, H.J., and Kleinschmidt, A.K. (1976).Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures. Proc. Nat. Acad. Sci. USA 73, 3852-3856. Sano, T., and Ishiguro, A. (1998). Viability and pathogenicity of intersubgroup viroid chimeras suggest possible involvement of the terminal right region in replication. Virology 240, 238-244. Sano, T. Candresse, T., Hammond, R.W., Diener, T.O., and Owens, R.A. (1992). Identification of multiple structural domains regulating viroid pathogenicity. Proc. Nat. Acad. Sci. USA 89, 10104-10108. Schmitz, A., and Riesner, D. (1998). Correlation between bending of the VM region and pathogenicity of different potato spindle tuber viroid strains. RNA 4, 1295-1303. Schneider, T.D., and Stephens, R.M. (1990). Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 18, 6097-6100. Schnölzer, M., Haas, B., Ramm, K., Hofmann, H., and Sänger, H.L. (1985). Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV). EMBO J. 4, 2181-2190.


Schröder, A.R., and Riesner, D. (2002). Detection and analysis of hairpin II, an essential metastable structural element in viroid replication intermediates. Nucleic Acids Res. 30, 3349-3359. Semancik, J.S., Szychowski, J.A., Rakowski, A.G., and Symons, R.H. (1993). Isolates of citrus exocortis viroid recovered by host and tissue selection. J. Gen. Virol. 74, 2427-2436. Semancik, J.S., Szychowski, J.A., Rakowski, A.G., and Symons, R.H. (1994). A stable 463 nucleotide variant of citrus exocortis viroid produced by terminal repeats. J. Gen. Virol. 75, 727-732. Spieker, R.L. (1996). The molecular structure of Iresine viroid, a new viroid species from Iresine herbstii (‘beefsteak plant’). J. Gen. Virol. 77, 2631-2635. Steger, G., Hofmann, H., Föärtsch, J., Gross, H.J., Randles, J.W., Sänger, H.L., and Riesner, D. (1984). Conformational transitions in viroids and virusoids: Comparison of results from energy minimization

algorithm and from experimental data. J. Biomol. Struct. Dyn. 2, 543-571. Visvader, J.E., and Symons, R.H. (1985). Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity. Nucleic Acids Res. 13, 2907-2920. Wassenegger, M., Heimes, S., and Sänger, H.L. (1994). An infectious viroid RNA replicon evolved from an in vitro-generated non-infectious viroid deletion mutant via a complementary deletion in vivo. EMBO J. 13, 6172-6177. Wassenegger, M., Spieker, R.L., Thalmeir, S., Gast, F.U., Riedel, L., and Sänger, H.L. (1996). A single nucleotide substitution converts potato spindle tuber viroid (PSTVd) from a noninfectious to an infectious RNA for Nicotiana tabacum. Virology 226, 191-197. Wimberly, B., Varani, G., and Tinoco, I., Jr. (1993). The conformation of loop E of eukaryotic 5 S ribosomal RNA. Biochemistry 32, 1078-1087.

29

PART II

CHAPTER 3

BIOLOGY ....................................................................................................


.................................................................................................................................................................................................................................................................

There are many reports of putative virus diseases of plants present early in the scientific literature which are currently known to be caused by viroids. The first cases to be partially characterized were the potato spindle tuber and citrus exocortis disease agents, which resulted in the existence of an unusual transmitted molecule as the causal agent which became known as the ‘viroid’. For more than three decades, over 30 viroid diseases have been reported and their causative agents fall into two structurally/functionally distinct families. In addition to causing disease directly, there are also a number of symptomless or latent viroid infections now known which might act as potential reservoirs of disease.

Avsunviroidae have narrower host ranges than most members of the family Pospiviroidae.

Transmission methods for viroid diseases are currently well understood in most cases. In terms of their worldwide distribution, man is probably the single biggest factor, spreading viroid infection in the field by the international exchange of germplasm and by vegetative propagation of many commercial crops. Viroids are the most efficient infectious agents. They are the smallest plant pathogens, encode no protein, bear no protective coat, have the widest distribution, have little or no tissue barriers and are able to change by recombination and mutation

Interestingly, two other viroids with hammerhead structure also have narrow host range. Chrysanthemum chlorotic mottle viroid (ChCMVd) is known only to infect chrysanthemum (Romaine and Horst 1975; Navarro and Flores 1997) and Peach latent mosaic viroid (PLMVd) infects only stone fruits and pears, which are all in the family Rosaceae, (Shamloul et al. 1995; Hadidi et al. 1997; Faggioli et al. 1997; Giunchedi et al. 1998; Osaki et al. 1999; Kyriakopoulou et al. 2001).

HOST RANGE Viroids differ greatly in the breadth of their reported host ranges, as shown in Table 3.1. Generally, viroids in the family

30

Family Avsunviroidae

Avocado sunblotch viroid (ASBVd) has an extremely narrow host range, infecting almost exclusively avocado or related species (da Graça and Van Vuuren 1980). It shows little overlap in host range with other viroids, apart from a single report of naturallyoccurring PSTVd in avocado in Peru (Querci et al. 1995) and of Citrus bent leaf viroid (CBLVd) or Citrus viroid Ib (CV-Ib) being passed in avocado seedlings (Ben-Shaul et al. 1995).

Family Pospiviroidae

Hop latent viroid (HLVd) infects only hops (Humulus lupulus) and has been detected in all 80 cultivars tested. It was assayed unsuccessfully in cucumber, tomato, Benincasa hispida, Gynura

BIOLOGY Table 3.1 Viroid

Host range of viroids. Main host(s)

Family

Others

Indicator

avocado

Lauraceae

cinnamon

avocado

PLMVd2,3

peach

Rosaceae

stonefruit: plum, apricot, cherry, pomefruit: pear

peach GF305

ChCMVd4

chrysanthemum

Compositae

Dendranthema grandiflora = Chrysanthemum morifolium

C. morifolium cv. ‘Mistletoe’

PSTVd5

potato

Solanaceae, and others

tomato, pepino, avocado

tomato, Scopolia sinensis, Solanum berthaultii

TCDVd6

tomato

Solanaceae

Nicandra physaloides, Physalis spp.

TPMVd7

tomato

Solanaceae

TASVd8

tomato

Solanaceae

S. pseudocapsicum

tomato

CEVd9

citrus spp.

Rutaceaea, Vitaceae, Compositae, Solanaceae

broad bean, carrot, chrysanthemum, eggplant, grapevine, petunia, potato, tomato, turnip, Zinnia elegans

Gynura aurantiaca

ITBTVd10

tomato

Solanaceae, Rutaceae

citrus, cucumber

cucumber, Rangpur lime, ‘Etrog’ citron

CSVd11

chrysanthemum

Compositae, Solanaceae

petunia, Ageratum, Cineraria

tomato, Gynura aurantiaca

CLVd12

Columnea

Solanaceae, Gesneriaceae

tomato, Brunfelsia undulata, potato, pouchflower

tomato, potato

IrVd13

beefsteak plant

Amaranthaceae

Iresine herbstii

none

CCCVd14

coconut palm

Palmae

Buri palm, African oil palm, Manila palm, Royal palm, Palmera

none

CTiVd15

coconut palm

Palmae

none

none

HLVd16

hop

Cannabaceae

none

none

HSVd17

hop

Cannabaceae, Rosaceae, Rutaceae, Vitaceae, Cucurbitaceae

citrus, grapevine, cucumber, plum, peach, pear

cucumber

CPFVd18

cucumber

Cucurbitaceae

wild cucurbit (Benincasa hispida)

cucumber

CVd-Ia

citrus

Rutaceae

citrus

citrus

CCaVd19

citrus

Rutaceae

citrus

citrus

Avsunviroidae Avsunviroid ASBVd1 Pelamoviroid

Pospiviroidae Pospiviroid

tomato tomato

Cocadviroid

Hostuviroid

31

R.P. Singh et al.

Table 3.1

Host range of viroids. (Continued)

Viroid

Main host(s)

Family

Others

Indicator

ASSVd20

apple

Rosaceae

pear

CVd-III

citrus

Rosaceae

citrus

21

grapefruit

Rutaceae, Lauraceae

citrus, avocado

GYSVd22

grapevine

Vitaceae

ADFVd d25

apple

Rosaceae

AFCVd28

apple

Rosaceae

PBCVd23

pear

Rosaceae

cucumber

Pyrus communis ‘A20’, and ‘Fieud 37’, and ‘Fieud 110’

Coleus blumei

Lamiaceae

Ocimum sanctum

none

Apscaviroid

CBLVd

none pear

Coleviroid CbVd24 Uncharacterized viroids BSVd26

burdock

OFYVd27

oil palm

PpMMVd29

pigeonpea

Leguminosae

NGSVd30

N. glutinosa

Solanaceae

Arctium spp. Palmae

References 1Da Graça and Van Vuuren 1980, 1981; 2Hadidi et al. 1997; Shamloul et al. 1995; Osaka et al. 1998; 3Kyriakopoulou et al. 2001; 4Romaine and Horst 1975; Navarro and Flores 1997; 5Singh and O’Brien 1970; Singh 1973, 1984b; Paliwal and Singh 1981; Puchta et al. 1990; Querci et al. 1995; Shamloul et al. 1997; 6Singh et al. 1999; 7Galindo et al. 1982; 8Candresse et al. 1987; Walter 1987; Spieker et al. 1996; 9Semancik et al. 1973; Flores et al. 1985; Fagaoga et al. 1995; Fagaoga and Duran-Vila 1996; 10Mishra et al. 1991; 11Dusi et al. 1990; Bianco and Vegetti 1991; Verhoeven et al. 1998; 12Singh 1983; Singh et al. 1992c; Spieker 1996d; 13Spieker 1996b; 14Randles 1975; Randles et al. 1980; Zelazny and Niven 1980; Imperial et al. 1985; 15Randles and Imperial 1984; Keese et al. 1988; 16Puchta et al. 1988b; 17Takahashi 1987; Sano et al. 1989; Hsu et al. 1994; 18van Dorst and Peters 1974; Sano et al. 1984; 19Duran-Vila et al. 1988b; 20Hadidi et al. 1991; Skrzeczkowski et al. 1993; Hurtt et al. 1996; 21Duran-Vila et al. 1988b; 22Koltunow et al. 1989; Rezaian et al. 1992; Szychowski et al. 1998; 23Flores et al. 1991; Ambrós et al. 1995b; 24Fonseca et al. 1989; Singh et al. 1991a; 25Di Serio et al. 1996; 26Wei et al. 1983, 1986; 27Singh et al. 1988a; 28Ito et al. 1993; 29Bhattiprolu 1993; 30Bhattiprolu 1991.

32

BIOLOGY

aurantiaca and Nicotiana glutinosa and was not observed to replicate in any of them (Puchta et al. 1988b). Only the commercial hop cultivar ‘Omega’ displays symptoms when infected with HLVd (see Chapter 30 on ‘Hop latent viroid’). Coconut cadang-cadang viroid (CCCVd) infects only plants in the family Palmae. It was detected in five species of palm trees (Randles et al. 1980; Imperial et al. 1985), while Coconut tinangaja viroid (CTiVd) and Oil palm fatal yellowing viroid (OFYDVd) have only been found in a single species (Randles and Imperial 1984; Singh et al. 1988a). Viroids with the widest known host range fall into the genus Pospiviroid, which includes Potato spindle tuber viroid (PSTVd) and Citrus exocortis viroid (CEVd). They have been studied more extensively than any other viroids and also have the broadest host range. PSTVd infects a variety of species within the Solanaceae, including potato and tomato (Singh and O’Brien 1970; Singh 1973; Singh and Slack 1984), pepino (Puchta et al. 1990; Shamloul et al 1997), as well as avocado (Lauraceae) (Querci et al. 1995) and Compositae (Singh 1973; Niblett et al. 1980), in addition to plant species belonging to the families Boraginaceae, Campanulaceae, Caryophyllaceae, Convolvulaceae, Dipsaceae, Sapindaceae, Scrophulariaceae, and Valerianaceae (Singh 1973). CEVd infects several citrus hosts but can also replicate in members of the Compositae (Niblett et al. 1980) and Solanaceae (Semancik et al. 1973; Singh and Clark 1973) . It has been shown capable of naturally infecting a range of vegetable crops, including tomato, broad bean, turnip and carrot (Fagoaga et al. 1995; Fagoaga and Duran-Vila 1996). In contrast to HLVd, Hop stunt viroid (HSVd) exhibits a fairly wide host range, infecting plants in five families and existing as three types: citrus type (Sano et al. 1988; Puchta et al. 1989), plum type (Sano et al. 1989) and hop type (Ohno et al. 1983). A grapevine isolate of HSVd (HSVd-g) differs from the hop type isolate in only one nucleotide (Sano et al. 1985, 1986; Puchta et al. 1988c). HSVd infecting peach and pear is of the plum type (Sano et al. 1989). Cucumber pale fruit viroid (CPFVd) is an isolate of HSVd (HSVd-c) (Sano et al. 1984; Puchta et al. 1988a).

SYMPTOMS The symptoms caused by viroids are generally similar to those caused by viruses in higher plants, with stunting as the most characteristic. Stunting itself is usually characterized by reduction in leaf size and shortening of internode length, as with Chrysanthemum stunt viroid (CSVd) (Lawson 1987; Verhoeven et al. 1998), PSTVd (Singh 1988) and HSVd (Momma and Takahashi 1984). However, reduced flower size by CSVd (Plate 1A); flower and fruit size by HSVd-c (Plate 1B and C) have also been observed (van Dorst and Peters 1974; Brunt 2001). Diseases are usually recognized in cases where the symptoms are

moderate to severe, whereas in nature it may be the mild strains that predominate (Singh et al. 1970; Singh and Boucher 1988a). The symptoms caused by PSTVd in potato include changes in leaf color, upright growth and twisting of the terminal leaflets. Leaves emerge more slowly, have ruffled margins and are smaller than normal. There are many shallow eyes on the tubers, which are elongated and pointed (spindle-shaped). Severe infections cause necrotic spotting on petioles and main stems and stunting (MacLachlan 1960; Singh et al, 1970; Singh 1970, 1984, 1988). The severity of the symptoms varies with the host species (Singh and Slack 1984) and cultivar (Herold et al. 1992). In a wide survey of 232 plant selections, 138 selections from 10 families were susceptible to PSTVd, but most were symptomless carriers (Singh 1973). Of 81 Solanum species surveyed, 38 showed no symptoms in at least some cases (Singh and Slack 1984). PSTVd caused latent infection in avocado (Querci et al. 1995). The wide host range of PSTVd (Diener and Hadidi 1977) also includes several indicator plants with different disease manifestations: Scopolia sinensis develops local lesions (Singh 1971) while Solanum berthaultii shows systemic necrosis (Singh 1984), neither of which are commonly seen in the natural host (Singh 1970; Singh et al. 1970). The effect of PSTVd infection on yield depended on viroid strain (Singh et al. 1971), as well as the cultivar of potato (Pfannenstiel and Slack 1980; Singh 1988) and tomato (Kryczynski et al. 1988). Viroid disease in citrus is caused by a number of viroid species. CEVd causes symptoms in trifoliate orange. It is asymptomatic in some citrus species (i.e. sweet orange, grapefruit, mandarin) unless scions are propagated on sensitive rootstock such as ‘Etrog’ citron (Broadbent and Dephoff 1992), in which case symptoms of bark scaling, leaf epinasty and rugosity, stunting and necrosis appear (Roistacher et al. 1977). Citrus cachexia viroid (CCaVd or CVd-IIb), a citrus isolate of HSVd, produces gumming, pegging and pitting, as well as a general decline in tree health (Broadbent and Dephoff 1992). Other citrus viroids (CVd-I, CVd-II, CVd-III) have been identified as part of the graft-transmissible dwarfing complex (GTDC) (Duran-Vila et al. 1988b; Gillings et al. 1991; Bar-Joseph 1993). The severity of symptoms induced by them varies, depending on environmental conditions and what combination of agents are present, but usually involve dwarfing or stunting, sometimes at no apparent detriment to the health of the tree (Duran-Vila et al. 1988b). The related viroids infecting coconut, CCCVd and CTiVd, both cause chlorotic spotting of the tree crown, reduction in crown size, decreased fertility, cause more severe disease in older trees, reduce the number and size of the nuts and ultimately result in the decline and early death of the tree (Reinking 1961;

33

R.P. Singh et al.

Keese et al. 1988; Hodgson et al. 1998). In the field, they can be distinguished only by their effect on coconut morphology, CCCVd causing rounded scarified nuts and CTiVd causing small mummified husks with no kernel in the nut (Reinking 1961; Keese et al. 1988). Other viroids cause milder symptoms. ASBVd infection is characterized mainly by discoloration of the fruit and leaves, with stunting only in severe cases (Schnell et al. 1997). Infected trees may be asymptomatic for long periods as a normal part of the later stages of the disease (Semancik and Szychowski 1994). The usual symptoms of apple scar skin (ASSVd) and related viroids (e.g. Dapple apple viroid) are blemishes on the fruit or bark (Hadidi et al. 1990, 1991; Hurtt et al. 1996; Zhu et al. 1995). Some viroid infections are completely symptomless in their natural hosts. These include HLVd (Puchta et al. 1988b), CLVd (Hammond et al. 1989; Owens et al. 1978; Singh et al. 1992c) and Coleus blumei viroid (CbVd) (Fonseca et al. 1989; Singh et al. 1991b). Others can be almost symptomless for long periods, such as PLMVd (Hernández and Flores 1992). Generally, viroids in woody plants have a latent period as symptoms may not appear unless the plant is producing fruit (i.e. mature). The physiological effects of viroid infection are not well studied. CEVd impaired the ability of the canopy for water uptake (Moresht et al. 1998) and inhibited the development of the root system (Flores and Rodriguez 1981). Infection by severe strains of PSTVd reduced pollen viability (Hooker et al. 1978).

SYMPTOM VARIANTS The nature and severity of symptoms in a viroid infected plant is a reflection of the presence or predominance of particular sequence variants within the viroid population. Five sequence variants of CCCVd were associated with severe brooming syndrome (Rodriguez and Randles 1993). Also, high molecular weight variants appeared in the later stages of disease progression, replacing the lower molecular weight forms that predominated in the early stages (Imperial et al. 1981). Distinct variants of ASBVd, isolated from tissues showing different symptoms, have been associated with three types of leaf symptoms occurring on infected avocado trees (Semancik and Szychowski 1994). One variant was associated with a bleached foliar reaction early in the onset of leaf symptoms. This reaction was acute and self-limiting. It was followed by persistent latent infection characterized by bleached and variegated symptoms. This involved a second variant population which could transmit those specific symptoms. The final stage of the disease was chronic asymptomatic infection with predominance of the third variant. Molecules associated with the bleached reaction were present in all three populations (Semancik and Szychowski 1994).

34

The ability of Cucumber pale fruit viroid (CPFVd) (HSVd-c) grown in the wild cucurbit, Benincasa hispida, to infect different hosts depends on the heterogeneity of the nucleic acid population. Transcripts from a cloned, and thus homogeneous, CPFVd-cDNA were less infectious in tomato and cucumber than the original heterogeneous RNA population (Puchta et al. 1988a). CEVd variants in field isolates from citrus fall into two classes that correlate with either mild or severe symptoms in tomato (Visvader and Symons 1985). CEVd isolates propagated in tomato showed a common pattern of high frequency nucleotide exchanges in both terminal domains (Semancik et al. 1993), which may be preferred sites of recombination (Semancik et al. 1994). The host used affected symptoms, viroid titer, sequence and mobility producing a ‘host signature’ (Semancik et al. 1993), which may reflect the selection of specific variants such that a specific set of symptoms continue to be expressed in that host. Mild and severe strains of PSTVd have exhibited differences in electrophoretic mobility as a consequence of 1–3 nucleotide changes in chain length (Gross et al. 1981; Herold et al. 1992). Analysis of PSTVd strains demonstrated a reciprocal correlation between symptom severity and both the thermal stability (Schnolzer et al. 1985; Owens et al. 1991) and conformation (Singh and Boucher 1987; Singh et al. 1991a; Owens et al. 1995, 1996) of the virulence modulating (VM) region of the pathogenicity (P) domain. However, this correlation does not hold for all PSTVd variants (Owens et al. 1996). The influence of the pathogen–host interaction on the sequence and structure of the viroid was demonstrated with PSTVd in tobacco. PSTVd replicated but produced no symptoms in tobacco (O’Brien and Raymer 1964; Hammond et al. 1989). Agrobacterium-mediated inoculation with wild or mutant constructs of PSTVd was used to eliminate possible defects in mechanical transmission (Hammond 1994; Wassenegger et al. 1994b). Even in cases where a deletion mutant construct was unable to replicate in tobacco, infectivity was restored, as demonstrated by Northern hybridization and transmissibility to tomato. This occurred as a result of a second and complementary deletion which shortened the viroid but maintained its overall secondary structure (Wassenegger et al. 1994b). Furthermore, a PSTVd isolate from tomato, which was not infectious in tobacco, became infectious after a single nucleotide substitution (C-U) at position 259 occurred in mechanically inoculated tobacco (Wassenegger et al. 1996). The new hostinduced variant was genetically stable in both species. The same substitution occurred in tobacco plants after Agrobacteriummediated transfection with Ti plasmid containing the original tomato-specific sequence (Wassenegger et al. 1996) and was also

BIOLOGY

present in the newly infectious double deletion mutant (Wassenegger et al. 1994b). A PSTVd variant isolated from three wild Solanum species, that was able to infect tomato, also contained a C-U substitution in the same position in an internal loop of the central conserved region (Owens et al. 1992). Incidence of the same substitution in tobacco transfected with two different constructs and in mechanically inoculated tobacco led Wassenegger et al. (1996) to suggest that this host-induced substitution represents a ‘tobacco signature’ of PSTVd. For comparison, it would be of interest to determine the nucleotide sequence of the original PSTVd isolate described by O’Brien and Raymer (1964), which infected tobacco plants in host range studies but produced no symptoms.

SYMPTOMS IN TISSUE CULTURE Symptoms of viroid infection exhibited in tissue culture are varied, as they are in vivo. With HSVd and CCaVd (CVd-IIb), no changes occurred in gross morphology of hop (Takahashi et al. 1992b) and citrus (Greño et al. 1988) plantlets in tissue culture. In contrast, the symptoms of CEVd infection in citron shoots in tissue culture were very similar to those observed in plants, including leaf curling, epinasty and necrosis (Greño et al. 1988). PSTVd caused changes in cell size and shape in callus culture, and tighter aggregation of cells in suspension culture (Stocker et al. 1993). Inhibition of growth is a common feature regardless of morphological effects. Overall growth rate in tissue culture was reduced by infection with PSTVd (Stocker et al. 1993) and HSVd (Takahashi et al. 1992a,b). Specific inhibition of root formation and growth was caused by HSVd (Takahashi et al. 1992a, 1992b), and of root and shoot formation by CEVd (Duran-Vila and Semancik 1982; Greño et al. 1988). Reduced response to auxins and increased tolerance to high temperature occurred with HSVd (Takahashi et al. 1992b) and CEVd (Marton et al. 1982) infection, even without morphological symptoms. Since indole acetic acid (IAA) is involved in cell wall expansion, this lack of response may account for some aberrations in cell wall morphology (Marton et al. 1982; Momma and Takahashi 1982). As with infected plants, symptoms in vitro vary according to host. CEVd produced morphological symptoms and reduced shoot number and size in plantlets derived from infected citron buds (Greño et al. 1988), but callus and suspension cultures from infected tomato appeared healthy (Marton et al. 1982).

ULTRASTRUCTURE Ultrastructural changes that occur during viroid replication appear to affect mainly the cell wall and, in some cases, chloro-

plasts. Distortions, undulations and variations in thickness of the cell wall occurred with HSVd and CEVd infection (Momma and Takahashi 1982, 1983), along with disorganization and deformation of the chloroplasts, which exhibited loosening of the thylakoid membranes and poor stacking of the grana (Momma and Takahashi 1982). Similar disorganization of the chloroplast occurred with PSTVd infection in tomato (Hari 1980) and in Scopolia sinensis (Paliwal and Singh 1981). Irregularities in the plasmalemmasomes near the cell membranes resulted from infection with ASBVd (da Graça and Martin 1981) PSTVd (Hari 1980) and CEVd (Semancik and Vanderwoude 1976). These structural changes manifested as distortions in the leaf surface on a macroscopic level (Momma and Takahashi 1982, 1983) and were presumably caused by changes in cell wall composition. In the case of CEVd, there were increased arabinosyl and galactosyl residues, fewer xylosyl, mannosyl and glucosyl residues, reduced protein levels, and an increase in hydroxyproline content in the wall of infected cells (Wang et al. 1986). The alterations in surface properties caused by these changes in composition were reflected in the packing arrangement of HSVd- and CEVd-infected cells (Momma and Takahashi 1982) and tight aggregation of PSTVd-infected cells in suspension culture (Stocker et al. 1993). No changes were observed in other organelles (Momma and Takahashi 1982). However, changes in phosphorylation of cellular proteins by Mn2+-dependent kinases coincided with the onset of symptoms in CEVd-infected cells (Vera and Conejero 1990). Insufficient work has been done on the effects of viroid infection on cells in vitro to know how much variations correspond to the presence or absence of symptoms in vivo. However, CEVd caused symptoms in tomato in vivo (Kano and Yamaguchi 1985) yet did not affect growth rate or gross morphology of tomato cells in callus or suspension culture (Marton et al. 1982). This illustrates that symptoms in vitro cannot be predicted from those in vivo, nor can the absence of symptoms in vitro be used to select viroid-free plantlets or cells.

SUBCELLULAR LOCALIZATION Localization of a viroid within infected cells has been achieved by in situ hybridization in conjunction with other techniques, with or without subcellular fractionation. All viroids appear to fall into one of two groups: those located primarily in the nucleus or in the chloroplast. In ASBVd-infected leaves, 80% of the viroid RNA was detected in the chloroplasts (Lima et al. 1994), mostly on the thylakoid membranes (Bonfiglioli et al. 1994) and not in the nuclei (Marcos and Flores 1990). It was replicated in purified chloroplasts (Navarro et al. 1999) by a nuclear-encoded chloroplast RNA

35

R.P. Singh et al.

polymerase (Navarro et al. 2000). PLMVd also accumulated mainly in chloroplasts (Bussière et al. 1999) suggesting that this organelle may represent a common subcellular location of all members of the family Avsunviroidae, which possess hammerhead structure. Viroids in the family Pospiviroidae, non-hammerhead structure, were located primarily in the nucleus of cells infected with HSVd (Takahashi et al. 1982), CCCVd (Bonfiglioli et al. 1994, 1996), CEVd (Bonfiglioli et al. 1996), Tomato planta macho viroid (TPMVd) (Galindo et al. 1982) and PSTVd (Harders et al. 1989). The nucleus was the replication site for CPFVd (Mühlbach and Sänger 1979) and the location of the majority of infectious material (Sänger 1979). CEVd was distributed evenly throughout the nucleus (Bonfiglioli et al. 1996), whereas CCCVd (Bonfiglioli et al. 1994, 1996) and PSTVd (Schumacher et al. 1983) were concentrated in the nucleolus. Low levels of viroid have been detected in the cytoplasm (Lima et al. 1994) and membrane fractions (Takahashi et al. 1982; Hadidi 1988), but were not associated with other organelles (Sänger 1979; Marcos and Flores 1990).

DISTRIBUTION WITHIN THE PLANT Viroids have been found in virtually all plant tissues by various methods, such as RT-PCR (Hadidi et al. 1991), R-PAGE (Weidemann 1987; Khoury et al. 1988), nucleic acid hybridization (dot blot) (Salazar et al. 1983) and bioassay on indicator plants (Hunter et al. 1969; Singh 1970, 1977, 1984). ASSVd and DAVd were detected in seeds (coat, cotyledon and embryo), floral parts (anthers, petals, receptacles), bark and roots (Hadidi et al. 1991). PSTVd was found in leaves, roots, tubers, sprouts (Singh 1977; Salazar et al. 1983; Singh 1984; Weidemann 1987; Shamloul et al. 1997), seed (Hunter et al. 1969; Singh 1970; Kryczynski et al. 1988; Singh et al. 1988b; Shamloul et al. 1997), pollen (Singh 1970; Kryczynski et al. 1988; Singh et al. 1992a; Shamloul et al. 1997), floral parts including sepals, petals, anthers and pistils (Khoury et al. 1988), stems (Galindo et al. 1982) and fruit pulp (Khoury et al. 1988). In potato tubers, PSTVd was found in periderm, contical parenchyma, external phloem, xylem ring, perimedullary starch storage parenchyma and pith regions (Shamloul et al. 1997). CbVd was found in seeds (Singh et al. 1991b) and both TPMVd and CEVd were found in stems, roots and leaves (Galindo et al. 1982). In the family Avsunviroidae, ASBVd was detected in shoots, leaves and branches, with and without symptoms (Palukaitis et al. 1981; Semancik and Szychowski 1994). Interestingly, ASBVd was transmissible in 92–100% of seeds, if such seeds came from symptomless infected plants but not from symptomatic plants (Wallace and Drake 1962).

36

ECONOMIC LOSSES, YIELD AND QUALITY The economic effects of viroid disease depend on the local and global economic importance of the crop, and on the specific effects of the disease on that crop, namely yield, product number and size, quality, and overall marketability. PSTVd

PSTVd in potato has received much attention due to its worldwide importance as a food staple and its effect on crop yield has been well documented. In PSTVd —infected field-grown potatoes, yields by weight were reduced 17–24% by mild strains and up to 64% by a severe strain in 3 cultivars (Singh et al. 1971; Singh 1988). While PSTVd elevated the number of tubers with growth cracks, its effects on yield were greater, reducing the number of tubers/plant as well as the tuber weight/plant (Pfannenstiel and Slack 1980). Generally, the reductions in tuber weight/plant became more acute with each successive year of infection, with losses up to 46% in the first year, up to 88% the following year and as high as 97% in the third year of infection, depending on the cultivar (Pfannenstiel and Slack 1980). The effect of PSTVd on the yields of tomato and avocado crops is similar. Tomato yields were reduced 40–45%, depending on the cultivar (Kryczynski et al. 1988). Latent infection occurred in many of the avocado trees naturally infected with PSTVd in Peru. When symptoms were present, yield reductions approached 90% in terms of both number and weight of fruit (Querci et al. 1995), which were often parthenocarpic. In some cases, infection led to death of the tree, especially in cases of coinfection with ASBVd (Querci et al. 1995), increasing the economic consequences. Thus, local economic losses generated by PSTVd may be worsened when susceptible species are intercropped, as sometimes occurs with potato and avocado in Peru (Querci et al. 1995) or might occur in other regions with potato and tomato. In TPMVd-infected tomato, the fruit became marble-sized and hence unmarketable. Consequently, crop losses can be total (Galindo et al. 1982). Tomato chlorotic dwarf viroid (TCDVd) in the tomato cultivar ‘Trust’ caused almost total losses because the plants became severely stunted and the fruits were cracked and reduced in size (Singh et al. 1999). HSVd and CPFVd

CPFVd (HSVd-c) represents the third most economically important viroid disease in the Netherlands, causing reduction in fruit size and quality. Losses have not been quantified but greenhouse seedlings are routinely monitored for signs of infection (Huttinga et al. 1987). Removal of infected plants does affect the overall cost of producing healthy, high quality fruit. In tomato, CPFVd reduced yields by 40–43%, depending on the cultivar (Kryczynski et al. 1988).

BIOLOGY

The effects of HSVd on hop yields are severe. Cone yields dropped by 50% or more (Sasaki and Shikata 1977). Quality was compromised by reduced levels of bitter and acidic substances in the cones (Sasaki and Shikata 1977). Specifically, the lupulin glands in the cones were withered, 40% fewer in number and contained lower levels of alpha-acids (Momma and Takahashi 1984). Similarly, HLVd infection caused reductions in cone yields of up to 35%, although numbers were unaffected, and up to 30% lower alpha-acid content, depending on the hop cultivar and duration of infection (Barbara et al. 1990b). In grapevines, neither HSVd nor Grapevine yellow speckle viroid (GYSVd) affected yields but both reduced acidity in the fruit (Momma and Takahashi 1984; Wolpert et al. 1996). Palm viroids

Viroid diseases in palms have limited economic impact in global terms, since these diseases (CCCVd, CTiVd, OFYDVd) are geographically restricted, but have a significant local economic effect. The size and number of coconuts produced by infected trees was greatly reduced once symptoms appeared (Keese et al. 1988; Hodgson et al. 1998). Losses in copra, the source of coconut oil, due to CCCVd infection were estimated at 22 000 tons/ year (Randles 1982). The greatest losses were due to the early decline (Zelazny and Niven 1980) and death of the trees. Over 70 000 oil palms were lost to OFYDVd from 1974–1988 (Singh et al. 1988a). As of 1982, over 30 million coconut palms had been lost to CCCVd/CTiVd, with ongoing losses estimated at approximately 500 000 trees per year (Randles 1982). Infection rates are high. In Guam, 25–30% of coconut palms were estimated to be infected with CTiVd (Wall and Wiecko 1997). Even with replacement, losses equivalent to about eight years production still occur for each tree replaced, due to reduced yields from diseased trees and no yields from immature replacement trees (Randles 1982). Citrus diseases

Citrus viroid diseases include a number of viroids and natural or field infections frequently include 4–5 agents (Duran-Vila et al. 1988b; Semancik et al. 1988, Visvader and Symons 1983, 1985). Losses due to citrus diseases in general, not just viroids, may be up to one-third (Broadbent and Dephoff 1992). In these natural infections, symptoms and losses cannot be attributed to a single agent and experimental evidence on yields from trees with a single infection is scarce. The reduced ability of the trees to utilize available water (Moresht et al. 1998) may increase costs and increase stress in areas where water is limiting and irrigation is required. In contrast to all other viroids, the citrus viroids are being investigated for their potential economic benefit (Semancik et al. 1997). Graft-transmissible dwarfing complex (GTDC) can cause symptomatic infection with bark scaling and gummy pits

but frequently exists in apparently healthy trees, depending on the scion-rootstock combination and viroid content. With citrus on trifoliate orange rootstock, the symptoms vary according to the nature of the heterogeneous infecting population (DuranVila et al. 1988b; Gillings et al. 1991; Owens et al. 1999; Moresht et al. 1998). If dwarfing could be maintained without detrimental effects on tree health, fruit quality and yield, the benefits would be substantial. In ‘Valencia’ orange, GTDC was reported to improve yields (Semancik et al. 1997). Closer spacing of trees would improve yields/acre and increase the efficiency of orchard work such as irrigation, harvesting and pesticide application (Gillings et al. 1991). However, yields might be reduced for certain scion types or for other susceptible crops. For example, GTDC reduces the health and vigour of lemon and mandarin. Therefore, dwarfing as an orchard practice requires containment (Gillings et al. 1991). Another concern is the stability of the infecting strain in terms of pathogenicity, since deliberate dwarfing is predicated on maintaining long-term asymptomatic infection. In this regard, the nature and disease status of nearby crops is important, as natural chimeras and viroid recombinants have been reported (Rezaian 1990; Ben-Shaul et al. 1995; Spieker 1996c; Kofalvi et al. 1997) and isolates of HSVd, a component of GTDC, infect other fruit and vegetable crops.

TRANSMISSION Mechanical methods of transmission have been reported for most viroids in both viroid families, including PSTVd, by contaminated machinery and tools (Manzer and Merriam 1961) and via sap from foliage, sprouts and tubers (Singh 1984); CPFVd by pruning and razor slashing (van Dorst and Peters 1974); ASSVd by rub inoculation or razor slashing (Koganezawa 1985); ASBVd by sap inoculation and razor slashing (Allen et al. 1981); and PLMVd via contaminated tools (Hadidi et al. 1997). The fact that PSTVd and ASSVd were detected in virtually all plant parts (Weidemann 1987; Khoury et al. 1988; Hadidi et al. 1991; Shamloul et al. 1997) means that the plants represent a major source of potential spread (Hadidi et al. 1991). On an experimental basis, HLVd was transmitted more efficiently by cutting than by abrasion (Adams et al. 1996). Therefore, transmission via tools or machinery is more likely than via contact between adjacent plants and would account for transmission in the field occurring early in the season, before plants are large enough for the leaves to rub together (Adams et al. 1996). Grafting as a mechanical means of inoculation is widely used experimentally and accounts for field transmission in citrus. Transmission through infected seed and pollen occurred with PSTVd (Hunter et al. 1969; Singh 1970; Kryczynski et al. 1988; Singh et al. 1992a), CbVd (Singh et al. 1991b), CPFVd

37

R.P. Singh et al.

(Kryczynski et al. 1988), grapevine viroids (Wah and Symons 1997) and ASBVd (Wallace and Drake 1962; Allen et al, 1981). Detectable levels of PSTVd in true potato seed (Singh et al. 1991c; Shamloul et al. 1997) remained even after 21 years storage at room temperature (Singh et al. 1991c) indicating that the longevity of the viroid may exceed that of viable seed. Nematodes do not appear to act as viroid vectors (van Dorst and Peters 1974). Attempts to transmit HLVd (Adams et al. 1996), CSVd (Hollings and Stone 1973), CPFVd (van Dorst and Peters 1974) and PSTVd (Singh et al. 1992b; Salazar et al. 1995) via aphids were unsuccessful. There have been isolated cases of aphid transmission of TPMVd at high efficiency by Myzus persicae (Galindo et al. 1986) and of Tomato apical stunt viroid (TASVd) at low efficiency by Aphis craccivora (Walter 1987). With PSTVd, aphid transmission depended on concurrent infection with Potato leafroll virus (PLRV) or both PLRV and Potato virus Y (PVY) (Salazar et al. 1995). The PSTVd-RNA was ‘transencapsidated’ by coat proteins of PLRV (Salazar et al. 1995; Querci et al. 1997; Singh and Kurz 1997) and Velvet tobacco mottle virus (VToMV) (Francki et al. 1986), but not by PVY protein (Singh et al. 1992b). Transencapsidation accounts for transmission by aphids occurring only in cases of double infection (Syller et al. 1997). In China, where most potato plants contained PLRV, transencapsidation of PSTVd RNA and subsequent transmission by aphids may contribute to PSTVd spread, especially over long distances, even though PSTVd represents less than 0.1% total encapsidated RNA (Querci et al. 1997). In general terms, the transmission of viroid diseases through seed and pollen, as well as from other tissues by mechanical means, is significant in that infected plants are the major source of spread, regardless of the propagation method. The source of infection of wild Solanum species with PSTVd in India (Owens et al. 1992) and Australia (Behjatnia et al. 1996) may have originally been from the use of infected germplasm, as no PSTVdinfected wild Solanum species have been found in South America (Owens et al. 1992). That all hop germplasm in some countries is apparently infected with HLVd accounts for infection rates of up to 80–100% and means that commercially-grown hops probably carry the viroid (Puchta et al. 1988b; Barbara et al. 1990a; Nelson et al. 1999).

SURVIVAL IN THE FIELD AND GREENHOUSE Repeated freezing and thawing of PSTVd resulted in loss of infectivity (Singh and Boucher 1988b). This indicates that there is little or no chance of the viroid over-wintering in infected tubers or plant parts left in the field, at least not in climates with sub-zero temperatures. Disinfection of knives and other tools with household bleach (1–3%) is an effective means to eliminate viroid transmission through crude sap (i.e. handling in the

38

greenhouse, cutting seed potatoes) (Singh et al. 1989a; Kesinger 1997). Another factor in persistence in the field may be the presence of susceptible wild species that can act as reservoirs of disease. Wild Solanum species infected with PSTVd in India may contribute to field infection of tomato (Owens et al. 1992). However, the main factor contributing to persistence in the field and greenhouse is the use of contaminated equipment and source material in commercial propagation, whether by seed or vegetative means.

ENVIRONMENTAL EFFECTS It is generally accepted that viroid replication and symptom development is enhanced as the temperature increases above 20°C, at least to 35°C (Singh 1983, 1989), with the result that viroid disease in the field is probably more prevalent in warmer climates (Singh 1983). This is not reflected in the published reports of viroid incidence because it is not where research money and efforts are concentrated globally. In natural infections, symptom severity due to Apple fruit crinkle viroid (AFCVd) was greater during warm summers (Ito et al. 1993). In field-grown hops in the UK, HLVd was not detectable in the cold weather of early spring, but was detected in all samples by mid-July, when temperatures were higher (Morton et al. 1993). In contrast, there was no seasonal variation in HLVd detection in greenhouse-grown hops at elevated temperature (Morton et al. 1993). In experimental infections, enhanced symptom expression (early onset/increased severity) at temperatures above 30°C was demonstrated for ASBVd (da Graça and Van Vuuren 1981), PSTVd (Sänger and Ramm 1974; Morris and Smith 1977; Harris and Browning 1980), CPFVd (van Dorst and Peters 1974), CEVd (Semancik et al. 1988), and Pigeonpea mosaic mottle viroid (PpMMVd) (Bhattiprolu 1993). Mixed infections with citrus CVd-Ib, CVd-IIa, CVd-IIb and CVD-IIIb produced more severe symptoms under conditions of high temperature and long day length (Duran-Vila et al. 1988b). In contrast, greatest accumulation of ASSVd RNA occurred at 18°C (Skrzeczkowski et al. 1993). However, a 24-hour photoperiod was used which may have affected the response to temperature. ‘Parson’s Special’ mandarin infected with CCaVd is usually symptomless at temperatures which produce symptoms for CEVd (daily range of 22–38°C). Symptoms of CCaVd may be better monitored at 17–29°C, where those of CEVd would be less visible (Semancik et al. 1988). The amount of light available is almost as important to symptom expression as the temperature. Leaf curling induced by a citrus viroid, which was found later to be a mixture of viroids, was enhanced at high temperature and light (Schlemmer et al. 1985). Symptoms expression was reduced under low light even

BIOLOGY

at high temperature for CSVd (Hollings and Stone 1973) and PSTVd (Harris and Browning 1980; Harris and James 1987) and was affected by both intensity and quality of light (Grasmick and Slack 1985). Light availability also affects the movement of viroid away from the site of inoculation, presumably due to transport in conjunction with photosynthetic activity (Palukaitis 1987). Shading of the tomato leaf inoculated with PSTVd delayed transport of the PSTVd out of the inoculated leaf (Palukaitis 1987) and is consistent with transport via the phloem. Pruning enhanced symptom expression by ASBVd (da Graça and Van Vuuren 1981), PpMMVd (Bhattiprolu 1993), and by PSTVd in tomato (Whitney and Peterson 1963; Grasmick and Slack 1985). This effect may be particularly helpful in the detection of mild, as opposed to severe strains. Symptom development due to PSTVd infection of the local lesion host Scopolia sinensis was improved by higher than normal levels of soil manganese (Lee and Singh 1972; Singh et al. 1974).

CROSS PROTECTION AND INTERFERENCE Cross protection is the phenomenon by which inoculation with a specific pathogen confers protection, particularly with respect to symptom expression, against subsequent infection or challenge by another strain of the same pathogen. Interference is often used to describe the effect of one agent on the replication or course of infection of another, particularly in co-infection. These phenomena have been likened to immunization in their potential application to disease control (de Zoeten and Fulton 1975). Some success has been achieved in the control of Citrus tristeza virus (Costa and Müller 1980), Cocoa swollen shoot virus and Papaya ringspot virus (Hughes and Ollennu 1994; Yeh and Gonsalves 1984) and in experimental field trials with Tobacco mosaic virus (Ahoonmanesh and Shalla 1981) but generally the effects have not been long lasting (Hamilton 1980). However, in the case of CTV in citrus, protection is maintained over time. With viroid diseases, cross protection is even less likely to be effective as a means of control due to potential recombination in the field, as has been demonstrated for HSVd (Kofalvi et al. 1997), CbVd (Hammond et al. 1989; Spieker 1996a,c), grapevine viroids (Rezaian 1990), and citrus viroids (Ben-Shaul et al. 1995). Cross protection by mild strains of PSTVd (m-PSTVd) delayed the onset of severe symptoms (Fernow 1967; Niblett et al. 1978; Singh et al. 1970; Singh 1971) after challenge with severe strain (s-PSTVd), especially if the challenge occurred 4 weeks postinoculation, rather than only 1–2 weeks (Khoury et al. 1988). Both strains replicated, but s-PSTVd replicated more slowly in plants previously inoculated with m-PSTVd (Khoury et al. 1988). Cross protection is incomplete, in that the protection

conferred by the initial infection was only temporary (Fernow 1967; Khoury et al. 1988) and depended on the inoculation route. Cross protection between strains of PSTVd in tomato was overcome by graft inoculation (Singh et al. 1989b, 1990) but not by pinpoint puncture (Singh et al. 1990). Similarly, two clones of S. berthaultii apparently resistant to infection from sap containing PSTVd were susceptible to graft-inoculation (Singh 1985), with latent infection demonstrated through bioassay and dot-blot hybridization (Singh 1985). The apparent resistance of tobacco to HSVd and S. acaule to PSTVd was overcome by Ti plasmid-mediated inoculation (Hammond et al. 1989, Wassenegger et al. 1994b, 1996), demonstrating that resistance was not due to inability of viroids to replicate. The reaction pattern of protection also depends on host susceptibility. With PSTVd, better protection was achieved in a highly susceptible cultivar (‘Russet Burbank’) than in the more tolerant potato cv. ‘BelRus’ (Singh et al. 1990). Partial protection against s-PSTVd was also conferred by transgenic introduction of Cucumber mosaic virus (CMV) satellite RNA or via CMV infection of tomato 14 days prior to challenge (Yang et al. 1996), producing only mild symptoms. This may be a form of non-specific systemic acquired resistance. Cross protection tests have been usefully applied to the detection of latent infections, where a symptomatic or severe strain exists. The test involves initial inoculation with the test sap or graft from a symptomless plant and challenge with the severe or symptomatic strain. If severe symptoms do not develop, then the presence of a cross protecting strain is indicated. Expression of severe symptoms confirms the absence of a cross protecting strain in the test inoculum. This type of assay is routinely used to detect latent strains of PLMVd in GF305 peach seedlings (Desvignes 1976; Flores et al. 1990) and latent ASBVd by graftinoculation onto healthy avocado rootstock, followed by challenge inoculation with bark from a symptomatic tree (Allen and Firth 1980). Co-inoculation of tomato plants with PSTVd and CLVd caused double infection with both viroids accumulating to comparable levels (Singh et al. 1992c). In cross protection tests, CLVd did protect plants against challenge with s-PSTVd. However, PSTVd did not completely protect against CLVd infection: CLVd simply accumulated to lower levels in challenged plants (Singh et al. 1992c). Cross protection by CEVd in tomato against PSTVd challenge occurred (Pallás and Flores 1989) but not vice versa (Niblett et al. 1978; Pallás and Flores 1989), although interference was shown in mixed infection (Pallás and Flores 1989). Development of severe CEVd symptoms in the presence of other citrus viroids (Duran-Vila et al. 1986; Schlemmer et al. 1985) indicates a lack of interference between them. In GTDC, symptom development

39

R.P. Singh et al.

varies. This has been attributed to the heterogeneous nature of the infecting RNA population and may involve interference (DuranVila et al. 1988b; Gillings et al. 1991; Bar-Joseph 1993). True interference, as distinct from cross protection, has been clearly demonstrated for HSVd in the presence of PSTVd (Branch et al. 1988). Co-inoculation with a mixture of transcripts reduced the levels of HSVd produced. Co-inoculation was also done using dual transcripts (generated from constructs containing both PSTVd and HSVd sequences). This ensured that each infected cell was infected simultaneously with both sequences. Symptom expression and replication of only PSTVd, but not HSVd, occurred (Branch et al. 1988). Whether or not cross protection or interference occurs may depend on competition for limiting host factors and whether affinity for those factors varies among viroids or with different hosts (Branch et al. 1988; Pallás and Flores 1989). Interestingly, CEVd offers partial protection against mal secco disease, a systemic fungal foot rot in ‘Etrog’ citron and ‘Rangpur’ lime which is caused by Phoma tracheiphila. CEVd infection reduced the systemic advance of the mycelium from the leaves into the branches (Solel et al. 1995) and probably accounts for field observations in Sicily and Israel of relative tolerance of old clones to mal secco disease (Solel et al. 1995). The mechanism by which this occurs is not known, but may involve the production of host pathogenesis-related (PR) proteins (Hadidi 1988; Belles et al. 1989; Garcia Breijo et al. 1990; Vera and Conejero 1989). In tomato, CEVd induced expression of two PR proteins identical to chitinases which are associated with hypersensitive responses and are implicated in host defence against fungal infections (Garcia Breijo et al. 1990).

CONTROL The primary and most effective method of control of viroid infections involves eradication of infected source material. The best example of viroid eradication has taken place in Canada. PSTVd had been widespread through most Canadian provinces from 1922 onwards (Singh et al. 1970, 1988b). Incidence peaked in the 1950s and was well established by the 1960s (Singh 1988). In 1969–1970 the disease occurred in 3–5% of tablestock fields in Eastern Canada, with mild strains predominating (92%) (Singh et al. 1970). In 1970, strict control measures were implemented and elimination of the disease occurred by 1980 (Singh and Crowley 1985; Singh 1988; Singh et al. 1988b). Extensive surveys of tablestock and seed fields in Prince Edward Island (Singh et al. 1988b) and New Brunswick (Singh and Crowley 1985) later confirmed that PSTVd was eradicated from the seed potato crop. The control measures taken to eradicate PSTVd included: development and use of faster, more sensitive detection methods; establishment of Elite seed farms to improve the produc-

40

tion of seed material free of virus and viroid diseases; certification regulations governing the source material used for seed potato production; zero tolerance for PSTVd in conjunction with widespread testing of field and seed material; testing of parental material by breeders to prevent the release of a new cultivar containing PSTVd; implementation of provincial disease eradication legislation to control transport, planting, inspection, isolation and disposal, and certification; the involvement of industry to encourage the use of high quality seed by their contract growers; improved disinfection during seed cutting; and the establishment of a tissue culture program in plant propagation centres as a source of pre-Elite seed for seed potato production (Singh 1988). In Europe, the European Economic Community (EEC) banned import of North American potatoes in 1978 (McDonald and Borrel 1991). Import of potatoes into EEC countries requires that material must initiate from PSTVd-free clones, from seed produced on farms that have not had a PSTVd outbreak for at least five years and that post-harvest testing be done (McDonald and Borrel 1991). These measures are effective in maintaining PSTVd-free potato crops in EEC nations. The eradication measures implemented in Canada allowed exports to EEC to resume in 1979 (McDonald and Borrel 1991). Since PLMVd shows worldwide distribution (Hadidi et al. 1997) it is likely that control of this disease in North America will be achieved only when similar measures are implemented governing quarantine, import and certification of stone fruit (Hadidi et al. 1997). Control of viroid infection by tissue culture methods is an important part of any general control program because of viroid transmission through vegetative and sexual propagation. Meristem culture was used to generate PSTVd-free plantlets from infected plants first subjected to six months cold treatment (6–8°C) (Lizarraga et al. 1980). Cold therapy was used to eliminate CSVd, ChCMVd and CPFVd (Paduch-Cichal and Kryczynski 1987). However, cold treatment was ineffective in eliminating HSVd (Momma and Takahashi 1984) and PLMVd (Howell et al. 1999). Since this method requires months, its use is limited. Conversely, heat treatment was reported effective in combination with shoot tip propagation, in eliminating PLMVd (Howell et al. 1999). Shoot tip culture was used to generate CEVd-free tomato plantlets (Duran-Vila and Semancik 1986) and plantlets freed of infection with grapevine viroids (Duran-Vila et al. 1988a). A combination of thermo-therapy and apical meristem culture was successfully utilized in eliminating ASSVd from pears (Postman and Hadidi 1995). Chemical methods of control can be aimed at eradicating infection from plants and preventing transmission. Household

BIOLOGY

bleach (1–3%) is effective at preventing mechanical transmission via crude sap during tissue culture and greenhouse practices, even when high concentrations of viroid are present (Singh et al. 1989a; Kesinger 1997). Piperonyl butoxide, found in some insecticidal sprays, prevented infection by PSTVd when sprayed on leaves prior to inoculation (Singh et al. 1975; Singh 1977). However, the effect was limited to certain hosts, was short-lived (~4 days), acted only on sprayed leaves, not new growth, and was phytotoxic above 1% (Singh 1977). Ribavirin, a broad spectrum antiviral agent (1-b-D-ribofuranosyl-1,2,4triazole-3-carboxamide) caused remission of symptoms in new leaves of Gynura aurantiaca and prevented establishment of infection when sprayed up to three days prior to inoculation with CEVd. Toxicity just above effective concentrations limits its use (Belles et al. 1986). Chitosan (1-4 glucosamine polymers) reduced PSTVd infection rates in tomato by 50–75%, when sprayed prior to or within three hours of inoculation or when added to inoculum (Pospieszny 1997). It might prove useful at times of mechanical activity in the field, such as pruning and pesticide application (Pospieszny 1997), but this would require evaluation of its effectiveness and expense in field trials. As control by chemical and culture methods or development of resistant cultivars has not been accomplished, transgenic approaches using antisense nucleic acids have been examined. Infection of tomato by PSTVd was almost prevented by mixing inoculum with antisense RNA corresponding to complete (-) monomers (Matousek et al. 1994a). This suggests that double stranded forms of viroid RNA were not infectious. In vivo, the (-) strand acts as template for replication. When antisense constructs targeting the CEVd (-) strand were expressed in transgenic tomato seedlings, accumulation of CEVd was reduced moderately (Atkins et al. 1995). Similar experiments with PSTVd, targetting the left half of the (-) strand, showed no significant inhibition of viroid accumulation (Matousek et al. 1994b). However, when hammerhead ribozyme sequences were used, a higher proportion of transgenic potato plants were resistant to challenge (Yang et al. 1997). The resistance was stable but was inefficient in tomato. PSTVd sequences integrated into tobacco by Agrobacteriummediated inoculation were methylated in cells where PSTVd replication occurred, thereby reducing their activity. No such methylation occurred in cells where no replication took place (Wassenegger et al. 1994a). The mechanism for inducing methylation is not understood. It may have implications for the limited effectiveness of antisense constructs in controlling viroid accumulation, although the question has not been addressed. Better results have been obtained by expression of a doublestranded RNA-specific ribonuclease in transgenic potato. The

gene product digested PSTVd in vitro and conferred partial resistance to PSTVd infection, reducing the accumulation of PSTVd and its transfer to progeny tubers. While similar approaches have been used to control experimental virus infections, performance in the field has been inconsistent (Hammond 1997; Sano et al. 1997). Field trials of such transgenic lines will have to show effectiveness and consistency in the field before they will have practical significance. References Adams, A.N., Barbara, D.J., Morton, A., and Darby, P. (1996). The experimental transmission of hop latent viroid and its elimination by low temperature treatment and meristem culture. Ann. Appl. Biol. 128, 37-44. Ahoonmanesh, A., and Shalla, T.A. (1981). Feasibility of cross-protection for control of tomato mosaic virus in fresh market field-grown tomatoes. Plant Dis. 65, 56-58. Allen, R.N., and Firth, D.J. (1980). Sensitivity of transmission tests for avocado sunblotch viroid and other pathogens. Aust. Plant Pathol. 9, 2-3. Allen, R.N., Palukaitis, P., and Symons, R.H. (1981). Purified avocado sunblotch viroid causes disease in avocado seedlings. Aust. Plant Pathol. 10, 31-32. Ambrós, S., Desvignes, J.C., Llácer, G., and Flores, R. (1995). Pear blister canker viroid: sequence variability and causal role in pear blister canker disease. J. Gen. Virol. 76, 2625-2629. Atkins, D., Young, M., Uzzell, S., Kelly, L., Fillatti, J., and Gerlach, W.L. (1995). The expression of antisense and ribozyme genes targeting citrus exocortis viroid in transgenic plants. J. Gen. Virol. 76, 1781-1790. Bar-Joseph, M. (1993). The use of viroids for dwarfing citrus trees. 9th International Congress of Virology Abstracts: 107. Barbara, D.J., Morton, A., and Adams, A.N. (1990a). Assessment of UK hops for the occurence of hop latent and hop stunt viroids. Ann. Appl. Biol. 116, 265-272. Barbara, D.J., Morton, A., Adams, A.N., and Green, C.P. (1990b). Some effects of hop latent viroid on two cultivars of hop (Humulus lupulus) in the UK. Ann. Appl. Biol. 117, 359-366. Behjatnia, S.A.A., Dry, I.B., Krake, L.R., Conde, B.D., Connelly, M.I., Randles, J.W., and Rezaian, M.A. (1996). New potato spindle tuber viroid and tomato leaf curl geminivirus strains from a wild Solanum sp. Phytopathology 86, 880-886. Belles, J.M., Hansen, A.J., Granell, A., and Conejero, V. (1986). Antiviroid effects of ribavirin on citrus exocortis viroid infection in ‘Gynura aurantiaca’ DC. Physiol. Mol. Plant Pathol. 28, 61-65. Belles, J.M., Vera, P., Duran-Vila, N., and Conejero, V. (1989). Ethylene production in tomato cultures infected with citrus exocortis viroid (CEV). Can. J. Plant Pathol. 11, 256-262. Ben-Shaul, A., Guang, Y., Mogilner, N., Hadas, R., Mawassi, M., Gafny, R., and Bar-Joseph, M. (1995). Genomic diversity among populations of two citrus viroids from different graft-transmissible dwarfing complexes in Israel. Phytopathology 85, 359-363. Bhattiprolu, S.L. (1991). Studies on a newly recognized disease of Nicotiana glutinosa of viroid etiology. Plant Dis. 75, 1068-1071. Bhattiprolu, S.L. (1993). Occurrence of mosaic mottle, a viroid disease of pigeonpea (Cajanus cajan) in India. J. Phytopathol. 137, 55-60.

41

R.P. Singh et al.

Bianco, P.A., and Vegetti, G.A. (1991). Detection and identification of chrysanthemum stunt viroid CSVd in Lombardy. Riv. Patol. Veg. 1, 43-50. Bonfiglioli, R.G., McFadden, G.I., and Symons, R.H. (1994). In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus. Plant J. 6, 99-103. Bonfiglioli, R.G., Webb, D.R., and Symons, R.H. (1996). Tissue and intra-cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy. Plant J. 9, 457-465. Branch, A.D., Benenfeld, B.J., Franck, E.R., Shaw, J.F., Varban, M.L., Willis, K.K., Rosen, D.L., and Robertson, H.D. (1988). Interference between coinoculated viroids. Virology 163, 538-546. Broadbent, P., and Dephoff, C.M. (1992). Virus indexing in the New South Wales citrus improvement scheme. Aust. J. Exp. Agric. 32, 493-502. Brunt, A.A. (2001). Plants and Seeds. Chrysanthemum stunt viroid. Department for Environment, Food, and Rural Affairs. http://.maff.gov.uk/planth/pestnote/csvd.htm. Bussière F., Lehoux, J., Thompson, D.A., Skrzeczkowski, L.J., and Perreault, J.-P. (1999). Subcellular localization and rolling circle replication of peach latent mosaic viroid: hallmarks of group A viroids. J. Virol. 73, 6353-6360. Candresse, T., Smith, D., and Diener, T.O. (1987). Nucleotide sequence of a full-length infectious clone of the Indonesian strain of tomato apical stunt viroid (TASV). Nucleic Acids Res. 15, 10597. Costa, A.S., and Müller, G.W. (1980). Tristeza control by cross protection. Plant Dis. 64, 538-541. da Graça, J.V., and Martin, M.M. (1981). Ultrastructural changes in avocado leaf tissue infected with avocado sunblotch. Phytopathol. Z. 102, 185-194. da Graça, J.V., and Van Vuuren, S.P. (1980). Transmission of avocado sunblotch disease to cinnamon. Plant Dis. 64, 475. da Graça, J.V., and Van Vuuren, S.P. (1981). Use of high temperature to increase the rate of avocado sunblotch symptom development in indicator seedlings. Plant Dis. 65, 46-57. Desvignes, J.C. (1976). The virus diseases detected in greenhouse and in field by the peach seedling GF305 indicator. Acta Hortic. 67, 315-323. de Zoeten, G.A., and Fulton, R.W. (1975). Letter to the editor: Understanding generates possibilities. Phytopathology 65, 221-222. Di Serio, F., Aparicio, F., Alioto, D., Ragozzino, A., and Flores, R. (1996). Identification and molecular properties of a 306 nucleotide viroid associated with apple dimple fruit disease. J. Gen. Virol. 77, 2833-2837. Diener, T.O., and Hadidi, A. (1977). Viroids. Pages 285-337 in: Comprehensive virology, XI. H. Fraenkel-Conrat, and R.R. Wagner, eds. Plenum Publishing Co.: New York. Diener, T.O., and Raymer, W.B. (1967). Potato spindle tuber virus: a plant virus with properties of a free nucleic acid. Science 158, 378-381. Duran-Vila, N., Flores, R., and Semancik, J.S. (1986). Characterization of viroid-like RNAs associated with the citrus exocortis syndrome. Virology 150, 75-84.

42

Duran-Vila, N., Juarez, J., and Arregui, J.M. (1988a). Production of viroid-free grapevines by shoot tip culture. Am. J. Enol. Vitic. 39, 217-220. Duran-Vila, N., Roistacher, C.N., Rivera-Bustamante, R., and Semancik, J.S. (1988b). A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69, 3069-3080. Duran-Vila, N., and Semancik, J.S. (1982). Effects of exogenous auxins on tomato tissue infected with the citrus exocortis viroid. Phytopathology 72, 777-781. Duran-Vila, N., and Semancik, J.S. (1986). Shoot-tip culture and the eradication of viroid-RNA. Scientia Hortic. 29, 199-203. Dusi, A.N., Fonseca, M.E.N., and de Avila, A.C. (1990). Occurence of a viroid in chrysanthemum in Brazil. Plant Pathol. 39, 636-637. Faggioli, F., Loreti, S., and Barba, M. (1997). Occurrence of peach latent mosaic viroid (PLMVd) on plum in Italy. Plant Dis. 81, 423. Fagoaga, C., and Duran-Vila, N. (1996). Naturally occurring variants of citrus exocortis viroid in vegetable crops. Plant Pathol. 45, 45-53. Fagoaga, C., Semancik, J.S., and Duran-Vila, N. (1995). A citrus exocortis viroid variant from broad bean (Vicia faba L.) infectivity and pathogenesis. J. Gen. Virol. 76, 2271-2277. Fernow, K.H. (1967). Tomato as a test plant for detecting mild strains of potato spindle tuber virus. Phytopathology 57, 1347-1352. Flores, R., Duran-Vila, N., Pallás, V., and Semancik, J.S. (1985). Detection of viroid and viroid-like RNAs from grapevine. J. Gen. Virol. 66, 2095-2102. Flores, R., Hernández, C., Desvignes, J.C., and Llácer, G. (1990). Some properties of the viroid inducing peach latent mosaic disease. Res. Virol. 141, 109-118. Flores, R., Hernández, C., Llácer, G., and Desvignes, J.C. (1991). Identification of a new viroid as the putative causal agent of pear blister canker disease. J. Gen. Virol. 72, 1199-1204. Flores, R., and Rodriguez, J.-L. (1981). Altered pattern of root formation on cuttings of Gynura aurantiaca infected by citrus exocortis viroid. Phytopathology 71, 964-966. Fonseca, M.E.N., Boiteux, L.S., Singh, R.P., and Kitajima, E.W. (1989). A small viroid in Coleus species from Brazil. Fitopatol. Bras. 14, 94-96. Francki, R.I.B., Zaitlin, M., and Palukaitis, P. (1986). In vivo encapsidation of potato spindle tuber viroid by velvet tobacco mottle virus particles. Virology 155, 469-473. Galindo, J., Lopez, M., and Aguilar, T. (1986). Significance of Myzus persicae in the spread of tomato planta macho viroid. Fitopatol. Bras. 11, 400-410. Galindo, J., Smith, D.R., and Diener, T.O. (1982). Etiology of planta macho, a viroid disease of tomato. Phytopathology 72, 49-54. Garcia Breijo, F.J., Garro, R., and Conejero, V. (1990). C7(P32) and C6(P34) PR proteins induced in tomato leaves by citrus exocortis viroid infection are chitinases. Physiol. Mol. Plant Pathol. 36, 249-260. Gillings, M.R., Broadbent, P., and Gollnow, B.I. (1991). Viroids in Australian citrus: Relationship to exocortis, cachexia and citrus dwarfing. Aust. J. Plant Physiol. 18, 559-570. Giunchedi, L., Gentit, P., Nemchinov, L., Poggi-Pollini, C., and Hadidi, A. (1998). Plum spotted fruit: a disease associated with peach latent mosaic viroid. Acta Hortic. 472, 571-579. Grasmick, M.E., and Slack, S.A. (1985). Symptom expression enhanced and low concentrations of potato spindle tuber viroid amplified in

BIOLOGY

tomato with high light intensity and temperature. Plant Dis. 69, 49-51. Greño, V., Navarro, L., and Duran-Vila, N. (1988). Influence of virus and virus-like agents on the development of citrus buds cultured in vitro. Plant Cell Tiss. Org. Cult. 15, 113-124. Gross, H.J., Liebl, U., Alberty, H., Krupp, A.G., Domdey, H., Ramm K., and Sänger, H.L. (1981). A severe and a mild potato spindle tuber viroid isolate differ in three nucleotide exchanges only. Biosci. Rep. 1, 235-241. Hadidi, A. (1988). Synthesis of disease-associated proteins in viroidinfected tomato leaves and binding of viroid to host proteins. Phytopathology 78, 575-578. Hadidi, A., Giunchedi, L., Shamloul, A.M., Poggi-Pollini, C., and Amer, M.A. (1997). Occurrence of peach latent mosaic viroid in stone fruits and its transmission with contaminated blades. Plant Dis. 81, 154-158. Hadidi, A., Hansen, A.J., Parish, C.L., and Yang, X. (1991). Scar skin and dapple apple viroids are seed-borne and persistent in infected apple trees. Res. Virol. 142, 289-296. Hadidi, A., Huang, C., Hammond, R.W., and Hashimoto, J. (1990). Homology of the agent associated with dapple apple disease to apple scar skin viroid and molecular detection of these viroids. Phytopathology 80, 263-268. Hamilton, R.I. (1980). Defences triggered by previous invaders: viruses. Plant Dis. 5, 279-303. Hammond, J. (1997). Repelling plant pathogens with ribonuclease. Nature Biotech. 15, 1247. Hammond, R., Smith, D.R., and Diener, T.O. (1989). Nucleotide sequence and proposed secondary structure of Columnea latent viroid: a natural mosaic of viroid sequences. Nucleic Acids Res. 17, 10083-10094. Hammond, R.W. (1994). Agrobacterium-mediated inoculation of PSTVd cDNAs onto tomato reveals the biological effect of apparently lethal mutations. Virology 201, 36-45. Harders, J., Lukacs, N., Robert-Nicoud, M., Jovin, T.M., and Riesner, D. (1989). Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy. EMBO J. 8, 3941-3949. Hari, V. (1980). Ultrastructure of potato spindle tuber viroid-infected tomato leaf tissue. Phytopathology 70, 385-387. Harris, P.S., and Browning, I.A. (1980). The effects of temperature and light on the symptom expression and viroid concentration in tomato of a severe strain of potato spindle tuber viroid. Potato Res. 23, 85-93. Harris, P.S., and James, C.M. (1987). Exclusion of viroids from potato resources and the modified use of a cDNA probe. EPPO Bull. 17, 51-60. Hernández, C., and Flores, R. (1992). Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures. Proc. Natl. Acad. Sci. 89, 3711-3715. Herold, T., Haas, B., Singh, R.P., Boucher, A., and Sänger, H.L. (1992). Sequence analysis of five new field isolates demonstrates that the chain length of potato spindle tuber viroid (PSTVd) is not strictly conserved but as variable as in other viroids. Plant Mol. Biol. 19, 329-333.

Hodgson, R.A.J., Wall, G.C., and Randles, J.W. (1998). Specific identification of coconut tinangaja viroid for differential diagnosis of viroids in coconut palm. Phytopathology 88, 774-781. Hollings, M., and Stone, O.M. (1973). Some properties of chrysanthemum stunt, a virus with the characteristics of an uncoated ribonucleic acid. Ann. Appl. Biol. 74, 333-348. Hooker, W.J., Tai, W., and Yang, T.C. (1978). Germination reduction in PSTV infected tomato pollen. Am. Potato J. 55, 378. Howell, W.E., Skrzeczowski, L.J., Burgess, J., and Eastwell, K.C. (1999). Peach latent mosaic viroid (PLMVd) therapy. NRSP-5/IR-2 and Washington State University. Available at http:// www.nrsp5.wsu.edu/nrspresl.html . Hsu, Y.-H., Chen, W., and Owens, R.A. (1994). Nucleotide sequence of a hop stunt viroid variant isolated from citrus growing in Taiwan. Virus Genes 9, 193-195. Hughes, J.D., and Ollennu, L.A.A. (1994). Mild strain protection of cocoa in Ghana against cocoa swollen shoot virus - a review. Plant Pathol. 43, 442-457. Hunter, D.E., Darling, H.M., and Beale, W.L. (1969). Seed transmission of potato spindle tuber virus. Am. Potato J. 46, 247-250. Hurtt, S.S., Podleckis, E.V., and Howell, W.E. (1996). Integrated molecular and biological assays for rapid detection of apple scar skin viroid in pear. Plant Dis. 80, 458-462. Huttinga, H., Mosch, W.H.M., and Treur, A. (1987). Comparison of bidirectional electrophoresis and molecular hybridization methods to detect potato spindle tuber viroid and chrysanthemum stunt viroid. EPPO Bull. 17, 37-43. Imperial, J.S., Bautista, R.M., and Randles, J.W. (1985). Transmission of the coconut cadang-cadang viroid to six species of palm by inoculation with nucleic acid extracts. Plant Pathol. 34, 391-401. Imperial, J.S., Rodriguez, J.B., and Randles, J.W. (1981). Variation in the viroid-like RNA associated with cadang-cadang disease: evidence for an increase in molecular weight with disease progress. J. Gen. Virol. 56, 77-85. Ito, T., Kanematsu, S., Koganezawa, H., Tsuchizaki, T., and Yoshida, K. (1993). Detection of viroid associated with apple fruit crinkle disease. Ann. Phytopathol. Soc. Jpn. 59, 520-527. Kano, T., and Yamaguchi, A. (1985). Indexing for citrus exocortis viroid using herbaceous plants. Bull. Fruit Tree Res. Station (Jap. Min. Agric.) 95-106. Keese, P., Osorio-Keese, M.E., and Symons R.H. (1988). Coconut tinangaja viroid: sequence homology with coconut cadang-cadang viroid and other potato spindle tuber viroid related RNAs. Virology 162, 508-510. Kesinger, M. (1997). Nursery tool sterilant. Citrus Nursery Newsletter, University of Florida 8: Available at www.imok.ufl.edu/citrus/news/ newslttr.htm. Khoury, J., Singh, R.P., Boucher, A., and Coombs, D.H. (1988). Concentration and distribution of mild and severe strains of potato spindle tuber viroid in cross-protected tomato plants. Phytopathology 78, 1331-1336. Kofalvi, S.A., Marcos, J.F., Cañizares, M.C., Pallás, V., and Candresse, T. (1997). Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. J. Gen.Virol. 78, 3177-3186.

43

R.P. Singh et al.

Koganezawa, H. (1985). Transmission to apple seedlings of a low molecular weight RNA extracted from apple scar skin diseased trees. Ann. Phytopathol. Soc. Jap. 51, 176-182. Koganezawa, H. (1986). Further evidence for viroid etiology of apple scar skin and dapple apple diseases. Acta Hortic. 193, 29-33. Koltunow, A.M., Krake, L.R., Johnson, S.D., and Rezaian, M.A. (1989). Two related viroids cause grapevine yellow speckle disease independently. J. Gen. Virol. 70, 3411-3419. Kryczynski, S., Paduch-Cichal, E., and Skrzeczkowski, L.J. (1988). Transmission of three viroids through seed and pollen of tomato plants. J. Phytopathol. 121, 51-57. Kyriakopoulou, P.E., Giunchedi, L., and Hadidi, A. (2001). Peach latent mosaic and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdaliformis: implications on possible origin of these viroids in the Mediterranean region. J. Plant Pathol. 183, 51-62. Kyriakou, A. (1992). Incidence in Cyprus of citrus exocortis viroid and its mechanical transmission. Plant Pathol. 41, 20-24. Lawson, R.H. (1987). Chrysanthemum stunt. Pages 247-259 in: The viroids. T. O. Diener, ed. Plenum Press: New York. Lee, C.R., and Singh, R.P. (1972). Enhancement of diagnostic symptoms of potato spindle tuber virus by manganese. Phytopathology 62, 516-520. Lima, M.I., Fonseca, M.E.N., Flores, R., and Kitajima, E.W. (1994). Detection of avocado sunblotch viroid in chloroplasts of avocodo leaves by in situ hybridization. Arch. Virol. 138, 385-390. Lizarraga, R.E., Salazar, S.F., Roca, W.M., and Schilde-Rentschler, L. (1980). Elimination of potato spindle tuber viroid by low temperature and meristem culture. Phytopathology 70, 754-755. MacLachlan, D.S. (1960). Potato spindle tuber in Eastern Canada. Am. Potato J. 37, 13-17. Manzer, F.E., and Merriam, D. (1961). Field transmission of the potato spindle tuber virus and virus X by cultivating and hilling equipment. Am. Potato J. 38, 346-352. Marcos, P., and Flores, R. (1990). Subcellular location of avocado sunblotch viroid in avocado leaves. Plant Sci. 67, 237-244. Marton, L., Duran-Vila, N., Lin, J.J., and Semancik, J.S. (1982). Properties of cell cultures containing the citrus exocortis viroid. Virology 122, 229-238. Matousek, J., Schroder, A.R.W., Trnena, L., Reimers, M., Baumstark, T., Dedic, P., Vlasak, J., Becker, I., Kreuzaler, F., Fladung, M., and Riesner, D. (1994a). Inhibition of viroid infection by antisense RNA expression in transgenic plants. Biol. Chem. (Hoppe-Seyler) 375, 765-777. Matousek, J., Trnena, L., Rakousky, S., and Riesner, D. (1994b). Inhibition of potato spindle tuber viroid (PSTVd) infectivity with antisense RNA transcripts. J. Phytopathol. 141, 10-24. McDonald, J., and Borrel, B. (1991). Development of post-harvest testing in Canada. Am. Potato J. 68, 99-121. Mishra, M.D., Hammond, R.W., Owens, R.A., Smith, D.R., and Diener, T.O. (1991). Indian bunchy top disease of tomato plants is caused by a distinct strain of citrus exocortis viroid. J. Gen. Virol. 72, 1-5. Momma, T., and Takahashi, T. (1982). Ultrastructure of hop stunt viroid-infected leaf tissue. Phytopathol. Z. 104, 211-221. Momma, T., and Takahashi, T. (1983). Cytopathology of shoot apical meristem of hop plants infected with hop stunt viroid. Phytopathol. Z. 106, 272-280.

44

Momma, T., and Takahashi, T. (1984). Developmental morphology of hop stunt viroid- infected hop plants and analysis of their cone yield. Phytopathol. Z. 110, 1-14. Moresht, S., Cohen, S., Assor, Z., and Bar-Joseph, M. (1998). Water relations of citrus exocortis viroid-infected grapefruit trees in the field. J. Exp. Bot. 49, 1421-1430. Morris, T.J., and Smith, E.M. (1977). Potato spindle tuber disease: procedures for the detection of viroid RNA and certification of disease-free potato tubers. Phytopathology 67, 145-150. Morton, A., Barbara, D.J., and Adams, A.N. (1993). The distribution of hop latent viroid within plants of Humulus lupulus and attempts to obtain viroid-free plants. Ann. Appl. Biol. 123, 47-53. Mühlbach, H.-P., and Sänger, H.L. (1979). Viroid replication is inhibited by alpha-amanitin. Nature 278, 185-188. Navarro, B., and Flores, R. (1997). Chrysanthemum chlorotic mottle viroid: unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes. Proc. Natl. Acad. Sci. USA 94, 11262-11267. Navarro, J., Daròs, J.-A., and Flores, R. (1999). Complexes containing both polarity strands of avocado sunblotch viroid: identification in chloroplasts and characterization. Virology 253, 77-85. Navarro, J., Vera, A., and Flores, R. (2000). A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid. Virology 268, 218-225. Nelson, M.E., Klein, R.E., and Skrzeczkowski, L.J. (1999). Occurrence of hop latent viroid (HLVd) in hops in Washington State. Available at www.nrsp5.wsu.edu/nrsphlvd.html. Niblett, C.L., Dickson, E., Fernow, K.H., Horst, R.K., and Zaitlin, M. (1978). Cross protection among four viroids. Virology 91, 198-203. Niblett, C.L., Dickson, E., Horst, R.K., and Romaine, C.P. (1980). Additional hosts and an efficient purification procedure for four viroids. Phytopathology 70, 610-615. O’Brien, M.J., and Raymer, W.B. (1964). Symptomless hosts of the potato spindle tuber virus. Phytopathology 54, 1045-1047. Ohno, T., Takamatsu, N., Meshi, T., and Okado, Y. (1983). Hop stunt viroid: molecular cloning and nucleotide sequence of the complete cDNA copy. Nucleic Acids Res. 11, 6185-6197. Osaki, H., Kudo, A., and Ohtsu, Y. (1996). Japanese pear fruit dimple disease caused by apple scar skin viroid. Ann. Phytopathol. Soc. Jpn. 62, 379-385. Owens, R.A., Chen, W., Hu, Y., and Hsu, Y.-H. (1995). Suppression of potato spindle tuber viroid replication and symptom expression by mutations which stabilize the pathogenicity domain. Virology 208, 554-564. Owens, R.A., Khurana, S.M.P., Smith, D.R., Singh, M.N., and Garg, I.D. (1992). A new mild strain of potato spindle tuber viroid isolated from wild Solanum spp. in India. Plant Dis. 76, 527-529. Owens, R.A., Smith, D.R., and Diener, T.O. (1978). Measurement of viroid sequence homology by hybridization with complementary DNA prepared in vitro. Virology 89, 388-394. Owens, R.A., Steger, G., Hu, Y., Fels, A., Hammond, R.W., and Riesner, D. (1996). RNA structural features responsible for potato spindle tuber viroid pathogenicity. Virology 222, 144-158. Owens, R.A., Thompson, S.M., Feldstein, P.A., and Garnsey, S.M. (1999). Effects of natural sequence variation on symptom induction by citrus viroid III. Ann. Appl. Biol. 134, 73-80.

BIOLOGY

Owens, R.A., Thompson, S.M., and Steger, G. (1991). Effects of random mutagenesis upon potato spindle tuber viroid replication and symptom expression. Virology 185, 18-31. Paduch-Cichal, E., and Kryczynski, S. (1987). A low temperature therapy and meristem-tip culture for eliminating four viroids from infected plants. J. Phytopathol. 118, 341-346. Paliwal, Y.C., and Singh, R.P. (1981). Cytopathological changes induced by potato spindle tuber viroid in Scopolia sinensis. Can. J. Bot. 59, 677-682. Pallás, V., and Flores, R. (1989). Interactions between citrus exocortis and potato spindle viroids in plants of Gynura aurantiaca and Lycopersicon esculentum. Intervirology 30, 10-17. Palukaitis, P. (1987). Potato spindle tuber viroid: Investigation of the long-distance, intra-plant transport route. Virology 158, 239-241. Palukaitis, P., Rakowski, A.G., Alexander, D.M., and Symons, R.H. (1981). Rapid indexing of the sunblotch disease of avocados using a complementary DNA probe to avocado sunblotch viroid. Ann. Appl. Biol. 98, 439-449. Pfannenstiel, M.A., and Slack, S.A. (1980). Response of potato cultivars to infection by the potato spindle tuber viroid. Phytopathology 70, 922-926. Pospieszny, H. (1997). Antiviroid activity of chitosan. Crop Prot. 16, 105-106. Postman, J.D., and Hadidi, A. (1995). Elimination of apple scar skin viroid from pears by in vitro thermo-therapy and apical meristem culture. Acta Hortic. 386, 536-543. Puchta, H., Herold, T., Verhoeven, K., Roenhorst, A., Ramm, K., Schmidt-Puchta W., and Sänger, H.L. (1990). A new strain of potato spindle tuber viroid (PSTVd-N) exhibits major sequence differences as compared to all other PSTVd strains sequenced so far. Plant Mol. Biol. 15, 509-511. Puchta, H., Ramm, K., Hadas, R., Bar-Joseph, M., Luckinger, R., Freimuller, K., and Sänger, H.L. (1989). Nucleotide sequence of a hop stunt viroid (HSVd) isolate from grapefruit in Israel. Nucleic Acids Res. 17, 1247. Puchta, H., Ramm, K., and Sänger, H.L. (1988a). Molecular and biological properties of a cloned and infectious new sequence variant of cucumber pale fruit viroid (CPFV). Nucleic Acids Res. 16, 8171. Puchta, H., Ramm, K., and Sänger, H.L. (1988b). The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops. Nucleic Acids Res. 16, 4197-4216. Puchta, H., Ramm, K., and Sänger, H.L. (1988c). Nucleotide sequence of a hop stunt viroid isolate from the German grapevine cultivar ‘Riesling’. Nucleic Acids Res. 16, 2730. Querci, M., Owens, R.A., Bartolini, I., Lazarte, V. , and Salazar, L.F. (1997). Evidence for heterologous encapsidation of potato spindle tuber viroid in particles of potato leafroll virus. J. Gen. Virol. 78, 1-5. Querci, M., Owens, R.A., Vargas, C., and Salazar, L.F. (1995). Detection of potato spindle tuber viroid in avocado growing in Peru. Plant Dis. 79, 196-202. Randles, J.W. (1975). Association of two ribonucleic acid species with cadang-cadang disease of coconut palm. Phytopathology 65, 163-167. Randles, J.W. (1982). Cadang-cadang disease — a potential threat to palms in South East Asia. Proc. Int. Conf. Plant Protect. in Tropics. 135-139.

Randles, J.W., Boccardo, G., and Imperial, J.S. (1980). Detection of the cadang-cadang associated RNA in African oil palm and Buri palm. Phytopathology 70, 185-189. Randles, J.W., and Imperial, J.S. (1984). Coconut cadang-cadang viroid. CMI/AAB Description of Plant Viruses. No. 287. Reinking, O.A. (1961). Yellow mottle decline of coconuts in the territory of Guam. Plant Dis. Rep. 45, 599-604. Rezaian, M.A. (1990). Australian grapevine viroid-evidence for extensive recombination between viroids. Nucleic Acids Res. 18, 1813-1818. Rezaian, M.A., Krake, L.R., and Golino, D.A. (1992). Common identity of grapevine viroids from USA and Australia revealed by PCR analysis. Intervirology 34, 38-43. Rodriguez, J.M.B., and Randles, J.W. (1993). Mutations in cadangcadang viroid associated with severe symptoms. 9th International Congress of Virology. Abstracts: 106. Roistacher, C.N., Calavan, E.C., Blue, R.L., Navarro, L., and Gonzales, R. (1977). A new more sensitive citron indicator for detection of mild isolates of citrus exocortis viroid (CEV). Plant Dis. Rep. 61, 135-139. Romaine, C.P., and Horst, R.K. (1975). Suggested viroid etiology for chrysanthemum chlorotic mottle disease. Virology 64, 86-95. Salazar, L.F., Owens, R.A., Smith, D.R., and Diener, T.O. (1983). Detection of potato spindle tuber viroid by nucleic acid spot hybridization: evaluation with tuber sprouts and true potato seed. Am. Potato J. 60, 587-597. Salazar, L.F., Querci, M., Bartolini, I., and Lazarte, V. (1995). Aphid transmission of potato spindle tuber viroid assisted by potato leafroll virus. Fitopatologia 30, 56-58. Sänger, H.L. (1979). Structure and function of viroids. Pages 291-341 in: Slow transmissible diseases of the nervous system: Volume 2. S.B. Prusiner, and W.J. Hadlow, eds. Academic Press: New York. Sänger, H.L., and Ramm, K. (1974). Radioactive labelling of viroid-RNA. 2nd John Innes Symposium, Norwich, England, pp. 229-252. Sano, T., Hataya, T., and Shikata, E. (1988). Complete nucleotide sequence of a viroid isolated from Etrog citron, a new member of hop stunt viroid group. Nucleic Acids Res. 16, 347. Sano, T., Hataya, T., Terai, Y., and Shikata, E. (1989). Hop stunt viroid strains from dapple fruit disease of plum and peach in Japan. J. Gen. Virol. 70, 1311-1319. Sano, T., Nagayama, A., Ogawa, T., Ishida, I., and Okada, Y. (1997). Transgenic potato expressing a double-stranded RNA-specific ribonuclease is resistant to potato spindle tuber viroid. Nature Biotech. 15, 1290-1294. Sano, T., Ohshima, K., Hataya, T., Uyeda, I., Shikata, E., Chou, T.-G., Meshi T., and Okada, Y. (1986). A viroid resembling hop stunt viroid in grapevines from Europe, the United States and Japan. J. Gen. Virol. 67, 1673-1678. Sano, T., Uyeda, I., Shikata, E., Meshi, T., Ohno, T., and Okada, Y. (1985). A viroid-like RNA isolated from grapevine has high sequence homology with hop stunt viroid. J. Gen. Virol. 66, 333-338. Sano, T., Uyeda, I., Shikata, E., Ohno, T., and Okada, Y. (1984). Nucleotide sequence of cucumber pale fruit viroid: homology to hop stunt viroid. Nucleic Acids Res. 12, 3427-3434. Sasaki, M., and Shikata, E. (1977). Studies on the host range of hop stunt disease in Japan. Jap. Acad. 53, 103-108.

45

R.P. Singh et al.

Sasaki, M., and Shikata, E. (1978). Studies on hop stunt disease II. Properties of causal agent, a viroid. Ann. Phytopathol. Soc. Jpn. 44, 570-577. Schlemmer, A., Roistacher, C.N., and Semancik, J.S. (1985). A unique, infectious RNA associated with citron showing symptoms typical of citrus exocortis disease. Phytopathology 75, 946-949. Schnell, R.J., Kuhn, D.N., Ronning, C.M., and Harkins, D. (1997). Application of RT-PCR for indexing avocado sunblotch viroid. Plant Dis. 81, 1023-1026. Schnolzer, M., Haas, B., Ramm, K., Hofmann, H., and Sänger, H.L. (1985). Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV). EMBO J. 4, 2181-2190. Schumacher, J., Sänger, H.L., and Riesner, D. (1983). Subcellular location of viroids in highly purified nuclei from tomato leaf tissue. EMBO J. 2, 1549-1555. Semancik, J.S., Magnuson, D.S., and Weathers, L.G. (1973). Potato spindle tuber disease produced by pathogenic RNA from citrus exocortis disease: evidence for the identity of causal agents. Virology 52, 292-294. Semancik, J.S., Rakowski, A.G., Bash, J.A., and Gumpf, D.J. (1997). Application of selected viroids for dwarfing and enhancement of production of ‘Valencia’ orange. J. Hortic. Sci. 72, 563-570. Semancik, J.S., Roistacher, C.N., Rivera-Bustamante, R., and Duran-Vila, N. (1988). Citrus cachexia viroid, a new viroid of citrus: relationship to viroids of the exocortis disease complex. J. Gen. Virol. 69, 3059-3068. Semancik, J.S., and Szychowski, J.A. (1994). Avocado sunblotch disease: a persistent viroid infection in which variants are associated with differential symptoms. J. Gen. Virol. 75, 1543-1549. Semancik, J.S., Szychowski, J.A., Rakowski, A.G., and Symons, R.H. (1993). Isolates of citrus exocortis viroid recovered by host tissue selection. J. Gen. Virol. 74, 2427-2436. Semancik, J.S., Szychowski, J.A., Rakowski, A.G., and Symons, R.H. (1994). A stable 463 nucleotide variant of citrus exocortis viroid produced by terminal repeats. J. Gen. Virol. 75, 727-732. Semancik, J.S., and Vanderwoude, W.J. (1976). Exocortis viroid: cytopathic effects at the plasma membrane in association with pathogenic RNA. Virology 69, 719-726. Semancik, J.S., and Weathers, L.G. (1972). Exocortis virus: an infectious free-nucleic acid plant virus with unusual properties. Virology 47, 456-466. Shamloul, A.M., Hadidi, A., Zhu, S.F., Singh, R.P., and Sagredo, B. (1997). Sensitive detection of potato spindle tuber viroid using RTPCR and identification of a viroid variant naturally infecting pepino plants. Can. J. Plant Pathol. 19, 89-96. Shamloul, A.M., Minafra, A., Hadidi, A., Giunchedi, L., Waterworth, H.E., and Allam, E.K. (1995). Peach latent mosaic viroid: nucleotide sequence of an italian isolate, sensitive detection using RT-PCR and geographic distribution. Acta Hortic. 386, 522-530. Singh, R.P. (1970). Seed transmission of potato spindle tuber virus in tomato and potato. Am. Potato J. 47, 225-227. Singh, R.P. (1971). A local lesion host for potato spindle tuber virus. Phytopathology 61, 1034-1035. Singh, R.P. (1973). Experimental host range of the potato spindle tuber ‘virus’. Am. Potato J. 50, 111-123. Singh, R.P. (1977). Piperonyl butoxide as a protectant against potato spindle tuber viroid infection. Phytopathology 67, 933-935.

46

Singh, R.P. (1983). Viroids and their potential danger to potatoes in hot climates. Can. Plant Dis. Surv. 63, 13-18. Singh, R.P. (1984). Solanum berthaultii, a sensitive host for indexing potato spindle tuber viroid from dormant tubers. Potato Res. 27, 163-172. Singh, R.P. (1985). Clones of Solanum berthaultii resistant to potato spindle tuber viroid. Phytopathology 65, 1432-1434. Singh, R.P. (1988). Occurrence, diagnosis and eradication of the potato spindle tuber viroid in Canada. Viroids of plants and their detection: International Seminar, 12–20 August 1986. Warsaw Agricultural University Press: Warsaw, Poland. pp. 37-50. Singh, R.P. (1989). Techniques in the study of viroid diseases of tropical and subtropical plants. Rev. Trop. Plant Pathol. 6, 81-118. Singh, R.P., and Bagnall, R.H. (1968). Infectious nucleic acid from host tissues infected with the potato spindle tuber virus. Phytopathology 58, 696-699. Singh, R.P., and Boucher, A. (1987). Electrophoretic separation of a severe from mild strains of potato spindle tuber viroid. Phytopathology 77, 1588-1591. Singh, R.P., and Boucher, A. (1988a). Comparative detection of mild strains of potato spindle tuber viroid from the dormant potato tubers by return-polyacrylamide gel electrophoresis and nucleic acid hybridization. Potato Res. 31, 159-166. Singh, R.P., and Boucher, A. (1988b). Loss of potato spindle tuber viroid from tuber tissues after repeated freezing. Am. Potato J. 65, 283-287. Singh, R.P., Boucher, A., Haas, B., and Sänger, H.L. (1991a). Differential migration during polyacrylamide gel electrophoresis suggests conformational differences among strains of potato spindle tuber viroid. Can. J. Plant Pathol. 13, 202-211. Singh, R.P., Boucher, A., and Singh, A. (1991b). High incidence of transmission and occurrence of a viroid in commercial seeds of Coleus in Canada. Plant Dis. 75, 184-187. Singh, R.P., Boucher, A., and Somerville, T.H. (1989a). Evaluation of chemicals for disinfection of laboratory equipment exposed to potato spindle tuber viroid. Am. Potato J. 66, 239-246. Singh, R.P., Boucher, A., and Somerville, T.H. (1990). Cross-protection with strains of potato spindle tuber viroid in the potato plant and other Solanaceous hosts. Phytopathology 80, 246-250. Singh, R.P., Boucher, A., and Somerville, T.H. (1992a). Detection of potato spindle tuber viroid in the pollen and various parts of potato plant pollinated with viroid-infected pollen. Plant Dis. 76, 951-953. Singh, R.P., Boucher, A., and Wang, R.G. (1991c). Detection, distribution and long-term persistence of potato spindle tuber viroid in true potato seed from Heilongjiang, China. Am. Potato J. 68, 65-74. Singh, R.P., Boucher, A., Wang, R.G., and Somerville, T.H. (1992b). Potato spindle tuber viroid is not encapsidated in vivo by potato virus Y particles. Can. J. Plant Pathol. 14, 18-21. Singh, R.P., and Clark, M.C. (1973). Similarity of host response to both potato spindle tuber and citrus exocortis viruses. FAO Plant Prot. Bull. 21, 121-125. Singh, R.P., and Crowley, C.F. (1985). Evaluation of polyacrylamide gel electrophoresis, bioassay and dot-blot methods for the survey of potato spindle tuber viroid. Can. Plant Dis. Surv. 65, 61-63. Singh, R.P., De Avila, A.C., Dusi, A.N., Boucher, A., Trindade, D.R., Van Slobbe, W.G., Ribeiro, S.G., and Fonseca, M.E.N. (1988a). Associ-

BIOLOGY

ation of viroid-like nucleic acids with the fatal yellowing disease of oilpalm. Fitopatol. Bras. 13, 392-394. Singh, R.P., DeHaan, T.-L., and Jaswal, A.S. (1988b). A survey of the incidence of potato spindle tuber viroid in Prince Edward Island using two testing methods. Can. J. Plant Sci. 68, 1229-1236. Singh, R.P., Finnie, R.E., and Bagnall, R.H. (1970). Relative prevalence of mild and severe strains of potato spindle tuber virus in Eastern Canada. Am. Potato J. 47, 289-293. Singh, R.P., Finnie, R.E., and Bagnall, R.H. (1971). Losses due to the potato spindle tuber virus. Am. Potato J. 48, 262-267. Singh, R.P., Khoury, J., Boucher, A., and Somerville, T.H. (1989b). Characteristics of cross-protection with potato spindle tuber viroid strains in tomato plants. Can. J. Plant Pathol. 11, 263-267. Singh, R.P., and Kurz, J. (1997). RT-PCR analysis of PSTVd aphid transmission in association with PLRV. Can. J. Plant Pathol. 19, 418-424. Singh, R.P., Lakshman, D.K., Boucher, A., and Tavantzis, S.M. (1992c). A viroid from Nematanthus wettsteinii plants closely related to Columnea latent viroid. J. Gen. Virol. 73, 2769-2774. Singh, R.P., Lee, C.R., and Clark, M.C. (1974). Manganese effect on the local lesion symptom of potato spindle tuber ‘virus’ in Scopolia sinensis. Phytopathology 64, 1015-1018. Singh, R.P., Michniewicz, J.J., and Narang, S.A. (1975). Piperonyl butoxide, a potent inhibitor of potato spindle tuber viroid in Scopolia sinensis. Can. J. Biochem. 53, 1130-1132. Singh, R.P., Nie, X., and Singh, M. (1999). Tomato chlorotic dwarf viroid: an evolutionary link in the origin of pospiviroids. J. Gen. Virol. 80, 2823-2828. Singh, R.P., and O’Brien, M.J. (1970). Additional indicator plants for potato spindle tuber virus. Am. Potato J. 47, 367-371. Singh, R.P., and Slack, S.A. (1984). Reactions of tuber-bearing ‘Solanum’ species to infection with potato spindle tuber viroid. Plant Dis. 68, 784-787. Skrzeczkowski, L.J., Howell, W.E., and Mink, G.I. (1993). Correlation between leaf epinasty symptoms on two apple cultivars and results of cRNA hybridization for detection of apple scar skin viroid. Plant Dis. 77, 919-921. Solel, Z., Mogilner, N., Gafny, R., and Bar-Joseph, M. (1995). Induced tolerance to mal secco disease in Etrog citron and Rangpur lime by infection with the citrus exocortis viroid. Plant Dis. 79, 60-62. Spieker, R.L. (1996a). In vitro-generated ‘inverse’ chimeric Coleus blumei viroids evolve in vivo into infectious RNA replicons. J. Gen.Virol. 77, 2839-2846. Spieker, R.L. (1996b). The molecular structure of Iresine viroid, a new viroid species from Iresine herbstii (‘beefsteak plant’). J. Gen. Virol. 77, 2631-2635. Spieker, R.L. (1996c). A new sequence variant of Coleus blumei viroid 1 from the Coleus blumei cultivar ‘Rainbow Gold’. Arch. Virol. 141, 2153-2161. Spieker, R.L. (1996d). A viroid from Brunfelsia undulata closely related to the Columnea latent viroid. Arch. Virol. 141, 1823-1832. Spieker, R.L., Marinkovic, S., and Sänger, H.L. (1996). A viroid from Solanum pseudocapsicum closely related to the tomato apical stunt viroid. Arch. Virol. 141, 1387-1395. Stocker, S., Guitton, M.-C., Barth, A., and Mühlbach, H.-P. (1993). Photosynthetically active suspension cultures of potato spindle tuber viroid infected tomato cells as tools for studying viroid-host cell interaction. Plant Cell Rep. 12, 597-602.

Syller, J., Marczewski, W., and Pawlowicz, J. (1997). Transmission by aphids of potato spindle tuber viroid encapsidated by potato leafroll luteovirus particles. Eur. J. Plant Pathol. 103, 285-289. Szychowski, J.A., Credi, R., Reanwarakorn, K., and Semancik, J.S. (1998). Population diversity in grapevine yellow speckle viroid-1 and the relationship to disease expression. Virology 248, 432-444. Takahashi, T. (1987). Plant viroid diseases occurring in Japan. Jap. Agric. Res. Quart. 21, 184-191. Takahashi, T., Chiba, K., Ozaki, R., Sadakata, H., Andoh, Y., and Yoshikawa, N. (1992a). Growth characteristics in cultured cucumber tissues infected with hop stunt viroid. J. Phytopathol. 136, 288-296. Takahashi, T., Fujiwara, S., Chiba, K., and Yoshikawa, N. (1992b). Comparison of plant hormone requirements in leaf tissues from hop stunt viroid-infected and uninfected hop plants. J. Plant Dis. Prot. 99, 62-70. Takahashi, T., Yaguchi, S., Oikawa, S., and Kamita, N. (1982). Subcellular location of hop stunt viroid. Phytopathol. Z. 103, 285-293. van Dorst, H.J.M., and Peters, D. (1974). Some biological observations on pale fruit, a viroid-incited disease of cucumber. Neth. J. Plant Pathol. 80, 85-96. Vera, P., and Conejero, V. (1989). The induction and accumulation of the pathogensis-related P69 proteinase in tomato during citrus exocortis viroid infection and in response to chemical treatments. Physiol. Mol. Plant. Pathol. 34, 323-334. Vera, P., and Conejero, V. (1990). Citrus exocortis viroid infection alters the in vitro pattern of protein phosphorylation of tomato leaf proteins. Mol. Plant- Microbe Interact. 3, 28-32. Verhoeven, N.J.T., Arts, M.S.J., Owens, R.A., and Roenhorst, T.J.W. (1998). Natural infection of petunia by chrysanthemum stunt viroid. Eur. J. Plant Pathol. 104, 383-386. Visvader, J.E., and Symons, R.H. (1983). Comparative sequence and structure of different isolates of citrus exocortis viroid. Virology 130, 232-237. Visvader, J.E., and Symons, R.H. (1985). Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity. Nucleic Acids Res. 13, 2907-2920. Wah, Y.F.W.C., and Symons, R.H. (1997). A high sensitivity RT-PCR assay for the diagnosis of grapevine viroids in field and tissue culture samples. J. Virol. Methods 63, 57-69. Wall, G.C., and Wiecko, A.T. (1997). Survey of tinangaja on Guam’s coconut populations. Phytopathology 87, S101. Wallace, J.M., and Drake, R.J. (1962). A high rate of seed transmission of avocado sun-blotch virus from symptomless trees and the origin of such trees. Phytopathology 52, 237-241. Walter, B. (1987). Tomato apical stunt. Pages 321-328 in: The viroids. T. O. Diener, ed. Plenum Press: New York. Wang, M.C., Lin, J.J., Duran-Vila, N., and Semancik, J.S. (1986). Alteration in cell wall composition and structure in viroid-infected cells. Physiol. Mol. Plant Pathol. 28, 107-124. Wassenegger, M., Heimes, S., Riedel, L., and Sänger, H.L. (1994a). RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567-576. Wassenegger, M., Heimes, S., and Sänger, H.L. (1994b). An infectious viroid RNA replicon evolved from an in vitro-generated non-infectious viroid deletion mutant via a complementary deletion in vivo. EMBO J. 13, 6172-6177.

47

R.P. Singh et al.

Wassenegger, M., Spieker, R.L., Thalmeir, S., Gast, F., Riedel, L., and Sänger, H.L. (1996). A single nucleotide substitution converts potato spindle tuber viroid (PSTVd) from a noninfectious to an infectious RNA for Nicotiana tabacum. Virology 226, 191-197. Wei, C., Bo, T., Xicai, Y., Yuxiang, Z., and Guodong, S. (1986). Study on burdock stunt viroid (BSV)- the denaturing behavior and occurrence in separate plant of BSV RNA-1 and RNA-2. Sci. Sinica (Ser. B) 29, 147-155. Wei, C., Tien, P., Xiang Zhu, Y., and Yong, L. (1983). Viroid-like RNAs associated with burdock stunt disease. J. Gen. Virol. 64, 409-114. Weidemann, H.L. (1987). The distribution of potato spindle tuber viroid in potato plants tubers. EPPO Bull. 17, 45-50. Whitney, E.D., and Peterson, L.C. (1963). An improved technique for inducing diagnostic symptoms in tomato infected by potato spindle tuber virus. Phytopathology 53, 893. Wolpert, J.A., Szychowski, J.A., and Semancik, J.S. (1996). Effect of viroids on growth, yield and maturity indices of cabernet sauvignon grapevines. Am. J. Enol. Vitic. 47, 21-24.

48

Yang, X., Kang, L., and Tien, P. (1996). Resistance of tomato infected with cucumber mosic virus satellite RNA to potato spindle tuber viroid. Ann. Appl. Biol. 130, 207-215. Yang X., Yie Y., Zhu F., Liu Y., Kang L., Wang X., and Tien P. (1997). Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes. Proc. Natl. Acad. Sci. USA 94, 4861-4865. Yeh, S.D., and Gonsalves, D. (1984). Evaluation of induced mutants of papaya ringspot virus for control by cross protection. Phytopathology 74, 1086-1091. Zelazny, B., and Niven, B.S. (1980). Duration of the stages of cadangcadang disease of coconut palm. Plant Dis. 64, 841-842. Zhu, S.F., Hadidi, A., Hammond, R.W., Yang, X., and Hansen, A.J. (1995). Nucleotide sequence and secondary structure of pome fruit viroids from dapple apple diseased apples, pear rusty skin diseased pears and apple scar skin symptomless pears. Acta Hortic. 386, 554-559.

PART II

CHAPTER 4

MOVEMENT ....................................................................................................


.................................................................................................................................................................................................................................................................

Systemic infection of a host plant by a viroid is a multi-step process: entry into a host cell is followed by viroid movement to the intracellular site of replication; following replication, the resulting progeny exit from this site and invade neighboring cells; and finally, the viroid moves from organ to organ to infect the whole plant. Despite their obvious importance to the establishment of a successful infection, these movement processes have received comparatively little attention — especially in comparison to the physical and structural properties of viroids. In this chapter, we discuss some basic features of viroid movement with emphasis on several currently unresolved issues.

INTRACELLULAR MOVEMENT Pospiviroid (and presumably other rod-shaped viroids) replicate in the nucleus. Only two viroids including Avocado sunblotch viroid (ASBVd) and Peach latent mosaic viroid (PLMVd) appear to replicate in the chloroplasts. Thus, viroids must enter the nucleus or chloroplasts prior to replication and exit these organelles after replication.

NUCLEAR IMPORT Woo et al. (1999) have studied the mechanism of Potato spindle tuber viroid (PSTVd) nuclear import in permeabilized proto-

plasts. Infectious, fluorescein-labeled potato spindle tuber viroid RNA transcripts synthesized in vitro (F-PSTVd) were shown to move into the nucleus of permeabilized tobacco BY2 cells within minutes after addition to the incubation medium (Figure 4.1a, b, c). In contrast, mRNA fragments of the same size remained in the cytoplasm. Nuclear import of F-PSTVd was inhibited by the presence of a ten-fold molar excess of nonfluorescent PSTVd but not by similar amounts of several control RNAs. PSTVd import thus appears to be a specific process mediated by as-yet-unidentified host factor(s). Although GTP hydrolysis is necessary for the nuclear import and export of many proteins and RNAs (Koepp and Silver 1996), it is not required for PSTVd import. The rapid concentration of F-PSTVd in the nucleoli of permeabilized BY2 cells is consistent with the results of Harders et al. (1989) who used in situ hybridization to demonstrate that PSTVd accumulates in the nucleoli of infected cells. Coconut cadang-cadang viroid (CCCVd) also accumulates in the nucleoli of infected cells (Bonfiglioli et al. 1994, 1996). Citrus exocortis viroid (CEVd), in contrast, is distributed throughout the nucleoplasm (Bonfiglioli et al. 1996). Presumably, multimeric PSTVd molecules synthesized in the nucleoplasm are transported into the nucleolus to be processed into monomeric molecules,

49


Figure 4.1 Intracellular and intercellular movement of PSTVd. (a–c) Nuclear import of fluorescein-labeled PSTVd in permeabilized tobacco BY2 protoplasts. All images are from the same cells. (a) 70 kilodalton rhodamine-labeled dextran (R-dex) enters the cytoplasm but is excluded from the nuclei. (b) DAPI staining shows location of nuclei. (c) F-PSTVd is concentrated in the nuclei (from Woo et al. 1999). (d and e) Cell-to-cell movement of fluorescent PSTVd in tobacco. (d) Microinjected PSTVd moves from cell to cell in mesophyll. Arrow indicates cell injected. Asterisk indicates intercellular space. N, nucleus. (e) PSTVd injected into a mature guard cell remains in the same cell. N, nucleus (from Ding et al. 1997). All scale bars = 20µm.

thereby accounting for the high nucleolar concentration (Harders et al. 1989). After replication, most viroid progeny remain in the nucleus. Some progeny, however, must exit the nucleus and move into neighboring cells in order to establish a systemic infection. How viroids are transported out of the nucleus is completely unknown.

CHLOROPLAST TRANSPORT Two viroids are localized predominantly in the chloroplasts of infected leaf tissues, suggesting that they replicate in these

50

organelles. These are ASBVd (Bonfiglioli et al. 1994; Lima et al. 1994; Navarro et al. 1999) and PLMVd (Bussière et al. 1999). Significantly, both viroids exhibit self-cleaving hammerhead ribozyme activities. Whether these viroids can replicate in other types of plastids (e.g. proplastids, amyloplasts, etioplasts, and chromoplasts) remains to be determined. How they enter and exit the chloroplast is also completely unknown. Resolution of this issue is important not only to understanding viroid pathogenicity, but also to understanding RNA trafficking between the cytoplasm and chloroplasts (Ding et al. 1999). The other member of the genus Pelamoviroid is Chrysanthemum chlorotic mottle viroid (CChMVd). CChMVd titers in infected plants are

MOVEMENT

Figure 4.2 Long-distance movement of PSTVd. (a) After inoculation onto the third leaf (shaded) of a tomato plant, PSTVd replicates and moves into all higher leaves and the root, as shown by dot blot analysis (top). The viroid does not move into leaves below the inoculated leaf (from Palukaitis 1987). (b) In situ hybridization reveals the presence of minus-sense PSTVd in the nuclei (arrows) of infected tomato phloem parenchyma/ companion cells. Scale bar=10µm (from Zhu et al. 2001). (c) PSTVd does not invade the shoot apical meristem in infected N. benthamiana. Scale bar=30µm (from Zhu et al. 2001).

very low (Navarro and Flores 1997), and whether this viroid replicates in the chloroplasts or other plastids remains to be determined.

INTERCELLULAR MOVEMENT After exiting the nucleus or chloroplast, viroids next move into neighboring cells. Conventional viruses encode one or more ‘movement proteins’ that interact with the viral genomes and cellular factors to potentiate viral intercellular movement (Carrington et al. 1996; Ding 1998; Lazarowitz and Beachy 1999). Because viroids encode no proteins, their movement likely involves distinct mechanisms. Indeed, PSTVd does not complement the cell-to-cell movement of a mutant tobacco mosaic virus (TMV) defective in the movement function (Kondakova et al. 1989).

Ding et al. (1997) have used microinjection to investigate cellto-cell movement of PSTVd. Upon injection into tomato or tobacco mesophyll cells, infectious PSTVd RNA transcripts rapidly moved into neighboring cells (Figure 4.1d). When such transcripts were injected into a mature guard cell, however, they remained localized in the injected cell (Figure 4.1e). Because mesophyll cells are interconnected by plasmodesmata and mature guard cells are not, these results suggest that PSTVd moves intercellularly through plasmodesmata. A control transcript containing only 1400 nt of vector sequences was unable to move out of the injected mesophyll cells, but when PSTVd was fused to this RNA, the resulting fusion RNA moved from cell to cell. Thus, PSTVd appears to have a specific sequence or structural motif that interacts with cellular factor(s) involved in the movement process.

51


upward to sink (but not source) leaves when inoculated onto the stem of a plant below all leaves, the viroid does not enter the shoot apical meristems (SAMs) (Figure 4.2c). In the flowers of infected plants, PSTVd was present in sepals but not in the petals, stamens, or ovary. Clearly, the developmental stage of a plant organ is a critical factor in controlling viroid movement. Although in situ hybridization did not detect PSTVd in the petals, stamens and ovary of tomato, this viroid was reported to be seed transmitted in tomato (Benson and Singh 1964; Singh 1970). Either current in situ hybridization protocols are not sensitive enough to detect very low levels of PSTVd in these organs, or particular growth conditions may enable the viroid to infect these organs. More extensive analyses are needed to resolve this issue. The absence of PSTVd in the SAMs of both tomato and N. benthamiana as determined by in situ hybridization, or Hop stunt viroid (HSVd) in infected hop plants as determined by bioassay and tip culture studies (Momma and Takahashi 1983) suggests that many viroids do not invade the SAM. This could be due to failure of these viroids either to replicate in the SAM or to move into the SAM. This is reminiscent of the situation in systemically acquired gene silencing, where a nucleotide sequence-specific signal generated ectopically in the lower part of a plant is able to trigger gene silencing in the upper part of the plant — with notable exception of the shoot apex and mature leaves. Available data suggest that the silencing signal fails to traffic into the shoot apex (Voinnet et al. 1998). Figure 4.3 A model for replication-sustained phloem transport of PSTVd. During movement through the sieve elements (SE), PSTVd enters companion cells (CC) or phloem parenchyma cells (PP) to replicate. Some PSTVd progeny re-enter the SE for further longdistance movement, while other molecules invade neighboring cells. Arrows indicate pathways of PSTVd movement (from Zhu et al. 2001).

LONG-DISTANCE MOVEMENT PSTVd spread in infected plants parallels photoassimilate transport, implying that the viroid moves long distances via the phloem (Figure 4.2a; Palukaitis 1987). Consistent with this observation, in situ hybridization has revealed the presence of PSTVd in vascular tissues of the stems and roots of infected tomato plants (Hammond 1994; Stark-Lorenzen et al. 1997). Coconut cadang-cadang viroid (CCCVd) and Citrus exocortis viroid (CEVd) are also localized in the vascular tissue of infected plants (Bonfiglioli et al. 1996). Minus-strand RNA, normally considered to be an indicator of active viroid replication, has also been detected in the phloem of PSTVd-infected plants (Figure 4.2b; Zhu et al. 2001). More detailed analysis of infection patterns revealed that PSTVd movement in tomato and Nicotiana benthamiana is also regulated by factors other than source-sink relationships (Zhu et al. 2001). For example, despite the fact that PSTVd replicates and moves

52

How is an infectious population of PSTVd maintained along the phloem during long-distance movement? Given the evidence for PSTVd replication in the nucleate phloem parenchyma and companion cells (see above), at least some PSTVd molecules may move laterally from the sieve elements into phloem parenchyma and companion cells. The additional PSTVd molecules could then re-enter the sieve elements for further long-distance movement. As shown in Figure 4.3, replication along the transport pathway would serve in part to propagate the PSTVd population for further trafficking while also providing infectious PSTVd molecules to invade neighboring nonvascular cells. Certain regulatory RNAs may utilize a similar strategy to amplify themselves and traffic systemically in plants; indeed, such a strategy could account for the observed long-distance relay of ectopically and locally produced gene silencing signals (Voinnet et al. 1998). This model raises the question of how viroids that replicate in the chloroplasts, such as ASBVd and PLMVd, move long distance, given that chloroplasts are absent from the phloem. Companion cells and even enucleate sieve elements contain other types of plastids, however. It will be of great interest to determine whether these plastids can support viroid replication. Within the sieve elements themselves, interaction with host protein(s) may regulate long-distance viroid movement. Recently,

MOVEMENT

HSVd has been shown to interact with phloem protein 2 (PP2) isolated from cucumber (Gómez and Pallás 2000; Owens and Ding 2000). Although the general biological functions of PP2 are still obscure, this protein has been shown to traffic over long distances within sieve tubes (Golecki et al. 1999). Interaction with PP2 could facilitate or restrict the long-distance transport of HSVd.

FUTURE PROSPECTS It is clear that our knowledge of viroid movement is still rudimentary. Nevertheless, such knowledge can serve as a basis for further studies to unravel the underlying mechanisms. Obvious areas for future research include identification of viroid movement motifs and host factors that facilitate or restrict viroid movement, as well as characterization of how these motifs and factors interact. Do viroids use the same structural motif(s) for intracellular (i.e. nuclear or chloroplast), intercellular, and longdistance movement? In particular, it will be most enlightening to compare cell-to-cell and long-distance movement patterns for viroids that replicate in the chloroplasts and those that replicate in the nucleus. Studies of viroid movement should benefit greatly from the well-characterized physical and structural properties of viroids as well as the availability of strains and mutants with different pathogenicities and even different host ranges. Knowledge of viroid movement mechanisms will have both fundamental and practical applications. Understanding how a pathogenic RNA has evolved the capacity to move systemically and how host specificity is determined at the level of movement should allow us to develop strategies for engineering crops that are resistant to viroid and viral infection by interfering with one or more of the movement steps. A detailed understanding of viroid movement mechanisms could also facilitate development of RNA-based strategies for regulating gene expression in plants. Acknowledgements

We thank Dr Ricardo Flores for discussions on CChMVd replication site. This work was supported in part by a grant from the United States Department of Agriculture National Research Initiative Competitive Grants Program (No. 97-35303-4519). References Benson, A.P., and Singh, R.P. (1964). Seed transmission of potato spindle tuber virus in tomato. Amer. Potato J. 41, 294. Bonfiglioli, R.G., McFadden, G.I., and Symons, R.H. (1994). In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang- cadang viroid in the nucleus. Plant J. 6, 99-103. Bonfiglioli, R.G., Webb, D.R., and Symons, R.H. (1996). Tissue and intra-cellular distribution of coconut cadang-cadang viroid and citrus exocortis viroid determined by in situ hybridization and

confocal laser scanning and transmission electron microscopy. Plant J. 9, 457-465. Bussière, F., Lehoux, J., Thompson, D.A., Skrzeczkowski, L.J., and Perreault, J.-P. (1999). Subcellular localization and rolling circle replication of peach latent mosaic viroid: hallmarks of group A viroids. J. Virol. 73, 6357-6360. Carrington, J.C., Kasschau, K.D., Mahajan, S.K., and Schaad, M.C. (1996). Cell-to-cell and long-distance transport of viruses in plants. Plant Cell 8, 1669-1681. Ding, B. (1998). Intercellular protein trafficking through plasmodesmata. Plant Mol. Biol. 38, 279-310. Ding, B., Kwon, M.-O., Hammond, R., and Owens, R. (1997). Cell-tocell movement of potato spindle tuber viroid. Plant J. 12, 931-936. Ding, B., Itaya, A., and Woo, Y.-M. (1999). Plasmodesmata and cell-tocell communication in plants. Internatl. Rev. Cytol. 190, 251-316. Golecki, B., Schulz, A., and Thompson, G.A. (1999). Translocation of structural P proteins in the phloem. Plant Cell 11, 127-140. Gómez, G., and Pallás, V. (2000). Identification of a ribonucleoprotein complex between Hop stunt viroid and phloem sap proteins from cucumber plants. EMBO Workshop ‘Plant Virus Invasion and Host Defense’, Kolymbari, Crete, Greece. Hammond, R.W. (1994). Agrobacterium-mediated inoculation of PSTVd cDNAs onto tomato reveals the biological effect of apparently lethal mutations. Virology 201, 36-45. Harders, J., Lukács, N., Robert-Nicoud, M., Jovin, T. M., and Riesner, D. (1989). Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy. EMBO J. 8, 3941-3949. Koepp, D.M., and Silver, P.A. (1996). A GTPase controlling nuclear trafficking: running the right way or walking RANdomly? Cell 87, 1-4. Kondakova, O.A., Malyshenko, S.I., Mozhaeva, K.A., Vasilieva, T.Ya., Taliansky, M.E., and Atabekov, J.G. (1989). Potato spindle tuber viroid does not complement tobacco mosaic virus temperature-sensitive transport function. J. Gen. Virol. 70, 1609-1612. Lazarowitz, S.G., and Beachy, R.N. (1999). Viral movement proteins as probes for intracellular and intercellular trafficking in plants. Plant Cell 11, 535-548. Lima, M.I., Fonseca, M.E.N., Flores, R., and Kitajima, E.W. (1994). Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization. Arch. Virol. 138, 385-390. Momma, T., and Takahashi, T. (1983). Cytopathology of shoot apical meristem of hop plants infected with hop stunt viroid. Phytopath. Z. 106, 272-280. Navarro, B., and Flores, R. (1997). Chrysanthemum chlorotic mottle viroid: unusual structural properties of a subgroup of viroids with hammerhead ribozymes. Proc. Natl. Acad. Sci. USA 94, 11262-11267. Navarro, J.A., Daròs, J.A., and Flores, R. (1999). Complexes containing both polarity strands of avocado sunblotch viroid: identification in chloroplasts and characterization. Virology 253, 77-85. Owens, R.A., and Ding, B. (2000). Long distance viroid movement: the role of viroid-protein interaction. EMBO Workshop ‘Plant Virus Invasion and Host Defense’, Kolymbari, Crete, Greece. Palukaitis, P. (1987). Potato spindle tuber viroid: investigation of the long-distance, intra-plant transport route. Virology 158, 239-241. Singh, R.P. (1970). Seed transmission of potato spindle tuber virus in tomato and potato. Am. Potato J. 47, 225-27.

53


Stark-Lorenzen, P., Guitton, M.-C., Werner, R., and Mühlbach, H.-P. (1997). Detection and tissue distribution of potato spindle tuber viroid in infected tomato plants by tissue print hybridization. Arch. Virol. 142, 1289-1296. Voinnet, O., Vain, P., Angell, S., and Baulcombe, D.C. (1998). Systemic spread of sequence-specific transgene RNA degradation in plants is inhibited by localized introduction of ectopic promoterless DNA. Cell 95, 177-187.

54

Woo, Y.-M., Itaya, A., Owens, R.A., Tang, L., Hammond, R.W., Chou, H.-C., Lai, M.M.C., and Ding, B. (1999). Characterization of nuclear import of potato spindle tuber viroid RNA in permeabilized protoplasts. Plant J. 17, 627-635. Zhu, Y., Green, L., Woo, Y.-M., Owens, R., and Ding, B. (2001). Cellular basis of potato spindle tuber viroid systemic movement. Virology 279, 69-77.

PART II

CHAPTER 5

REPLICATION ....................................................................................................

R. Flores, J.A. Daròs, and J.A. Navarro

.................................................................................................................................................................................................................................................................

Integration of molecular and cellular data about a given biological phenomenon is critical for its full understanding. That this view was soon appreciated in viroid research is revealed by early studies on the subcellular accumulation site of these RNAs as a first step to unravel their mode of replication. Experiments using conventional cell fractionation methods based on differential centrifugation showed that Potato spindle tuber viroid (PSTVd), the type species of family Pospiviroidae, accumulates predominantly in the nucleus (Diener 1971), in contrast to Avocado sunblotch viroid (ASBVd), the type species of family Avsunviroidae, that accumulates in the chloroplast (Mohamed and Thomas 1980). Re-examination of the question using alternative and more resolutive approaches combining in situ hybridization with confocal laser scanning and transmission electron microscopy, confirmed these observations. Using purified nuclear preparations, PSTVd was found in the nucleolus (Harders et al. 1989) and using thin sections of infected tissue, Citrus exocortis viroid (CEVd) and Coconut cadang-cadang viroid (CCCVd), two other members of the family Pospiviroidae, were also located in the nucleus (Bonfiglioli et al. 1996). Similar experiments with sections of ASBVd-infected leaves showed the preferential accumulation of this viroid in the chloroplast (Lima et al. 1994) and more specifically in the thylakoid membranes

(Bonfiglioli et al. 1994), and recent results indicate that Peach latent mosaic viroid (PLMVd), which also belongs to the family Avsunviroidae, also accumulates predominantly in the chloroplast (Bussière et al. 1999). Although the accumulation of viroid in an organelle does not necessarily indicate the site of replication, additional data on the localization of the viroid complementary strands that are synthesized during replication support the notion that PSTVd replicates in the nucleus (Spiesmacher et al. 1983), and ASBVd (Bonfiglioli et al. 1994; Navarro et al. 1999) and PLMVd (Bussière et al. 1999) in the chloroplast.

ROLLING-CIRCLE MECHANISM OF REPLICATION: ASYMMETRIC AND SYMMETRIC PATHWAYS

The circular structure of the viroid RNA template that initiates the replication cycle and the oligomeric nature of some RNA intermediates found in different viroid-infected tissues, support the view that these pathogens replicate through a rolling-circle mechanism (Branch and Robertson 1981 and 1984; Owens and Diener 1982; Hutchins et al. 1985) (Figure 5.1), with only RNA intermediates (Grill and Semancik 1978; Hadidi et al. 1982). The most abundant infecting monomeric circular RNA, to which the (+) polarity is assigned by convention, is transcribed by an RNA-dependent RNA polymerase into oligomeric (-) and

55

R. Flores et al.

Figure 5.1 Rolling-circle mechanism proposed for replication of viroids. Symmetric (upper) and asymmetric (lower) pathways with two and one rolling circles that presumably operate in members of the families Avsunviroidae and Pospiviroidae, respectively. Solid and open lines refer to plus (+) and minus (-) polarities, respectively, and processing sites are marked by arrowheads. Self-cleavage mediated by hammerhead ribozymes (RZ) leads to linear monomeric RNAs with 5'-hydroxyl and 2'-3'-cyclic phosphate termini, the same termini being also most likely generated in cleavage catalyzed by host enzymes (HE).

subsequently (+) strands, which after proper cleavage and ligation via an RNase and an RNA ligase respectively, lead to the monomeric (+) circular RNA, the final product of the cycle. The oligomeric strands resulting from the first RNA-RNA transcription step, but not their monomeric circular counterparts, have been identified in tissues infected by PSTVd and closely related viroids as the major (-) intermediate that serves as the template for the second RNA-RNA transcription step (Branch and Robertson 1984; Branch et al. 1988; Feldstein et al. 1998). Based on these observations, PSTVd and other members of its family are assumed to follow the asymmetric pathway of the rolling-circle mechanism (Figure 5.1, lower half). Conversely, the monomeric (-) circular RNA has been characterized in ASBVdinfected avocado (Hutchins et al. 1985; Daròs et al. 1994; Navarro et al. 1999) and more recently in PLMVd-infected peach (Bussière et al. 1999) and, consequently, members of the family Avsunviroidae are assumed to replicate through the alternative symmetric pathway of the rolling-circle mechanism (Figure 5.1, upper half). Therefore, cleavage and ligation occur in both polarities in the symmetric version with two rolling circles, but only in the (+) polarity in the asymmetrical version with a single rolling circle. One final point in this context concerns the nature of the three catalytic activities required in viroid replication. They must be host-coded enzymes or, alternatively, ribozymes contained in the viroid RNA itself because the available evidence indicates that these minimal pathogens do not function as messenger RNAs.

bach 1992) have shown that replication of PSTVd, CEVd and Hop stunt viroid (HSVd), is inhibited by the low concentrations of α-amanitin that typically inhibit RNA polymerase II, strongly suggesting that an RNA polymerase II-like enzyme, acting on an RNA template, is involved. The same enzyme appears to catalyze both RNA-RNA transcription steps because elongation of (+) and (-) PSTVd RNAs, and of genuine polymerase II transcripts, is similarly inhibited by α-amanitin (Schindler and Mühlbach 1992). Recent results using a monoclonal antibody against a conserved domain in the largest subunit of RNA polymerase II have shown that in chromatinenriched fractions of CEVd-infected tissue with CEVd RNA synthesis activity, this subunit co-purifies with the (+) and (-) strands of the viroid, thus proving the association in vivo between RNA polymerase II and CEVd (Warrilow and Symons 1999).

INITIATION AND ELONGATION OF VIROID STRANDS

Regarding viroids replicating in chloroplasts, at least two DNAdependent RNA polymerases have been characterized in this organelle: a plastid-encoded polymerase (PEP) with a multisubunit structure similar to the E. coli enzyme, and a single-unit nuclear-encoded polymerase (NEP) that resembles T3, T7 and SP6 phage RNA polymerases (see for a review Stern et al. 1997), posing again the question of which enzyme catalyzes elongation of viroid strands. This has been tackled by studying the effects

Because there are three nuclear DNA-dependent RNA polymerases (I, II and III), the first question in this context would be which enzyme is implicated in the synthesis of PSTVd and other members of the family Pospiviroidae. This issue has been addressed by analyzing the effects of the fungal toxin αamanitin. Experiments in vivo (Mühlbach and Sänger 1979) and in vitro (Flores and Semancik 1982; Schindler and Mühl-

56

A second question pertains as to whether transcription of viroid strands is initiated at defined positions or at random, a conceivable alternative considering that the circular nature of the (+) template and the oligomeric nature of the (-) template, enables transcription of oligomeric viroid RNAs irrespective of where synthesis is initiated. Based on in vitro transcription studies with a nuclear extract from potato cells supplemented with the monomeric (+) circular PSTVd RNA, initiation of PSTVd (-) strands has been proposed to occur at two different positions located 15-16 nt downstream of two GC boxes in the proposed rod-like structure (Riesner et al. 2000).

REPLICATION

Figure 5.2 Secondary structures predicted for the ASBVd (+) and (-) monomeric circular RNAs. Sequences involved in forming the hammerhead ribozymes are delimited by flags, with the conserved residues of the catalytic core residues on a black background and the selfcleavage sites indicated by arrows. The initiation sites for (+) and (-) strands (U121 and U119, respectively) are marked by arrowheads. Numbering is the same in both polarities.

on a chloroplastic transcription system from ASBVd-infected leaves of tagetitoxin, which thus parallels the use of α-amanitin in the case of nuclear viroids. Since PEP is sensitive to tagetitoxin whereas this inhibitor has no effect on NEP-transcribed genes or on ASBVd, this suggests that polymerization of viroid strands is mediated by a NEP-like activity acting on an RNA template (Navarro et al. 2000). On the other hand, the transcription initiation sites of ASBVd strands have been recently mapped (Navarro and Flores 2000) taking advantage of evidence that in chloroplasts, the 5’ termini of primary transcripts, but not those resulting from their processing, have a free triphosphate group that can be specifically capped in vitro with [α32P]GTP and guanylyltransferase. This labeling, combined with RNase protection assays (RPA) using ASBVd-specific riboprobes, has been applied to the linear monomeric (+) and (-) ASBVd RNAs isolated from infected tissue. Moreover, these results were confirmed by primer-extension experiments on the same templates. Both ASBVd strands begin with a UAAAA sequence that maps to similar A+U-rich right terminal loops in their predicted quasi-rod-like secondary structures (Figure 5.2). Therefore, a short segment of the ASBVd molecule contains in the (+) and (-) polarities both ini-

tiation sites separated by only 2 nt, an economical solution for such a small RNA as ASBVd (247 nt). Since the sequences forming the central region of the quasi-rod-like secondary structures proposed for the ASBVd (+) and (-) RNAs have a functional role in the self-cleavage of both polarity strands mediated by hammerhead ribozymes (Hutchins et al. 1986; Daròs et al. 1994; see below), these results associate a second ASBVd structural domain, the A+U-rich right terminal loops, with a defined function: determining where synthesis of viroid strands starts (Figure 5.2). Interestingly, the two best characterized NEP promoters (Liere and Maliga 1999), exclusively composed of a short motif (15 to 19 nt) placed immediately upstream of the transcription start sites, have extensive similarity with the sequences around both ASBVd initiation sites, thus reinforcing the involvement of a NEP-like activity in transcription of ASBVd RNAs (Navarro et al. 1999; Navarro and Flores 2000).

PROCESSING OF THE OLIGOMERIC VIROID STRANDS TO THE CIRCULAR MONOMERIC FORMS

Within the family Pospiviroidae, the RNase and RNA ligase activities catalyzing cleavage of the oligomeric (+) RNAs and circularization of the resulting linear monomers, are generally

57

R. Flores et al.

assumed to be host coded-enzymes. Potato nuclear extracts obtained with different protocols are able to in vitro process an oligomeric (+) PSTVd RNA (Tsagris et al. 1987), or a monomeric (+) PSTVd RNA with a short repeat of the upper central conserved region (CCR) (Baumstark et al. 1997), into the infectious monomeric circular form. However, since a nonspecific fungal RNase can also catalyze the same in vitro cleavage and ligation reactions leading to the infectious circular PSTVd (Tabler et al. 1992), the question is whether a RNase mediates both reactions in vivo or, alternatively, an RNA ligase is required for the second step. Recent results indicate that two different host activities are responsible for the cleavage and ligation reactions of PSTVd (+) strands (Riesner et al. 2000). Moreover, typical RNases generate at least transiently 5’-hydroxyl and 2’,3’cyclic phosphodiester termini, and an RNA ligase has been characterized from wheat germ with the ability to join such specific termini. In fact, this RNA ligase can catalyze the in vitro circularization of monomeric linear PSTVd forms isolated from infected tissue (Branch et al. 1982). The specificity of the cleavage reaction or, in other words, the location of the processing/ligation site, is most probably dictated by a defined conformation of the oligomeric viroid RNA. In this context it has been proposed that the enzymatic cleavage and ligation of the PSTVd (+) strand is driven by a switch from a branched structure containing a tetraloop to an extended conformation with an E loop (Baumstark et al. 1997). However, other data indicate the existence of alternative cleavage sites in the CCR lower strand of PSTVd (Hammond et al. 1989) and of CCCVd with the additional proposition in this latter case that cleavage is autocatalytic (Liu and Symons 1998). Moreover, results obtained with a series of RNA transcripts from monomeric CEVd cDNA clones, with the cloning site in different regions of the viroid molecule, have correlated their infectivity with the ability to form short double-stranded regions between the viroid and vector sequences at the junction of the two termini (Rakowski and Symons 1994). Within the family Avsunviroidae, the cleavage reaction is autocatalytic and mediated by hammerhead ribozymes that can be adopted by both polarity strands of these viroids (see Chapter 56 on ‘Ribozyme Reactions of Viroids’ for a detailed description). Regarding ligation, hammerhead ribozymes produce the same 5’-hydroxyl and 2’,3’-cyclic phosphodiester termini as conventional RNases, suggesting that an RNA ligase with properties similar to the wheat germ RNA ligase may catalyze this step of the replication cycle. Alternatively, on the basis of experiments that show that linear monomeric PLMVd RNA resulting from self-cleavage are able to self-ligate in vitro, it has been proposed that not only cleavage but also ligation may be autocatalytic in this viroid (Lafontaine et al. 1995). However, since most of the phosphodiester bonds produced in PLMVd self-ligation are

58

atypical (2’,5’ instead of the 3’,5’ characteristically found in RNA) (Côte and Perrault 1997), the significance of the reaction in vivo is unclear. Moreover, in vitro self-ligation has been also observed in the case of PSTVd (Baumstark et al. 1997), showing that this is not a peculiarity of members of the family Avsunviroidae.

CONCLUDING REMARKS Considerable progress has been made on understanding the process of how viroids replicate. The general outlines of the replication cycle appear well established: an RNA-based rollingcircle mechanism with two pathways, asymmetrical and symmetrical, followed by members of families Pospiviroidae and Avsunviroidae, respectively. Their different subcellular replication sites have a reflection on the enzymes involved. Polymerization of viroid strands is initiated at specific sites, and catalyzed by the nuclear RNA polymerase II and by a NEP-like chloroplastic RNA polymerase in the case of PSTVd and ASBVd, respectively. Nuclear RNA polymerases and their genes have been known for a long time. However, this is not the case with chloroplastic RNA polymerases some of which, as for the NEPlike activity, have been recently described leaving open the possibility that other(s) may still remain to be discovered. Last but not least, there is an important question that should not be ignored: under normal physiological conditions RNA polymerases catalyze transcription of DNA templates, as opposed to their functioning on viroid replication in which they must transcribe RNA templates. It is likely, therefore, that an additional factor may be required to facilitate this template switch of the RNA polymerases. The study of the cleavage reaction has led to the discovery of hammerhead ribozymes in ASBVd and then in the two other members of the family Avsunviroidae (see Chapter 56 on ‘Ribozyme Reactions of Viroids’). This is a hallmark not only in plant virology but also in molecular biology with deep functional, evolutionary and practical consequences. However, discrepancies concerning the enzymology of the cleavage reaction and the processing site(s) that await to be convincingly solved still exist in PSTVd and other viroids in its family. A similar situation also occurs with the third step, RNA ligation, with conflicting alternatives regarding this process as autocatalytic or mediated by a host enzyme. Acknowledgements

This work was partially supported by grants PB95-0139 and PB98-0500 from the Comisión Interministerial de Ciencia y Tecnología de España (to R. Flores). J. A. Daròs and J. A. Navarro were recipients of a postdoctoral contract and a predoctoral fellowship from the Ministerio de Educación y Cultura and from the Generalidad Valenciana (España), respectively.

REPLICATION

References Baumstark, T., Schröder, A.R.W., and Riesner, D. (1997). Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation. EMBO J. 16, 599-610. Bonfiglioli, R.G., McFadden, G.I., and Symons, R.H. (1994). In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus. Plant J. 6, 99-103. Bonfiglioli, R.G., Webb, D.R., and Symons, R.H. (1996). Tissue and intra-cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy. Plant J. 9, 457-465. Branch, A.D., and Robertson H.D. (1981). Longer-than-unit-length viroid minus strands are present in RNA from infected plants. Proc. Natl. Acad. Sci. USA 78, 6381-6385. Branch, A.D., Robertson, H.D., Greer, C., Gegenheimer, P., Peebles, C., and Abelson, J. (1982). Cell-free circularization of viroid progeny RNA by an RNA ligase from wheat germ. Science 217, 1147-1149. Branch, A.D., and Robertson, H.D. (1984). A replication cycle for viroids and other small infectious RNAs. Science 223, 450-455. Branch, A.D., Benenfeld, B.J., and Robertson, H.D. (1988). Evidence for a single rolling circle in the replication of potato spindle tuber viroid. Proc. Natl. Acad. Sci. USA 85, 9128-9132. Bussière, F., Lehoux, J., Thompson, D.A., Skrzeczkowski, L.J., and Perreault, J.-P. (1999). Subcellular localization and the rolling circle replication of peach latent mosaic viroid: hallmarks of group A viroids. J. Virol. 73, 6353-6360. Côte, F., and Perrault, J.P. (1997). Peach latent mosaic viroid is locked by a 2',5'-phosphodiester bond produced by in vitro self-ligation. J. Mol. Biol. 273, 533-543. Daròs, J.A., Marcos, J.F., Hernández, C., and Flores, R. (1994). Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing. Proc. Natl. Acad. Sci. USA 91, 12813-12817. Diener, T.O. (1971). Potato spindle tuber ‘virus’: a plant virus with properties of a free nucleic acid. III. Subcellular location of PSTVRNA and the question of whether virions exist in extracts or in situ. Virology 43, 75-89. Feldstein, P.A., Hu, Y., and Owens, R.A. (1998). Precisely full length, circularizable, complementary RNA: an infectious form of potato spindle tuber viroid. Proc. Natl. Acad. Sci. USA 95, 6560-6565. Flores, R., and Semancik, J.S. (1982). Properties of a cell-free system for synthesis of citrus exocortis viroid. Proc. Natl. Acad. Sci. USA 79, 6285-6288. Grill, L.K., and Semancik, J.S. (1978). RNA sequences complementary to citrus exocortis viroid in nucleic acid preparations from infected Gynura aurantiaca. Proc. Natl. Acad. Sci. USA 75, 896-900. Hadidi, A., Hashimoto, J., and Diener, T.O. (1982). Potato spindle tuber viroid — specific double-stranded RNA in extracts from infected leaves. Ann. Virol. 133E, 15-31. Hammond, R., Diener, T.O., and Owens, R.A. (1989). Infectivity of chimeric viroid transcripts reveals the presence of alternative processing sites in potato spindle tuber viroid. Virology 170, 486-495. Harders, J., Lukacs, N., Robert-Nicoud, M., Jovin, J.M., and Riesner, D. (1989). Imaging of viroids in nuclei from tomato leaf tissue by in situ

hybridization and confocal laser scanning microscopy. EMBO J. 8, 3941-3949. Hutchins, C.J., Keese, P., Visvader, J.E., Rathjen, P.D., McInnes, J.L., and Symons, R.H. (1985). Comparison of multimeric plus and minus forms of viroids and virusoids. Plant Mol. Biol. 4, 293-304. Hutchins, C.J., Rathjen, P.D., Forster, A.C., and Symons, R.H. (1986). Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 14, 3627-3640. Lafontaine, D., Beaudry, D., Marquis, P., and Perrault, J.P. (1995). Intraand inter-molecular non enzymatic ligations occur within transcripts derived from the peach latent mosaic viroid. Virology 212, 705-709. Liere, K., and Maliga, P. (1999). In vitro characterization of the tobacco rpoB promoter reveals a core sequence motif conserved between phage-type plastid and plant mitochondrial promoters. EMBO J. 18, 249-257. Lima, M.I., Fonseca, M.E.N., Flores, R., and Kitajima, E.W. (1994). Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization. Arch. Virol. 138, 385-390. Liu, Y.-H., and Symons, R.H. (1998). Specific RNA self-cleavage in coconut cadang cadang viroid: potential for a role in rolling circle replication. RNA 4, 418-429. Mühlbach, H.P., and Sänger, H.L. (1979). Viroid replication is inhibited by α-amanitin. Nature 278, 185-188. Mohamed, N.A., and Thomas, W. (1980). Viroid-like properties of an RNA species associated with the sunblotch disease of avocados. J. Gen. Virol. 46, 157-167. Navarro, J.A., Daròs, J.A., and Flores, R. (1999). Complexes containing both polarity strands of avocado sunblotch viroid: identification in chloroplasts and characterization. Virology 253, 77-85. Navarro, J.A., Vera, A., and Flores, R. (2000). A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid. Virology 268, 218-225. Navarro, J.A., and Flores, R. (2000). Characterization of the initiation sites of both polarity strands of a viroid RNA reveals a motif conserved in sequence and structure. EMBO J. 19, 2662-2670. Owens, R.A., and Diener, T.O. (1982). RNA intermediates in potato spindle tuber viroid replication. Proc. Natl. Acad. Sci. USA 79, 113-117. Rakowski, A.G., and Symons, R.H. (1994). Infectivity of linear monomeric transcripts of citrus exocortis viroid: terminal sequence requirements for processing. Virology 203, 328-335. Riesner, D., Fels, A., Hu, K., Klümper, S., Schröder, A., Schrader, O., Dingley, A., and Grzesiek, S. (2000). Molecular viroid-host interactions involved in transcription, processing, and pathogenesis. EMBO Workshop ‘Plant Virus Invasion and Host Defense’ , Kolymbari, Greece, 11. Schindler, I.M., and Mühlbach, H.P. (1992). Involvement of nuclear DNA-dependent RNA polymerases in potato spindle tuber viroid replication: a reevaluation. Plant Sci. 84, 221-229. Spiesmacher, E., Mühlbach, H.P., Schnölzer, M., Haas, B., and Sänger, H.L. (1983). Oligomeric forms of potato spindle tuber viroid (PSTV) and of its complementary RNA are present in nuclei isolated from viroid-infected potato cells. Biosci. Rep. 3, 767-774. Stern, D.B., Higgs, D.C., and Yang, J. (1997). Transcription and translation in chloroplasts. Trends Plant Sci. 2, 308-315.

59

R. Flores et al.

Tabler, M., Tzortzakaki, S., and Tsagris, M. (1992). Processing of linear longer-than-unit-length potato spindle tuber viroid RNAs into infectious monomeric circular molecules by a G-specific endoribonuclease. Virology 190, 746-753. Tsagris, M., Tabler, M., Mühlbach, H.P., and Sänger, H.L. (1987). Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately

60

processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells. EMBO J. 6, 2173-2183. Warrilow, D., and Symons, R.H. (1999). Citrus exocortis viroid RNA is associated with the largest subunit of RNA polymerase II in tomato in vivo. Arch. Virol. 144, 2367-2375.

PART II

CHAPTER 6

PATHOGENESIS ....................................................................................................

J.S. Semancik

.................................................................................................................................................................................................................................................................

With the well described physical properties of viroids as a uniquely small, non-translated RNA and the generally accepted pathway of replication as totally dependent on the host cell, the one compelling feature of viroid pathogenesis that can be accepted is that of a totally host-generated process. The process(es) by which the host is coerced to express either an obvious symptom or no apparent response in the presence of comparable viroid titers can be conjectured as centered anywhere from a primary effect on expression of the host genome to any number of anomalies detected in intermediary metabolites. As a result, a varied number of diverse observations have been made over the years of viroid ‘existence’ that have been considered linked to viroid pathogenesis. Unfortunately, none of these has succeeded in presenting a single or comprehensive explanation for the varied expression of viroid pathogenesis. In spite of the basic simplicity of the viroid structure coupled with the absence of introduction of any additional translatable genetic information in this minimal form of transmissible pathogenic molecule, the intrinsic complexity of the interaction with the host remains just as cryptic as that of more sophisticated viral and microbial pathogens. Since the inception of the viroid concept as a class of unusual disease-inducing agents, several overviews have attempted to

integrate observations of the effect of viroids on host metabolic functions into scenarios to explain pathogenesis (Diener 1981b; Semancik and Conejero 1987; Sänger 1982; Riesner 1991). For the most part these have relied more on correlation and conjecture with well-defined sequences of physiological and structural events in host development. Nevertheless, the ability of the viroid to influence host development cannot be denied. From this, it is reasonable to speculate that the viroid molecule may interfere with or mimic the function of a constitutive host element as in signaling (Conejero et al. 1990) as well as the recently reported evidence for post-transcriptional gene silencing (Papaefthimiou et al. 2001). It has been recently suggested that the trafficking of Potato spindle tuber viroid (PSTVd) and gene silencing signals may be under similar molecular control by host-specific factors (Zhu et al. 2001). The existence of this property of the viroid molecule has already been acknowledged in the replication process in recruiting the activity of a DNAdirected RNA polymerase II (RNAPII)-like enzyme (Mühlbach and Sänger 1979). As an organizational scheme to offer a perspective of viroid pathogenesis, a consideration of the components in the interaction might be appropriate. This discussion is not presented as a comprehensive review of related observations for insufficient

61

J.S. Semancik

evidence is available to present a clear and complete understanding of the course of events leading to scenario(s) for the varied expression of viroid–host interactions.

THE VIROID Structural implications in pathogenesis

With the ‘domain’ concept for the organization of the viroid genome, Keese and Symons (1985) introduced the ‘P’ or pathogenic domain as characterized by a physical oligo A site and an association with symptom expression. Changes as minor as one to three nucleotides were shown to affect infectivity (Wassenegger et al. 1996) as well as symptom severity (Gross et al. 1981) presumably by affecting distinct loci such as the VM or ‘virulence modulating’ region (Schnolzer et al. 1985). Specific structures such as a premelting loop (PM1) (Steger et al. 1984) by virtue of specific conformation and/or bending (Owens et al. 1996) may interact with host components resulting in symptoms (Hammond 1992). However, with data from the characterization of additional viroid species, the specificity of this focus for pathogenicity was later shown to encompass additional domains until at present virtually the entire viroid genome for different host–viroid combinations can be implicated (Sano et al. 1992). When sequence analyses became routine, homology with host nucleic acids was first thought to hold the key to the understanding of viroid pathogenesis. Thus, small nuclear RNAs (snRNA U1, U3 and U5) were linked to viroids in a possible common relationship as nuclear elements (Dickson 1981; Kiss and Solymosy 1982; Kiss et al. 1983). In addition, a sequence similarity to rRNA (Meduski and Velten 1990) might support the earlier suggestions for a possible interference or interaction with processing of mRNA or rRNA (Haas et al. 1988; Jakab et al. 1986). Structural characteristics were also employed to introduce possible relationships as precursors or products of introns (Diener 1981a; Dinter-Gottlieb 1986; Hadidi 1986) and transposable genetic elements (Kiefer et al. 1983). No subsequent experimental data has been offered to substantiate these early theoretical models. From another perspective, the viroid might be seen as a ‘passive’ pathogen for it is only by the host recognition and responses that symptoms sometimes occur. And occurrence among hosts of symptomless carriers of viroids is not uncommon. How should these diverse reactions be possible from the simplest transmissible molecule void of any translated information for imposing its genome? It is obvious as has been presented in so many forums previously that the key must reside in the structural complexity of the viroid (Langowski et al. 1978; Flores 1984). The often used term ‘native’ structure as an absolute cannot be well defined when the conformation of the viroid in situ and

62

compartmentalized in association with host components for the purpose of replication and/or homologous biological functions remains completely unknown. As representative of many biologically active molecules, viroids generally exist as a population of minor variants (Gora et al. 1994; Gora-Sochacka et al. 1997) or quasispecies (Holland et al. 1992). Changes in a few nucleotides can result in as dramatic symptom differences as evidenced among three variants of Avocado sunblotch viroid (ASBVd) in which localized bleached or generalized variegated can be induced as well as the complete absence of any visible reaction with a reduction of genome in the TR domain of 251 to 247 nucleotides (Semancik and Szychowski 1994). Although the minimal free energy form of a viroid sequence has been usually introduced as the primary viroid structure, any number of theoretically possible suboptimal conformers might exist in various locations in planta and more importantly act as the preferred agents of biological activity. With these considerations, the viroid cannot be viewed as deficient in the complexity required to drive the diverse expressions of pathogenicity observed. However, sorting among the accumulated progeny of viroid infection for the molecular form(s) responsible for pathogenicity remains a formidable challenge. Viroid associations with host components

Implicit in the process of viroid replication in the absence of a translated genome is the acceptance of a RNA-protein interaction by which a host-specified polymerase (or polymerases) is recruited to complete synthesis of a template as well as progeny. Experimental evidence for this association can be drawn from studies of Citrus exocortis viroid (CEVd) replication in nuclearrich preparations that indicated that viroid in the process of synthesis could be detected with properties of a viroid-ribonuclearprotein particle (RNP) (Rivera-Bustamante and Semancik 1989). Additional evidence for the binding of viroid-RNA to specific host proteins and the possible implication to movement of the viroid in planta was introduced in the binding of Hop stunt viroid (HSVd) to phloem protein 2 (PP2) of cucumber (Gómez and Pállas 2001). Recently, a chloroplast protein that binds ASBVd RNA in vivo and facilitates its hammerheadmediated self-cleavage has been characterized in avocado (Daròs and Flores 2002). Generic binding of viroid RNA with host plant histones (Wolff et al. 1985) as well as the more specific reaction between PSTVd and a 33 kD tomato protein (Hadidi 1988) and a 43 kD protein and PSTVd (Klaff et al. 1989) support the importance of interactive event(s) between viroid RNA and host components. The search for sequence homology of significant consequence between viroid RNA and host genomes has been largely unsuccessful. However, partial homology with U1 snRNA (Dickson 1981) and tomato 7S RNA (Haas et al. 1988) offer some evidence for competition and/or interference with processing systems for

PATHOGENESIS

constitutive host nucleic acids. Direct interactions of viroid RNA with host nucleic acid components may result in the induction of enzymatic activities such as the phosphorylation of certain host proteins (Hiddinga et al. 1988) or methylation of sequencespecific host DNA (Wassenegger et al. 1994a) which might then trigger the cascade resulting in pathogenic responses. However, further development of the biological significance for any of these complexes has yet to be been constructed.

THE HOST In any description of a viroid host, it is implicit that the capacity for viroid replication must precede any pathogenic response displayed by the host. That is not to say that pathogenesis is inexorably linked to replication, for the occurrence of non-symptomatic hosts is not uncommon. Perhaps the most curious of these relationships is displayed with ASBVd possibly as a result of virtually a single natural host, avocado, in which the viroid may be dormant and undetectable for decades (Semancik and Szychowski 1994). Viroids have the ability to coexist in a quiescent condition within plants in the absence of any obvious deleterious effects to host metabolism resembling the properties of 7S RNA (Lin and Semancik 1985). Retention in perennial or vegetatively propagated plant materials can assure the survival of viroids for extended periods, some perhaps of historic proportions. However, the ability to observe pathological responses is limited by technical approaches as well as the limits of detection of available probes. The gross visible modifications of plant growth and development as observed in different host–viroid combinations surely result from a wide range of the metabolic functions affected by the presence of viroids. But even in the absence of these expressions, the assumption cannot be made that no host process has been affected. As a consequence, viroid-induced diseases have been commonly described as ‘developmental diseases’. Receptive centers within the host

Some yet to be defined conditions for and/or properties of ‘recognition’ or ‘lack of recognition’ between the host and a viroid must be triggered to account for the directing of host activities to viroid replication and pathogenesis. Nevertheless, it is possible to feature some properties of plants receptive to viroid infection. Although symptoms of viroid infection can be seen in virtually every plant part, the intrinsic affinity of viroids for meristematic tissues for initiation of infection is striking. The implication suggests a ‘target’ cell with essential recognition factors made available perhaps by a receptive stage of growth or development. The nucleus as the locus of replication and accumulation for most viroids (Harders et al. 1989; Bonfiglioli et al. 1994) also suggests that specific process or structural events associated with mitotic activity may be critical to successful viroid infection. The association of ASBVd with chloroplasts is

a notable exception and may be related to the pre-cellular origin of the association between the viroid and this organelle (Semancik and Duran-Vila 1999). Established viroid infections are characteristically in association with vascular tissues that provide for circulative movement via plasmadesmata of a specific conformational structure (Ding et al. 1997). This property coupled with viroid retention within the nucleus with progressive cell division assures the survival of the free RNA viroid progeny even in the absence of a protective coating such as the viral protein. The small target size, highly structured conformation and possible association with host components describe properties of an unusually successful pathogen and adequately compensate for the absence of a structural protein as well as movement proteins. That is not to say that the selection of apex tissues in advance of vascular development from infected plants can be employed to generate viroid-free material by shoot tip culturing. By contrast, mature or fully developed cells appear to be either immune or unreceptive to the initiation phase of viroid infection. It is only following the establishment of a successful infection that replication in more mature cells is detected. Even though mature tissues can express symptoms of viroid infection, it is possible that these centers had an origin in more primordial cells that then developed under the influence of viroids as ancillary elements governing developmental processes. And, in fact, some of these developmental aberrations have been viewed not in terms of a pathogenic response but as positive consequences in terms of agricultural or economic impact. Host pressures on viroid populations

Selection pressure applied to viroid populations by different hosts is evidenced in the range of variants of a single viroid species that can be recovered. HSVd characterized by the widest host range among viroids presents a dramatic view of the diversity possible within a collection of viroid quasispecies (Kofalvi et al. 1997). Simple passage through an alternate host can significantly alter the symptom severity and possibly transmissibility. Thus, when CEVd from a citrus source was transmitted to Gynura aurantiaca, tomato as well as tomato cells in culture, distinct progeny variants with altered biological properties became dominant (Semancik et al. 1993). The striking segregation of ASBVd variants from tissues expressing different forms of the sunblotch disease can be observed within a single plant (Semancik and Szychowski 1994). Host-specific processing of viroid synthesis can result in an alteration in the physical as well as biological properties of the viroid. An infectious 341 nt variant of the native 359 nt PSTVd was generated in planta from a non-infectious 350 bp PSTVd cDNA by removal of a nucleotide cluster with symmetry to the 9 nt deletion (Wassenegger et al. 1994b). The right terminal

63

J.S. Semancik

(RT) repeated sequence first noted for Coconut cadang-cadang viroid (CCCVd) (Haseloff et al. 1982) as more a curiosity of an atypical viroid with a very restricted host range has now been extended to include CEVd (Semancik et al. 1994). With the more tractable multi-host experimental system available with CEVd, it is interesting to note that the enlargement of the CEVd genome from 371 to 462 nucleotides was detected not in the primary disease host species, Citrus, but in a hybrid of Lycopersicon esculentum × L. peruvianum. Although the enlarged variant, CEVd D-92, is independently transmissible to Gynura, it has never been generated in this species. In addition, CEVd D-92, although containing the entire CEVd genome is no longer transmissible to citron.

THE INTERACTION — PATHOGENESIS Much of the effort that has been devoted to the understanding of viroid pathogenesis has been directed to the detection and characterization of secondary metabolites and induced hostspecified products. Thus, a diverse collection of data on metabolic aberrations has been accumulated over the years. However, just as the sorting of biologically active form(s) of the viroid from a progeny population remains a formidable venture, so too the relationship of metabolic and structural change following infection to a defined pathway of ‘pathogenesis’ remains a challenge. Putative events and related metabolic observations

Nevertheless, an outline, broadly defined, can be proposed for a generalized sequence of events that appear to have gained a consensus. With viroid introduction, the primary event of pathogenesis most probably involves some interaction between the viroid and the host genome affecting gene expression. Recent data provided for the initiation of post-transcriptional gene silencing following infection by PSTVd (Papaefthimiou et al. 2001) may be taken as evidence for interaction with the host genome. This may encompass the viroid functioning directly as a genetic element perhaps even in a fashion similar to some yet to be defined snRNA constitutive host component or as a ‘signal’ or elicitor of a secondary signal such as ethylene, salicylic acid, or gentisic acid (Belles et al. 1999). This sets in place a series of reactions altering the composition or regulation of interdependent metabolic pathways. Examples of possible secondary metabolites in this process can be drawn from the appearance of host-directed pathogenesis-related (PR) proteins (Conejero et al. 1979), modification of specific enzymes (Hiddinga et al. 1988), and observations of hormonal imbalance (Duran-Vila and Semancik 1982) resulting in changes in tissue composition and development. Early reports of the appearance of host-specified PR proteins following viroid infection have been defined to describe enhanced enzymatic activity including an alkaline cysteine proteinase (Vera

64

and Conejero 1988), chitinases (Garcia-Breijo et al. 1990) and β1,3-glucanase. Succeeding studies have addressed the characterization of these proteins with an implicit assumption of a relationship to viroid pathogenesis. Thus, two 69-kDa proteinases (PRP69 and PR-P69B) have been identified as members of the subtilisin-like proteinase family (Tornero et al. 1996; 1997) which have been identified as constitutive host enzymes that might be expressed at specific stages of development and also as a response to pathogen or stress-related conditions. It is possible that any number of these observations may be duplicated by infection by bacterial or fungal pathogens or even by abiotic agents (Granell et al. 1987) as induced host-specified defense responses with no clearly defined link to viroid infection (Dixon et al. 1994). This can be illustrated by the question why should chitinase and other antimicrobial activities be enhanced as a response to the invasion of a small, transmissible, nontranslated RNA? Environmental considerations in viroid pathogenicity

As a practical observation, the expression of viroid diseases in the field is many times facilitated by increased symptom severity apparent under conditions of higher temperature. In fact, viroid diseases are more common to semi-tropical than temperate climates. This property can be extended to the extreme of cells cultured under laboratory conditions in which CEVd-containing tomato cells displayed a higher optimum for growth as well as a survival advantage at temperatures above 30°C (Marton et al. 1982). The intimate host–viroid relationship resulting in pathogenesis coupled with the effect of environmental conditions is described in the temperature-sensitive expression of the CEVd-M variant that occurs exclusively in Gynura (Skoric et al. 2001). Although CEVd-M differs for the severe source by only 5 nucleotides, virtually no symptoms are expressed under standard glasshouse conditions but can be altered to severe when plants are grown at 40°C. This severe symptom expression is reversed to mild to symptomless when glasshouse-growing conditions are restored. These observations indicate that not only host selection but also environmental conditions affecting the physiology of a specific host may be instrumental in the expression of viroid symptoms and from that the detection of a viroid disease. Host structural alterations

A survey of cytopathic effects has provided a structural perspective for the whole plant symptoms observed as a result of viroid infection. The most striking feature observed in viroid-infected cells was a disturbance of the cell membrane system manifest as paramural bodies of internal invaginations and plasmalemmasomes (Semancik and Vanderwoude 1976; Wahn et al. 1980). These were accompanied by major changes in the cell wall structure and composition (Marton et al. 1982; Wang et al. 1986)

PATHOGENESIS

reflecting more of a developmental anomaly than a viroidspecific response. In addition to the obvious disruption of chloroplasts associated with disease development, dedifferentiation of the organelle resulting in leucoplasts in sunblotch-infected avocado are characterized by high titers of ASBVd. The accumulation of viroid seen in the chloroplast (Bonfiglioli et al. 1994) may be related to the chloroplast as the subcellular site of ASBVd replication (Marcos and Flores 1992). However, replication and accumulation of the variant SB-1 (Symons 1981) in high titers can be detected in symptomless carrier tissues with no obvious cytopathic effects. This absence of any specific viroid-induced cellular aberration is best illustrated by the absence of any effects on the nucleus as the primary site of replication and accumulation for most viroids. References Belles, J.M., Garro, R., Fayos, J., Navarro, P., Primo, J., and Conejero, V. (1999). Gentisic acid as a pathogen-inducible signal, additional to salicylic acid for activation of plant defense in tomato. Molec. Plant Microbe Interactions 12, 227-235. Bonfiglioli, R.G., McFadden, G.I., and Symons, R.H. (1994). In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus. Plant J. 6, 99-103. Conejero, V., Belles, J.M., Garcia-Breijo, F., Garro, R., Hernández-Yago, J., Rodrigo, I., and Vera, P. (1990). Signalling in viroid pathogenesis. Pages 233-261 in: Recognition and response in plant-virus interactions. R.S.S. Fraser, ed. Springer-Verlag: Berlin. Conejero, V., Picazo, I., and Segado, P. (1979). Citrus exocortis viroid (CEV): protein alterations in different hosts following viroid infection. Virology 97, 454-456. Daròs, J.A., and Flores, R. (2002). A chloroplast protein binds a viroid RNA in vivo and facilitates its hammerhead-mediated self-cleavage. EMBO J. 21, 749-759. Dickson, E. (1981). A model for the involvement of viroids in RNA splicing. Virology 115, 216-221. Diener, T.O. (1981a). Are viroids escaped introns? Proc. Natl. Acad. Sci. USA 78, 5014-5015. Diener, T.O. (1981b). Viroids: abnormal products of plant metabolism. Ann. Rev. Plant Physiol. 32, 313-325. Diener, T.O. (1999). Viroids and the nature of viroid diseases. Arch. Virol. [Suppl] 15, 203-220. Ding, B., Kwon, M-O., Hammond, R., and Owens, R.A. (1997). Cell-tocell movement of potato spindle tuber viroid. The Plant J. 12, 931-936. Dinter-Gottlieb, G. (1986). Viroids and virusoids are related to group I introns. Proc. Natl. Acad. Sci. USA 83, 6250-6254. Dixon, R.A., Harrison, J.J., and Lamb, C.J. (1994). Early events in the activation of plant defense responses. Ann. Rev. Phytopath. 32, 479-501. Duran-Vila, N., and Semancik, J.S. (1982). Differential response of tomato tissue infected with the citrus exocortis viroid to exogenous auxins. Phytopathology 72, 777-781.

Flores, R. (1984). Is the conformation of viroids involved in their pathogenicity? J. Theor. Biol. 108, 519-527. Garcia-Breijo, F.J., Garro, R., and Conejero, V. (1990). C 7(P32) and C6(P34) PR proteins induced in tomato leaves by citrus exocortis viroid infection are chitinases. Physiol. Mol. Plant Pathol. 36, 249-260. Gómez, G., and Pállas, V. (2001). Identification of an in vitro ribonucleoprotein complex between a viroid RNA and a phloem protein from cucumber plants. Molec. Plant Microbe Interactions 14, 910-913. Gora, A., Candresse, T., and Zagorski, W. (1994). Analysis of the population structure of three phenotypically different PSTVd isolates. Arch. Virol. 138, 233-245. Gora-Sochacka, A., Kierzek, A., Candresse, T., and Zagorski, W. (1997). The genetic stability of potato spindle tuber viroid (PSTVd) molecular variants. RNA 3, 68-74. Granell, A., Belles, J.M., and Conejero, V. (1987). Induction of pathogenesis related proteins in tomato by citrus exocortis viroid, silver ion and ethephon. Physiol. Mol. Plant Path. 31, 83-90. Gross, H.J., Domdey, H., and Lossow, C. (1981). A severe and mild potato spindle tuber viroid isolate differ in three nucleotide exchanges only. Biosci. Rep. 1, 235-241. Haas, B., Klanner, A., Ramm, K., and Sänger, H.L. (1988). The 7S RNA from tomato leaf tissue resembles a signal recognition particle RNA and exhibits a remarkable sequence complementary to viroids. EMBO J. 7, 4063-4074. Hadidi, A. (1986). Relationship of viroids and certain other plant pathogenic nucleic acids to group I and II introns. Plant Mol. Biol. 7, 129-142. Hadidi, A. (1988). Synthesis of disease-associated proteins in viroidinfected tomato leaves and binding of viroid to host proteins. Phytopathology 78, 575-578. Hammond, R.W. (1992). Analysis of the virulence modulating region of potato spindle tuber viroid (PSTVd) by site-directed mutagenesis. Virology 187, 654-662. Harders, J., Lukacs, N., Robert-Nicoud, M., Jovin, T.M., and Riesner, D. (1989). Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy. EMBO J. 8, 3941-3949. Haseloff, J., Mohamed, N.A., and Symons, R.H. (1982). Viroid RNAs of cadang-cadang disease of coconuts. Nature 299, 316-321. Hiddinga, H.J., Crum, C.J., Hu, J., and Roth, D.A. (1988). Viroid-induced phosphorylation of a host protein related to a dsRNA-dependent protein kinase. Science 241, 451-453. Holland, J.J., De La Torre, J.C., and Steinhauer, D.A. (1992). RNA virus populations as quasispecies. Curr. Top. Microbiol. Immunol. 176, 1-20. Jakab, G., Kiss, T., and Solymosy, F. (1986). Viroid pathogenicity and pre-rRNA processing: a model amenable to experimental testing. Biochim. Biophys. Acta 950, 455-458. Keese, P., and Symons, RH. (1985). Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proc. Natl. Acad. Sci. USA 82, 4582-4586. Kiefer, M.C., Owens, R.A., and Diener, T.O. (1983). Structural similarities between viroids and transposable genetic elements. Proc. Natl. Acad. Sci. USA 80, 6234-6238.

65

J.S. Semancik

Kiss, T., and Solymosy, F. (1982). Sequence homologies between a viroid and a small nuclear RNA (snRNA) species of mammalian origin. FEBS Lett. 144, 318-320. Kiss, T., Posfai, J., and Solymosy, F. (1983). Sequence homology between potato spindle tuber viroid and U3B snRNA. FEBS Lett. 163, 217-220. Klaff, P., Gruner, R., Hecker, R. Sattler, A., Theissen, G., and Riesner, D. (1989). Reconstituted and cellular viroid-protein complexes. J. Gen. Virol. 70, 2257-2270. Kofalvi, S.A., Marcos, J.F., Cañizares, M.C., Pállas, V., and Candresse, T. (1997). Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. J. Gen. Virol. 78, 3177-3186. Langowski, J., Henco, K., Riesner, D., and Sänger, H.L. (1978). Common structural features of different viroids: serial arrangement of double helical sections and internal loops. Nucleic Acids Res. 5, 1589-1610. Lin, J.J., and Semancik, J.S. (1985). Coordination between host nucleic acid metabolism and citrus exocortis viroid turnover. Virus Res. 3, 213-230. Marcos, J.F., and Flores, R. (1992). Characterization of RNAs specific to avocado sunblotch viroid synthesized in vitro by a cell-free system from infected avocado leaves. Virology 186, 481-488. Marton, L., Duran-Vila, N., Lin, J.J., and Semancik, J.S. (1982). Properties of cell cultures containing the citrus exocortis viroid. Virology 122, 229-238. Meduski, C.J., and Velten, J. (1990). PSTV sequence similarity to large rRNA. Plant Mol. Biol. 14, 625-627. Mühlbach, H.P., and Sänger, H.L. (1979). Viroid replication is inhibited by alpha-amanitin. Nature 278, 185-188. Owens, R.A., Steger, G., Hu, Y., Fels, A., Hammond, R.W., and Riesner, D. (1996). RNA structural features responsible for potato spindle tuber viroid pathogenicity. Virology 222, 144-158. Papaefthimiou, I, Hamilton, A.J., Denti, M.A., Baulcombe, D.C., Tsagris, M., and Tabler, M. (2001). Replicating potato spindle tuber viroid RNA is accompanied by short RNA fragments that are characteristic of post-transcriptional gene silencing. Nucleic Acids Res. 29, 2395-2400. Riesner, D. (1991). Viroids: from thermodynamics to cellular structure and function. Molec. Plant Microbe Interactions 4, 122-131. Rivera-Bustamante, R., and Semancik, J.S. (1989). Properties of a viroid replicating complex solubilized from nuclei. J. Gen. Virol. 70, 2707-2716. Sänger, H.L. (1982). Biology, structure, functions and possible origin of viroids. Pages 368-454 in: Encyclopedia of Plant Physiology 14B. Springer-Verlag: Berlin. Sano, T., Candresse, T., Hammond, R.W. Diener, T.O., and Owens, R.A. (1992). Identification of multiple structural domains regulating viroid pathogenicity. Proc. Natl. Acad. Sci. USA 89, 10104-10108. Schnolzer, M., Haas, B., Ramm, K., Hofmann, H., and Sänger, H.L. (1985). Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV). EMBO J. 4, 2181-2190. Semancik, J.S., and Conejero, V. (1987). Viroid pathogenesis and expression of biological activity. Pages 71-126 in: Viroids and viroidlike pathogens. J.S. Semancik, ed. CRC Press: Boca Raton, FL. Semancik, J.S., and Duran-Vila, N. (1999). Viroids in plants: shadows and footprints of a primitive RNA. Pages 37-64 in: Origin and evo-

66

lution of viruses. E. Domingo, R. Webster, and J. Holland, eds. Academic Press: New York. Semancik, J.S., and Szychowski, J.A. (1994). Avocado sunblotch disease: a persistent viroid infection in which variants are associated with differential symptoms. J. Gen. Virol. 75, 1543-9. Semancik, J.S., and Vanderwoude, W.J. (1976). Exocortis viroid: cytopathic effects at the plasmamembrane in association with pathogenic RNA. Virology 69, 719-726. Semancik, J.S., Szychowski, J.A., Rakowski, A.G., and Symons, R.H. (1993). Isolates of citrus exocortis viroid recovered by host and tissue selection. J. Gen. Virol. 74, 2427-2436. Semancik, J.S., Szychowski, J.A., Rakowski, A.G., and Symons, R.H. (1994). A stable 463 nucleotide variant of citrus exocortis viroid produced by terminal repeats. J. Gen. Virol. 75, 727-732. Skoric, D., Conerly, M., Szychowski, J.A., and Semancik, J.S. (2001). CEVd-induced symptom modification as a response to a hostspecific temperature-sensitive reaction. Virology 280, 115-123. Steger, G., Hoffmann, H., Fortsch, J., Gross, H.J., Randles, J.W., Sänger, H.L., and Riesner, D. (1984). Conformational transitions in viroids and virusoids: comparison of results from energy minimization algorithm and from experimental data. J. Biomol. Struct. Dyn. 2, 543-571. Symons, R.H. (1981). Avocado sunblotch viroid: primary sequence and proposed secondary structure. Nucleic Acids Res. 9, 6527. Tornero, P., Conejero, V., and Vera, P. (1996). Primary structure and expression of a pathogen-induced protease (PR-P69) in tomato plants: similarity of functional domains to subtilisin-like endoproteases. Proc. Natl. Acad. Sci. USA 93, 6332-6337. Tornero, P., Conejero, V., and Vera, P. (1997). Identification of a new pathogen-induced member of the subtilisin-like processing protease family from plants. J. Biol. Chem. 272, 14412-14419. Vera, P., and Conejero, V. (1988). Pathogenesis-related proteins of tomato. P69 as an alkaline endoproteinase. Plant Physiol. 87, 58-63. Wahn, K., Rosenberg de Gómez, F., and Sänger, H.L. (1980). Cytopathic changes in leaf tissue of Gynura aurantiaca infected with the viroid of citrus exocortis disease. J. Gen. Virol. 49, 358-365. Wang, M.C., Lin. J.J., Duran-Vila, N., and Semancik, J.S. (1986). Alteration in cell wall composition and structure in viroid-infected cells. Physiol. Mol. Plant Pathol. 28, 107-127. Wassenegger, M., Heimes, S., and Sänger, H.L. (1994b). An infectious viroid RNA replicon evolved from an in vitro-generated noninfectious viroid deletion mutant via a complementary deletion in vivo. The EMBO J. 13, 6172-6177. Wassenegger, M., Heimes, S., Riedel, L., and Sänger, H.L. (1994a). RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567-576. Wassenegger, M., Spieker, R.L., Thalmeir, S., Gast, F.-U., Riedel, L., and Sänger, H.L. (1996). A single nucleotide substitution converts potato spindle tuber viroid (PSTVd) from a noninfectious to an infectious RNA for Nicotiana tabacum. Virology 226, 191-197. Wolff, P. Gilz, R., Schumaker, J., and Riesner, D. (1985). Complexes of viroids with histones and other proteins. Nucleic Acids Res. 13, 355-367. Zhu, Y., Green, L., Woo, Y-M., Owens, R.A., and Ding, B. (2001). Cellular basis of potato spindle tuber viroid systemic movement. Virology 279, 69-77.

PART II

CHAPTER 7

VIROIDS AND GENE SILENCING ....................................................................................................

V. Conejero

.................................................................................................................................................................................................................................................................

Viroids have the lowest biological complexity of known pathogens (naked RNAs, with a molecular mass of about 105 Da). Their informational content is about one-tenth that of a minimal virus without the capacity to encode any protein. All the in vitro or in vivo attempts to demonstrate the capacity of viroids to code for specific proteins were unsuccessful (Davies et al. 1974; Hall et al. 1974; Semancik et al. 1977; Conejero et al. 1979). On this basis, it was thought that their pathogenicity had to be exerted through direct interference with some critical cellular targets (Diener 1979; Semancik and Conejero 1987; Conejero et al. 1979). From this, and taking into account the replication and location of most viroids in the nucleus (Riesner 1987; Robertson and Branch 1987; Sänger 1987), the obvious strategy was to look for possible interference of viroid RNA molecules or their complementary replicative forms, with either host nucleic acids or proteins, as the primary pathogenic event. Thus, it was suggested that viroids express their pathogenicity by altered regulation of gene expression (Rackwitz et al. 1981; Diener 1981; Dickson 1981; Solymosy and Kiss 1985) and altered translocation of proteins through base pairing with RNA signal recognition particles (Haas et al. 1988). These interpretations were supported by theoretical considerations. A different type of approach in our laboratory (Conejero and Granell 1986)

led to evidence indicating that viroids seem to be elicitors of a general response system of the host plant (Conejero et al. 1990). Very soon after the discovery of viroids, any new finding or idea concerning the role of RNA in any of the steps of gene expression (transcription, RNA maturation, translation) and/or their regulation, has been used to help interpretation of viroid pathogenicity. In the last decade the emergence of the new concept of gene silencing (GS), and its specific signaling by small RNA molecules, again could help to understand how viroids produce developmental distortions and/or the plants defend themselves, specifically, against the systemic infection of viroids. It is interesting to note, that, in its turn, viroid infection has served as a model system to support the new concept of RNA modulation of gene silencing through DNA-methylation as a normal mechanism of epigenetic regulation (Wassenegger and Pélissier 1998; Wassenegger 2000; Matzke and Matzke 2000; Wang and Waterhouse 2002). The evidence of RNA-directed DNA methylation (RdDM) was first discovered in tobacco plants that contained multimeric genome-integrated copies of the Potato spindle tuber viroid (PSTVd) cDNA (Wassenegger et al. 1994). Later, the observation that the heavy methylation was essentially co-extensive with the length of the PSTVd cDNA sequences provided evidence that a direct RNA-DNA interaction can act

67

V. Conejero

as a strong and highly specific signal for de novo DNA methylation (Pélissier et al. 1999). Homology-dependent gene silencing (HDGS), is a type of epigenetic inactivation that is based on interactions between homologous nucleic acid sequences. HDGS phenomena have been described in diverse organisms and are probably common to most eukaryotes. In plants, HDGS can occur at the transcriptional or postranscriptional levels, and may involve DNADNA, RNA-DNA or RNA-RNA associations, respectively. While the mechanisms of these silencing effects are still under investigation, it is clear that they are revealing unanticipated ways in which interactions between nucleic acid sequences can regulate gene expression in the nucleus and in the cytoplasm (Matzke and Matzke 2000).

GENETIC SILENCING AS POSSIBLY INVOLVED IN VIROID PATHOGENICITY

RNA silencing can be subclassified into RNA-mediated transcriptional gene silencing (TGS) and postranscriptional gene silencing (PTGS). RNA-mediated TGS occurs when double stranded (ds) RNA with sequence homology to a promoter is produced, leading to de novo DNA methylation of the promoter region of the structural gene (Mette et al. 1999). PTGS, on the other hand: i

yields reduced steady state levels of targeted host or viral cytoplasmic RNA, and to a lesser, but sometimes observable reduction of nuclear RNA; and

ii

may be mediated through host-encoded RNA-dependent RNA polymerase (RdRp) and RNA helicase.

A hallmark of RNA-mediated TGS and PTGS is the production of small RNAs of 21 to 23 nucleotides (nt) with sequence specificity to the silenced gene (reviewed in Itaya et al. 2001; Wang and Waterhouse 2002). The idea that viroids could exert their pathogenicity through RNA-directed gene silencing was proposed by Sänger et al. (1996). The experimental trigger of their working hypothesis was the finding that PSTVd-specific cDNA integrated into the genome of Nicotiana tabacum SRI becomes specifically methylated as soon as an autonomous viroid RNA-directed RNA replication has taken place in these plants (Wassenegger et al. 1994). From these observations it was inferred that RNA, in general, is capable of inducing and directing sequence-specific de novo methylation of genomic DNA. Since DNA methylation had been associated with gene silencing, it was conceivable that a viroid-induced RNA-directed DNA methylation resulted in a subsequent plant gene silencing. This interference in the normal regulation and expression of important plant host’s genes could be responsible for initiation and phenotypic expression of disease symptoms. According to the authors, such a mechanism

68

would demand: plant-specific sequences complementary to the PSTVd RNA whose hypermethylation, upon viroid infection, would lead to silencing. Unfortunately, there is not yet experimental evidence for any of these conditions. Later, in transgenic plants, RdDM was considered as an attractive mechanism to account for the frequent association of DNA methylation with PTGS (Wassenegger 2000). Also, it was shown that RdDM is not peculiar to the viroid system (Jones et al. 1998; Mette et al. 1999) and that transgenes and cytoplasmic RNA viruses could both activate PTGS, resulting in a sequencespecific degradation of potentially all the RNA species with sufficient homology to the triggering RNA molecules (Stam et al. 1997; Ruiz et al. 1998). All this reinforced the speculation that RdDM represents a broadly important mechanism for gene regulation (Wassennegger and Pélissier 1998). The high efficiency of this regulatory process was demonstrated by the fact that a DNA target with as few as 30 nt of sequence complementarity to the PSTVd RNA is specifically methylated (Pélissier and Wassenegger 2000). Thus, it was proposed that a host gene(s), exhibiting as few as 30 nt of sequence complementarity with the PSTVd RNA, could be partially methylated after viroid infection. Methylation-mediated silencing of the corresponding gene(s) would then initiate disease symptom expression consistently to the model previously proposed (Sänger et al. 1996). The authors considered that although mature viroid molecules adopt a highly double-stranded rod-like secondary structure, thermodynamic studies show that viroids can undergo structural transitions from this rod-like structure to a metastable, branched structure with a marked loss of base pairing (Loss et al. 1991). This structure displays three particularly stable hairpins which, at higher temperatures, dissociate independently from each other according to their individual thermal stability. Assuming that such metastable, partially denatured PSTVd-RNA molecules interact with genomic DNA, the occurrence of viroid cDNA subfragment-specific methylation patterns can be explained. They also pointed out that it is possible that the binding of protein-like histones, or RNA polymerase II, to the viroid RNA, may also contribute to stabilization or destabilization of particular structures (Pélissier and Wassenegger 2000). As has been noted before, this plausible and attractive hypothesis for the genesis of the viroid-induced diseases still awaits the experimental confirmation of the predicted specific silencing. This silencing, triggered by some viroid RNA species, should have to be mediated through DNA methylation. The gene(s) silenced should have also to be critical for disease development.

GENE SILENCING AS A DEFENCE MECHANISM We have focused our attention on the possibility that was first proposed by Sänger et al. (1996) of the involvement of gene silencing in viroid pathogenesis: the production of a disease syn-

VIROIDS AND GENE SILENCING

drome. Nevertheless, if the most generally admitted defensive function for RNA silencing (Ding 2000; Bender 2001) is considered, then, there is a second possible role for PTGS: the defense against the invading infectious viroid RNA. This aspect came into the scenario of viroid pathogenesis very recently (Papaefthimiou et al. 2001; Itaya et al.,2001), when two groups working independently, found the first experimental evidence that PSTVd-infected tomato plants accumulate the small viroid-specific RNAs (≅25-nt) that are considered (Hamilton and Baulcombe 1999) to be an indication of RNA silencing. According to the results obtained in tomato cv. ‘Rentita’ (Papaefthimiou et al. 2001), the small RNAs (22–23 nt) of both polarities represent different domains of the viroid molecule. In this work, no difference was found in the PTGS response produced in the host by viroid strains with different virulence, as determined by the concentration of the PTGS-associated small RNAs. Thus, it was not possible to correlate aggressiveness of the viroid isolates with PTGS. This is consistent with the observation that virulence of different viroid isolates is not correlated with the titer of accumulating viroid RNA (Gruner et al. 1995). The authors concluded that it is unlikely, therefore, that the variation in aggressiveness is related to differential effects of PTGS on the overall accumulation of the viroid RNA. On the other hand, the work with tomato cv. ‘Rutgers’ (Itaya et al. 2001) revealed that the accumulation of the small RNAs induced by the severe isolate, was higher than that elicited by the mild isolate. These results were, again, unexpected under the assumption of a defensive role for PTGS. However, they become coherent, if the possibility that viroid-induced PTGS is directly involved with symptom development in infected plants, as it has been proposed (Sänger et al. 1996; Papaefthimiou et al. 2001). More recently, Martínez de Alba, Flores and Hernández (unpublished results) have found an inverse correlation between the viroid accumulation levels and the presence or absence of the small RNAs. In this case, the study was carried out with three different viroids belonging to the family Avsunviroidae: Avocado sunblotch viroid (ASBVd), Peach latent mosaic viroid (PLMVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) that, contrary to PSTVd, the type species of family Pospiviroidae, do not have a central conserved region (CCR) but self-cleave through hammerhead ribozymes. Furthermore, the three viroids replicate and accumulate in the chloroplast (Flores et al. 2000). In this study the small RNAs of 21–22 nt were found in the the case of PLMVd- and-CChMVd-infected tissue, but not in ASBVd-infected avocado leaves. Interestingly enough, viroid accumulation was high in the case of ASBVd and much lower in the other two cases. These results thus being consistent with the possible involvement of the viroid-specific small RNAs in a defensive PTGS host response.

CONCLUSIONS AND PROSPECTS The results presented and discussed in this chapter constitute an indication that gene silencing is involved in viroid–host interactions. These are very important findings that have permitted viroid research to become an active participant of such an explosive movement, as it represents the research on novel epigenetic silencing phenomena, in modern plant science. Furthermore, they have opened a new and exciting path in viroid research. Because of that, I would like to pose some questions and make some remarks. i

If RNA-directed gene silencing has a defensive role against viroid infection, then how does the viroid manage to counteract or suppress this mechanism? How do the same viroid molecules activate their proper replication and the mechanism that impairs their accumulation in the host plant? Perhaps the outcome of these opposite processes might condition the degree of success of a certain viroid in a given host.

ii

If suppression of RNA silencing using viral-encoded proteins is widely used as counter-defensive strategy by plant viruses (Li and Ding 2001), what is the alternative for viroids that do not code for any protein?

iii

Is it possible for GS to be implicated, at the same time in the production of symptoms and in the defensive response of the host plant to viroid infection? The answer might be yes, provided that the host has some gene(s) critical to these processes and susceptible to being specifically silenced by any viroid-specified sequence. As has already been discussed, this question awaits experimental demonstration. On the other hand, can the complexity of a disease syndrome, as that induced by viroids, be explained by interference of the expression of a few genes? That could also be possible, as the interference(s) could lead to activation of a response programmed by the host (Conejero et al. 1990).

A more accurate knowledge of the basic component steps of viroid–host interactions will allow us to establish meaningful structure–function relationships. Moreover, even if we know the complete sequence of a nucleic acid or of the whole genome we have yet to relate their information with the structures, functions and the interactions and regulations that this genomic information encodes in the biological system under study. We need precise knowledge of details at the molecular, cellular and physiological levels, of metabolic signaling and regulatory pathways and networks, and their changes along the normal developmental stages and, also, those associated with the response to internal and external signals. This is a formidable task and I hope that the new high-throughput global functional analyses and approaches newly emerged around genomics (mainly proteomics and metabolomics) will help us in this challenge.

69

V. Conejero

References Bender, J. (2001). A vicious cycle: RNA silencing and DNA methylation in plants. Cell 106, 129-132. Conejero, V., and Granell, A. (1986). Stimulation of a viroid-like syndrome and the impairment of viroid infection in Gynura aurantiaca DC plants by treatment with silver ions. Physiol. Mol. Plant. Pathol. 29, 317-323. Conejero, V., Picazo, I., and Segado, P. (1979). Citrus exocortis viroid (CEV): protein alterations in different hosts following viroid infection. Virology 97, 454-456. Conejero, V., Bellés, J.M., García-Breijo,F., Garro, R., Hernández-Yago, J., Rodrigo, I. , and Vera, P. (1990). Signalling in viroid pathogenesis. Pages 233-261 in: Recognition and response in plant-virus interactions. R.S.S. Fraser, ed. Springer-Verlag: Berlin. Davies, J.W., Kaesberg, P., and Diener, T.O. (1974). Potato spindle tuber viroid. XII. An investigation of viroid RNA as messenger for protein synthesis. Virology 61, 281-286. Dickson, E. (1981). A model for the involvement of viroids in RNA splicing. Virology 115, 216-221. Diener, T.O. (1979). Viroids and viroid diseases. Wiley: New York. Diener, T.O. (1981). Are viroids escaped introns? Proc. Natl. Acad. Sci. USA 78, 5014-5015. Ding, S.W. (2000). RNA silencing. Cur. Opin. Biotechnol. 11, 152-156. Flores, R., Daròs, J.A., and Hernández, C. (2000). The Avsunviroidae family: viroids containing hammerhead ribozymes. Adv. Virus Res. 55, 271-323. Gruner, R., Fels, A., Qu, F., Zimmat, R., Steger, G., and Riesner, D. (1995). Interdependence of pathogenicity and replicability with potato spindle tuber viroid. Virology 209, 60-69. Hall, T.C., Wrepprich, R.K., Davies, J.W., Weathers, L.G., and Semancik, J.S. (1974). Functional distinctions between the ribonucleic acids from citrus exocortis viroid and plant viruses: cell-free translation and aminoacylation reactions. Virology 1, 486-492. Haas, B., Klanner, A., Ramm, K., and Sänger, H.L. (1988). The 7S RNA from tomato leaf tissue resembles a signal recognition particle RNA and exhibits a remarkable sequence complementary to viroids. EMBO J. 7, 4063-4074. Hamilton, A.J. , and Baulcombe, D.C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950-952. Itaya, A., Folimonov, A., Matsuda, Y., Nelson, R.S., and Ding, B. (2001). Potato spindle tuber viroid as inducer of RNA silencing in infected tomato. Mol. Microbe Interact. 14, 1332-1334. Jones, A.L., Thomas, C.L., and Maule, A.J. (1998). De novo methylation and co-supression induced by a cytoplasmically replicating plant RNA virus. EMBO J. 17, 6385-6393. Li, W.X., and Ding, S.W. (2001). Viral suppressors of RNA silencing. Curr. Opin. Biotechnol. 12, 150-154. Loss, P., Schmitz, M. Steger, G., and Riesner, D. (1991). Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA. EMBO J. 10, 719-727. Matzke, M.A., and Matzke, A.J.M. (2000). Plant gene silencing. Plant Mol. Biol. 43, 7-10.

70

Mette, M.T., van der Winden, J., Matzke, M.A., and Matzke, A.J.M. (1999). Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans. EMBO J. 18, 241-248. Papaefthimiou, J., Hamilton, A.J., Denti, M.A., Baulcombe, D.C., Tsagris, M., and Tabler, M. (2001). Replicating potato spindle tuber viroid RNA is accompanied by short RNA fragments that are characteristic of post-transcriptional gene silencing. Nucleic Acids Res. 29, 2395-2400. Pélissier, T., and Wassenegger, M. (2000). A DNA target of 30 bp is sufficient for RNA-directed DNA methylation. RNA 6, 55-65. Pélissier, T. Thalmeir, S., Kempe, D., Sänger H.L., and Wassenegger, M. (1999). Heavy de novo methylation and non-symetrical sites is a hallmark of RNA-directed DNA methylation. Nucleic Acids Res. 27, 1625-1634. Rackwitz, H.R., Rhode, W., and Sänger, H.L. (1981). DNA-dependent RNA polymerase II of plant origin transcribes viroid RNA into fulllength copies. Nature 291, 297-301. Riesner, D. (1987). Structure formation. Pages 63-98 in: The viroids. T.O. Diener, ed. Plenum: New York. Robertson, H.D., and Branch, A. (1987). The viroid replication process. Pages 50-69 in: Viroids and viroid-like pathogens. J. S. Semancik, ed. CRC Press: Boca Raton, FL. Ruiz, T., Voinet, O., and Baulcombe, D.C. (1998). Initiation and maintenance of virus-induced gene silencing. Plant Cell 10, 937-946. Sänger, H.L. (1987). Viroid replication. Pages 117-166 in: The viroids. T.O. Diener, ed. Plenum: New York. Sänger, H.L., Schiebel, L., Riedel, T. Pélissier, T., and Wassenegger, M. (1996). The possilble links between RNA-directed DNA methylation (RdDM), sense and antisense RNA, gene silencing, symptominduction upon microbial infections and RNA-directed RNA polymerase (RdRP). Proc. 8th Intern. Symp. Molecular Plant-Microbe Interactions. Knoxville, Tennesee, July 1996. Semancik, J.S., and Conejero, V. (1987). Viroid pathogenesis and expression of biological activity. Pages 71-126 in: Viroids and viroidlike pathogens. J.S. Semancik, ed. CRC Press: Boca de Raton, FL. Semancik, J.S., Conejero, V., and Gerhart, J. (1977). Citrus exocortis viroid: survey of protein synthesis in Xenopus laevis oocytes following addition of viroid RNA. Virology 80, 218-221. Solymosy, F., and Kiss, T. (1985). Viroids and snRNAs. Pages 183-199 in: Subviral pathogens of plants and animals: viroids and prions. K. Maramorosch, and J.J. McKelvey, eds. Academic Press: New York. Stam, M., Mol, J.N.M., and Kooter, J.M. (1997). The silence of genes in transgenic plants. Ann. Bot. 78, 3-12. Wang, M.W., and Waterhouse, P.M. (2002). Application of gene silencing in plants. Curr. Opin. Plant Biol. 5, 146-150. Wassenegger, M. (2000). RNA-directed DNA methylation. Plant Mol. Biol. 43, 203-220. Wassenegger, M., and Pélissier, T. (1998). A model for RNA-mediated gene silencing in higher plants. Plant Mol. Biol. 37, 349-362. Wassenegger, M., Heimes, S., Riedel, L., and Sänger, L.C. (1994). RNAdirected de novo methylation of genomic sequences in plants. Cell 76, 567-576.

PART II

CHAPTER 8

CLASSIFICATION ....................................................................................................

R. Flores, J.W. Randles, and R.A. Owens

.................................................................................................................................................................................................................................................................

The aim of this chapter is to present a summary of viroid classification and nomenclature according to the guidelines established by the International Committee on Taxonomy of Viruses (ICTV). A preliminary proposal was offered for discussion previously (Flores et al. 1998), and an updated and more detailed description of this topic can be found in the ‘7th Report of the ICTV’ (Flores et al. 2000). Although viroids resemble viruses in certain aspects [e.g. the type of symptoms that they induce in their hosts (see for a review Garnsey and Randles 1987; see also Part 4)], they also differ in fundamental aspects that include structure, function and, what is particularly relevant in a taxonomic context, evolutionary origin. In contrast to viruses, viroids: i

have a considerably smaller genome;

ii

do not code for any protein;

iii

are (in certain cases) endowed with ribozyme activity; and

iv

may have originated in the precellular RNA world that ultimately gave rise to our present cellular world based on DNA and proteins (Diener 1989).

Despite the fact that viroids should be in principle considered as a taxon independent from viruses, they are classified into families, genera and species following some of the criteria adopted for viruses. Other criteria, such as particle morphology or protein characteristics, are not applicable to viroids.

FAMILIES The 28 viroid species described so far (Figure 8.1), can be allocated into two families according to three criteria (Figure 8.2). Members of the family Pospiviroidae, whose type species is Potato spindle tuber viroid (PSTVd) (Diener 1971; Gross et al. 1978), have a central conserved region (CCR) in their proposed most stable rod-like secondary structure (Keese and Symons 1985; Sano et al. 1992), do not display self-cleavage mediated by hammerhead ribozymes, and replicate (and accumulate) in the nucleus through an asymmetric rolling-circle mechanism. Members of the family Avsunviroidae, whose type species is Avocado sunblotch viroid (ASBVd) (Symons 1981; Hutchins et al. 1986), lack a CCR, their strands of both polarities self-cleave via hammerhead ribozymes, and replicate (and accumulate) in the chloroplast through a symmetric rolling-circle mechanism (see

71

R. Flores et al.

FAMILY

GENUS

POSPIVIROID

POSPIVIROIDAE

AVSUNVIROIDAE

SPECIES PSTVd (potato spindle tuber) TCDVd (tomato chlorotic dwarf) MPVd (mexican papita) TPMVd (tomato planta macho) CSVd (chrysanthemum stunt) CEVd (citrus exocortis) TASVd (tomato apical stunt) IrVd (iresine 1) CLVd (columnea latent)

HOSTUVIROID

HSVd (hop stunt)

COCADVIROID

CCCVd (coconut cadang-cadang) CTiVd (coconut tinangaja) HLVd (hop latent) CVd-IV (citrus IV)

APSCAVIROID

ASSVd (apple scar skin) CVd-III (citrus III) ADFVd (apple dimple fruit) GYSVd 1 (grapevine yellow speckle 1) GYSVd 2 (grapevine yellow speckle 2) CBLVd (citrus bent leaf) PBCVd (pear blister canker) AGVd (Australian grapevine)

COLEVIROID

CbVd 1 (coleus blumei 1) CbVd 2 (coleus blumei 2) CbVd 3 (coleus blumei 3)

AVSUNVIROID

ASBVd (avocado sunblotch)

PELAMOVIROID

PLMVd (peach latent mosaic) CChMVd (chrysanthemum chlorotic mottle)

Figure 8.1 Classification scheme of the 28 sequenced viroids. The type species from which genus and family names derive are underlined.

Chapter 2 ‘Molecular characteristics’, Chapter 5 ‘Replication’ and Chapter 56 ‘Ribozyme reactions of Viroids’). The third criterion (nuclear or chloroplastic site of replication and accumulation) should be considered as tentative at the present stage, but it is fulfilled by those viroids in which this particular aspect has been examined: PSTVd, Tomato planta macho viroid (TPMVd), Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Coconut cadang-cadang viroid (CCCVd), ASBVd and Peach latent mosaic viroid (PLMVd) (see Semancik and Conejero-Tomás 1987 for a review on early results; see also Chapter 5 ‘Replication’). Most viroids belong to the family Pospiviroidae, with the second family, Avsunviroidae, containing only three members: ASBVd, PLMVd and Chrysanthemum chlorotic mottle viroid (CChMVd).

72

GENERA Within each family, viroids are grouped into genera according to different criteria. In the case of family Pospiviroidae, five genera are distinguished depending primarily on the sequences that form the CCR (Figure 8.1). A second criterion is the presence or absence of two other conserved motifs: the terminal conserved region (TCR) (Koltunow and Rezaian 1988), found in all members of genera Pospi- and Apscaviroid as well as in the two largest members (CbVd2 and CbVd3) of genus Coleviroid, and the terminal conserved hairpin (TCH) (Flores et al. 1997) found in HSVd and in all members of genus Cocadviroid. Within the family Avsunviroidae, PLMVd is more closely related to CChMVd than to ASBVd because:

CLASSIFICATION

Viroid families

Pospiviroidae

Avsunviroidae

Central Conserved Region (CCR)

No Central Conserved Region (CCR)

No hammerhead ribozymes

Hammerhead ribozymes

Nuclear replication via asymmetric rolling circle

Chloroplastic replication via symmetric rolling circle

Figure 8.2

Demarcating criteria for the two viroid families.

i

PLMVd and CChMVd have relatively high G+C contents (55.4% and 52.5%, respectively) whereas ASBVd contains only 38% G+C;

ii

the predicted lowest free energy secondary structures of PLMVd and CChMVd are branched in contrast to that of ASBVd which is quasi-rod-like;

iii

the single hammerhead structures that PLMVd and CChMVd can adopt are stable as opposed to those of ASBVd which are unstable (see Chapter 56 ‘Ribozyme Reactions of Viroids’); and

iv

PLMVd and CChMVd, but not ASBVd, are insoluble in 2 M LiCl (Navarro and Flores 1997).

On this basis, PLMVd and CChMVd have been grouped in genus Pelamoviroid whereas ASBVd forms the monospecific genus Avsunviroid. Names of genera are derived from those of their corresponding type species following the same rules used for plant viruses: Pospiviroid (from Potato spindle tuber viroid, PSTVd) Hostuviroid (from Hop stunt viroid, HSVd) Cocadviroid (from Coconut cadang-cadang viroid, CCCVd) Apscaviroid (from Apple scar skin viroid, ASSVd) Coleviroid (from Coleus blumei viroid 1, CbVd1) Avsunviroid (from Avocado sunblotch viroid, ASBVd) Pelamoviroid (from Peach latent mosaic viroid, PLMVd)

SPECIES Sequence data has been previously used as the only criterion for this purpose, with the arbitrary level of 90% of nucleotide identity separating different viroid species from variants of the same viroid species, but the ICTV now recommends the use of at least a second demarcating criterion. Consider, for example, the fact that Mexican papita viroid (MPVd) and Tomato planta macho viroid (TPMVd) share a nucleotide identity around 90%. In this case the biological properties, particularly host range

including natural and experimental hosts (Martínez-Soriano et al. 1996), allow their classification as two different viroid species. A second especially difficult issue concerns citrus viroids (Duran-Vila et al. 1988), whose diversity is reflected in the existence of species belonging to genera Pospi-, Hostu-, Cocad- and Apscaviroid. Point mutations and, particularly, RNA recombination events that seem to occur between some of these viroids co-infecting the same host plant, contribute to the frequent identification of new viroid sequences in citrus with nucleotide identity close to the borderline separating species from variants (Owens et al. 2000). A detailed study of their corresponding biological properties will help to decide into which taxon these viroid sequences should be allocated.

VARIANTS Although this is not a formal taxon within the classification scheme of the ICTV, it deserves some consideration on the basis of several factors. First, like other RNA replicons, viroids are complex populations of closely related but not identical sequence variants, which emerge mostly during replication as a result of the error-prone nature of RNA polymerases and form what is known as a quasi-species (Domingo et al. 1996). Usually, one or a limited number of variants predominate, but this should not imply that the population is uniform. Complexity of viroid quasi-species may also arise from recombination events, as well as from repeated infections of an individual plant, an especially plausible scenario for long-lived fruit trees which are the subject of frequent agronomic manipulation (e.g. pruning and grafting) that facilitate re-infection. Second, variants are phytopathologically relevant because minimal changes affecting approximately 1% of the viroid genome may have major effects on symptom expression. For example, a severe and a mild variant of PSTVd differ only in three nucleotide exchanges (Gross et al. 1981), lamina-depleting forms of CCCVd show point mutations at one or two sites (Rodriguez and Randles 1993), HSVd variants inducing citrus cachexia differ from other variants that do not cause the disease in only six nucleotide residues (Levy and Hadidi 1993; Reanwarakorn and Semancik 1998), and ASSVd variance that induce dapple apple and pear rusty skin symptoms differ from the prototype isolate of ASSVd at 9

73

R. Flores et al.

and 25 sites, respectively (Zhu et al. 1995). And third, passage through an experimental host may result in selection of variants of the initial population as a consequence of a filtering effect. This is illustrated by the observation that a single nucleotide substitution converts PSTVd from a noninfectious to an infectious RNA for Nicotiana tabacum (Wassenegger et al. 1996).

FINAL CONSIDERATIONS Taxonomy always entails decisions that to some extent are arbitrary. This is particularly evident in the case of viroids, in which the very limited number of demarcating criteria makes it difficult in some instances to separate species from variants. However, the present viroid classification scheme appears overall well founded and is additionally supported by phylogenetic analyses carried out using complete viroid sequences instead of the conserved motifs alone (Flores et al. 2000). These studies also indicate that members of genera Pospi-, Hostu- and Cocadviroid are more closely related to each other than to members of genera Apsca- and Coleviroid, a feature that is also inferred from sequence comparisons restricted to their corresponding CCRs. A compilation of viroid sequences that includes numerous variants and their accession numbers for easy retrieval from databases is available (Baxevanis 2001; Viroids database 2002). As already indicated, viroids seem to have an evolutionary origin independent from that of viruses. They may, however, be related to viroid-like satellite RNAs, which possess features resembling viroids (e.g. small size and replication through a rolling-circle mechanism), although they are functionally dependent on a helper virus with single-stranded RNA genome for replication and encapsidation. The presence of hammerhead structures in one or both polarity strands of all viroid-like satellite RNAs described so far (Symons 1997), is the strongest argument for such a relationship. Acknowledgements

This work was partially supported by grants PB95-0139 and PB98-0500 from the Comisión Interministerial de Ciencia y Tecnología de España (to R. Flores), and by the United Nations Development Program and the Food and Agriculture Organization, and grants from the Australian Centre for International Agricultural Research and the Australian Research Council (to J.W. Randles). References Baxevanis, A.D. (2001). The molecular biology database collection: an updated compilation of biological database resources. Nucleic Acids Res. 29, 1-10. Diener, T.O. (1971). Potato spindle tuber ‘virus’ IV. A replicating low molecular weight RNA. Virology 45, 411-428. Diener, T.O. (1989). Circular RNAs: relics of precellular evolution? Proc. Natl. Acad. Sci. USA 86, 9370-9374.

74

Domingo, E., Escarmís, C., Sevilla, N., Moya, A., Elena, S.F., Quer, J., Novella, I. S., and Holland. J.J. (1996). Basic concepts in RNA virus evolution. FASEB J. 10, 859-864. Duran-Vila, N., Roistacher, C.N., Rivera-Bustamante, R., and Semancik, J.S. (1988). A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69, 3069-3080. Flores, R., Di Serio, F., and Hernández, C. (1997). Viroids: the noncoding genomes. Semin. Virol. 8, 65-73. Flores, R., Randles, J.W., Bar-Joseph, M., and Diener, T.O. (1998). A proposed scheme for viroid classification and nomenclature. Arch. Virol. 143, 623-629. Flores, R., Randles, J.W., Bar-Joseph, M., and Diener, T.O. (2000). Subviral agents: viroids. Pages 1009-1024 in: Virus taxonomy, Seventh Report of the International Committee on Taxononomy of Viruses. M.H.V. van Regenmortel, C.M. Fauquet, D.H.L. Bishop, E.B. Carstens, M.K. Estes, S.M. Lemon, D.J. McGeoch, J. Maniloff, M.A. Mayo, C.R. Pringle, and R.B. Wickner, eds. Academic Press: San Diego, CA. Garnsey, S.M., and Randles, J.W. (1987). Pages 127-160 in: Viroids and viroid-like pathogens. J.S. Semancik, ed. CRC Press: Boca Raton, FL. Gross, H.J., Domdey, H., Lossow, C., Jank, P., Raba, M., Alberty, H., and Sänger, H.L. (1978). Nucleotide sequence and secondary structure of potato spindle tuber viroid. Nature 273, 203-208. Gross, H.J., Liebl, U., Alberty, H., Krupp, G., Domdey, H., Ramm, K., and Sänger, H.L. (1981). A severe and a mild potato spindle tuber viroid isolate differ in three nucleotide exchanges only. Biosci. Rep. 1, 235-241. Keese, P., and Symons, R.H. (1985). Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proc. Natl. Acad. Sci. USA 82, 4582-4586. Hutchins, C.J., Rathjen, P.D., Forster, A.C., and Symons, R.H. (1986). Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 14, 3627-3640. Koltunow, A.M., and Rezaian, M.A. (1988). Grapevine yellow speckle viroid. Structural features of a new viroid group. Nucleic Acids Res. 16, 849-864. Levy, L., and Hadidi, A. (1993). Direct nucleotide sequencing of PCRamplified DNAs of the closely related citrus viroids IIa and IIb (cachexia). Proceedings of the XIIth International Conference on Citrus Virology, pp. 180-186. Martínez-Soriano, J.P., Galindo-Alonso, J., Maroon, C.J.M., Yucel, I., Smith, D.R., and Diener, T.O. (1996). Mexican papita viroid: putative ancestor of crop viroids. Proc. Natl. Acad. Sci. USA 93, 9397-9401. Navarro, B., and Flores, R. (1997). Chrysanthemum chlorotic mottle viroid: unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes. Proc. Natl. Acad. Sci. USA 94, 11262-11267. Owens, R.A., Yang, G., Gudersen-Rindal, D., Hammond, R.W., Candresse, T., and Bar-Joseph, M. (2000). Both point mutation and RNA recombination contribute to sequence diversity of citrus viroid III. Virus Genes 20, 243-252. Reanwarakorn, K., and Semancik, J.S. (1998). Regulation of pathogenicity in hop stunt viroid-related group II citrus viroid. J. Gen. Virol. 79, 3163-3171. Rodriguez, M.J.B., and Randles, J.W. (1993). Coconut cadang-cadang viroid (CCCVd) mutants associated with severe disease vary in both the pathogenicity domain and the central conserved region. Nucleic Acids Res. 21, 2271.

CLASSIFICATION

Sano, T., Candresse, T., Hammond, R.W., Diener, T.O., and Owens, R.A. (1992). Identification of multiple structural domains regulating viroid pathogenicity. Proc. Natl. Acad. Sci. USA 89, 10104-10108. Semancik, J.S., and Conejero-Tomás V. (1987). Pages 71-126 in: Viroids and viroid-like pathogens. J.S. Semancik, ed. CRC Press: Boca Raton, FL. Symons, R.H. (1981). Avocado sunblotch viroid: primary sequence and proposed secondary structure. Nucleic Acids Res. 9, 65276537. Symons, R.H. (1997). Plant pathogenic RNAs and RNA catalysis. Nucleic Acids Res. 25, 2683-2689.

Viroids database. (2002). http://nt.ars-grin.gov/subviral Wassenegger, M., Spieker, R.L., Thalmeir, S., Gast, F.U., Riedel, L., and Sänger, H.L. (1996). A single nucleotide substitution converts potato spindle tuber viroid (PSTVd) from a noninfectious to an infectious RNA for Nicotiana tabacum. Virology 226, 191-197. Zhu, S.F., Hadidi, A., Hammond, R.W., Yang, X., and Hansen, A.J. (1995). Nucleotide sequence and secondary structure of pome fruit viroids from dapple apple disease of apples, pear rusty skin diseased pears and apple scar skin symptomless pears. Acta Hortic. 386, 554-559.

75

PART II

CHAPTER 9

VIROID-LIKE SATELLITE RNAS ....................................................................................................

L. Rubino, F. Di Serio, and G.P. Martelli

.................................................................................................................................................................................................................................................................

Viroid-like satellite RNAs (VL-satRNAs), together with viroids, are the smallest infectious RNAs known, so far. These two groups of subviral RNAs share several structural and biochemical properties, but, at the same time, display different biological features. In this chapter, the main traits of viroid-like satellite RNAs are briefly described highlighting similarities and differences with respect to viroids. For more detailed descriptions of viroid-like satellite RNAs readers are referred to previous reviews (Francki 1987; Kaper and Collmer 1988; Symons 1997; Symons and Randles 1999).

BIOLOGICAL FEATURES VL-satRNAs are small subviral molecules 220–457 nucleotides (nt) in size that assume a covalently-closed circular conformation in one or more steps of their life cycle. They are functionally dependent on a ‘helper’ virus, by the coat protein of which are encapsidated, do not possess coding capacity, and share little, if any, sequence similarity with the supporting virus and with the host genome. VL-satRNAs can be grouped into two main clusters, which are associated, respectively, with:

76

i

members of the genus Sobemovirus, also called ‘virusoids’, which are encapsidated as circular molecules with a highly base-paired rod-like secondary structure; and

ii

members of the genera Polerovirus and Nepovirus, which are encapsidated in linear form; in both cases circular and linear molecules are found in infected tissues.

The two types of satRNAs will be distinguished by a small ‘v’ and a small ‘s’ preceding the abbreviation of the helper virus (Table 9.1). Other RNA molecules functionally dependent on a helper virus are the linear satellite RNAs of some cucumoviruses, tombusviruses, nepoviruses and carmoviruses, and the defective interfering (DI) RNAs found in plants infected by certain members of tombusviruses, carmoviruses and tospoviruses. However, all these RNAs clearly differ from viroid-like satellite RNAs because both DI RNAs and linear satellite RNAs never occur in a circular form at any point of their life cycle. Moreover, the largest linear satellite RNAs have coding capacity (for a review see Mayo et al. 1999; Taliansky and Palukaitis 1999), whereas DI RNAs share complete sequence homology with their parent virus, from the genome of which they are generated de novo

VIROID-LIKE SATELLITE RNAS

Table 9.1

Viroid-like satellite RNAs.

Helper virus

VL-sat RNA abbreviation

Size (nt)

Accession number

Helper virus genus

Encapsidated RNA form

Ribozyme in the (+) strand

Ribozyme in the (-) strand

Lucerne transient streak virus

vLTSV

324

X01984

Sobemovirus

circular

Hammerhead

Hammerhead

Subterranean clover mottle virus

vSCMoV

332 388

M33000, M33001

Sobemovirus

circular

Hammerhead

-

Velvet tobacco mottle virus

vVTMoV

366

J02439

Sobemovirus

circular

Hammerhead

-

Solanum nodiflorum mottle virus

vSNMV

377

J02386

Sobemovirus

circular

Hammerhead

-

Rice yellow mottle virus

vRYMV

220

AF039909

Sobemovirus

circular

Hammerhead

-

Tobacco ringspot virus

sTRSV

359

M14879

Nepovirus

linear

Hammerhead

Hairpin

Chicory yellow mottle virus

sCYMV

457

D00721

Nepovirus

linear

Hammerhead

Hairpin

Arabis mosaic virus

sArMV

300

M21212.

Nepovirus

linear

Hammerhead

Hairpin

Cereal yellow dwarf virus-RPV

sCYDV-RPV

322

M63666.

Polerovirus

linear

Hammerhead

Hammerhead

during replication following repeated deletions (for a review see White 1996). VL-satRNAs are not necessary for helper virus genome replication (Jones et al. 1983; Jones and Mayo 1984; Francki et al. 1986; Piazzolla and Rubino 1984), but can interfere with the replication process, modulating symptoms, so as to increase or attenuate their severity. Thus, viroid-like satellite RNAs of some sobemoviruses, like for instance that of Velvet tobacco mottle virus (vVTMoV), intensify the symptoms induced by the supporting virus, while the satellite of Cereal yellow dwarf virusRPV (sCYDV-RPV, formerly known as Barley yellow dwarf virus satellite RNA) decreases helper virus accumulation and attenuates symptom expression (Taliansky and Palukaitis 1999). Within the genus Nepovirus, VL-satRNAs of Tobacco ringspot virus (TRSV) and Chicory yellow mottle virus (CYMV) decrease the severity of symptoms induced by their supporting viruses (Kaper and Collmer 1988; Piazzolla et al. 1986), whereas the viroid–like satellite RNA of Arabis mosaic virus (sArMV) enhances symptom severity in Chenopodium quinoa and hop (Davies and Clark 1983). The ability of VL-satRNAs to attenuate symptoms induced by their helper viruses has been successfully used as a tool for virus disease control. Gerlach et al. (1987), for instance, reported that symptoms and virus accumulation in transgenic tobacco plants expressing TRSV sat-RNA sequences in both orientations were reduced with respect to those shown in non-transformed plants.

STRUCTURAL FEATURES Because of the small size (220–457 nt) and absence of any apparent coding capacity, the genetic information required to support the biological activity of VL-satRNAs must be extremely compressed and perhaps be contained not only in the primary structure, but also in higher order structural levels. At the primary level, short consensus sequences are conserved in all known VL-satRNAs corresponding mainly to structural

domains of the ribozymes involved in the self–cleavage of one or both polarity strands of the satRNA molecules (see next paragraph). Only virusoids of Lucerne transient streak virus (vLTSV) and Rice yellow mottle virus (vRYMV) share a long segment corresponding to the left terminal domain in their proposed secondary structure, whereas only short sequences are conserved among other virusoids (Collins et al. 1998). VL-satRNAs are characterized by highly ordered secondary structures, where regions of intramolecular complementarity are intermingled with loops formed by unpaired nucleotides. The secondary structure of lowest free energy of those satRNAs associated with sobemoviruses have a rod-like conformation (Francki 1987; Keese and Symons 1987), similar to that of members of the viroid family Pospiviroidae (see also Chapter 2 ‘Molecular characteristics’ of viroids in this volume), whereas secondary structures of VL-satRNAs associated with nepoviruses and poleroviruses are more branched (Kaper et al. 1988; Rubino et al. 1990) (Figure 9.1). The spatial conformation is also important for VL-satRNAs since elements of tertiary structure have been identified in hammerhead and hairpin ribozymes involved in the self-cleavage of replication intermediates (McKay 1996; Rupert and FerreD’Amare 2001; Klostermeier and Millar 2001).

REPLICATION OF VIROID-LIKE SATELLITE RNAS AND ROLE OF RIBOZYMES

Due to the presence of circular forms in infected tissues, all VLsatRNAs are thought to replicate by a RNA-based rolling circle mechanism (Branch and Robertson 1984; Hutchins et al. 1985). As for viroids, two pathways (symmetrical and asymmetrical) were proposed for VL-satRNAs replication (Forster and Symons 1987). By convention, the encapsidated infectious strand of a VLsatRNA that accumulates at higher concentration in plant tissues is designed as the positive strand. In the symmetrical pathway of the model, the circular positive sense RNA is transcribed into a

77

L. Rubino et al.

Figure 9.1 Proposed secondary structure of Chicory yellow mottle virus satellite (sCYMV) RNA. Flags delimit positive (solid flags) and negative (open flags) self-cleavage domains involved in the formation of the hammerhead and hairpin ribozymes; the predicted positive and negative selfcleavage sites are marked by solid and open arrows, respectively. The sequence involved in the formation of both hammerhead and hairpin ribozymes of sCYMV RNA is boxed (modified from Rubino et al. 1990).

multimeric negative strand, which is then cleaved to generate the corresponding linear monomeric forms. Ligation of these forms originates a circular template that serves for the synthesis of the positive multimeric strands, which are finally cleaved and ligated to give the progeny of monomeric circular RNAs. The minus multimeric strands of some VL-satRNAs are neither processed in vitro and, most likely, nor in vivo. In these cases, it is presumed to operate the asymmetric pathway of the model in which the multimeric negative RNAs are the template for the synthesis of the multimeric positive strands, which then undergo cleavage and ligation to monomers. Autolytic processing without the assistance of any protein seems to be involved in the replication of VL-satRNAs. This hypothesis was put forward because it was shown that one or both polarity strands of VL-satRNAs self-cleave in vitro. Evidence for the in

78

vitro self-cleaving activity of a VL-satRNA was first obtained by Prody et al. (1986), who demonstrated that purified oligomeric RNAs of sTRSV self-cleaved in vitro in absence of any protein, generating infectious natural linear monomers. At the same time, it was shown that in vitro-synthesized RNA transcripts containing positive and negative dimeric RNAs of Avocado sunbloch viroid (ASBVd) and of sTRSV undergo specific self-cleavage reactions generating monomeric forms (Hutchins et al. 1986; Buzayan et al. 1986a). Two ribozyme structures, called hammerhead and hairpin, were shown to be involved in the VL-satRNA self-splicing (Figure 9.2 and Table 9.1). In vitro self-cleavage via hammerhead structures was demonstrated for both polarity strands of sCYDV-RPV (Miller et al. 1991) and of vLTSV (Forster and Symons 1987). The other virusoids of Solanum nodiflorum mottle virus (vSNMV), Subterraneam clover mottle virus (vSCMoV), Vel-


Figure 9.2 Ribozyme structures found in the positive (hammerhead, panel A) and negative (hairpin, panel B) strands of Tobacco ringspot virus satellite (sTRSV), Chicory yellow mottle virus satellite (sCYMV) and Arabis mosaic virus satellite (sArMV), respectively. In each ribozyme structure the conserved nucleotides are in bold and the self-cleavage sites are indicated by an arrow. Lines denote sequences functional for cleavage of both plus and minus RNA strands. The same numbers are used in the positive and in the negative polarity (modified from Rubino et al. 1990).

vet tobacco mottle virus (vVTMoV) and Rice yellow mottle virus (vRYMV) and the VL-satRNAs associated with nepoviruses contain active hammerhead ribozymes only in the positive polarity strand. No self-cleaving activity was observed in the negative RNA of these virusoids, while hairpin ribozyme-mediated selfcleavage was observed in the negative strands of VL-satRNAs associated with nepoviruses (Feldstein et al. 1989; Etscheid et al. 1995; Deyoung et al. 1995). Evidence for the in vivo function of ribozyme-mediated selfcleavage in VL-satRNAs replication stems from a number of observations. The relative accumulation of positive and negative monomeric and multimeric RNA forms of vLTSV and other virusoids in infected tissues is consistent with the in vitro selfcleavage data (Hutchins et al. 1985; Davies et al. 1990). Moreover, as expected for an in vivo activity of the hammerhead selfcleavage, multimeric negative RNAs of vLTSV were only

detected in infected tissues when the negative ribozyme was inactivated by mutations at different sites. Moreover, in part of the progeny, the mutant reverted and self-cleaving RNAs were generated, providing further evidence for the predicted role of the ribozyme in vivo (Sheldon and Symons 1993). In the case of viroids, the role of hammerhead ribozymes in vivo is supported by other convincing experimental data (reviewed by Flores et al. 2000, 2001; see also Chapter 56 ‘Ribozyme reactions of viroids’ in this volume). Although ribozyme-mediated self-cleavage is an RNA-based splicing mechanism, the involvement of a host protein in facilitating the hammerhead autolytic reaction in vitro, and possibly in vivo, has been recently reported for a viroid (Daròs and Flores 2002). Therefore, it can be speculated that host or viral proteins may be also involved in stimulating VL-satRNA self-cleavage and/or ligation in vivo.

79

L. Rubino et al.

STRUCTURE AND CATALYTIC ACTIVITY OF HAMMERHEAD AND HAIRPIN RIBOZYMES

Comparing nucleotide sequences of several self-cleaving RNAs, Forster and Symons (1987) proposed a structural model for the active site of hammerhead ribozymes. It consists of a contiguous stretch of about 50 nt, forming three stable stems connected by unpaired regions (Figure 9.2A). A consensus sequence of 13 nt mainly located in the single-stranded regions is conserved in almost all natural hammerhead ribozymes (Figure 9.2A). Stability of the helices is preserved, although nucleotides involved in their formation differ in the natural hammerhead structures. A GUC sequence precedes the self-cleaving site in most natural hammerhead structures. This is the case of all VL-satRNAs of nepoviruses and of most virusoids. However, a GUA triplet precedes the cleavage site of the negative strand of vLTSV and a AUA sequence precedes that of the positive polarity strand of sCYDV-RPV. Hammerhead-mediated self-cleavage has been observed in other small circular RNAs, including three viroids (Hutchins et al. 1986; Hernández and Flores 1992; Navarro and Flores 1997), and two viroid-like RNAs isolated from carnation and cherry (Hernández et al. 1992; Di Serio et al. 1997; see also other viroid-like RNAs from plants and animals in this chapter). Self-cleaving of minus sTRSV RNA was discovered at the same time as that mediated by hammerhead ribozymes (Buzayan et al. 1986a), but the structure of the catalytic core was determined a few years later (Feldstein et al. 1989; Hampel and Tritz 1989). It consists of a stretch of nucleotides with a size similar to that of the hammerhead ribozyme, but the secondary structure of the active conformation is different. Contrary to natural hammerhead ribozymes, which are usually formed by nucleotides located in a contiguous RNA sequence, the formation of hairpin structures in the negative strand of viroid-like satellite RNAs associated with nepoviruses requires two distant segments folding into two double-stranded segments interrupted by unpaired regions (Figure 9.2B). Selection and mutation studies have shown that most nucleotides located in the single-stranded regions (loop a and b in Figure 9.2B) are essential for catalytic activity (Chowrira et al. 1991; Berzal-Herranz et al. 1993; Joseph et al. 1993). The only hairpin ribozymes discovered so far in natural molecules are those found in the VL-satRNAs associated with nepoviruses. In these natural hairpin ribozymes the self-cleaving site occurs in the consensus sequence 5’-A‚GU– 3’ (Figure 9.2B). Cleavage reactions promoted by both hammerhead and hairpin ribozymes generate linear RNAs having a 5’-hydroxyl and a cyclic 2’-3’ phosphate termini (reviewed by Fedor 2000). The catalytic reaction mediated by hairpin ribozymes is reversible,

80

because spontaneous ligation generating a normal 3’-5’ bond was observed in vitro with the minus sTRSV linear forms (Buzayan et al. 1986a; 1986b). The hairpin ribozyme is a better ligase than it is a nuclease while the hammerhead reaction favors cleavage over ligation of bound products by nearly 200-fold (Fedor 2000). Self-ligation of RNAs resulting from hammerheadmediated self-cleavage has been reported for Peach latent mosaic viroid in vitro (Lafontaine et al. 1995) and in vivo (Côté et al. 2001). However, instead of the 3’-5’ bonds usually present in RNAs, 2’-5’ phosphodiester bonds were generated at the ligation site (Côté and Perreault 1997). Therefore, the involvement of this process in the in vivo circularization of hammerhead containing RNAs still remains to be fully demonstrated (Flores et al. 2000; Diener 2001). Cis and trans cleaving activity of hairpin and hammerhead ribozymes

Natural ribozymes of viroid-like satellite RNAs and viroids catalyze cleavage in cis. However, self-cleavage of circular monomeric molecules is not favored as in their secondary structure of lowest free energy the sequences forming part of the ribozymes are not folded in the active conformation but in alternative stable structures. During replication, multimeric forms are synthesized and the ribozymes fold in their active conformations that promote self-cleavage. In vLTSV, positive and negative hammerhead domains are located in opposite positions in the proposed secondary structures (Keese et al. 1983), as observed also with some viroids (Hernández and Flores 1992; Navarro and Flores 1997) and viroid-like RNAs (Di Serio et al. 1997). In VL-satRNAs of nepoviruses, some of the sequences forming the hammerhead and hairpin ribozymes are partially overlapping. This is the case of a stretch of seven nucleotides (located just upstream of the selfcleavage site of the negative strand) which is involved in both self-cleaving structures (Figure 9.2). Thus, the same structural domain, in one and the other polarity, is functional for cleavage of both strands, illustrating that genetic information in these small RNAs is extremely compacted. Partial overlapping of positive and negative self-cleavage domains was also observed in sLTSV (Forster and Symons 1987) and in a small circular RNA from carnation (Hernández et al. 1992). In trans processing activity of both hammerhead (Uhlenbeck 1987; Haseloff and Gerlach 1988) and hairpin ribozymes (Feldstein et al. 1989; Hampel and Tritz 1989) was soon demonstrated after their discovery. Ribozyme structures engineered to act in trans have major biotechnological implications as they can be used in gene therapy to inhibit function of oncogenes or to target degradation of viral RNAs in vivo (reviewed by Couture and Stinchcomb 1996; Shippy et al. 1999).


OTHER VIROID-LIKE RNAS FROM PLANTS AND ANIMALS

Several other small RNAs, sharing many structural features with VL-satRNAs and viroids, have been isolated from plants. Since their biological nature has not been completely unraveled or is clearly different from that of viroids and of VL-satRNAs, they will be briefly described here and referred to as viroid-like RNAs. At least two small viroid-like RNAs were isolated from cherry (Di Serio et al. 1996). Molecular characterization of the most abundant cherry small circular RNA (cscRNA1) showed that both its polarity strands can adopt hammerhead structures and self-cleave accordingly in vitro (Di Serio et al. 1997). The hammerhead structures of cscRNA1 present several peculiarities, prominent among which is the AUA triplet preceding the selfcleavage site in the hammerhead of the positive polarity strand (Di Serio et al. 1997). The same triplet in the same position was previously observed in only one other natural hammerhead structure: that of the positive strand of sCYDV-RPV. The biological nature of cscRNA1 has not been determined, although it does not seem to be a viroid, because transmission attempts using purified circular RNAs have been unsuccessful (J.C. Desvignes, F. Di Serio and R. Flores, unpublished information). Moreover, the close association of cscRNAs with a series of dsRNAs of possible viral origin, together with other structural features, suggested a satellite nature for these cscRNAs (Di Serio et al. 1997). Carnation small viroid-like RNA (CarSV RNA) is a non-infectious RNA containing active self-cleaving hammerhead structures in both polarity strands (Hernández et al. 1992; Daròs and Flores 1995). It is a unique example of a special biological system termed retroviroid-like element because a DNA homologous to CarSV RNA has been identified in carnation plants (Daròs and Flores 1995). Tandemly repeated CarSV DNA monomers fused to sequences of Carnation etched ring virus (CERV) have been found forming part of an extrachromosomal element (Daròs and Flores 1995). The CarSV-CERV DNA fusion sites were recently analyzed and the involvement of the viral reverse transcriptase in the origin of such a retroviroid-like elements was suggested (Vera et al. 2000). Viroid-like RNAs have been also found in animals. Hepatitis delta virus (HDV) is a small RNA satellite virus of Hepatitis B virus (HBV) that can cause fulminant hepatitis in infected patients (reviewed by Lai 1995; Taylor 1999). Although the single-stranded HDV RNA has properties in common with viroid and viroid-like satellite RNAs, it differs in coding for short and long form of the δ antigen, which has a crucial role in the viral life cycle. HDV RNA is circular, shows a high degree of secondary structure, and contains specific ribozymes in both polarity strands thought to be critical for the viral rolling circle

replication in vivo, which is nuclear and mediated by the DNAdependent RNA polymerase II (reviewed by Lai 1995; Taylor 1999). Finally, the small transcript from newt satellite DNA and the schistosome satellite DNA transcript are the only animal RNAs known to contain hammerhead ribozymes active in vitro and perhaps in vivo (Epstein and Gall 1987; Ferbeyre et al. 1998).

SIMILARITIES AND DIFFERENCES BETWEEN VIROIDS AND VIROID-LIKE SATELLITE RNAS As indicated above, VL-satRNAs are similar to viroids in many structural aspects, including the small size, high content of selfcomplementary regions in the predicted secondary structure, and existence of covalently closed circular forms. They also share biochemical features, like the RNA-based replication through a rolling circle model, the self-splicing activity and the absence of protein coding capacity. The presence of ribozyme structures in these and other small RNAs suggested the hypothesis that these molecules may be remnants of a precellular RNA world, in which genetic information was contained exclusively in free RNA molecules. Selfcleavage activity was conserved in some viroids and in viroidlike RNAs when they became intracellular pathogens. Taken all together, these considerations suggested the possibility of a common origin for these molecules (Diener 1989). Phylogenetic analysis supported this hypothesis (Elena et al. 1991). Jenkins et al. (2000) has recently questioned these conclusions, but when obvious sequence similarities and rearrangements are considered in the alignment and an appropriate estimator of the genetic distances is used, the phylogenetic relationships between viroids and viroid-like satellite RNAs are confirmed (Elena et al. 2001). In this phylogenetic reconstruction the groups cluster according to their structural and biological properties and members of the family Avsunviroidae (which are self-cleaving viroids) appear in an intermediate position between the non-selfcleaving viroids (family Pospiviroidae) and the VL-satRNAs. Therefore, the hammerhead ribozyme, found in both Avsunviroidae and in the VL-satRNAs, can be considered the structural and functional link between viroids and viroid-like RNAs (Elena et al. 2001). Interestingly, a non-active hammerhead-like sequence was recently found in all sequence variants of a member of family Pospiviroidae (Amari et al. 2001). VL-satRNAs associated with nepoviruses are slightly different from the others, because they contain a hairpin ribozyme in the negative polarity strand which has never been identified in viroids nor in viroid-like RNAs. Nevertheless, a common origin has been recently proposed for several ribozymes, including hammerhead and hairpin (Harris and Elder 2000). Main differences between viroids and VL-satRNAs reside in their biological properties. Whereas the intracellular behavior of

81

L. Rubino et al.

VL-satRNAs depends on a helper virus, replication of both nuclear and chloroplast viroids is mediated by host DNAdependent RNA polymerases, and their movement is dependent on cellular mechanisms (Diener 2001 and references therein). Both viroids and VL-satRNAs contain sequences and structural signals needed for infectivity. In the case of viroids, some of these signals have recently been characterized, more specifically the initiation sites and possible promoter sequences of Potato spindle tuber viroid and ASBVd (Navarro and Flores 2000; Fels et al. 2001). Less is known about molecular signals of VLsatRNAs. However, the existence of such recognition signals can be presumed considering that sobemoviruses support replication of a number of different virusoids (Jones and Mayo 1983, 1984; Paliwal 1984; Dall et al. 1990; Sehgal et al. 1993). It was also demonstrated that in contrast to VL-satRNAs, viroids and the HDV RNA have both many polypurine and polypyrimidine tracts. Although the meaning of such a structural difference is not understood, a relation with difference in replication strategy was suggested (Branch et al. 1993). From the epidemiological point of view, strong differences exist between viroids and VL-satRNAs, the epidemiology of the latter being closely related to that of the helper viruses. In the case of viroids the involvement of vectors is rare and the major role in viroid spread is played by agricultural practices (Diener 2001). References Amari, K., Gómez, G., Myrta, A., Di Terlizzi, B., and Pallás, V. (2001). The molecular characterization of 16 new sequence variants of Hop stunt viroid reveals the existence invariable regions and a conserved hammerhead-like structure on the viroid molecule. J Gen Virol. 82, 953-962. Berzal-Herranz, A., Joseph, S., Chowrira, B.M., Butcher, S.E., and Burke, J.M. (1993). Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. EMBO J. 12, 2567-2573. Branch, A.D., and Robertson, H.D. (1984). A replication cycle for viroids and other small infectious RNAs. Science 223, 450-455. Branch, A.D., Lee, S.E., Neel, O.D., and Robertson, H.D. (1993). Prominent polypurine and polypyrimidine tracts in plant viroids and in RNA of the human hepatitis delta agent. Nucleic Acids Res. 21, 3529-3535. Buzayan, J.M., Gerlach, W.L., and Bruening, G. (1986a). Non-enzymatic cleavage and ligation of RNAs with sequences that are complementary to a plant virus satellite RNA. Nature 323, 349-353. Buzayan, J.M., Hampel, A., and Bruening, G. (1986b). Nucleotide sequence and newly formed phosphodiester bond of spontaneously ligated satellite tobacco ringspot virus RNA. Nucl. Acids. Res. 14, 9729-9743. Chowrira, B.M., Berzal-Herranz, A., and Burke, J.M. (1991). Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature 354, 320-322.

82

Collins, R.F., Gellantly, D.L., Sehgal, O.P., and Abounhaida, M.G. (1998). Self-cleaving circular RNA associated with rice yellow mottle virus is the smallest viroid-like RNA. Virology 241, 269-275. Côté, F., and Perreault, J.P. (1997). Peach latent mosaic viroid is locked by a 2',5'-phosphodiester bond produced by in vitro self-ligation. J. Mol. Biol. 273, 533-543. Côté, F., Lévesque, D., and Perreault, J.P. (2001). Natural 2',5'-phosphodiester bonds found at the ligation sites of peach latent mosaic viroid. J. Virol. 75, 19-25. Couture, L.A., and Stinchcomb, D.T. (1996). Anti-gene therapy: the use of ribozymes to inhibit gene function. Trends Genet. 12, 510-515. Dall, D.J., Graddon D.J., Randles J.W., and Francki R.I. (1990). Isolation of subterranean clover mottle virus-like satellite RNA from lucerne infected with lucerne transient streak virus. J. Gen. Virol. 71, 1873-1875. Daròs, J.A., and Flores, R. (1995). Identification of a retroviroid-like element from plants. Proc. Natl. Acad. Sci. USA 92, 6856-6860. Daròs, J.A., and Flores, R. (2002). A chloroplast protein binds a viroid RNA in vivo and facilitates its hammerhead-mediated self-cleavage. EMBO J. 21, 749-759. Davies, C., Haseloff, J., and Symons, R.H. (1990). Structure, self-cleavage, and replication of two viroid-like satellite RNAs (virusoids) of subterranean clovermottle virus. Virology 177, 216-224. Davies, D.L., and Clark, M.F. (1983). A satellite-like nucleic acid of arabis mosaic virus associated with hop nettlehead disease. Ann. Appl. Biol. 103, 439-448. Deyoung, M., Siwkowski, A.M., Lian Y., and Hampel, A. (1995). Catalytic properties of hairpin ribozymes derived from chicory yellow mottle virus and arabis mosaic virus satellite RNAs. Biochemistry 34, 15785-157891. Di Serio, F., Daròs, J.A., Ragozzino, A., and Flores, R. (1997). A 451nucleotide circular RNA from cherry with hammerhead ribozymes in its strands of both polarities. J. Virol. 71, 6603-6610. Di Serio, F., Flores, R., and Ragozzino, A. (1996). Cherry chlorotic rusty spot: description of a new virus-like disease from cherry and studies on its etiologic agent. Plant. Dis. 80, 1203-1206. Diener, T.O. (1989). Circular RNAs: relicts of precellular evolution. Proc. Natl. Acad. Sci. USA 86, 9370-9374. Diener, T.O. (2001).The viroid: biological oddity or evolutionary. Adv. Virus Res. 57, 137-184. Elena, S.F., Dopazo, J., Flores, R., Diener, T.O., and Moya, A. (1991). Phylogeny of viroids, viroidlike satellite RNAs, and the viroidlike domain of hepatitis d virus RNA. Proc. Natl. Acad. Sci. USA 88, 5631-5634. Elena, S.F., Dopazo, J., de la Peña, M., Flores, R., Diener, T.O., and Moya, A. (2001). Phylogenetic analysis of viroid and viroid-like satellite RNAs from plants: a reassessment. J. Mol. Evol. 53, 155-159. Epstein, L.M., and Gall, J. (1987). Self-cleaving transcripts of satellite DNA from the newt. Cell 48, 535-543. Etscheid, M., Tousingant, M.E., and Kaper, J.M. (1995). Small satellite of arabis mosaic virus: autolytic processing of in vitro transcripts of (+) and (-) polarity and infectivity of (+) strand transcripts. J. Gen. Virol. 76, 271-282. Fedor, M.J. (2000). Structure and function of the hairpin ribozyme. J. Mol. Biol. 297, 269-291. Feldstein, P.A., Buzayan, J.M., and Bruening, G. (1989). Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA. Gene 82, 53-61.


Fels, A., Hu, K., and Riesner, D. (2001). Transcription of potato spindle tuber viroid by RNA polymerase II starts predominantly at two specific sites. Nucleic Acids Res. 29, 4589-4597. Ferbeyre, G., Smith, J.M., and Cedergren, R. (1998). Schistosome satellite DNA encodes active hammerhead ribozymes. Mol. Cell Biol. 18, 3880-3888. Flores, R., Daròs, J.A., and Hernández, C. (2000). Avsunviroidae family: viroids containing hammerhead ribozymes. Adv. Virus Res. 55, 271-323. Flores, R., Hernández, C., de la Peña, M., Vera, A., and Daròs J.A. (2001). Hammerhead ribozyme structure and function in plant RNA replication. Methods Enzymol. 341, 540-552. Forster, A.C., and Symons, R.H. (1987). Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell 49, 211-220. Francki, R.I.B. (1987). Encapsidated viroidlike RNA. Pages 205-218 in: The viroids. T.O. Diener, ed. Plenum: New York. Francki, R.I.B., Grivell, C.J., and Gibbs, K.S. (1986). Isolation of velvet tobacco mottle virus capable of replication with and without a viroid-like RNA. Virology 148, 381-384. Gerlach, W.L., Llewellyn, D.Y., and Haseloff, J. (1987). Construction of a plant disease resistance gene for the satellite RNA of tobacco ringspot virus. Nature 328, 802-805. Hampel, A., and Tritz, R. (1989). RNA catalytic properties of the minimum (-) sTRSV sequence. Biochemistry 28, 4929-4933. Harris, R.J., and Elder, D. (2000). Ribozyme relationships: the hammerhead, hepatitis delta, and hairpin ribozymes have a common origin. J. Mol. Evol. 51, 182-184. Haseloff, J., and Gerlach, W.L. (1988). Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 334, 585-591. Hernández, C., and Flores, R. (1992). Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures. Proc. Natl. Acad. Sci. USA 89, 3711-3715. Hernández, C., Daròs, J.A., Elena, S.F., Moya, A., and Flores R. (1992). The strands of both polarities of a small circular RNA from carnation self-cleave in vitro through double- and single-hammerhead structures. Nucleic Acids Res. 20, 6323-6329. Hutchins, C.J., Keese, P., Visvader, J.E., Rathjen, P.D., McInnes, J.L., and Symons, R.H. (1985). Comparison of plus and minus forms of viroid and virusoids. Plant Mol. Biol. 4, 292-304. Hutchins, C.J., Rathjen, P.D., Forster, A.C., and Symons, R.H. (1986). Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 14, 3627-3640. Jenkins, G.M., Woelk, C.H., Rambaut, A., and Holmes, E.C. (2000). Testing the extent of sequence similarity among viroids, satellite RNAs, and hepatitis delta virus. J. Mol. Evol. 50, 98-102. Jones, A.T., Mayo, M.A., and Duncan, G.H. (1983). Satellite-like properties of small circular RNA molecules in particles of lucerne transient streak virus. J. Gen. Virol. 64, 1167-1173. Jones, A.T., and Mayo, M.A. (1983). Interaction of lucerne transient streak virus and the viroid-like RNA-2 of Solanum nodiflorum mottle virus. J. Gen. Virol. 64, 1771-1774. Jones, A.T., and Mayo, M.A. (1984). Satellite nature of the viroid-like RNA-2 of Solanum nodiflorum mottle virus and the ability of other plant viruses to support the replication of viroid-like RNA molecules. J. Gen. Virol. 65, 1713-1721.

Joseph, S., Berzal-Herranz, A., Chowrira, B.M., Butcher, S.E., and Burke, J.M. (1993). Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. 7, 130-138. Kaper, J.M., and Collmer, C.W. (1988). Modulation of viral plant diseases by secondary RNA agents. Pages 171-194 in: RNA genetics. Variability of RNA genomes. E. Domingo, J.J. Holland, and P. Alquist, eds. CRC Press: Boca Raton, FL. Kaper, J.M., Tousignant, M.E., and Steger, G. (1988). Nucleotide sequence predicts circularity and self-cleavage of 300-ribonucleotide satellite of arabis mosaic virus. Biochem. Biophys. Res. Commun. 154, 318-325. Keese, P., and Symons, R.H. (1987). The structure of viroids and virusoids. Pages 1-47 in: Viroid and viroid-like pathogens. J.S. Semancik, ed. CRC Press: Boca Raton, FL. Keese, P., Bruening, G., and Symons, R.H. (1983). Comparative sequence and structure of circular RNAs from two isolates of lucerne transient streak virus. FEBS lett. 159, 185-190. Klostermeier, D., and Millar, D.P.( 2001). Tertiary structure stability of the hairpin ribozyme in its natural and minimal forms: different energetic contributions from a ribose zipper motif. Biochemistry 40, 11211-11218. Lafontaine, D., Beaudry, D., Marquis, P., and Perreault, J.P. (1995). Intraand intermolecular nonenzymatic ligations occur within transcripts derived from the peach latent mosaic viroid. Virology 212, 705-709. Lai, M.M. (1995). The molecular biology of hepatitis delta virus. Annu. Rev. Biochem. 64, 259-286. Mayo, M.A., Taliansky, M.E., and Fritsch, C. (1999). Large satellite RNA: molecular parasitism or molecular symbiosis. Curr. Top. Microbiol. Immunol. 239, 65-79. McKay, D.B. (1996). Structure and function of the hammerhead ribozyme: an unfinished story. RNA 2, 395-403. Miller, W.A., Hercus, T., Waterhouse, P.M., and Gerlach, W.L. (1991). A satellite RNA of barley yellow dwarf virus contains a novel hammerhead structure in the self-cleavage domain. Virology 183, 711-720. Navarro, B., and Flores, R. (1997). Chrysanthemum chlorotic mottle viroid: unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes. Proc. Natl. Acad. Sci. USA 94, 11262-11267. Navarro, J.A., and Flores, R. (2000). Characterization of the initiation sites of both polarity strands of a viroid RNA reveals a motif conserved in sequence and structure. EMBO J. 19, 2662-2670. Paliwal, Y.C. (1984). Interaction of the viroid-like RNA-2 of lucerne transient streak virus with southern bean mosaic virus. Can. J. Plant Pathol. 6, 93-97. Piazzolla, P., and Rubino, L. (1984). Evidence that the low molecular weight RNA associated with chicory yellow mottle virus is a satellite. J. Phytopathol. 111, 199-202. Piazzolla, P., Vovlas, C., and Rubino, L. (1986). Symptom regulation induced by chicory yellow mottle virus satellite-like RNA. J. Phytopathol. 115, 124-129. Prody, G.A., Bakos, J.T., Buzayan, J.M., Schneider, I.R., and Bruening, G. (1986). Autolytic processing of dimeric plant virus satellite RNA. Science 231, 1577-1580. Rubino, L., Tousignant, M.E., Steger, G., and Kaper, J.M. (1990). Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus. J. Gen. Virol. 71, 1897-1903.

83

L. Rubino et al.

Rupert, P.B., and Ferre-D’Amare, A.R. (2001). Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis. Nature 410, 780-786. Sehgal, O.P., Sinha, R.C., Gellatly, D.L., Ivanov, I., and AbouHaidar, M.G. (1993). Replication and encapsidation of the viroid-like satellite RNA of lucerne transient streak virus are supported in divergent hosts by cocksfoot mottle virus and turnip rosette virus. J. Gen. Virol. 74, 785-788. Sheldon, C.C., and Symons, R.H. (1993). Is hammerhead self-cleavage involved in the replication of a virusoid in vivo? Virology 194, 463-474. Shippy, R., Lockner, R., Farnsworth, M., and Hampel, A. (1999). The hairpin ribozyme. Discovery, mechanism, and development for gene therapy. Mol. Biotechnol. 12, 117-129. Symons, R.H., and Randles, J.W. (1999). Encapsidated circular viroidlike satellite RNAs (virusoids) of plants. Curr. Top. Microbiol. Immunol. 239, 81-105.

84

Symons, RH. (1997). Plant pathogenic RNAs and RNA catalysis. Nucleic Acids Res. 25, 2683-2689. Taliansky, M.E., and Palukaitis, P.F. (1999). Satellite RNAs and satellite viruses. Pages 1607-1615 in: Encyclopedia of virology, 2nd Edition. B.G. Webster, and A. Granoff, eds. Academic Press: San Diego, CA. Taylor, JM. (1999). Hepatitis delta virus. Intervirology 42, 173-178. Uhlenbeck, O.C. (1987). A small catalytic oligoribonucleotide. Nature 328, 596-600. Vera, A., Daròs, J.A., Flores, R., and Hernández, C. (2000). The DNA of a plant retroviroid-like element is fused to different sites in the genome of a plant pararetrovirus and shows multiple forms with sequence deletions. J. Virol. 74, 10390-10400. White, K.A. (1996). Formation and evolution of Tombusvirus defective interfering RNAs. Sem. Virol. 7, 409-416.

............................................................................................................................................................................................................................................................................................

PART III

DETECTION OF VIROIDS

...........................................................................................................................................................................................................................................................................


PART III

CHAPTER 10

HOST CONSIDERATIONS ....................................................................................................

J.S. Semancik

.................................................................................................................................................................................................................................................................

VIROID-INDUCED DISEASE EXPRESSIONS The interaction between viroids and specific plant species has been critical to not only the origin and recognition of this unusual group of plant pathogens but also to the fundamental understanding of nucleic acid biosynthesis (Symons 1997; Flores et al. 1997) and structure (Riesner 1991). Without the stimulus of a definitive disease as noted for potato spindle tuber and citrus exocortis, the detection of the unusual nature of these causal agents (Diener 1971; Semancik and Weathers 1972) would certainly have been delayed or perhaps not even addressed. This is supported by the frequency in the host range of symptomless carriers of several viroids where the viroid is transmissible but latent. Reactions registered by primary hosts sustaining viroid infection can range from very severe resulting in death of extensive plantings, as in the case of cadang-cadang disease of coconut palms (Bigornia 1977), to a symptomless condition as with the five viroids found in sweet orange (Duran-Vila et al. 1988) as well as four in grapevine (Rezaian 1990; Semancik and Szychowski 1992). In addition, the host range for a viroid can be highly restricted to a single or few experimental species as in the case of Coconut cadang-cadang viroid (CCCVd) and Avocado sunblotch

viroid (ASBVd) or broadly distributed across a diverse collection of herbaceous and woody host species as with Hop stunt viroid (HSVd) which include hop, cucumber, grapevine, citrus, plum, peach, pear, apricot, almond and pomegranate (Shikata 1990; Hadidi et al. 1992; Astruc et al. 1996).

BIOASSAY AND INDEXING Secondary hosts are frequently more useful than the primary host for indexing or the production of higher titers of viroid for molecular detection and characterization. However, genetic variation and host selection are active processes in the dominance of any viroid variant in a population. A subtle difference of a few nucleotides as exists between the causal agents of citrus cachexia disease (CVd-IIb), and CVd-IIa can mark the distinction between a pathogenic variant and a transmissible yet nonpathogenic (latent) variant (Levy and Hadidi 1993; Reanwarakorn and Semancik 1998; Reanwarakorn and Semancik 1999). The importance of host selection was evidenced with the genome of Citrus bent leaf viroid (CBLVd) (Ashulin et al. 1991), previously detected in citron and named Citrus viroid Ib (CVdIb) (Duran-Vila et al. 1988). The initial nucleotide sequence of CBLVd recovered from avocado was later shown to be distinctly

87

J.S. Semancik

different from a number of variants sequenced from citron (Ben-Shaul et al. 1995). A reduction in severity on citron with the citrus exocortis agent (CEVd) was noted with passage through Gynura aurantiaca and a loss of transmissibility with passage through a hybrid tomato (Lycopersicon esculentum × L. peruvianum) (Semancik et al. 1993). Thus, although the sequence of the viroid genome is accepted as the fundamental characteristic for description, the variation in the population of variants accepted within that genome may display dramatically different biological properties. As a result, confirmation of the biological properties of a viroid in the disease host of origin is critical to any comprehensive description. With but a single case of a local lesion host, that of Scopolia sinensis inoculated with Potato spindle tuber viroid (PSTVd) (Singh 1973) reported, diseases induced by viroids can be characterized virtually exclusively as systemic infections. As a consequence, bioassay and indexing protocols must rely on systemic reactions in many cases requiring prolonged incubations of weeks in many herbaceous hosts to years in woody indicators. Specific growing conditions such as elevated temperatures for plant growth favor viroid replication and infection. In many cases, as indicated by the descriptive names of viroids, ‘stunting’ is the most typical reaction, yet a wide range of foliar, fruit, stem and bark symptoms may also characterize a viroid infection. Specific host responses to different viroids can be found in the chapters reporting descriptions of the different viroids.

BIOAMPLIFICATION Much of the progress in viroid research has been accomplished with a relatively small number of host species because of the ease of culture and purification compared with diseased sources. These include tomato, cucumber, chrysanthemum, and citron. In general, preferred hosts have been selected from the Solanaceae, Cucurbitaceae, and Rutaceae families. This fact can probably be ascribed to the pioneering descriptions and subsequent scope of research activity with PSTVd, HSVd and Citrus exocortis viroid (CEVd). Secondary hosts also provide a vehicle to ‘amplify’ viroids normally found in very low titers in field specimens. Citron has been especially valuable in the bioamplification of the five viroids found in citrus species at levels difficult to detect by any molecular protocol. Attesting to the value and broad application of citron as an amplification host is the diverse collection of viroids recovered from citrus sources representing four genera of viroids. Included are CEVd (Pospiviroid), CBLVd or CVd-I and CVd-III (Apscaviroid), CVd-II (Hostuviroid) and CVdIV(Cocadviroid).

88

REFERENCES Ashulin, L., Lachman, O., Hadas, R., and Bar-Joseph, M. (1991). Nucleotide sequence of a new viroid species, citrus bent leaf viroid (CBLVd) isolated from grapefruit in Israel. Nucleic Acids Res. 19, 4767. Astruc, N., Marcos, J.F., Macquaire, G., Candresse T., and Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. Eur. J. Plant Pathol. 102, 837-846. Ben-Shaul, A., Guang, Y., Mogilner, N., Hadas, R., Mawassi, M., Gafney, R., and Bar-Joseph, M. (1995). Genomic diversity among populations of two citrus viroids from different graft-transmissible dwarfing complexes in Israel. Phytopathology 85, 359-364. Bigornia, A.E. (1977). Evaluation and trends of researches on the coconut cadang-cadang disease. Phil. J. Coconut Studies 2, 5-33. Diener, T.O. (1971). Potato spindle tuber ‘virus’. A replicating, low molecular weight RNA. Virology 45, 411-428. Duran-Vila, N., Roistacher, C.N., Rivera-Bustamante, R., and Semancik, J.S. (1988). A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69, 3069-3080. Flores, R., Di Serio, F., and Hernández, C. (1977). Viroids: the noncoding genomes. Sem. in Virol. 8, 65-73. Hadidi, A., Terai, Y., Powell, C.A., Scott, S.W., Desvignes, J.C., Ibrahim, L.M., and Levy, L. (1992). Enzymatic cDNA amplification of hop stunt viroid variants from naturally infected fruit crops. Acta Hortic. 309, 339-344. Levy, L., and Hadidi, A. (1993). Direct nucleotide sequencing of PCRamplified DNAs of the closely related citrus viroids IIa and IIb (cachexia). Proc. of the XIIth Conference of the International Organization of Citrus Virologists. pp. 180-186. Reanwarakorn, K., and Semancik, J.S. (1998). Regulation of pathogenicity in hop stunt viroid-related group II citrus viroids. J. Gen. Virol. 79, 3163-3171. Reanwarakorn, K., and Semancik, J.S. (1999). Correlation of hop stunt viroid variants to cachexia and xyloporosis diseases of citrus. Phytopathology 89, 568-574. Rezaian, M.A. (1990). Australian grapevine viroid: evidence for extensive recombination between viroids. Nucleic Acids Res. 18, 18131818. Riesner, D. (1991). Viroids: drom thermodynamics to cellular structure and function. Mol. Plant-Microbe Inter. 4, 122-131. Semancik, J.S., and Weathers, L.G. (1972). Exocortis disease: evidence for a new species of ‘infectious’ low molecular weight RNA in Plants. Nature (New Biology) 237, 242-244. Semancik, J.S., and Szychowski, J.A. (1992). Relationships among the viroids derived from grapevines. J. Gen. Virol. 73, 1465-1469. Semancik, J.S., Szychowski, J.A., Rakowski, A.G., and Symons, R.H. (1993). Isolates of citrus exocortis viroid recovered by host and tissue selection. J. Gen. Virol. 74, 2427-2436. Shikata, E. (1990). New viroids from Japan. Sem. Virol. 1, 107-115. Singh, R.P. (1973). Experimental host range of the potato spindle tuber ‘virus’. Amer. Potato J. 50, 111-123. Symons, R.H. (1997). Plant pathogenic RNAs and RNA catalysis. Nucleic Acids Res. 25, 2683-2689.

PART III

CHAPTER 11

BIOLOGICAL INDEXING ....................................................................................................


.................................................................................................................................................................................................................................................................

Indexing is any test that will identify a disease or its causative agent and which will positively and reliably confirm either the presence or absence of a transmissible pathogen (Roistacher 1998). This includes transmission by graft, vector, mechanical or propagative means. Biological indexing involves assay in indicator plants rather than with molecular methods and therefore requires an available indicator species, i.e. a host in which the pathogen consistently produces symptoms and which allows the pathogen to be reliably distinguished from other pathogens. Biological indexing or bioassay was the earliest method of identifying diseases caused by viroids (Raymer and O’Brien 1962) and still represents an important step in detection and identification of viroid infections (Hodgson et al. 1998). It offers the advantage of providing a definitive visual assay of biological activity (i.e. potential for transmission, replication and the production of disease symptoms) but is not always practical or possible. Consequently, molecular methods are sometimes used in conjunction with or instead of bioassay (Huttinga 1996).

phoresis, nucleic acid hybridization and reverse transcriptionpolymerase chain reaction (RT-PCR). These include reliability of symptom recognition, time required for symptom development, variation in symptom severity, sensitivity, environmental effects, effect of multiple pathogens, as well as the scale and expense of testing. Symptom recognition

The first requirement for successful biological indexing is a knowledge of the host range of the viroid in question, the nature of the symptoms produced and the factors affecting their development. Conclusive demonstration of the absence of a pathogen, a most difficult consideration in theory, can be as important as demonstration of its presence, especially when applied in certification or quarantine programs. Knowledge of symptoms produced by various pathogens in shared host species is also required in order to rule out other viroids or recognize the possibility of multiple infection. Symptom variation with viroid and host

FACTORS AFFECTING BIOLOGICAL INDEXING Several factors must be considered in the use of biological indexing in contrast to molecular detection methods, such as electro-

Variations in the symptoms produced by viroids infecting a common host can provide the basis for biological indexing, but the use of more than one indicator species may be required. Citrus viroid (CVd) I–IV and Citrus exocortis viroid (CEVd),

89


have been indexed according to a combination of the production and characteristic symptoms in citron, cucumber, chrysanthemum and Gynura aurantiaca (Duran-Vila et al. 1988). The presence or absence of floral variegation after infection of Nicotiana glutinosa allowed Tomato planta macho viroid (TPMVd) to be distinguished from Potato spindle tuber viroid (PSTVd) or Mexican papita viroid (MPVd) (Martinez-Soriano et al. 1996). However, this method is not sufficient to index all viroids infecting members of the Solanaceae unless at least four indicator species are used (see Chapter 15 ‘Viroids of solanaceous species’). Viroid strain

Symptom severity in certain indicator plants may depend on the dominant strain of the viroid present in the population of an isolate. Although in the field, mild strains of PSTVd predominate (Singh et al. 1970, 1971), they can be diagnosed in potato because of their characteristic symptoms. In contrast, the symptoms of Peach latent mosaic viroid (PLMVd) infection can only be indexed in peach and depend on the isolate: mild or latent strains produce symptoms that are more difficult or impossible to recognize and index than severe strains (Flores et al. 1990). Different strains of a viroid may also differ in their host range, as is the case with Hop stunt viroid (HSVd) (Sano et al. 1984, 1985, 1986, 1988a, b; 1989; Hsu et al. 1994), CEVd (Flores et al. 1985; Semancik and Szychowski 1992; Fagoaga et al. 1995; Fagoaga and Duran-Vila 1996) and other citrus viroids (Gillings et al. 1991; Broadbent and Dephoff 1992; Reanwarakorn and Semancik 1998). The use of several host species in biological indexing can allow discrimination between viroids infecting a common host. Niblett et al. (1980) used differences in the type and severity of symptoms of viroid infection in G. aurantiaca, Lycopersicon esculentum cv. ‘Rutgers’ and Chrysanthemum morifolium to distinguish between six viroids: CEVd, Chrysanthemum stunt viroid (CSVd), mild and severe strains of PSTVd, and symptomatic and asymptomatic strains of Chrysanthemum chlorotic mottle viroid (ChCMVd). However, distinguishing between strains of a viroid which originate from different hosts is more difficult. Biological assay in cucumber does not differentiate between strains of HSVd and a molecular probe is therefore required (Sano et al. 1988b). Host species or cultivar

Field symptoms provide the usual basis for first reports of a disease, except in cases where new, unexpected or symptomless infections are revealed during a systematic disease survey. The appearance of symptoms in the field may allow diagnosis of disease but will not necessarily allow detection of all infected plants.

90

The severity of the symptoms in field infections may depend on the species or cultivar infected. In specific pear cultivars, the symptoms of Apple scar skin viroid (ASSVd) infection may be milder than in apple (Hurtt et al. 1996). However, severe symptoms of ASSVd in pears have also been reported (Osaki et al. 1996; Kyriakopoulou et al. 2001). With CSVd, symptoms vary with the species and cultivar infected. While many species in the family Compositae are susceptible to CSVd infection, only Chrysanthemum and two species of Senecio produce symptoms. For this reason, biological indexing is done using either cv. ‘Mistletoe’ or ‘Blanche’ (Hollings and Stone 1973; Lawson 1987). Whether or not CEVd produces symptoms depends on the use of a susceptible or tolerant rootstock. If symptomless, infected budwood from a CEVd-tolerant rootstock, such as sweet orange or rough lemon, is grafted onto a sensitive rootstock, such as trifoliate orange, then symptoms of CEVd infection will develop, as was the case with grapefruit imported into New South Wales in 1949 (see Broadbent and Dephoff 1992). The symptoms of PSTVd also vary in severity depending on the species, the viroid strain (see Figure 15.1C, Chapter 15 ‘Viroids of solanaceous species’), and cultivar infected (Singh and O’Brien 1970; Singh 1971, 1973). Biological indexing of some infections may require an experimental host. For example, Columnea latent viroid (CLVd) was first isolated from a symptomless ornamental species (Owens et al. 1978) but was later found to produce PSTV-like symptoms in potato and tomato (Hammond et al. 1989). Multiple infection

Symptoms can be altered in the presence of coinfective agents. Infection of potato by PSTVd and Potato virus Y (PVY) produces a synergistic response resulting in severe necrosis not observed with either pathogen alone (Singh and Somerville 1987). Distinctive ‘crinkle’ symptoms develop when chrysanthemum cv. ‘Blanche’ is infected with both CSVd and Chrysanthemum virus B, which is normally masked in this cultivar. This phenomenon improves the reliability of bioassay for CSVd in this cultivar (Hollings and Stone 1973). Infected citrus represents an excellent example of multiple virus and viroid infections in the field. Indexing in Australia revealed plants infected with up to four viroids at a time and required the use of several citrus species and sequential polyacrylamide gel electrophoresis (Broadbent and Dephoff 1992). Infected citrus frequently contain 4–5 viroids and symptoms vary depending on the composition of the viroid population (Duran-Vila et al. 1988; Semancik et al. 1988; Gillings et al. 1991; Moresht et al. 1998). Environmental factors

The course of infection and appearance of symptoms is affected by environmental conditions, including temperature, light

BIOLOGICAL INDEXING

quality and intensity, day length and nutritional status of the plant. Symptoms of Avocado sunblotch viroid (ASBVd) infection develop faster at 28–30°C than at 18–20°C (da Graça and Van Vuuren 1981). The symptoms induced by Apple fruit crinkle viroid are more severe in warm summers (Ito et al. 1993) as typical of most viroid infections. Symptom expression in PSTVdinfected plants is more frequent and more severe with daytime temperatures of 30–39°C than at lower temperatures (Harris and Browning 1980; Kryczynski et al. 1980; Grasmick and Slack 1985), is delayed at low light intensity even under high temperature conditions (Harris and Browning 1980), and may be enhanced with improved light intensity and quality (Grasmick and Slack 1985). Rapid onset of severe symptoms caused by Cucumber pale fruit viroid (CPFVd) occurs only at temperatures of at least 30°C (van Dorst and Peters 1974). Symptoms are also masked under conditions of low light regardless of the temperature (Hollings and Stone 1973). Pigeonpea mosaic mottle viroid (PpMMVd) produces most severe symptoms at temperatures of 35–45°C with long daylight hours (Bhattiprolu 1993).

bark or fruit, and thus will not appear until the plant is mature, i.e. ASSVd (Hurtt et al. 1996). Bioassay for CVd-IIb in ‘Parsons’ Special’ mandarin and for ASBVd in avocado cv. ‘Haas’ or ‘Collins’ can take up to two years (Allen and Firth 1980; da Graça and Van Vuuren 1981). Indexing of PLMVd and citrus viroids in their natural hosts is also very slow. Although assay of CEVd in the herbaceous species G. aurantiaca is faster, it is less sensitive than citron (Horst et al. 1983).

Differences in the effect of temperature on replication of CEVd and Citrus cachexia viroid (CVd-IIb) may affect the results of biological indexing for cachexia in plants with multiple infection. Specifically, CEVd replication in ‘Etrog’ citron is enhanced at high temperature whereas that of CVd-IIb in ‘Parsons’ Special’ mandarin is not (Semancik et al. 1988). Consequently, if multiple infection is a possibility, then the bioassay for CVd-IIb may be more reliable if conducted at the lower temperature where the replication of CVd-IIb remains efficient (Semancik et al. 1988) and interference by other citrus viroids may be reduced (Duran-Vila et al. 1988; Gillings et al. 1991; Bar-Joseph 1993). Similarly, indexing of PSTVd in Scopolia sinensis is most efficient at low temperature where local lesions form, rather than at high temperatures where symptoms become systemic (Singh 1973), and leaf epinasty symptoms in ASSVd-infected apple occur only at low temperatures (Skrzeczkowski et al. 1993).

The route of inoculation can affect symptom development. Cutting or slashing is more effective than rub inoculation for infecting with Hop latent viroid (HLVd) (Adams et al. 1996) Generally, graft-inoculation appears to be the most efficient method. Distinctive symptoms are produced by sap inoculation of chrysanthemum with CSVd, but full symptom expression can take up to six months and transmission is often unreliable. Efficiency is improved and expression time decreased with graftinoculation instead of sap inoculation (Hollings and Stone 1973). Similarly, some clones of Solanum berthaultii are susceptible to PSTVd infection by graft-inoculation, but not sap (mechanical) inoculation (Singh 1985), and cross-protection in PSTVd-infected tomato can be overcome by graft-inoculation, but not pin-point puncture (Singh et al. 1989, 1990).

Pruning to force new growth also enhances symptom expression in plants infected with PSTVd (Whitney and Peterson 1963; Grasmick and Slack 1985), ASBVd (da Graça and Van Vuuren 1981) and PpMMVd (Bhattiprolu 1993). The development of lesions in Scopolia sinensis, a local lesion host for PSTVd, is enhanced by the presence of higher than normal levels of Mn2+ in the soil (Lee and Singh 1972; Singh et al. 1974). As well, the effects of environmental stress are accentuated in viroid-infected plants (Horst et al. 1983). Time for symptom development

Biological indexing may be performed in an experimental host that develops symptoms more rapidly than the natural host. This may be especially true for natural infections in woody plants or those where symptoms are expressed primarily in the

Inoculation method

One advantage of biological indexing is sensitivity, as long as the assay is not conducted near the dilution end-point of the pathogen (Diener and Hadidi 1977). Except in rare cases where a local lesion host is available, most biological indexing for viroids is done in systemic hosts, in which the response cannot be accurately related to titer (Diener and Hadidi 1977). Sensitivity and time required for symptom expression can vary with different hosts (Schlemmer et al. 1985) and with the source or concentration of inoculum (Singh 1984).

CROSS-PROTECTION ASSAY Cross-protection assay is an indirect method of biological indexing suitable for detecting infection by mild or latent strains, and confirming the absence of infection. It was first used by Fernow (1967) to overcome a high rate of false negative results in a bioassay for a mild strain of PSTVd in tomato. The symptoms were so mild in tomato as to be frequently overlooked. In the doubleinoculation or cross-protection assay, the presence of the mild strain was demonstrated by the fact that it would confer temporary resistance to challenge with a severe strain of the same viroid (Fernow 1967). A similar technique has been used to demonstrate the presence of latent strains of PLMVd (Flores et al. 1990) and to confirm negative results in assays for ASBVd infection (Allen and Firth 1980). In a conventional bioassay, a negative result occurs with a healthy sample. In a certification or quarantine program, this

91


may result in a long delay before uninfected material is approved for use. The advantage of the cross-protection assay lies in the fact that the absence of the pathogen in question will be confirmed by a positive result, as no protection against challenge will be conferred. The only delay will occur in cases where the pathogen is present and confers temporary protection against challenge (Allen and Firth 1980).

DISADVANTAGES OF BIOLOGICAL INDEXING Biological indexing has a number of disadvantages. It is generally not practical for large scale testing. In most cases it requires a great deal of greenhouse space and is time consuming and labor-intensive (Huttinga 1996). This would make the cost prohibitive, except in regions where the low cost of labor outweighs a shortage of highly trained personnel or lack of sophisticated facilities and equipment. Otherwise, molecular methods such as electrophoresis, nucleic acid hybridization and RT-PCR may be preferred. The time required for a bioassay is at least several weeks (e.g. PSTVd), and periods of months or years are required for some viroids (i.e. ASBVd, ASSVd, CVd-IIb). Molecular methods are much faster. In Florida, RT-PCR is used to detect asymptomatic ASBVd infection in commercial avocado groves (Schnell et al. 1997). Biological indexing of Coconut cadang-cadang viroid (CCCVd) and Coconut tinangaja viroid (CTiVd) in palms is impractical because no suitable herbaceous host is known, mechanical inoculation can only be achieved by high pressure injection (CCCVd) and symptom expression may not begin for a period of 1–4 years (Hodgson et al. 1998). Sophisticated laboratory facilities are not common in areas where these infections occur. Even though electrophoretic methods are less sensitive than bioassay or dot-blot hybridization (Singh and Crowley 1985), they are less expensive, require less training, and represent the most practical technique to confirm the presence of CTiVd infection in palms showing apparent symptoms (Hodgson et al. 1998). However, in order to detect very low levels of the viroid reliably or to distinguish it from CCCVd, oligonucleotide hybridization or RT-PCR is necessary (Hodgson et al. 1998). In some cases, there is no symptomatic host known. For example, no biological indexing methods exist for Iresine viroid (Spieker 1996), Coleus blumei viroid (CbVd) (Fonseca et al. 1989; Singh et al. 1991), and HLVd (Puchta et al. 1988) and molecular methods provide the only means of detection.

USE OF COMBINED BIOLOGICAL AND MOLECULAR ASSAY

Grapevine yellow speckle viroid (GYSVd) is difficult to index biologically because there are often no symptoms except in very hot weather and no other host exists. Therefore, dot-blot hybridiza-

92

tion represents a good alternative or complement to bioassay (Wah and Symons 1997). Citrus may be infected with CEVd and HSVd. Reliable distinction between CEVd and HSVd by bioassay or molecular methods is possible due to differences in size, sequence and pathogenicity. Molecular hybridization and RT-PCR assays are particularly useful for indexing these viroids (Hadidi et al. 1992; Sano et al. 1988b, Yang et al. 1992). For a while, the only suitable indicator for Pear blister canker viroid (PBCVd) infection is pear, Pyrus communis cv. ‘A20’, in which symptoms can take two years to develop (Flores et al. 1991). More recently, two other pear indicators, ‘Fieud 37’ and ‘Fieud 110’, have been developed in which symptoms only take three months to appear (Desvignes et al. 1999). Reliable RTPCR assays for the detection of PBCVd have been developed (A. Hadidi, personal communication). In other cases, the number of hosts required to allow conclusive identification of a viroid, as distinct from related viroids, may be impractical. Thus, a determination of symptomatic and asymptomatic hosts can be made, improving the usefulness and scope of biological indexing. This has been used for viroids infecting members of the Solanaceae (see Chapter 15 ‘Viroids of solanaceous species’; Hammond et al. 1989; Martinez-Soriano et al. 1996).

PRACTICAL APPLICATIONS OF BIOLOGICAL INDEXING

Biological indexing can provide an accurate indication of the pathogens present in a region in the course of diagnostic survey, allow the detection of new pathogens and is an important component of programs to improve plant health and crop yields (Roistacher 1998). Specific applications occur in certification of propagative materials which sometimes require bioassay. For example, regulations in New Brunswick (Canada) used to require biological testing during the certification process for seed potatoes (Singh 1988) but molecular methods now provide an acceptable alternative. European and Mediterranean Plant Protection Organization (EPPO) certification of ornamental species, such as chrysanthemum, and fruit tree propagative material requires viroid testing but this can be done by bioassay or molecular probe, which shortens the time involved (Huttinga 1996; Krczal 1998). Other applications include quarantine and importation of plant materials, eradication of pathogens from plant tissue culture programs and germplasm collections, disease survey, and widespread disease eradication. Even as rapid and sensitive molecular techniques become more widely available and less expensive,

BIOLOGICAL INDEXING

indexing infectious agents in host species remains the only conclusive demonstration of biological activity. References Adams, A.N., Barbara, D.J., Morton, A., and Darby, P. (1996). The experimental transmission of hop latent viroid and its elimination by low temperature treatment and meristem culture. Ann. Appl. Biol. 128, 37-44. Allen, R.N., and Firth, D.J. (1980). Sensitivity of transmission tests for avocado sunblotch viroid and other pathogens. Aust. Plant Pathol. 9, 2-3. Bar-Joseph, M. (1993). The use of viroids for dwarfing citrus trees. 9th International Congress of Virology Abstracts. p. 107. Bhattiprolu, S.L. (1993). Occurrence of mosaic mottle, a viroid disease of pigeonpea (Cajanus cajan) in India. J. Phytopathol. 137, 55-60. Broadbent, P., and Dephoff, C.M. (1992). Virus indexing in the New South Wales citrus improvement scheme. Aust. J. Exp. Agric. 32, 493-502. da Graça, J.V., and Van Vuuren, S.P. (1981). Use of high temperature to increase the rate of avocado sunblotch symptom development in indicator seedlings. Plant Dis. 65, 46-47. Desvignes, J.C., Cornaggia, D., Grasseau, N., Ambrós, S., and Flores, R. (1999). Pear blister canker viroid: studies on host range and improved bioassay with two new pear indicators, Fieud 37 and Fieud 110. Plant Dis. 83, 5419-5422. Diener, T.O., and Hadidi, A. (1977). Viroids. Pages 285-337 in: Comprehensive virology. H. Fraenckel-Conrat, and R. Wagner, eds. Plenum Press: New York. Duran-Vila, N., Roistacher, C.N., Rivera-Bustamante, R., and Semancik, J.S. (1988). A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69, 3069-3080. Fagoaga, C., and Duran-Vila, N. (1996). Naturally occurring variants of citrus exocortis viroid in vegetable crops. Plant Pathol. 45, 45-53. Fagoaga, C., Semancik, J.S., and Duran-Vila, N. (1995). A citrus exocortis viroid variant from broad bean (Vicia faba L.) infectivity and pathogenesis. J. Gen. Virol. 76, 2271-2277. Fernow, K.H. (1967). Tomato as a test plant for detecting mild strains of potato spindle tuber virus. Phytopathology 57, 1347-1352. Flores, R., Duran-Vila, N., Pallás, V., and Semancik, J.S. (1985). Detection of viroid and viroid-like RNAs from grapevine. J. Gen. Virol. 66, 2095-2102. Flores, R., Hernández, C., Desvignes, J.C., and Llácer, G. (1990). Some properties of the viroid inducing peach latent mosaic disease. Res. Virol. 141, 109-118. Flores, R., Hernández, C., Llácer, G., and Desvignes, J.C. (1991). Identification of a new viroid as the putative causal agent of pear blister canker disease. J. Gen. Virol. 72, 1199-1204. Fonseca, M.E.N., Boiteux, L.S., Singh, R.P., and Kitajima, E.W. (1989). A small viroid in Coleus species from Brazil. Fitopatol. Bras. 14, 94-96. Gillings, M.R., Broadbent, P., and Gollnow, B.I. (1991). Viroids in Australian citrus: relationship to exocortis, cachexia and citrus dwarfing. Aust. J. Plant Physiol. 18, 559-570. Grasmick, M.E., and Slack, S.A. (1985). Symptom expression enhanced and low concentrations of potato spindle tuber viroid amplified in tomato with high light intensity and temperature. Plant Dis. 69, 49-51.

Hadidi, A., Terai, Y., Powell, C.A., Scott, S.W., Desvignes, J.C., Ibrahim, L.M., and Levy, L. (1992). Enzymatic cDNA amplification of hop stunt viroid variants from naturally infected fruit crops. Acta Hortic. 309, 339-344. Hammond, R., Smith, D.R., and Diener, T.O. (1989). Nucleotide sequence and proposed secondary structure of Columnea latent viroid: a natural mosaic of viroid sequences. Nucleic Acids Res. 17, 10083-10094. Harris, P.S., and Browning, I.A. (1980). The effects of temperature and light on the symptom expression and viroid concentration in tomato of a severe strain of potato spindle tuber viroid. Potato Res. 23, 85-93. Hodgson, R.A.J., Wall, G.C., and Randles, J.W. (1998). Specific identification of coconut tinangaja viroid for differential diagnosis of viroids in coconut palm. Phytopathology 88, 774-781. Hollings, M., and Stone, O.M. (1973). Some properties of chrysanthemum stunt, a virus with the characteristics of an uncoated ribonucleic acid. Ann. Appl. Biol. 74, 333-348. Horst, R.K., Kawamoto, S.O., Schumann, G.L., and Dietert, M.F. (1983). Chlorosis in healthy and viroid-infected plants exposed to the steam additive diethyl-l-aminoethanol. Sci. Hortic. 19, 1-8. Hsu, Y.-H., Chen, W. , and Owens, R.A. (1994). Nucleotide sequence of a hop stunt viroid variant isolated from citrus growing in Taiwan. Virus Genes 9, 193-195. Hurtt, S.S., Podleckis, E.V., and Howell, W.E. (1996). Integrated molecular and biological assays for rapid detection of apple scar skin viroid in pear. Plant Dis. 80, 458-462. Huttinga, H. (1996). Sensitivity of indexing procedures for viruses and viroids. Adv. Bot. Res. 23, 59-71. Ito, T., Kanematsu, S., Koganezawa, H., Tsuchizaki, T., and Yoshida, K. (1993). Detection of viroid associated with apple fruit crinkle disease. Ann. Phytopathol. Soc. Japan 59, 520-527. Krczal, G. (1998). Virus certification in ornamental plants — the European strategy. Pages 277-287 in: Plant virus disease control. A. Hadidi, R.K. Khetarpal, and H. Koganezawa, eds. APS Press: St. Paul, MN. Kryczynski, S., Stawiszynska, A., Kowalska, A., Skrzeczowska, S., Szkutnicka, K., and Bielecka-Pluta, D. (1980). Methods of detecting infection of plants with severe and mild isolates of potato spindle tuber viroid. Ziemniak (The Potato) 1980, 33-61. Krriakopoulou, P.E., Giunchedi, L., and Hadidi, A. (2001). Peach Latent mosaic and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdalifironis: implications on possible origin of these viroids in the Mediterranean region. J. Plant Pathol. 83, 51-62. Lawson, R.H. (1987). Chrysanthemum stunt. Pages 247-259 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Lee, C.R., and Singh, R.P. (1972). Enhancement of diagnostic symptoms of potato spindle tuber virus by manganese. Phytopathology 62, 516-520. Martinez-Soriano, J.P., Galindo-Alonso, J., Maroon, J.M., Yucel, I., Smith, D.R., and Diener, T.O. (1996). Mexican papita viroid: putative ancestor of crop viroids. Proc. Natl. Acad. Sci. USA 93, 9397-9401. Moresht, S., Cohen, S., Assor, Z., and Bar-Joseph, M. (1998). Water relations of citrus exocortis viroid-infected grapefruit trees in the field. J. Exp. Bot. 49, 1421-1430.

93


Niblett, C.L., Dickson, E., Horst, R.K., and Romaine, C.P. (1980). Additional hosts and an efficient purification procedure for four viroids. Phytopathology 70, 610-615. Osaki, H., Kudo, A., and Ohtsu, Y. (1996). Japanese pear fruit dimple disease caused by apple scar skin viroid. Ann. Phytopathol. Soc. Japan 62, 379-385. Owens, R.A., Smith, D.R., and Diener, T.O. (1978). Measurement of viroid sequence homology by hybridization with complementary DNA prepared in vitro. Virology 89, 388-394. Puchta, H., Ramm, K., and Sänger, H.L. (1988). The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops. Nucleic Acids Res. 16, 4197-4216. Raymer, W.B., and O’Brien M.J. (1962). Transmission of potato spindle tuber virus to tomato. Am. Potato J. 39, 401-408. Reanwarakorn, K., and Semancik, J.S. (1998). Regulation of pathogenecity in hop stunt viroid-related group II citrus viroids. J. Gen. Virol. 79, 3163-3171. Roistacher, C.N. (1998). Indexing for viruses in citrus. Pages 301-319 in: Plant virus disease control. A. Hadidi, R.K. Khetarpal, and H. Koganezawa, eds. APS Press: St. Paul, MN. Sano, T., Hataya, T., and Shikata, E. (1988a). Complete nucleotide sequence of a viroid isolated from Etrog citron, a new member of hop stunt viroid group. Nucleic Acids Res. 16, 347. Sano, T., Hataya, T., Terai, Y., and Shikata, E. (1989). Hop stunt viroid strains from dapple fruit disease of plum and peach in Japan. J. Gen. Virol. 70, 1311-1319. Sano, T., Kudo, H., Sugimoto, T., and Shikata, E. (1988b). Synthetic oligonucleotide hybridization probes to diagnose hop stunt viroid strains and citrus exocortis viroid. J. Virol. Methods 19, 109-120. Sano, T., Ohshima, K., Hataya, T., Uyeda, I., Shikata, E., Chou, T.-G., Meshi T., and Okada, Y. (1986). A viroid resembling hop stunt viroid in grapevines from Europe, the United States and Japan. J. Gen. Virol. 67, 1673-1678. Sano, T., Uyeda, I., Shikata, E., Meshi, T., Ohno, T., and Okada, Y. (1985). A viroid-like RNA isolated from grapevine has high sequence homology with hop stunt viroid. J. Gen. Virol. 66, 333-338. Sano, T., Uyeda, I., Shikata, E., Ohno, T., and Okada, Y. (1984). Nucleotide sequence of cucumber pale fruit viroid: homology to hop stunt viroid. Nucleic Acids Res. 12, 3427-3434. Schlemmer, A., Roistacher, C.N., and Semancik, J.S. (1985). A unique, infectious RNA associated with citron showing symptoms typical of citrus exocortis disease. Phytopathology 75, 946-949. Schnell, R.J., Kuh, D.N., Ronning, C.M., and Harkins, D. (1997). Application of RT-PCR for indexing avocado sunblotch viroid. Plant Dis. 81, 1023-1026. Semancik, J.S., Roistacher, C.N., Rivera-Bustamante, R., and Duran-Vila, N. (1988). Citrus cachexia viroid, a new viroid of citrus: relationship to viroids of the exocortis disease complex. J. Gen. Virol. 69, 3059-3068. Semancik, J.S., and Szychowski, J.A. (1992). Relationships among the viroids derived from grapevines. J. Gen. Virol. 73, 1465-1469. Singh, R.P. (1971). A local lesion host for potato spindle tuber virus. Phytopathology 61, 1034-1035.

94

Singh, R.P. (1973). Experimental host range of the potato spindle tuber ‘virus’. Am. Potato J. 50, 111-123. Singh, R.P. (1984). Solanum berthaultii, a sensitive host for indexing potato spindle viroid from dormant tubers. Potato Res. 27, 163-172. Singh, R.P. (1985). Clones of Solanum berthaultii resistant to potato spindle tuber viroid. Phytopathology 65, 1432-1434. Singh, R.P. (1988). Occurrence, diagnosis and eradication of the potato spindle tuber viroid in Canada. Viroids of Plants and their Detection: International Seminar, August, 1986. Warsaw Agricultural University Press, Warsaw, Poland. pp. 37-50. Singh, R.P., Boucher, A., and Singh, A. (1991). High incidence of transmission and occurrence of a viroid in commercial seeds of Coleus in Canada. Plant Dis. 75, 184-187. Singh, R.P., Boucher, A., and Somerville, T.H. (1990). Cross-protection with strains of potato spindle tuber viroid in the potato plant and other Solanaceous hosts. Phytopathology 80, 246-250. Singh, R.P., and Crowley, C.F. (1985). Evaluation of polyacrylamide gel electrophoresis, bioassay and dot-blot methods for the survey of potato spindle tuber viroid. Can. Plant Dis. Surv. 65, 61-63. Singh, R.P., Finnie, R.E., and Bagnall, R.H. (1970). Relative prevalence of mild and severe strains of potato spindle tuber virus in Eastern Canada. Am. Potato J. 47, 289-293. Singh, R.P., Finnie, R.E., and Bagnall, R.H. (1971). Losses due to the potato spindle tuber virus. Am. Potato J. 48, 262-267. Singh, R.P., Khoury, J., Boucher, A., and Somerville, T.H. (1989). Characteristics of cross-protection with potato spindle tuber viroid strains in tomato plants. Can. J. Plant Pathol. 11, 263-267. Singh, R.P., Lee, C.R., and Clark, M.C. (1974). Manganese effect on the local lesion symptom of potato spindle tuber ‘virus’ in Scopolia sinensis. Phytopathology 64, 1015-1018. Singh, R.P., and O’Brien, M.J. (1970). Additional indicator plants for potato spindle tuber virus. Am. Potato J. 47, 367-371. Singh, R.P., and Somerville, T.H. (1987). New disease symptoms observed on field- grown potato plants with potato spindle tuber viroid and potato virus Y infections. Potato Res. 30, 127-132. Skrzeczkowski, L.J., Howell, W.E., and Mink, G.I. (1993). Correlation between leaf epinasty symptoms on two apple cultivars and results of cRNA hybridization for detection of apple scar skin viroid. Plant Dis. 77, 919-921. Spieker, R.L. (1996). The molecular structure of Iresine viroid, a new viroid species from Iresine herbstii (‘beefsteak plant’). J. Gen. Virol. 77, 2631-2635. van Dorst, H.J.M., and Peters, D. (1974). Some biological observations on pale fruit, a viroid-incited disease of cucumber. Neth. J. Plant Pathol. 80, 85-96. Wah, Y.F.W.C., and Symons, R.H. (1997). A high sensitivity RT-PCR assay for the diagnosis of grapevine viroids in field and tissue culture samples. J. Virol. Methods 63, 57-69. Whitney, E.D., and Peterson, L.C. (1963). An improved technique for inducing diagnostic symptoms in tomato infected by potato spindle tuber virus. Phytopathology 53, 893. Yang, X., Hadidi, A., and Garnsey, S.M. (1992). Enzymatic cDNA amplification of citrus exocortis and cachexia viroids from infected citrus hosts. Phytophathology 82, 279-285.

PART III

CHAPTER 12

POLYACRYLAMIDE GEL ELECTROPHORESIS ....................................................................................................

D. Hanold, J.S. Semancik, and R.A. Owens

.................................................................................................................................................................................................................................................................

From the beginning, polyacrylamide gel electrophoresis (PAGE) has played a key role in viroid research. The pioneering studies of Diener (1971) and Semancik and Weathers (1972) used a combination of sucrose density gradient centrifugation and polyacrylamide gel electrophoresis to show that the molecular weight of the Potato spindle tuber viroid (PSTVd) and Citrus exocortis viroid (CEVd) genomes was only ca. 1 × 105 — far smaller than those of conventional RNA viruses. RNA sedimentation is strongly conformation-dependent (i.e. tight or loose, single- or double-stranded); thus, sedimentation rate alone was not sufficient to unambiguously determine genome size. Because the amounts of viroid present were too low for direct detection by staining or UV absorbance, the observations leading to the discovery of PSTVd and CEVd were based on infectivity measurements alone. Determination of the complete nucleotide sequence of PSTVdintermediate strain (Gross et al. 1978) required the isolation and purification of milligram amounts of viroid. Preparative PAGE under denaturing conditions was used to obtain RNA of sufficient purity for direct RNA sequence analysis. Not long thereafter, analytical PAGE protocols (e.g. Schumacher et al. 1986; Singh and Boucher 1987, Hadidi et al. 1990) began to displace the slower, more cumbersome bioassay for routine viroid diag-

nosis. Protocols in which electrophoresis under nondenaturing conditions is followed by electrophoresis under denaturing conditions (i.e. the so-called S(equential)- or R(eturn)-PAGE techniques) are particularly useful (Rivera-Bustamante et al. 1986; Singh and Boucher 1987; Hadidi et al. 1990). Although molecular hybridization and polymerase chain reaction (PCR) techniques (see Chapters 13 ‘Molecular hybridization’ and 14 ‘Polymerase chain reaction’) have largely replaced PAGE for routine detection of well characterized viroids, electrophoresis remains an essential tool for the detection of unknown viroidlike molecular species. In contrast to other techniques, PAGE requires no nucleotide sequence information. This chapter provides only an introduction to the many uses of PAGE in viroid research. Detailed descriptions of actual experimental protocols can be found in several widely used laboratory manuals (e.g. Sambrook et al. 1989). We begin with a brief description of several sample preparation protocols; next, we describe how PAGE is used to establish the viroid nature of a pathogen. Descriptions of two widely used and two specialized PAGE protocols constitute the core of the chapter. PAGE can be adapted to a wide variety of research environments, and we end by describing a ‘mobile laboratory’ that has been developed to bring PAGE into the field for

95

D. Hanold et al.

and water-saturated phenol followed by centrifugation, the resulting aqueous phase is made to 35% ethanol-1x STE buffer (0.1 M NaCl, 0.05 M Tris-HCl, 1 mM EDTA). To this solution is added CF-11 cellulose to which the viroid RNA becomes bound. After washing with 30% ethanol-STE to remove host impurities retained in the cellulose, the bound nucleic acids comprising principally low molecular weight RNAs along with viroids are eluted with STE buffer and concentrated by ethanol precipitation.

Coconut cadang-cadang viroid (CCCVd) diagnosis and epidemiology studies in the Philippines.

SAMPLE PREPARATION PAGE, especially in combination with silver staining, is a highly sensitive and specific detection method. Unfortunately, the high gel strengths required to detect RNA molecules as small as viroids make the method very susceptible to impurities in the nucleic acid extracts to be analyzed, causing either distortion of banding patterns or heavy background staining that may mask the presence of viroid bands. The challenge is to prepare viroid RNAs that are undegraded by various agents, such as RNases, in sufficient concentration to be visible on the gels yet containing a low enough level of impurities so as not to interfere with PAGE analysis.

iii

The protocol of Owens and Diener (1984) uses yet another extraction buffer [0.5 M K2HPO4, 1% bentonite, 0.1% sodium diethyldithiocarbamate, 0.1% ascorbic acid, 2% SDS], homogenization is carried out in the presence of a 1:1 (v/v) mixture of buffer-saturated phenol containing 0.1% 8-hydroxyquinoline and chloroform, and contaminating polysaccharides are removed by extraction with 2-methoxyethanol and precipitation with cetyltrimethylammonium bromide (CTAB).

iv

The protocol of Astruc et al. (1996) uses a nucleic extraction procedure based on non-organic solvents. Leaf tissue — 2 g are homogenized in 10 ml of 0.1 M Tris-HCl pH 8.0, 50 mM EDTA, 0.5 M NaCl and 10 mM MCE. After homogenization, 0.5 ml of 20% SDS are added and the mixture is incubated at 65°C for 20 min. Subsequently, 2.5 ml of 5 M potassium acetate are added and incubation proceeds at 0°C for another 20 min. Samples are centrifuged at 12000 × g for 15 min and the nucleic acids in the supernatant are ethanol precipitated.

No single extraction protocol is applicable to all possible viroid/ host combinations. The protocols briefly described below have been shown to work well with a variety of hosts and provide a good starting point for further optimization: i

Singh (1991) has described a very simple extraction protocol in which each gram of tissue is homogenized with 3 ml of extraction buffer (0.53 M NH4OH, 13 mM EDTA adjusted to pH 7.0 with Tris-HCl, 4 M LiCl, 1% bentonite) and 4 ml of Tris-HCl-saturated phenol containing 0.1% 8-hydroxyquinoline. After centrifugation to separate the aqueous and organic phases, nucleic acids are recovered from the aqueous (i.e. upper) phase by ethanol precipitation and dissolved in 100-200 ul 1x TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) prior to electrophoresis.

ii

For plants with low concentrations of deleterious natural products which might interfere with viroid recovery, shoot tips containing young leaves and stem tissue can also be homogenized in an extraction medium [0.4 M TrisHCl pH 8.9, 1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 4% 2-mercaptoethanol] containing water-saturated phenol (Semancik et al. 1975). Total nucleic acid preparations are then partitioned in 2 M LiCl, and the soluble fraction is concentrated by ethanol precipitation. Viroid RNAs can be further concentrated by chromatography on CF-11 in the presence of ethanol (Semancik 1986). A second protocol is preferred for extraction of tissues of either poor condition because of age or growing conditions or high concentrations of phenolic, mucilaginous, or acidic host elements such as encountered with grapevines (Szychowski et al. 1988). After homogenization in a medium containing equal volumes of 0.5 M sodium sulfite, 1% sodium dodecyl sulfate, 4% 2-mercaptoethanol

96

MOLECULAR CHARACTERIZATION OF SUSPECTED VIROIDS

To establish the viroid nature of a nucleic acid, PAGE is used to carry out the following tests: RNA or DNA Alkali such as 0.3–0.5M KOH degrades RNA, but not DNA. DNase-free RNase specifically digests RNA, leaving DNA intact. Selective degradation of DNA by RNase-free DNase (Sambrook et al. 1989) requires Mg2+, and RNasin (placental ribonuclease inhibitor) is always added to the reaction mix. Single- or double-stranded S1 nuclease degrades only single-stranded (ss) nucleic acids. RNase A degrades only ssRNA in the presence of 0.3M NaCl, while in the absence of salt all RNA is degraded. Acridine orange fluoresces red when bound to ss nucleic acids and green when bound to double-stranded (ds) RNA or DNA (McMaster and Carmichael 1977), and this dye can be used in place of ethidium bromide to stain gels. Double-stranded nucleic acids are much

POLYACRYLAMIDE GEL ELECTROPHORESIS

Figure 12.1 Sequential PAGE analysis. (A) Schematic diagram of the S-PAGE process. The first electrophoresis (3 hrs, 55 mA constant current) is carried out at 4°C in a 150 × 120 × 1.5 mm slab of 5% polyacrylamide containing TAE buffer (12 mM Tris-HCl, 6 mM sodium acetate, 1 mM EDTA adjusted to pH 7.2 with glacial acetic acid). As the xylene cyanol marker dye reaches the middle of the gel, the portion of the gel that contains the viroid-sized RNAs is excised and placed on top of a second 5% denaturing gel containing TAE buffer (pH 6.5) and 8M urea. The second electrophoresis (4–4.5 hrs, 15 mA constant current) is carried out at room temperature using 0.25x TBE (22.5 mM Tris-HCl, 22.5 mM boric acid, 0.5 mM EDTA, pH 8.3) as the running buffer. Shadowed areas indicate the positions of host RNAs in unpurified samples. (B) S-PAGE analysis of nucleic acid extracts from citron infected with different citrus viroid field sources. Lane 1, the severe isolate E117 containing CEVd and CVd-Ia, -IIa, and –IIId. Lane 3, a mild field source containing CVd-IIa, -IIIa, and -IV. Lane 5, CVd-IIa, -IIIa, and -IIIc. Lane 6, isolate E804 containing CVd-IIIa and -IV. Lane 2, samples as in lanes 1 and 3 mixed before electrophoresis; lane 4, samples as in lanes 1 and 5 mixed before electrophoresis; lane 7, samples as lanes 1 and 6 mixed before electrophoresis. Arrows indicate the positions of CVd-IIIa and -IIIb (lane 2) and CVd-IIIa and -IIIc, (lane 4). [Reprinted from Duran-Vila et al. (1988) with permission.]

more rigid than the corresponding ss molecules; thus, by plotting relative electrophoretic mobility versus polyacrylamide concentration, it is possible to differentiate ss from ds nucleic acids (Riesner et al. 1982 and references cited therein; Gast and Sänger 1994). Due to their strong secondary structure, viroids behave anomalously in such analyses; i.e. they migrate like ss molecules in non-denaturing gels containing ≤10% acrylamide and like ds molecules in gels containing acrylamide concentrations greater than 10% (Semancik 1979). Circular or linear Two-dimensional non-denaturing/denaturing PAGE provides a powerful means to detect small circular RNA molecules because they migrate more slowly than the linear RNAs forming the diagonal (Schumacher et al. 1983). The S(equential)PAGE

method described by Rivera-Bustamante et al. (1986) utilizes a discontinuous pH system between the gel and running buffer to enhance the separation between circular and linear molecules, and bidirectional electrophoresis (Feldstein et al. 1997) can also be used. If purified preparations are available, the presence of circular molecules can be confirmed by electron microscopy of nucleic acid spreads under native and denaturing conditions (Randles and Hatta 1979). Molecular weight Electrophoresis in polyacrylamide gels under denaturing conditions is the most convenient method to estimate the size of a new viroid. In view of their circularity and extensive secondary structure, comparison to well-characterized markers (preferably including a range of known viroids) is essential.

97

D. Hanold et al.

an excellent introduction to the principles of nucleic acid electrophoresis, especially the analysis of single-stranded RNA and DNA molecules under denaturing conditions. Their small size (i.e. 246–399 nucleotides) make viroids ideal candidates for analysis in polyacrylamide gels, and many different protocols involving both denaturing or non-denaturing conditions have been reported. Resolution of specific molecules can be optimized by adjusting the gel concentration, the acrylamide:N,N’methylene-bis-acrylamide (i.e. crosslinking) ratio, or the running conditions. Below, four different protocols for viroid purification and analysis are described. Protocols developed by Rivera-Bustamante et al. (1986) and Singh and Boucher (1987) are widely used for viroid detection. Both involve a two-dimensional analysis in which an initial fractionation under nondenaturing conditions is followed by electrophoresis under denaturing conditions. Depending on the sensitivity required, the viroid RNA band(s) can be visualized by staining with either toluidine blue, ethidum bromide, or silver. Electrophoresis of glyoxal-denatured RNAs in agarose gels has only rarely been used in viroid research (e.g. for the characterization of replicative intermediates; Hutchins et al. 1985) and will not be described further. This section ends with brief descriptions of temperature gradient gel electrophoresis (TGGE, Riesner et al. 1989) and two-dimensional denaturing PAGE (Feldstein et al. 1997), two specialized types of denaturing PAGE specially suited to viroid analysis.

Figure 12.2 Return PAGE analysis. (A) Schematic diagram of the RPAGE process. The first electrophoresis is carried out from top to bottom under native conditions (i.e. 20°C, 1 × TBE buffer). When the xylene cyanol marker dye nears the bottom of the gel, the direction of electrophoresis is reversed and buffer conditions changed to those favoring RNA denaturation (i.e. ≥60°C, 0.11 × TBE buffer). Relative positions of circular (C) and linear (L) viroid molecules are indicated. [Modified from Singh (1991).] (B) Separation of mild (M) and severe (S) strains of Potato spindle tuber viroid. Only their circular forms are visible. [Modified from Singh and Boucher (1987).]

Secondary and tertiary structure Denaturing gradient electrophoresis, using either temperature or chemicals (urea and/or formamide) as the denaturant, is the method of choice to reveal the characteristic highly cooperative melting profile of viroids (Riesner 1987; see Figure 12.3).

METHODS FOR ELECTROPHORESIS AND RECOVERY OF VIROID RNAS The electrophoretic mobility of a macromolecule is determined by its charge, size, and shape. Sambrook et al. (1989) provides

98

S(equential) PAGE This method, developed by Semancik and colleagues (RiveraBustamante et al. 1986), has been extensively used to characterize the members of viroid complexes that affect citrus and grapes (e.g. Duran-Vila et al. 1986, 1988). Figure 12.1 contains a schematic diagram illustrating the S-PAGE process together with a gel photograph demonstrating the resolution achievable. R(eturn) PAGE This method, originally developed by Schumacher et al. (1983, 1986) and later modified by Singh and Boucher (1987), eliminates the need to transfer the viroid from an urea-free to a ureacontaining gel; instead, denaturation is achieved by simultaneously raising the temperature and lowering the ionic strength. Figure 12.2 presents a schematic illustration of the entire process. Temperature gradient gel electrophoresis Separation of the small amount of viroid RNA present in infected tissues from the much larger amounts of contaminating host RNAs by either S- or R-PAGE depends on the relatively low mobility of circular molecules under denaturing conditions. If the denaturation process is induced by a temperature gradient established perpendicular to the direction of electrophoresis,


ualization by hybridization or antibody-based techniques, respectively. Two-dimensional denaturing PAGE Most two-dimensional PAGE systems for RNA analysis are based on the effect of increasing the concentration of either acrylamide or urea between the first and second dimension (e.g. Schumacher et al. 1983). The system developed by Feldstein et al. (1997), in contrast, relies upon an increase in the degree of crosslinking rather than acrylamide concentration to preferentially retard migration of circular molecules. Because the buffer conditions are identical (i.e. 1 × TBE – 7M urea), both dimensions can be cast before the sample is applied. The gel is simply rotated 90° between the first and second dimensions.

Figure 12.3 Temperature gradient gel electrophoresis. The sample to be analyzed is loaded into a slot extending across the width over which the gradient will be formed (step 1) and then run a short distance into the gel slab under nondenaturing conditions (step 2). The current is then turned off and the thermostatically controlled circulating waterbaths are connected to establish a linear temperature gradient (step 3). Electrophoresis is resumed after the gradient has come to equilibrium, and fractionation continues until the desired degree of separation between denatured and nondenatured molecules is attained (step 4). [Figure provided by P.A. Feldstein.]

conversion of the rod-like structure to an open circular conformation is visualized as a continuous S-shaped transition (Riesner et al. 1989). Temperature gradient gel electrophoresis (TGGE) is carried out in a horizontal polyacrylamide gel resting on an electrically insulated metal plate that is heated at one edge and cooled at the other edge. The entire process is schematically illustrated in Figure 12.3. TGGE is a versatile technique that can be used to analyze many different phenomena; e.g. sequence variation and conformational transitions in RNA and DNA molecules as well as protein-nucleic acid interactions. The latter may be relevant to studies of viroid replication. After electrophoresis, samples containing purified nucleic acids or proteins are visualized by staining with either silver or ethidium bromide. A major advantage of TGGE is the ability to carry out high resolution studies using unpurified cell extracts. In such cases, nucleic acids or proteins are transferred to a nitrocellulose or nylon membrane before vis-

Figure 12.4 illustrates the ability of this PAGE system to resolve a complex mixture of circular and linear Coconut cadang-cadang viroid (CCCVd) RNAs. Note, in particular, that the use of denaturing conditions in both dimensions allows certain linear RNAs to be identified as products of intragel cleavage of their respective circular forms. The resulting cross-peak phenomenon allows identification of sets of linear and circular RNAs and can provide a considerable analytical advantage over other gel systems where the first dimension is non-denaturing and the second is denaturing. Visualization and recovery of viroid RNAs Following electrophoresis, viroids can be visualized by staining with either 0.5 mg/ml ethidium bromide in electrophoresis buffer (Sambrook et al. 1989), toluidine blue (0.05% in water; Palukaitis and Symons 1980), or silver nitrate (e.g. Sammons 1981; Igloi 1983). Three factors govern the choice of staining protocol. First, the amount of viroid RNA present (i.e. silver staining is the most sensitive, toluidine blue the least); second, the amount of time required (i.e. ethidium bromide staining can be completed in as little as 10–15 minutes with no destaining); and third, the intended use of the RNA being analyzed. For diagnostic assays involving R-PAGE and other applications where maximum sensitivity is important, silver staining is the method of choice. Protocols described by Sammons et al. (1981) and Igloi (1983) are widely used. Purification of microgram amounts of viroid RNAs by preparative electrophoresis, in contrast, usually involves staining with ethidium bromide followed by excision of the appropriate band(s). Ethidum bromide can be removed from nucleic acids after staining, but silver staining is irreversible — an important consideration when RNA recovery from the gel or confirmation of biological activity is the objective. Various suppliers (e.g. Molecular Probes — Eugene, OR) offer a series of intercalating dyes including SYBR Gold and SYBR Green II that are approximately 25-fold more sensitive than ethidium bromide. Other dyes such as SYBR

99

D. Hanold et al.

Green I preferentially stain dsDNA rather than ssDNA or ssRNA. Viroids can be recovered after preparative electrophoresis using the ‘crush and soak’ protocol described by Sambrook et al. (1989). Gel slices containing viroid RNA are either crushed or chopped into small pieces and then incubated overnight in an appropriate volume of 500 mM NH4 acetate – 0.1% SDS at room temperature. The viroid RNA is then recovered from the eluate by ethanol precipitation (Puchta et al. 1990). Alternatively, viroid RNA can be recovered from ethidium bromidestained bands by electroelution (Semancik et al. 1988).

FIELD APPLICATION OF PAGE Since 1988, a ‘mobile laboratory’ for CCCVd field surveys has been successfully used for the routine detection of CCCVd by staff of the Philippine Coconut Authority (PCA). This unit was developed by J.W. Randles in collaboration with the Albay Research Centre with funding from the United Nations Food and Agricultural Organisation (FAO) and the Australian Centre for International Agricultural Research (ACIAR). It consists of a custom built container (ca. 1.80 × 1.80 × 3m) mounted on the back of a commercial 4WD truck. Power is supplied by a portable diesel generator, and the truck is usually accompanied on field trips by a smaller vehicle to carry personnel.

Figure 12.4 Analysis of circular RNAs by two-dimensional PAGE under denaturing conditions. (A) Preparation of the gel and schematic analysis of six linear and six circular RNAs. (B) Fractionation of the complex mixture of monomeric and dimeric viroid RNAs present in CCCVd-infected palms. Unlabeled CCCVd RNAs were fractionated by two-dimensional PAGE and transferred to a positively-charged nylon membrane before hybridization with a 32P-labeled RNA probe complementary to CCCVd. In addition to circular and linear forms of the monomeric fast (CCCVdF, 246–247 nt) and slow (CCCVdS, 296–297 nt) forms of CCCVd RNA, three dimeric RNAs could also be resolved; i.e. fast–fast (CCCVDFF), fast–slow (CCCVdFS), and slow–slow (CCCVdSS) molecules. [Reprinted from Feldstein et al. (1997) with permission.]

100

In the mobile laboratory, a short extraction protocol followed by 20% non-denaturing PAGE and silver staining is used to monitor epidemiology plots and disease boundaries. Equipment (i.e. balance, unrefrigerated table-top centrifuge, electrophoresis equipment, fume hood, and refrigerated chemical storage) has been both minimized and miniaturized. The small-scale extraction protocol is suitable for use at temperatures approaching 40°C, and adjustments of the crosslinking ratio and running conditions permit high resolution of viroid bands after only 6 hr of electrophoresis (Randles et al. 1992). With several workers performing the extractions, the capacity of this facility is 80 samples in ca. 4 hr followed by the PAGE run and staining procedure (Rodriguez et al. 1998). It is independent of unreliable local power supplies and has even been used at the main laboratory in the PCA Albay Research Centre during typhoon-related power failures. Successful operation of a mobile facility requires a wellestablished, reliable method of analysis and an experienced and capable staff, because trouble shooting under field conditions is often very difficult. This may lead to the mission turning into only a sample collection trip. Furthermore, transport of a number of chemicals — some hazardous and requiring appropriate handling under field conditions or in road emergencies — is necessary. Nevertheless, the mobile laboratory has proven extremely successful under the local conditions, because it solves a range of problems. It eliminates the risk of sample spoilage


during transport at ambient tropical temperatures or storage in the main laboratory, therefore avoiding expensive and timeconsuming re-sampling. Use of fresh tissue within hours of sampling provides better reproducibility than transport and storage under uncontrolled conditions. The availability of on-the-spot results allows optimization of sampling and survey patterns, and re-sampling of individual trees in case of ambiguity is easy. Because the mobile laboratory returns from field trips with silver-stained preserved gels ready for storage and information ready for analysis, resources of the main laboratory are freed up for basic research.

CONCLUSIONS Of all the molecular techniques currently used in viroid research, only PAGE requires no nucleotide sequence information. Using one or more of the techniques described above, it is possible to rapidly prove/disprove the suspected viroid nature of an RNA molecule and purify it to homogeneity based solely on the unusual physical properties of these small, circular RNAs. The viroid nature of the unknown RNA, however, has to be confirmed by infectivity studies in order to eliminate the satellite nature of the RNA. When the target is known, sequencebased techniques such as molecular hybridization and RT-PCR analysis may be advantageous, especially if sensitivity is critical or impurities cannot be removed from the extracts sufficiently to allow silver staining. Versatile and easy-to-use, PAGE analysis remains an indispensable technique in viroid research. References Astruc, N., Marcos, J.F., Macquaire, G., Candresse, T., and Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. Eur. J. Plant Pathol. 102, 837-846. Diener, T.O. (1971). Potato spindle tuber ‘virus’ IV. A replicating, low molecular weight RNA. Virology 45, 411-428. Duran-Vila, N., Flores, R., and Semancik, J.S. (1986). Characterization of viroid-like RNAs associated with the citrus exocortis syndrome. Virology 150, 75-84. Duran-Vila, N., Roistacher, C.N., Rivera-Bustamante, R., and Semancik, J.S. (1988). A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69, 3069-3080. Feldstein, P.A., Levy, L., Randles, J.W., and Owens, R.A. (1997). Synthesis and two-dimensional electrophoretic analysis of mixed populations of circular and linear RNAs. Nucleic Acids Res. 25, 4850-4854. Gast, F-U., and Sänger, H.L. (1994). Gel dependence of electrophoretic mobilities of double-stranded and viroid RNA and estimation of the contour length of a viroid by gel electrophoresis. Electrophoresis 15, 1493-1498. Gross, H.J., Domdey, H., Lossow, C., Jank, P., Raba, M., Alberty, H., and Sänger, H.L. (1978). Nucleotide sequence and secondary structure of potato spindle tuber viroid. Nature 273, 203-208. Hadidi, A., Huang, C., Hammond, R.W., and Hashimoto, J. (1990). Homology of the agent associated with dapple apple disease to

apple scar skin viroid and molecular detection of these viroids. Pathology 80, 263-268. Hutchins, C.J., Keese, P., Visvader, J.E., Rathjen, P.D., McInnes, J.L., and Symons, R.H. (1985). Comparison of multimeric plus and minus forms of viroids and virusoids. Plant Mol. Biol. 4, 293-304. Igloi, G.L. (1983). A silver stain for the detection of nanogram amounts of tRNA following two-dimensional electrophoresis. Analytical Biochem. 134, 184-188. McMaster, G., and Carmichael, G. (1977). Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74, 4835-4838. Owens, R.A., and Diener, T.O. (1984). Spot hybridization for detection of viroids and viruses. Pages 173-187 in: Methods in virology Vol. VII. K. Maramorosch, and H. Koprowski, eds. Academic Press: New York. Palukaitis, P., and Symon, R.H. (1980). Purification and characterisation of the circular and llinear forms of chrysanthemum stunt viroid. J. Gen. Virol. 46, 477-489. Puchta, H., Luckinger, R., Yang, X., Hadidi, A., and Sänger, H.L. (1990). Nucleotide sequence and secondary structure of apple scar skin viroid (ASSVd) from China. Plant Molec. Biol. 14, 1065-1067. Randles, J.W., and Hatta, T. (1979). Circularity of the ribonucleic acids associated with cadang-cadang disease. Virology 96, 47-53. Randles, J.W., Hanold, D., Pacumbaba, E.P., and Rodriguez, M.J.B. (1992). Cadang-cadang disease of coconut palm. Pages 277-295 in: Plant diseases of international importance. Vol. IV. A.N. Mukhopadhyay, J. Kumar, H.S. Chaube, and U.S. Singh, eds. Prentice Hall: Englewood Cliffs, NJ. Riesner, D. (1987). Physical-chemical properties. Pages 63-98 in: The viroids. T.O. Diener, ed. Plenum Press: New York, NY. Riesner, D., Kaper, J.M., and Randles, J.W. (1982). Stiffness of viroids and viroid-like RNA in solution. Nucleic Acids Res. 10, 5587-5598. Riesner, D., Steger, G., Zimmat, R., Owens, R.A., Wagenhöfer, M., Hillen, W., Vollbach, S., and Henco, K. (1989). Temperature-gradient gel electrophoresis of nucleic acids: Analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions. Electrophoresis 10, 377-389. Rivera-Bustamante, R., Gin, R., and Semancik, J.S. (1986). Enhanced resolution of circular and linear molecular forms of viroid and viroid-like RNA by electrophoresis in a discontinuous-pH system. Anal. Biochem. 156, 91-95. Rodriguez, M.J.B., Namia, M.T.I., and Estioko, L.P. (1998). The mobile diagnostic laboratory. Pages 47-52 in: Report on ACIAR-funded research on viroids and viruses of coconut palm and other tropical monocotyledons 1985-1993. ACIAR Monograph No. 45. D. Hanold, and J.W. Randles, eds. Australian Centre for International Agricultural Research: Canberra. http://www.aciar.gov.au/publications/monographs/45/index.htm] Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning: a laboratory manual. 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor: NY. Sammons, D.W., Adams, L.P., and Nishazawa, E.E. (1981). Ultrasensitive silver-based colour staining of polypeptides in polyacrylamide gels. Electrophoresis 2, 135-141. Schumacher, J., Randles, J.W., and Riesner, D. (1983). A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids. Anal. Biochem. 135, 228-295.

101

D. Hanold et al.

Schumacher, J., Meyer, N., Riesner, D., and Weideman, H.L. (1986). Diagnostic procedure for detection of viroids and viruses with circular RNAs by ‘return’-gel electrophoresis. J. Phytopathol. 115, 332-343. Semancik, J. S. (1979). Small pathogenic RNA in plants — the viroids. Ann. Rev. Phytopathol. 17, 461-484. Semancik, J.S. (1986). Separation of viroid RNAs by cellulose chromatography indicating conformational distinction. Virology 155, 39-45. Semancik, J.S., and Weathers, L.G. (1972). Exocortis disease: evidence for a new species of ‘infectious’ low molecular weight RNA in plants. Nature New Biology 237, 242-244. Semancik, J.S., Morris, T.J., Weathers, L.G., Rodorf, B.F., and Kearns, D.R. (1975). Physical properties of a minimal infectious RNA (viroid) associated with the exocortis disease. Virology 63, 160-167.

102

Semancik, J.S., Roistacher, C.N., and Duran-Vila, N. (1988). A new viroid is the causal agent of the citrus cachexia disease. Proc. Int. Org. Citrus Virol. 10, 125-135. Singh, R.P. (1991). Return-polyacrylamide gel electrophoresis for the detection of viroids. Pages 89-107 in: Viroids and satellites: molecular parasites at the frontier of life. K. Maramorosch, ed. CRC Press: Boca Raton, FL. Singh, R.P., and Boucher, A. (1987). Electrophoretic separation of a severe from mild strains of potato spindle tuber viroid. Phytopathology 77, 1588-1591. Szychowski, J.A., Goheen, A.C., and Semancik, J.S. (1988). Mechanical transmission and rootstock reservoirs as factors in the widespread distribution of viroids in grapevines. Am. J. Enol. Viticul. 39, 213-216.

PART III

CHAPTER 13

MOLECULAR HYBRIDIZATION ....................................................................................................

H-P. Mühlbach, U. Weber, G. Gómez, V. Pallás, N. Duran-Vila, and A. Hadidi

.................................................................................................................................................................................................................................................................

Viroids are the smallest pathological entities which are composed exclusively of RNA, and therefore, detection procedures rely either on bioassays or on the direct detection of the RNA. Bioassays have been largely used as a detection tool even before the viroid etiology of some diseases was demonstrated. In spite of their general sensitivity, the utilization of bioassays for screening large numbers of samples is time consuming, expensive, and indicator plants, especially for temperate fruit tree viroids, may not show symptoms or show weak symptoms in 3–5 years. Likewise, gel electrophoresis techniques, based on the distinct mobility of small circular viroid RNAs, have also been used for detection purposes but they are not suitable when numerous samples need to be analyzed. The extraordinary progress made on nucleic acid research during the last few decades and the application of recombinant DNA technology to plant virology have expanded the use of diagnostic methods based on the genomic component of viruses and viroids. Among them, molecular hybridization and polymerase chain reaction (PCR) have received great interest lately and have been incorporated into the diagnostic field of Plant Virology. The potential of molecular hybridization for detection purposes was first demonstrated for Potato spindle tuber viroid (PSTVd) (Owens and Diener 1981). In addition,

most of the progress on viroid research, would not have been possible without the availability of molecular hybridization technologies that allow the identification of the nucleic acids that act as templates during viroid replication, ribozyme activities, etc. In this chapter the presently established filter membrane hybridization procedures in viroid detection will be briefly summarized, including dot blot, RNA gel blot (Northern blot) and tissue print hybridization. Application of in situ hybridization in viroid research will also be presented The basic principles of molecular hybridization are beyond the scope of this chapter and will only be briefly discussed here. A detailed analysis of the physicochemical and biochemical aspects of hybridization of nucleic acids on solid supports is given in Meinkoth and Wahl (1984).

BASIC PRINCIPLES Molecular hybridization is based on the specific interaction between complementary purine and pyrimidine bases forming A–U and G–C base pairs, which results in a stable hybrid formed by part (or the totality) of the nucleic acid sequence of the pathogen to be detected (the target molecule) and the

103

H-P. Mühlbach et al.

labeled complementary sequence (the probe). The stability of the hybrid molecules depends on the number of hydrogen bonds formed and on both electrostatic and hydrophobic interactions. Electrostatic interactions rely on the phosphate residues of the nucleic acid backbone whereas hydrophobic interactions are maintained between the staggered bases. The molecular hybridization approaches that have been developed are based on the same principles but they differ in: i

the way the target molecule is applied and immobilized onto a solid support;

ii

the molecule selected as a probe (cDNA or cRNA);

iii

the type of probe labeling (radioactive or chemical); and

iv

the strategy to detect the viroid-probe hybrids.

When a reliable and sensitive technique for viroid-specific hybridization is to be set up, a test system has to be used whose biological properties are well known and allow rapid assessment of the infection status of the test plants. In studies on PSTVd detection tomato plants proved to be very well suited, since PSTVd-infected tomato plants provide the advantage of visible disease symptoms in the leaves within about a couple of weeks post inoculation (p.i.). Similarly, Gynura aurantiaca and tomato for Citrus exocortis viroid (CEVd) and cucumber for Hop stunt viroid (HSVd), have also been used as test systems. With the exception of HSVd, pome and stone fruit viroids do not have herbaceous hosts as indicators. Several aspects affecting the different steps of the molecular hybridization technique (which include the synthesis of the labeled probe, sample preparation, hybridization and detection), are discussed below. Detailed protocols for all these steps can be found in previous reviews (Hull 1993; Pallás et al. 1998).

PROBE SYNTHESIS The choice of nucleic acid to be used as a probe is mainly dependent on the availability and source of material for labeling. When the sequence of the viroid to be detected is unknown, synthesis of radiolabeled cDNA by random priming is the method of choice. The advent of recombinant DNA technologies has offered different alternatives for the preparation of probes. In routine protocols either synthetic oligodeoxyribonucleotides, cloned or PCR-generated double stranded (ds)DNA fragments, or single stranded (ss)RNA (riboprobes) prepared by phage polymerase directed in vitro transcription of appropriate cDNA clones are used as hybridization probes. In early studies, radioisotopes (i.e. 32P, 35S, and 125I) were used for labeling probes and visualizion was by autoradiography (Owens and Diener 1981; Hadidi et al. 1981, 1982; Branch et al. 1981; Rohde and Sänger 1981; Spiesmacher et al. 1983;

104

Baker et al. 1985; Faustmann et al. 1986; Hadidi 1988). The use of radioactive precursors has been the only choice of labeling for many years. However, the availability of non-radioactive precursors to label nucleic acids has made molecular hybridization more accessible. Non-isotopic probes were developed to overcome the obvious disadvantages of the radioactive labeled probes such as the difficulty in handling, which requires stringent radiosafety precautions, and instability for long-term storage. In our hands, non-radioactive RNA probes, diluted in hybridization buffer containing formamide and stored at –20°C, remained stable for up to two years depending on the frequency of application. Similarly PCR-generated non-radioactive DNA probes can be readily stored at –20°C without losing their properties. Among non-isotopic probes, those labeled with biotin and digoxigenin (DIG) are the most widely used. The biotin-labeled nucleic acids are bound with great efficiency by avidin or its microbial analogue, streptavidin, because of the exceptionally high affinity constant of the avidin-biotin complex. The first non-isotopic probes used for routine diagnosis of viroids were developed by biotinylation with photobiotin of plasmid vectors containing full-length monomeric viroid inserts. Photobiotin-labeled DNA probes were developed for diagnosis of Avocado sunblotch viroid (ASBVd), Coconut cadangcadang viroid (CCCVd), Chrysanthemum stunt viroid (CSVd), and PSTVd (McInnes et al. 1989). The sensitivity obtained was similar to that provided by radioactive probes. The main disadvantage of this system is that when sap extracts are used, the endogenous biotin may cause false positives. The presence of glycoproteins that bind avidin or biotin-binding proteins can also give unacceptable backgrounds. The chemiluminescent detection of viroids by the hapten digoxigenin (DIG)-labeled dot blot and tissue blot hybridization was first introduced by Podleckis et al. (1993). It was used for diagnosis of PSTVd and Apple scar skin viroid (ASSVd). The sensitivity of DIG-labeled or radioactive-labeled cRNA probe was very similar (Podleckis et al. 1993, 1994). DIG which is bound via a spacer arm of eleven carbon atoms to uridine and incorporated enzymatically into nucleic acids by standard methods, has become increasingly common. DNA probes

Single-stranded DNA probes complementary to the viroid sequence can be synthesized by the random priming method even when the sequence of the viroid to be detected is unknown. Therefore, this is the choice for detection of uncharacterized viroids. The synthesis of viroid cDNAs is performed by a reverse transcription reaction using oligonucleotides of random sequence to prime the reaction and a highly purified viroid preparation as the template. With the addition of the four dNTPs one of which is α-32P-labeled (specific activity 3000 Ci/ mol) or non-radioactively labeled (with DIG-UTP or biotin-

MOLECULAR HYBRIDIZATION

11-UTP) in the reaction mixture, the cDNA obtained becomes labeled. Since the random primers can theoretically bind to various sites of the viroid molecule, the cDNAs obtained will be of different sizes but all with good probing activity provided that the viroid RNA used as template was devoid of other contaminant RNAs. However, as proposed by Francis et al. (1995), the compact conformation of viroids seem to favor the synthesis of cDNAs more homogenous than expected. When recombinant plasmids containing viroid sequences are available, DNA probes can be easily synthesized following various approaches. dsDNA probes can be readily obtained by nick translation a process based on the ability of DNA polymerase I to incorporate nucleotide residues to the 3’-hydroxyl terminus of nicks in dsDNA generated by low concentrations of DNase I with the concomitant sequential removal of nucleotides from the adjacent 5’-phosphoryl terminus. By adding one of the four dNTPs as α-32P-labeled, unlabeled nucleotides of each DNA strand are replaced by labeled ones. These probes are easily synthesized but being double stranded, they must be denatured prior to their incorporation into the hybridization solution. dsDNA probes were used by Owens and Diener (1981) to detect PSTVd in clarified plant sap at a level of 83 to 250 pg. More recently, PCR-generated dsDNA fragments are obtained from viroid cDNA cloned into appropriate vectors and labeled during PCR, either with radioisotopes, biotin, or digoxigenin, following established protocols. A simple PCR reaction using as a template a recombinant plasmid containing the viroid sequence and in the presence of the four dNTPs one of which is α-32P-labeled (specific activity 3000 Ci/mol) or nonradioactively labeled (DIG-dUTP) can generate uniformly labeled probes with high specific activity (Palacio et al. 2000 a, b). These probes are easy to handle and when labeled with DIG can be stored for long periods of time. As indicated above, probes generated by PCR are double stranded and therefore are less efficient than cDNA or cRNA probes, and must be conveniently denatured before being added to the hybridization solution. Modifications of the standard PCR reaction by using a single primer have been shown to be a suitable strategy to generate ssDNA probes (Konat et al. 1994) but they have not been tested for viroid detection. cRNA probes (riboprobes)

Since RNA–RNA hybrids are more stable than RNA–DNA hybrids, riboprobes offer the possibility to perform hybridization under more stringent conditions which will help to increase specificity and reduce background. In addition, a non-specifically bound probe can be removed by digestion with RNase A, which is highly specific for ssRNA. Riboprobes, being single stranded, have higher probing efficiencies when compared to nick translated DNA probes or dsDNA probes since there is no competition for the complementary strands. In addition, since

radiolabeled riboprobes can be synthesized at very high specific activity, they are the best choice to detect RNAs that may be present at very low concentrations. Viroid specific riboprobes are generated by in vitro transcription of viroid cDNAs cloned into suitable vectors which contain phage RNA polymerase promoters flanking the multiple cloning site. Cloning vectors containing SP6, T3 and T7 RNA polymerase promoters are commercially available for the production of highly specific ssRNA probes. In order to obtain RNA transcripts of a defined length, it is necessary to linearize the recombinant plasmid at a unique site immediately downstream of the inserted DNA sequence (Melton et al. 1984; Hadidi et al. 1990; Hiruki 1991; Podleckis et al. 1993). Transcription in the presence of the four NTPs, one of which is α32 P-labeled (specific activity 3000 Ci/mol) or non-radioactively labeled (with DIG–UTP or biotin-11-UTP) proceeds from the promoter through the length of the probe sequence. The transcript will be either complementary or homologous to the viroid molecule depending upon the orientation of the insert. Once the reaction is completed, the DNA template and the unincorporated nucleotides can be removed by standard methods. In cases, where oligomeric viroid cDNA is cloned into the transcription vectors, highly specific and strongly labeled probes are produced. The high sensitivity of oligomeric riboprobes was demonstrated by Singh et al. (1994): 0.48 pg of PSTVd were detectable and in end point dilution experiments PSTVd was still detected in extracts from PSTVd-infected plants at 16,384fold dilution. In other experiments, PSTVd-specific RNA was synthesized by transcription with SP6 RNA polymerase from the vector pSP62–PL, with six tandem repeats of PSTVd cDNA (Tabler and Sänger 1985). Such probes were successfully used in RNA gel blot hybridization to detect oligomeric (+) and (-) PSTVd forms in potato protoplasts after liposome mediated infection (Faustmann et al. 1986). In vitro synthesized viroid RNAs are also excellent tools to study several aspects of nature of viroids such as structural requirements for infectivity (Tabler and Sänger 1985), processing (Tsagris et al. 1987), and interaction with host plant proteins (Werner et al. 1995). Non-radioactive riboprobes, and particularly DIG-labeled probes, have become increasingly common for routine detection purposes. To check the integrity and/or the yield of the riboprobe, the electrophoretic mobility in TBE-agarose gels of the transcription products obtained in the presence and absence of the precursor DIG–UTP should be compared. If digoxigenin has been incorporated into the cRNA, the electrophoretic mobility of the transcript should be slower than that of its unlabeled counterpart. Non-radioactive riboprobes have been obtained for detecting PSTVd (Roy et al. 1989; Podleckis et al. 1993), ASSVd (Podleckis et al. 1993; Kyriakopoulou and

105


A

B

C

Figure 13.1 Different applications of the molecular hybridization technique in viroid research. A) Non-isotopic dot-blot hybridization applied to routine diagnosis of HSVd in apricot trees. Samples c5, d1 and d3 were negatives. B) Northern blot analysis of viroid-enriched fractions of almond samples probed against a HSVd riboprobe. Samples 1 and 4 were positives and circular and linear forms of the viroid molecule are visible. C) Application of the non isotopic tissue printing technology to large-scale diagnosis of HSVd in fruits of apricot trees. Samples a9, a10, a11 and d10 were considered to be negatives.

Hadidi 1998; Kyriakopoulou et al. 2001), Pear blister canker viroid (PBCVd) (Kyriakopoulou et al. 2001), Peach latent mosaic viroid (PLMVd) (Ambrós et al. 1995; Badenes and Llácer 1998; Hadidi et al. 1997; Kyriakopoulou et al. 2001), HSVd (Romero-Durbán et al. 1995; Astruc et al. 1996; Amari et al. 2001), CEVd (Romero-Durbán et al. 1995; Palacio et al. 2000a), ASBVd (Romero-Durbán et al. 1995), and citrus viroids (Palacio et al. 2000a). Comparative studies indicate that the biotinylated cDNA and cRNA probes are 15 and 6 times less sensitive than radiolabeled probes, respectively (Candresse et al. 1990). On the other hand, digoxigenin-labeled cRNA probe for PSTVd or ASSVd (Podleckis et al. 1993, 1994) or for HSVd (Astruc et al. 1996), was found to be as sensitive as its equivalent radiolabeled probe. Mutimeric cRNA probes, labeled with digoxigenin have shown

106

a 4-fold higher sensitivity than monomeric probes (Singh et al. 1994). For routine indexing purposes, the method of choice will depend on the simplicity and cost without impairing the reliability of detection. Oligonucleotide probes

Synthetic oligonucleotide probes were reported for the detection of ASBVd (Bar- Joseph et al. 1985), HSVd and CEVd (Sano et al. 1988) and PSTVd (Welnicki et al. 1989). In studies on PSTVd replication, 32P-labeled oligonucleotide probes (14mer and 15-mer) have been successfully used to detect (+) and (-) PSTVd RNAs in nuclei isolated from PSTVd-infected potato cell suspension cultures (Spiesmacher et al. 1983). The probes were designed to bind to regions of low self complementarity in the PSTVd sequence to allow clear discrimination between (+) and (-) PSTVd strands.


For routine diagnostics, a mixture of synthetic non-radioactively labeled oligonucleotides was reported to detect PSTVd in potato cultivars with high sensitivity (Nakahara et al. 1998). With this approach, a well-known disadvantage of single oligonucleotide probes, namely their comparably low sensitivity, could be overcome. The five different oligonucleotides (24-mer to 26-mer) in the mixture were designed to hybridize to different regions in the PSTVd secondary structure model of Gross et al. (1978), thus also avoiding annealing among the probes.

SAMPLE PREPARATION Dot-blot hybridization

Owens and Diener (1981) showed that the membrane filter hybridization of non-fractionated plant sap is a powerful tool to analyze the infection status of plants in viroid investigations as well as in diagnostic programs. Since then, the most common method used for routine detection purposes is the dot-blot hybridization technique (Figure 13.1A), which involves the direct application of a nucleic acid solution to a solid support, such as nitrocellulose or nylon membranes. Moreover, using specifically designed and commercially available equipment (Hybri-Dot, BRL and Minifold II, Schleicher and Schuell), this approach gives a reliable estimation of the concentration of viroid RNA in the sample under study. Due to the defined area at which RNA is loaded onto the membrane, the intensity of the hybridization signal can be easily determined and compared to that of appropriate standards. In tissue print and Northern blot hybridization quantification is more difficult because of the irregular shape of the hybridization signal. There are no universal conditions for preparing samples for dotblot molecular hybridization analysis. These will depend on the viroid to be detected, the host and even the kind of probe and the method used for detecting the viroid–probe hybrids. Clarified sap extracts and partially purified preparations have been successfully used but some adjustments may be needed to avoid high backgrounds (Flores 1986). In some instances, the natural green-brownish color of sap extracts from fruit trees may interfere with the colorimetric detection of DIG-labeled probes, probably due to the reduction of the nitroblue tetrazolium by components of the plant sap, while the light emission is not altered by the presence of these components (Más et al. 1993; Pallás et al. 1998). Most methods generally used for viroid extraction require the use of phenol or other toxic organic solvents, making them undesirable for diagnostic laboratories that process large number of samples. An extraction method that avoids the use of phenolics, previously described for obtaining plant genomic DNA (Dellaporta et al. 1983), has been used to enrich partially purified extracts of viroid-like RNAs (Pallás et al. 1987) and viroids (Astruc et al. 1996; Cañizares et al. 1998).

A simple, rapid, and reliable protocol for the small-scale extraction of RNA based on inactivation of nucleic acids by guanidium thiocyanate and binding properties of silica (SiO2) particles to nucleic acids was described (Boom et al. 1990). This technique was used for the isolation of RNA from virus-infected plant material (Rott and Jelkmann 2001). A modified version of that method using 6M guanidium-HCL instead of 4M guanidium thiocyanate, which is highly toxic, was established in Dr Mühlbach’s laboratory to extract RNA for Northern blot analyses from 20 mg of viroid-infected tomato and potato tissue. Total RNAs may be extracted from viroid-infected tissues by non-organic solvents using commercially available RNA extraction kits such as BIO 101 FastRNA Green Protocol (BIO 101, Carlsbad, CA) or QIAGEN RNeasy plant mini kit (Qiagen Inc., Valencia, CA). Viroid RNA extracted by these methods may be suitable for viroid detection by molecular hybridization and by RT-PCR (A. Hadidi, unpublished). These commercially available kits were successfully used for the isolation of RNA from virus-infected tissue for RT-PCR detection of plum pox and prune dwarf viruses from fruit trees (Youssef et al. 2002). Alkaline denaturation of crude sap extracts from PLMVdinfected tissue was reported to be the best method for routine dot-blot detection of PLMVd because of its simplicity and minimal manipulation (Truturo et al. 1998). Organic solvents were completely avoided in this method. Northern and Southern Hybridization

The well-established procedure of RNA gel blot hybridization known as Northern blot hybridization (Alwine et al. 1977) was applied for studying the presence of viroid RNA forms in plants or plant cells. Northern blot hybridization relies on the electrophoretic separation of the viroid target prior to being transferred to the membrane (Figure 13.1B). After electrophoretic separation of total RNA from plant tissue in polyacrylamide or agarose gels, all RNA is transferred to membrane filters and hybridized with an excess of appropriate probes. Due to the compact secondary structure of viroid RNAs, a crucial step in Northern blot hybridization is their complete denaturation which is usually achieved by treatment with glyoxal (McMaster and Carmichael 1977) or formaldehyde prior to electrophoretic separation. Alternatively, RNA in polyacrylamide gels is denatured by alkaline (Na OH) treatment for a short time just after electrophoresis and before electrophoretic transfer of the RNA onto a membrane (Hadidi et al. 1982, 1990; Hadidi 1988). However in some instances positive hybridization has been successfully accomplished even without a previous denaturation step (Romero-Durban et al. 1995; Francis et al. 1995). This previous electrophoretic separation step introduces a powerful approach to analyse viroid molecules with a high sequence similarity but with different size or conformation. Thus,

107


A

B

C

Figure 13.2 Northern blot (A, B) and dot-blot (C) hybridization to detect PSTVd(+) and (-)RNA synthesis in tomato protoplasts after in vitro inoculation. A and B) Protoplasts from Lycopersicon peruvianum were inoculated with PSTVd and samples collected after 6h (1), 18h (2), 30h (3), 42h (4), 54h (5), 66h (6), 78h (7), 90h (8), 102h (9), 114h (10), 126h (11), and 138h (12). RNA was separated in 5% PAA gels, blotted and hybridized with 32P labeled riboprobes specific for PSTVd-(+)-RNA (A) and for PSTVd-(-)-RNA (B) V: Circular monomeric PSTVd(+)RNA; S: Hinf I fragments of pBR322 DNA as size standard. c) Dot-blot hybridization with dilution series to assess PSTVd(+) and (-)RNA quantities. RNA was extracted from protoplasts immediately after inoculation (0) and 1, 2, and 3 days p.i. and from regenerated cells 70 days p.i.

Northern and/or Southern (DNA gel blot) hybridization is an essential tool for basic research on viroids and it has been especially used to study the intermediates of the replication cycle of these pathogens (e.g. Hadidi et al. 1981, 1982; Hutchins et al. 1985; Daròs et al. 1994). In studies on the replication of PSTVd in tomato plants, Northern blot hybridization allowed to detect the complementary longer-than-unit-length PSTVd(-)RNA strands in the presence of large amounts of PSTVd(+)RNA (Branch et al. 1981; Rohde and Sänger 1981; Owens and Diener 1982). It also allowed the detection of PSTVd longer-than-unitlength (+) strands (Hadidi and Hashimoto 1981) and PSTVdspecific dsRNA (Hadidi et al. 1982).These findings led to the cur-

108

rent well-established concept of viroid replication through a rolling-circle mechanism (Branch and Robertson 1984). The high sensitivity and reliability of strand specific hybridization is also demonstrated by studies on PSTVd replication in isolated protoplasts, where minimum amounts of RNA were available. Using dot blots and Northern blots the time course of synthesis of (+) and (-) PSTVd strands could be analysed in tomato protoplasts even during the early hours following in vitro infection (Mühlbach et al. 1992; see Figure 13.2). In addition, these two techniques have been very useful for phytopathological purposes. For instance, Northern blot analysis


has been applied to distinguish between two different viroids, ASSVd and Apple dimple fruit viroid (ADFVd), with such a high sequence similarity that would impede discrimination by dotblot hybridization (Di Serio et al. 2001). Similarly, this approach was initially used to establish sequence similarities among viroid-like RNAs with distinct electrophoretic mobilities found in citrus (Duran-Vila et al. 1988; Semancik et al. 1988), which were later demonstrated to be variants of the same viroid species. Another interesting application in the diagnosis field is its use to demonstrate the presence of a known viroid in a new host, e.g. PLMVd in apricot and plum (Hadidi et al. 1997) or HSVd in almond (Cañizares et al. 1999). Imprint hybridization

For routine analysis sample manipulation must be reduced to a minimum. This can be easily achieved by using the tissue-printing technique that avoids sample extraction and only requires the direct transfer of the plant material (stem, cutting, leaf, fruit) to a nylon or nitrocellulose membrane (Figure 13.1C). This technique was first described to detect proteins by immunocytolocalization (Cassab and Varner 1987) and was later used for RNA detection (McClure and Guilfoyle 1989). The same approach was then adapted for detection and localization of plant viruses (Mansky et al. 1990; Chia et al. 1992; Más and Pallás 1995) and viroids (e.g. Podleckis et al. 1993; RomeroDurbán et al. 1995; Hooftman et al. 1996; Astruc et al. 1996; Hurtt et al. 1996; Palacio et al. 2000a, b). The imprint hybridization technique can be applied not only for diagnostic purposes with the obvious advantage of reducing the test times (see previous references) but also to study viroid distribution within the infected plant (e.g. Stark-Lorenzen et al. 1997; Amari et al. 2001), as can be seen below.

HYBRIDIZATION AND DETECTION Samples must be fixed to the membrane by baking for 2 h at 80°C, at 120°C for 30 min, or by UV cross-linking (in the last two cases only Nylon membranes positively charged can be used). This last method gives a 5- to 10-fold increase of the hybridization signals over the baking methods. The hybridization process depends on several factors such as the complexity (length and composition of the nucleic acid) and concentration of the probe, temperature, salt concentration, base mismatches and hybridization accelerators. The temperature at which half of the strands are disassociated is the melting temperature (Tm). The stringency of the hybridization conditions and the stability of the viroid–probe hybrid complexes will determine the specificity of hybrid formation. In general higher temperatures and a lower salt concentration increase stringency of hybridization. The presence of formamide in the hybridization solution, besides increasing stringency, favors correct base pairing and decreases background. For detection of viroids and

Figure 13.3 Comparison of chemoluminescent and colorimetric detection of PSTVd on the same membrane. Tissue imprints from tomato plants were hybridized with a Dig-labeled riboprobe. A) Chemoluminescent detection with CSPD as substrate, recorded by exposure to X-ray film. B) Colorimetric detection of the same membrane using X-phosphate/NBT as substrate. The left three rows are from infected plants, followed by two rows from uninfected plants. The spots of RNA standard on the right side correspond to a concentration range from 100 ng (top) to 1 pg (bottom).

viroid-related molecules hybridizations are often carried out at 70–72°C in 50% formamide which provides a good signal/ background ratio. However when using DNA probes lower temperatures (50–55°C) produce the best results. Once the hybridized membranes have been washed to remove the unbound probe, the strategy to detect the viroid–probe hybrids will depend on how the probe had been labeled. Radiolabeled probes are detected by direct exposure of an X-ray film to the membrane. The intensity of the hybridization signal obtained will depend on the exposure time and the specific activity of the probe. DIG labels are detected by an immunological procedure using Fab fragments of an anti-digoxigenin-alkaline phosphatase conjugate that can cleave either chromogenic or chemiluminescent substrates. The chromogenic substrate (5-bromo-4-chloro3-indolyl phosphate, called X-phosphate, together with nitroblue tetrazolium) will finally form an indigo dye precipitate, which is directly visible on the hybridization membrane, while through dephosphorylation of the chemiluminescent substrate (i.e. CSPD: disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2´(5´chloro)tricyclo [3.3.1.13,7] decan-4-yl) phenyl phosphate) light will be emitted that is recorded by exposure on a highly sensitive X-ray film. The chemiluminescence method is preferred when a prolonged storage of the results in the form of X-ray films is desired. Moreover, exposure times as short as 1 min are sufficient to detect PSTVd in Northern blots. The colorimetric

109


Figure 13.4 Tissue print hybridization to analyze the intra-plant distribution of PSTVd(+) and (-)RNA in tomato. a–d) Combination of conventional tissue prints and squeegee blots of leaves. a, b) detection of (+)-PSTVd; c, d) detection of (-)-PSTVd; a and c) infected plant; b and d) uninfected (mock inoculated) plant. The five spots visible in d represent PSTVd(-)RNA transcripts used as hybridization control and indicate a detection limit of 1 pg (arrow). e) Comparison of tissue print images with corresponding tissue sections. TS: Tissue Section; TP: tissue print; Inf: infected plant; Uninf: uninfected (mock inoculated) plant; Rh: rachis; St: stem; Ro: root.

110


son (Amari et al. 2000) or to study the etiology of the apricot ‘viruela’ disease (Cañizares et al. 2001). This technique has also been successfully used for indexing of CSVd in several chrysanthemum cultivars and allowed the identification and eradication of a contaminated source in 1995 (Duran-Vila et al. 1996). There is a limitation for direct detection of citrus viroids from infected plants using tissue imprinting hybridization assays due to seasonal fluctuations of viroid titers below detection levels. This limitation was overcome using citron as an amplification host in which viroids accumulate to levels detectable either by sPAGE or dot-blot hybridization (Duran-Vila et al. 1993; Palacio et al. 2000b). Recently, the analysis of inoculated citrons has been simplified using the tissue imprinting hybridization method and it is the method used for routine indexing in Spain (Palacio et al. 2000a, b). Tissue imprinting hybridization and viroid replication Figure 13.5 Detection of (+)-PSTVd in tomato fruits. Rut: (L.e. cv. Rutgers). GK: (L.e. cv. Goldkugel). Inf: infected plant; Uninf: uninfected (mock inoculated) plant.

detection is applicable with simpler laboratory equipment, but provides comparable sensitivity and equal if not higher resolution. Additionally, the development of hybridization signals can be continuously monitored. Both techniques can be applied consecutively for the same membrane (see Figure 13.3).

APPLICATION OF MOLECULAR HYBRIDIZATION Routine indexing

As stated above, viroids do not code for any protein so serological methods can not be applied for their diagnosis. Thus, only biological (bioassays), biochemical (sequential PAGE) or molecular methods (molecular hybridization, RT-PCR etc.) can be used. The time scale for these three different methods is months, days and hours, respectively and therefore, molecular hybridization, in its non-isotopic version, must be considered as the method of choice for large-scale indexing, especially if a very high level of sensitivity is not needed. Non-isotopic dot-blot hybridization has been applied in field conditions to study the incidence of HSVd and/or PLMVd in several countries of the Mediterranean Region (Cañizares et al. 1998; Badenes and Llácer 1998; Loreti et al. 1998; Amari et al. 2000; Al-Rwahnih et al. 2001; Ismaeil et al. 2001) and in other parts of the world (Hadidi et al. 1997). Tissue-imprinting hybridization, has been applied in field conditions for large-scale indexing for the presence of HSVd in apricot trees (Astruc et al. 1996; Cañizares et al. 2001) or for rapid detection of ASSVd in pear (Hurtt et al. 1996), to monitor the infection of HSVd in apricot trees over a whole growing sea-

As stated above, imprint hybridization has been used to monitor tissue specific accumulation of the (+)-strand viroid as well as its replicative (-)-strand intermediate. The PSTVd molecules accumulate predominantly in the younger parts of the plants and, even in the early stages of the infection, in the root. The blots pictured in Figure 13.4 are combinations of conventional tissue prints obtained from stem, rachis and petiole, together with squeegee blots from leaves. They show a more detailed view of the intra-plant distribution of PSTVd. In addition to the PSTVd-(+)-strand the replicative intermediate, the (-)-strand has been detected. Comparing single prints of the different plant organs with the corresponding tissue sections (Figure 13.4e) revealed the tissue-specific distribution of the pathogen. In rachis or stem cross-sections PSTVd (+)- and (-)-strands were found associated with the vascular tissue. In stem cross-sections (St) a ring-shaped hybridization signal is seen with occasional dark spots located in the pith parenchyma, while in the rachis (Rh) the horseshoe-shaped hybridization signal was predominant with only very faint additional signals that could be assigned to the cortical layer. Tissue prints of the root (Ro) showed the abundance of (+)-and (-)-PSTVd-molecules in this organ. We could detect the pathogen and its replicative intermediates predominantly in the outer parts of the central cylinder, containing endodermis, pericycle and the vascular tissue, leaving out the pith parenchyma. The hybridization of tissue prints of tomato fruits from two cultivars (Rutgers and Goldkugel) showed a homogenous distribution of (+)-PSTVd throughout the fruit pulp in both cultivars (Figure 13.5). In situ hybridization

Harders et al. (1989) used in situ hybridization to demonstrate that PSTVd accumulates in the nuclei of infected cells. Presumably multimeric PSTVd molecules synthesized in the nucleoplasm are transported into the nucleolus to be processed

111


into monomeric molecules, thereby accounting for the high nuclear concentration (Harders et al. 1989). CCCVd also accumulates in the nucleoli of infected cells (Bonfiglioli et al. 1994, 1996). CEVd in contrast is distributed throughout the nucleoplasm (Bonfiglioli et al. 1996). The three above viroids are members of the family Pospiviroidae. In situ hybridization has also revealed the presence of PSTVd in vascular tissues of the stems and roots of infected tomato plants (Hammond 1994; Stark-Lorenzen et al. 1997). CCVd and CEVd are also localized in the vascular tissue of infected plants (Bonfiglioli et al. 1996). Minus-strand, normally considered to be indicator of active viroid replication, has also been detected in the phloem of PSTVd-infected plants (Zhu et al. 2001). Detection of ASBVd in the thylakoid membranes (Bonfiglioli et al. 1994) of avocado leaf chloroplasts (Lima et al. 1994) has been made by in situ hybridization. Localization of intermediates in the replication of ASBVd in chloroplasts suggests a site for in vivo synthesis (Navarro et al. 1999). PLMVd accumulates also in the chloroplasts (Bussière et al. 1999). ASBVd and PLMVd belong to the family Avsunviroidae.

CONCLUSIONS Molecular hybridization has become an important tool in viroid research. It has been shown to be essential not only in basic research but also in applied purposes as in studying the epidemiology of viroid diseases and in controlling viroids by their detection in certification and quarantine programs. It has allowed researchers on one hand to improve their knowledge of the molecular biology of these pathogens and on the other hand to determine their incidence in different crops and areas on a very large scale. The incorporation of non-isotopic precursors to label the corresponding probes has allowed this methodology to extend to non-specialized laboratories. The simplicity and sensitivity of the non-isotopic molecular hybridization is good enough to detect most of the known viroids at levels even below economic thresholds. Since serological methods can not be applied to detect viroids, molecular hybridization technique may be considered among the best options for detecting these pathogens and with PCR technology as the natural way to introduce molecular tools at the commercial level. References Al-Rwahnih, M., Myrta, A., Abou-Ghanem N., Di Terlizzi, B., and Savino, V. (2001). Viruses and viroids of stone fruits in Jordan. EPPO Bull. 31, 95-98. Alwine, J.C., Kemp, D.J., and Stark, G.R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350-5354. Amari, K., Cañizares, M.C., Myrta, A., Sabanadzovic, S., Srhiri, M., Gavriel, L., Caglayan, K., Varveri, C., Gatt, M., Di Terlizzi, B., and Pal-

112

lás, V. (2000). First report on Hop Stunt Viroid (HSVd) from some Mediterranean countries. Phytopathol. Medit. 39, 271-276. Amari, K., Cañizares, M.C., Myrta, A., Sabanadzovic, S., Di Terlizzi, B., and Pallás, V. (2001). Tracking hop stunt viroid (HSVd) infection in apricot trees during a whole year by non-isotopic tissue printing hybridization. Acta Hortic. 550, 315-320. Ambrós, S., Desvignes, J.C., Llácer, G., and Flores, R. (1995). Peach latent mosaic and pear blister canker viroids: detection by molecular hybridization and relationship with specific maladies affecting peach and pear trees. Acta Hortic. 386, 515-521. Astruc, N., Marcos, J.F., Macquaire, G., Candresse, T., and Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. Eur. J. Plant Pathol. 102, 837-846. Badenes, M.L., and Llacer, G. (1998). Ocurrence of peach latent mosaic viroid in American peach and nectarine cultivars. Acta Hortic. 472, 565-570. Barker, J.M., McInnes, J.L., Murphy, P.J., and Symons, R.H. (1985). Dotblot procedure with [32P]DNA probes for the sensitive detection of avocado sunblotch and other viroids in plants. J. Virol. Methods 10, 87-98. Bar-Joseph, M., Segev, D., Twizer, S., and Rosner, A. (1985). Detection of avocado sunblotch viroid by hybridization with synthetic oligonucleotide probes. J. Virol. Methods 10, 69-73. Bonfiglioli, R.G., McFaddan, G.I., and Symons, R.H. (1994). In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus. Plant J. 6, 99-103. Bonfiglioli, R.G., Webb, D.R., and Symons, R.H. (1996). Tissue and intracellular distribution of coconut cadang cadang viroid and cirus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy. Plant J. 9, 457-465. Boom, R., Sol, C.J.A., Salimans, M.M.M., Jansen, C.L., Werthheim van Dillen, M.E., and van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495-503. Branch, A.D., and Robertson, H.D. (1984). A replication cycle for viroids and other small infectious RNAs. Science 223, 450-455. Branch, A.D., Robertson, H.D., and Dickson E.C. (1981). Longer-thanunit-length viroid minus strands are present in RNA from infected plants. Proc. Natl. Acad. Sci. USA 78, 6381-6385. Bussière, F., Lehoux, J., Thompson, D.A., Skrzeczkowski, L.J., and Perreault, J.-P. (1999). Subcellular localization and rolling circle replication of peach latent mosaic viroid: hallmarks of group A viroids. J. Virol. 73, 6357-6360. Candresse, T., Macquaire, G., Brault, V., Monsion, M., and Dunez, J. (1990). 32P and biotin-labelled in vitro transcribed cRNA probes for the detection of potato spindle tuber viroid and chrysanthemum stunt viroid. Res. Virol. 141, 97-107. Cañizares, M.C., Marcos, J.F., and Pallás, V. (1998). Studies on the incidence of hop stunt viroid in apricot trees by using an easy and short extraction method to analyze a large number of plants. Acta Hortic. 472, 581-585. Cañizares, M.C., Marcos, J.F., and Pallás, V. (1999). Molecular characterization of an almond isolate of hop stunt viroid (HSVd) and conditions for eliminating spurious hybridization in its diagnosis in almond samples. Eur. J. Plant Pathol. 105, 553-558.


Cañizares, M.C., Aparicio, F., Amari, K., and Pallás, V. (2001). Studies on the aetiology of Apricot ‘Viruela’ disease. Acta Hortic. 550, 249-258. Cassab, G.I., and Varner, J.E. (1987). Immunocytolocalization of extensin in developing soybean seed coats by immuno gold-silverstaining and by tissue printing on nitrocellulose paper. J. Cell Biol. 105, 2581-2588. Chia, T.F., Chan, Y.S., and Chua, N.H. (1992). Detection and localization of viruses in orchids by tissue-print hybridization. Plant Pathol. 41, 355-361. Daròs, J.E., Marcos, J.F., Hernández, C., and Flores, R. (1994). Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing. Proc. Natl. Acad. Sci. USA 91, 12813-12817. Dellaporta, S., Wood, J., and Hicks, J. (1983). A plant DNA minipreparation: Version II. Plant Mol. Biol. Rep. 1, 19-21. Di Serio, F., Malfitano, M., Alioto, D., Ragozzino, A., Desvignes, J.C., and Flores, R. (2001). Apple dimple fruit viroid: fulfillment of Koch’s postulates and symptom characteristics. Plant Dis. 85, 179-182. Duran-Vila, N., Pina, J.A., and Navarro, L. (1993). Improved indexing of citrus viroids. Pages 202-211 in: Proc. 12th Conf. Int. Organ. of Citrus Virol. P. Moreno, J.V. da Graça, and L.W. Timmer, eds. IOCV: Riverside, CA. Duran-Vila, N., Roistacher, C.N., Rivera-Bustamante, R., and Semancik, J.S. (1988). A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69, 3069-3080. Duran-Vila, N., Romero-Durbán, J., and Hernández, M. (1996). Detection and eradication of chrysathemum stunt in Spain. EPPO Bull. 26, 399-405. Faustmann O., Kern R., Sänger H.L., and Mühlbach H.-P. (1986). Potato spindle tuber viroid (PSTV) RNA oligomers of (+) and (-) polarity are synthesized in potato protoplasts after liposome mediated infection with PSTV. Virus Res. 4, 213-227. Flores, R. (1986). Detection of citrus exocortis viroid in crude extracts by dor blot hybridization: conditions for reducing spurious hybridization results and for enhancing the sensitivity of the technique. J Virol. Methods 13, 309-319. Francis, M.I., Szychowski, J.A., and Semancik, J.S. (1995). Structural sites specific to viroid groups. J. Gen. Virol. 76, 1081-1089. Gross, H.J., Domdey, H., Lossow, C., Jank, P., Raba, M., Alberty, H., and Sänger, H.L. (1978). Nucleotide sequence and secondary structure of potato spindle tuber viroid. Nature 273, 203-208. Hadidi, A. (1988). Synthesis of disease-associated proteins in viroidinfected tomato leaves and binding of viroid to host proteins. Phytopathology 78, 575-578. Hadidi, A., Cress, D.E., and Diener, T.O. (1981). Nuclear DNA from uninfected or potato spindle tuber viroid-infected tomato plants contains no detectable sequences complementary to cloned double-stranded viroid cDNA. Proc. Natl. Acad. Sci. USA 78, 6932-6935. Hadidi, A., Giunchedi, L., Shamloul, A.M., Poggi-Pollini, C., and Amer, M.A. (1997). Occurrence of Peach latent mosaic viroid in stone fruits and its transmission with contaminated blades. Plant Dis. 81, 154-158. Hadidi, A., and Hashimoto, J. (1981). Viroid-specific ribonucleic acid in cells infected with potato spindle tuber viroid. Phytopathology 71, 222. Hadidi, A., Hashimoto, J., and Diener, T.O. (1982). Potato spindle tuber-specific double-stranded RNA in extracts from infected leaves. Ann. de Virol. 133E, 15-31.

Hadidi, A., Huang, C., Hammond, R.W., and Hashimoto, J. (1990). Homology of the agent associated with dapple apple disease to apple scar skin viroid and molecular detection of these viroids. Phytopathology 80, 263-268. Hammond, R.W. (1994). Agrobacterium-mediated inoculation of PSTVd cDNA onto tomato reveals the biological effect of apparently lethal mutations. Virology 201, 36-45. Harders, J., Lukacs, N., Robert-Nicoud, M., Jovin, T.M., and Riesner, D. (1989). Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy. EMBO J. 8, 3941-3949. Hiruki, C. (1991). Use of complementary RNA as diagnostic probes for viroids. Pages 79-88 in: Viroids and satellites: molecular parasites at the frontier of life. K. Maramorosch, ed. CRC Press: Boca Raton, FL. Hooftman, R., Arts, M-J., Shamloul, A.M., Van Zaayen, and Hadidi, A. (1996). Detection of chrysanthemum stunt viroid by reverse transcription-polymerase chain reaction and by tissue blot hybridization. Acta Hortic. 432, 120-128. Hull, R. (1993). Nucleic acid hybridization procedures. Pages 79-88 in: Diagnosis of plant virus diseases. R.E.F. Matthews, ed. CRC Press, Inc.: Boca Raton, FL. Hurtt, S.S., Podlekis, E.V., and Howell, W.E. (1996). Integrated molecular and biological assays for rapid detection of apple scar skin viroid in pear. Plant Dis. 80, 458-462. Hutchins, C.J., Keesse, P., Visvader, J.E., Rathjen, P.D., McInnes, J.L., and Symons, R.H. (1985). Comparison of multimeric plus and minus forms of viroids and virusoids. Plant Mol. Biol. 4, 293-304. Ismaeil, F., Abou Ghanem-Sabanadzovic, N., Myrta, A. Di Terlizzi, B., and Savino, V. (2001). First record of Peach latent mosaic viroid and Hop stunt viroid in Syria. J. Plant Path. 83, 147-148. Konat, G.W., Laszkiewicz, I., Grubinska, B., and Wiggins, R.C. (1994). Generation of labeled DNA probes by PCR. Pages 37-42 in: PCR technology, current innovation. G.H. Griffin, and A.M. Griffin, eds. CRC Press: Boca Raton, FL. Kyriakopoulou, P.E., and Hadidi, A. (1998). Natural infection of wild and cultivated pears with apple scar skin viriod in Greece. Acta Hortic. 472, 617-625. Kyriakopoulou, P.E., Giunchedi, L., and Hadidi, A. (2001). Peach latent mosaic viroid and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdaliformis: implications on possible origin of these viroids in the Mediterranean region. J. Plant Pathol. 83, 51-62. Lima, M.I., Fonseca, M.E.N., Flores, R., and Kitajima, E.W. (1994). Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization. Arch. Virol. 138, 385-390. Loreti, S., Fagiolli, F., Barrale, R., and Barba, M. (1998). Occurrence of viroids in temperate fruit trees in Italy. Acta Hortic. 472, 555-560. Mansky, L.M., Andrew, R.E., Durand, D.P., and Hill, J.H. (1990). Plant virus location in leaf tissue by press blotting. Plant Mol. Biol. Rep. 8, 13-17. Más, P., and Pallás, V. (1995). Non-isotopic tissue-printing hybridization: a new technique to study long-distance plant virus movement. J. Virol. Methods 52, 7-32. Más, P., Sánchez-Navarro, J.A., Sánchez-Pina, M.A., and Pallás, V. (1993). Chemiluminescent and colorigenic detection of cherry leaf roll virus with digoxigenin-labelled RNA probes. J. Virol. Methods 45, 93-102.

113


McClure, B.A., and Guilfoyle, T.J. (1989). Tissue print hybridization. A simple technique for detecting organ- and tissue-specific Mic gene expression. Plant Mol. Biol. 12, 517-524. McInnes, J.L., Habili, N., and Symons, R.H. (1989). Nonradioactive, photobiotin-labelled DNA probes for routine diagnosis of viroids in plant extracts. J. Virol. Methods 23, 299-312. McMaster, G.K., and Carmichael, G.G. (1977). Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74, 4835-4838. Meinkoth, J., and Wahl, G. (1984). Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138, 267-284. Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., and Green, M.R. (1984). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes for plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035-7056. Mühlbach H.-P., Barth A., and Tank C. (1992). Efficient transfer of infectious potato spindle tuber viroid cDNA and ß-glucuronidase gene to tomato protoplasts by polyethyleneglycol. Molecular Biology (Life Science Adv.) 11, 79-90. Nakahara, K., Hataya, T., Hayashi, Y., Sugimoto, T., Kimura, I., and Shikata, E. (1998). A mixture of synthetic oligonucleotide probes labeled with biotin for the sensitive detection of potato spindle tuber viroid. J. Virol. Methods 71, 219-227. Navarro, J.A., Daros, J.A., and Flores, R. (1999). Complexes containing both polarity strands of avocado sunblotch viroid: identification in chloroplasts and characterization. Virology 253, 77-85. Owens, R.A., and Diener, T.O. (1981). Sensitive and rapid diagnosis of potato spindle tuber viroid disease by nucleic acid hybridization. Science 213, 670-672. Owens, R.A., and Diener, T.O. (1982). RNA intermediates in potato spindle tuber viroid replication. Proc. Natl. Acad. Sci. USA 79, 113-117. Palacio, A., Foissac, X., and Duran-Vila, N. (2000a). Indexing of citrus viroids by imprint hybridisation. Eur. J. Plant Pathol. 105, 897-903. Palacio, A., Foissac, X., and Duran-Vila, N. (2000b). Indexing of citrus viroids by imprint hybridization: comparison with other detection methods. Pages 294-301 in: Proc. 14th Conf. Int. Org. Citrus Virol. J.V. da Graça, R.F. Lee, and R.K. Yokomi, eds. IOCV: Riverside, CA. Pallás, V., Más, P., and Sánchez-Navarro, J.A. (1998). Detection of plant RNA viruses by non-isotopic dot-blot hybridization. Pages 461468 in: Plant virus protocols: from virus isolation to transgenic resistance. G. Foster, and S. Taylor, eds. Humana Press, Totowa. Pallás, V., Navarro, A., and Flores, R. (1987). Isolation of a viroid-like RNA from hop different from hop stunt viroid. J. Gen. Virol. 68, 3201-3205. Podleckis, E.V., Hammond, R.W., Hurtt, S.S., and Hadidi, A. (1993). Chemiluminescent detection of potato and pome fruit viroids by digoxigenin-labelled dot blot and tissue blot hybridization. J. Virol. Methods 43, 147-158. Podleckis, E.V., Hammond, R.W., Hurtt, S.S., and Hadidi, A. (1994). A comparison of digoxigenin-labeled and 32P-labeled probes: dot blot detection of plant viroids. B. M. Biochemica 10, 10-12. Rohde, W., and Sänger, H.L. (1981). Detection of complementary RNA intermediates of viroid replication by Northern blot hybridization. Bioscience Reports 1, 327-336.

114

Romero-Durban, J., Cambra, M., and Duran-Vila, N. (1995). A simple imprint hybridization method for detection of viroids. J. Virol. Methods 55, 37-47. Rott, M.E., and Jelkmann, W. (2001). Characterization and detection of several filamentous viruses of cherry: adaptation of an alternative cloning method (DOP-PCR), and modification of an RNA extraction protocol. Eur. J. Plant Pathol. 107, 411-420. Roy, B.P., Abouhaider, M.G., and Alexander, A. (1989). Biotinylated RNA probes for the detection of potato spindle tuber (PSTVd) in plants J. Virol. Methods 23, 149-156. Sano, T., Kudo, H., Sugimoto, T., and Shikata, E. (1988). Synthetic oligonucleotide hybridization probes to diagnose hop stunt viroid strains and citrus exocortis viroid. J. Virol. Methods 19, 109-120. Semancik, J.S., Roistacher, C.N., Rivera-Bustamante, R., and Duran-Vila, N. (1988). Citrus cachexia viroid, a new viroid of citrus: relationship to viroids of the exocortis disease complex. J. Gen.Virol. 69, 3059-3068. Singh, R.P., Boucher, A., Lakshman, D.K., and Tavantzis, S.M. (1994). Multimeric non-radioactive cRNA probes improve detection of potato spindle tuber viroid (PSTVd). J. Virol. Methods 49, 221-233. Spiesmacher, E., Mühlbach, H.-P., Schnölzer, M., Haas, B., and Sänger, H.L. (1983). Oligomeric forms of potato spindle tuber viroid (PSTV) and of its complementary RNA are present in nuclei isolated from viroid-infected potato cells. Bioscience Reports 3, 767-774. Stark-Lorenzen, P., Guitton, M.-C., Werner, R., and Mühlbach, H.-P. (1997). Detection and tissue distribution of potato spindle tuber viroid in infected tomato plants by tissue print hybridization. Arch. Virol. 142, 1289-1296. Tabler, M., and Sänger, H.L. (1985). Infectivity studies on different potato spindle tuber viroid (PSTV) RNAs synthesized in vitro with the SP6 transcription system. EMBO J. 4, 2191-2199. Truturo, C., Minafra, A., Hong, N., Wang, G.P., Di Terlizzi, B., and Savino, V. (1998). Occurrence of peach latent mosaic viroid in China and development of improved detection method. J. Plant Pathol. 80, 165-169. Tsagris M., Tabler M., Mühlbach H.-P., and Sänger H.L. (1987). Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells. EMBO J. 6, 2173-2183. Welnicki, M., Skrzeczkowski, J., Soltynska, A., Jonzyk, P., Markiewicz, W., Kierzek, R., Imiolzyk, B., and Zagorsky, W. (1989). Characterisation of synthetic DNA probes detecting potato spindle tuber viroid. J. Virol. Methods 24, 141-152. Werner, R., Mühlbach, H.-P., and Guitton, M.-C. (1995). Isolation of viroid-RNA-binding proteins from an expression library with non radioactive labelled RNA probes. BioTechniques 19, 218-221. Youssef, S.A., Shalaby, A.A., Mazyad, H.M., and Hadidi, A. (2002). Detection and identification of prune dwarf virus and plum pox virus by standards and multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA). J. Plant Pathol. 84, 113-119. Zhu, Y., Green, L., Woo, Y.-M., Owens, R. , and Ding, B. (2001). Cellular basis of potato spindle tuber viroid systemic movement. Virology 279, 69-77.

PART III

CHAPTER 14

POLYMERASE CHAIN REACTION ....................................................................................................


.................................................................................................................................................................................................................................................................

The roots of the polymerase chain reaction (PCR) began as early as 1955 with Nobel Laureate Arthur Kornberg’s discovery of a cellular enzyme called DNA polymerase. DNA polymerases serve several natural functions, including the repair and replication of DNA. It was not until the winter of 1983–1984, however, that the PCR, utilizing a thermostable DNA polymerase, was developed by Kary Mullis who received the Nobel Prize for Chemistry in 1993 for this discovery. The PCR is best described in the words of US patent number 4,683,202: ‘The process comprises treating separate complementary strands of the (target) nucleic acid with a molar excess of two oligonucleotide primers and extending the primers to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence’. Currently, the PCR process is covered by patents owned by Hoffmann-La Roche, Inc. (Nutley, NJ) and F. Hoffmann-La Roche Ltd (Basel, Switzerland); collectively called Roche. Roche has granted exclusive and nonexclusive licenses for various applications of PCR, and Roche and its licensees provide end-user licenses within their designated fields. For over a decade, especially after the introduction of the automated DNA thermal cyclers in 1989, PCR has been established as one of the most substantial technical advances in molecular

biology. Its current applications are in the areas of disease diagnosis, detection of pathogens, detection of DNA in small samples, DNA comparisons and mutations detection, high efficiency cloning of genomic sequences and numerous other areas. PCR has thus impacted on basic molecular, biological, and clinical research as well as forensics, evolutionary studies, genome projects, and plant pathology.

IMPORTANCE The significance of PCR lies in its ability to amplify in vitro a specific DNA or cDNA sequences from trace amounts in a complex mixture of templates. It is possible to amplify specific DNA or cDNA sequences, from as short as 50 bp to over 10,000 bp in length, more than a million fold in a few hours (106 to 109fold amplifications in 3–4 hours or less), in a reaction that is carried out in an automated DNA thermal cycler. Because of its great sensitivity, the PCR provides a good alternative to other diagnostic methods and can speed up diagnosis, reduce the sample size required, and often eliminates the need for radioactive probes. Another important criterion of PCR is its very high specificity and fidelity, which allows for the discrimination of sequences that differ by as little as single nucleotide mutations.

115


Table 14.1 Family/Genus

Major viroid species for which RT–PCR-based detection systems have been reported. Species

Host

Reference

Avsunviroid

Avocado sunblotch viroid

Avocado

Semancik and Szychowski 1994; Schnell et al. 1997

Pelamoviroid

Peach latent mosaic viroid

Peach

Shamloul et al. 1995; Hadidi et al. 1997

Non-Peach

Giunchedi et al. 1998; Hadidi et al. 1997; Osaki et al. 1999; Kyriakopoulou et al. 2001

Chrysanthemum

De la Peña and Flores, unpublished

Apple

Hadidi and Yang 1990b; Hadidi et al. 1991; Zhu et al. 1995, 1998; Shamloul and Hadidi 1999; Osaki et al. 1996, 1998; Faggioli et al. 2001; Di Serio et al. 2002; Shamloul et al. 2002

Pear

Hadidi and Yang 1990b

Apple dimple fruit viroid

Apple

Di Serio et al. 1998, 2002; Faggioli et al. 2001; Shamloul et al. 2002

Apple fruit crinkle viroid

Apple

Ito et al. 1998

Australian grapevine viroid

Grapevine

Rezaian et al. 1992; Wah and Symons 1997

Grapevine yellow speckle viroid

Grapevine

Rezaian et al. 1992; Wah and Symons 1997

Pear blister canker viroid

Pear, Quince

Hadidi, in Shamloul et al. 2002; Faggioli et al. 2001

Coconut cadang-cadang viroid

Coconut

Hanold and Randles 1991; Hodgson et al. 1998

Coconut tinangaja viroid

Coconut

Hodgson et al. 1998

Hop latent viroid

Hop

Hataya et al. 1992; Cajza et al. 1996; Nakahara et al. 1999; Knabel et al. 1999; Seigner 2000

Hop stunt viroid

Hop

Sano et al. 2001

Citrus

Hadidi et al. 1992; Yang et al. 1992; Francis et al. 1995; Villalobos et al. 1997

Cucumber

Yang et al. 1992

Avsunviroidae

Chrysanthemum chlorotic mottle viroid Pospiviroidae Apscaviroid

Cocadviroid

Hostuviroid

Pospiviroid

Apple scar skin viroid

Potato spindle tuber viroid

Citrus exocortis viroid

Chrysanthemum stunt viroid

Grapevine

Hadidi et al. 1992; Wah and Symons 1997

Plum, Peach

Hadidi et al. 1992; Kusano and Shimomura 1997; Shamloul et al. 2002

Potato

Shamloul et al. 1997

Avocado

Querci et al. 1995

Pepino


Tomato


Citrus

Yang et al. 1992; Francis et al. 1995; Ben-Shaul et al. 1995; Ito and Ieki 1996; Villalobos et al. 1997

Grapevine

Wah and Symons 1997

Broad bean

Fagoaga et al. 1995

Chrysanthemum

Hooftman et al. 1996; Nakahara et al. 1999; Mumford et al. 2000

Argyranthemum

Menzel and Maiss 2000

Following the initial publications demonstrating the feasibility of the adaptation of PCR to the detection of genetic diseases such as sickle cell anemia and human viruses such as hepatitis B and rhinoviruses, Dr Hadidi’s laboratory in the late 1980s promptly attempted to adapt this technique for the detection and identification of viroids, with the first oral presentation of

116

their findings in Berlin during the VIIIth International Congress of Virology (Hadidi and Yang 1990a) and the first paper reporting the details of the findings published in the same year (Hadidi and Yang 1990b). Since then, rapid developments on the detection and characterization of viroids by PCR has strongly influenced diagnostic and quarantine practices, epide-

POLYMERASE CHAIN REACTION

woody or lignified material. Since by definition viroids are circular RNA molecules, using primers that anneal to the central conserved region of the viroid with 3’ ends facing away from each other, full-length cDNA copies of a number of viroids (Figure 14.1) have been successfully amplified in a fashion reminiscent of the inverse PCR technique (see for example Hadidi and Yang 1990b; Yang et al. 1992). In addition to the diagnostic field, PCR amplification of viroids has been used for screening transgenic plants, for the molecular cloning and sequencing of viroids as well as for the studying of host–viroid interactions (Candresse et al. 1998; Hadidi et al. 1995). Moreover, it has been used for the study of viroid infection (Zhu et al. 1998).

THE PCR REACTION

Figure 14.1 RT of full length viroid RNA to cDNA and subsequent amplification of the cDNA by PCR. DNA primers used are designed from the nucleotide sequence of the upper central conserved region (CCR) of the viroid and the adjacent segment.

miology and variability studies, as well as studies of viroid–host interactions. Since the initial report in 1990 of the feasibility of adapting PCR for the detection of viroids, the number of papers reporting such results for viroids as well as plant viruses has increased steadily (see for example Candresse et al. 1998). This has been facilitated by the increased availability of nucleotide sequence data for many viroids and plant viruses. Table 14.1 lists the major viroids for which PCR assays have been reported. Viroids (circular, single-stranded RNA) require the additional preliminary step of reverse transcription (RT) to convert their sequences from RNA to cDNA before the amplification process. The RT–PCR assay transcribes viroid RNA and amplifies its cDNA in total nucleic acid extracts of infected tissue with high specificity and fidelity (Hadidi and Yang 1990a, b; Hadidi et al. 1995; Candresse et al. 1998; Shamloul and Hadidi 1999; Shamloul et al. 2002). The detection of viroids by RT–PCR requires 1 to 100 pg of total nucleic acids from infected tissue and generally is 10 to 100-fold more sensitive than viroid detection by hybridization using cRNA probes and 2500-fold more sensitive than return gel electrophoresis analysis (Hadidi and Yang 1990b). PCR amplication of whole viroid genomes has been successfully achieved from a variety of monocotyledonous and dicotyledonous plant hosts, including tissues of varied composition such as

As shown above, viroids require the introduction of an RT step before the PCR amplification process (RT–PCR). The two most frequently used enzymes to perform this RT step are the reverse transcriptases (RTases) isolated from either avian myeloblastosis virus (AMV–RT) or from Moloney murine leukemia virus (MMLV–RT), and their derivatives in which the RNase H activity has been inactivated. The PCR reaction is based on the annealing and enzymatic extension of two oligonucleotide primers (each usually 16 to 30 nucleotides in length) that flank the target region in a duplex DNA by means of a ‘thermostable’ DNA polymerase which will retain enzymatic activity at high temperatures. Typically, the reaction mixture is first heated (DNA denaturation) and subsequently cooled (DNA annealing) in cycles of 30 seconds to a few minutes in length for each heating or cooling period. Heating the mixture separates the double-stranded DNA into two single strands. As the mixture cools, each primer hybridizes to its separated, complementary DNA strand. Each of the annealed primers is then enzymatically extended on the template strand into a new DNA strand by the thermostable DNA polymerase. These three steps (denaturation, primer annealing, and primer extension) which are carried out at discrete temperature ranges (for example, 94°C to 98°C, 37°C to 65°C, and 72°C respectively) represent a single PCR cycle. The primer annealing and/ or extension temperature may depend on the DNA enzyme used and on the specific sequence/length of the primers used. The next cycle of heating separates the copies from the original strands and both become templates for a new round of DNA synthesis. As a result of repeated cycles, the target DNA is amplified exponentially in a chain reaction. In a few minutes to a few hours, 30–40 cycles of PCR can amplify a molecular signal that was too small to detect before PCR, more than several million fold. The length of the product generated during the PCR is equal to the sum of the length of the two primers plus the flanked target sequence. Double- or single-stranded DNA, or RNA (after RT into a cDNA copy) can serve as templates.

117


A number of parameters, such as magnesium ion concentration, primer annealing temperature, choice of primers, ‘hot starting’ the PCR reaction, number of PCR cycles, presence of various additives to increase specificity, purity of the nucleic acid extract, inclusion of an RNA denaturation step before reverse transcription, and the choice and concentration of enzyme(s) can dramatically influence the final sensitivity of a PCR assay. Therefore, in order to achieve maximal sensitivity of detection, these parameters should be carefully optimized. Such optimization is, however, usually very specific for a given target/primer pair combination. A number of thermostable DNA polymerases are now available, however, the Taq polymerase isolated from Thermus aquaticus is by far the most frequently used enzyme for PCR assays. Recently, however, the AmpliTaq DNA polymerase has become more frequently used in many laboratories as this enzyme also ‘hot starts’ the PCR reaction which significantly reduces the synthesis of unspecific PCR products. In a few cases, other thermostable enzymes have been used and sometimes compared to the Taq polymerase. The use of such enzymes has at least two applications: i

the use of enzymes possessing both a reverse transcriptase (RT) and a DNA polymerase activity, allowing the use of a single enzyme in RT–PCR assays (Shamloul et al. 1997; Faggioli et al. 2001); and

ii

the use of DNA polymerases having a 3' to 5' exonuclease (proof-reading) activity, which results in an increased fidelity of the amplification process.

The use of proof-reading thermostable polymerases and AmpliTaq DNA polymerase has clear advantages over Taq polymerase for PCR-based assays aimed at the study of the molecular variability of viroids. One of the major advantages of the PCR-based assays is the ability to select the level of specificity of the amplification procedure. Primers may be selected for the amplification of genomic sequences of members of a viroid genus i.e. Apscaviroid (Faggioli et al. 2001; Di Serio et al. 2002; Shamloul et al. 2002). Primers of such wide specificity are usually targeted to conserved regions of the viroid genome that are likely to be highly conserved among members of a genus. Most often, primers have been selected to allow amplification of all isolates of a given species. Primers could potentially also be selected for the discrimination of viroid variants or strains within a viroid species. Such an approach has, however, not yet been reported. Another very interesting and important approach for PCRbased detection assays is the development of ‘Multiplex PCR’, a technique which allows the simultaneous amplification of more than one viroid using a mixture of specific primer pairs (Levy et al. 1992; Di Serio et al. 2002; Shamloul et al. 2002). Multiplex standard, fluorescent, and ELISA PCR have been utilized (Levy

118

et al. 1992; Di Serio et al. 2002; Shamloul et al. 2002, respectively). Multiplex PCR has the potential to dramatically reduce the diagnostic cost by reducing the number of assays to perform if detection of several viroids is needed. Primer design is critical to the success of viroid PCR assay. Generally primers are between 16 to 30 nucleotides in length and 50 to 60% G + C, with closely matched melting points. The 3’ end should contain GG, CC, GC, or CG. Mispriming in G + C rich regions may result from runs of Cs or Gs at the 3’ end of primers. Complementarity between the 3’ ends of primers should be avoided to prevent the annealing and extension of primer pairs resulting in the synthesis of primer-dimer artefacts. Full-length cDNA copies of several viroids have been successfully amplified by using primers that anneal to the central conserved region of the viroid with the 3’ ends facing away from each other (see above). Although primers may be selected manually, several good computer programs are commercially available to aid in the selection of the primers. These programs allow for the analysis of primer pairs to decrease the level of complementarity between primers. This can be important especially for multiplex PCR where multiple primer pairs are contained in single reactions. Some of these programs also determine annealing temperatures and unexpected priming sites in other regions of the target sequence which could generate additional unpredicted PCR products.

ANALYSIS OF PCR AMPLIFIED PRODUCTS Generally, in viroid or plant virus research, amplified DNA (amplicons) is detected by staining with ethidium bromide, Sybr Green™, or silver nitrate following agarose or polyacrylamide gel electrophoresis, by Southern blotting hybridization with labeled probes, or by colorimetric assay after affinity binding. Amplified DNA may also be digested with restriction endonucleases before electrophoresis to analyze the restriction fragment length polymorphism (RFLP) pattern of the amplified products. PCR amplified products labeled by incorporating biotin-11-dUTP, or biotin-14-dATP during the PCR reaction or labeled by the use of biotinylated primers can be detected by spotting on nitrocellulose or nylon membranes followed by a colorimetric or chemiluminescent assay. Nucleotides or oligonucleotide primers labeled with fluorescent molecules have also been used in conjunction with laser-excited fluorescence detection on an automated DNA sequencer. Among the methods described above, the most common method for the detection of amplified viroid DNA is staining of the amplicon in polyacrylamide gels with silver nitrate or ethidium bromide or, in agarose gels, with ethidium bromide or Sybr Green™; silver nitrate cannot be used to stain nucleic acids in agarose gel. Several PCR techniques and equipment have been described for amplification and detection of desirable amplicons from


different biological systems such as the fluorogenic 5' nuclease detection assay using the TaqMan™ System (Applied Biosystems, Foster City, CA). Other fluorogenic PCR assays may include molecular beacons, double-strand specific DNA dyes, ‘sunrise’ or Scorpion™ PCR primers, energy-transfer minisequencing, the peptide nucleic acid (PNA)-based light-up probes, real-time PCR/hybridization probe systems (TaqMan™, LightCycler), and oligoligation assays. All of these assays rely on fluorescent dyes, and several rely on energy transfer or fluorescence quenching which may allow the use of multiple probes in one tube (Tyagi et al. 1998; Lee et al. 1999). Among fluorogenic PCR assays to date, only the 5’ nuclease (TaqMan™) assay was used for the detection of Chrysanthemum stunt viroid (Mumford et al. 2000). The other fluorogenic PCR assays have the potential to be used for viroid detection. In 1999, a combination of a RT–PCR and an ELISA-type assay has been described for the detection of the PCR-amplified nucleic acid of viroids (Shamloul and Hadidi 1999). Biotinylated cDNA oligonucleotides or cRNA (capture probes) are hybridized to digoxigenin-11 dUTP (DIG-11dUTP) labeled RT–PCR amplicons from viroid-infected tissue. The amplicon/ capture probe hybrid is captured on the surface of a streptavidin-coated microtiter plate (solid phase) via avidin-biotin interaction. The hybridized amplicon is then detected with an enzyme-conjugated anti-DIG antibody. The PCR-ELISA assay for viroid detection is generally reported to be 10–100-fold more sensitive than analysis of the PCR product by gel electrophoretic analysis. The increase in sensitivity is usually in the 10× range, but larger increases (100×) have also been obtained (Shamloul and Hadidi 1999). Recently, a multiplex RT-PCRELISA assay for the simultaneous detection of six viroids in four genera has been developed (Shamloul et al. 2002). The viroids detected by this multiplex assay include Potato spindle tuber viroid (Pospiviroid); Peach latent mosaic viroid (Pelamoviroid); Apple scar skin viroid, Apple dimple fruit viroid, and Pear blister canker viroid (Apscaviroid); and Hop stunt viroid (Hostuviroid) (Figure 14.2). PCR-ELISA has the potential to provide quantitative or semi-quantitative results and can be adapted for high throughput automation requiring only limited specialized equipment. These qualities, coupled with the specificity, sensitivity, speed, and low cost of the assay, makes this technique an attractive tool in viroid basic research and in viroid detection programs worldwide.

PLANT SAMPLE PREPARATION FOR PCR In PCR, as in other assays, a critical step in the procedure is sample preparation. Also plant tissues often pose specific problems not encountered when working with human or animal tissues. Viroid-infected plant tissues may require a grinding step before the RT step. Plant tissues, especially those of woody plants, are very frequently rich in poorly characterized compounds (polysac-

Figure 14.2 Specificity of biotin-labeled capture probe for the colorimetric detection of homologous and heterologous DIG-labeled amplified viroid cDNA product. Each viroid capture probe was hybridized to amplified product from: Potato spindle tuber viroid-infected tomato (A1, PSTVd), Hop stunt viroid-infected peach (B2, HSVd), Peach latent mosaic viroid-infected peach (C3, PLMVd), Pear blister canker viroidinfected quince (D4, PBCVd), Apple dimple viroid-infected apple (E5, ADFVd), and Apple scar skin viroid-infected apple (F6, ASSVd), and all of the above samples (G1–6, six viroids). Standard RT-PCR-ELISA (A–F, 1–6), positive samples: A1; B2; C3; D4; E5; F6. Mutiplex RT-PCR-ELISA (G, 1–6), six positive samples: G 1–6. Negative controls for standard RT-PCR-ELISA: healthy uninfected tomato; peach, quince, and apple leaves (wells 1–4) labeled negative controls. Negative controls for multiplex RT-PCR-ELISA: a mixture of healthy uninfected tomato, peach, quince, and apple leaves (well 5), and buffer control (well 6) labeled negative controls. (From Shamloul et al. 2002, with permission.)

charides, phenolics, etc.) that may interfere with the RT and/or PCR reactions of viroids (Hadidi and Yang 1990b; Yang et al. 1992; Hadidi et al. 1995, 1997; Candresse et al. 1998; Shamloul et al. 1995, 1997). The simplest approach to remove the inhibitors is to initially treat the crude nucleic acid extracts with resins such as DEAE cellulose or to prepare crude plant homogenates in simple buffers (or in sterile water) and then dilute the extract. Although appealing in their simplicity, these sample preparation protocols have the disadvantage of lowering the overall detection sensitivity by way of diluting the target nucleic acids. The most

119


widely applied approach is the use of purified total plant nucleic acids or total plant RNAs as starting material for RT and PCR. In some instances, an additional step may be needed to completely remove RT and/or PCR inhibitors.

evaluation of transgenic plants; the determination of specific sequence information with or without cloning from crude total nucleic acids; and the generation of pathogen-specific cDNA clones without extensive viroid purification.

A wide variety of protocols have been published for the preparation of purified nucleic acids. Several protocols involving organic solvent or non-organic solvent extraction methods have been reported. The most frequently used approach is to homogenize the plant material in an extraction buffer followed by phenol and/or chloroform extraction (e.g. Shamloul et al. 1997; Faggioli et al. 2001). Since all viroids are RNA molecules, special precautions must be taken during extraction in order to prevent degradation by nucleases. Typically, liquid nitrogen is used to reduce the plant material to a powder without allowing thawing of the tissue. Then, to prevent cellular RNases from becoming active after the tissue thaws, the tissue is homogenized in a high ionic strength, high pH extraction buffer followed by one or more rounds of phenol and/or chloroform extraction to remove plant proteins and contaminating substances. The nucleic acids are then recovered by ethanol or isopropanol precipitation, resuspended in sterile water and used for amplification. Total RNAs may be extracted from viroid-infected tissues by non-organic solvents using commercially available RNA extraction kits such as BIO 101 FastRNA Green Protocol (BIO 101, Carlsbad, CA) or Qiagen RNeasy plant mini kit (Qiagen Inc., Valencia, CA) (Shamloul et al. 2002). Additional purification steps are frequently added since these protocols may not always succeed in removing interfering substances from the nucleic acid preparation. These include such diverse procedures such as commercial nucleic acid minicolumn chromatography (Yang et al. 1992), treatment with CF-11 cellulose (Kyriakopoulou and Hadidi 1998; Pallás et al. 1987), or even further purification of total nucleic acids by RNA extraction kits such as Qiagen RNeasy plant mini kit (Shamloul et al. 2002). A last alternative is the use of the GeneReleaser™ matrix. This commercially available resin is used directly on crude plant homogenates, total nucleic acids, or total RNAs and is apparently able to remove interfering substances from a variety of host plants including tobacco, apple, peach, apricot, grapevine and periwinkle (Levy et al. 1994).

PCR is primer directed. Thus primers can be designed to specifically amplify viroid cDNA from heterogeneous samples. This eliminates the need to purify the viroid from infected plant tissue and therefore can be performed on very small biological samples.

ADVANTAGES AND DRAWBACKS OF PCR PCR allows for the rapid and sensitive detection of low titer viroids which elude conventional detection methods such as dot-blot hybridization or that are currently detected by lengthy bioassays. It also allows the identification of unknown pathogens that are possibly of viroid nature, as well as detection of multiple and unrelated viroids in a single PCR reaction or the identification of the components of mixed infections or disease complexes. As such, it is extremely useful for the rapid and sensitive evaluation of plants after viroid elimination therapy; the

120

The major drawbacks include: the initial expense in PCR laboratory set-up; the requirement for trained personnel; the careful laboratory practices that must be followed to prevent possible cross-samples or the contamination of samples by PCR products. In addition, primer design requires some knowledge of the target sequence or of a related sequence and positive identification of a specific target sequence may require the availability of a specific or related cDNA clone for hybridization or the sequencing of the amplified product. Also, the cost of PCRbased assays is still substantially higher than that of more classical detection techniques such as molecular hybridization. For reliable PCR assays the following elements are suggested: A

Laboratory design: 1. Separate laboratories or clearly defined work areas for sample handling and preparation, PCR reaction set-up and PCR products construction and analysis. 2. Avoid air flows back from the post-PCR laboratory to the pre-PCR laboratory. 3. Dedicated equipment for each laboratory.

B

Stock solution preparation: Use fresh ‘PCR-dedicated’ chemicals (including water) and aliquot all solutions.

C

Operating procedures: 1. Set up a ‘master RT and PCR mix’, then aliquot into each tube. 2. Use positive-displacement pipettes or plugged (barrier) tips. 3. Use multiple coprocessed, negative controls. 4. Set up the known positive controls last. 5. Handle only one tube at a time. 6. Use gloves and change often.

D

Assay and detection procedures: 1. Use multiple primer pairs, corresponding to different regions of the viroid genome (one primer pair per assay). 2. Ensure, if needed, positive confirmation of the PCR target product by a subsequent hybridization step or by sequencing.

FUTURE PCR APPLICATIONS The GeneChip™ Microarray System, developed during the last few years by Affymetrix of Santa Clara, CA, and other biotechnology companies, is a powerful tool for genetic analysis. It is currently being applied in three broad areas: biomedical research of cancer, and infectious and genetic diseases; genomics research such as sequence analysis, gene expression studies, gene


typing, and large-scale polymorphism screening; and clinical diagnosis such as diagnosis of HIV in blood. The GeneChip™ Microarray System is comprised of instrumentation and computer software capable of nucleotide sequence specific assays. A ‘gene chip’ probe system is the core of the assay in which tens to hundreds of thousands of oligonucleotide probes are synthesized on the surface of a small chip. This will allow the simultaneous detection of multiple pathogens, including viroids, by hybridization of PCR-amplified nucleic acids with the oligonucleotide probes in one microfluidic chamber. This capability has the potential to decrease the amount of labor required for sample preparation, time required for data analysis, the total time required to run the assay, and the total cost. Many multiplex PCR methods that will be available in the near future may allow amplification of as many as 50–100 separate sequences in a single tube. To detect plant pathogens, including viroids that are infecting a specific crop, a single PCR may thus be needed to amplify all pathogens known to infect that crop (probably in the range of 10–100 pathogens) followed by typing of the amplification products on a chip to reveal the presence or absence of a specific pathogen. This technology, or similar ones, may be available in the next few years for the detection of viroids and other pathogens, especially in large-scale certification and quarantine programs. Acknowledgement

This work was supported in part by USAID grant no. PCE-G00-98-00009-00 to A. Hadidi. References Ben-Shaul, A., Guang, Y., Mogilner, N., Hada, R., Mawassi, M., Gafny, R., and Bar-Joseph, M. (1995). Genomic diversity among populations of two citrus viroids from different graft-transmissible dwarfing complexes in Israel. Phytopathology 85, 359-364. Cajza, M., Wypijewski, K., and Pospiesny, H. (1996). The use of reverse transcription and polymerase chain reaction (RT-PCR) for the detection of hop latent viroid (HLVd). J. Plant Protec. Res. 37, 52-58. Candresse, T., Hammond, R.W., and Hadidi, A. (1998). Detection and identification of plant viruses and viroids using polymerase chain reaction. Pages 399-416 in: Plant virus disease control. A. Hadidi, R.K. Khetarpal, and H. Koganezawa, eds. APS Press: St Paul, MN. Di Serio, F., Alioto, D., Ragozzino, A., Giunchedi, L., and Flores, R. (1998). Identification of apple dimple fruit viroid in different commercial varieties of apple grown in Italy. Acta Hortic. 472, 595-601. Di Serio, F., Malfitano, M., Alioto, D., Ragozzino, A., and Flores, R. (2002). Apple dimple fruit viroid: sequence variability and its specific detection by multiplex fluorescent RT-PCR in the presence of Apple scar skin viroid. J. Plant Pathol. 84, 27-34. Faggioli, F., Ragozzino, E., and Barba, M. (2001). Simultaneous detection of pome fruit viroids by single tube - RT-PCR. Acta Hortic. 550, 59-63. Fagoaga, C., Semancik, J. S., and Duran-Vila, N. (1995). A citrus exocortis variant from broad bean (Vicia faba L.): infectivity and pathogenesis. J. Gen. Virol. 76, 2271-2277.

Francis, M.I., Szychowski, J.A., and Semancik, J.S. (1995). Structural sites specific to citrus virus groups. J. Gen. Virol. 76, 1081-1089. Giunchedi, L., Gentit, P., Nemchinov, L., Poggi-Pollini, C., and Hadidi, A. (1998). Plum spotted fruit: a disease associated with peach latent mosaic viroid. Acta Hortic. 472, 571-579. Hadidi, A., Giunchedi, L., Shamloul, A.M., Poggi-Pollini, C., and Amer, A.M. (1997). Occurrence of peach latent mosaic viroid in stone fruits and its transmission with contaminated blades. Plant Dis. 81, 154-158. Hadidi, A., Hansen, A.J., Parish, C.L., and Yang, X. (1991). Scar skin and dapple apple viroids are seed borne and persistent in infected apple trees. Res. Virol. 142, 289-296. Hadidi, A., Levy, L., and Podleckis, E.V. (1995). Polymerase chain reaction technology in plant pathology. Pages 167-187 in: Molecular methods in plant pathology. R.P. Singh, and U.S. Singh, eds. CRC Press: Boca Raton, FL. Hadidi, A., Terai, Y., Powell, C.A., Scott, S.W., Desvignes, J.C., Ibrahim, L.M., and Levy, L. (1992). Enzymatic cDNA amplification of hop stunt viroid variants from naturally infected fruit crops. Acta Hortic. 309, 339-344. Hadidi, A., and Yang, X. (1990a). Detection of pome fruit viroids by enzymatic cDNA amplification. VIII International Congress of Virology, Berlin, Germany. Page 21 (Abstract). Hadidi, A., and Yang, X. (1990b). Detection of pome fruit viroids by enzymatic cDNA amplification. J. Virol. Methods 30, 261-270. Hanold, D., and Randles, W.J. (1991). Coconut cadang-cadang disease and its viroid agent. Plant Dis. 75, 330-335. Hataya, T., Hikage, K., Suda, N., Nagata,T., Li, S., Itoga, Y., Tanikoshi, T., and Shikata, E. (1992). Detection of hop latent viroid (HLVd) using reverse transcription and polymerase chain reaction (RT-PCR). Ann. Phytopathol. Soc. Jpn. 58, 677-684. Hodgson, R.A.J., Wall, G.C., and Randles, J.W. (1998). Specific identification of coconut tinangaja viroid (CTiVd) for differential field diagnosis of viroids in coconut palm. Phytopathology 88, 774-781. Hooftman, R., Arts, M-J., Shamloul, A.M., Van Zaayen, A., and Hadidi, A. (1996). Detection of chrysanthemum stunt viroid by reverse transcription-polymerase chain reaction and by tissue blot hybridization. Acta Hortic. 432, 120-128. Ito, T., and Ieki, H. (1996). Detection of citrus exocortis viroid and citrus viroids I, II, III, and IV by reverse transcription and polymersae chain reaction (RT-PCR). Ann. Phytopathol. Soc. Jpn. 62, 614-615. Ito, T., Sano, T., and Yoshida, K. (1998). Nucleotide sequence of apple fruit crinkle viroid (AFCVd). Ann. Phytopathol. Soc. Jpn. 64, 424-425 (Abstract in Japanese). Knabel, S., Seigner, L., and Wallnofer, P.R. (1999). Detection of hop latent viroid (HLVd) using the polymerase chain reaction (PCR). Gesunde Pflanzen 51, 234-239. Kusano, N., and Shimomura, K. (1997). Selection of PCR primers and a simple extraction method for detection of hop stunt viroid-plum in plum by reverse transcription-polymerase chain reaction. Ann. Phytopathol. Soc. Jpn. 63, 119-123. Kyriakopoulou, P.E., Giunchedi, L., and Hadidi, A. (2001). Peach latent mosaic and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdaliformis: Implications on possible origin of these viroids in the Mediterranean region. J. Plant Pathol. 83, 51-62.

121


Kyriakopoulou, P.E., and Hadidi, A. (1998). Natural infection of wild and cultivated pears with apple scar skin viroid in Greece. Acta Hortic. 472, 555-559. Lee, L.G., Livak, K.J., Mullah, B., Graham, R.J., Vinayak, R.S., and Woundenberg, T.M. (1999). Seven-color, homogeneous detection of six PCR products. Biotechniques 27, 342-349. Levy, L., Hadidi, A., and Garnsey, S.M. (1992). Reverse transcriptionpolymerase chain reaction assays for the rapid detection of citrus viroids using multiplex primer sets. Proc. Int. Soc. Citriculture 2, 800-803. Levy, L., Lee, I. M., and Hadidi, A. (1994). Simple and rapid preparation of infected plant tissue extracts for PCR amplification of virus, viroid, and MLO nucleic acids. J. Virol. Methods 49, 295-304. Menzel, W., and Maiss, E. (2000). Detection of chrysanthemum stunt viroid (CSVd) in cultivars of Argyranthemum frutescens by RT-PCRELISA. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 107, 548-552. Mumford, R.A., Walsh, K., and Boonham, N. (2000). A comparison of molecular methods for the routine detection of viroids. EPPO Bull. 30, 341-346. Nakahara, K., Hataya, T., and Uyeda, I. (1999). A simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and RT-PCR. J. Virol. Methods 77, 47-58. Osaki, H., Kudo, A., and Ohtsue, Y. (1996). Japanese fruit dimple disease caused by apple scar skin viroid. Ann. Phtopathol. Soc. Japan 62, 379-385. Osaki, H., Ohtsu, Y., and Kudo, A. (1998). Two rapid extraction methods to detect apple scar skin and hop stunt viroids. Acta Hortic. 472, 603-611. Osaki, H., Yamamuchi, Y., Sato, Y., Tomita, Y., Kawai, Y., Miyamoto, Y., and Ohtsu, Y. (1999). Peach latent mosaic viroid isolated from stone fruits in Japan. Ann. Phytopathol. Soc. Jpn. 65, 3-8. Pallás, V., Navarro, A., and Flores, R. (1987). Isolation of a viroid-like RNA from hop different from hop stunt viroid. J. Gen. Virol. 68, 3201-3205. Querci, M., Owens, R., Vargas, C., and Salazar, L.F. (1995). Detection of potato spindle tuber viroid in avocado growing in Peru. Plant Dis. 79, 196-202. Rezaian, M.A., Krake, L.R., and Golino, D.A. (1992). Common identity of grapevine viroids from U.S.A. and Australia revealed by PCR analysis. Intervirology 34, 38-43. Sano, T., Mimura, R., and Ohshima, K. (2001). Phylogenetic analysis of hop stunt viroid supports a grapevine origin for hop stunt disease. Virus Genes 21, 53-59.

122

Schnell, R.J., Kuhn, D.N., Ronning, C.M., and Harkins, D. (1997). Application of RT-PCR for indexing avocado sunblotch viroid. Plant Dis. 81, 1023-1026. Seigner, L. (2000). Aktueller Stand des Einsatzes der Polymerase-Kettenreaktion (PCR) zur Pathogendiagnostik an der LBP. Schule und Beratung; Bayerisches Staatsministerium für Emahrung, Landwirtschaft und Forsten. 5, 17-21. Semancik, J.S., and Szychowski, J.A. (1994). Avocado sunblotch disease: a persistent viroid infection in which variants are associated with differential symptoms. L. Gen. Virol. 75, 1543-1549. Shamloul, A.M., Faggioli, F., Keith, J.M., and Hadidi, A. (2002). A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid. J. Virol. Methods 105, 115-121. Shamloul, A.M., and Hadidi, A. (1999). Sensitive detection of potato spindle tuber and temperate fruit tree viroids by reverse transcription - polymerase chain reaction-probe capture hybridization. J. Virol. Methods 80, 145-155. Shamloul, A.M., Hadidi, A., Zhu, S.F., Singh, R.P., and Sagredo, B. (1997). Sensitive detection of potato spindle tuber viroid using RTPCR and identification of a viroid variant naturally infecting pepino plants. Can. J. Plant Pathol. 19, 89-96. Shamloul, A.M., Minafra, A., Hadidi, A., Giunchedi, L., Waterworth, H.E., and Allam, E.K. (1995). Peach latent mosaic viroid: nucleotide sequence of an Italian Isolateisolate, sensitive detection using RTPCR and geographic distribution. Acta Hortic. 386, 522-530. Tyagi, S., Bratu, D.P., and Kramer, F.R. (1998). Multicolor molecular beacons for allele discrimination. Nature Biotechnology 16, 49-53. Villalobos, W., Rivera, C., and Hammond, R. (1997). Occurrence of citrus viroids in Costa Rica. Rev.Trop. Biol. 45, 983-987. Wah, Y.F.W.C., and Symons, R.H. (1997). A high sensitivity RT-PCR assay for the diagnosis of grapevine viroids in field and tissue culture samples. J. Virol. Methods 63, 57-69. Yang, X., Hadidi, A., and Garnsey, S.M. (1992). Enzymatic cDNA amplification of citrus exocortis and cachexia viroids from infected citrus hosts. Phytopathology 82, 279-285. Zhu, S.F., Hadidi, A., Hammond, R.W., Yang, X., and Hansen, A.J. (1995). Nucleotide sequence and secondary structure of pome fruit viroids from dapple apple diseased apples, pear rusty skin diseased pears and apple scar skin symptomless pears. Acta Hortic. 386, 554-559. Zhu, S.F., Hammond, R.W., and Hadidi, A. (1998). Agroinfection of pear and apple with dapple apple viroid results in systemic infection. Acta Hortic. 472, 613-616.

............................................................................................................................................................................................................................................................................................

PART IV

DISEASES AND VIROIDS ASSOCIATED WITH PLANT SPECIES

...........................................................................................................................................................................................................................................................................


PART IV

VIROIDS OF SOLANACEOUS SPECIES VIROIDS OF SOLANACEOUS SPECIES R.P. Singh, K.F.M. Ready, and X. Nie

....................................................................................................

CHAPTER 15

.................................................................................................................................................................................................................................................................

In addition to Potato spindle tuber viroid (PSTVd), there are six viroids infecting Solanaceae that cause symptoms which are similar to or indistinguishable from those induced by PSTVd: Mexican papita viroid (MPVd) (Martinez-Soriano et al. 1996), Tomato apical stunt viroid (TASVd) (Walter 1981, 1987; Spieker et al. 1996), Tomato bunchy top viroid (TBTVd) (McLean 1948; Mishra et al. 1991), Tomato chlorotic dwarf viroid (TCDVd) (Singh et al. 1999), and Tomato planta macho viroid (TPMVd) (Galindo et al. 1982). Unlike the above viroids, Nicotiana glutinosa stunt viroid (NgSVd) (Bhattiprolu 1991) did not infect tomato plants; potato plants have not yet been tested. Besides these viroids naturally infecting solanaceous plants, Citrus exocortis viroid (CEVd) (Semancik et al. 1973; Singh and Clark 1973) and Columnea latent viroid (CLVd) (Hammond et al. 1989; Singh et al. 1992b), Chrysanthemum stunt viroid (CSVd) (Verhoeven et al. 1998) cause PSTVd-like symptoms in solanaceous plants or infect solanaceous plants and remain symptomless under experimental and field conditions (Mishra et al. 1991; Spieker 1996).

ECONOMIC EFFECTS Economic losses due to PSTVd have been well documented in potato crops. Yields were reduced 17–24% for mild strains and

up to 64% for severe strains (Singh et al. 1971). Losses were lower in the first year of infection, became more severe in subsequent generations grown from infected seed potatoes, and were close to 100% by the third generation of infection with severe PSTVd (Pfannenstiel and Slack 1980; Singh 1988). The potential economic effects of PSTVd in tomato crops have not been documented. TPMVd caused severe losses in commercial tomato crops in Mexico. Plants produced only marble-sized fruit that are completely unmarketable (Galindo et al. 1982). TCDVd also caused very small fruit in tomato, as well as cracked tubers in infected potato. TCDVd was reported only in commercial greenhouse tomatoes (Singh et al. 1999) and not in the field. However, an infected tomato crop could be totally unmarketable (R.P. Singh, unpublished observations). In contrast, no economic losses have been reported to result from MPVd, CLVd or TASVd infection. MPVd was isolated from symptomless infection of a wild species, Solanum cardiophyllum, but has not been found in any crop species. Infection in tomato was only in an experimental setting (Martinez-Soriano et al. 1996) and thus, MPVd has had no economic consequence to date. Similarly, CLVd was isolated from symptomless infection

125

R.P. Singh et al.

of the ornamentals Columnea erythrophae (Owens et al. 1978; Hammond et al. 1989) and Nematanthus wettsteinii (Singh et al. 1992b), and a similar viroid was found in Brunfelsia undulata (Spieker 1996). CLVd has not been found in natural infections in any other species. There have been only isolated reports of natural TASVd infection and no reports of economic losses due to this disease (Walter 1987).

may be advisable to exercise caution in comparing results from separate studies, bearing in mind that the course of symptom onset can be radically affected by the environmental conditions, particularly temperature and light intensity (see Chapter 11 ‘Biological indexing’). If the information so far available is to be used for indexing, then a comparison of several species infected with this group of viroids under controlled conditions is merited.

Indian TBTVd was isolated during a survey of cultivated tomato (Mishra et al. 1991) and from field plantings of citrus (Ramachandran et al. 1992). Economic effects were not reported in either case. Symptoms described for experimentally infected tomato concerned herbaceous tissue and the presence or absence of infection, not effects on fruit yields or quality. This viroid is a strain of Citrus exocortis viroid (CEVd) (Mishra et al. 1991) and is probably not the same as the South African TBTVd, as will be discussed. We will refer to these isolates with different prefixes (I-TBTVd and SA-TBTVd) to avoid confusion.

I-TBTVd is the only member of this group to have been found infecting citrus, consistent with its identity as a strain of CEVd (Mishra et al. 1991). Its ability to infect citrus species would not be useful as a rapid means of biological indexing, due to the time required for symptom development. It could be distinguished from PSTVd and TASVd by its ability to produce symptoms in cucumber (Singh 1973; Walter 1987; Ramachandran et al. 1992). However, CLVd also caused symptoms in cucumber (Hammond et al. 1989), as well as in Scopolia sinensis, Gynura aurantiaca and tomato, but did not infect tobacco (Hammond et al. 1989; Singh et al. 1992b).

SYMPTOMS The viroids infecting the solanaceous species produce similar symptoms in cases of severe infection in tomato. Stunting, epinasty, leaf distortions or discolorations and necrosis, usually of the veins but sometimes of petioles and stems (Plate 2A and B), have been reported for all of these viroids (PSTVd: Singh 1973; TPMVd: Galindo et al. 1982, Martinez-Soriano et al. 1996; MPVd: Martinez-Soriano et al. 1996; TCDVd: Singh et al. 1999; TASVd: Owens 1990; Owens et al. 1990; Walter 1987; ITBTVd: Mishra et al. 1991; SA-TBTVd: McClean 1948). Severity of symptoms in tomato varied with the PSTVd strains (Plate 2C). Floral variegation occurred for PSTVd, TPMVd, MPVd, and TCDVd (Martinez-Soriano et al. 1996; Singh et al. 1999) and was host-dependent. Apical proliferation (PSTVd: Singh 1973; TASVd: Owens 1990; Owens et al. 1990; Walter 1987; ITBTVd: Mishra et al. 1991), tuber elongation and cracking (PSTVd: Werner 1924; TCDVd: Singh et al. 1999) (Plate 2D and E) and reduction in tomato fruit size (PSTVd: Behjatnia et al. 1996; TPMVd: Galindo et al. 1982; TCDVd: Singh et al. 1999) have also occurred, but not for all of these viroids.

HOST RANGE Host range was examined in a wide range of species for PSTVd, TASVd and SA-TBTVd, and in fewer species for TPMVd, MPVd, TCDVd, CLVd and I-TBTVd (Table 15.1; Plate 3A and B). These viroids replicate and show PSTVd-like symptoms in tomato (Lycopersicon esculentum ) and with the exception of CLVd, replicate in Nicotiana glutinosa but vary in the symptoms produced. There are conflicting reports of the ability of PSTVd to replicate in Gomphrena globosa. Therefore, its utility in distinguishing PSTVd from TPMVd, MPVd and TCDVd is questionable. It

126

SA-TBTVd can no longer be located but its host range and pattern of symptom production suggest that it may have been a strain or very close relative of PSTVd (McClean 1948; Singh 1973). It would be useful to have more extensive information on the host range of MPVd. Most assays were negative (see Table 15.1) and correspond to species in which PSTVd does not produce symptoms. Furthermore, particular attention to environmental conditions in experimental assay is required. For example, temperature has been shown to be critical to the distribution of field infections of TPMVd (Galindo 1987). Martinez-Soriano et al. (1996) suggested that PSTVd, MPVd and TMPVd could be differentiated on the basis of their replication and symptom production in N. glutinosa and G. globosa. In Table 15.2, we propose that biological indexing in four species would allow distinction between all members of this group, while placing less reliance on the performance of PSTVd in G. globosa.

TRANSMISSION All seven viroids infecting the Solanaceae are transmissible by mechanical means, such as abrasion and graft-inoculation. Seed transmission is well-documented for PSTVd (Singh 1970; Singh and Finnie 1973) and was reported for SA-TBTVd in certain species (McClean 1948). Seed transmission has not been demonstrated for TPMVd (Galindo 1987) or TCDVd (Singh et al. 1999), and has not been investigated for TASVd. PSTVd is the only member of this group known to be transmitted by pollen (Singh 1970; Singh et al. 1992a). Low level of aphid-mediated transmission of PSTVd occurred by transencapsidation. In cases of co-infection with either Potato leafroll virus (PLRV)

VIROIDS OF SOLANACEOUS SPECIES

Table 15.1

Host range of viroids infecting Solanacea.

Plant family or species Amaranthaceae Gomphrena globosa Campanulaceae Campanula medium Chenopodiaceae Beta vulgaris Chenopodium spp. Cucurbitaceae spp. Compositae Chrysanthemum carinatum Gynura aurantiaca Zinnia elegans Scrophulariaceae Nemesia floribunda Penstemon hirsutus Torenia fournieri Rutaceae citron Poncirus trifoliata Rangpur lime Sweet orange Solanaceae Browallia demissa Capsicum annuum Datura inoxia D. metel D. stramonium Lycopersicon esculentum Nicandra physaloides Nicotiana alata N. benthamiana N. bigelovii N. clevelandii N. debneyi N. edwardsonii N. forgetiana N. glauca N. glutinosa N. goodspeedii N. langsfordii N. longiflora N. megalosiphon N. nudicaulis N. physaloides N. plumbaginifolia N. quadrivalvis

PSTVd1

TPMVd2

MPVd3

+/+

+/-

-/-

Replication/Symptoms TCDVd4 TASVd5

SA-TBTVd6

I-TBTVd7

CLVd8

+/+

+/+

-/-

+/-

-/-

-/-/-

+/-/-/+/+

+/+ -/-

+/-

+/+ +/? +/+m +/+m +/+/± +/± +/+ +/+

+/+/-/+/+ +/+

-/+/+

-/-/+/+

-/+/+ +/+

+/+/+/+/+

+/+/+ +/+/+/+/+/-

+/-

-/+/+

+/+

+/+m -/+/+m +/+m -/+/+

+/+

+/+/+ +/+

+/+

+/+

+/+/+ +/+m -/+/+/+m +/+m +/+nec +/+/+/+m +/+ +/+top

+/+

+/+ +/+/-

+/+/-

127

R.P. Singh et al.

Table 15.1

Host range of viroids infecting Solanacea. (Continued)

Plant family or species PSTVd1 +/-

MPVd3

Replication/Symptoms TCDVd4 TASVd5 +/+/+/-/+/+ +/+ +/+/+ +/+m +/+ +/+

SA-TBTVd6 I-TBTVd7 CLVd8 N. repanda N. rustica N. sylvestris +/N. tabacum +/+ -/+/-/Petunia +/+ +/+flwr Physalis alkekengi +/P. angulata +/+/P. curassavica P. floridana P. foetens +/m -/P. peruviana +/+m +/P. philadelphica +/+/P. pubescens +/+/+m P. viscosa +/-/Scopolia corniolica +/+nec S. lurida +/+nec S. sinensis +/+loc +/+ S. tanguticus +/+nec Solanum aviculare +/+ S. berthaultii +/+nec +/+ S. cardiophyllum +/S. carolinense +/+/S. cervantesii +/-/S. demissum +/+ S. dulcamara +/+/+ S. dupolisinuatum +/S. ehrenbergii -/S. eleagnifolium -/S. giganteum +/± S. heterodoxum -/-/S. incanum +/S. indicum +/+m +/S. lycopersicum +/+m S. melongena +/± +/+/+/S. nigrescens +/m S. nigrum +/+/S. pseudocapsicum +/+/S. quitoense +/S. rostratum +/+nec +/m -/S. sisymbrifolium +/+/+/S. sodamaeum L. +/+/S. topiro +/+ S. tuberosum +/+ +/+/+ flwr = symptoms show on flower; loc = local lesions; m = mild symptoms; nec = necrosis; top = symptoms only at top of plant. References: 1Singh and Bagnall 1968; Singh 1971, 1973; 1984; 2Galindo et al. 1982, 1986; Galindo 1987, 1988; 3Martinez-Soriano et al. 1996; 4Singh et al. 1999; 5Walter 1987; Owens 1990; 6McClean 1948; 7Pandey and Summanwar 1982; Mishra et al. 1991; Ramachandran et al. 1992; 8Hammond et al. 1989; 9Singh et al. 1992; 10O’Brien and Raymer 1964; 11Wassenegger et al. 1994.

128

TPMVd2


Table 15.2

Potential biological indexing using four indicator species.

Host

Replication/Symptoms PSTVd1

TPMVd2

MPVd3

TCDVd4

TASVd5

SA-TBTVd6

CLVd7

Datura stramonium

++

-/-

-/-

NT

-/-

+/-

NT

Gomphrena globosa

+/±

+/-

-/-

-/-

NT

NT

NT

Nicotiana glutinosa

+/fv

+/-

+/fv

+/fv

+/nec

+/fv

NT

N. tabacum

+/+

-/-

NT

-/-

+/+

+/-

-/-

Abbreviations: fv = floral variegation, nec = necrotic lesions, NT = not tested, ± = reports of both symptomatic and symptomless infection, or replication/no replication. References: 1Singh 1973; 2Galindo 1987; Galindo et al. 1982, 1986; 3Martinez-Soriano et al. 1996; 4Singh et al. 1999; 5Walter 1987; 6McClean 1948; 7 Hammond et al. 1989.

orVelvet tobacco mottle virus (VTMoV) (Francki et al. 1986), the viroid RNA was encapsidated at low frequency by the viral coat protein. Low efficiency transmission of TASVd by Aphis craccivora was reported but not proven (Galindo et al. 1986) and may be due to mechanical transmission by the insects, rather than true feeding-associated transmission (Walter 1987). High efficiency transmission of TPMVd by Myzus persicae ranged from 33% on Solanum nigrescens to close to 100% on Physalis foetens (Galindo et al. 1986). TPMVd was shown to persist in M. persicae for at least 8 days (Galindo 1988). Since aphids are known not to act as true vectors of viroid transmission in any other cases studied, these data warrant confirmation.

GEOGRAPHICAL DISTRIBUTION With the exception of PSTVd (see Table 3.1, Chapter 3 ‘Biology’), which is widely distributed in some parts of the world and has been successfully eradicated in others, the geographic distribution of other viroids infecting Solanaceae is so far limited. TPMVd and MPVd have been reported only in Mexico (Galindo et al. 1982, 1986; Martinez-Soriano et al. 1996). TPMVd occurred only in fields with an annual mean temperature above 22°C (Galindo 1987). TCDVd occurred in Canada in commercial greenhouse tomato seedlings, grown from seed imported from the Netherlands via the US (Singh et al. 1999). It therefore has the potential for wide distribution but there have been no other reports of this viroid. TASVd was reported in Ivory Coast (Walter 1981), Niger (Walter 1987) and Indonesia (Candresse et al. 1987). It was found in Germany in greenhouse specimens of the ornamental plant S. pseudocapsicum, in which it produces no symptoms (Spieker et al. 1996). This indicates that, like TCDVd, it has the potential for widespread distribution. Tomato bunchy top disease was reported in tomato in South Africa between 1930–1950 (McClean 1948; Diener 1987). ITBTVd was isolated from field plantings of tomato and citrus in India (Pandey and Summanwar 1982; Ramachandran et al. 1992) and shown to be a strain of CEVd (Mishra et al. 1991).

This is the only reported case of natural infection of tomato by CEVd (Mishra et al. 1991). The relationship of I-TBTVd to SA-TBTVd cannot be evaluated due to loss of material infected with SA-TBTVd ( Diener 1987), but we suggest that SATBTVd is more likely to have been a strain or close relative of PSTVd, based on its host range. The spread of at least one of these diseases appears restricted by climate (TPMVd). Both PSTVd and MPVd have been found naturally in wild Solanum species (Behjatnia et al. 1996; Martinez-Soriano et al. 1996). All of these viroids are able to infect at least one crop species (Lycopersicon esculentum ) and all except TCDVd produce latent or symptomless infection in at least one species. Both PSTVd and TASVd have been found in several countries and TCDVd originated from seed available in at least three countries (Singh et al. 1999). Consequently, these agents have the potential for more widespread occurrence and substantial economic effect on a global basis.

DETECTION Since these viroids all produce similar symptomatic infections in tomato, it is likely that visual inspection and roguing of infected plants may occur based on the assumption that the plants are infected with PSTVd, especially in countries where PSTVd is known to occur. Currently, with the exception of PSTVd, routine testing for these viroids does not occur. The potential for biological indexing exists, as shown in Table 15.2. Implementation would require experimental confirmation of its utility under uniform conditions. The inclusion of ITBTVd is questionable, as it is a strain of CEVd (Mishra et al. 1991) but is probably warranted due to the fact that TASVd is related to CEVd (see Figure 15.1) (Keese and Symons 1985) and would use a different set of host species than those used to index CEVd and other citrus viroids (Duran-Vila et al. 1988). Detection of these agents could also be done by molecular methods, since all except SA-TBTVd have been sequenced. Oligonucleotide probes could be prepared for common or distinct regions, depending on the requirement for a general or specific

129

R.P. Singh et al.

Figure 15.1 Primary and most probable secondary structures of viroids infecting solanaceous plants. Mexican papita viroid (MPVd), Tomato planta macho viroid (TPMVd), PSTVd, Tomato chlorotic dwarf viroid (TCDVd), Citrus exocortis viroid (CEVd), Tomato apical stunt viroid (TASVd), and Columnea latent viroid (CLVd). Accession numbers are indicated in parentheses.

test. Successful RT-PCR assays for direct detection of PSTVd from nucleic acid or sap extracts of infected potato tubers, true seeds, and pollen, as well as from other infected hosts have been reported (Shamloul et al. 1997).

and symptomless infections. It will be particularly difficult for disease harbored silently in wild hosts.

TAXONOMIC POSITION AND NUCLEOTIDE SEQUENCE

CONTROL Incidence of these diseases has been limited and control has involved removal of infected plants from commercial plantings in the single case of TCDVd (Singh et al. 1999), and widespread eradication, as with PSTVd in Canada (see Chapters 3 and 11 — ‘Biology’ and ‘Biological indexing’). Control measures have not been documented for TPMVd, have probably not been attempted for I-TBTVd at least not in citrus plantings, and are likely impossible in the case of MPVd, as it infects a wild species without showing symptoms. However, natural transmission from wild Solanum species to commercial species has not been reported. If these infections are or become widespread, control will require effective detection and elimination of symptomatic

130

Nucleotide sequences have been determined for PSTVd (Gross et al. 1978; Puchta et al. 1990; Herold et al. 1992), TPMVd (Kiefer et al. 1983), MPVd (Martinez-Soriano et al. 1996), TCDVd (Singh et al. 1999), TASVd (Kiefer et al. 1983) and ITBTVd (Mishra et al. 1991) (Figure 15.1) making determination of taxonomic positions possible for this group. Distinct species are those exhibiting less than 90% sequence similarity whereas variants of the same species show greater than 90% sequence similarity (Flores et al. 1998). Phylogenetic relationships of solanaceous viroids based on current information for Pospiviroids are shown in Figure 15.2. Sequence data were used to show that I-TBTVd is a strain of CEVd (Mishra et al. 1991). Although no sequencing informa-


interference or cross protection by PSTVd infection (Singh et al. 1999). Sequence similarity is not uniform throughout the genome (Singh et al. 1999). The left terminal domain (TL), pathogenicity domain (P) and central conserved region (CCR) show over 97% identity to PSTVd. Similarity is only 89% for the right terminal domain (TR) and a surprising 59% in the variable (V) domain, which contains sequences important for replication and accumulation of the viroid, as well as the premelting region (Riesner 1991; Hu et al. 1996). However, the right terminal domain (TR) of TCDVd is 93% similar to the TR of TASVd and Columnea latent viroid (CLVd) (Singh et al. 1999). CLVd was originally isolated from symptomless infection of an ornamental species (Owens et al. 1978), but produces PSTVd-like symptoms in experimental infection of potato and tomato (Hammond et al. 1989). CLVd is similar to viroids in the PSTVd-group in the TL , TR , and P domains, but carries a CCR identical to that of HSVd (Hammond et al. 1989). In contrast, TCDVd shows 93% sequence similarity to MPVd in the TR domain, even though their overall similarity is only 80%. This indicates a closer relationship in the TR domain than to PSTVd -TR, and suggests that TCDVd-TR may be of different origin than the TL-P-C regions.

Figure 15.2 GrowTree phylogram of members of the genus Pospiviroid, indicating the distinct position of the TCDVd in comparison to PSTVd. Distances are estimated number of base substitutions per 100 bases. MPVd, Mexican papita viroid; TPMVd, tomato planta macho viroid; CEVd, citrus exocortis viroid; TASVd, tomato apical stunt viroid; CSVd, chrysanthemum stunt viroid; CLVd, Columnea latent viroid; and IrVd, Iresine viroid. (Reprinted with permission from Singh et al. 1999.)

tion is available for SA-TBTVd, its high rate of seed transmission in some species (McClean 1948), lack of aphid transmission and similarity of host range and symptom expression (McClean 1948; Singh 1973) indicate that it is not related to I-TBTVd, but may have been an isolate of PSTVd. Overall, viroids infecting solanaceous species show greater similarity in their right than left halves (Kiefer et al. 1983, Loss et al. 1991), have a highly conserved central conserved region (CCR) and exhibit strict conservation of the terminal conserved region (TCR) within the left terminal domain (TL) (Koltunow and Rezain 1988) and the core of hairpin II (Loss et al. 1991). TCDVd is the most closely related to PSTVd in this group, with 85–89% sequence similarity, depending on the PSTVd isolate to which it was compared (Singh et al. 1999). This indicates very close relationship but as a distinct species, illustrated as a separate branch in Figure 15.1. This is confirmed by the lack of

MPVd and TPMVd are most closely related to each other, as illustrated by the separate branch of the phylogram (Figure 15.2). They show 89–92% sequence similarity, but their biological properties suggest that they are distinct. Their sequence similarities to PSTVd are low, at 78–80% and 83%, respectively (Kiefer et al. 1983; Martinez-Soriano et al. 1996). A mild strain of PSTVd confers partial cross protection against TPMVd (Galindo et al. 1982), even though PSTVd is more closely related to TCDVd, against which it confers no protection (Singh et al. 1999). TPMVd is even less closely related to TASVd (75%), CEVd (74%) and CSVd (72%) (Kiefer et al. 1983). These latter three are more closely related to each other than to TPMVd. Overall sequence similarity of TASVd to CEVd and CSVd is only 78% and 74%, respectively (Kiefer et al. 1983). However, the sequence similarity between TASVd and CEVd is high in the TL and C regions, at 91% and 99%, respectively (Keese and Symons 1985). In vitro-generated TASVd recombinants containing the TL and P regions of CEVd developed symptoms characteristic of CEVd, demonstrating at least partial exchangeability of these domains (Owens et al. 1990). TASVd strains show 91% sequence similarity but variability is evident in the P and V domains (Candresse et al. 1987; Keese and Symons 1985), particularly in the regions of P adjacent to the CCR (Owens 1990).

131

R.P. Singh et al.

Figure 15.2 reflects the overall relationships between these viroids. Those relationships vary according to domain: the highest sequence conservation in this group occurs in the CCR, then the TR domain, followed by the TL and P domains. References Behjatnia, S.A.A., Dry, I.B., Krake, L.R., Conde, B.D., Connelly, M.I., Randles, J.W., and Rezaian, M.A. (1996). New potato spindle tuber viroid and tomato leaf curl geminivirus strains from a wild Solanum sp. Phytopathology 86, 880-886. Bhattiprolu, S.L. (1991). Studies on a newly recognized disease of Nicotiana glutinosa of vioroid etiology. Plant Dis. 75, 1068-1071. Candresse, T., Smith, D., and Diener, T.O. (1987). Nucleotide sequence of a full- length infectious clone of the Indonesian strain of tomato apical stunt viroid (TASV). Nucleic Acids Res. 15, 10597. Diener, T.O. (1987). Tomato bunchy top. Pages 329-331 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Duran-Vila, N., Roistacher, C.N., Rivera-Bustamante, R., and Semancik, J.S. (1988). A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69, 3069-3080. Flores, R., Randles, J.W., Bar-Joseph, M., and Diener, T.O. (1998). A proposed scheme for viroid classification and nomenclature. Arch. Virol. 143, 623-629. Francki, R.I.B., Zaitlin, M., and Palukaitis, P. (1986). In vivo encapsidation of potato spindle tuber viroid by velvet tobacco mottle virus particles. Virology 155, 469-473. Galindo, J. (1987). Tomato planta macho. Pages 315-320 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Galindo, J. (1988). Ecology and vector of tomato planta macho viroid (TPMV). Pages 28-33 in: Abstracts of Papers, Yamanashi Viroid Disease Workshop, “Possible Viroid Etiology and Detection”, Second meeting of the International Viroid Working Group, August 16-19, 1988, Yamanashi, Japan. Galindo, J., Lopez, M., and Aguilar, T. (1986). Significance of Myzus persicae in the spread of tomato planta macho viroid. Fitopatol. Bras. 11, 400-410. Galindo, J., Smith, D.R., and Diener, T.O. (1982). Etiology of planta macho, a viroid disease of tomato. Phytopathology 72, 49-54. Gross, J.H., Domdey, H., Lossow, C., Jank, P., Raba, M., Alberty, H., and Sänger, H.L. (1978). Nucleotide sequence and secondary structure of potato spindle tuber viroid. Nature 273, 203-208. Hammond, R., Smith, D.R., and Diener, T.O. (1989). Nucleotide sequence and proposed secondary structure of Columnea latent viroid: a natural mosaic of viroid sequences. Nucleic Acids Res. 17, 10083-10094. Herold, T., Haas, B., Singh, R.P., Boucher, A., and Sänger, H.L. (1992). Sequence analysis of five new field isolates demonstrates that the chain length of potato spindle tuber viroid (PSTVd) is not strictly conserved but as variable as in other viroids. Plant Mol. Biol. 19, 329-333. Hu, Y., Feldstein, P.A., Bottino, P.J., and Owens, R.A. (1996). Role of the variable domain in modulating potato spindle tuber viroid replication. Virology 219, 45-56. Keese, P., and Symons, R.H. (1985). Domains in viroids: Evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proc. Natl. Acad. Sci. USA 82, 4582-4586.

132

Kiefer, M.C., Owens, R.A., and Diener, T.O. (1983). Structural similarities between viroids and transposable genetic elements. Proc. Natl. Acad. Sci. USA 80, 6234-6238. Loss, P., Schmitz, M., Steger, G., and Riesner, D. (1991). Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA. EMBO J. 10, 719-727. Martinez-Soriano, J.P., Galindo-Alonso, J., Maroon, J.M., Yucel, I., Smith, D.R., and Diener, T.O. (1996). Mexican papita viroid: putative ancestor of crop viroids. Proc. Natl. Acad. Sci. USA 93, 9397-9401. McClean, A.P.D. (1948). Bunchy-top disease of the tomato: additonal host plants, and the transmission of the virus through the seed of infected plants. South African Department of Agriculture, Botany and Pathology Series No. 11 #256, 28 pages. Mishra, M.D., Hammond, R.W., Owens, R.A., Smith, D.R., and Diener, T.O. (1991). Indian bunchy top disease of tomato plants is caused by a distinct strain of citrus exocortis viroid. J. Gen. Virol. 72, 1-5. O’Brien, M.J., and Raymer, W.B. (1984). Symptomless hosts of the potato spindle tuber virus. Phytopathology 54, 1045-1047. Owens, R.A. (1990). Mutational analysis of viroid pathogenicity: tomato apical stunt viroid. Mol. Plant-Microbe Interact. 3, 374-380. Owens, R.A., Candresse, T., and Diener, T.O. (1990). Construction of novel viroid chimeras containing portions of tomato apical stunt and citrus exocortis viroids. Virology 175, 238-246. Owens, R.A., Smith, D.R., and Diener, T.O. (1978). Measurement of viroid sequence homology by hybridization with complementary DNA prepared in vitro. Virology 89, 388-394. Pandey, P.K., and Summanwar, A.S. (1982). Occurrence of tomato bunchy top virus in India. Curr. Sci. 51, 96. Pfannenstiel, M.A., and Slack, S.A. (1980). Response of potato cultivars to infection by the potato spindle tuber viroid. Phytopathology 70, 922-926. Puchta, H., Herold, T., Verhoeven, K., Roenhorst, A., Ramm, K., SchmidtPuchta W., and Sänger, H.L. (1990). A new strain of potato spindle tuber viroid (PSTVd-N) exhibits major sequence differences as compared to all other PSTVd strains sequenced so far. Plant Mol. Biol. 15, 509-511. Ramachandran, P., Pandey, P.K., Ahlawat, Y.S., Varma, A., and Kapur, S.P. (1992). Viroid diseases of citrus in India. 12th Proceedings, International Organisation of Citrus Virologists, New Delhi. Riesner, D. (1991). Viroids: from thermodynamics to cellular structure and function. Mol. Plant-Microbe Interact. 4, 122-131. Semancik, J.S., Magnuson, D.S., and Weathers, L.G. (1973). Potato spindle tuber disease produced by pathogenic RNA from citrus exocortis disease: evidence for the identity of the causal agents.Virology 52, 292-294. Shamloul, A.M., Hadidi, A., Zhu, S.F., Singh, R.P., and Sagredo, B. (1997). Sensitive detection of potato spindle tuber viroid using RTPCR and identification of a viroid variant naturally infecting pepino plants. Can. J. Plant Pathol. 19, 89-96. Singh, R.P. (1970). Seed transmission of potato spindle tuber virus in tomato and potato. Am. Potato J. 47, 225-227. Singh, R.P. (1971). A local lesion host for potato spindle tuber virus. Phytopathology 61, 1034-1035. Singh, R.P. (1973). Experimental host range of the potato spindle tuber 'virus'. Am. Potato J. 50, 111-123.


Singh, R.P. (1984). Solanum berthaultii, a sensitive host for indexing potato spindle tuber viroid from dormant tubers. Potato Res. 27, 163-172. Singh, R.P. (1988). Occurrence, diagnosis and eradication of the potato spindle tuber viroid in Canada. Pages 37-50 in:Viroids of Plants and their Detection: International Seminar, August 12-20, 1986. Warsaw Agricultural University Press, Warsaw, Poland. Singh, R.P., and Bagnall, R.H. (1968). Solanum rostratum Dunal., a new test plant for the potato spindle tuber virus. Am. Potato J. 45, 335-336. Singh, R.P., Boucher, A., and Somerville, T.H. (1992a). Detection of potato spindle tuber viroid in the pollen and various parts of potato plant pollinated with viroid-infected pollen. Plant Dis. 76, 951-953. Singh, R.P., and Clark, M.C. (1973). Similarity of host response to both potato spindle tuber and citrus exocortis viruses. FAO Plant Prot. Bull. 21, 121-125. Singh, R.P., and Finnie, R.E. (1973). Seed transmission of potato spindle tuber metavirus through the ovule of Scopolia sinensis. Can. Plant Dis. Surv. 53, 153-154. Singh, R.P., Finnie, R.E., and Bagnall, R.H. (1970). Relative prevalence of mild and severe strains of potato spindle tuber virus in eastern Canada. Am. Potato J. 47, 289-293.

Singh, R.P., Finnie, R.E., and Bagnall, R.H. (1971). Losses due to the potato spindle tuber virus. Am. Potato J. 48, 262-267. Singh, R.P., Lakshman, D.K., Boucher, A., and Tavantzis, S.M. (1992b). A viroid from Nematanthus wettsteinii plants closely related to Columnea latent viroid. J. Gen. Virol. 73, 2769-2774. Singh, R.P., Nie, X., and Singh, M. (1999). Tomato chlorotic dwarf viroid: an evolutionary link in the origin of pospiviroids. J. Gen. Virol. 80, 2823-2828. Spieker, R.L. (1996). A viroid from Brunfelsia undulata closely related to the Columnea latent viroid. Arch. Virol. 141, 1823-1832. Spieker, R.L., Marinkovic, S., and Sänger, H.L. (1996). A viroid from Solanum pseudocapsicum closely related to the tomato apical stunt viroid. Arch. Virol. 141, 1387-1395. Walter, B. (1981). Un viroïde de la tomate en Afrique de l’ouest: identité avec le viroïde du “Potato Spindle Tuber”? C. R. Acad. Sci. 292, 537. Walter, B. (1987). Tomato apical stunt. Pages 321-328 in: The viroids. T. O. Diener, ed. Plenum Press: New York. Wassenegger, M., Spieker, R.L., Thalmeir, S., Gast, F., Riedel, L., and Sänger, H.L. (1996). A single nucleotide substitution converts potato spindle tuber viroid (PSTVd) from noninfectious to an infectious RNA for Nicotiana tabacum. Virology 226, 191-197. Werner, H.O. (1924). Spindle-tuber, the cause of ‘run out’ potatoes. Nebraska Potato Improv. Assoc. 6, 57-79.

133

PART IV

CUCUMBER VIROIDS

....................................................................................................

CHAPTER 16

HOP STUNT VIROID IN CUCUMBER T. Sano

.................................................................................................................................................................................................................................................................

Cucumber pale fruit disease was first recognized in 1963 in two greenhouses in the western part of the Netherlands, and has been observed in different cucumber growing areas over the whole country. The disease spread slowly and the number of affected plants in a greenhouse was usually less than 0.1% (Van Dorst and Peters 1974). The severity of the symptom expression strongly depends on a high incubation temperature. The most distinctive symptom is found on the fruits. The infected fruits are pale green in color, slightly pear-shaped and retarded in growth (Plate 1C). The infected flowers are also abnormal in shape; the edge of the petal is slightly notched and looks shriveled (see Plate 1B). The leaf blades are small and undulated, their edges turned downward. The internodes of the younger parts of the infected plants are shorter than those of healthy plants, and the whole plant becomes stunted (Van Dorst and Peters 1974).

HOST RANGE The natural host of the viroid [which was previously known as cucumber pale fruit viroid (CPFVd)] is cucumber. The viroid infects experimentally 30 cucurbitaceous, two composite, and 11 solanaceous plants (Van Dorst and Peters 1974; Peters and Runia 1985). Cucumber (Cucumis sativus) is the most sensitive

134

host plant for the viroid, and Benincasa hispida may be the most useful test plant for the viroid because its incubation period is short and symptoms are the most pronounced (Van Dorst and Peters 1974). As described later in this chapter, the nucleotide sequence of the viroid is very similar or almost identical to that of Hop stunt viroid found in citrus (HSVd-citrus or CVd-II) (Sano et al. 1984b, 1988b; Puchta et al. 1988, 1989). Based on this similarity, citrus may also be added to the natural host plants of the viroid. Moreover, the viroid in cucumber was recognized as a strain of HSVd.

TRANSMISSION Most diseased plants are found from the beginning of April through May, which seemingly are infected at the end of January to March. Occasionally some diseased plants are found in June (having been planted in May); however, no infection has ever been found in those planted in July or August (Van Dorst and Peters 1974). The viroid is transmissible by crude sap and can be more efficiently transmitted by slashing the stems with a razor blade. Infectivity is also enhanced by addition of bentonite to the homogenate (Yoshizaki et al. 1985). The viroid is easily transmitted during pruning and by grafting. Since diseased

HOP STUNT VIROID IN CUCUMBER

plants usually occur in the same row near those that first developed symptoms, transmission through pruning seems to play an important role for the spread of the viroid in a greenhouse. The viroid is also transmitted experimentally by dodder (Cuscuta subinclusa) from cucumber to some cucurbitaceous species. Since the first diseased plants are often found near the outer walls of the greenhouse or close to the main walk, Van Dorst and Peters (1974) suggested insect transmission of the disease. The disease, however, was not transmitted either by Myzus persicae after a short (3 hours) or a long (one week) feeding period or through soil samples collected around the first diseased plants. The fact that the location of the greenhouse where the disease is found usually differs from year to year also supports the conclusion that the agent is not soil-borne. Preliminary experiments also indicated that the disease was not transmitted through seeds (Van Dorst and Peters 1974).

GEOGRAPHICAL DISTRIBUTION AND EPIDEMIOLOGY The disease was first observed in the western part of the Netherlands in 1963. The number of greenhouses with affected plants increased gradually from 1965, and it was recognized in all the cucumber growing areas in the Netherlands by 1972 (Van Dorst and Peters 1974).

DETECTION The viroid can be detected by inoculation to cucumber (Sano et al. 1981). Molecular hybridization or RT-PCR is also a useful detection method (Yang et al. 1992). The viroid can be discriminated from HSVd-hop or -grapevine, the closely related variants, by denaturing polyacrylamide gel electrophoresis in the presence of 8M urea (Sano et al. 1984a), or by a variant-specific synthetic oligonucleotide probe (Sano et al. 1988a).

CONTROL Since the disease spreads in the greenhouse mainly by pruning, the diseased plants including several healthy-looking neighbors in the same row should be eliminated as quickly as possible. To eradicate the disease, it is necessary to know the source of the pathogen and the route of invasion from the source to cucumber, because the disease incidence is relatively high in a greenhouse where the disease has occurred in previous years. It is a mystery where the causal agent survives for the next invasion to cucumber, because cucumber is an annual seed-propagated crop and no insect-, soil- and seed-transmission of the causal agent has been observed.


Comparative analysis of the host range, symptomatology and gel electrophoretic nature of the causal agent of cucumber pale

fruit disease and of HSVd reported in hop plants in Japan, suggested that these viroids are very similar (Sano et al. 1981; Ohshima et al. 1988). The dissimilarity of these viroids was first indicated in the molecular size by gel electrophoretic analysis under denaturing conditions (Uyeda et al. 1984; Sano et al. 1984a), and later by nucleotide sequencing (Sano et al. 1984b). The viroid from cucumber is composed of 303 nucleotides, 6 nucleotides larger than the HSVd type (Ohno et al. 1983), and differs from the HSVd at 16 positions, resulting in about 95% sequence homology. Later, a cucumber variant of 301 nucleotide in length was reported (Puchta et al. 1988). Although the cucumber viroid and HSVd were isolated independently from different diseases affecting different host species in different countries, the cucumber viroid is now considered to be a strain (or isolate) of HSVd based on the similarity of the nucleotide sequence (Sano et al. 1984b). Furthermore, similar or almost identical HSVd variants are found latently infecting citrus trees all around the world (Sano et al. 1988b; Puchta et al. 1989). The isolate from ‘Etrog’ citron (Sano et al. 1988b), for example, differs in two positions from the cucumber viroid of 303 nucleotides (Sano et al. 1984b), and only in one deletion from the cucumber viroid of 301 nucleotides (Puchta et al. 1989). References Ohno, T., Takamatsu, N., Meshi, T., and Okada, Y. (1983). Hop stunt viroid; molecular cloning and nucleotide sequence of the complete cDNA copy. Nucleic Acids Res. 11, 6187-6197. Ohshima, K., Sano, T., Uyeda, I., and Shikata, E. (1988). Comparative studies on host range and the infectivity of hop stunt viroid — cucumber isolate (cucumber pale fruit viroid) native RNA and its cDNA. Arch. Phytopathol. Pflanzenschuts, Berlin 24, 475-484. Peters, D., and Runia, W.T. (1985). The host range of viroids. Pages 21-38 in: Subviral pathogens of plants and animals: viroids and prions. K. Maramorosch, and J. J. McKelvey, eds. Academic Press: New York. Puchta, H., Ramm, K., and Sänger, H.L. (1988). Molecular and biological properties of a cloned and infectious new sequence variant of cucumber pale fruit viroid (CPFV). Nucleic Acids Res. 16, 8171. Puchta, H., Ramm, K., Hadas, R., Bar-Joseph, M., Luckinger, R., Freimuller, K., and Sänger, H. L. (1989). Nucleotide sequence of a hop stunt viroid (HSVd) isolate from grapefruit in Israel. Nucleic Acids Res. 17, 1247. Sano, T., Sasaki, M., and Shikata, E. (1981). Comparative studies on hop stunt viroid, cucumber pale fruit viroid and potato spindle tuber viroid. Ann. Phytopathol. Soc. Jpn. 47, 599-605. Sano, T., Uyeda, I., and Shikata, E. (1984a). Comparative studies of hop stunt viroid and cucumber pale fruit viroid by polyacrylamide gel electrophoretic analysis and electron microscopic examination. Ann. Phytopathol. Soc. Jpn. 50, 339-345. Sano, T., Uyeda, I., Shikata, E., Ohno, T., and Okada, Y. (1984b). Nucleotide sequence of cucumber pale fruit viroid: homology to hop stunt viroid. Nucleic Acids Res. 12, 3427-3434. Sano, T., Kudo, H., Sugimoto, T., and Shikata, E. (1988a). Synthetic oligonucleotide hybridization probes to diagnose hop stunt viroid strains and citrus exocortis viroid. J. Virol. Methods 19, 109-120.

135

T. Sano

Sano, T., Hataya, T., and Shikata, E. (1988b). Complete nucleotide sequence of a viroid isolated from Etrog citron, a new member of hop stunt viroid group. Nucleic Acids Res. 16, 347. Uyeda, I., Sano, T., and Shikata, E. (1984). Purification of cucumber pale fruit viroid. Ann. Phytopathol. Soc. Jpn. 50, 331-339. Van Dorst, H. J. M., and Peters, D. (1974). Some biological observations on pale fruit, a viroid-incited disease of cucumber. Neth. J. Pl. Pathol. 80, 85-96.

136

Yang, X., Hadidi, A., and Garnsey, S. M. (1992). Enzymatic cDNA amplification of citrus exocortis and cachexia viroids from infected citrus hosts. Phytopathology 82, 279-285. Yoshizaki, T., Sano, T., Uyeda, I., and Shikata, E. (1985). The effects of some chemicals on the infectivity of cucumber isolate of hop stunt viroid (cucumber pale fruit viroid). Ann. Phytopathol. Soc. Jpn. 51, 405-412.

PART IV

POME FRUIT VIROIDS APPLE SCAR SKIN VIROID IN APPLE H. Koganezawa, X. Yang, S.F. Zhu, J. Hashimoto, and A. Hadidi

....................................................................................................

CHAPTER 17

.................................................................................................................................................................................................................................................................

Apple scar skin disease was first reported as Manchurian apple ‘Sabika’ disease from Manchuria, China (Ohtsuka 1935, 1938). In 1986, Koganezawa suggested the viroid etiology of the disease. The causal agent, Apple scar skin viroid (ASSVd) (Hashimoto and Koganezawa 1987; Puchta et al. 1990; Yang et al. 1992), is now recognized as the prototype of the genus Apscaviroid (Flores et al. 2000). Dapple apple disease was first described in the 1950s from an apple orchard in New Hampshire (Smith et al. 1956; Barrat et al. 1958). Subsequently, the disease was reported from Canada, Japan, China and UK (Welch and Keene 1961; Yamaguchi et al. 1975; Campbell and Sparks 1976). Hadidi et al. (1990) presented evidence which indicated that dapple apple disease is caused by a viroid that is highly homologous to ASSVd. These results were confirmed by RT-PCR, nucleotide sequence analysis of the infectious agent, and the successful agro-infection of pear and apple seedlings with cloned viroid (Hadidi and Yang 1990; Zhu et al. 1995, 1998). Thus dapple apple disease is caused by a variant of ASSVd.

ECONOMIC IMPACT AND SYMPTOMATOLOGY Apple scar skin disease caused serious economic losses in China. According to surveys conducted in 1950s in China, nearly

10,000 apple trees were affected with the disease around Liadong Bandao peninsula area and approximately 360,000 apple trees were affected in Liaoning Province. In some counties of Shanxi, Hebei and Shenxi Provinces, more than 50% of apple trees were affected with the disease (Liu et al. 1957). In Japan, the disease was first found in 1953 (Ushirozawa et al. 1968). In the 1970s, the disease was observed in every major apple growing area of Japan, though its occurrence is sporadic. The entire crop from apple scar skin affected apple trees is unmarketable. Fruit scar skin or dapple symptoms (see Plate 3 lower figures), as well as both symptoms together can appear depending on the apple cultivar. Dapple symptoms generally appear on redskinned cultivars like ‘Jonathan’ and ‘Red Gold’ and become obvious near harvesting time. Scar skin symptoms appeared on ‘Ralls Janet’ and ‘Indo’, and both appeared on ‘Starking Delicious’ and ‘Red Delicious’. In most cases, these symptoms are concentrated near the calyx end of the fruit. On ‘Indo’, one of the most susceptible varieties, scars appear around the calyx end of the fruit beginning about 5 weeks after the full-bloom stage of bud development. The scarring gradually increases until by harvest it may cover more than 50% of the fruit surface. On ‘Ralls Janet’, water-soaked blotches appear first, followed by scar tissue, and finally a cracking of the blotches. The scar skin symptoms become more pronounced each year, while the dappling

137

H. Koganezawa et al.

becomes less obvious (Yamaguchi and Yanase 1976). Fruits of the cultivar ‘Ohrin’ express green depressed spots with fresh necrosis that resemble symptoms caused by Apple fruit crinkle viroid and Apple dimple fruit viroid (T. Ito, personal communication). The severity of the diseases depends on cultivars. Liu et al. (1962) classified 83 apple cultivars into 5 groups from highly tolerant to highly susceptible. Desvignes et al. (1999) also classified 42 apple cultivars into 5 groups ranging from inconspicuous spots to severely scarred skin and cracking. According to the authors, tolerant cultivars are ‘Golden Delicious’, ‘Granny Smith’, ‘Pink Lady’ and others. The symptoms are usually restricted to the fruit in most cultivars, but leaf roll or leaf epinasty symptoms develop on certain apple cultivars under specific conditions. The cultivars ‘Stark’s Earliest’ and ‘Sugar Crab’ express leaf epinasty when maintained for 24-hr photoperiods at 18 or 28°C (Skrzeczkowski et al. 1993). Similar symptoms appear on ‘Ralls Janet’ (Liu et al. 1957). Ito and Yoshida (1993) analyzed the factors required for the expression of the symptoms on ‘Ralls Janet’. The symptoms were obvious when i

the shoot of the indicator extends to more than 80 cm in final length;

ii

double grafting was made in April to May to increase viroid titer to a certain level; and

iii

indicator plants were maintained at 25°C. Higher temperatures near 30°C tended to suppress symptom expression.

HOST RANGE Probably all Malus and Pyrus species are susceptible to ASSVd infection (Desvignes et al. 1999). Most commercial pear cultivars do not develop symptoms, but variants of ASSVd may cause pear rusty skin in China (Zhu et al. 1995), pear fruit dimple in Japan (Osaki et al. 1996), pear rusty skin-like symptoms on Italian pear, pear fruit russetting, scarring, and/or cracking in Greece (Kyriakopoulou et al. 2001), and pear fruit crinkle in China (X. Yang, and A. Hadidi, unpublished).

slashing (Koganezawa 1985). Several investigators observed that the disease spread naturally to neighboring trees (Ohtsuka 1935; Ushirozawa et al. 1968; Desvignes et al. 1999). The transmission to neighboring trees may be due to root grafts. ASSVd was detected in apple seed, especially seed coat and subcoat, which indicated that the viroid is seed-borne (Hadidi et al. 1991). Vertical transmission of ASSVd from seed to seedling, however, has not yet been elucidated. Ushirozawa et al. (1968) first examined the seed-transmissibility of ASSVd. They produced 27 seedlings from 3 cultivars of affected trees. None of the seedlings showed viroid symptoms. Howell et al. (1995) collected apple seeds from fruit affected by 4 isolates of ASSVd in 2 growing seasons (1994 and 1995) from 5 seed sources and examined seed-transmissibility of ASSVd. All of more than 400 seedlings were free of ASSVd. Desvignes et al. (1999) also obtained similar results using 400 apple seedlings originated from ‘Starkrimson’ and ‘Indo’ fruits with typical ASSVd symptoms. These observations suggest that ASSVd is seed-borne but not seed-transmitted.

GEOGRAPHICAL DISTRIBUTION AND EPIDEMIOLOGY Apple scar skin disease is widespread in East Asia. Recently the disease was reported from India (Behl et al. 1998; Thakur et al. 1995). The disease has also been reported from the USA (Smith et al. 1956; Millikan and Martin 1956), Canada (Welsh and Keene 1961; Hadidi et al. 1991), the UK (Campbell and Sparks 1976), but its incidence is relatively rare in North America and Europe. In France, ASSVd has never been observed in commercial orchards, indicating that this viroid is absent or extremely infrequent (Desvignes et al. 1999). ASSVd was only detected in Japanese sources imported to serve as positive controls. Dapple apple was once reported from Italy (Giunchedi 1976), but now it is attributed to Apple dimple fruit viroid (Di Serio et al. 1998; Loreti et al. 1998).

Desvignes et al. (1999) inoculated ASSVd to 4 Prunus, 13 Malus, 17 Pyrus, and 17 other pomaceous species. All the species tested of Malus, Pyrus, Sorbus, Chaenomeles, Cydonia, and Pyronia were susceptible to ASSVd. The viroid was not detected in the tested species of Amelanchier, Aronia, Cotoneaster, Crataegus, Prunus, and Pyracantha. Although several attempts have been made, no herbaceous host has been identified (Hadidi et al. 1991).

Many pear trees which originated in China are latently infected with ASSVd (Liu et al. 1962, 1985; Zhu et al. 1995; Hurtt and Podleckis 1995). Surveys conducted from 1960 to 1962 in China showed that nearly 90% of pear trees carry ASSVd (Liu et al. 1962). High infection of ASSVd was observed in apple trees neighboring pear orchards (Liu et al. 1957). Ohtsuka (1935) noted that diseased apple trees were found as a line in a field adjacent to a pear orchard, suggesting that transmission of ASSVd from pear to apple might occur. In particular apple trees mix-planted with pear were highly affected with scar skin (Liu et al. 1957). ASSVd in pear has been recently identified in Italy and Greece (Kyriakopoulou et al. 2001).

TRANSMISSION

DETECTION

There are no known vectors of ASSVd. ASSVd is easily transmitted by grafting. Experimentally it is also transmitted by razor

ASSVd can be indexed by graft-inoculation to the fruit-bearing apple trees such as ‘Red Delicious’, ‘Sugar Crab’ and ‘NY11894’

138

APPLE SCAR SKIN VIROID IN APPLE

Figure 17.1 Nucleotide sequence and secondary structure of dapple apple variant of ASSVd. (From Zhu et al. 1995.)

in an orchard or in a greenhouse. These assays require 2 or more years to complete. Howell and Mink (1992) described a more rapid bioassay in which the inocula are budded to the indicator ‘Stark’s Earliest’ or ‘Sugar Crab’ grown in growth chambers under a 24-hr photoperiod. Positive reactions are the induction of leaf curling or leaf epinasty within 2 months. As the titer of ASSVd in apples is relatively high, it can be detected easily by return, two-dimensional, or bidirectional polyacrylamide gel electrophoresis (PAGE) analysis of the extracted RNA. Molecular hybridization assays using 32P- or digoxigeninlabeled ASSV cRNA probes have been developed for detection of the viroid from apple tissue (Hadidi et al. 1990; Podleckis et al. 1993). These assays are rapid, accurate and easy to perform. Li et al. (1995) compared the reliability of three methods, return PAGE, hybridization using DIG-labeled DNA or RNA probe. The return-PAGE using 0.5 g tissue was reliable for diagnosis of ASSVd in apple, because of a relatively high viroid titer. Although DIG-labeled DNA probe was 2.5 to 25 times more sensitive than return-PAGE, the practical reliability of this method was not so superior to return-PAGE. DIG-labeled cRNA probe was 25–125 fold and 100–625 fold more sensitive than DIG-labeled cDNA probe and return-PAGE, respectively for ASSVd detection. RT-PCR is the most sensitive method for the detection of ASSVd (Hadidi and Yang 1990; Hadidi et al. 1990; Zhu et al. 1995). ASSVd may also be detected by RT-PCR-ELISA (Shamloul and Hadidi 1999).

CONTROL Quarantine regulations in most countries require ASSVd assay in imported pome fruit germplasm to prevent introduction of this viroid into imported countries. The major local control

measure for the disease is to propagate the nursery stock from ASSVd-indexed mother trees and to remove affected trees from orchards to avoid viroid spread to neighboring trees. ASSVd was eliminated from most infected apple plants when plants were subjected to a dormant stage followed by thermotherapy (38°C for 70 days or at 36 –37°C for 48 days) and shoot tip grafting (Howell et al. 1998; Desvignes et al. 1999). Temperature of 36–37°C for 41 days or 99 days, without a dormant stage, eliminated viruses but not ASSVd (Desvignes et al. 1999).


Family: Pospiviroidae Genus: Apscaviroid Species: Apple scar skin viroid (ASSVd) Variants of ASSVd consist of 299–234 nucleotides (Hashimoto and Koganezawa 1987; Puchta et al. 1990; Yang et al. 1992; Zhu et al. 1995). Nucleotide sequence of each variant can be arranged into the rod-like secondary structure characteristics of Pospiviroidae. The Canadian dapple apple variant of ASSVd (301 nucleotides, Figure 17.1) shares 97% identity with the 330 nucleotide Japanese type strain of ASSVd (Hashimoto and Koganezawa 1987) and differs at 9 sites (Zhu et al. 1995). Most of the deletions, insertions and changes among variants of ASSVd occur in regions corresponding to the pathogenicity and left terminal domains of ASSVd (Hashimoto and Koganezawa 1987; Puchta et al. 1990; Yang et al. 1992; Zhu et al. 1995) which are responsible for pathogenecity variation in Potato spindle tuber viroid (Sano et al. 1992). References Barrat, J. G., Smith, W. W., and Rich, A. E. (1958). Transmission of the dapple apple virus. Phytopathology 48, 260.

139

H. Koganezawa et al.

Behl, M. K., Khurana, S. M. P., and Parakh, D. B. (1998). Bumpy fruit and other viroid and viroid-like diseases of apple in HP, India. Acta Hortic. 472, 627-629. Campbell, A. I., and Sparks, T. R. (1976). Experiments with dapple apple virus. Acta Hortic. 67, 261-264. Desvignes, J. C., Grasseau, N., Boyé, R., Cornaggia, D., Aparicio, F., Di Serio, F., and Flores, R. (1999). Biological properties of apple scar skin viroid: isolates, host range, different sensitivity of apple cultivars, elimination, and natural transmission. Plant Dis. 83, 768-772. Di Serio, F., Alioto, D., Ragozzino, A., Giunchedi, L., and Flores, R. (1998). Identification of apple dimple fruit viroid in different commercial varieties of apple grown in Italy. Acta Hortic. 472, 595-601. Flores, R., Randles, J. W., Bar-Joseph, M., and Diener, T. O. (2000). Subviral agents: Viroids. Pages 1009-1024 in: Virus taxonomy, 7th Report of the International Committee on Taxonomy of Viruses. M. H. V. van Regenmortel, C. M. Fauquet, D. H. L., Bishop, E. P., Carstens, M. K., Estes, S. M., Lemon, J., Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle, and R. B. Wickner, eds. Academic Press: San Diego, CA. Giunchedi, L. (1976). Transmission experiments on the virus disease dapple apple. Acta Hortic. 67, 255-260. Hadidi, A., Hansen, A. J., Parish, C. L., and Yang, X. (1991). Scar skin and dapple apple viroids are seed-borne and persistent in infected apple trees. Res. Virol. 142, 289-296. Hadidi, A., Huang, C., Hammond, R.W., and Hashimoto, J. (1990). Homology of the agent associated with dapple apple disease to apple scar skin viroid and molecular detection of these viroids. Phytopathology 80, 263-268. Hadidi, A., and Yang, X. (1990). Detection of pome fruit viroids by enzymatic cDNA amplification. J. Virol. Methods 30, 261-270. Hashimoto, J., and Koganezawa, H. (1987). Nucleotide sequence and secondary structure of apple scar skin viroid. Nucleic Acids Res. 15, 7045-7051. Howell, W. E., Burgess, J., and Mink, G. I., and Zhang, Y. P. (1998). Elimination of apple fruit and bark deforming agents by heat therapy. Acta Hortic. 472, 641-646. Howell, W. E., and Mink, G. I. (1992). Rapid biological detection of apple scar skin viroid. Acta Hortic. 309, 291-295. Howell, W. E., Skrzeczkowski, L. J., Wessels, T., Mink, G. I., and Nunez, A. (1995). Non-transmission of apple scar skin viroid and peach latent mosaic viroid through seed. Acta Hortic. 472, 635-639. Hurtt, S. S., and Podleckis, E. V. (1995). Apple scar skin viroid is not seed transmitted or transmitted at a low rate in oriental pear. Acta Hortic. 386, 544-550. Ito, T., and Yoshida, K. (1993). Factors for expression of leaf roll symptoms on young tree of apple cv. ‘Ralls Janet’ caused by apple scar skin viroid. Bull. Fruit Tree Res. Stn 25, 87-100. Koganezawa, H. (1985). Transmission to apple seedlings of low molecular weight RNA from apple scar skin diseased trees. Ann. Phytopathol. Soc. Jpn. 51, 176-182. Koganezawa, H. (1986). Further evidence for viroid etiology of apple scar skin and dapple diseases. Acta Hortic. 193, 29-33. Koganezawa, H., Yanase, H., and Sakuma, T. (1982). Viroid-like RNA associated with apple scar skin (or dapple apple) disease. Acta Hortic. 130, 193-197. Kyriakopoulou, P. E., Giunchedi, L., and Hadidi, A. (2001). Peach latent mosaic and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdaliformis: Implications on

140

possible origin of these viroids in the Mediterranean region. J. Plant Pathol. 83, 51-62. Li, S. F., Sano, T., Yoshida, K., Wang, G. P., and Shikata, E. (1995). Gene diagnosis of viroids: Comparisons of return-PAGE and hybridization using DIG-labeled DNA and RNA probes for practical diagnosis of hop stunt, citrus exocortis and apple scar skin viroids in their natural host plants. Ann. Phytopathol. Soc. Jpn. 61, 381-390. Liu, F.-C, Chen, R.-F, and Chen, Y.-X. (1957). Apple scar skin disease. Academia Sinica Printery, Beijing (in Chinese). Liu, F.-C., Wang, S.-Y, and Tian, C.-F., and Chen, R,-F. (1962). Studies on apple scar skin disease (1960–1962). Ann Rep. Fruit. Inst., Chinese Acad. Agr. Sci. 1962, 71-79 (in Chinese). Liu, F.-C., Wang, S.-Y., Chen, C., and Chen, Y.-X. (1985). Research on the relationship between apple scar skin disease and pear trees. China Fruit 1, 36-39 (in Chinese). Loreti, S., Faggioli, F., Barrale, R., and Barba, M. (1998). Occurrence of viroids in temperate fruit trees in Italy. Acta Hortic. 472, 555-559. Milikan, D. F., and Martin, W. R. Jr. (1956). An unusual fruit symptom in apple. Plant Dis. Reptr. 40, 229-230. Ohtsuka, Y. (1935). A new disease of apple, on the abnormality of fruit. J. Jap. Soc. Hort. Sci. 6, 44-53 (in Japanese). Ohtsuka, Y. (1938). On Manshu-sabika-byo of apple, graft transmission and symptom variation in cultivars. J. Jap. Soc. Hort Sci. 9, 282-286 (in Japanese). Osaki, H., Kudo, A., and Ohtsu, Y. (1996). Japanese pear fruit dimple disease caused by apple scar skin viroid (ASSVd). Ann. Phytopathol. Soc. Jpn. 62, 379-385. Podleckis, E. V., Hammond, R. W., Hurtt, S. S., and Hadidi, A. (1993). Chemiluminescent detection of potato and pome fruit viroids by digoxigenin-labeled dot blot and tissue blot hybridization. J. Virol. Methods. 43, 147-158. Puchta, H., Luckinger, R., Yang, X., Hadidi, A., and Sänger, H. L. (1990). Nucleotide sequence and secondary structure of apple scar skin viroid (ASSVd) from China. Plant Mol. Biol. 14, 1065-1067. Sano, T., Candresse, T.,Hammond, R. W., Diener, T. O., and Owens, R. A. (1992). Identification of multiple structural domains regulating viroid pathogenecity. Proc. Natl. Acad. Sci. USA 89, 10104-10108. Shamloul, A., M., and Hadidi, A. (1999). Sensitive detection of potato spindle tuber viroid and temperate fruit tree viroids by reverse transcription-polymerase chain reaction-probe capture hybridization. J. Virol. Methods 80, 145-155. Skrzeczkowski, L. J., Howell, W. E., and Mink, G. I. (1993). Correlation between leaf epinasty symptoms on two apple cultivars and results of cRNA hybridization for detection of apple scar skin viroid. Plant Dis. 77, 919-921. Smith, W. W., Barrat, J. G., and Rich, A. E. (1956). Dapple apple, unusual fruit symptom of apples in New Hampshire. Plant Dis. Reptr. 40, 765-766. Thakur, P. D., Ito, T., and Sharma, J. N. (1995). Natural occurrence of a viroid disease of apple in India. Indian J. Virol. 11, 73-75. Ushirozawa, K., Tojo, Y., Takemae, S., and Sekiguchi, A. (1968). Studies on apple scar skin disease (1) On transmission experiments. Bull. Nagano Hort. Res. Stn., Jpn. 9, 1-12 (in Japanese with English summary). Welsh, M. F., and Keene, F. W. L. (1961). Diseases of apple in British Columbia that are caused by viruses or have characteristics of virus diseases. Can. Plant Dis. Surv. 41, 123-147.

APPLE SCAR SKIN VIROID IN APPLE

Yamaguchi, A., and Yanase, H. (1976). Possible relationship between the causal agent of dapple apple and scar skin. Acta Hortic. 67, 249-254. Yamaguchi, A., Yanase, H., and Koganezawa, H. (1975). Graft transmission of dapple apple. Bull. Fruit. Tree Res. Stn. Ser. C2, 73-79. Yang, X., Hadidi, A., and Hammond, R. W. (1992). Nucleotide sequence of apple scar skin viroid reverse-transcribed in host extracts and amplified by the polymerase chain reaction. Acta Hortic. 309, 305-309.

Zhu, S. F., Hadidi, A., Hammond, R. W., Yang, X., and Hansen, A. J. (1995). Nucleotide sequence and secondary structure of pome fruit viroids form dapple diseased apples, pear rusty skin diseased pears and apple scar skin symptomless pears. Acta Hortic. 386, 554-559. Zhu, S. F., Hammond, R. W., and Hadidi, A. (1998). Agroinfection of pear and apple with dapple apple viroid result in systemic infection. Acta Hortic. 472, 613-616.

141

PART IV

CHAPTER 18

APPLE SCAR SKIN VIROID IN PEAR ....................................................................................................

P.E. Kyriakopoulou, H. Osaki, S.F. Zhu, and A. Hadidi

.................................................................................................................................................................................................................................................................

Pear is a host of Apple scar skin viroid (ASSVd). The viroid may infect pear trees without showing symptoms on infected fruit (Hadidi et al. 1991; Hurtt et al. 1992) or it may cause a variety of symptoms on the diseased fruit. Pear rusty skin disease was first reported from China in the late 1980s (Chen et al. 1987). The agent associated with the disease was found to be related to ASSVd by RT-PCR (Hadidi and Yang 1990); nucleotide sequence analysis of the viroid demonstrated that it is a distinct variant of ASSVd (Zhu et al. 1995). Abnormal fruit disorders consisting of dimpling of mature fruit surface of Japanese pear cultivars ‘Nitaka’ and ‘Yoshino’ were observed in Chiba, Ibaraki, and Oita Prefectures (Ohtsu et al. 1990). ASSVd was identified as the causal agent of the fruit disorder (Osaki and Kudo 1995; Osaki et al. 1996). In the late 1980s a disease similar to pear rusty skin disease in China (Zhu et al. 1995) was observed on fruits of 6–8 year old trees of cultivar ‘Passacrassana’ grafted on quince rootstocks in Emilia-Romagna in central Italy (Kyriakopoulou et al. 2001). Moreover, in recent years in Greece, it has been recognized that pear (Pyrus communis Linn) and wild pear (P. amygdaliformis Vill) trees have diseases of uncertain etiology that affect the quality and yield of fruits (Kyriakopoulou and Hadidi 1998).

142

ASSVd was found to be associated with diseased cultivated pear trees in Italy and Greece and with wild pear trees in Greece (Kyriakopoulou et al. 2001).

ECONOMIC IMPACT AND SYMPTOMATOLOGY Many pear trees originating from China are latently infected with ASSVd (Liu et al. 1962, 1985; Hadidi et al. 1991; Hurtt et al. 1992). These trees are a source of ASSVd infection to apple trees in a high viroid infection rate indicated by developing scar skin symptoms on infected apple fruits. These symptoms were observed in apple trees neighboring pear orchards and apple trees mix-planted with pear trees (Liu et al. 1957) and made their fruit unmarketable. Rusty skin disease renders pear fruit unmarketable. The disease symptoms are restricted to the fruit. Chinese pears such as cultivar ‘Muoli’ shows wide lines and patches of rusty appearance on the skin of the fruit. Pear fruit dimple disease causes pear fruits to lose their market value. As its occurrence is relatively rare in Japan, the disease is not economically important. The characteristic symptoms consist of dimpling of the mature fruit surface (see Plate 4A–C), and this symptomology seems to be more severe in a hot summer.

APPLE SCAR SKIN VIROID IN PEAR

The symptoms are restricted to fruits, whereas other tree parts show no symptoms. Consequently, farmers cannot identify this disorder until Japanese pear trees are at the productive stage. Symptoms of European and wild pears induced by ASSVd infection are usually serious. Cases are reported in Greece where young pear orchards have been abandoned due to this disease. Symptoms include fruit russeting, scarring, and/or cracking of pear (Pyrus communis Linn) and wild pear (P. amygdaliformis Vill) (see Plate 4E and F) which reduce fruit value or render it unmarketable. Symptoms on other parts of pear trees are not known. Pear and wild pear trees are often also infected with Pear blister canker viroid whose symptoms develop on stems (trunk, branches, twigs, pedicels). ASSVd was experimentally transmitted to several pear species: Pyrus amygdaliformis, P. betulifolia, P. calleryana, P. canescens, P. communis, P. nivalis, P. persica, P. pyrifolia, P. salicifolia, P. ussuriensis, P. X michauxii, and Pyrus hybrids with Malus, Sorbopyrus, and Sorbaronia (Desvignes et al. 1999). For additional hosts of ASSVd, see Chapter 17 on ASSVd in apple.

TRANSMISSION ASSVd is transmitted to pear seedlings by grafting. It is also transmitted by agroinfection (Zhu et al. 1998). The viroid is not transmitted by the direct knife cut method in Japanese pear (Osaki et al. 1996). ASSVd is not seed transmitted, or transmitted at a low rate, in oriental pear (Hurtt and Podleckis 1995).

GEOGRAPHICAL DISTRIBUTION AND EPIDEMIOLOGY ASSVd-infected symptomless pear and pear fruit with rusty skin symptoms originated in China (Zhu et al. 1995). Pear fruits infected with ASSVd and with symptoms similar to that of rusty skin on the Italian cultivar ‘Passacrassana’ were reported from the Emilia-Romagna region of central Italy (see Plate 4D) (Kyriakopoulou et al. 2001). Pear fruit dimple disease was reported from Japan (Osaki et al. 1996). It was identified in Japanese pear cultivars ‘Niitaka’ and ‘Yoshino’, in Chiba, Ibaraki and Oita Prefectures. The viroid was thought to be spread by grafting. European and wild pear with fruits showing russetting, cracking and/or scarring symptoms were identified in Greece. Disease symptoms are widespread in Greece and with high frequency in the spontaneous wild pear shrubs/trees and pear orchards or isolated pear trees traditionally grafted onto wild pear or on modern rootstocks such as quince. Several commercial or local Greek cultivars, like the high valued early ‘Kontoula’ and other local cultivars such as ‘Vaviza’, ‘Aleurapidia’, ‘Cheimonapidia’, and ‘Theristapidia’ of Olympia Prefecture, as well as the international cultivars ‘Bartlet’, ‘Krystalli’ (Napoleon), and ‘Santa Maria’ bear affected fruit and are infected with ASSVd (Kyria-

kopoulou et al. 2001). The viroid is thought to be transmitted by grafting onto affected wild pear rootstock, the traditional pear and other pome fruit rootstock in Greece and by using infected scions, and spread through using infected propagation material. Its widespread and highly frequent occurrence in Greece in wild pear sites unapproachable to man indicate that Greece and probably other Mediterranean places are a second known origin of ASSVd after NE Asia. It also indicates that there is some effective means of natural transmission.

DETECTION Field indexing of ASSVd from pear trees is similar to that described in Chapter 17 for indexing of ASSVd from apple trees. Greenhouse indexing of ASSVd from pear trees may be done as described by Hurtt et al. 1992. The pear inoculum sources can be indexed by bark chip grafting below dormant buds of the pear cultivar ‘Nouveau Poiteau’ on pear seedling rootstocks. Six weeks after forcing, the ‘Nouveau Poiteau’ shoots are cut off 2–3 nodes above the graft to force a second flush of growth. Nucleic acid or crude sap extracts of petiole and midrib tissue from the shoots are analyzed by dot hybridization with a cRNA probe of ASSVd. Dot, tissue, and Northern blot hybridizations using 32P- or digoxigenin-labeled ASSVd cRNA probes were utilized for the detection of ASSVd from pear germplasm, surveys of ASSVd in cultivated and wild pears in Greece, and/or monitoring of viroid agroinfection in pear seedlings (Hurtt et al. 1992; Hurtt and Podleckis 1995; Zhu et al. 1998; Kyriakopoulou and Hadidi 1998; Kyriakopoulou et al. 2001). RT-PCR is the most sensitive method for the detection of ASSVd in symptomless Chinese pear, Chinese pear with rusty skin disease, Japanese pear with fruit dimple disease, Italian pear with rusty skin-like disease, and Greek cultivated and wild pear with russetting, cracking, and/or scarring symptoms (Hadidi and Yang 1990; Zhu et al. 1995, 1998; Osaki et al.1996, 1998; Kyriakopoulou and Hadidi 1998; Kyriakopoulou et al. 2001).

CONTROL Most quarantine programs, especially in developed countries, require testing for ASSVd in imported pear germplasm to prevent the viroid introduction to local susceptible plant species. Another major control measure is to propagate pear nursery stock from ASSVd-indexed mother trees. Infected trees in orchards should be removed to avoid spreading the viroid to neighboring trees by root grafting. ASSVd may be eliminated from pears by in vitro therapy and apical meristem culture (Postman and Hadidi 1995). Application of heat therapy (temperature alternating between 30°C and

143

P.E. Kyriakopoulou et al.

Figure 18.1 Nucleotide sequence and secondary structure of pear rusty skin variant of ASSVd (from Zhu et al. 1995).

38°C every 4 hours) or cold therapy (plants cold hardened for 7 days at temperatures alternating between 22°C for 8 hours and –1°C for 16 hours, and then moved to a cold room and grown at 4°C) to ASSVd-infected pear plantlets in vitro for 8 weeks followed by meristem tip culture. This procedure resulted in a high percentage of viroid-free pear trees as shown by RT-PCR assays.


Family: Pospiviroidae Genus: Apscaviroid Species: Apple scar skin viroid The nucleotide sequence of ASSVd from symptomless pear is 330 nucleotides and differs from the Japanese type strain from apple (Hashimoto and Koganezawa 1987) at only 4 sites (Zhu et al. 1995). Similarly, the nucleotide sequence of the viroid variant that causes the Japanese fruit dimple disease is 330 nucleotides and differs from the Japanese type strain from apple at only 3 sites (Osaki et al. 1996). The pear rusty skin variant of ASSVd consists of 334 nucleotides, shares 92% identity with the Japanese ASSVd type strain, and differs from it at 25 sites (Zhu et al. 1995) (Figure 18.1). The nucleotide sequence of this viroid variant confirms our previous findings which suggested that it is a member of the ASSVd group as indicated by RT-PCR (Hadidi and Yang 1990). The nucleotide sequence of each variant of ASSVd in pears can be arranged into the rod-like secondary structure characteristic of Pospiviroidae. References Chen, W., Yang, X.C., and Po, T. (1987). Pear rusty skin, a viroid disease. In Abstracts of the 7th Intern. Virology Conf., p. 300. Edmonton, Canada. Desvignes, J.C., Grasseau, N., Boyé, R., Cornaggia, D., Aparicio, F., Di Serio, F., and Flores, R. (1999). Biological properties of apple scar

144

skin viroid: Isolates, host range, different sensitivity of apple cultivars, elimination and natural transmission. Plant Dis. 83, 768-772. Hadidi, A., and Yang, X. (1990). Detection of pome fruit viroids by enzymatic cDNA amplification. J. Virol. Methods 30, 261-270. Hadidi, A. Hansen, A.J., Parish, C.L., and Yang, X. (1991). Scar skin and dapple apple viroids are seed-borne and persistent in infected apple trees. Res. Virol. 142, 289-296. Hashimoto, J., and Koganezawa, H. (1987). Nucleotide sequence and secondary structure of apple scar skin viroid. Nucleic Acids Res. 15, 7045-7052. Hurtt, S.S., and Podleckis, E.V. (1995). Apple scar skin viroid is not seed transmitted or transmitted at a low rate in oriental pear. Acta Hortic. 386, 544-550. Hurtt, S. S., Podleckis, E. V., Ibrahim, L. M., and Hadidi, A. (1992). Early detection of apple scar skin group viroids from imported pear germplasm. Acta Hortic. 309, 311-318. Kyriakopoulou, P.E., Giunchedi, L., and Hadidi, A. (2001). Peach latent mosaic and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdaliformis: implications on possible origin of these viroids in the Mediterranean region. J. Plant Pathol. 83, 51-62. Kyriakopoulou, P.E., and Hadidi, A. (1998). Natural infection of wild and cultivated pears with apple scar skin in Greece. Acta Hortic. 472, 617-625. Liu, F.–C, Chen, R.–F., and Chen, Y.–X. (1957). Apple scar skin disease. Academia Sinica Printery: Beijing (in Chinese). Liu, F.–C., Wang, S.–Y., Tian, C.–F., and Chen, R.–F. (1962). Studies on apple scar skin disease (1960-1962). Ann. Rep. Fruit Inst. Chinese Acad. Agr. Sci. 1962, 71-79 (in Chinese). Liu, F.–C., Wang, S.–Y., Chen, C., and Chen, Y.–X. (1985). Research on the relationship between apple scar skin disease and pear trees. China Fruit 1, 36-39 (in Chinese). Ohtsu,Y., Sakuma, T., Tanaka, Y.,Takanashi, K., Isoda, T., Sekimoto, Y., Matsuura, E., and Taniguchi, N. (1990). A few symptoms of ‘Kubomi’ on fruits of Japanese pear. Ann. Phytopathol. Soc. Jpn. 56, 101 (Abstract, in Japanese). Osaki, H., and Kudo, A. (1995). Identification of a viroid associated with fruit disorder of Japanese pear. Bull. Fruit Tree Res. Stn. 27, 65-77.

APPLE SCAR SKIN VIROID IN PEAR

Osaki, H., Kudo, A., and Ohtsu, Y. (1996). Japanese pear fruit dimple disease caused by Apple scar skin viroid (ASSVd). Ann. Phytopathol. Soc. Jpn. 62, 379-385. Osaki, H., Kudo, A., and Ohtsu, Y. (1998). Two rapid extraction methods to detect apple scar and hop stunt viroids by RT-PCR. Acta Hortic. 472, 603-611. Postman, J. D., and Hadidi, A. (1995). Elimination of apple scar skin viroid from pears by in vitro thermotherapy and apical meristem culture. Acta Hortic. 386, 536-543.

Zhu, S.F., Hadidi, A., Hammond, R.W., Yang, X., and Hansen, J.A. (1995). Nucleotide sequence and secondary structure of pome fruit viroids from dapple apple diseased apples, pear rusty skin diseased pears and apple scar skin symptomless pears. Acta Hortic. 386, 554-559. Zhu, S. F., Hammond, R. W., and Hadidi, A. (1998). Agroinfection of pear and apple with dapple apple viroid result in systemic infection. Acta Hortic. 472, 613-616.

145

PART IV

CHAPTER 19

APPLE DIMPLE FRUIT VIROID ....................................................................................................

F. Di Serio, M. Malfitano, D. Alioto, A. Ragozzino, and R. Flores

.................................................................................................................................................................................................................................................................

Apple dimple fruit (ADF) disease was first observed in a commercial orchard of the cv. ‘Starking Delicious’ in Southern Italy (Di Serio et al. 1996). Symptomatic fruits displayed roundish and depressed green spots of 3–4 mm in diameter scattered on the red skin (see Plate 5A–D) and necrotic areas in the underlying flesh. In some cases, the spots fused and large discolored skin areas were observed predominantly around the calyx end. Fruits with yellow or green spots on the skin but without depressions and necrosis in the flesh were also observed in the close vicinity of the more deeply damaged and deformed ones. However, experimental inoculations on cv. ‘Starkrimson’, an apple indicator closely related to ‘Starking Delicious’, using electrophoretically pure preparations of the causal agent of this disease, Apple dimple fruit viroid (ADFVd) (see below), have shown that the internal necrotic regions underlying the spots found in the original field material probably resulted from other undetermined biotic or abiotic stresses (Di Serio et al. 2001). Apple cvs. such as ‘Gala’, ‘Pink Lady®’ and ‘Braeburn’ react to ADFVd with similar symptoms, which in the latter case are occasionally accompanied by scar skin (see Plate 5), whereas other apple cvs. such as ‘Golden’ are tolerant (ADFVd replicates without eliciting symptoms) (Di Serio et al. 2001).

146

ADF symptoms are similar although generally milder than those typical of dapple apple (DA) disease induced by ASSVd in some apple varieties (Koganezawa 1989) and are restricted to fruits, with no alterations observed in other plant organs (Di Serio et al. 1996, 2001). The resemblance between the symptoms characterizing ADF and DA diseases suggests the possibility that these two maladies may have been confused in the past. More specifically, the fruit symptoms observed occasionally in Italy and presumed to be the expression of DA disease (Giunchedi 1976), may have been actually incited by ADFVd considering that ASSVd has not been identified in apple in this country so far. ASSVd, however, has been recently reported in pear in Italy (Kyriakopoulou et al. 2001). The economic impact of ADF disease appears limited for the moment. No interference in symptom expression or viroid accumulation has been observed in plants inoculated first with ASSVd and then with ADFVd (Di Serio et al. 2001).

HOST RANGE Apple (Malus pumila Mill.) is the only known natural host of ADFVd. The viroid can be experimentally transmitted to the ‘Fieud 37’ pear (Pyrus communis L.), an indicator developed recently for Pear blister canker viroid (PBCVd) (Desvignes et al.

APPLE DIMPLE FRUIT VIROID

1999a). However, ADFVd replicates in this indicator without causing discernible symptoms (Di Serio et al. 2001).

TRANSMISSION ADFVd may have been inadvertently transmitted by interchange of propagative material of apple cultivars, some of which are tolerant. ADFVd can be experimentally transmitted by grafting and budding, and mechanically by slashing apple and pear stems with a razor blade wetted with inoculum (Di Serio et al. 2001). Although there is no direct evidence, ADFVd is unlikely to be transmitted through seed because the closely related ASSVd is either not seed-transmitted or it is at a very low rate in apple (Howell et al. 1998; Desvignes et al. 1999b) and oriental pear (Hurtt and Podleckis 1995). A similar situation occurs in pear with PBCVd that also belongs to the same genus Apscaviroid (Desvignes, unpublished data; Postman and Skrzeczkowski, personal communication). There is no information regarding transmission of ADFVd by insect vectors.

GEOGRAPHICAL DISTRIBUTION AND EPIDEMIOLOGY ADFVd has been reported so far only in Italy. In addition to ‘Starking Delicious’, typical symptoms of ADF disease in natural infections have been observed in fruits of ‘Royal Gala’ (Di Serio et al. 1998) and ‘Annurca’ (Di Serio et al. 2000) in close association with ADFVd. The rusty skin symptoms observed previously in fruits of ‘Golden Delicious’ naturally infected by ADFVd (Di Serio et al. 1998), are probably not caused by this viroid because experimental inoculations with purified preparations of ADFVd have shown that this cultivar is tolerant (Di Serio et al. 2001). Considering that besides ‘Golden Delicious’ other commercial apple cultivars, including ‘Golden’, ‘Smoothee®’, ‘Granny Smith’, ‘Baujade’ and ‘Reinette Grise du Canada’ are tolerant to ADFVd (Di Serio et al. 2001), this viroid may well have a wider occurrence.

DETECTION ADFVd can be detected by bioassay on sensitive apple cvs. like ‘Starkrimson’ and ‘Braeburn’, although a long waiting time (2–3 years) is required between inoculation and onset of fruit symptoms. However, the close resemblance between the symptoms induced by ADFVd and ASSVd makes a reliable differential diagnosis between both viroids difficult. ADFVd accumulates in infected tissue to levels comparable to those of ASSVd. ADFVd can be detected by polyacrylamide gel electrophoresis (PAGE) and ethidium bromide staining (and/or Northern blot hybridization) using fruit, leaf or bark RNA preparations obtained by phenol extraction and chromatography on non-ionic cellulose (Di Serio et al. 1996, 2001). ADFVd could be in principle revealed by dot-blot hybridization of apple

extracts and most likely of tissue imprints of fruit sections following protocols as those reported for ASSVd (Hurtt et al. 1996; Desvignes et al. 1999b). However, due to the sequence similarity between ADFVd and ASSVd (see below), full-length cRNA probes from one of them are able to hybridize, although with lower sensitivity, with the other one. Therefore, a differential molecular diagnosis between ASSVd and ADFVd must be based on: i

denaturing PAGE and Northern blot hybridization to distinguish their distinct sizes (Di Serio et al. 1996, 2001),

ii

dot-blot hybridization with a full ADFVd-specific RNA probe, treating the membrane after hybridization with ribonuclease under high ionic conditions to remove the imperfect hybrids that the probe could form with ASSVd (Di Serio et al. 2000),

iii

development of partial-length specific probes derived from regions in which ADFVd and ASSVd sequences differ; and

iv

reverse transcription and polymerase chain reaction amplification (RT-PCR).

In this latter case, primers corresponding to regions conserved between ASSVd, ADFVd and Pear blister canker viroid (PBCVd) have been used for the simultaneous detection of these viroids, which can be discriminated by PAGE analysis of the size of their PCR products (different for each viroid) (Faggioli et al. 2001). On the other hand, a multiplex fluorescent RT-PCR method has been recently reported in which viroidspecific primers, each labeled with different fluorescent dye, have been designed on the basis of sequence conservation between certain regions of ADFVd and ASSVd, as well as on sequence divergence in other regions. Detection is achieved by agarose gel elecrophoresis, being ADFVd- and ASSVd-specific PCR products discriminated by both their different size and color fluorescence emitted when UV-irradiated. An additional advantage is that ethidium bromide, a powerful mutagen, is not needed for gel staining (Di Serio et al. 2002). ADFVd, ASSVd, and PBCVd may also be detected and differentiated by multiplex RT-PCR probe capture hybridization (ELISA) (Shamloul et al. 2002).

CONTROL Thermotherapy combined with shoot tip grafting should presumably provide ADFVd-free material, because a protocol of this kind has been proven successful for eliminating ASSVd from infected apple (Desvignes et al. 1999b). In the meantime, only preventive measures limited to the use of viroid-free propagation material can be implemented. Pear, which replicates ADFVd without displaying any symptomatology (Di Serio et al. 2001), should be considered as a potential reservoir for this

147

F. Di Serio et al.

Figure 19.1 Primary and proposed secondary structure of the lowest free energy for the original ADFVd isolate from the apple cultivar ‘Starking Delicious’. The upper and lower strands of the central conserved region (CCR) typical of genus Apscaviroid are delimited by filled and open circles respectively, and the terminal conserved region (TCR) by flags (from Di Serio et al. 1986).

viroid. Pruning tools should also be regularly disinfected with diluted commercial bleach to avoid tree-to-tree transmission.


A viroid nature for the causal agent of ADF disease was advanced when a small circular RNA was isolated from symptomatic fruits and the analysis of its sequence showed significant similarities with ASSVd (Di Serio et al. 1996). However, the autonomous replication of this novel RNA, then tentatively termed Apple dimple fruit viroid (ADFVd), and the symptoms incited when inoculated free from other pathogens that might be present in the original source, have been demonstrated only recently (Di Serio et al. 2001). Cloning and sequencing of ADFVd has revealed a size of 306–307 nt in consonance with its electrophoretic mobility in denaturing PAGE intermediate between those of PBCVd and hop stunt viroids (315 and 297 nt, respectively) (Di Serio et al. 1996, 1998). The reference sequence of the original ADFVd isolate from ‘Starking Delicious’ (EMBL accession number X99487) has 306 nt consisting of 61 A (20%), 87 C (28.5%), 92 G (30.0%) and 66 U (21.5%), and adopts a quasi-rod-like most stable secondary structure (Di Serio et al. 1996) (Figure 19.1). Other molecular variants from ‘Golden Delicious’, ‘Royal Gala’, ‘Red Delicious’ and ‘Annurca’ isolates have 306–307 residues and minor sequence changes (Di Serio et al. 1998, 2002). The nine polymorphic positions observed so far in ADFVd variants are distributed along the viroid molecule and no major variable region has been found (Di Serio et al. 2002). ADFVd belongs to the family Pospiviroidae (see Chapter 8 ‘Classification’) and contains the central conserved region (CCR) characteristic of genus Apscaviroid whose type species is ASSVd (Hashimoto and Koganezawa 1987), as well as the terminal conserved region (TCR) located in the left-terminal domain of members of this genus and of other viroid genera (see Figure 19.1 above) (Di Serio et al. 1996). ADFVd has the highest sequence similarity with ASSVd and with citrus viroid III (Rakowski et al. 1994; Stasys et al. 1995), sharing with the latter an almost identical left terminal domain (Di Serio et al. 1996).

148

Acknowledgement

Work in R. Flores’ laboratory was partially supported by grants PB95-0139 and PB98-0500 from the Comisión Interministerial de Ciencia y Tecnología de España. References Desvignes, J. C., Cornaggia, D., Grasseau, N., Ambrós, S., and Flores, R. (1999a). Pear blister canker viroid: studies on host range and improved bioassay with two new pear indicators, Fieud 37 and Fieud 110. Plant Dis. 83, 419-422. Desvignes, J. C., Grasseau, N., Boyé, R., Cornaggia, D., Aparicio, F., Di Serio, F., and Flores, R. (1999b). Biological properties of apple scar skin viroid: Isolates, host range, different sensitivity of apple cultivars, elimination, and natural transmission. Plant Dis. 83, 768-772. Di Serio F., Alioto D., Ragozzino A., and Flores R. (1996). Identification and molecular properties of a 306 nucleotide viroid associated with apple dimple fruit disease. J. Gen. Virol. 77, 2833-2837. Di Serio, F., Alioto D., Ragozzino, A., Giunchedi, L., and Flores, R. (1998). Identification of apple dimple fruit viroid in different commercial varieties of apple grown in Italy. Acta Hortic. 472, 595-601. Di Serio, F., Alioto, D., and Ragozzino, A. (2000). Segnalazione in Campania di un focolaio d’infezione del viroide della maculatura crateriforme della mela sulle cv Annurca e Starking Delicious. Inf. Fitopat. 6, 53-56. Di Serio, F., Malfitano, M., Alioto D., Ragozzino A., Desvignes, J. C., and Flores, R. (2001). Apple dimple fruit viroid: Fulfillment of Koch postulates and symptom characteristics. Plant Dis. 85, 179-182. Di Serio, F., Malfitano, M., Alioto, D., Ragozzino, A., and Flores, R. (2002). Apple dimple fruit viroid: sequence variability and its specific detection by multiplex fluorescent RT-PCR in the presence of Apple scar skin viroid. J. Plant Pathol. 84, 27-34. Faggioli, F., Ragozzino, E., and Barba, M. (2001). Simultaneous detection of stone or pome fruit viroids by single tube RT-PCR. Acta Hortic. 550, 59-63. Giunchedi, L. (1976). Transmission experiments on the virus disease dapple apple. Acta Hortic. 67, 255-260. Hashimoto, J., and Koganezawa, H. (1987). Nucleotide sequence and secondary structure of apple scar skin viroid. Nucleic Acids Res. 15, 7045-7052. Howell, W. E., Skrzeczkowski, L. J., Mink, G. I., Nunez, A., and Wessels, T. (1998). Non-transmission of apple scar skin viroid and peach latent mosaic viroid through seed. Acta Hortic. 472, 635-639. Hurtt, S. S., and Podleckis, E. V. (1995). Apple scar skin viroid is not seed transmitted or transmitted at a low rate in oriental pear. Acta Hortic. 386, 544-550.

APPLE DIMPLE FRUIT VIROID

Hurtt, S. S., Podleckis, E. V., and Howell, W. E. (1996). Integrated molecular and biological assays for rapid detection of apple scar skin in pear. Plant Dis. 80, 458-462. Koganezawa, H. (1989). Apple scar skin viroid. AAB Descrip. Plant Viruses, number 349. Kyriakopoulou, P. E., Giunchedi, L., and Hadidi, A. (2001). Peach latent mosaic viroid and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdaliformis: implications on possible origin of these viroids in the Mediterranean region. J. Plant Pathol. 83, 51-62.

Rakowski, A. G., Szychowski, J. A., Avena, Z. S., and Semancik, J. S. (1994). Nucleotide sequence and structural features of the Group III citrus viroids. J. Gen. Virol. 75, 3581-3584. Shamloul, A. M., Faggioli, F., Keith, J. M., and Hadidi, A. (2002). A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid. J. Virol. Methods 105, 115-121. Stasys, R. A., Dry, I. A., and Rezaian, M. A. (1995). The termini of a new citrus viroid contain duplications of the central conserved region of two viroid groups. FEBS Lett. 358, 182-184.

149

PART IV

CHAPTER 20

APPLE FRUIT CRINKLE VIROID ....................................................................................................


.................................................................................................................................................................................................................................................................

In the mid-1970s, two graft-transmissible diseases of apple were found independently in Japan. An abnormal fruit disorder of apple cv. ‘Mutsu’ (Crispin) was found in Iwate Prefecture, and named as apple fruit crinkle (‘Yuzuka-byo’ in Japanese) based on fruit symptoms (Koganezawa et al. 1989). A bark disorder of cv. ‘Nero 26’ was found in Aomori Prefecture (Matsunaka and Machita 1987). Later both diseases were shown by cross-transmission experiments to be caused by the same agent (Ito et al. 1999). Ito et al. (1993) detected a viroid in infected apple trees and the viroid etiology of the disease was established by backtransmission experiments (Ito and Yoshida 1998). The sequence of the causal Apple fruit crinkle viroid (AFCVd) was determined (GenBank accession number E29032) and revealed that it is a distinct viroid belonging to the genus Apscaviroid (Ito et al. 1998).

ECONOMIC IMPACT AND SYMPTOMATOLOGY The characteristic symptoms are the roughened and crinkled appearance of the fruit. Fruit crinkling appears on cvs. ‘Ohrin’, ‘Mutsu’, ‘Hokuto’, ‘Senshu’, ‘Jonathan’, ‘Fuji’, ‘Tsugaru’, ‘Sansa’, ‘Yoko’ and ‘Blaxstayman’. The ‘Ohrin’, which ranks as the fourth highest in apple production in Japan, shows the severest symptoms (see Plate 5E–G). Brown necrotic pithy areas

150

are scattered in the flesh of the ‘Ohrin’ fruit. Dapple symptoms which are similar to dapple apple caused by Apple scar skin viroid (ASSVd) appear on red apple cultivars like ‘Jonathan’, ‘Hokuto’, ‘Senshu’, ‘Fuji’ and ‘Mutsu’. On ‘Jonathan’ dapple symptoms appear mainly at the calyx end of the fruit (see Plate 5F). Fruit symptoms become obvious in the middle of August and clearer near harvesting time. The severity seems to be reduced by low temperatures in summer. Affected fruits, in particular of cv. ‘Ohrin’, tend to drop prematurely. ‘Starking Delicious’ do not show the fruit symptoms mentioned above except for small fruit, but show blister bark (see Plate 5G) accompanying twig dieback on the branches (Koganezawa et al. 1989; Iijima 1990, 1993). This bark disorder resembles apple measles caused by boron deficiency or manganese excess (Nagai et al. 1966; Shannon 1954). ‘Nero 26’, ‘Winesap’ and a crab apple ‘NY58-22’ also show blister bark symptoms (Ito and Yoshida 1998). In ‘NY58-22’, severe fruit crinkling with necrotic pithy areas in the flesh and dappling develop in addition to bark symptoms. This appears to be the most sensitive host of the disease. ‘Golden Delicious’ and ‘Granny Smith’ produce slightly crinkled fruit in some seasons. Cultivars ‘Indo’ and ‘Spartan’ are symptomless. Generally, shoots and roots on infected trees, especially dwarfed trees, are less vigorous. No diagnostic leaf

APPLE FRUIT CRINKLE VIROID

symptoms are observed. The latent period in graft-transmission experiments was usually two to three years, and more for bark symptoms. Occasionally apple trees are doubly infected with AFCVd and ASSVd. Double-infected ‘Fuji’ shows severer fruit crinkling than that infected singly with either AFCVd or ASSVd (Ito and Yoshida 1997). A survey conducted in Nagano Prefecture in 1988 showed that 178 apple trees were affected with the disease (Iijima 1990). Since affected trees were removed from the orchard, occurrence of the disease at present is sporadic. However, all modern cultivars in Japan are susceptible to the disease. There is a possibility that the disease may spread, because propagation of scions by top-working is a common practice in Japan.

HOST RANGE The disease has been observed only in commercial apple cultivars and in crab apples such as ‘NY58-22’, ‘Tartan’ and ‘Zao Sheng Ping Guo’ (Ito and Yoshida 1991). No attempt to inoculate with AFCVd to woody plants other than Malus species has been made. Transmission tests of AFCVd to tomato cv. ‘Rutgers’, cucumber cv. ‘Suyo’, cowpea cv. ‘Kurodane-sanjyaku’, Gynura aurantiaca, Chenopodium quinoa and Nicotiana occidentalis 37B were negative (Ito et al. 1993).

TRANSMISSION AFCVd is easily transmitted by budding, grafting and chip budding. Experimentally it is also transmitted by razor slashing (Ito et al. 1993). Natural spread to neighboring trees has not been observed so far, indicating that no natural vector is present.

GEOGRAPHICAL DISTRIBUTION AND EPIDEMIOLOGY The disease has been reported only from Japan so far (Koganezawa et al. 1989). The disease has been observed in every apple growing area of Japan. Several graft-transmissible bark disorders resembling apple fruit crinkle were reported previously on apple cv. ‘Delicious’ and its sports from USA. They are called blister bark (Parish 1981) or pustule canker (Cheney et al. 1970). Bark symptoms reproduced on ‘Starking Delicious’ and ‘Nero 26’ (a progeny of ‘Richard Delicious’) by AFCVd resemble those of blister bark and pustule canker. The relationship between these diseases has not been examined. Wide occurrence of the disease is mostly due to artificial propagation of scions on infected trees. In one case, a grower used infected ‘Starking Delicious’ trees for propagation because of lower productivity of the tree and top-grafted onto healthy apple trees. Consequently, all the renewed apple trees were infected. In another case, ‘Senshu’ was also propagated on ‘Starking Delicious’ trees, and infected scions were distributed through a local agricultural cooperative.

DETECTION AFCVd can be indexed by graft-inoculation to fruit-bearing apple trees such as ‘Ohrin’ and ‘NY58-22’ in an orchard or easily by top-grafting scions (virus and viroid free) on trees to be indexed. In the latter case, bioassay using ‘NY58-22’ can be completed within two years, though it also develops similar fruit symptoms without bark symptoms by ASSVd infection. AFCVd can be detected by return, two-dimensional or bidirectional PAGE analysis of the extracted RNA from 1–5 g of petiole or bark. The use of gel electrophoresis to diagnose the disease is incorporated into the certification program for mother fruit trees by the Plant Protection Service of Japan in 1998. At present RT-PCR is the most sensitive method for detecting AFCVd.

CONTROL The major control procedure of the disease is to propagate the nursery stock from viroid-indexed mother trees. It is important to avoid the propagation of scions on non-indexed trees. References Cheney, P. W., Lindner, R. C., and Parish, C. L. (1970). Two graft-transmissible bark diseases of apple. Plant Dis. Reptr. 54, 44-48. Iijima, A. (1990). Occurrence of a new viroid-like disease, apple fruit crinkle. Plant Protection, Jpn. 44, 130-132 (in Japanese). Iijima, A. (1993). Graft transmission and fruit symptoms of apple fruit crinkle disease and apple scar skin disease. Proc. Kanto-Tosan Plant Protec. Soc. 40, 119-121 (in Japanese). Ito, T., Kanematsu, S., Koganezawa, H., Tsuchizaki, T., and Yoshida, Y. (1993). Detection of a viroid associated with apple fruit crinkle disease. Ann. Phytopathol. Soc. Jpn. 59, 520-527. Ito, T., Sano, T., and Yoshida, K. (1998). Nucleotide sequence of apple fruit crinkle viroid. Abstract of Int’l Congress of Plant Pathology, Abstract No. 3.7.6. Ito, T., Suzaki, K., Nakahara, K., Machita, I., Matsunaka, K., and Yoshida, K. (1999). Apple fruit crinkle viroid (AFCVd) causes a graft-transmissible blister bark on apple cv. Nero 26. Ann. Phytopathol. Soc. Jpn. 65, 394 (abstract in Japanese). Ito, T., and Yoshida, K. (1991). Possible woody indicator plants for short-term indexing of three graft-transmissible fruit disorders of apple. Tohoku Agric. Res. 44, 213-214 (in Japanese). Ito, T., and Yoshida, K. (1997). The effect of double-infection with apple scar skin and apple fruit crinkle viroids on fruit symptom expression of apple trees. Ann. Rept. Plant Prot. North Japan 48, 219 (abstract in Japanese). Ito, T., and Yoshida, K. (1998). Reproduction of apple fruit crinkle disease symptoms by apple fruit crinkle viroid. Acta Hortic. 472, 587-594. Koganezawa, H., Ohnuma, Y., Sakuma, T., and Yanase, H. (1989). ‘Apple fruit crinkle’, a new graft-transmissible fruit disorder of apple. Bull. Fruit Tree Res. Stn. 16, 57-62 (in Japanese with English summary). Matsunaka, K., and Machita, I. (1987). A graft-transmissible blister bark occurring on apple cv. Nero 26. Ann. Rept. Plant Prot. North Japan 38, 186 (abstract in Japanese).

151


Nagai, K., Ichiki, S., Izumiya, A., Seito, M., Sakurada, S., and Kamada, C. (1966). Studies on the nutritional disorders of apple bark. 1. On ‘Sohibyo’ caused by manganese excess. J. Jpn. Soc. Hort. Sci. 34, 265271 (in Japanese with English summary).

152

Parish, C. L. (1981). Graft-transmission of blister bark and internal bark necrosis in Delicious apples. Hort. Science 16, 52-54. Shannon, L. M. (1954). Internal bark necrosis of the Delicious apple. Proc. Amer. Soc. Hort. Sci. 64, 165-174.

PART IV

CHAPTER 21

PEAR BLISTER CANKER VIROID ....................................................................................................

R. Flores, S. Ambrós, G. Llácer, and C. Hernández

.................................................................................................................................................................................................................................................................

The disease termed ‘pear blister canker’ (PBC) refers to a bark alteration observed in the pear indicator ‘A20’ (Ambrós et al. 1995a), and should not be confused with other bark disorders reported previously in different pear cultivars with the same name (Cropley 1960), and with others as pear rough bark (Thomsen 1961), pear bark split and bark necrosis (Kegler 1965), and pear bark measles (Cordy and MacSwan 1961). Whereas there is convincing evidence that PBC disease is incited by Pear blister canker viroid (PBCVd) (see below), the causal agents of the other bark disorders of pear remain poorly defined.

number and size as if they were caused by different strains. With the four reference isolates of PBCVd, the symptom severity increases from P1914T to P2098T, P10433T and P47A (Desvignes et al. 1999).

Symptoms of the PBC disease in the pear cultivar ‘A20’ are restricted to bark, with leaves and fruits not exhibiting any pathological alteration. Bark symptoms usually appear two years after infection as pustules or superficial cracks on the epidermis that turn progressively into scattered cankers and scaly bark or deep bark splits (see Plate 6A and B), causing plant death within 5–8 years. Commercial pear cultivars such as ‘Williams’, ‘Comice’ and ‘Beurré Hardy’ can be infected but do not develop the typical bark symptoms (Ambrós et al. 1995a; Desvignes, unpublished data).

PBCVd can be experimentally transmitted to cucumber (Cucumis sativus L.) in which it induces mild leaf rugosity or no symptoms at all (Flores et al. 1991). With the aim of finding a more convenient indicator, PBCVd was inoculated to a series of plants but it was not detected in any of the tested species of genera Amelanchier, Aronia, Cotoneaster, Crataegus and Pyracantha; however, some species of genera Chaenomeles, Cydonia and Sorbus, 5 out of 13 species of Malus, 15 Pyrus species and 16 commercial pear cultivars, were susceptible to PBCVd although none developed symptoms (Desvignes et al. 1999).

The pustule bark cankers induced on ‘A20’ by several PBCVd sources are similar but in some cases differ consistently in their

In view of these negative results, the search for an improved PBCVd indicator was extended to several populations of pear

HOST RANGE Pear (Pyrus communis L.) and quince (Cydonia oblonga Mill.) are the only known natural hosts of PBCVd. The latter, which is often used in Europe as a pear rootstock, may be a reservoir of the viroid.

153

R. Flores et al.

seedlings. No symptoms appeared on any of the 108 Pyrus serotina seedlings analyzed while 2 of the 54 ‘A20’ seedlings analyzed exhibited bark cankers, although two years after inoculation. However, 3 of 250 ‘Fieudière’ seedlings exhibited necrosis on petioles, leaves and young shoots 3–5 months after inoculation in the greenhouse. To confirm these results, non-inoculated material from these three seedlings was grafted onto pear seedling rootstocks that were simultaneously inoculated with different PBCVd isolates. The characteristic PBCVd necrosis appeared after 3–4 months on ‘Fieud 37’ and ‘Fieud 110’ selections, and after 5 months on ‘Fieud 186’ (which was discarded). Necrosis began at the base of the petioles and extended to the leaves and bark (see Plate 6B). Grafted or in vitro-propagated young plants of ‘Fieud 37’ and ‘Fieud 110’ selections present the same symptoms throughout the year in the greenhouse (Desvignes et al. 1999).

TRANSMISSION PBCVd may have been inadvertently transmitted by agricultural practices such as the use of quince rootstocks that may be infected but symptomless, or by interchange of propagation material of pear cultivars, most of which are tolerant. PBCVd can be experimentally transmitted by grafting and budding, and mechanically by slashing pear stems with a razor blade wetted with inoculum or by applying the inoculum to carborundum-dusted leaves of cucumber. PBCVd is unlikely to be transmitted through seed because when the indicator ‘A20’ was propagated onto 200 pear seedlings, which had been obtained from a source originally infected with the PBCVd isolate P2098T, no symptoms were observed up to 4 years after inoculation (Desvignes, unpublished data). Similar results have been independently obtained by other authors (J. Postman and L.K. Skrzeczkowski, personal communication). There is no information regarding transmission of PBCVd by insect vectors.

Skrzeczkowski, personal communication; A. Hadidi, unpublished data) and China (A. Hadidi, unpublished data).

DETECTION The finding that the pear cultivar ‘A20’, selected initially as an indicator for pear vein yellow virus (PVYV) and some strains of apple chlorotic leaf spot virus (ACLSV), also developed pustule bark cankers presumably caused by an unknown pathogen (Desvignes 1970), prompted the characterization of a pathogen that was subsequently shown to be PBCVd. Therefore, ‘A20’ served as a first diagnostic host for PBCVd, although the long time elapsing between inoculation and onset of symptoms (2–3 years), promoted the search for alternative hosts. Cucumber, which can replicate the viroid, was discarded because symptoms were mild and erratic (Flores et al. 1991). More recently two pear selections, ‘Fieud 37’ and ‘Fieud 110’, that develop specific symptoms in 3–4 months (see Plate 6B), have been proposed as PBCVd indicators to replace ‘A20’ (Desvignes et al. 1999). PBCVd reaches relatively high titers in infected tissue and can be detected in leaf or bark RNA preparations obtained by phenol extraction and chromatography on non-ionic cellulose by polyacrylamide gel electrophoresis and silver staining (Flores et al. 1991). The viroid can also be detected by dot-blot hybridization with radioactive and non-radioactive cRNA probes following a simplified protocol that includes mild extraction of the tissue, removal of cellular debris, phenol-mediated deproteination, and recovery of nucleic acids by ethanol precipitation (Ambrós, et al. 1995a). This procedure, which facilitates the manipulation of a larger number of samples, has also shown that PBCVd replicates efficiently in pear varieties that do not develop symptoms, and that PBCVd is not involved in bark alterations like ‘Williams’ bark split, ‘Williams’ bark necrosis and ‘Comice’ bud drop which, therefore, probably have a different etiology. PBCVd is also detectable by RT-PCR with PBCVd-specific primers (A. Hadidi, personal communication).

CONTROL GEOGRAPHICAL DISTRIBUTION AND EPIDEMIOLOGY PBCVd has been reported in France, Spain and Italy (Desvignes 1970; Hernández et al. 1992b; Ambrós et al. 1995a; Loreti et al. 1997). A survey of more than 150 old French pear cultivars revealed that approximately 10% were infected (Desvignes et al. 1999). The distribution and pathogenic effects of PBCVd have probably been underestimated because pear and quince cultivars are generally tolerant, and when they are sensitive, the infected trees decline quickly or exhibit bark cankers as if they were infected by bacteria or fungi, which are then presumed to be the causal agent (Desvignes et al. 1999). Therefore, PBCVd may well exist in other areas of the world outside Europe and in fact this may well be the case in the USA (J. Postman and L.K.

154

Only preventive measures, limited to the use of viroid-free propagating material, can be implemented because attempts to eliminate PBCVd by thermotherapy have not been successful (Desvignes, unpublished data; J. Postman and L.K. Skrzeczkowski, personal communication). Pruning tools should also be regularly desinfected with diluted commercial bleach to avoid any risk of tree to tree transmission.


The viroid nature of the causal agent of PBC disease was suspected following the lack of success in eliminating it by thermotherapy. A viroid hypothesis was confirmed when a small

PEAR BLISTER CANKER VIROID

Figure 21.1 Primary and proposed secondary structure of the lowest free energy for the reference sequence from PBCVd isolate P2098T. The upper and lower strands of the central conserved region (CCR) typical of the genus Apscaviroid are delimited by filled and open circles respectively, and the terminal conserved region (TCR) by flags (from Hernández et al. 1992a).

circular RNA was found in infected tissue (Flores et al. 1991; Hernández et al. 1992a and b), and when purified preparations of this RNA induced the characteristic symptoms after mechanical inoculation into the ‘A20’ indicator (Ambrós et al. 1995b). Cloning and sequencing of PBCVd revealed a size of 315–316 nt (Hernández et al. 1992a; Ambrós et al. 1995b), in conformity with its electrophoretic mobility in denaturing polyacrylamide gels (Flores et al. 1991). The reference sequence of PBCVd isolate P2098T (EMBL accession number S46812) has 315 nt consisting of 54 A (17.1%), 92 C (29.2%), 99 G (31.4%) and 70 U (22.2%), and adopts a quasi-rod-like most stable secondary structure (Hernández et al. 1992a) (Figure 21.1). Other molecular variants detected in isolates P1914T, P47A and in an Italian source, have 315–316 residues with minor sequence changes (Ambrós et al. 1995b; Loreti et al. 1997). This sequence heterogeneity might explain the different symptom severity observed on ‘A20’. PBCVd belongs to the family Pospiviroidae (see Chapter 8 ‘Classification’) and contains the central conserved region (CCR) characteristic of genus Apscaviroid whose type species is Apple scar skin viroid (ASSVd), as well as the terminal conserved region (TCR) located in the left-terminal domain of members of this genus and of other viroid genera (Figure 21.1) (Hernández et al. 1992a). Acknowledgements

This work was partially supported by grants PB95-0139 and PB98-0500 from the Comisión Interministerial de Ciencia y Tecnologia de España to R. Flores. S. Ambrós was recipient of a predoctoral fellowship from the Generalidad Valenciana (España). We thank J. C. Desvignes for providing photographs illustrating PBCVd symptoms.

References Ambrós, S., Desvignes, J. C., Llácer, G., and Flores, R. (1995a). Peach latent mosaic and pear blister canker viroids: detection by molecular hybridization and relationships with specific maladies affecting peach and pear trees. Acta Hortic. 386, 515-521. Ambrós, S., Desvignes, J. C., Llácer, G., and Flores, R. (1995b). Pear blister canker viroid: sequence variability and causal role in pear blister canker disease. J. Gen. Virol. 76, 2625-2629. Cordy, C. B., and MacSwan, J. C. (1961). Some evidence that pear bark measles is seed-borne. Plant Dis. Reptr. 45, 891. Cropley, R. (1960). Pear blister canker: A virus disease. Annu. Rep. East Malling Res. Stn. 43, 104. Desvignes, J. C. (1970). Les maladies à virus du poirier et leur détection. CTIFL Doc. 26, 1-12. Desvignes, J. C., Cornaggia, D., Grasseau, N., Ambrós, S., and Flores, R. (1999). Pear blister canker viroid: studies on host range and improved bioassay with two new pear indicators, Fieud 37 and Fieud 110. Plant Dis. 83, 419-422. Flores, R., Hernández, C., Llácer, G., and Desvignes, J. C. (1991). Identification of a new viroid as the putative causal agent of pear blister canker disease. J. Gen. Virol. 72, 1199-1204. Hernández, C., Elena, S. F., Moya, A., and Flores, R. (1992a). Pear blister canker viroid is a member of the apple scar skin viroid subgroup (apscaviroids) and also has sequence homologies with viroids from other subgroups. J. Gen. Virol. 73, 2503-2507. Hernández, C., Llácer, G., Desvignes, J. C., and Flores, R. (1992b). Evidence supporting a viroid etiology for pear blister canker viroid. Acta Hortic. 309, 319-324. Kegler, H. (1965). Bark split and decline in Beurré Hardy pear trees. Zast. Bilja 16, 311-316. Loreti, S., Faggioli, F., and Barba, M. (1997). Identification and characterization of an Italian isolate of pear blister canker viroid. J. Phytopathol. 145, 541-544. Thomsen, A. (1961). Split bark of pears (rough bark of pears). Tidsskr. Planteavl 65, 69-72.

155

PART IV

STONE FRUIT VIROIDS

....................................................................................................

CHAPTER 22

PEACH LATENT MOSAIC VIROID IN PEACH R. Flores, C. Hernández, G. Llácer, A.M. Shamloul, L. Giunchedi, and A. Hadidi

.................................................................................................................................................................................................................................................................

Peach latent mosaic (PLM) disease was first reported in France in the course of graft indexing of peach germplasm imported from the US and Japan on peach ‘GF-305’ indicator (Desvignes 1976). The disease is economically important because it affects fruit quality, reduces the lifespan of trees, and causes peach trees to be more susceptible to other biotic and abiotic stresses. PLM disease is induced by Peach latent mosaic viroid (PLMVd). Symptoms of PLM on leaves are rare (as implied by the term ‘latent’ in the name of the disease). Occasionally, alterations on the foliage are observed, or blurred chlorotic blotches (peach blotch), yellow-creamy mosaics (peach yellow mosaic) and, in most severe cases, white patterns that may cover most or all of the leaf area (peach calico). Diseases with these names, peach blotch, peach yellow mosaic and peach calico, were reported previously in the US and Japan (Nemeth 1986; Kishi et al. 1973), and most likely are distinct manifestations of PLMVd infections. Under field conditions the most conspicuous PLMVd symptoms become visible two years after planting infected material and it may include delays in foliation, flowering, and ripening, deformations of fruits that usually are discolored with cracked sutures and flattened stones, bud necrosis, open habit, and rapid aging of the trees. Sporadically, pink

156

streaks on flowers and wood grooving are observed (see Plate 6C–F). In the greenhouse, natural PLMVd isolates are divided into severe or latent strains depending on whether they induce leaf symptoms on seedlings of the peach indicator ‘GF-305’ (Desvignes 1976). When preinoculated, the latent strains have a cross-protection effect against their severe counterparts that has been used for diagnosis purposes.

HOST RANGE Using the peach indicator ‘GF-305’, PLMVd has been naturally found in peach (Prunus persica, Batsch.) but not in any tested tree of other Prunus species (almond, plum, cherry and apricot) of different origins examined within the French fruit tree certification program (Desvignes 1986, and personal communication). In another study using dot-blot hybridization, PLMVd was found widely spread in the US in peach and nectarine trees, but it was not detected in tested samples of P. tomentosa, Thunb., P. salicina Lindl., P. avium L., and P. serrulata Lindl (Skrzeczkowski et al. 1996). PLMVd was graft-transmitted to peach hybrids (i.e. almond × peach and plum × peach), but attempts to transmit the viroid to other Prunus species were unsuccessful (Desvignes 1982, 1986).

PEACH LATENT MOSAIC VIROID IN PEACH

The above results are in contrast with those reported recently by other investigators who detected PLMVd in hosts other than peach or nectarine. PLMVd was detected by Northern-blot hybridization, dot-blot hybridization, and/or RT-PCR in naturally infected sweet cherry, plum, apricot (Hadidi et al. 1997), plum (Faggioli et al. 1997; Giunchedi et al. 1998), Japanese plum, apricot, mume, sweet cherry (Osaki et al. 1999), and wild and cultivated pears (Kyriakopoulou et al. 2001). The identity of PLMVd in cherry, plum, and pear was confirmed by nucleotide sequence analysis. Unlike peach, the titer and distribution of PLMVd in the other hosts is relatively low. PLMVd was reported to be graft transmissible from infected plum to peach ‘GF-305’ (Faggioli et al. 1997) and from infected cherry to cherry and from infected cherry to peach ‘GF-305’ (A. Crescenzi, personal communication).

TRANSMISSION PLMVd is readily transmissible by grafting and budding but not through seed (Desvignes 1986). PLMVd has also been experimentally transmitted, although at a low rate, by Myzus persicae, whereas parallel experiments with Aphis gossypii and A. spiraecola did not provide conclusive results (Flores et al. 1992). PLMVd was mechanically transmitted with blades which were either wetted with purified preparations of PLMVd (Flores et al. 1990) or contaminated with the viroid by slashing infected plants (Hadidi et al. 1997). This latter result indicates that contaminated pruning tools may play a role in viroid spread in commercial orchards.

GEOGRAPHICAL DISTRIBUTION AND EPIDEMIOLOGY PLMVd has been found in North and South America, Asia (Japan and China), the Mediterranean basin (France, Spain, Italy, Austria, Greece, Romania, Yugoslavia, Algeria and Morocco) and, more recently, in Australia (Desvignes 1986; Flores and Llácer 1988; Flores et al. 1990; Albanese et al. 1992; Flores et al. 1992; Shamloul et al. 1995; Di Serio and Ragozzino 1995; Skrzeczkowski et al. 1996; Di Serio et al. 1999; Pelchat et al. 2000). The disease seems to be prevalent in the US because an extensive survey in 5 states, in which samples from more than 1000 peach and nectarine trees grown in fields and approximately 300 trees grown in screenhouses were tested, revealed that more than 50% of the samples were infected with PLMVd (Skrzeczkowski et al. 1996). Similarly, out of 134 peach varieties analyzed in Spain, mostly imported from the US, 110 (about 80%) were infected with PLMVd (Badenes and Llácer 1998); this high value, however, may also reflect bad nursery habits in the receiving country. The interchange of propagative material of infected peach and nectarine cultivars has most probably been the major factor in PLMVd epidemiology, particularly considering that this viroid

does not incite conspicuous leaf symptoms. In a survey of peach germplasm from Europe, Asia, North America, and South America, PLMVd was found widely distributed (approximately 55%) (Hadidi et al. 1997).

DETECTION Before the identification and characterization of PLMVd as a viroid, the control of the disease relied on the cross-protection assay performed in ‘GF-305’ peach seedlings grown in the greenhouse (Desvignes 1976). ‘GF-305’ seedlings are first inoculated by chip budding with material from the trees to be tested (which, even if infected, do not usually exhibit leaf symptoms), and approximately two months later they are challenge-inoculated with a severe PLMVd strain. If the seedlings have been infected with the first inoculation, they do not develop the characteristic leaf symptoms induced by the severe strain. The severe strains are only partially stable (some infected plants revert to a symptomless condition) and have to be maintained in the greenhouse by periodical inoculation of new indicator plants with symptomatic tissue. The duration of the bioassay is approximately three months, and it has enabled the selection and distribution in France of PLMVd-tested peach cultivars for the last 25 years (Desvignes 1986). PLMVd can be commonly detected by polyacrylamide gel electrophoresis and silver staining of leaf or fruit RNA preparations obtained by phenol extraction and chromatography on nonionic cellulose, but this is not a procedure to be recommended for general use because the viroid accumulates in infected tissue at relatively low titers (Flores et al. 1990 and 1992; Hadidi, unpublished). Molecular cloning of PLMVd (Hernández and Flores 1992; Shamloul et al. 1995) paved the way for reliable detection of the viroid by dot-blot hybridization with radioactive and non-radioactive cRNA probes, following simplified protocols that include mild extraction of the tissue, removal of cellular debris, phenol-mediated deproteination, and recovery of nucleic acids by ethanol precipitation (Ambrós et al. 1995; Hadidi et al. 1997), or direct extraction of the tissue with buffersaturated phenol and chloroform (Loreti et al. 1995). Nucleic acids can be directly applied to nylon membranes (Ambrós et al. 1995) or, alternatively, following denaturation with formaldehyde (Loreti et al. 1995; Hadidi et al. 1997). PLMVd can also be detected by RT-PCR (Shamloul et al. 1995) using primers derived from the reference sequence (Hernández and Flores 1992). Primers derived from an Italian isolate of PLMVd (Shamloul et al. 1995) can also be used with equal efficiency for PLMVd detection. For this purpose, total nucleic acids extracted from leaf, bark or fruit tissue, are partially fractionated and purified by chromatography through an RNA binding matrix or non-ionic cellulose, or by precipitation with 6% polyethylene glycol and 0.75 M NaCl (Shamloul et al.

157

R. Flores et al.

Figure 22.1 Primary and predicted secondary structure of lowest free energy of the reference variant of PLMVd RNA (Hernández and Flores 1992). Plus and minus self-cleavage domains are delimited by flags, residues conserved in most natural hammerhead structures are indicated by bars, and the self-cleavage sites by arrows. Solid and open symbols refer to plus and minus polarities, respectively. Residues involved in a pseudoknot between the residues around positions 180 and 215, proposed on the basis of in vitro mapping assays with nucleases (Bussière et al. 2000), are indicated by broken lines. (Inset) Hammerhead structures of plus and minus strands of PLMVd. Residues conserved in most natural hammerhead ribozymes are on a black background and the self-cleavage sites are denoted by arrows (adapted from Hernández and Flores 1992 and Bussière et al. 2000, with modifications).

1995). Alternatively, GeneReleaser-treated leaf extracts may also be used for RT-PCR detection of the viroid (Shamloul et al. 1995). RT-PCR capture hybridization (RT-PCR-ELISA) has been successfully applied for the detection of PLMVd (Shamloul and Hadidi 1999). It is about 100-fold more sensitive than standard RT-PCR. These molecular tools have expedited the simultaneous manipulation of multiple samples and, additionally, have enabled the establishment of the relationship of PLMVd with specific maladies affecting peach trees on a firm experimental basis. Peach latent mosaic, peach yellow mosaic and peach

158

mosaic diseases, described in France, Japan and the US respectively (Nemeth 1986), were initially presumed to be caused by the same pathogen on the basis of many common biological characteristics (Desvignes 1986). Data from molecular hybridization have demonstrated that PLMVd is the causal agent of the two first diseases (Flores et al. 1990; Ambrós et al. 1995). In contrast, RT-PCR analysis using PLMVd-specific primers showed that PLMVd is not involved in peach mosaic disease (Shamloul et al. 1995; Hadidi et al. 1997), which is now known to be caused by a virus (Gispert et al. 1998; James and Howell 1998).

PEACH LATENT MOSAIC VIROID IN PEACH

CONTROL The most effective preventive procedure is the use of viroidtested propagating material. PLMVd may be eliminated from infected peach material by heat treatment at 37°C for 35–45 days followed by shoot tip grafting (Desvignes 1986). In vitro micrografting on Nemaguard seedlings has been also proved as effective in eliminating PLMVd, although the fraction of viroidfree plants obtained depends on the variety and size of the excised apex (Barba et al. 1995). Pruning tools should be periodically disinfected with diluted commercial bleach to prevent tree to tree contamination.


The heat resistance of the causal agent of the PLM disease and the impossibility to identify viral particles associated with the malady suggested a viroid etiology. Confirmation of this hypothesis was obtained when a viroid-like RNA was detected in nucleic acid extracts of peach samples infected with different isolates of the PLM agent (Flores and Llácer 1988), and subsequent work demonstrated that such an RNA was able to infect the peach indicator ‘‘GF-305’’ and induce the characteristic symptoms of the PLM disease (Flores et al. 1990). Cloning and sequencing of PLMVd from peach has revealed a size of 335–342 nt (Hernández and Flores 1992; Shamloul et al. 1995, Ambrós et al. 1998, 1999; Pelchat et al. 2000) and the presence of hammerhead structures in both polarity strands. The reference sequence of PLMVd (GenBank accession number M83545) has 338 nt consisting of 91 G (26.9%), 87 C (25.7%), 80 A (23.6%) and 80 U (23.6%) residues, and adopts a highly branched secondary structure (Figure 22.1) according to computer predictions and in vitro assays (Hernández and Flores 1992; Bussière et al., 2000). Moreover, analysis of sequence variability gives strong support to the existence of such a branched conformation in vivo (Ambrós et al. 1998, 1999; Pelchat et al. 2000). The PLMVd reference sequence was obtained from the peach cultivar ‘Armking S5615’, which was infected with a latent isolate of the viroid as indicated by field symptoms and cross-protection bioassay in the greenhouse. Other molecular variants from isolates D168 (severe), LS35 (latent), Esc76906 (latent) and from Italian and North American sources have 335–342 residues and a considerable number of point mutations (Shamloul et al. 1995; Ambrós et al. 1998; Pelchat et al. 2000). Despite the relatively high polymorphism found for PLMVd, some constraints seem to limit sequence heterogeneity in this viroid as the preservation of the stability of the hammerhead ribozymes, the conservation of a branched secondary structure and the formation of one or more pseudoknots (Ambrós et al. 1998; Bussière et al. 2000; Pelchat et al. 2000).

Data on the biological properties of the molecular variants that form different PLMVd isolates have been obtained by mechanical inoculation of dimeric cDNA clones on the peach indicator ‘GF-305’. PLMVd variants from latent isolates gave rise to symptomless infections. In contrast, the effects induced by variants from a severe isolate were variable indicating the existence of a mixture of sequence variants with different pathogenicity. Modifications of the balance between the distinct kinds of variants in the course of the infection may determine to a large extent the fluctuations in symptoms observed in the case of PLMVd severe isolates (Ambrós et al. 1998). On the other hand, analysis of the progenies from individual PLMVd cDNA clones showed a quick generation of sequence heterogeneity suggesting that modifications of the sequence spectrum, due to the quasi species, was a natural consequence of the population dynamic, observed in natural PLMVd infections (Ambrós et al. 1999). Moreover, the high polymorphism of PLMVd progenies also suggests that the genetic variability found in natural isolates of this viroid is not the consequence of repeated infections of the same plant but rather an intrinsic property of this RNA to evolve rapidly. PLMVd belongs to family Avsunviroidae (see Chapter 8 ‘Classification’ for a detailed description) which includes two other members, Avocado sunblotch viroid (ASBVd) (Hutchins et al. 1986) and Chrysanthemum chlorotic mottle viroid (CChMVd) (Navarro and Flores 1997). The three species of this family contain hammerhead structures in both polarity strands that mediate in vitro, and most probably in vivo, ribozymatic self-cleavage (see Chapter 56 ‘Ribozyme reactions of viroids’). Within family Avsunviroidae, PLMVd shows more features in common with CChMVd than with ASBVd (Navarro and Flores 1997) and, therefore, the two former viroids have been clustered in the genus Pelamoviroid, a name derived from PLMVd which is the type species of this genus. Acknowledgements

This work was partially supported by grants PB95-0139 and PB98-0500 from the Comisión Interministerial de Ciencia y Tecnología de España to R. Flores, and by grant PCE-G-00-9800009-00 from USAID to A. Hadidi. We thank J.C. Desvignes for providing photographs illustrating PLMVd symptoms. References Albanese, G., Giunchedi, L., La Rosa, R., and Poggi-Pollini, C. (1992). Peach latent mosaic viroid in Italy. Acta Hortic. 309, 331-338. Ambrós, S., Desvignes, J. C., Llácer, G., and Flores, R. (1995). Peach latent mosaic and pear blister canker viroids: detection by molecular hybridization and relationships with specific maladies affecting peach and pear trees. Acta Hortic. 386, 515-521. Ambrós, S., Hernández, C., Desvignes, J. C., and Flores, R. (1998). Genomic structure of three phenotypically different isolates of peach latent mosaic viroid: implications of the existence of

159

R. Flores et al.

constraints limiting the heterogeneity of viroid quasi-species. J. Virol. 72, 7397-7406. Ambrós, S., Hernández, C., and Flores, R. (1999). Rapid generation of genetic heterogeneity in progenies from individual cDNA clones of peach latent mosaic viroid in its natural host. J. Gen. Virol. 80, 22392252. Badenes, M.L., and Llácer, G. (1998). Occurrence of peach latent mosaic viroid in American peach and nectarine cultivars in Valencia, Spain. Acta Hortic. 472, 565-570. Barba, M., Cupidi, A., Loreti, S., Faggioli, F., and Martino, L. (1995). In vitro micrografting: a technique to eliminate peach latent mosaic viroid from peach. Acta Hortic. 386, 531-535. Bussière, F., Ouellet, J., Côté, F., Lévesque, D., and Perreault, J. P. (2000). Mapping in solution shows the peach latent mosaic viroid to possess a new pseudoknot in a complex, branched secondary structure. J. Virol. 74, 2647-2654. Desvignes, J. C. (1976). The virus diseases detected in greenhouse and in the field by the peach seedling GF 305 indicator. Acta Hortic. 67, 315-323. Desvignes, J. C. (1982). Resistance of some Prunus species to peach latent mosaic virus disease. Acta Hortic. 130, 89-91. Desvignes, J. C. (1986). Peach latent mosaic and its relation to peach mosaic and peach yellow mosaic virus diseases. Acta Hortic. 193, 51-57. Di Serio, F., and Ragozzino, A. (1995). Indagini sulla presenza del viroide del mosaico latente del pesco (PLMVd) in Campania. Inform. Fitopatol. 9, 57-61. Di Serio, F., Malfitano, M., Flores, R., and Randles, J. W. (1999). Detection of PLMVd in Australia. Aust. Plant Pathol. 28, 80-81. Faggioli, F., Loreti, S., and Barba, M. (1997). Occurrence of peach latent mosaic viroid (PLMVd) on plum in Italy. Plant Dis. 81, 423. Flores, R., and Llácer, G. (1988). Isolation of a viroid-like RNA associated with peach latent mosaic disease. Acta Hortic. 235, 325-332. Flores, R., Hernández, C., Desvignes, J. C., and Llácer, G. (1990). Some properties of the viroid inducing the peach latent mosaic disease. Res. Virol. 141, 109-118. Flores, R., Hernández, C., Avinent, L., Hermoso, A., Llácer, G., Juárez, J., Arregui, J.M., Navarro. L., and Desvignes, J. C. (1992). Studies on the detection, transmission and distribution of peach latent mosaic viroid in peach trees. Acta Hortic. 309, 325-330. Gispert, C., Perring, T. M., and Creamer, R. (1998). Purification and characterization of peach mosaic virus. Plant Dis. 82, 905-908. Giunchedi, L., Gentit, P., Nemchinov, L., Poggi-Pollini, C., and Hadidi, A. (1998). Plum spotted fruit: a disease associated with peach latent mosaic viroid. Acta Hortic. 472, 571-579. Hadidi, A., Giunchedi, L., Shamloul, A. M., Poggi-Pollini, C., and Amer, M. A. (1997). Occurrence of peach latent mosaic viroid in stone

160

fruits and its transmission with contaminated blades. Plant Dis. 81, 154-158. Hernández, C., and Flores, R. (1992). Plus and minus RNAs of peach latent mosaic viroid self cleave in vitro via hammerhead structures. Proc. Natl. Acad. Sci. USA 89, 3711-3715. Hutchins, C., Rathjen, P. D., Forster, A. C., and Symons, R. H. (1986). Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 14, 3627-3640. James, D., and Howell, W. E. (1998). Isolation and partial characterization of a filamentous virus associated with peach mosaic. Plant Dis. 82, 909-913. Kishi, K., Takanashi, K., and Abiko, K. (1973). New virus diseases of peach, yellow mosaic, oil blotch and star mosaic. Bull. Hort. Res. Sta. Japan, Ser A. 12, 197-208. Kyriakopoulou, P. E., Giunchedi, L., and Hadidi, A. (2001). Peach latent mosaic viroid and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdaliformis: implications on possible origin of these viroids in the Mediterranean region. J. Plant Pathol. 83, 51-62. Loreti, S., Faggioli, F., and Barba M. (1995). A rapid extraction method to detect peach latent mosaic viroid by molecular hybridization. Acta Hortic. 386, 560-564. Navarro, B., and Flores, R. (1997). Chrysanthemum chlorotic mottle viroid: unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes. Proc. Natl. Acad. Sci. USA 94, 11262-11267. Nemeth, M. (1986). Virus, mycoplasma and rickettsia diseases of fruit trees. Martinus Nijhoff Publishers: Dordrecht, The Netherlands. Osaki, H., Yamamuchi, Y., Sato, Y., Tomita, Y., Kawai, Y., Miyamoto, Y., and Ohtsu, Y. (1999). Peach latent mosaic viroid isolated from stone fruits in Japan. Ann. Phytopathol. Soc. Japan 65, 3-8. Pelchat, M., Levesque, D., Ouellet, J., Laurendeau, S., Levesque, S., Lehoux, J., Thompson, D. A., Eastwell, K. C., Skrzeczkowski, L. J., and Perreault, J. P. (2000). Sequencing of peach latent mosaic viroid variants from nine North American peach cultivars shows that this RNA folds into a complex secondary structure. Virology 271, 37-45. Shamloul, A.M., and Hadidi, A. (1999). Sensitive detection of potato spindle tuber and temperate fruit tree viroids by reverse transcription-polymerase chain reaction-probe capture hybridization. J. Virol. Methods 80, 145-155. Shamloul, A. M., Minafra, A., Hadidi, A., Giunchedi, L., Waterworth, H. E., and Allam, E. K. (1995). Peach latent mosaic viroid: nucleotide sequence of an Italian isolate, sensitive detection using RT-PCR and geographic distribution. Acta Hortic. 386, 522-530. Skrzeczkowski, L. J., Howell, W. E., and Mink, G. I. (1996). Occurrence of peach latent mosaic viroid in commercial peach and nectarine cultivars in the U.S. Plant Dis. 80, 823.

PART IV

CHAPTER 23

PEACH LATENT MOSAIC VIROID IN TEMPERATE FRUIT A. Hadidi, L. Giunchedi, H. Osaki, A.M. Shamloul, A. Crescenzi, P. Gentit, L. Nemchinov, P. Piazzolla, and P.E. Kyriakopoulou

....................................................................................................

HOSTS

.................................................................................................................................................................................................................................................................

For almost two decades PLMVd was presumed to be restricted to peach or peach hybrid trees. During the last few years, however, it has been shown that other stone fruit trees, such as sweet cherry, P. avium; plum, P. domestica; and apricot, P. armeniaca, as well as Japanese plum, P. salicina; and mume, P. mume, are natural hosts of PLMVd (Faggioli et al. 1997; Hadidi et al. 1997; Giunchedi et al. 1998; Osaki et al. 1999). In addition, natural infection of cultivated pear, Pyrus communis, and wild pear, P. amygdaliformis, with PLMVd has been reported recently (Kyriakopoulou et al. 2001). With the exception of PLMVd infection in plum that is associated with plum spotted fruit disease in Italy (see below), the economic impact of PLMVd in stone fruits other than peach and in cultivated and wild pears has not yet been critically determined. Plum fruits with spot symptoms in Italy may be of reduced value as they may not be suitable for export.

Emilia-Romagna region of central Italy (Giunchedi et al. 1998). Plum fruit symptoms consist of numerous small areas of the epidermis with a lighter color than that of the surrounding skin. The areoles on the deep purple face of the plums are pale red, while those on the shaded side are pale green. These areoles appear to have similar dimensions, a round shape with a diameter of 1–2 mm, with an irregular border but quite clearly defined (see Plate 7A). There are normally a large number of these on the same fruit, irregularly distributed, but with a tendency to be concentrated in the petiole area. Discolored areas may merge to form spots 6–8 mm in diameter clearly visible during July–August but with the tendency to be masked as the fruit matures by the black color and the presence of the fruit’s waxy coating. If, however, the fruit’s waxy coating is removed, the spots become evident in the form of marks slightly lighter than the basic purple-black color of the normal surrounding areas. In some fruits, the discolored marks are so numerous that they give the fruits a distinct spotty appearance. The disease is widespread in the ‘Angeleno’ plum orchards affecting up to 50% in an orchard with as many as 60–70% of fruit showing disease symptoms.

PLMVd is associated with a fruit disease of ‘Angeleno’ plum, termed plum spotted fruit, observed in plum orchards in the

PLMVd is latent in infected sweet cherry, apricot, Japanese plum, and mume.

Peach latent mosaic viroid (PLMVd) in peach, Prunus persica, is presented in the previous chapter. This chapter deals with currently known hosts of PLMVd other than peach.

ECONOMIC IMPACT AND SYMPTOMATOLOGY

161

A. Hadidi et al.

Mixed infection of PLMVd with other viroids in wild or cultivated pear show symptoms that appear on fruits as brown rusty circular or irregular patches on the skin that cover the whole fruit.

HOST RANGE In addition to peach and peach hybrids (see previous chapter), PLMVd was detected in naturally infected plum (Hadidi et al. 1997; Faggioli et al. 1997; Giunchedi et al. 1998), apricot and sweet cherry (Hadidi et al. 1997; Osaki et al. 1999), Japanese plum and mume (Osaki et al. 1999). PLMVd also naturally infects cultivated pear in Greece and Italy and wild pear in Greece (Kyriakopoulou et al. 2001).

TRANSMISSION PLMVd in a symptomless naturally infected sweet cherry tree was successfully transmitted by grafting to peach ‘GF-305’ seedlings as indicated by dot blot hybridization of leaf nucleic acid extracts from the two plant species with a DIG- labeled PLMVd cRNA probe (Figure 23.1). Also, PLMVd in a symptomless naturally infected plum tree was successfully transmitted by grafting to peach ‘GF-305’ seedlings as shown by polyacrylamide gel electrophoretic analysis of extracted leaf RNA from the naturally infected plum tree and from graft-infected peach seedlings (Faggioli et al. 1997).

GEOGRAPHICAL DISTRIBUTION AND EPIDEMIOLOGY PLMVd was detected at a low rate in quarantined germplasm at the USDA plant quarantine facility in Beltsville, MD in the following plant species: apricot (from France, Nepal, and Japan), plum (from Romania, and former Yugoslavia), sweet cherry (from Romania, and Italy) (Hadidi et al. 1997). It was also detected in sweet cherry from Canada (Hadidi et al. 1997), and in plum from Italy (Faggioli et al. 1997; Giunchedi et al. 1998). In Japan, PLMVd was detected at a low rate in sweet cherry, Japanese plum, and mume from local orchards and in apricot germplasm located at the National Institute of Fruit Tree Science, Fujimoto, Tsukuba (Osaki et al. 1999). PLMVd naturally infects cultivated pear in Greece and it has been found in a few pear trees cv. ‘Passacrassana’ in Italy and wild pear in Greece (Kyriakopoulou et al. 2001). About one-third of pear samples from Peloponesus, Greece were found to be infected with PLMVd.

DETECTION PLMVd in a naturally infected Italian symptomless plum tree was detected by polyacrylamide gel electrophoresis analysis of RNA extracts of plum leaves and leaves of grafted peach ‘GF305’ seedlings (Faggioli et al. 1997). PLMVd in naturally infected quarantined plum, apricot, and cherry germplasm in Beltsville, MD was detected by dot-blot and Northern-blot molecular hybridization of nucleic acid extracts of infected leaf tissue using a DIG-labeled PLMVd cRNA probe (Hadidi et al. 162

Figure 23.1 Dot-blot hybridization detection of PLMVd in leaf nucleic acid extracts of naturally infected Italian sweet cherry and grafted peach ‘GF-305’ seedlings. A DIG-labeled PLMVcRNA probe was used for hybridization. a) peach ‘GF-305’, PLMVd positive control; b) peach ‘GF-305’, healthy control; c) cherry plant P1 (from field); d) cherry plant P2 (from field), PLMVd infected; e) cherry plant P3 (from field), PLMVd infected; f) peach ‘GF-305’, experimentally infected (by graft inoculation) from cherry plant P3; g) cherry, healthy control.

1997). PLMVd naturally infected Italian plum showing plum spotted fruit disease (Giunchedi et al. 1998) was detected by reverse transcription-polymerase reaction (RT-PCR) using DNA primers specific for RT-PCR amplification of PLMVd (Shamloul et al. 1995; Hadidi et al. 1997) and the identity of the viroid was confirmed by molecular cloning and nucleotide sequencing of the amplified cDNA product (Figure 23.2). PLMVd in naturally infected Italian sweet cherry was detected by dot-blot molecular hybridization analysis of extracted RNA of cherry leaves and leaves of grafted peach ‘GF-305’ seedlings using a DIG-labeled PLMVd cRNA probe (Figure 23.1). PLMVd was directly detected from Canadian sweet cherry infected leaves by RT-PCR and the viroid identity was confirmed by molecular cloning and nucleotide sequencing of RTPCR product (Hadidi et al. 1997). In Japan, PLMVd was detected by RT-PCR at a low rate in Japanese plum, mume, and sweet cherry from local orchards and in apricot from a germplasm collection. PLMVd in Italian and Greek pear and in Greek wild pear was detected by molecular hybridization of nucleic acid extracts

PEACH LATENT MOSAIC VIROID IN TEMPERATE FRUIT HOSTS

Figure 23.2 The predicted optimal secondary structure at minimum free energy for PLMVd plum (from Giunchedi et al. 1998).

with a DIG-labeled PLMVd cRNA probe. RT-PCR detection of PLMVd from Greek wild pear and Italian cultivated pear followed by molecular cloning and nucleotide sequencing of the amplified cDNA products confirmed the identity of PLMVd (Kyriakopoulou et al. 2001). PLMVd from the above hosts may also be detected by RT-PCRprobe capture hybridization (ELISA) assay which was applied successfully for the detection of PLMVd from peach (Shamloul and Hadidi 1999).

CONTROL Use of viroid-tested propagated material.


Family: Avsunviroidae Genus: Pelamoviroid Species: Peach latent mosaic viroid Nucleotide sequence analysis of cloned viroids from sweet cherry plants indicated that the cherry variant of PLMVd is 337 nt long. It differs from the 336– and 337-nt-long Italian and French peach isolates of PLMVd at 25 and 30 sites, respectively.

The sweet cherry variant of PLMVd shares 91–92% identity with the French and Italian isolates of PLMVd from peach (Hadidi et al. 1997). Nucleotide sequence analysis of cloned viroids from symptomless plum plants from Romania or from plum plants from Italy showing plum spotted fruit disease demonstrated that the plum variant is 338 nucleotides (Figure 23.2) and it differs from the Italian and French peach isolates of PLMVd at 25 and 26 sites, respectively. The plum variant of PLMVd shares 92% identity with the peach isolates (Giunchedi et al. 1998). Nucleotide sequence analysis of cloned viroids from the Italian cultivated pear and the Greek wild pear showed the presence of PLMVd variants with the same chain length of 338 nucleotides. The Italian and Greek pear variants differ from the Italian and French peach isolates at 36–37 and 32–33 sites, respectively. The sequences of these two variants share 89–90% identity with the French and Italian isolates from peach (Kyriakopoulou et al. 2001). References Faggioli, F., Loreti, S., and Barba, M. (1997). Occurrence of peach latent mosaic viroid (PLMVd) on plum in Italy. Plant Dis. 81, 423.

163

A. Hadidi et al.

Giunchedi, L., Gentit, P., Nemchinov, L., Poggi-Pollini, C., and Hadidi, A. (1998). Plum spotted fruit: a disease associated with peach latent mosaic viroid. Acta Hortic. 472, 571-579. Hadidi, A., Giunchedi, L., Shamloul, A. M., Poggi-Pollini, C., and Amer, M. A. (1997). Occurrence of peach latent mosaic viroid in stone fruits and its transmission with contaminated blades. Plant Dis. 81, 154-158. Kyriakopoulou, P.E., Giunchedi, L., and Hadidi, A. (2001). Peach latent mosaic viroid and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdaliformis: implications on possible origin of these viroids in the Mediterranean region. J. Plant Pathol. 83, 51-62.

164

Osaki, H., Yamamuchi, Y., Sato, Y., Tomita, Y., Kawai, Y., Miyamoto, Y., and Ohtsu, Y. (1999). Peach latent mosaic isolated from stone fruits in Japan. Ann. Phytopathol. Soc. Jpn. 65, 3-8. Shamloul, A.M., and Hadidi, A. (1999). Sensitive detection of potato spindle tuber and temperate fruit tree viroids by reverse transcription-polymerase chain reaction-probe capture hybridization. J. Virol. Methods 80, 145-155. Shamloul, A. M., Minafra, A., Hadidi, A., Giunchedi, L., Waterworth, H. E., and Allam, E. K. (1995). Peach latent mosaic viroid: nucleotide sequence of an Italian isolate, sensitive detection using RT-PCR and geographic distribution. Acta Hortic. 386, 522-530.

PART IV

CHAPTER 24

HOP STUNT VIROID IN PLUM AND PEACH ....................................................................................................

T. Sano

.................................................................................................................................................................................................................................................................

Dapple fruit disease of plum and peach is one of the viroid diseases endemic to Japan. A disease which farmers called ‘Kirinka’, meaning a fruit with giraffe skin-like patches, was first discovered in 1986 on plum (Prunus salicina) cv. ‘Taiyo’ at an orchard in Enzan city, Yamanashi prefecture, Japan (Terai 1985, 1990). The disease problem intensified in the 1980s as it became prevalent on the other main cultivars such as ‘Ohishiwase-Sumomo’, ‘Beauty’, and ‘Santa Rosa’. Another fruit disorder, of cv. ‘Soldam’— which is now known as Soldam yellow fruit disease — was also recognized from earlier times by farmers in the same prefecture. It was called ‘Abura-Soru’, meaning a ‘Soldam’ with greasy pericarp, however, the cause of the disorder had remained unsolved. Generally it was not considered as an infectious disease before Terai (Terai et al. 1987; Terai 1990) reported that it was incited by the same pathogen causing plum dapple fruit disease. Furthermore, a similar fruit disorder was also recognized on a peach (Prunus persica) cv. ‘AsamaHakutou’ in Yamanashi prefecture. More recently, a new fruit disorder showing crinkling on the mature fruit surface has been reported on peach cv. ‘Hikawa-Hakutou’ in Yamanashi prefecture (Y. Terai, personal communication). Since infection with hop stunt viroid (HSVd-plum) was confirmed by nucleotide

sequencing, the disease appeared to be another example of HSVd-inducing syndromes (T. Sano, unpublished data). Similar diseases were prevalent in plum orchards in Fukushima and Fukuoka prefectures. In Fukuoka, after 1978, plum production has been encouraged by the regional government as a specialty of the region. Due to an expansion of the acreage, similar fruit disorders gradually became conspicuous on cvs. ‘Ohishiwase-Sumomo’ and ‘Soldam’ after around 1987 (Hirashima et al. 1994). From the symptoms and route of introduction of the scion material, it seemed that the history of the disease was identical to that of plum dapple fruit disease found in Yamanashi prefecture.

ECONOMIC IMPACT AND SYMPTOMATOLOGY Symptoms of plum and peach diseases are restricted to the fruit and vary according to the species (plum or peach) and cultivars. On plum (cvs. ‘Taiyo’ and ‘Ohishiwase-Sumomo’), irregular reddish blotches are produced on the pericarp which results in the dapple fruit symptom (plum dapple fruit disease) (see Plate 7B). In cv. ‘Taiyo’, which produces a typical dapple fruit, the surface of immature green fruit becomes irregular. At the initial stage of coloring, irregular reddish blotches appear on the light

165

T. Sano

green fruit surface. Since it resembles the pattern of giraffe’s skin, farmers called it ‘Kirin-ka’ (giraffe fruit). The dappling of fruit becomes less distinct as the fruit ripens with the color of the fruit skin progressing from light green to red. The maturation of infected fruits is retarded by one week or so. The infected fruit flesh becomes a little hard which somehow results in an improvement of preservation quality. Although the eating quality is normal or not so good, the commodity value of the infected fruit is diminished significantly by the abnormal appearance. There are no discernible symptoms on the foliage or tree structure. On the plum cv. ‘Soldam’, the dapple fruit symptom does not appear, but the pericarp gets a polished appearance due to poor formation of the wax layer on the surface, and the normal crimson color of the fruit flesh turns to yellowish red (Soldam yellow fruit disease). A reciprocal graft-inoculation experiment between ‘Taiyo’ and ‘Ohishiwase-Sumomo’ clearly showed that both diseases were caused by the same pathogen (Terai et al. 1987). The symptoms of peach dapple fruit disease on cv. ‘AsamaHakutou’ are characterized by chlorotic blotches on the pericarp of mature fruits (see Plate 7C). On the peach cv. ‘HikawaHakutou’, the symptom is characterized by crinkling on the mature fruit surface (Plate 7D).

CHARACTERISTICS OF THE CAUSAL VIROID The varieties of fruit disorders described above were all incited by infection with Hop stunt viroid (HSVd) isolates (HSVd-plum and -peach) (Sano et al. 1989) and back-inoculation experiments induced the typical dapple fruit symptom (Terai et al. 1990). These HSVd isolates consist of 297 nucleotides with a few minor sequence variations. They are considered to be a type mainly infecting stone fruits, because other isolates of identical or very similar nucleotide sequence were commonly found in symptomless apricot and almond in Europe (Astruc et al. 1996; Kofalvi et al. 1997). The viroid can be transmitted mechanically to several cucurbitaceous plants. Thus isolates from plum (cv. ‘Taiyo’) induced symptoms of leaf curling, stunting, or necrosis on Benincasa hispida, Cucumis melo, C. sativus, Lagenaria siceraria, Luffa cylindrica and Momordica charantia, but latently infected Citrullus vulgaris, Cucurbita mochata, and Cucurbita maxima (Sano et al. 1989). The same isolate is also transmissible to hops (Humulus lupulus) and has a potential to induce hop stunt disease (Sano et al. 1999).

TRANSMISSION The plum and peach dapple fruit disease agent is mainly transmitted in nature by grafting (Terai 1985; Terai et al. 1990).

166

However, the possibility of mechanical transmission through contaminated implements such as grafting knives can not be excluded. Terai et al. (1990) inoculated by razor slashing 3-yearold plum seedlings with HSVd extracts prepared from an infected plum tree. Buds collected from the inoculated seedlings in the fall were graft-inoculated on the branches of an adult healthy plum tree; similar inoculations using bud-tip grafting were repeated once again in the next fall. Typical dapple fruit symptoms were observed in the summer three years post-inoculation on the tree which received the grafted buds. Hirashima et al. (1994) carried out a mechanical transmission experiment to examine whether the viroid could be transmitted by contaminated knives to healthy trees from the infected ones. Healthy trees were repeatedly injured, three times in two years, by a 30time reciprocating slashing using a knife which had previously slashed an infected tree. When the experiment was performed using cv. ‘Soldam’, the three inoculated trees became HSVdpositive a year post-slashing, suggesting that the causal viroid could be transmitted from affected to healthy plants of this cultivar by contaminated implements. However, when the experiment was performed using cv. ‘Ohishiwase-Sumomo’ as donor and peach seedlings as acceptors, none of the seedlings inoculated became HSVd-positive. Although the possibility of viroid transmission among trees appears to be variable depending on the cultivars, it is concluded that the viroid could be transmitted by implements such as a grafting knife which had been contaminated with infected plant sap. Hirashima et al. (1994) examined whether the agent was transmissible through root contact. To this aim, an infected plum cv. ‘Ohishiwase-Sumomo’ was grafted on a peach seedling and planted in a pot with eight peach seedlings which are commonly used as a rootstock for plum cultivation in Japan. The roots of these plants were tightly intertwined with one another. A year later, however, none of the eight peach seedlings were HSVdpositive until two years after mix-planting. These results may suggest that viroid transmission through root contact is low, though much longer-term observations would be needed. In addition, it will also be necessary to examine cases in which plum or Japanese plum seedlings are used as rootstocks, because although they are not as common in Japan, they are much more sensitive to the viroid than peach.

DIAGNOSIS Plum cvs ‘Taiyo’, ‘Soldam’ and ‘Ohishiwase-Sumomo’ appear to be the most sensitive indicators, but it takes at least 2–3 years to express typical disease symptoms. Symptom expression on plum is also influenced by cultivars and the weather conditions. Furthermore, symptoms on peach are not only obscure but in many cases are not apparent at all indicating that peach can be regarded as a symptomless carrier. For the reasons described above, the application of accurate diagnostic techniques is indis-

HOP STUNT VIROID IN PLUM AND PEACH

pensable for disease control. One of the most sensitive methods to detect the plum and peach dapple fruit agent is the ‘cucumber assay’ which is also used to diagnose hop stunt disease. Low molecular weight RNAs extracted from bark or shoots of infected trees is the optimum inoculum for the assay. Infected plants show symptoms of leaf curling, vein clearing and finally stunting 3–4 weeks post-inoculation. The assay is sensitive enough to detect HSVd in symptomless plum and peach. However, the assay is no longer used for diagnostic purposes in Japan, since it requires the greenhouse space kept at high temperature. Techniques using gel electrophoresis such as return-, two dimensional-, or double-polyacrylamide gel electrophoresis (PAGE) are useful, but not always satisfactory when the titer of the viroid is low (Sano et al. 1989; Hirashima et al. 1994). Dot-blot hybridization or double-PAGE followed by Northern hybridization using a digoxigenin (DIG)-labeled HSVd cRNA probe is highly sensitive and one of the most reliable diagnostic techniques which is almost independent of the cultivars or samples use for the diagnostic testing. Disadvantages of the method are the requirement for highly skilled operators and that it is not suitable for handling a large number of samples. Polymerase chain reaction in combination with reverse transcription (RT-PCR) is another highly reliable technique, which can overcome some of the disadvantages mentioned above, for the detection of plum and peach dapple fruit disease agent (Hadidi et al. 1992). Since plum and peach leaves contain relatively large amounts of polysaccharides and polyphenolic compounds which may inhibit the PCR reaction, these inhibitors must be removed by using ELUTIP-r mini columns (Hadidi et al. 1992), CF-11 cellulose (Whatman) in the extraction procedure or a nucleic acid extraction kit such as SepaGene (Sanko Chemical) (Kusano et al. 1997). Nucleic acid preparations extracted from bark or roots are preferable for RT-PCR amplification than those from leaves. RT-PCR under the best conditions is 10,000 times more sensitive than the PAGE based analysis (Kusano et al. 1997).

CONTROL Though the plum and peach dapple fruit diseases are mainly transmitted by grafting in orchards (Terai 1985, 1990; Terai et al. 1990), the experimental data clearly indicated that the causal agent could be transmitted through contaminated implements such as grafting knives or pruning shears (Hirashima et al.

1994). Based on this knowledge, the following control measures are now recommended: i

routine diagnosis of HSVd-plum and -peach in the laboratory by molecular hybridization or RT-PCR for earlier detection and elimination of the diseases; and

ii

production and provision of viroid-free plum and peach trees as replanting materials.

References Astruc, N., Marcos, J.F., Macquaire, G., Candresse, T., and Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. Eur. J. Plant Pathol. 102, 837-846. Hadidi, A., Terai, Y., Powell, C.A., Scott, S.W., Desvignes, J.C., Ibrahim, L.M., and Levy, L. (1992). Enzymatic cDNA amplification of hop stunt viroid variants from naturally infected fruit crops. Acta Hortic. 309, 339-344. Hirashima, K., Noguchi, Y., Ushijima, K., and Kusano, N. (1994). Diagnosis of plum dapple fruit disease by polyacrylamide gel electrophoresis and mechanical transmission. Bull. Fukuoka. Agric. Res. Cent. B-13, 65-68. Kofalvi, S. A., Marcos, J. F., Cañizares, M. C., Pallás, V., and Candresse, T. (1997). Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. J. Gen. Virol. 78, 3177-3186. Kusano, N., and Shimomura, K. (1997). Selection of PCR primers and a simple extraction method for detection of hop stunt viroid-plum in plum by reverse transcription-polymerase chain reaction. Ann. Phytopathol. Soc. Jpn. 63, 119-123. Sano, T., Hataya, T., Terai, Y., and Shikata, E. (1989). Hop stunt viroid strains from dapple fruit disease of plum peach in Japan. J. Gen. Virol. 70, 1311-1319. Sano, T., Ito, S., Narita, M., Murakami, A., and Shikata, E. (1999). Assessment of potential risks of hop stunt viroid isolates harboring in grapevine, plum, citrus and hop. In Abstracts of XIth International Congress of Virology, August 9-13, Sydney, Australia. Terai, Y. (1985). Symptoms and graft-transmission of plum dapple fruit disease. Ann. Phytopathol. Soc. Jpn. 51, 363-364. (Abstract in Japanese). Terai, Y., Sano, T., Hataya, T., and Shikata, E. (1987). The relationship between plum dapple fruit disease and yellow fruit disease of Soldam. Ann. Phytopathol. Soc. Jpn. 53, 423. (Abstract in Japanese). Terai, Y. (1990). Occurrence of a new viroid disease, plum dapple fruit. Shokubutu Boeki. 44, 127-129. Terai, Y., Sano, T., and Shikata, E. (1990). Back-inoculation of plum dapple fruit disease and graft-transmission of peach dapple fruit disease. Ann. Phytopathol. Soc. Jpn. 56, 428. (Abstract in Japanese).

167

PART IV

CHAPTER 25

HOP STUNT VIROID IN APRICOT AND ALMOND ....................................................................................................

V. Pallás, G. Gómez, K. Amari, M.C. Cañizares, and T. Candresse

.................................................................................................................................................................................................................................................................

Although Hop stunt viroid (HSVd) was described in the 1970s as the causal agent of a peculiar disease characterized by severe stunting on Japanese commercial hop (Humulus lupulus) (Sasaki and Shikata 1977), it was later reported in a wide range of hosts including cucumber, grapevine, citrus, plum, peach and pear (Shikata 1990). More recently, it was described in apricot (Flores et al. 1990; Astruc et al. 1996; Kofalvi et al. 1997) and almond (Cañizares et al. 1999). In some hosts, such as grapevine (Shikata 1990; Polivka et al. 1996) and apricot (Astruc et al. 1996), the infection seems to be latent; in others it is associated with serious disorders of economic importance i.e. hop stunt (Shikata 1990), dapple fruit disease of plum and peach (Sano et al. 1989) and citrus cachexia (Diener et al. 1988; Semancik et al. 1988; Reanwarakorn and Semancik 1999). Apricot and almond are two of the most important fruit crops in the world with a total world production of 2,763,855 and 1,633,036 tons, respectively (FAO 1999). Recent studies (Cañizares et al. 1998, 2001) revealed HSVd in 81% of the apricot trees tested in southeastern Spain. In these studies samples were taken from four different growing areas of the Murcia tegion and HSVd found to be uniformly distributed. This survey included five commercially important apricot cultivars. Although the number of samples analyzed for each cultivar var-

168

ied from 11 to 81 (the number of samples analyzed was highest for cv. ‘Bulida’ which is the most extensively grown variety in the Murcia area), there were no significant differences in infection levels between the different cultivars. A lower HSVd incidence in apricot was reported for other Mediterranean countries: 10.4% for Cyprus, 10.3% for Morocco, 5% for Greece and 2% for Turkey (Amari et al. 2000b). Of the 65 apricot varieties tested in these countries, 12 were found infected, including 5 local varieties. Thus, although HSVd is latent in apricot, this host could represent a natural reservoir from which the viroid can potentially be transmitted to other susceptible host crops, including other stone fruits. Although HSVd has also been isolated and characterized from almond (Cañizares et al. 1998), no significant data were collected to know the real incidence of this viroid in this important crop.

DETECTION HSVd can be detected by biological, and molecular methods. The viroid can be detected by mechanical transmission to cucumber (Shikata 1990). Alternatively, indexing of HSVdinfected Prunus can be performed on Prunus salicina in which the viroid reaches high titers, although the lack of symptoms in this host makes it necessary to detect by other methods. Thus,

HOP STUNT VIROID IN APRICOT AND ALMOND

this assay is too expensive and lengthy for large-scale indexing. HSVd has also been detected in apricot by sequential PAGE and ethidium bromide staining using RNA preparations obtained by phenol extraction and chromatography on non-ionic cellulose (Flores et al. 1990). Recently, a nucleic acid extraction method that avoids the use of phenol (Pallás et al. 1987; Astruc et al. 1996) in conjunction with the use of non-isotopic molecular hybridization with a cRNA probe (Pallás et al. 1998a, and b) has been shown to be very sensitive and to allow the processing of a large number of samples (Cañizares et al. 1998, 1999; Amari et al. 2000). However, in almond, a considerable number of false positives were observed when this methodology was used. A treatment with RNase A at high ionic strength after hybridization was effective in eliminating these spurious hybridizations and allowed efficient indexing (Cañizares et al. 1999). It was determined that the spurious hybridization signals were due to host RNAs with sequence similarity to HSVd. This kind of interference was never observed in apricot samples. Tissue-imprinting hybridization, a technique that avoids sample extraction by the direct transfer from the cut surface of plant materials (fruits, stem, cuttings, leaves) has been applied to the detection of HSVd in apricot trees (Astruc et al. 1996; Cañizares et al. 2001; Amari et al. 2001a). The application of petiole imprint was used to monitor infection with HSVd in apricot trees throughout a growing season (Amari et al. 2001a) whereas the application of imprint of fruit pieces was successfully used for large-scale indexing of this pathogen in this crop (Cañizares et al. 2001). RT-PCR detection of HSVd from stone fruits (plum and peach) and other fruit trees was first reported in 1992 (Hadidi et al. 1992). Recently, RT-PCR has also been used for the detection of HSVd in apricot and almond tissues (Amari et al. 2000; Cañizares et al. 1999). For this purpose, PCR primers hybridizing to the central conserved region of the viroid molecule were designed in order to allow the detection of sequence variants (Astruc et al. 1996). All the known apricot and almond HSVd isolates have been successfully amplified using this primer pair (Kofalvi et al. 1997; Cañizares et al. 1999; Amari et al. 2000; Amari et al. 2001b).

detailed phylogenetic analysis of the existing HSVd sequences, together with the new HSVd sequences of Prunus isolates determined by Kofalvi et al. (1997) resulted in a redefinition of the grouping of variants of this viroid. A bias towards the presence of certain sequences and/or structures in some hosts was observed, although no host-determinants could be found conclusively. This analysis also revealed that a number of HSVd isolates might have been derived from recombination events and that the hop-type group itself could have resulted from a recombination event between members of the plum-type and citrustype groups (Kofalvi et al. 1997; Candresse et al. 1997). Most of the apricot sequence variants are grouped in the Plum-type group or in one of the minor recombinant groups (see Plate 8) (P-C group of Amari et al. 2001). In fact the high number of new sequence variants grouped in this recombinant P-C group indicates that these putative recombinant variants are more frequent than previously thought. The two almond sequence variants were also grouped in the Plum-type group. Interestingly, neither apricot nor almond sequence variants were found to belong to the Citrus-type group. Analysis of the geographical distribution of the apricot sequence variants revealed that most of the Moroccan variants are phylogenetically related to the Spanish apricot variants whereas the Greek variants were more closely related to the German grapevine variants suggesting a different geographical origin, presumably by importation, for these two groups of apricot HSVd isolates (Plate 8) (Amari et al. 2001b). Finally, it is worth noting that most of the apricot sequence variants from Cyprus are grouped into the recombinant P-C group which may suggest that other HSVd variants were derived from an intra-specific recombination event; alternatively, this event occurred more often in Cyprus than in other countries. Acknowledgements

Work in the V. Pallás and T. Candresse laboratories was partially supported by a collaborative AIR2 project of the European Community (#93-1567). In addition, work in V. Pallás laboratory was also supported by grant BIO96-0459 of the Spanish granting agency CICYT. References

MOLECULAR VARIABILITY AND PHYLOGENETIC STUDIES

Sequence homologies (Shikata 1990) and phylogenetic analysis (Hsu et al. 1994) first indicated that HSVd isolates can be separated into three groups (plum-type, hop-type and citrus-type). The fact that these groups often contained isolates coming from a limited number of isolation hosts prompted the suggestion that the group-discriminating sequence variations could in fact represent host-specific sequence determinants which may facilitate or be required for replication in a given host. Later, a

Amari, K., Cañizares M.C., Myrta, A., Sabanadzovic, Di Terlizzi, B., and Pallás, V. (2001a). Tracking Hop stunt viroid infection in apricot trees during a whole year by non-isotopic tissue printing hybridization. Acta Hortic. 550, 315-320. Amari, K., Cañizares M.C., Myrta, A., Sabanadzovic, S., Srhiri, M., Gavriel, I., Çaglayan, K., Varveri, C., Gatt, M., Di Terlizzi, B., and Pallás, V. (2001b). First report on Hop stunt viroid from some Mediterranean countries. Phytopathol. Medit. 39, 271-276. Amari, K., Gómez, G., Myrta, A., Di Terlizzi, B., and Pallás, V. (2001). The molecular characterization of 16 new sequence variants of Hop stunt viroid reveals the existence of invariable regions and a

169

V. Pallás et al.

conserved hammerhead-like structure on the viroid molecule. J. Gen. Virol. 82, 953-962. Astruc, N., Marcos, J.F., Macquaire, G., Candresse, T., and Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. European J. Plant Pathol. 102, 837-846. Candresse, T., Revers, F., Le Gall, O., Kofalvi, S.A., Marcos, J.F., and Pallás, V. 1997. Systematic search for recombination events in plant viruses and viroids. Pages 20-25 in: Virus resistant transgenic plants: potential ecological impact. M. Tepfer, and E. Balázs, eds. Springer Press: Paris. Cañizares, M.C., Aparicio, F., Amari, K., and Pallás, V. (2001). Studies on the aetiology of Apricot ‘Viruela’ disease. Acta Hortic. 550, 249-258. Cañizares, M.C., Marcos, J.F., and Pallás, V. (1998). Studies on the incidence of hop stunt viroid in apricot trees (Prunus armeniaca) by using an easy and short extraction method to analyze a large number of samples. Acta Hortic. 472, 581-585. Cañizares, M.C., Marcos, J.F., and Pallás, V. (1999). Molecular characterization of an almond isolate of hop stunt viroid (HSVd) and conditions for eliminating spurious hybridization in its diagnosis in almond samples. Eur. J. Plant Pathol. 105, 553-558. Desvignes, J.C. (ed.). (1999). Virus diseases of fruit trees. Ctifl Editions: Paris. Diener, T., Smith, D., Hammond, R., Albanese, G., La Rosa, R., and Davino, M. (1988). Citrus B viroid identified as a strain of hop stunt viroid. Plant Dis. 72, 691-693. FAO. (1999). FAO statistics. In: www.fao.org. Flores, R., Hernández, C., García, S., and Llácer, G. (1990). Is apricot ‘viruela’ (pseudopox) induced by a viroid? In: Proceedings of the 8th Congress of the Mediterranean Phytopathological Union, Agadir. Hadidi, A., Terai, Y., Powell, C.A., Scott, S.W., Desvignes, J.C., Ibrahim, L.M., and Levy, L. (1992). Enzymatic cDNA amplification of hop stunt viroid variants from naturally infected fruit crops. Acta Hortic. 309, 339-344. Hsu, Y-H., Chen, W., and Owens, R. A. (1994). Nucleotide sequence of a hop stunt viroid variant isolated from citrus growing in Taiwan. Virus Genes 9, 193-195.

170

Kofalvi, S.A., Marcos, J.F., Cañizares, M.C., Pallás, V., and Candresse, T. (1997). Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. J. Gen. Virol. 78, 3177-3186. Pallás V., Navarro, A., and Flores, R. (1987). Isolation of a viroid-like RNA from hop different from hop stunt viroid. J. Gen. Virol. 68, 3201-3205. Pallás V., Más, P., and Sánchez-Navarro, J.A. (1998a). Detection of plant RNA viruses by non-isotopic dot-blot hybridization. Pages 461-468 in: Plant virus protocols: from virus isolation to transgenic resistance. G. Foster, and S. Taylor, eds. Humana Press: Totowa, NY, USA. Pallás, V., Sánchez-Navarro, J.A., Más, P., Cañizares, M.C., Aparicio, F., and Marcos, J.F. (1998b). Molecular diagnostic techniques and their potential role in stone fruit certification schemes. Pages 191-207 in: Stone fruit viruses and certification in the Mediterranean countries: problems and prospects. B. Di Terlizzi, A. Myrta, and V. Savino, eds. Options Méditerranéennes Série B. nº 19: Bari, Italy. Polivka, H., Staub, U., and Gross, H.J. (1996). Variation of viroid profiles in individual grapevine plants: novel grapevine yellow speckle viroid 1 mutants show alterations of hairpin I. J. Gen. Virol. 77, 155-161. Reanwarakorn, K., and Semancik, J.S. (1998). Regulation of pathogenicity in hop stunt viroid-related group II citrus viroids. J. Gen. Virol. 79, 3163-3171. Reanwarakorn, K., and Semancik, J.S. (1999). Correlation of hop stunt viroid variants to cachexia and xyloporosis disease of citrus. Phytopathology 89, 568-574. Sano, T., Hataya, T., Terai, Y., and Shikata, E. (1989). Hop stunt viroid strains from dapple fruit disease of plum and peach in Japan. J. Gen. Virol. 70, 1311-1319. Sasaki, M., and Shikata, E. (1977). On some properties of hop stunt disease agent, a viroid. Proceedings of the Japan Academy 53B, 109-112. Semancik, J., Roistacher, C., Rivera-Bustamante, R., and Durán-Vila, N. (1988). Citrus cachexia viroid, a new viroid of citrus: relationship to viroids of the exocortis disease complex. J. Gen. Virol. 69, 30593068. Shikata, E. (1990). New viroids from Japan. Semin. in Virol. 1, 107-115.

PART IV

AVOCADO VIROIDS AVOCADO SUNBLOTCH VIROID J.S. Semancik

....................................................................................................

CHAPTER 26

.................................................................................................................................................................................................................................................................

Avocado sunblotch viroid (ASBVd) is the only viroid of economic importance reported to occur in avocado. Avocado sunblotch disease was first believed to be a physiological (Coit 1928) or genetic (Horne 1929) disorder, but following transmission studies, a virus etiology was proposed (Horne and Parker 1931; Whitsell 1952; Wallace and Drake 1953; Wallace 1958). Minor variations of the term ‘sunblotch’ for the causal agent as well as the disease such as sun-blotch, and sun blotch can be found in the literature. Names for the sunblotch disease and ASBVd in other languages include: i

Spanish: viroide de la mancha amarilla del aguacate, mancha de sol de los aguacate, and monchado sol del aquacate;

ii

Hebrew: kitmey shemesh; and

iii

Afrikaans: avocado sonvlek.

It is interesting to note that although avocado displays disease symptoms with only ASBVd, a natural infection with Potato spindle tuber viroid (PSTVd) was found in Peru (Querci et al. 1995). In addition, the unrelated Apscaviroid, Citrus viroid-Ib (CVd-Ib) (Duran-Vila et al. 1988), was experimentally transmitted to avo-

cado and subsequently the sequence of this variant was named Citrus bent leaf viroid (CBLVd) (Ashulin et al. 1991).

ECONOMIC IMPACT AND SYMPTOMATOLOGY No comprehensive evaluation of economic impact of ASBVd on avocado production has been made. However, it has been observed that trees affected with sunblotch disease are less productive. An estimate of yield reductions of about 30% were noted for the cultivar ‘Fuerte’ with sunblotch (da Graca et al. 1983). In addition, more than half of the fruit was downgraded on quality standards. Other cultivars such as ‘Edranol’ appear less affected with yield reduced by only about 18% (da Graca 1985). Fruits from the cultivar ‘Edranol’ with symptoms of sunblotch, although smaller in size than fruit from non-symptomatic trees, retained similar oil content (da Graca and van Vuuren 1977). With the specialized case of trees acting as symptomless carriers of ASBVd, a dramatic reduction (95%) in yield can occur as with the varieties ‘Caliente’ and ‘Reed’ (Desjardins 1987). Field symptoms of the sunblotch disease are most typically displayed as characteristic stem streaks and discolored lesions on fruit of avocado (Plate 9A and B) and thus were initially viewed as an expression of a physiological (Coit 1928) or genetic

171

J.S. Semancik

(Horne 1929) disorder. Depressed stem streaks with white, yellow or pink discoloration and grooving on older branches can be observed. Prominent white to yellow lesions on fruit of irregular size and shape result in a distortion of the fruit. Sunblotchinfected trees have a diminished canopy with a decumbent growth appearance. Mature trees with established infections display browning and cracking with possible crocodile skin checking of bark (Whitsell 1952).

‘Topa-Topa’ and ‘Todd’ produce normal crops. Fruit from the cultivars ‘Topa-Topa’, ‘Todd’, ‘Caliente’ and ‘Reed’ exhibit an unusual increase in seed transmission of sunblotch from 5% to 95% (Desjardins 1987). Seedlings propagated from such trees become themselves symptomless carrier trees. The specific variant found associated with this condition, ASBVd-Sc, accumulates to a high titer that can be detected in virtually every tissue sample.

Leaf symptoms (Plate 9C and D) although not common in the field can be observed at irregular intervals of growth flushes under most greenhouse conditions. Initially, tissue defined by bleached or chlorotic areas usually appears to be associated with vascular elements. A more generalized variegation may develop in later stages of infection. Symptoms are most striking on the cultivars ‘Hass’, ‘Bacon’ and ‘Fuerte’ which have been employed as bioassay hosts for indexing of sunblotch.

The disease syndrome can display properties of both an acute and persistent form of infection with the characteristic symptoms replaced by a ‘recovery’ to the symptomless carrier form of the disease. This transition may result from a changing profile in the viroid population since distinct variants have been found associated with bleached (ASBVd-B), variegated (ASBVd-V), and symptomless (ASBVd-Sc) tissues (Semancik and Szychowski 1994). The initial acute form of the disease identified by the bleached symptoms develops into a persistent infection with irregular and infrequent expressions of both the primary symptoms and variegation. It seems that the disease assumes a chronic behavior with the continuous production of a high titer of the predominating variant, ASBVd-Sc, throughout the plant in the absence of any disease symptom expression other than a marked reduction in fruit production but an increase in seed transmission (Desjardins 1987).

Cytological observations of disorganized and swollen chloroplasts with reduced grana, rearrangement of lamellae and the appearance of vesicles have been related to the leaf symptoms (daGraca and Martin 1981). With a severe reaction, leaf distortion, stunting and premature senescence can occur on apex tissue. This abnormal development might be a result of the depressed levels of indole acetic acid (IAA) oxidase and a higher titer of IAA detected (da Graca and van Lelyveld 1978). The relationships between the titer of ASBVd and the tissues expressing distinctly different symptoms as well as from symptomless carrier hosts is unusual for viroid-infected tissues. In leaves with ‘bleached’ symptoms, an extremely high titer of ASBVd, approaching levels of host 5S RNA, is concentrated in bleached portions of infected leaves (Semancik and Desjardins 1980). Non-symptomatic areas of the same leaf contain significantly lower titers of the viroid that were undetected by some detection protocols. The viroid is more uniformly distributed throughout both the variegated and healthy appearing areas of leaves with variegated symptoms. The most stable and uniform titers of ASBVd can be recovered from virtually every sample taken from symptomless carrier trees. The isolation and characterization of distinct variants of ASBVd from bleached (ASBVdB), variegated (ASBVd-V) and symptomless carrier (ASBVd-Sc) tissues, suggested a serial relationship among these variants during disease development (Semancik and Szychowski 1994). The term ‘recovered’ described the appearance of apparent normal growth on known sunblotch-infected trees (Wallace 1967; Wallace and Drake 1962) and the viroid was retained in this symptomless carrier reservoir of ASBVd. While these trees do not express any characteristic foliar, stem or fruit symptoms, the viroid is uniformly distributed throughout the tree and fruit production can be severely reduced in some cultivars such as the commercial cultivars ‘Hass’ and ‘Fuerte’ whereas the varieties

172

‘Stumping’ or severe pruning of older trees for top working to different scions may stimulate the appearance of sunblotch symptoms when apparently healthy symptomless carrier trees are involved. The newly grafted scions may even respond by rapid senescence. This latent expression has prompted field workers to describe sunblotch as a ‘sleeping’ disease that may remain quiescent for years. This pattern of symptom expression might suggest a serial or progressive emergence of distinctly different dominant variants of ASBVd with uneven rates of replication and/or accumulation.

HOST RANGE Among the viroids, ASBVd is characterized by having the most restricted host range. Natural transmission and disease expression is limited to avocado, Persea americanum Mill. Transmission under experimental conditions to other members of the family Lauraceae has been possible by bark patch grafting (daGraca and van Vuuren 1980b, 1981b). These include: Persea schiedeana Nees, Cinnamomum zeylanicum Blume, Cinnamomum camphora (Li) Nees and Eberm, and Ocotea bullata (Burch) Benth. Attempted transmission to C. liebertiana and P. indica Spreng, in addition to the herbaceous indicators of several viroids, Lycopersicon esculentum Mill and Gynura aurantiaca DC were unsuccessful.

AVOCADO SUNBLOTCH VIROID

TRANSMISSION

DETECTION

The principal modes of infection are by graft transmission during propagation or tissue implant and the introduction of ASBVd infected seedling sources for rootstocks. Severe outbreaks of sunblotch are possible when seedlings used as rootstock are derived from symptomless carrier trees in which seed transmission of ASBVd is very high (95%). Although root graft transmission has been reported (Whitsell, 1952), the frequency of infection in the field is unknown and probably of minor importance.

The most obvious detection of sunblotch in the field for practical diagnosis is the appearance of the symptoms of characteristic fruit lesions. In addition, when an apparently normal tree displays a markedly reduced yield, it may be considered as a candidate for symptomless carrier of ASBVd. In the absence of fruit production, reduced vegetative growth and stem grooving are also observed as expressions of sunblotch disease.

Mechanical transmission by either slash inoculation and/or leaf rub with extracts from infected tissues is possible (Desjardins et al. 1980; Allen et al. 1981) although much less efficient than graft transmission. Considering the extremely high titers of ASBVd approaching the levels of host 5S RNA (Semancik and Desjardins 1980) even in partially purified extracts, the low efficiency of mechanical inoculation suggests a problem with either the technical approach of introducing the viroid to vascular tissues of avocado or a very low intrinsic biological activity. The demonstration of specific variants associated with tissues of different symptom types as well as symptomless carrier tissues (Semancik and Szychowski 1994), suggests that successful infection may be possible with only some variants contained in the population of ASBVd quasi-species recovered during extraction. Experimental transmission of pollen from sunblotch-infected trees by a biological vector, the bee Apis mellifera, was recorded at 1–4% (Desjardins et al. 1979). It was later reported that both symptomatic and symptomless carrier trees could serve as pollen donors (Desjardins et al. 1984). However, it is doubtful if vector transmission is a major factor in the field spread of sunblotch since seed transmission (Whitsell 1952) of about 5% is common from disease expressing trees (Wallace and Drake 1953).

GEOGRAPHICAL DISTRIBUTION AND EPIDEMIOLOGY Although first reported from North America in California (Coit 1928; Horne and Parker 1931; Whitsell 1952; Wallace 1958) and later in Florida, no reports of the disease have been made from the world’s largest producer of avocado, Mexico, contributing about 35–40% of the world crop. However, the sunblotch disease has been detected in other Central American countries, Costa Rica and Guatemala as well as South America including Peru (Vargas et al. 1991) and Venezuela (Rondon and Figueroa 1976). Reports from additional avocado producing areas throughout the world have been made. These include Africa: South Africa (Loest and Stofberg 1954; da Graca and Mason 1983); Asia: Israel (Spiegel et al. 1984); Europe: Spain (LopezHerrera et al. 1987); Oceania: Australia (Thomas and Mohamed 1979; Dale and Allen 1979).

Before the viroid nature of the causal agent was recognized, indexing for the sunblotch disease initially employed a bioassay test on avocado (Whitsell 1952; Wallace 1958). Although variations of the protocol were introduced (Allen and Firth 1980), all suffered from the requirement of lengthy incubation periods of 2 to 36 months. A successful registration program for the production of sunblotch-tested trees in California was introduced by Wallace and Drake (1971) that included graft transmission and cross protection tests with an observation period of 2 years. When growth flushes of indicator plants was stimulated by topping, the incubation period for symptom expression could be reduced. Seedlings of the cultivar ‘Collison’ maintained at high temperatures (30°C) and cut back 3 months after graft inoculation displayed sunblotch symptoms in 8 months (da Graca and van Vuuren 1981a). Continuous light combined with high temperature was also found to promote symptoms (Desjardins 1987). Detection of the causal agent as a viroid and identification of ASBVd RNA by polyacrylamide gel electrophoresis (PAGE) (Palukaitis et al. 1979) introduced the application of gel electrophoresis as an indexing tool (Utermohlen and Ohr 1981) for sunblotch detection. Extracts from flower buds provided a rapid and more accurate PAGE detection procedure (da Graca and Mason 1983). More sensitive identification of the sunblotch agent was facilitated by the conservative genome of the viroid. A variety of probes could be used with molecular hybridization with both radioactive and more recently non-radioactive probes having been effectively employed in detection of ASBVd in nucleic acid extracts (Palukaitis et al. 1981; Allen 1982; Barker et al. 1985). Photobiotin-labeled DNA probes containing full-length monomers inserted in plasmid vectors detected the viroid in extracts spotted to nitrocellulose membranes (McInnes et al. 1989). Chemiluminescent RNA probes with a simple imprint hybridization procedure was effective for routine indexing (RomeroDurban et al. 1995). This procedure has the advantage of testing stems from field samples without the necessity to perform any extraction procedures. Although hybridization is generally accepted to be a more sensitive protocol than PAGE and adequate for detection of sunblotch, some plants known to be infected were not detected by these tests (Allen and Dale 1981;

173

J.S. Semancik

Figure 26.1 Nucleotide sequences and conformations of a) avocado sunblotch viroid type variant, ASBVdSB-1 (Symons 1981) and b) variants associated with symptomatic bleached (ASBVd-B) and variegated (ASBVd-V) as well as symptomless carrier (ASBVd-Sc) tissues (Semancik and Szychowski 1994).

Spiegel et al. 1984). This may result from a sampling problem and the uneven distribution of some variants of ASBVd throughout the infected plant rather than a deficiency in these techniques. Detection of ASBVd by reverse transcription-polymerase chain reaction (RT-PCR) has been described (Semancik and Szychowski 1994; Schnell et al. 1997). The conservative genome of ASBVd of 247–251 nucleotides confirmed from sequencing a

174

number of variants isolated from different avocado growing regions render a range of primer pairs effective for RT-PCR application. Those from the central conserved domain of the molecule located near nucleotide #40–80 and 160–200 (Rakowski and Symons 1989) have found the greatest application. Detection of ASBVd in the thylakoid membranes (Bonfiglioli et al. 1994) of avocado chloroplasts (Lima et al. 1994) has been made by in situ hybridization using RNA probes complemen-


tary to the viroid RNA. Localization of intermediates in the replication of ASBVd in chloroplasts suggests a site for in vivo synthesis (Navarro et al. 1999). All other viroid–host systems tested have implicated the nucleus as the primary site of replication and accumulation, with the exception of Peach latent mosaic viroid (PLMVd), which belongs to the same family as ASBVd (see below), and that recent evidence indicates that it accumulates also in the chloroplasts (Bussière et al. 1999). A serious problem in detection may be encountered with the possible sampling error introduced by the uneven distribution of ASBVd throughout infected trees. As much as a 1000-fold variation in the ASBVd content of leaves can be found on the same tree (Allen and Dale 1981). Thus infected trees expressing symptoms can be more difficult to index for ASBVd because of the unpredictable viroid distribution. On the other hand, symptomless carrier trees in which symptoms are never expressed but where ASBVd can be detected in virtually every tissue sample are easily detected by most techniques.

CONTROL An indexing program for the propagation and dissemination of registered rootstock and scion materials tested for ASBVd offers the best approach for control of sunblotch. Sanitation by removal of sunblotch expressing or symptomless carrier trees is the primary mode to control spread of the disease in the field. Mechanical transmission of extracts from infected tissues by razor slashing (Desjardins et al. 1980) or graft inoculation with filter paper containing extracts from infected trees (Allen et al. 1981) is possible but of very low efficiency. Chemical inactivation of ASBVd on pruning and propagating tools is highly effective with dip solutions of 20% sodium hypochlorite, 2% formaldehyde + 2% sodium hydroxide, or 6% hydrogen peroxide (Desjardins et al. 1987). In addition, ASBVd was reported to be inactivated in infected tissue with exposure to 56°C for 15 minutes (da Graca and van Vuuren 1980a). This treatment might be employed for the retention of valuable propagation materials. Also, seed treatment has not been reported to be effective in the reduction of infection, although exposure to the conditions noted above for infected tissues might present an effective procedure for testing.


Family: Avsunviroidae Genus: Avsunviroid Species: Avocado sunblotch viroid (ASBVd) Nucleotide sequence: see Figure 26.1. The initial indication for the viroid nature of the causal agent of sunblotch disease was the detection of an unusual RNA associ-

ated with the disease (Dale and Allen 1979; Palukaitis et al. 1979; Thomas and Mohamed 1979; Desjardins et al. 1980; Mohamed and Thomas 1980). Symons (1981) reported ASBVd as a covalently closed single-stranded, circular RNA molecule of 247 nucleotides with a high degree (67%) of antiparallel base pairing with 34% G:C, 52% A:U, and 14% G:U. The AU-rich property of the ASBVd genome is unique among the exclusively GC-rich viroids. The RNA is not associated with any coat protein and therefore cannot be recovered in vitro packaged as any regular geometric structure. Unlike the replication of other viroids, synthesis of ASBVdspecific RNAs was not affected by concentrations of alphaamanitin known to inhibit DNA-directed RNA polymerase II and III. Thus, a RNA polymerase I-like enzyme has been implicated in the replication process (Marcos and Flores 1992), and more recent data suggests the involvement of a choloroplastic RNA polymerase (Navarro et al. 2000). Replication is accomplished via a monomeric or multimeric template of the reverse polarity to the viroid as described by Bruening et al. (1982). Minor variations in the nucleotide sequence of two isolates of ASBVd were first reported by Palukaitis et al. (1981). However, additional variants of ASBVd occur (Pallas et al. 1988) within different isolates. The conserved number of residues for 16 variants within a range of 247–251 is marked by nucleotide changes found principally in the terminal loops of the molecules (Rakowski and Symons 1989). Similarly, the most striking changes found in the variants isolated from bleached (ASBVdB), variegated (ASBVd-V), and symptomless carrier (ASBVdSc) tissues were found in the right terminal loop (Semancik and Szychowski 1994). The nucleotide sequence of ASBVd-Sc is virtually identical to the sequence described originally by Symons (1981) as ASBVd SB-1. Structural periodicity of repeating units of 80 nucleotides for ASBVd similar to the shorter units of 11–12 for PSTVd and 60 for Apple scar skin viroid was suggested as an indication of possible protein-binding ability (Juhasz et al. 1988). ASBVd was the first viroid to be characterized with a ribozyme site for self-cleavage in vitro (Hutchins, et al. 1986; Davies et al. 1991; Marcos and Flores 1993), a property proposed to be related to in vivo replication. This property is now shared with Peach latent mosaic viroid (PLMVd) (Hernandez and Flores 1992; Flores et al. 1998) and Chrysanthemum chlorotic mottle viroid (CChMVd) (Navarro and Flores 1997) which are also characterized by the presence of hammerhead ribozyme sites. However, ASBVd is soluble in 2M LiCl as typical of most viroids whereas PLMVd and CChMVd are insoluble because of a more complex molecular conformation. Biological activity of ASBVd as the causal agent of sunblotch was demonstrated by graft inoculation of filter paper pieces moistened with a purified extract containing circular viroid

175

J.S. Semancik

molecules (Allen et al. 1981). The circular form of ASBVd can be cleaved in vivo as a result of extraction procedures to produce linear forms that are detected by PAGE as well as electron microscopy (Palukaitis et al. 1979). Although not verified for ASBVd, it may be assumed from data with other viroid systems, as with PSTVd (Owens et al. 1977) that both the circular and linear forms are biologically active. References Allen, R.N. (1982). A reaction rate constant for avocado sunblotch viroid and its complementary DNA with application to viroid detection. Intervirology 18, 76-82. Allen, R.N., and Dale, J.L. (1981). Application of rapid biochemical methods for detecting avocado sunblotch disease. Ann. Appl. Biol. 98, 451-461. Allen, R.N., and Firth, D.J. (1980). Sensitivity of transmission tests for avocado sunblotch viroid and other pathogens. Aust. Plant Pathol. 9, 2-3. Allen, R.N., Palukaitis P., and Symons, R.H. (1981). Purified avocado sunblotch viroid causes disease in avocado seedlings. Aust. Plant Pathol. 10, 31-32. Ashulin, L., Lachman, O., Hadas, R., and Bar-Joseph, M. (1991). Nucleotide sequence of a new viroid species, citrus bent leaf viroid (CBLVd) isolated from grapefruit in Israel. Nucleic Acids Res. 19, 4767. Barker, J.M., McInnes, J.L., Murphy, P.J., and Symons, R.H. (1985). Dotblot procedure with [32P]DNA probes for the sensitive detection of avocado sunblotch and other viroids in plants. J. Virol. Methods 10, 87-98. Bonfiglioli, R.G., McFadden, G.I., and Symons, R.H. (1994). In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus. Plant J. 6, 99-103. Bruening, G., Gould, A.R., Murphy, P.J., and Symons, R.H. (1982). Oligomers of avocado sunblotch viroid are found in infected avocado leaves. FEBS Letters 148, 71–78. Bussière, F., Lehoux, J., Thompson, D.A., Skrzeczkowski, L.J., and Perreault, J.-P. (1999). Subcellular localization and the rolling circle replication of peach latent mosaic viroid: hallmarks of group A viroids. J. Virol. 73, 6353-6360. Coit, J.E. (1928). Sunblotch of the avocado. California Avocado Society Yearbook 1928, 27. Dale, J.L., and Allen, R.N. (1979). Avocado affected by sunblotch disease contains low molecular weight ribonucleic acid. Aust. Plant Pathol. 8, 3. Davies, C., Sheldon, C.C., and Symons, R.H. (1991). Alternative hammerhead structures in the self-cleavage of avocado sunblotch viroid RNAs. Nucleic Acids Res. 19, 1893-1898. da Graca, J.V. (1985). Sunblotch associated reduction in fruit yield in both symptomatic and symptomless carrier trees. South Africa Avocado Growers’ Association Yearbook 8, 59. da Graca, J.V., and Martin, M.M. (1981). Ultrastructural changes in avocado leaf tissue infected with avocado sunblotch. Phytopathol. Z. 102, 185-194.

176

da Graca, J.V., and Mason, T.E. (1983). Detection of avocado sunblotch viroid in flower buds by polyacrylamide gel electrophoresis. Phytopathol. Z. 108, 262-266. da Graca, J.V., Mason, T.E., and Antel, H.J. (1983). Effect of avocado sunblotch disease on fruit yield. South African Avocado Growers’ Association Yearbook 6, 86-87. da Graca, J.V., and van Lelyveld, L.J. (1978). Peroxidase and indole 3 acetic acid oxides activities and isoenzymes in the mature bark of sunblotch infected avocado (Persea Americana). Phytopathol. Z. 92, 143-149. da Graca, J.V., and van Vuuren, S.P. (1977). Effects of avocado sunblotch disease on the mass, size and oil content of the fruit. Citrus and Subtropical Fruit J. 526, 10-11. da Graca, J.V., and van Vuuren, S.P. (1980a). Thermal inactivation of the avocado sunblotch agent and its rate of movement into indicator plants. Phytopathol. Z. 97, 82-84. da Graca, J.V., and van Vuuren, S.P. (1980b). Transmission of avocado sunblotch disease to cinnamon. Plant Dis. 64, 475. da Graca, J.V., and van Vuuren, S.P. (1981a). Use of high temperature to increase the rate of avocado sunblotch symptom development in indicator seedlings. Plant Dis. 65, 46-47. da Graca, J.V., and van Vuuren, S.P. (1981b). Host range studies on avocado sunblotch. South African Avocado Growers’ Association Yearbook 4, 80. Desjardins, P.R. (1987). Avocado sunblotch. Pages 299-313 in: The viroids. T.O. Diener, ed. Plenum Publishing: New York, NY. Desjardins, P.R., Drake, R.J., Atkins, E.L., and Bergh, O. (1979). Pollen transmission of avocado sunblotch virus experimentally demonstrated. California Agriculture 33, 14-15. Desjardins, P.R., Drake, R.J., Sasaki, P.J., Atkins, E.L., and Bergh, B.O. (1984). Pollen transmission of avocado sunblotch viroid and the fate of the pollen recipient tree. Phytopathology 74, 845. Desjardins, P.R., Drake, R.J., and Swiecki, S.A. (1980). Infectivity studies of avocado sunblotch disease causal agent, possibly a viroid rather than a virus. Plant Dis. 64, 313-315. Desjardins, P.R., Sasaki, P.J., and Drake, R.J. (1987). Chemical inactivation of avocado sunblotch viroid on pruning and propagation tools. California Avocado Society Yearbook 71, 259-262. Duran-Vila, N., Roistacher, C.N., Rivera-Bustamante, R., and Semancik, J.S. (1988). A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69, 3069-3080. Flores, R., Ambros, S., and Hernandez, C. (1998). Peach latent mosaic viroid: structural and functional properties. Acta Hortic. 472, 789794. Hernandez, C., and Flores, R. (1992). Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures. Proc. Natl. Acad. Sci. USA 89, 3711-3715. Horne, W.T. (1929). Progress in the study of certain disease of avocado. Phytopathology 19, 1144. Horne, W.T., and Parker, E.R. (1931). The avocado disease called sunblotch. Phytopathology 21, 235. Hutchins, C.J., Rathjen, P.D., Forster, A.C., and Symons, R.H. (1986). Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 14, 3627-3640. Juhasz, A., Hegyi, H., and Solymosy, F. (1988). A novel aspect of the information content of viroids. Biochim. Biophys. Acta 950, 455458.


Lima, M.I., Fonseca, M.E.N., Flores, R., and Kitajima, E.W. (1994). Detection for avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization. Arch. Virol. 138, 385-390. Loest, F.C., and Stofberg, F.J. (1954). Avocado diseases. Farming in South Africa 29, 517-520. Lopez-Herrera, C., Pliego, F., and Flores, R. (1987). Detection of avocado sunblotch viroid in Spain by double polyacrylamide gel electrophoresis. J. Phytopathol. 119, 184-189. Marcos, J.F., and Flores, R. (1992). Characterization of RNAs specific to avocado sunblotch viroid synthesized in vitro by a cell free system from infected avocado leaves. Virology 186, 481-488. Marcos, J.F., and Flores, R. (1993). The 5’ end generated in the in vitro self-cleavage reaction of avocado sunblotch viroid RNAs is present in naturally occurring linear viroid molecules. J. Gen. Virol. 74, 907910. McInnes, J.L., Habili, N., and Symons, R.H. (1989). Nonradioactive, photobiotin labeled DNA probes for routine diagnosis of viroids in plant extracts. J. Virol. Methods 23, 299-312. Mohamed, N.A., and Thomas, W. (1980). Viroid-like properties of an RNA species associated with sunblotch disease of avocado. J. Gen. Virol. 46, 157-167. Navarro, B., and Flores, R. (1997). Chrysanthemum chlorotic mottle viroid: unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes. Proc. Natl. Acad. Sci. USA 94, 11262-11267. Navarro, J.A., Daros, J.A., and Flores, R. (1999). Complexes containing both polarity strands of avocado sunblotch viroid: identification in chloroplasts and characterization. Virology 253, 77-85. Navarro, J.A., Vera, A., and Flores, R. (2000). A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid. Virology 268, 218-225. Owens, R.A., Erbe, E., Hadidi, A., Steere, R.L., and Diener, T.O. (1977). Separation and infectivity of circular and linear forma of potato spindle tuber viroid. Proc. Natl. Acad. Sci. USA 74, 3859-3863. Pallàs, V., Garcia-Luque, I., Domingo, E., and Flores, R. (1988). Sequence variability in avocado sunblotch viroid (ASBV). Nucleic Acids Res. 16, 9864. Palukaitis, P., Hatta, T., Alexander, D.M., and Symons, R.H. (1979). Characterization of a viroid associated with avocado sunblotch disease. Virology 99, 145-151. Palukaitis, P., Rakowski, A.G., Alexander, D.M., and Symons, R.H. (1981). Rapid indexing of the sunblotch disease of avocados using a complementary DNA probe to avocado sunblotch viroid. Ann. Appl. Biol. 98, 439-449. Querci, M., Owens, R., Vargas, C., and Salazar, L.F. (1995). Detection of potato spindle tuber viroid in avocado growing in Peru. Plant Dis. 79, 196-202.

Rakowski, A., and Symons, R.H. (1989). Comparative sequence studies of variants of avocado sunblotch viroid. Virology 173, 352-356. Romero-Durban, J., Cambra, M., and Duran-Vila, N. (1995). A simple imprint hybridization method for detection of viroids. J. Virol. Methods 55, 37-47. Rondon, A., and Figueroa, M. (1976). Sun blotch of avocados in Venezuela. Agronomia Tropical 26, 463-466. Schnell, R.J., Kuhn, D.N., Ronning, C.M., and Harkins, D. (1997). Application of RT-PCR for indexing avocado viroid. Plant Dis. 81, 10231026. Schroeder, C.A. (1993). Aberrant avocado leaf forms. California Avocado Society Yearbook 77, 121-124. Semancik, J.S., and Desjardins, P.R. (1980). Multiple small RNA species and the viroid hypothesis for the sunblotch disease of avocado. Virology 104, 117-121. Semancik, J.S., and Szychowski, J.A. (1994). Avocado sunblotch disease: a persistent viroid infection in which variants are associated with differential symptoms. J. Gen. Virol. 75, 1543-1549. Spiegel, S., Alper, M., and Allen, R.N. (1984). Evaluation of biochemical methods for the diagnosis of the avocado sunblotch viroid in Israel. Phytoparasitica 12, 37-43. Symons R.H. (1981). Avocado sunblotch viroid: primary sequence and proposed secondary structure. Nucleic Acids Res. 9, 6527–6537. Thomas, W., and Mohamed, N.A. (1979). Avocado sunblotch – a viroid disease? Aust. Plant Pathol. 8, 1-3. Vargas, C.O., Querci, M., and Salazar, L.F. (1991). Identification and dissemination of avocado sunblotch viroid in Persea Americana L. in Peru and the presence of another viroid in avocado. Fitopatologia 26, 23-27. Utermohlen, J.G., and Ohr, H.D. (1981). A polyacrylamide gel electrophoresis index method for avocado sunblotch. Plant Dis. 65, 800802. Wallace, J.M. (1958). The sunblotch disease of avocado. J. Rio Grande Val. Horticultural Society 12, 69. Wallace, J.M. (1967). Infected symptomless avocado trees and their possible use in avoiding sunblotch disease. California Avocado Society Yearbook 51, 187. Wallace, J.M., and Drake, R.J. (1953). Seed transmission of the avocado sunblotch virus. Citrus Leaves 33, 18. Wallace, J.M., and Drake, R.J. (1962). A high rate of seed transmission of avocado sunblotch virus from symtomless trees and the origin of such trees. Phytopathology 52, 237. Wallace, J.M., and Drake, R.J. (1971). Report on the program for production of avocado nursery trees free from sunblotch. California Avocado Society Yearbook 55, 120. Whitsell, R. (1952). Sunblotch disease of avocado. California Avocado Society Yearbook 37, 216.

177

PART IV

CITRUS VIROIDS

....................................................................................................

CHAPTER 27

CITRUS VIROIDS N. Duran-Vila and J.S. Semancik

.................................................................................................................................................................................................................................................................

GENERALIZED FEATURES COMMON TO THE VIROIDS FOUND IN CITRUS

Identification of the Citrus exocortis viroid (CEVd) as the causal agent of the exocortis disease

The exocortis disease was described in 1948 as a bark scaling disorder affecting trees grown on the trifoliate orange (Poncirus trifoliata) rootstock (Fawcet and Klotz 1948) and soon afterward it was demonstrated to be a graft transmissible disease (Benton et al. 1949, 1950). Similar disorders were also reported as ‘scaly butt’ of trifoliate orange in Australia (Benton et al. 1949) and in ‘Rangpur’ lime (Citrus limonia) in Brazil (Moreira 1955, 1959), and later demonstrated to be caused by the same agent. As a graft transmissible disease, for many years it was considered as probably induced by a virus. In 1972, it was demonstrated to be caused by an ‘infectious low molecular weight RNA’ (Semancik and Weathers 1972a, b) and the term ‘viroid’ was adopted to refer to these unusual types of infectious molecules (Diener 1971). In the 1970s, nucleic acid technologies were extremely limited as compared to the tools available today, and plant virologists relied on the availability of easy to handle herbaceous hosts yielding high titers of the target pathogenic agent. At that time, the identification of Citrus exocortis viroid (CEVd) as the causal

178

agent of the exocortis disease was possible through the use of Gynura aurantiaca as an experimental host displaying characteristic symptoms of stunting, epinasty and leaf distortion (Weathers and Greer Jr. 1972), and yielding high titres of the viroid molecule. Presently, CEVd is one of the best characterized viroids in terms of structure, replication, host range and biological properties. However, the reliance on alternate herbaceous hosts for viroid studies and especially Gynura hindered the identification of other citrus viroids with narrower host ranges than CEVd until the following decade. Identification of additional citrus viroids

‘Etrog’ citron (Citrus medica) adopted as an indicator for the biological indexing of exocortis, displayed a variety of symptoms ranging from severe to very mild (Calavan 1968; Roistacher et al. 1977). This range of symptoms was considered for many years as evidence for the existence of strains of a single viroid species responsible for exocortis. However, Gynura aurantiaca plants when inoculated with sources inducing mild and moderate symptoms on citron remained symptomless and CEVd was undetectable by PAGE analysis.

CITRUS VIROIDS

Table 27.1

Classification of citrus viroids and their relationship with citrus diseases. Citrus viroids

Family

Pospiviroidae

Genus

Species

Citrus diseases

Pospiviroid

CEVd

Exocortis

Apscaviroid

CVd-I (CBLVd)

Hostuviroid

CVd-II (HSVd)

Apscaviroid

CVd-III

Cocadviroid

CVd-IV

Cachexia1

1Only

specific variants of CVd-II (HSVd) originally named CCaVd (Semancik et al. 1988; Reanwarakorn and Semancik 1998) are causal agents of the cachexia disease.

The initial observation for the implication of viroid(s) distinct from CEVd was obtained with analysis by PAGE under standard and denaturing conditions of extracts from citrons displaying symptoms of leaf epinasty and midvein necrosis which occurred as a variable or intermittent pattern (Schlemmer et al. 1985). With the development and application of the more sensitive sequential polyacrylamide gel electrophoresis (sPAGE) and silver staining procedures, additional viroids were detected from citrons inoculated with different field sources (Duran-Vila et al. 1986, 1988a, b). Distinct viroid-like RNA bands with electrophoretic mobilities faster than CEVd were consistently identified from citrons displaying mild and moderate symptoms. Extensive analysis of different citrus sources revealed that commercial citrus usually contained mixtures of viroid-like RNA bands (Duran-Vila et al. 1988b; La Rosa et al. 1988; Gillings et al. 1991). Catalogues of citrus viroids were proposed taking into consideration the biological and molecular information available: i

electrophoretic migration in 5% sPAGE;

ii

sequence homology determined by molecular hybridization against specific cDNA probes;

iii

host range; and

iv

symptoms induced on the ‘Etrog’ citron indicator.

Based on this information citrus viroids were organized into five different groups (Semancik and Duran-Vila 1991). Sequencing information accumulated over the years demonstrated that the proposed groups were consistent with the viroid species concept proposed to the Executive Committee of International Committee on Taxonomy of Viruses (Flores et al. 1998; 2000) (Table 27.1). The association of other citrus symptoms with viroids will be discussed later in this chapter. Citrus, like grapevines (Szychowski et al. 1991), appear to harbor several viroids as compared with other crops, and should be considered as unusual hosts in terms of their ability to sustain the infection and replication of different viroid species. This is further supported by the recent characterization of a viroid

closely related to CVd-I that has only 82–85% sequence homology with CVd-I sequences (Ito et al. 2000). Therefore, the possibility that additional viroids may be described in the future must be entertained.

ECONOMIC IMPACT AND SYMPTOMATOLOGY Citrus viroids have a broad host range infecting species of Citrus as well as citrus relatives. Most of these viroid/host combinations are symptomless and a disease condition is only perceived when a specific viroid infects a sensitive species. Koch’s postulates have been successful for showing that the exocortis and the cachexia diseases are caused by CEVd and specific citrus variants of HSVd (CVd-II) respectively. Following the first description of the exocortis disease as a bark shelling and scaling disorder on trifoliate orange rootstock (Plate 10A), a range of symptoms including vertical cracking of the bark, yellow blotching of twigs and stunting have also been considered as exocortis symptoms. However, since some viroid sources employed as mild strains of CEVd have been shown to contain other single viroids or combinations of viroid species, the relationship between viroid infection and the development of specific symptoms on sensitive species was reconsidered. As illustrated in Table 27.2, bark cracking symptoms and some forms of mild and moderate dwarfing cannot be considered as exocortis symptoms. Exocortis-sensitive species include also the citrange hybrids (P. trifoliata × Citrus sinensis) and ‘Rangpur’ lime used as rootTable 27.2

Effect of citrus viroids on trifoliate orange. Symptoms

Viroids

Bark

Twigs

Stunting

CEVd

scaling

Yellow blotching

severe

CVd-I

pitting

–

mild

CVd-II

cracking

–

mild

CVd-III

–

–

moderate

CVd-IV

–

–

–

179

N. Duran-Vila and J.S. Semancik

Table 27.3

Experimental host range of citrus viroids1. Citrus viroids

Host

CEVd

CVd-I

CVd-II

CVd-III

CVd-IV

Aster grandiflorus

+*

NT

-

NT

NT

Benicasa híspida

NT

NT

+

NT

+*

Capsicum annum

+*

NT

-

NT

NT

Chrysanthemum morifolium

+

-

+

-

+

Cucumis sativus

+*

-

+

-

+*

Cucurbita pepo

+

NT

+

NT

NT

Dalia variabilis

+*

NT

-

NT

NT

Datura stramonium

+*

-

-

-

+

Gomphrena globosa

+*

-

-

-

NT

Gynura aurantiaca

+

-

-

-

-

Gynura sarmentosa

+

NT

NT

NT

NT

NT

NT

+

NT

NT

Luffa cylindrical Lycopersicon esculentum

+

-

-

-

+

Lycopersicon peruvianum

+

NT

NT

NT

+

Oscimum basilicum

+*

NT

-

NT

NT

Persea americana

-

+

-

-

-

Petunia axillaris

+

NT

NT

NT

NT

Petunia hybrida

+

NT

-

NT

NT

Petunia violacea

+

NT

NT

NT

NT

Physalis floridana

+

NT

NT

NT

NT

Physalis ixocarpa

+

NT

NT

NT

NT

Physalis peruviana

+

NT

NT

NT

NT

Solanum aculeatiisium

+

NT

NT

NT

NT

Solanum dulcamara

+

NT

NT

NT

NT

Solanum hispidum

+

NT

NT

NT

NT

Solanum integrifolium

+

NT

NT

NT

NT

Solanum marginatum

+

NT

NT

NT

NT

Solanum melongena

+*

-

+*

-

NT

Solanum quitoense

+

NT

NT

NT

NT

Solanum topiro

+

NT

NT

NT

NT

Solanum tuberosum

+

NT

NT

NT

NT

Tagetes patula

+*

NT

NT

NT

NT

Zinnia elegans

+

NT

-

NT

NT

1Weathers

1965; Weathers et al. 1967; Weathers and Greer, Jr 1968, 1972; Semancik and Weathers 1972c; Semancik and Weathers 1973; Niblett et al. 1980; Albanese et al. 1988; Duran-Vila et al. 1988 a,b; Puchta et al. 1989, 1991; Ashulin et al. 1991; Semancik and Duran-Vila 1991; Hadas et al. 1992; Fonseca and Kitajima 1993. Abbreviations: * = species symptomless for CEVd and CVd-IV; + = positive; – = negative; NT = not tested.

stocks, and some citrons, limes and lemons (Weathers and Calavan 1961). Although most commercial species and cultivars are tolerant, if they are budded on sensitive rootstocks, bark scaling is still evident on the rootstock. The bark initially shows longitudinal splits and eventually patches of dead scales of outer bark appear whereas the inner bark remains alive. Tolerant cultivars grafted on sensitive rootstocks may also show

180

yellowing of the canopy and general decline, even in the absence of bark scaling. Symptom severity, onset and position above or below ground level are influenced by the CEVd strain, the age of the tree at infection and environmental conditions. Although fruit quality is not affected, economic losses in production are the result of reduced tree size and poor fruit bearing in declining trees.

CITRUS VIROIDS

Table 27.4

Symptoms induced by citrus viroids on Etrog citron1,2.

Viroid

Stunting

Leaf epinasty

Necrosis Petiole

Leaf tip

CEVd

severe

severe

general

+

-

CVd-I (CBLVd)

mild

random

local

-

-

CVd-II (HSVd)

-

-

-

+/-

+/-

CVd-III

moderate

general

general

+

-

CVd-IV

moderate

random

general

+

-

1The Etrog citron indicator 2Duran-Vila et al. 1988a, b.

Mid-vein

was of ‘Arizona 861-S1’ (Roistacher et al. 1977).

The cachexia disease was described in 1948 as discoloration, gumming and browning of phloem tissues, wood pitting and bark cracking on ‘Orlando’ tangelo (C. paradisi × C. reticulata) (Childs 1950). Its identity to a similar disorder described earlier as xyloporosis in Palestine sweet lime (C. limettioides) (Reichert and Perlberger 1934) has been demonstrated by Reanwarakorn and Semancik (1999a). The disease is caused by variants of CVd-II containing a specific sequence motif in the V domain (Reanwarakorn and Semancik 1998). Most commercial species and cultivars are tolerant but mandarins, some mandarin hybrids (tangors and tangelos) and kumquats (Fortunella sp.) are sensitive. ‘Alemow’ (C. macrophylla), rough lemon (C. jambhiri) and ‘Rangpur’ lime used as rootstocks are also sensitive. The degree of injury can vary from a slight to severe stunting, chlorosis, tree decline and death. ‘Tahiti’ lime (C. lattifolia), a commercial species highly valued for its fruits in Mexico, Brazil and Cuba, has been reported as sensitive to exocortis showing longitudinal bark cracks in the branches that break easily (Salibe and Moreira 1965). However, the observation that trees with bark symptoms on the scion never showed bark-scaling symptoms on ‘Rangpur’ lime rootstock, suggests that citrus viroids other than CEVd may be the causal agents of the ‘Tahiti’ lime symptoms (Salibe 1980). Other observations indicating the development of cachexia-like symptoms on ‘Cleopatra’ mandarin (Ochoa et al. 1996) and bark scaling on citrumelo (Bello et al. 2000) should be further studied and their association with specific viroids confirmed.

HOST RANGE The first herbaceous species described as an alternate host for CEVd was Petunia hybrida (Weathers 1965). The results of transmission experiments performed from infected petunias evidenced the wide host range of CEVd (Weathers and Calavan 1961; Weathers et al. 1967; Weathers and Greer 1968; Semancik and Weathers 1972c; 1973). In these early assays, infectivity by CEVd was demonstrated by the expression of symptoms on the inoculated plants, typically Gynura, but later the availability of nucleic acid detection methods allowed the identification of

symptomless hosts (Table 27.3) (Albanese et al. 1988; DuranVila et al. 1988a, b; Puchta et al. 1991; Semancik and DuranVila 1991; Hadas et al. 1992). HSVd has been reported naturally infecting herbaceous and woody species (Astruc et al. 1996). In Table 27.3, only the results of transmission assays from citrus sources have been listed. However, since the term CVd-II refers to HSVd variants isolated from Citrus a broader range of species are probably hosts for CVd-II. The information available on the host range of other citrus viroids is limited and is also summarized in Table 27.3. Since transmission assays are usually conducted in the search for herbaceous hosts more suitable for research, information on woody species as experimental hosts of plant pathogens is very limited. In citrus viroids, a single report shows the successful transmission of CVd-I to avocado by heterologous grafting (Hadas et al. 1992). The results of indexing tests conducted in several countries illustrate the worldwide occurrence of the five citrus viroids in commercial cultivars. However, specific information regarding the transmission of viroids to other citrus species is rather limited. In this regard, the discovery of citron as a symptomatic host displaying a wide range of symptoms after a relatively short incubation period with most citrus viroids resulted in its general acceptance as an indicator for biological indexing. Although the biological indexing using the specific selection of ‘Etrog’ citron, ‘Arizona 861-S1’, as the indicator designed for exocortis detection (Roistacher et al. 1977), symptom specificity to each citrus viroid has been verified (Table 27.4). Most of the information available on the sensitivity of commercial citrus species has been gathered from field observations and performance assays conducted in different locations. Assays conducted on non-commercial species of citrus and related genera indicate that all are susceptible but most are symptomless hosts. No information is available on the occurrence of viroids in wild citrus.

181


TRANSMISSION

DETECTION

Citrus viroids are readily graft-transmitted and dissemination occurs principally through propagation of symptomless, viroidinfected budwood. Citrus viroids are mechanically transmitted as contaminants on cutting and pruning tools (Garnsey and Jones 1967; Wutscher and Shull 1975). Originally viroid-free budwood sources may become infected via contamination in nursery, budwood source plantings and commercial orchards. The efficiency of mechanical transmission varies with the donor and receptor hosts involved, and the degree of phloem wounding (Garnsey and Weathers 1972). Seed transmission and vector transmission have not been demonstrated in citrus.

Biological indexing

The CEVd molecule was reported to be highly resistant to heat inactivation and to many chemicals used to inactivate viruses (Roistacher et al. 1969). It can remain infectious for long periods in dry tissue or as a contaminant on dry surfaces (Allen 1968). Hydrolysis, ribonuclease treatment, or hypochlorite have practical application for inactivation (Roistacher et al. 1969). Although these studies were conducted with CEVd sources, similar properties would also apply to the other citrus viroids.

GEOGRAPHICAL DISTRIBUTION AND EPIDEMIOLOGY Since citrus viroids are graft-transmitted, dissemination occurs principally through propagation of contaminated budwood and international exchange of plant materials. Except for some Asian countries, the citrus industry throughout the world is based on a limited number of commercial species and cultivars with which viroids were probably disseminated. The earliest circumstantial evidence of citrus viroid infections has been found in a floor mosaic from an ancient synagogue in Israel (Bar-Joseph 1996) showing as decorations citron fruits with malformations characteristic of CEVd infection (Bitters et al. 1987). This suggests that citrus and viroids had co-existed at least for 1.5 to 2 millennia. The results of early indexing tests conducted in many parts of the world provide good evidence for the widespread occurrence of citrus viroids. Today the routine application of more specific indexing tests associated with sanitation, certification and quarantine programs has provided additional information on the occurrence of specific viroids in different areas of the world (Duran-Vila et al. 1988b; Gillings et al. 1991; Villalobos et al. 1997; Pagliano et al. 2000; Pérez et al. 2000). This information indicates that infection with CVd-II and CVd-III, many times as a mixture, is very frequent, whereas CVd-I is found less frequently and CVd-IV is rare. The frequency of CEVd in the sources analyzed appears to be intermediate.

182

The first detection tests were developed before the etiology of exocortis and cachexia was established. These early biological indexing procedures were based on the observation of symptoms of graft-inoculated sensitive species. ‘Rangpur’ lime and trifoliate orange grown as seedlings or as topworked rootstocks were the species of choice for indexing of exocortis, and ‘Orlando’ tangelo for cachexia. These long-term diagnosis tests were abandoned with the development of more sensitive and rapid indexing procedures with other indicator species. The use of citron as a rapid indicator was first proposed by Salibe and Moreira (1965) and a method based on forcing a citron bud on a vigorous seedling stock was developed as a quick and sensitive procedure for detection of exocortis (Calavan et al. 1964; Calavan 1968). Over the years, a number of citron selections have been made, and the clonal propagation of ‘Arizona 861-S1’ is considered the most sensitive choice that is currently being used worldwide (Roistacher et al. 1977). Although this test was initially designed for detection of exocortis, mild and moderate symptoms may be produced by citrus viroids other than CEVd (Table 27.4). Thus, the test should be regarded as a general indexing procedure for all known citrus viroids (Duran-Vila et al. 1993). With the experience accumulated over the years and the results of the assays performed with the aim of enhancing sensitivity, a number of recommendations regarding inoculation, growing and care of indicator plants, temperature requirements and symptom observation have been made (Roistacher 1991). ‘Parson’s Special’ mandarin forced as a scion on a vigorous rootstock under warm conditions is the accepted biological indexing test for cachexia (Roistacher et al. 1973). ‘Clemelin 11-20’, a hybrid of clementine and sweet orange (Pérez et al. 1992) as well as ‘Mapo’ tangelo, a hybrid of ‘Avana’ mandarin and ‘Duncan’ grapefruit (Terranova et al. 1991) have also been shown to be good cachexia indicators. In both indicators cachexia induces the formation of gum deposits that develop within 6 to 12 months near the base of the scion. Although high and/or alternating incubation temperatures and light intensity appear to be important factors for enhancing symptom expression, these parameters have not been fully evaluated and the cachexia tests require long incubation periods to be reliable (Roistacher 1991). Biological indexing tests using the ‘Arizona 861-S1’ citron selection and ‘Parson’s Special’ mandarin as recommended for detection of exocortis and cachexia, respectively, can be used reliably to detect all the citrus viroids described (Table 27.5). Whereas a positive reaction on ‘Parson’s Special’ mandarin or on ‘Clemelin 11-20’ is considered a definitive proof for cachexia-inducing sources of CVd-II, the range of symptoms displayed by inoculated citrons is rather non-specific unless the viroid sources to be

CITRUS VIROIDS

Table 27.5

Biological indexing of citrus viroids.

Citrus viroids

Reactions on citrus viroids indicators Citron

Parson’s Special

CEVd

severe

-

CVd-I

mild

-

CVd-II: non cachexia

very mild

-

-

+

CVd-III

moderate

-

CVd-IV

moderate

-

CVd-II: cachexia

tested contain a single viroid species. Since most field sources contain several viroids, the symptoms observed are unlike those outlined in Table 27.4 as a result of synergistic and inhibitory interactions which may cause delayed or enhanced symptoms, pronounced dwarfing and epinasty or even variable leaf symptoms (Semancik and Duran-Vila 1991). In such cases, the specific viroids present in a field isolate can only be demonstrated by additional molecular analysis. Molecular analysis

Nucleic acid analysis procedures based on the identification of viroid species have been reported for detection of CEVd and other citrus viroids. These include polyacrylamide gel electrophoresis (PAGE), hybridization of nucleic acid extracts or tissue imprints against viroid specific probes, and retrotranscription and polymerase chain reaction (RT-PCR) of extracts using specific primers. The sensitivity of these procedures depends on the test conditions employed, the viroid titers in extracts or within tissues of different cultivars and the presence of interfering natural products. After the discovery of CEVd as the causal agent of the exocortis disease attempts were made to use PAGE analysis for detection purposes, but this approach was abandoned because it failed to detect what were considered at that time to be mild sources of CEVd (Barksh et al. 1984; Boccardo et al. 1984). The resolution achieved with the utilization of two-electrophoresis systems exploiting the unique properties of viroid molecules under the denaturing conditions used in the second run, coupled with silver staining is critical for the visualization of the circular forms of all the citrus viroids. The procedure commonly used for viroid analysis involves the migration of nucleic acid preparations into standard PAGE followed by excision of the gel segment containing the host 7S RNA band and CEVd that is then placed in contact with a second gel containing 8M urea. Placement of the gel segment on the bottom (bi-directional PAGE) or the top (sequential PAGE, sPAGE) of the urea containing gel produces similar results and the different citrus viroids can be identified by their characteristic mobility (Schumacher et al. 1983; Duran-Vila et al. 1986). When the urea containing gel is cast at a lower pH than the running buffer the separation of the

circular forms is enhanced thus facilitating the identification of specific viroids present in a given source (Rivera-Bustamante et al. 1986). Other procedures (return PAGE, R-PAGE) in which the second run is based on the denaturing properties of temperature and low ionic strength buffer have been also infrequently used (Singh and Boucher 1987; Mishra et al. 1991). The initial attempts to use molecular hybridization for detection of citrus viroids were based on radioactively labeled randomly primed cDNA probes (Owens and Diener 1981) and mainly used for experimental purposes. Hybridization assays were primarily conducted to identify homologies among different viroids and viroid sources (Duran-Vila et al. 1988a; Davino et al. 1991). However, even cDNA probes generated by random priming displayed an unusual specificity to all variants tested within a single species of citrus viroid. A specific stem-loop structure was included within the mapping site for the specific cDNA probes for CEVd, CVd-II and CVd-IV (Francis et al. 1995). The availability of cloned viroid sequences facilitated the synthesis of labeled probes either through the incorporation of 32 P on purified plasmid DNA by nick translation or by in vitro transcription in the presence of 32P-, biotin- or DIG-labeled nucleotides and subsequently were evaluated for diagnostic purposes. However, until recently most attempts have been restricted to the detection of CEVd and HSVd (CVd-II) from experimental and commercial hosts (Gillings et al. 1988; Albanese et al. 1991; Fonseca et al. 1996). The utilization of RT-PCR strategies was first described for CEVd and CVd-II (Yang et al. 1992) but today they have become the preferred tool to obtain DNA from full-length viroid sequences for cloning purposes. Their suitability as diagnostic tools to detect viroids from different citrus hosts has also been explored using single or multiplex primer sets (Levy et al. 1992; Tessitori et al. 1996; Tururo et al. 1998) but it has not been adopted when large numbers of tests need to be routinely performed. Improved indexing procedures The availability of sensitive indexing procedures is essential to control viroid-induced diseases through the implementation of quarantine, sanitation and certification programs. The routine indexing tests being conducted have evolved as a result of the search for rapid and more sensitive procedures. The biological indexing using the ‘Arizona 861-S1’ citron selection and ‘Parson’s Special’ mandarin as recommended for detection of exocortis and cachexia, respectively, is a sensitive procedure to detect all the citrus viroids described (Table 27.5), but the incubation period required for symptom expression and the lack of specificity are the major drawbacks. Molecular analysis approaches have been proposed as alternatives for quick detection of viroids directly from commercial species grown under field conditions, but the seasonal and year-to-year fluctuations

183


Table 27.6

Natural hosts of CEVd outside the Rutaceae family.

Host

Symptoms

Reference

Brassica napus

-

Fagoaga and Duran-Vila 1996

Daucus carota

-


Lycopersicon esculentum

Bunchy top

Mishra et al. 1991

Lycopersicon esculentum

-


Solanum melongena

-


Vicia faba

-

Fagoaga et al. 1995

Vitis sp.

-

Flores et al. 1985; García-Arenal et al. 1987

of viroid titers even below the detection levels presents a serious limitation (Albanese et al. 1991; Tessitori et al. 1996; Palacio et al. 2000b). This can be theoretically overcome by the utilization of RT-PCR approaches following adequate evaluation in terms of suitability of hosts, efficacy and cost. The use of citron as a bioamplification host coupled with molecular analysis has been adopted as the most sensitive and specific procedure for indexing purposes. The method combines the property of citron to replicate the five citrus viroids at titers above detection levels and the specificity of either PAGE or hybridization analysis (Duran-Vila et al. 1993; Palacio et al. 2000b). The hybridization approach has been further simplified through the use of non-radioactive probes generated by PCR and avoiding the need to perform nucleic acid extraction procedures (Palacio et al. 2000a, b). When identification beyond viroid species level is desired, additional tests may be needed. In this regard, hybridization against synthetic oligonucleotide probes or RT-PCR procedures using specific primer pairs under defined conditions have been developed (La Rosa et al. 1993; Reanwarakorn and Semancik 1999b).

CONTROL Viroid-induced disease can only be controlled by prevention measures. The availability of viroid-free budwood as a source of propagation material is essential and requires the utilization of adequate indexing procedures. Existing clones can be easily freed of viroids by shoot-tip grafting in vitro (Navarro et al. 1975) and quarantine measures are desirable when importing plant materials from other countries. Untested field trees should not be used as budwood sources if the trees are to be grown on viroid-sensitive rootstocks. Spread of viroids by contamination in nursery operations should be avoided by separation of infected and healthy plants, by appropriate sanitation, and by treatment of cutting tools (budding knives, secateurs) with a sterilant such as sodium hypochlorite (2% available chlorine). Spread of viroids in orchards by hedging and harvesting equipment can also be avoided by treatment of tools with sodium hypochlorite. Top-

184

working with susceptible cultivars or onto susceptible rootstocks should be avoided.

SPECIFIC PROPERTIES OF THE FIVE CITRUS VIROIDS Citrus exocortis viroid (CEVd)

CEVd is the causal agent of the exocortis disease. With the indexing of the exocortis disease by citron bioassay, the identification of five citrus viroids introduced the question whether all induced exocortis or exocortis-like symptoms exist in trifoliate orange. Field trials demonstrated that CEVd is the only agent of the exocortis disease as initially described for trifoliate orange. Under mild environmental conditions, CEVd infected trees grafted on Citrange carrizo rootstock may show yellowing of the canopy, general decline and poor performance, even in the absence of bark scaling. The economic impact depends on the effect on the specific scion/rootstock combination. In countries where the tolerant sour orange rootstock has been substituted by trifoliate orange or citrange hybrids, strategies for making viroid-free sources available to the growers have been implemented. CEVd has a wide experimental host range (see Table 27.3), with sensitive hosts displaying symptoms of stunting, epinasty and leaf distortion, in addition to a number of species as symptomless carriers. A CEVd variant has been found as a natural infection in commercial tomato plantings affected with a disease characterized by extensive apical proliferation, stunting, epinasty, leaf distortion and veinal necrosis in the Pune region of India (Mishra et al. 1991). CEVd has also been reported as natural infections in symptomless grapevine, tomato, eggplant, turnip, carrot and broad bean (see Table 27.6). The first CEVd sequences of 371 nucleotides reported in 1982 (Gross et al. 1982; Visvader et al. 1982) differed by only four bases at nucleotides 264, 278, 301 and 313 (see Figure 27.1). These were not characterized from citrus but from the alternate hosts, Gynura and tomato, which yielded higher viroid titers. The proposed secondary structure is a highly base paired rodlike structure with 67% of the residues base paired (34 A:U, 72 G:C and 18 G:U base pairs). Following the viroid species concept proposed to the Executive Committee of International

CITRUS VIROIDS

Figure 27.1 Nucleotide sequence of Citrus exocortis viroid variant CEVd-C (Gross et al. 1982) with changes found in variant CEVd-A (Visvader et al. 1982) boxed. Structure is represented as the minimum free energy form at 24°C by mFold (Zuker 1989).

Committee on Taxonomy of Viruses (Flores et al. 1998, 2000) CEVd is a species of the genus Pospiviroid belonging to the family Pospiviroidae (Table 27.1). Cloning strategies and automated sequencing have facilitated the molecular characterization of additional CEVd sources. Extensive sequence analysis of CEVd isolates has shown that infected plants contain complex populations of sequence variants (Visvader and Symons 1985; Palacio and Duran-Vila 1999; Gandía et al. 2000) that follow the ‘quasispecies’ model proposed to describe heterogeneous populations of RNA molecules with the high frequency variants representing the most fit individuals (Eigen and Biebricher 1988; Eigen 1993). The finding that host and tissue selection can identify CEVd variants that differ in symptom expression, titer and electrophoretic mobility from the source inoculum (Semancik et al. 1993) is compatible with this dynamic model. Similarly, the effect of passage through tomato as an intermediate host on the severity of the symptoms induced by CEVd isolate on broad bean can be viewed as a shift of the population of variants towards those which are more fit in the tomato host (Fagoaga et al. 1995). A relatively large number of CEVd sequence variants and susceptible hosts have been reported. This can be regarded as evidence of the ability to incorporate mutations and changes without impairing transmission and replication. The polymorphism potential of CEVd is illustrated by the identification of a stable 463 nucleotide variant (CEVd D-92) produced by the duplication of a 92 nucleotide right terminal domain (Semancik et al. 1994). This structural feature, shared only by CCCVd, has been suggested to occur as a consequence of discontinuous transcription during replication. The importance of structure to the biological activity of the viroid molecule is reflected in the loss of transmissibility to citron by CEVd D-92 with enlargement of the CEVd genome while infectivity to tomato is retained. With the recovery of CVd-IV following infection with CEVd D-92, it has been suggested that stable variants resulting from partial duplications of the viroid genome may act as transient progenitors of new viroid or viroid-like species (Semancik and DuranVila 1999). With tomato as an experimental host, a classification of CEVd sequences based on their biological properties into severe ‘Class A’ and mild ‘Class B’ inducing symptoms was proposed (Visvader and Symons 1985). These two classes of sequences differ

by a minimum of 26 nucleotides mainly affecting two regions, PL and PR located in the P and V domains, respectively. Experimental evidence obtained from infectivity assays conducted with artificial chimeric constructs suggested that the specific changes in the P domain were responsible for symptom modulation (Visvader and Symons 1986). However, since the specific changes in the P domain discriminating ‘Class A’ and ‘Class B’ appear to be always associated in naturally occurring sequences of CEVd with the changes in the V domain, an interdependence between both regions may exist. The role of the P domain in modulating symptom expression on inoculated tomato seedlings was also observed upon inoculation of chimeric constructs containing portions of TASVd and CEVd (Owens et al. 1990) as well as larger portions of the viroid secondary structure including the TL region (Sano et al. 1992). However, the studies performed also using interspecies chimeras between CEVd and HSVd (Sano and Ishiguro 1998) suggest the importance of the TR in pathogenicity. Thus, determinants for pathogenicity may exist throughout the viroid genome in discrete structural units. Studies performed using Gynura aurantiaca as an experimental host, identified in the P domain a set of 5 nucleotides discriminating variants that induced severe and mild symptoms (Skoric et al. 2001), which were not consistent with those associated with differential responses in tomato (Visvader and Symons 1983, 1985). This can be considered as an indication that different hosts may show different responses in terms of symptom modulation or that the 5-nucleotide differences described in Gynura is only associated with a specific temperature-sensitive response which modulates a variable expression of symptoms. No information is available regarding the modulation of symptom expression of the exocortis disease as described in citrus, but since the bark scaling symptoms observed in affected trees do not resemble those of experimental hosts, the information available on experimental hosts should not be readily extrapolated to exocortis-sensitive citrus. Citrus viroid I (CVd-I) and Citrus bent leaf viroid (CBLVd)

CVd-I as characterized from citron was initially described as two independently transmissible viroids termed CVd-Ia and CVdIb that migrated as distinct bands in sPAGE analysis (DuranVila et al. 1988b). CVd-I induces a very specific yet inconsistently appearing symptom in ‘Etrog’ citron of a necrotic lesion

185


Figure 27.2 Nucleotide sequence of (a) Citrus viroid Ia (CVd-Ia) with changes in the variant CVd-LSS boxed and (b) Citrus viroid Ib (CVd-Ib) with changes in the variant CBLVd boxed. Structures are represented as the minimum free energy form at 24°C by mFold.

on the underside of the leaf midvein resulting in an abrupt bending alternating with flushes of symptomless leaves and pinholing under the bark (Duran-Vila et al. 1993). Deep pits with corresponding pegs in the bark of the trifoliate orange rootstock (see Plate 10B) associated with a significant reduction of tree canopy have been described as symptoms in CVd-Ia infected trees (Roistacher et al. 1993). Canopy reduction as a result of CVd-Ia infection was further confirmed demonstrating that in spite of the reduced size and net yield, the yield/canopy volume was slightly improved (Semancik et al. 1997). The host range is restricted almost exclusively to rutaceous hosts with the exception of a heterologous graft transmission to avocado as an experimental host (Hadas et al. 1992). Hybridization analysis indicated sequence homology (DuranVila et al. 1988a) between CVd-Ia and -Ib that was later confirmed by nucleotide sequence determination (Figure 27.2). A source of CVd-Ib from Citrus macrophylla was transmitted to avocado by heterologous grafting (Hadas et al. 1992) and the sequence of 318 nucleotides reported under a new designation citrus bent leaf viroid (CBLVd) (Ashulin et al. 1991) while CVdIa was later sequenced at 327 nucleotides (Semancik et al. 1997). It should also be clarified that the descriptive term ‘bent leaf’ describes the symptom that occurs only in the indexing host ‘citron’ and not in ‘citrus’ species as a disease symptom. Thus, ‘Citron bent leaf viroid’ might have been more appropriate for the acronym ‘CBLVd’. Considerable variability was found in the sequence of CBLVd as recovered from avocado and succeeding variants sequenced from citrus sources including differences in six positions that were found in CBLVd exclusively (Ben-Shaul et al. 1995).

186

Thus, designation of CBLVd as the ‘type strain’ by virtue of the first available sequence as it was derived from the experimental host avocado could at times not present a representative standard of comparison for the range in size and sequence of CVd-I variants characterized from natural infections in rutaceous species. For this reason, a strict adherence to the proposal for a 90% homology in defining variants within a viroid species (Flores et al. 1998) might be employed as a guide in keeping with the nature of a ‘proposal’ especially in the application of CBLVd as a ‘type’ strain. Nevertheless, CVd-Ia, CVd-Ib and CBLVd share more than 90% sequence homology and should be considered as variants of CVd-I existing as populations of sequence variants (Foissac and Duran-Vila 2000). In noting the insertion of two 5-nucleotide clusters in the TR domain of CVd-Ia, Hataya et al. (1998) proposed that CVd-Ia might be considered as a derivative of CVd-Ib. Recently, two additional citrus viroids belonging to the genus Apscaviroid have been described from Japan. Although displaying a sequence homology of only 82–85% with CVd-I variants, the acronym CVd-I-LSS (low sequence similarity) was proposed for one thus implying the relationship to the CVd-I species (Ito et al. 2000). Inclusion as a variant of CVd-I is supported by the size of 327 nucleotides as well as the leaf-bending symptom in citron characteristic of CVd-I variants. The identification of a new citrus viroid has become a very real possibility with the description of CVd-OS (original sample) (Ito et al. 2001). The 330 nucleotide genome has only a 68% homology with CVdIII, the closest related citrus viroid (Figure 27.3). Although similar to other apscaviroids in citrus, the severity of very mild leaf bending and petiole necrosis symptoms is unlike any other citrus viroid. Thus, CVd-OS may be considered as the first truly

CITRUS VIROIDS

Figure 27.3 Nucleotide sequence of Citrus viroid-OS (CVd-OS) with the structure represented as the minimal free energy form at 24°C by mFold.

new viroid species identified since the original ‘citrus viroid catalogue’ was introduced about 15 years ago (Duran-Vila et al. 1988). Citrus viroid II (CVd-II) or Hop stunt viroid (HSVd) citrus variants

The initial description of two viroid bands termed CVd-IIa and CVd-IIb with close but yet distinct electrophoretic mobilities (Duran-Vila et al. 1988a) led to the subsequent nucleotide sequence determination (Figure 27.4) of the two highly homologous variants (Sano et al. 1988; Levy and Hadidi 1992). Sequence analysis of CVd-IIa samples from throughout the world indicates a highly conserved genome (Sano et al. 1988; Puchta et al. 1989; Hsu et al. 1994). Since the titer of all CVdII variants is lower than that of any other citrus viroid even in extracts from the indexing and amplification host, citron, and CVd-IIa was not associated with any disease of economic importance, many old lines as well as even some plant materials tested in certification programs contained CVd-IIa as a symptomless carrier. In the oldest samples available from the parent ‘Washington navel’, only CVd-IIa is detected suggesting that this viroid may have been introduced into California with the original plant materials from which all subsequent propagations were made and that commercial performance may be improved by CVd-IIa (Semancik et al. 1997). The non-cachexia-inducing

variants (CVd-IIa) appear to affect the performance of trees grown in the trifoliate orange rootstock in terms of induction of mild bark cracking (see Plate 10C) and growth modulation of the tree canopy (Roistacher et al. 1993). The demonstration that the cachexia disease (Childs 1950) was caused by CVd-IIb (CCaVd) (Semancik et al. 1988) and not CVd-IIa defined the distinct biological activity of the two closely related genomes. Further analysis of cachexia isolates indicated the existence of additional variability in electrophoretic mobility with an additional more rapidly migrating viroid band termed CVd-IIc (Semancik and Duran-Vila 1991). CVd-II variants demonstrated to induce a positive reaction in the cachexia indexing host, ‘Parson’s Special’ mandarin, with the exception of the very mild variant Ca-909 are characterized by a 6 nucleotide cluster spanning the C and V domains proposed as the locus determining pathogenicity (Reanwarakorn and Semancik 1998, 1999a). The implications of the specific nucleotide differences identified in the V domain in the overall secondary structure of the viroid molecule are discussed in these proceedings. Following the identification of HSVd-related sequences by molecular hybridization, nucleotide sequencing identified CVd-II as a variant of HSVd (Sano et al. 1988). HSVd displays

Figure 27.4 Nucleotide sequence of (a) a non-cachexia variant Citrus viroid IIa (CVd-IIa) and (b) two cachexia variants Citrus viroid IIb (CVd-IIb) or Citrus cachexia viroid (CCaVd) with the changes in Citrus viroid IIc (CVd-IIc) boxed. Structures are represented as the minimum free energy form at 24°C by mFold.

187


Figure 27.5 Nucleotide sequence of (a) Citrus viroid IIIb (CVd-IIIb) with changes in Citrus viroid IIIa (CVd-IIIa) boxed and (b) CVd-IIIb with changes in Citrus viroid IIIc (CVd-IIIc) boxed. Structures are represented as the minimum free energy form at 24°C by mFold.

among the viroids the largest host range and accompanying variants that may be organized into three types (Sano et al. 1989) by host affinity. It has been suggested that extensive recombination may have occurred between the different variants to accomplish such an extensive host diversity (Kofalvi et al. 1997). No experimental evidence from mixed infection has been offered in support and by virtue of viroid dependence for replication, the role of the host in variant selection must be considered. However, from the perspective of biological activity, two distinct groups of HSVd variants should be considered, those that act as latent infections in cachexia sensitive hosts (initially termed as CVd-IIa) and those that are cachexia-inducing pathogens (initially termed as CVd-IIb or CCaVd). All CVd-II variants induce the characteristic symptoms of stunting and leaf rugosity in cucumber to a varying degree of severity and very mild symptoms in ‘Etrog’ citron, however, only the cachexia-inducing variants display a response in ‘Parson’s Special’ mandarin, ‘Tangelo Orlando’ and ‘Alemow’ (see Plate 10D). Citrus viroid III (CVd-III)

CVd-III was initially described as several independently transmissible viroids, CVd-IIIa and CVd-IIIb, migrating as distinct bands in sPAGE analysis (Duran-Vila et al. 1988b). Initial hybridization analysis showed that they shared sequence homology (Semancik and Duran-Vila 1991) and sequencing demonstrated that they were actually highly homologous variants (Rakowski et al. 1994) of a single viroid (Figure 27.5). The sequence of the terminal regions of CVd-III appears to be derived from the conserved regions of the Potato spindle tuber

188

viroid (PSTVd) and the Apple scar skin viroid (ASSVd) (Stasys et al. 1995). CVd-III has a very restricted host range and causes very specific symptoms in ‘Etrog’ citron, characterized by petiole ringing and necrosis and general leaf drooping as a result of petiole bending. Finger imprint or trunk strangling symptoms which were initially described as a result of CVd-III infection in trees grafted on trifoliate orange rootstock (Roistacheret al. 1993) did not persist and it was suggested that they might have resulted from constrictions introduced during propagation or young tree training. Similar symptoms were never observed in field trials conducted in California or in Spain and Corsica (unpublished). A significant reduction of trunk section and canopy volume was described in CVd-IIIb-infected ‘Valencia’ sweet orange trees grafted on trifoliate orange rootstock (Roistacher et al. 1993) and further confirmed showing that in spite of the reduced size and net yield, the yield/volume was considerably superior (Semancik et al. 1997). The survey of viroids of independently selected ‘dwarfing factors’ from different countries revealed that CVd-III had actually been selected for the potential to reduce tree size (Gillings et al. 1991; Semancik et al. 1997; Villalobos et al. 1997; Owens et al. 2000). Although CVd-IIIb occurs as a very conservative genome in nature throughout citrus growing, it has been suggested that other variants that have been characterized as members of the population of quasi-species may present additional viroid species that might be employed in citrus dwarfing (Owens et al. 1999). Unfortunately, these have been tested only on the indexing host citron for transmissibility and have yet to be tested for dwarfing and performance in field trials.

CITRUS VIROIDS

Figure 27.6 Nucleotide sequence of Citrus viroid IV (CVd-IV) with the structure represented as the minimum free energy form at 24°C by mFold.

Citrus viroid IV (CVd-IV)

CVd-IV appears to be the least widespread of the citrus viroids. It was initially described as a component of a viroid source from California (Duran-Vilaet al. 1988b) and later described as a component of ‘graft-transmissible dwarfing factors’ selected in Israel (Hadas et al. 1989) and Turkey (Önelge et al. 2000). Although CVd-IV has been recovered from ‘graft-transmissible dwarfing factors’, the description of the effect of CVd-IV infection is very limited and its effect on citrus species other than citron has not been fully evaluated. The nucleotide sequence was first achieved after transmission of the CVd-IV source from Israel to Benicassia hispida (Puchta et al. 1991). CVd-IV has 284 residues and the proposed secondary structure is a highly base paired rod-like structure with 71% of the residues base paired (32 A:U, 63 G:C and 8 G:U base pairs) (Figure 27.6). Additional sequencing information obtained from two sources isolated from California (Francis et al. 1995) and Turkey (Önelge et al. 2000) indicates that CVd-IV is a highly conserved viroid. A section of 80–90 nucleotides in the V and TR domains of the molecule are identical to CEVd (Puchta et al. 1991) confirming the weak but yet positive hybridization reaction against CEVd specific probes (DuranVila et al. 1988b). On the basis of the homology between CVd-IV and CEVd and the similarity of the left terminal region with HSVd, it has been suggested that CVd-IV is a novel mosaic-type chimeric viroid (Puchta et al. 1991). However, there is evidence indicating that stable variants resulting from partial duplications of CEVd genome (Semancik et al. 1994) may act as transient progenitors of new viroids or viroid-like species similar to CVd-IV (Semancik and Duran-Vila 1999). In the classification proposed by the Executive Committee of International Committee on Taxonomy of Viruses (Flores et al. 1998, 2000), on the basis of the identity of a subset of nucleotides within the central conserved region and the similarity in the terminal conserved hairpin of the TL domain with Coconut cadang-cadang viroid (Flores et al. 1997), CVd-IV has been suggested as a new species of the genus Cocadviroid belonging to the family Pospiviroidae (Table 27.1). However, in this proposal no consideration was given to the block of 80–90 nucleotides concentrated in the V and TR domains shared by CVd-IV and CEVd as describing a relationship between these two viroids. This relationship suggests that CVd-IV is a possible member of the genus Pospiviroid, family Pospiviroidae. This section also comprises a major portion of the

terminal repeated sequence in CEVd D-92 (Semancik et al. 1994). Strengthening this suggested alternative relationship is the biological evidence linking CEVd-related variants as possible progenitors of CVd-IV (Semancik and Duran-Vila 1999).

CITRUS DISEASES WITH POSSIBLE VIROID ETIOLOGY Wood pitting, gum pocket, gummy pitting

The terms ‘wood pitting’, ‘gum pocket’, and ‘gummy pitting’ have been used to describe a characteristic disorder of trifoliate orange. Wood pitting was the name given in Argentina to the cachexia-like disorder characterized by the presence of wood indentations with gum deposits accumulating both in the xylem and the bark at the site of the pit of trees grafted on trifoliate orange rootstock (Fernandez-Valiela et al. 1965). These symptoms were usually found associated with the ‘bud-union crease’ and ‘laminate shelling’ disorders also of unknown origin and were shown to be unrelated to the cachexia disease (Foguet and Oste 1968). Gum pocket and gummy pitting were the names given in South Africa (Schwarz and McClean 1969) and Australia (Fraser et al. 1976). They are similar disorders characterized by the development of gum-impregnated pits and gum pockets on the trunk of the trifoliate orange rootstocks. Similar symptoms have also been reported in Italy (Cartia et al. 1984) and Turkey (Azeri 1985) but their association with a graft-transmissible agent was not demonstrated. Since these characteristic symptoms were commonly seen in the dwarfing trials conducted in Australia to evaluate the effect of ‘graft-transmissible dwarfing factors’ and those are now known to be caused by specific viroids or viroid combinations (Hutton et al. 2000), a viroid etiology was considered as a likely hypothesis (Marais et al. 1996). The viroid hypothesis was consistent with earlier observations indicating that the severity of the symptoms varied with locality, scion variety, and dwarfing strain (Fraser et al. 1976). The results of the transmission assays conducted with the disease source from South Africa supports the involvement of a graft-transmissible RNA that appears to have the electrophoretic mobility similar to CVd-III (Marais et al. 1996) but since it has not been positively identified as a viroid, Koch’s postulates cannot be considered fulfilled. Since this type of symptom has not been observed in field assays conducted to evaluate the effect of the variant CVd-IIIb on trees grafted on trifoliate orange (Semancik et al. 1997) a cause–effect relationship between all variants of CVd-III and the disease cannot be inferred. If the viroid hypothesis for the disease can be

189


established, it seems probable that an unusual variant of CVdIII or a new viroid with a size similar to CVd-III might be implicated. Yellow corky vein

Yellow corky vein disease of citrus is a disorder that was first described in 1974 in declining sweet orange trees (Reddy et al. 1974). The typical symptoms are yellowing of the veins followed by vein corking on the underside of the leaves and curling. The symptoms resemble those described earlier as ‘yellow vein’ in California (Weathers 1957), but the vein corking was not reported as characteristic of this disease. In addition, nutrient deficiency, especially boron, results in a similar foliar response. The disease appears to be graft-transmissible by mechanical means and dodder but not through seed (Reedy and Naidu 1989). The host range appears to be restricted to citrus species (Rustem and Ahlawat 1999) with an unusually large number of hosts developing symptoms. The detection of an electrophoretic band of mobility characteristic for a small circular RNA similar to CVd-III in nucleic acid extracts from symptomatic plants has been presented as evidence for the involvement of a viroid as the causal agent (Rustem and Ahlawat 1999). Kassala disease

Bark gumming of grapefruit is a disorder characterized by the presence of gum impregnations and gum streaks in the bark of grapefruit trees (Bové 1995). The symptoms resemble those of cachexia on mandarin and gummy bark on sweet orange. The symptoms were first observed on ‘Foster’ grapefruit in Kassala (Sudan) but later numerous cases were found in other locations of Sudan. Cultivars other than ‘Foster’ were also affected regardless of the rootstock used. The disease seems to be unrelated to the agents of cachexia or gummy bark as no evidence of graft transmission has been presented. However, similarity of symptoms with those of the cachexia disease support a viroid etiology. Since the symptoms are always observed in old trees from very early introduced materials, the finding that trees were infected with viroids should be regarded with caution (Semancik and Duran-Vila 1991). Gummy bark

See Chapter 51 ‘Citrus gummy bark’ in this volume. References Albanese, G., LaRosa, R., Davino, M., Hammond, R.M., Smith, D.R., and Diener, T.O. (1988). A viroid different form citrus exocortis viroid found in commercial citrus in Sicily. Pages 165-172 in: Proc 10th Conf. Int. Org. Citrus Virol. L. W. Timmer, S. M. Garnsey, and L. Navarro, eds. IOCV: Riverside, CA. Albanese, G., Renis, M., Grimaldi, V., La Rosa, R., Polizzi G., and Diener, T.O. (1991). Hybridization analysis of citrus viroids with citrus exo-

190

cortis viroid- and hop stunt viroid-specific probes. Pages 202-205 in: Proc 11th Conf. Int. Org. Citrus Virol. R. H Brlansky, R. F. Lee, and L. W. Timmer, eds. IOCV: Riverside, CA. Allen, R.M. (1968). Survival time of exocortis virus of citrus on contaminated knife blades. Plant. Dis. Reptr. 52, 935-939. Ashulin, L., Lachman, O., Hadas, R., and Bar-Joseph, M. (1991). Nucletide sequence of a new viroid species, citrus bent leaf viroid (CBLVd) isolated from grapefruit in Israel. Nucleic Acids Res. 19, 4767. Astruc, N, Marcos, J.F., Macquaire, G., Candresse, T., and Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. Eur. J. Plant Pathol. 102, 837-846. Azeri, T. (1985). Gummy pitting, a destructive virus disease of Poncirus trifoliate rootstock in Izmir province of Turkey. J. Turkish Phytopathol. 14, 45-52. Bar-Joseph, M. (1996). A contribution to the natural history of viroids. Pages 226-229 in: Proc 13th Conf. Int. Org. Citrus Virol. J. V. da Graça, P. Moreno, and R.K. Yokomi, eds. IOCV: Riverside, CA. Barksh, N., Lee, R.F., and Garnsey, S.M. (1984). Detection of citrus exocortis viroid by polyacrylamide gel electrophoresis. Pages 343352 in: Proc 9th Conf. Int. Org. Citrus Virol. S.M. Garnsey, L.W. Timmer, and J.A. Dodds, eds. IOCV: Riverside, CA. Bello, L., Pérez, R., and González, H. (2000). Performance of clementine mandarins infected with exocortis disease on nine rootstocks. Pages 384-385 in: Proc 14th Conf. Int. Org. Citrus Virol. J.V. da Graça, R.F. Lee, and R.K. Yokomi, eds. IOCV: Riverside, CA. Ben-Shaul, A., Guang, Y., Mogilner, Hadas, R., Mawassi, M., Gafney, R., and Bar-Joseph, M. (1995). Genomic diversity among populations of two citrus viroids from different graft-transmissible dwarfing complexes in Israel. Phytopathology 85, 359-364. Benton, R. J., Bowman, F. T., Fraser, L., and Kebby, R. G. (1949). Selection of citrus budwood to control scaly butt in trifoliata rootstock. Agr. Gaz. N. S. Wales 60, 31-34. Benton, R. J., Bowman, F. T., Fraser, L., and Kebby, R. G. (1950). Stunting and scaly butt associated with Poncirus trifoliata rootstock. N. S. Wales, Dept. Agr., Sci. Bull. 70, 1-20. Bitters, W.P., Duran -Vila, N., and Semancik, J.S. (1987). Effect of exocortis viroid on flower and fruit structure and development on Etrog citron. Plant. Dis. 71, 397-399. Boccardo, G., La Rosa, R., and Catara A. (1984). Detection of citrus exocortis viroid by polyacrylamide gel electrophoresis of nucleic acid extracts from galsshouse citrus. Pages 357-361 in: Proc 9th Conf. Int. Org. Citrus Virol. S.M. Garnsey, L.W. Timmer, and J.A. Dodds, eds. IOCV: Riverside, CA. Bové, J.M. (1995). Virus and virus-like diseases of citrus in the Near East region. FAO, Rome. 518pp. Calavan, E.C. (1968). Exocortis. Pages 23-34 in: Indexing procedures for 15 citrus diseases of citrus trees. Agricultural Handbook No.33. ARS: USDA. Calavan, E.C., Frolich, E.F., Carpenter, J.B., Roistacher, C.N., and Christiansen, D.W. (1964). Rapid indexing for exocortis of citrus. Phytopathology 54, 1359-1362. Cartia, G., La Rosa, R., and Catara, A. (1984). A gummy pitting of trifoliate orange in Italy. Pages 184-187 in: Proc. 9th Conf. Int. Organ.

CITRUS VIROIDS

Citrus Virol. S.M. Garnsey, L.W. Timmer and J.A. Dodds, eds. IOCV: Riverside, CA. Childs, J.F.L. (1950). The cachexia disease of Orlando tangelo. Plant Dis. Rep. 34, 295-298. Davino, M., Pelicani, L., Renis, M. , and Albanese, G. (1991). Homology of hop stunt viroid with citrus cachexia viroid. Pages 196-201 in: Proc. 11th Conf. Int. Organ. Citrus Virol. R.H. Brlansky, R.F. Lee, and L.W. Timmer, eds. IOCV: Riverside, CA. Diener, T. O. (1971). Potato spindle tuber ‘virus’ IV. A replicating, low molecular weight RNA. Virology 45, 411-428. Duran -Vila, N., Flores, R., and Semancik, J. S. (1986). Characterization of viroid-like RNAs associated with the citrus exocortis syndrome. Virology 150, 75-84. Duran-Vila, N., Roistacher, C. N., Rivera-Bustamante, R., and Semancik, J. S. (1988a). A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69, 3069-3080. Duran-Vila, N., Pina, J .A.., Ballester, J. F., Juárez, J., Roistacher, C. N., Rivera-Bustamante, R., and Semancik, J.S. (1988b). The citrus exocortis disease: a complex of viroid RNAs. Pages 152-164 in: Proc 10th Conf. Int. Org. Citrus Virol. L. W. Timmer, S. M. Garnsey, and L. Navarro, eds. IOCV: Riverside, CA. Duran-Vila, N., Pina, J.A., and Navarro, L. (1993). Improved indexing of citrus viroids. Pages 202-211 in: Proc. 12th Conf. Int. Organ. of Citrus Virol. P. Moreno, J.V. da Graça, and L.W. Timmer, eds. IOCV: Riverside, CA. Eigen, M. (1993). The origin of genetic information: Viruses as models. Gene 135, 37-47. Eigen, M., and Biebricher, C.K. (1988). Sequence space and quasispecies distribution. Pages 211-245 in: RNA genetics. Vol. 3. E. Domingo, J.J. Holland, and P. Ahlquist, eds. CRC: Boca Raton, FL. Fagoaga, C., and Duran-Vila, N. (1996). Naturally occurring variants of citrus exocortis viroid in vegetable crops. Plant Pathol. 45, 45-53. Fagoaga, C., Semancik, J.S., and Duran-Vila, N. (1995). A citrus exocortis viroid variant from broad bean (Vicia faba L.): Infectivity and pathogenesis. J. Gen. Virol. 76, 2271-2277. Fawcett, H. S., and Klotz, L. J. (1948). Exocortis on trifoliate orange. Citrus Leaves 28, 8. Fernández-Valiela, M.V., Fortugno, C., and Corizzi, F. (1965). Incidence of bud-union crease in citrus trees grafted on trifoliata rootstock in the Delta del Paraná and San Pedro areas of Argentina. Pages 182186 in: Proc. 3d Conf. Int. Organ. Citrus Virol. W.C. Price, ed. IOCV: Riverside, CA. Flores, R. (1986). Detection of citrus exocortis viroid in crude extracts by dot blot hybridization: conditions for reducing spurious hybridization results and for enhancing the sensitivity of the technique. J Virol. Methods 13, 309-319. Flores, R., Duran-Vila, N., Pallás, V., and Semancik, J.S. (1985). Detection of viroid and viroid like RNAs from grapevine. J. Gen. Virol. 66, 2095-2102. Flores, R., Di Serio, F., and Hernández, C. (1997). Viroids: The noncoding genomes. Sem. Virol. 8, 65-73. Flores, R., Randles, J. W., Bar-Joseph, M., and Diener, T. O. (1998). A proposed scheme for viroid classification and nomenclature. Arch. Virol. 143, 623-629. Flores, R., Randles, J. W., Bar-Joseph, M., and Diener, T. O. (2000). Subviral agents: Viroids. Pages 1009-1024 in: Virus taxonomy, Seventh Report of The International Committee on Taxonomy of Viruses.

M.H.V. van Regenmortel, C.M. Fauquet, D.H.L. Bishop, E.B. Carstens, M.K. Estes, S.M. Lemon, D.J. McGeoch, J. Maniloff, M.A. Mayo, C.R. Pringle, and R.B. Wickner, eds. Academic Press: San Diego, CA. Foguet, J.L., Oste, C.A. (1968). Disorders of trifoliate orange rootstock in Tucumán, Argentina. Pages 183-189 in: Proc. 4th Conf. Int. Organ. Citrus Virol. J.F.L. Childs, ed. IOCV: Jacksonville, FL. Foissac, X., and Duran-Vila, N. (2000). Characterization of two citrus apscaviroids isolated in Spain. Arch. Virol. 145, 1975-1983. Fonseca, M.E., and Kitajima, E.W. (1993). French marigold (Tagetes patula): a new experimental host of citrus exocortis viroid. Plant Dis. 77, 953. Fonseca, M.E., Marcellino, L.H., and Garder, E. (1996). A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes. J. Virol. Methods 57, 203-207. Francis, M., Szychowski, J.A., and Semancik, J.S. (1995). Structural sites specific to citrus viroid groups. J. Gen. Virol. 76, 1081-1089. Fraser, L.R., Broadbent, P., and Cox, J.E. (1976). Gummy pitting of Poncirus trifoliata: Its association with dwarfing of citrus in New South Wales. Pages 147-151 in: Proc. 7th Conf. Int. Organ. of Citrus Virol. E.C. Calavan, ed. IOCV: Riverside, CA. Gandía, M., Palacio, A., and Duran-Vila, N. (2000). Variability of citrus exocortis viroid (CEVd). Pages 265-272 in: Proc. 14th Conf. Int. Organ. Citrus Virol. J.V. da Graça, R.F. Lee, and R.K. Yokomi, eds. IOCV: Riverside, CA. García-Arenal, F., Pallás, V., and Flores, R. (1987). The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid. Nucleic Acids Res. 15, 4203-4210. Garnsey, S.M., and Jones, J.W. (1967). Mechanical transmission of exocortis virus with contaminated budding tools. Plant. Dis. Reptr. 51, 410-413. Garnsey, S.M., and Weathers, L.G. (1972). Factors affecting mechanical spread of exocortis virus. Pages 105-111 in: Proc. 5th Conf. Int. Organ. Citrus Virol. W.C. Price, ed. University of Florida Press: Gainesville, FL. Gillings, M.R., Broadbent, P., and Gollnow, B.I. (1988). Biochemical indexing for citrus exocortis viroid. Pages 178-187 in: Proc. 12th Conf. Int. Organ. Citrus Virol. P. Moreno, J.V. da Graça, and L.W. Timmer, eds. IOCV: Riverside, CA. Gillings, M.R., Broadbent, P., and Gollow, B.I. (1991). Viroids in Australian Citrus: Relationship to exocortis, cachexia and citrus dwarfing. Aust. J. Plant Physiol. 18, 559-570. Gross, H.J., Krupp, G., Domdey, H., Raba, M., Jank, P., Lossow, C., Alberty, H., Ramm, K., and Sänger, H.L. (1982). Nucleotide sequence and secondary structure of citrus exocortis and chrysanthemum stunt viroid. Eur. J. Biochem. 121, 249-257. Hadas, R., Bar-Joseph, M., and Semancik, J.S. (1989). Segregation of a viroid complex from a graft-transmissible dwarfing agent source for grapefruit trees. Ann. Appl. Biol. 115, 515-520. Hadas, R., Ashulin, L., and Bar-Joseph, M. (1992). Transmission of a citrus viroid to avocado by heterologous grafting. Plant Dis. 76, 357-359. Hataya, T., Nakahara, K., Ohara, T. Ieki, H., and Kano, T. (1998). Citrus viroid Ia is a derivative of citrus bent leaf viroid (CVd-Ib) by partial sequence duplications in the right terminal region. Arch. Virol. 143, 971-980.

191


Hsu, Y.-H, Chen, W., and Owens, R.A. (1994). Nucleotide sequence of a hop stunt viroid variant isolated from citrus growing in Taiwan. Virus Genes 9, 193-195. Hutton, R.J., Broadbent, P., and Bevington, K.B. (2000). Viroid dwarfing for high density plantings. Horticultural Reviews 24, 277-317. Ito, T., Ieki, H., and Ozaki K. (2000). A population of variants of a viroid closely related to citrus viroid-I in citrus plants. Arch. Virol. 145, 2105-2114. Ito, T., Ieki, H., Ozaki, K., and Ito, T. (2001). Characterization of a new citrus viroid species tentatively termed citrus viroid OS. Arch. Virol. 146, 975-982. Kofalvi, S. Marcos, J.F., Cañizares, M.C., Pallás, V., and Candresse, T. (1997). Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. J. Gen. Virol. 78, 3177-3186 La Rosa, R., Albanese, G., Azzaro, A., Sesto, F., and Domina, F. (1988). Suitability of nucleic acid analysis to diagnose viroid infections in citrus. Pages 188-191 in: Proc. 10th Conf. Int. Organ. Citrus Virol. S. Garnsey, and L. Navarro, eds. IOCV: Riverside, CA. La Rosa, R., Tessitori, M., Albanese, G., Catara, A., and Davino, M. (1993). Diagnosis of citrus exocortis and hop stunt-homologous citrus viroids by oligonucleotide probes. Pages 435-437 in: Proc. 12th Conf. Int. Organ. Citrus Virol. P. Moreno, J.V. da Graça, and L.W. Timmer, eds. IOCV: Riverside, CA. Levy, L., Hadidi, A., and Garnsey, S.M. (1992). Reverse-transcriptionpolymerase chain reaction assays for the rapid detection of citrus viroids using multiplex primer sets. Proc. Int. Soc. Citriculture 7, 800-803. Levy, L., and Hadidi, A. (1992). Direct nucleotide sequencing of PCRamplified DNAs of the closely related citrus viroids IIa and IIb (cachexia). Pages 180-186 in: Proc. 12th Conf. Int. Organ. Citrus Virol. P. Moreno, J.V. da Graça, and L.W. Timmer, eds. IOCV: Riverside, CA. Marais, J., Lee, R.F., Breytenbach, J.H.J., Manicom, B.Q., and Van Vuuren, S.P. (1996). Association of a viroid with gum pocket disease of trifoliate orange. Pages 236-244 in: Proc. 13th Conf. Int. Organ. of Citrus Virol. J.V. da Graça, P. Moreno, R.K. Yokomi, eds. IOCV: Riverside, CA. Mishra, M.D., Hammond, R.W., Owens, R.A., Smith, D.R., and Diener, T.O. (1991). Indian bunchy top disease of tomato plants is caused by a distinct strain of citrus exocortis viroid. J. Gen. Virol. 72, 1781-1785. Moreira, S. (1955). Sintomas de “exocortis” em limoneiro cravo. Bragantia 14, 19-21. Moreira, S. (1959). Rangpur lime disease and its relationship to exocortis. Pages 135-140 in: Citrus virus diseases. J. M. Wallace, ed. Univ. Calif. Div. Agr. Sci.: Berkeley, CA. Navarro, L., Roistacher, C.N., and Murashige, T. (1975). Improvement of shoot tip grafting in vitro for virus-free citrus. J. Am. Soc. Hortic. Sci. 100, 471-479. Niblett, C.L., Dickson, E., Horst, K.K., and Romaine, C.P. (1980). Additional hosts and an efficient purification procedure for four viroids. Phytopathology 70, 610-615. Ochoa, F., La Rosa, R., Albanesse, G., Tessitori, M., and Fuggetta, E. (1996). Survey of citrus viroids in Venezuela. Pages 354-356 in: Proc. 13th Conf. Int. Organ. Citrus Virol. J.V. da Graça, P. Moreno, and R.K. Yokomi, eds. IOCV: Riverside, CA. Önelge, N., Çinar, A., Kersting, U., and Semancik, J.S. (1996). Viroids associated with Citrus Gummy bark disease of sweet orange in Tur-

192

key. Pages 245-248 in: Proc. 13th Conf. Int. Organ. Citrus Virol. J.V. da Graça, P. Moreno, and R.K. Yokomi, eds. IOCV: Riverside, CA. Önelge, N., Kersting, U., Guang, Y., Bar-Joseph, M., and Bozan, O. (2000). Nucleotide sequence of citrus viroids CVd-IIIa and CVd-IV obtained from dwarfed Meyer lemon trees grafted on sour orange. J. Plant Dis. and Protection 107, 387-391. Owens, R.A., and Diener, T.O. (1981). Sensitive and rapid diagnosis of potato spindle tuber viroid disease by nucleic acid hybridization. Science 213, 670-672. Owens, R.A., Candresse, T., and Diener, T.O. (1990). Construction of novel viroid chimeras containing portions of tomato apical stunt and citrus exocortis viroids. Virology 175, 238-246. Owens, R.A., Thompson, S.M., Feldstein, P.A., and Garnsey, S.M. (1999). Effects of natural sequence variation on symptom inducion by citrus viroid III. Ann. Appl. Biol. 134, 73-80. Owens, R.A., Yang, G., Gundersen-Rindal, D., Hammond, R.W., Candresse, T., and Bar-Joseph, M. (2000). Both point mutations and RNA recombination contribute to the sequence diversity of citrus viroid III. Virus Genes 20, 243-252. Pagliano, G., Peyrou, M., Del Campo, L., Orlando, A., Gravina, R., Wettsttein, R., and Francis, M. (2000). Detection and characterization of citrus viroids in Uruguay. Pages 282-287 in: Proc 14th Conf. Int. Org. Citrus Virol. J.V. da Graça, R.F. Lee, and R.K. Yokomi, eds. IOCV: Riverside, CA. Palacio, A., and Duran-Vila, N. (1999). Single-strand conformation polymorphism (SSCP) analysis as a tool for viroid characterisation. J. Virol. Methods 77, 27-36. Palacio, A., Foissac, X., and Duran-Vila, N. (2000a). Indexing of citrus viroids by imprint hybridisation. Eur. J. Plant Pathol. 105, 897-903. Palacio, A., Foissac, X., and Duran-Vila, N. (2000b). Indexing of citrus viroids by imprint hybridization: Comparation with other detection methods. Pages 294-301 in: Proc 14th Conf. Int. Org. Citrus Virol. J.V. da Graça, R.F. Lee, and R.K. Yokomi, eds. IOCV: Riverside, CA. Pérez, J.M., Peña, I., and Pérez, R. (2000). Comparative viroid detection of Cuban viroid isolates using biological indexing and sequential PAGE. Pages 302-303 in: Proc 14th Conf. Int. Org. Citrus Virol. J.V. da Graça, R.F. Lee, R.K. Yokomi, eds. IOCV: Univ. Calif., Riverside, CA. Pérez, R., González, A., Bello, L., and Marrero, A. (1992). ‘Clemelin 1120’ a new indicator plant for cachexia. Proc. Int. Soc. Citriculture 7, 786-787. Puchta, H., Ramm, K., Hadas, R., Bar-Joseph, M., Luckinger, R., Freimüller, K., and Sänger, H. L. (1989). Nucleotide sequencing of hop stunt viroid (HSVd) isolated from grapefruit in Israel. Nucleic Acids Res. 17, 1247. Puchta, H., Ramm, K., Luckinger, R., Hadas, R., Bar-Joseph, M., and Sänger, H.L. (1991). Primary and secondary structure of citrus viroid IV (CVd IV), a new chimeric viroid present in dwarfed grapefruit in Israel. Nucleic Acids Res 19, 6640. Rakowski, A.G., Szychowski, J.A., Avena, Z.S., and Semancik, J.S. (1994). Nucleotide sequence and structural features of the Group III citrus viroids. J. Gen. Virol. 75, 3581-3584. Reanwarakorn, K., and Semancik, J. S. (1998). Regulation of pathogenicity in hop stunt viroid related group II citrus viroids. J. Gen. Virol. 79, 3163-3171. Reanwarakorn, K., and Semancik, J.S. (1999a). Correlation of hop stunt viroid variants to cachexia and xyloporosis diseases of citrus. Phytopathology 89, 568-574.

CITRUS VIROIDS

Reanwarakorn, K., and Semancik, J.S. (1999b). Discrimination of cachexia disease agents among citrus variants of hop stunt viroid. Ann. Appl. Biol. 135, 481-487. Reddy, G.S., Dakshinamurti, V., and Reddy, V.R.K. (1974). Yellow corky-vein: First report of a new graft-transmissible disorder of Sathgudi in Andhra Pradesh. Indian Phytopathol. 27, 82-84. Reedy, M.R.S., and Naidu, H. (1989). Transmission of citrus corky vein. Indian Phytopathol. 42, 455-456. Reichert, I., and Perlberger, P. (1934). Xyloporosis, the new citrus disease. Agr. Exp. Sta. Rehovoth, Palestine Bull. 12, p. 44. Rivera-Bustamante, R.F., Gin, R., and Semancik, J.S. (1986). Enhanced resolution of circular and linear molecular forms of viroid and viroid-like RNA by electrophoresis in a discontinuous-pH system. Anal. Biochem. 156, 91-95. Roistacher, C.N. 1991. Graft-transmissible diseases of citrus. Handbook for detection and diagnosis. (Ed. C.N.Roistacher.) IOCV-FAO: Rome. 286 pages. Roistacher, C.N., Bash, J.A., and Semancik, J.S. (1993). Distinct disease symptoms in Poncirus trifoliata induced by three citrus viroids from three specific groups. Pages 173-179 in: Proc. 12th Conf. Int. Organ. Citrus Virol. P. Moreno, J.V. da Graça, and L.W. Timmer, eds. IOCV: Riverside, CA. Roistacher, C.N., Blue, R.L., and Calavan, E.C. (1973). A new test for citrus cachexia. Citrograph 58, 261-262. Roistacher, C.N., Calavan, E.C., and Blue, R.L. (1969). Citrus exocortis virus – chemical inactivation on tools and tolerance to heat and separation of isolates. Plant. Dis. Rep. 53, 333-336. Roistacher, C.N., Calavan, E.C., Blue, R.L., Navarro, L., and Gonzales, R. (1977). A new more sensitive citron indicator for the detection of mild isolates of citrus exocortis viroid (CEV). Plant Dis. Rep. 61, 135-139. Rustem, F.A., and Ahlawat, Y.S. (1999). Yellow corky vein – a viroid disease of citrus in India. Indian J. Agr. Sci. 69, 789-794. Salibe, A.A., and Moreira, S. (1965). Tahiti lime bark disease is caused by exocortis virus. Pages 143-147 in: Proc 3rd. Conf. Int. Org. Citrus Virol. W.C. Price, ed. University of Florida Press, Gainesville, FL. Salibe, A.A. (1980). Further studies on exocortis disease of citrus. Pages 215-219 in: Proc 8th Conf. Int. Org. Citrus Virol. E.C. Calavan, S. M. Garnsey, and L. W. Timmer, eds. IOCV: Riverside, CA. Sano, T., Candresse, T., Hammond, R.W., Diener, T.O., and Owens, R.A. (1992). Identification of multiple structural domains regulating viroid pathogenicity. Proc. Natl. Acad. Sci. USA 89, 1014-1018. Sano, T., and Ishiguro, A. (1998). Viability and pathogenicity of intersubgroup viroid chimeras suggest possible involvement of the terminal right region in replication.Virology 240, 238-244. Sano, T., Hataya, T., and Shikata, E. (1988). Complete nucleotide sequence of a viroid isolated from Etrog citron, a new member of hop stunt viroid group. Nucleic Acids Res. 16, 347. Sano, T., Hataya, T., Terai, Y., and Shikata, T. (1989). Hop stunt viroid strains from dapple fruit disease of plum and peach in Japan. J. Gen Virol. 70, 1311-1319. Schlemmer, A., Roistacher, C.N., and Semancik, J.S. (1985). A unique, infectious RNA associated with citron showing symptoms typical of citrus exocortis disease. Phytopathology 75, 946-949. Schumacher, J., Randles, J.W., and Riesner, D. (1983). A two dimensional electrophoretic technique for detection of circular viroids and virusoids. Anal. Biochem. 135, 288-295.

Schwarz, R.E, and McClean, A.P.D. (1969). Gum-pocket, a new viruslike disease of Poncirus trifoliata. Plant Dis Rep. 53, 336-339. Semancik, J. S., and Duran-Vila, N. (1991). The grouping of citrus viroids: Additional physical and biological determinants and relationship with disease of citrus. Pages 178-188 in: Proc. 11th Conf. Int. Org. Citrus Virol. R. H Brlansky, R. F. Lee, and L. W. Timmer, eds. IOCV: Riverside, CA. Semancik, J.S. and Duran-Vila, N. (1999). Viroids in plants: Shadows and footprints of primitive RNA. Pages 37-64 in: Origin and evolution of viruses. E. Domingo, R. Webster, and J. Holland, eds. Academic Press: New York. Semancik, J.S., and Weathers, L.G. (1972a). Exocortis disease: Evidence for a new species of “infectious” low molecular weight RNA in plants. Nature New Biology 237, 242-244. Semancik, J.S., and Weathers, L.G. (1972b). Exocortis virus: An infectious free-nucleic acid plant virus with unusual properties. Virology 46, 456-466. Semancik, J.S., and Weathers, L.G. (1972c). Pathogenic 10 s RNA from exocortis disease recovered from tomato bunchy-top plants similar to potato spindle tuber infection. Virology 49, 622-625. Semancik, J.S., and Weathers, L.G. (1973). Potato spindle tuber disease produced by pathogenic RNA from citrus exocortis disease: evidence for the identity of the causal agents. Virology 52, 292-294. Semancik, J.S., Rakowski, A.G., Bash, J.A., and Gumpf, D.J. (1997). Application of selected viroids for dwarfing and enhancement of production of “Valencia” orange. J. Hort. Sci. 72, 563-570. Semancik, J.S., Roistacher, C.N., Rivera-Bustamante, R., and Duran-Vila N. (1988). Citrus cachexia viroid, a new viroid of citrus: Relationship to viroids of the exocortis disease complex. J. Gen. Virol. 69, 3059-3068. Semancik, J.S., Szychowski, J.A., Rakowski, A.G., and Symons, R.H. (1993). Isolates of citrus exocortis viroid recovered by host and tissue selection. J. Gen. Virol. 74, 2427-2436. Semancik, J.S., Szychowski, J.A., Rakowski, A.G., and Symons, R.H. (1994). A stable 436 nucleotide variant of citrus exocortis viroid produced by terminal repeats. J. Gen. Virol. 75, 727-732. Singh, R. P., and Boucher, A. (1987). Electrophoretic separation of a severe from mild isolates of potato spindle tuber viroid. Phytopathology 77, 1588-1591. Skoric, D., Conerly, M., Szychowski, J.A., and Semancik, J.S. (2001). CEVd-induced symptom modification as a response to hostspecific temperature-sensitive reaction. Virology 280, 115-123. Stasys, R.A., Dry, I.B., and Rezaian, M.A. (1995). The termini of a new citrus viroid contain duplications of a central conserved regions from two viroid groups. FEBS Letters 358, 182-184. Szychowski, J.A., Doazan, J.P., Lecalir, P., Garnier, M., Credi, R., Minafra, A., Duran-Vila N., Wolpert, J.A., and Semancik, J.S. (1991). Relationship and patterns of distribution among grapevine viroids from California and Europe. Vitis 30, 25-36. Terranova, G., Caruso, A., and Reforgiato Recupero, G. (1991). Susceptibility and symptomatology of Mapo tangelo to xyloporosis. Pages 209-213 in: Proc. 11th Conf. Int. Org. Citrus Virol. R. H Brlansky, R. F. Lee, and L. W. Timmer, eds. IOCV: Riverside, CA. Tessitori, M., La Rosa, R., Albanese, G., and Catara, A. (1996). PCR diagnosis of citrus viroids in field samples. Pages 230-235 in: Proc. 13th Conf. Int. Organ. Citrus Virol. J.V. da Graça, P. Moreno, and R.K. Yokomi, eds. IOCV. Riverside, CA.

193


Turuto, C., D’Onoghia, A.M., Minafra A., and Savino, V. (1998). PCR detection of citrus exocortis and citrus cachexia viroids. Phytopathol. Medit. 37, 99-105. Villalobos, W., Rivera, C., and Hammond, R.W. (1997). Occurrence of citrus viroids in Costa Rica. Rev. Biol. Trop. 45, 983-987. Visvader, J.E., and Symons, R.H. (1983). Comparative sequence and structure of different isolates of citrus exocortis viroid. Virology 130, 232-237. Visvader, J.E., and Symons, R.H. (1985). Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity. Nucleic Acids Res. 13, 2907-2920. Visvader, J., and Symons, H. (1986). Replication of in vitro constructed viroid mutants: location of the pathogenicity modulating domain of citrus exocortis viroid. EMBO J. 5, 2051-2055. Visvader, J.E., Gould, A.R., Bruening, G.E., and Symons, R.H. (1982). Citrus exocortis viroid: Nucleotide sequence and secondary structure of an Australian isolate. FEBS Lett. 137, 288-292. Weathers, L.G. (1957). A vein-yellowing disease of citrus caused by a graft transmissible virus. Plant Dis. Reptr. 41, 741-742. Weathers, L. G. (1965). Petunia as a herbaceous host of exocortis virus of citrus. Phytopathology 55, 1081.

194

Weathers, L. G., and Calavan, (1961). Additional indicator plants for exocortis and evidence for strain differences in the virus. Phytopathlogy 51, 262-264. Weathers, L. G., and Greer, F. C. Jr. (1968). Additional herbaceous hosts of the exocortis virus of citrus. Phytopathology 58, 1071. Weathers, L. G., and Greer, F. C. Jr. (1972). Gynura as a host for exocortis virus of citrus. Pages 95-98 in: Proc. 5th Conf. Int. Organ. Citrus Virol. W.C. Price, ed. University of Florida Press: Gainesville, FL. Weathers, L.G., Greer, F.C., Jr, and Harjungs, M.K. (1967). Transmission of exocortis virus of citrus to herbaceous plants. Plant Dis. Rep. 51, 868-871. Wei, P., Yu-an, Z., Xue-qian, Z., and Gang, L. (1996). Survey and indexing for citrus exocortis and other viroids in Sichuan province. Pages 357-359 in: Proc 13th Conf. Int. Org. Citrus Virol. J.V. da Graça, P. Moreno, and R.K. Yokomi, eds. IOCV: Riverside, CA. Wutscher, H.K., and Shull, A.V. (1975). Machine-hedging of citrus trees and transmission of exocortis and xyloporosis virus. Plant Dis. Reptr. 59, 368-369. Yang, X., Hadidi, A., and Garnsey, S.M. (1992). Enzymatic cDNA amplification of citrus exocortis and cachexia viroids from infected citrus hosts. Phytopathology 82, 279-285.

PART IV

GRAPEVINE VIROIDS CHAPTER 28 ....................................................................................................

GRAPEVINE VIROIDS A. Little and M.A. Rezaian

.................................................................................................................................................................................................................................................................

The first viroid reported in grapevines was an isolate of Hop stunt viroid (HSVd-g) from Japan (Shikata et al. 1984; Sano et al. 1985). Subsequent research revealed the widespread occurrence of grapevine viroid infection in various countries. As these viroids were characterised and their molecular structure determined the picture became simpler. As a result five viroids were identified (see Table 28.1). Two of these were found to be Grapevine yellow speckle viroids 1 and 2 (GYSVd-1 and GYSVd2) the independent causal agents of yellow speckle disease (Koltunow et al. 1988, 1989a) and another was Australian grapevine viroid (AGVd) (Rezaian 1990). These three viroids were unique to grapevines. The other two were identified as grapevine isolates of HSVd (Sano et al. 1985) and Citrus exocortis viroid (CEVd-g) (García-Arenal et al. 1987). Unlike some viroids that cause serious plant diseases, grapevine viroids produce little or no obvious disease symptoms and often replicate in the host unnoticed. Of the five grapevine viroids only yellow speckle viroids 1 and 2 have been shown to induce disease symptoms. There is no published evidence of a significant adverse effect due to yellow speckle disease or the other three viroids. Mixed infection of viroids in grapevines is common. Many clones of cultivars are infected with one or more viroid species, but seem to produce acceptable yield and quality

without any obvious signs of degeneration. For this reason grapevine viroids are not generally viewed as an economic problem. An exception is the synergism that exists between Grapevine fanleaf virus and yellow speckle disease resulting in the more severe vein-banding disease.

GRAPEVINE YELLOW SPECKLE DISEASE Symptoms

Taylor and Woodham (1972) first described grapevine yellow speckle (YS) disease in Australia and it has remained the only grapevine disease to be caused by viroids in grapevine to date (Koltunow and Rezaian 1988; Koltunow et al. 1989a). The foliar symptoms of yellow speckle are often absent in affected vines or are difficult to see because the symptoms are confined to a few yellowish spots or flecks scattered in tissue along major or minor veins of leaves. In some years in Australia, the symptoms can be spectacular causing yellow blotches and vein banding patterns (see Plate 11A–E). Depending on weather conditions, the symptoms may develop as early as October, but can occur during December and January, and as late as March if the vines are still in active growth. When the symptoms occur early, the speckles bleach and by late summer they appear white. Speckles that develop mid-season are a paler yellow and those

195


Table 28.1

Viroids known to infect grapevine.

Viroids

Size (nt)

Genus

Pathogen-icity in grapevine

Herbaceous host

Synonyms

Reference

GYSVd-1

366

Apscaviroid

yellow speckle

none known

GVd-f, GV-1

Koltunow and Rezaian 1988

GYSVd-2

363

Apscaviroid

yellow speckle

none known

GV-2, GV-1B

Koltunow and Rezaian 1989a

AGVd

369

Apscaviroid

no symptoms

cucumber

none

Rezaian 1990

HSVd-g

297

Hostuviroid

no report

cucumber

GV-3

Sano et al. 1985

CEVd-g

369

Pospiviroid

no report

tomato

GV-s

García-Arenal et al. 1987

that develop during late summer tend to be light green. Yellow speckle symptoms are more likely to occur on exposed leaves and on basal leaves of exposed lateral growth (Taylor and Woodham 1972). The effect of CYSVd-1, CYSVd-2, and HSVd on growth, yield, and maturity indices of ‘Cabernet Sauvignon’ grapevines was analyzed comprehensively by Wolpert et al. (1996) in California. Growth and yield components were measured over a threeyear period (1991–1993) in own-rooted vines and shoot tipcultured vines inoculated with GYSVd-1 and GYSVd-2. Infection by these viroids appeared to have no significant affect on the parameters observed (Wolpert et al. 1996). However, it is unclear whether any yellow speckle disease symptoms were actually observed in the viroid-infected ‘Cabernet Sauvignon’ variety during the three-year period. Occasionally in Australia, severe cases of yellow speckle disease occur that result in a significant reduction in healthy leaf area. It remains a possibility that severe grapevine yellow speckle disease symptoms may result in a lower level of photosynthesis from infected leaves, which could have a significant effect on grapevine growth, yield or quality. Causal agents

Two closely related circular RNA molecules, Grapevine yellow speckle viroid 1 (GYSVd-1) and GYSVd-2, were associated with yellow speckle symptom expression. It was suggested from observations of their molecular properties (Koltunow and Rezaian 1988, 1989a) that GYSVd-1 and GYSVd-2 were viroids. To confirm that GYSVd-1 and GYSVd-2 were true viroids, it was necessary to demonstrate that purified GYSVd-1 and GYSVd2 RNA could replicate autonomously when inoculated into viroid-free plants. When RNA extracts of grapevine were inoculated onto cucumber and tomato, disease symptoms developed, but neither GYSVd-1 nor GYSVd-2 could be detected showing that GYSVd-1 and GYSVd-2 could not replicate in these herbaceous hosts (Rezaian et al. 1988; Koltunow and Rezaian 1988). This observation led to the detection of other viroids in grapevine. The infectivity of circular RNAs associated with yellow speckle was tested subsequently by inoculating viroid-free grapevine seedlings with various combinations of in vitro GYSVd-1 and GYSVd-2 transcripts (Koltunow et al. 1989a). The inoculated plants were observed for symptom

196

development and for the presence of viroids, which was determined by probe hybridization, using the GYSVd-1 and GYSVd-2 probes. The use of individual probes also acted as a check for the purity of the initial inocula used to infect plants. Both GYSVd-1 and GYSVd-2 could be transmitted independently to viroid-free grapevines. When a mixed inoculum containing both viroids was injected into grapevines, both viroids replicated showing GYSVd-1 and GYSVd-2 can replicate autonomously in the same plant (Koltunow et al. 1989a). All of the inoculated and uninoculated plants were maintained under natural summer conditions in South Australia and observed for the expression of yellow speckle disease symptoms (see Plate 11A–D). Only plants in which GYSVd-1, GYSVd-2 or both viroids had replicated expressed yellow speckle disease symptoms. GYSVd-1 and GYSVd-2 could be isolated from the infected plants, thus fulfilling Koch’s postulates. Therefore GYSVd-1 and GYSVd-2 could independently induce symptoms resembling the grapevine yellow speckle disease (Koltunow et al. 1989a). Occurrence

The disease agents, GYSVd-1 and GYSVd-2, are widespread and appear to occur worldwide (Bovey and Martelli 1992).

GRAPEVINE YELLOW SPECKLE VIROID 1 (GYSVd-1) Family

Pospiviroidae Genus

Apscaviroid Original source

Grapevine yellow speckle viroid 1 was first isolated from the grapevine cultivar, ‘Cabernet Franc’, infected with yellow speckle disease containing a single circular RNA revealed after 2D electrophoresis (Koltunow and Rezaian 1988). The circular RNA migrated as a single RNA band away from other infecting viroids after prolonged electrophoresis under denaturing conditions. As with the other circular RNAs isolated from grapevine, the yields of GYSVd-1 were low, approximately 1 ug of RNA/ kg of leaf tissue (Rezaian et al. 1988).

GRAPEVINE VIROIDS

TCR

A. AGVd

P

20

1

40

CCR

60

80

100

140

120

160

180

G GA A AA UA A A G C A C G A C A U U C C A C G A A A G A C C C CG G A U U AC A G AG U AA C C C A G U U GGC CCAA UAG GGUUCC GGUA UCAC GG CGC CGUAGAAAGA GAUAGA AAAGCUGG GACU CU GGCGA UCGU UCGAC GGG CC CAGC AGC CC GCAGG CGCUA GCAGG CG UAGGGGU CU GCGGAG GAAGAAAC C U CCG GGUU GUC UCGAGG CCAU GGUG CC GCG GCGUCCUUUU CUAUUU UUUUGACC CUGG GA CUGCU GGCG AGCUG CCC GG GUCG UCG GG CGUCC GCGGU CGUUC GU GUCCCUA GG UGUCUC CUUCUUUG C C GA U CC UC U C G C G A C C A C C CC CA A G U A A G A A A GC U A U A A U G UG U AC UC A A CG C U U

360

340

B. GYSVd-1

320

300

280

T1

P

20

1

U CC A C G CU GG U G GA UC G A A C

UU

CCC

C

UU

AA C

GC

40

U U CC G U A UUG GG U UGUG UU C GGC CC A C U

360

CC

ACGU AG G G C C

220

V

100

T2 140

120

160

180

G

CC

C

C UG U G U AC UUUCUUUUUC G CC C GC C CC A A CG G A A CU U C AG CA A

320

300

280

P 40

60

CUUCU UG UUC GG AGC G U CCUC CGU AGG C CAC G CUCGGC CA UGCGG CGG G CU C G UGC A C A CC G UCUUC CAG C C G A CG U A CG A C UU G A UC A U C G CU UU U A A U U

260

CCR 80

100

240

220

V 120

200

T2

140

G A C A G G G AA U UC G G A U C A C C G CG A C A A G A CU U U U A C G A C C C CU G A GGG GAA GAG CC GUC UCGAC GGGG GCAUU GAG UGG CU GCGU UC AGA GAAGGG CC C GAUC UUUUC UG GGUUCC GGUU CAC UCG GGC UC GGAC GC AGA U

200

GA A G CC C GG CC C A A G CA A A C CG U G C A U C A C U CG CA CC UC A C C C CU G A U U G GC G C A UG AAAGAAGAAG U GG G GGGGG GU GAG CU UCG C A GGGG GCA UCC G GUG C GAGCUG GU ACGUC GGU C GG G C ACG U G U GG C GGAAG GUC U CG G

340

20

1

80

A

240

CCR

60

T1

C. GYSVd-2

260

160 CU C CGG CC UCG ACCG

A C C

180

G C C C CGGAG UAGAA AAGGUC U

CCUC CGUAG CUC GUC GG CGCA AG GCC GG AGU UGGC GCCUC AUCUU UUUCAG C UUU UUUUCC GG CCC CUU CUC GG CAG AGCUG CUGG AAAAG GU CCAAGG CUAA GUG AGC CCG AG CUUG CG UCU A G A C U U C G GC C UU G G A CG C U C A A AG A C C C GA C CC C C G G C C C UU U C G C UC AU GA AU C C AA

363

340

320

300

280

260

240

220

200

Figure 28.1 (A) The proposed sequence and secondary structure of AGVd. Boxed residues are the upper and lower central conserved regions of the ASSVd group. The pathogenicity (P) domain and the terminal conserved sequence (TCR) are identified. The arrows show the sequences which can form a palindromic structure. (B) The proposed sequence and secondary structure of GYSVd-1. T1 and T2 represent terminal domains, P represents the pathogenicity region, V the variable region, and CCR the central conserved region. The area spanned by the arrows indicates sequences which are not conserved between GYSVd and ASSVd, but in each case contribute to the formation of the stem loop structure. (C) The proposed sequence and secondary structure of GYSVd-2. Positions of the five domains are indicated. Arrows are drawn above the inverted repeat residues which flank the CCR (boxed in solid lines). Dotted boxes contain residues which are also conserved in ASSVd and GYSVd-1. Flags indicate the borders of the GYSVd-2 sequence which is almost identical to that found in TPMVd.

Genome sequence

Partial sequencing of GYSVd-1 was achieved by the direct enzymic method and generated three non-overlapping fragments of sequence (Koltunow and Rezaian 1988). However, the identity of some of the residues in each fragment was not certain, a problem inherent in direct RNA sequencing. Data obtained from the enzymic sequencing was used to synthesize specific primers for the construction of full-length cDNA clones. The complete nucleotide sequence of GYSVd-1 and its proposed secondary structure is presented in Figure 28.1B, which shows GYSVd-1 can potentially form a rod-like structure with a high degree of base pairing which is characteristic of viroids. In total, 62% of the residues are base paired and the paired residues consist of 53% G:C, 26% A:U and 21% G:U. The G+C content is 60%, which is comparable to that of other viroids (Keese and Symons 1987). The GYSVd-1 sequence was analyzed for putative translation products in both the plus and minus strands using AUG as the possible initiation codon. No AUG codons are present in the minus strand and the single

AUG codon present in the plus strand (residues 254–256) is followed by a reading frame potentially encoding a polypeptide of 68 amino acids. The significance of this putative translation product is not known. Structure

GYSVd-1 has 37% nucleotide sequence homology with Apple scar skin viroid (ASSVd). The homologous residues in ASSVd and GYSVd-1 are not randomly distributed, but occur as blocks of base paired residues in the secondary structures of both viroids. There is also some homology between GYSVd-1, ASSVd and members of the genus Pospiviroid, which is limited to three blocks of residues in the T1 domain, A-rich region of the pathogenicity (P) domain and the U-rich region of the P domain (Koltunow and Rezaian 1988). The sequence comparisons between GYSVd-1, ASSVd and the Pospiviroid genus show that GYSVd-1 structure conforms to the viroid domain model proposed by Keese and Symons (1985).

197


There is a stretch of 17 nucleotide residues in the T1 region of the PSTVd group, which is also found in a similar position of the proposed secondary structure of GYSVd-1 (Position 17 to 34). No sequence in the proposed T2 region is shared with viroids of the PSTVd group, but the region is defined by stretches of sequence common to GYSVd and ASSVd. A characteristic of the PSTVd group P domain is the presence of an adenine dominated oligopurine sequence of 15 to 17 residues on one strand and an oligo (U4–7) sequence on the opposite strand (Keese and Symons 1987). There is a 19-residue oligopurine sequence in GYSVd-1 located between residues 61 and 90 with an adenine rich portion lying between residues 63 and 73. In the proposed secondary structure of GYSVd-1, the adenine rich sequence base pairs strongly with an oligo U sequence in the opposite strand (residues 299–309). This structural feature is also present in ASSVd (Hashimoto and Koganezawa 1987). A portion of the U rich sequence found in the GYSVd-1 is also present in ASSVd and members of the PSTVd group. GYSVd-1, like ASSVd, did not contain the central conserved region (CCR) sequence common to all of the other viroids reported previously. However, these two viroids share a sequence of 16 residues in a structural location corresponding to the PSTVd CCR. These sequences are potentially involved in the formation of two structures, which resemble those on the CCR of the genus Pospiviroid (Koltunow and Rezaian 1988; Keese and Symons 1985, 1987). The upper portion of the CCR of GYSVd-1 and ASSVd can potentially assume a stem loop conformation with the 16 perfectly conserved bases capping the top of the structure. Residues flanking the conserved sequences contribute to the stem in both GYSVd-1 and ASSVd. A remarkably similar structure can also be found in the CCR of all the genus Pospiviroid (Keese and Symons 1985). It has been postulated that a stem loop structure is involved in the transition between the native viroid structure and structures important in viroids replication (Keese and Symons 1985). The finding of a common CCR in GYSVd-1 and ASSVd led to the identification of a new viroid genus represented by ASSVd. In GYSVd-1, 36 nucleotide residues of the CCR are involved in the formation of the stem loop structure. These 36 residues also have the potential to form a stable palindromic duplex with another GYSVd-1 molecule in linear form. Alternatively, the palindromic duplex structure could be formed within multimeric GYSVd-1 molecules. The 16 residues perfectly conserved between ASSVd and GYSVd-1 form the central core of the duplex. ASSVd also has the potential to form a palindromic structure (Hashimoto and Koganezawa 1987). Similar structures have been postulated for oligomers of all the pospiviroids (Diener 1986). It was also proposed that the stable duplex may

198

be involved in the processing of oligomeric viroid intermediates into monomeric progeny (Diener 1986). In most of the members of the genus Pospiviroid, the V region is characterized by an oligopurine:oligopyrimidine helix with a minimum of 3 G:C base pairs (Keese and Symons 1987). In the proposed secondary structure of GYSVd-1 (Figure 28.1B), the region between residues 122 and 145 has short stretches of oligopurine:oligopyrimidine helices with a minimum of 2 G:C base pairs. Sequence variation

There is considerable variation in the intensity of yellow speckle symptom expression, which may relate to the grapevine cultivar, plant age and environmental factors. Examples of this variation are cultivars that exhibit yellow speckle symptoms in Australia, but are symptomless in California (Taylor and Woodham 1972). The existence of GYSVd-1 sequence variants may play a role in variations of yellow speckle disease symptoms. In the initial cloning of GYSVd-1 (Koltunow and Rezaian 1988) partial length cDNA clones contained residue changes suggesting that the RNA population was heterogeneous. Subsequently, the majority of sequence variation in GYSVd-1 was found to occur in the pathogenicity domain of the viroid (Rigden and Rezaian 1993). The GYSVd-1 variants could be divided into two types each containing a distinct secondary structure within the P domain. Monomeric exact-length RNA transcripts produced from individual clones belonging to both types were infectious in viroid-free grapevines and produced homogenous populations of progeny viroids in vivo (Rigden and Rezaian 1993). General surveys of viroid variant composition in a vineyard and within single plants in Germany showed the independent evolution of GYSVd-1 variants (Polivka et al. 1996). While the GYSVd-1 population was variable, two prominent groups of sequence variants, type 1 and type 2, could be identified (Rigden and Rezaian 1993). To determine if the variants of GYSVd-1 were responsible for the variability of symptom expression observed in the field, the specific biological activity of each type variant was investigated (Szychowski et al. 1998). The Australian ‘type 1’ variant has been established as the non-symptom-inducing form of GYSVd-1 and ‘type 2’ as the symptom-inducing variant (Szychowski et al. 1998). Szychowski et al. (1998) also identified a ‘type 3’ variant from Italy, which induces distinct symptoms. So far the sequence of 35 variants of GYSV-1, ranging from 366 nt to 369 nt, has been reported (http://nt.ars-grin.gov/subviral/viroids/gysv1.html). The occurrence of new variants of GYSV-1 is of special interest because the nature of variation as well as the combination of variants present may influence yellow speckle symptom expression.

GRAPEVINE VIROIDS

GRAPEVINE YELLOW SPECKLE VIROID 2 (GYSVd-2) Family

Pospiviroidae Genus

Apscaviroid Original source

Grapevine yellow speckle viroid 2 was purified from the grapevine cultivar ‘Kyoto’ grafted onto ‘Dogridge’ rootstock, infected with yellow speckle disease. The viroid could be resolved from other grapevine viroids of similar size only after prolonged electrophoresis under denaturing conditions (Koltunow and Rezaian 1989a). Genome sequence

Partial length cDNA clones were made by the RNase H method (Gubler and Hoffman 1983). The complete sequence of GYSVd-2 was determined by the overlapping sequence of four cDNA clones (Koltunow and Rezaian 1989a). The sequence of GYSVd-2 and its proposed secondary structure is presented in Figure 28.1C. GYSVd-2 contains 363 nucleotide residues consisting of 68 A, 77 U, 106 G and 112 C. In total, 67% of the nucleotides are base-paired. The bases paired, expressed as a percentage of the total, include 33% A:U, 55% G:C and 12% G:U base pairs. GYSVd-2 is most closely related to GYSVd-1 showing 73% overall sequence similarity, which accounts for the cross-hybridisation which has been observed between the two RNAs (Koltunow and Rezaian 1988). Structure

The structure of GYSVd-2 is consistent with the domain model proposed by Keese and Symons (1985) in that the CCR, P, and T1 domains can be defined easily (see Figure 28.1C). The existence of a V domain is debatable because an oligopurine:oligopyrimidine helix containing a minimum of 3 G:C base pairs as defined by Keese and Symons (1985) is not evident in the usual position between the CCR and T2 regions. The P domain of GYSVd-2 is purine rich in the upper strand and uridine rich in the bottom strand, which results in a relatively high sequence similarity when the P domain of GYSVd-2 is compared with the P domains of other viroids (Koltunow and Rezaian 1989a). GYSVd-2 contains the central conserved sequence of the Apscaviroid genus consisting of an inverted repeat sequence (Figure 28.1C) (Koltunow and Rezaian 1988; Hashimoto and Koganezawa 1987). An additional 10-base inverted repeat sequence flanks the core conserved central sequence. This inverted repeat sequence is very similar to the 10-base sequence, which flanks the conserved central sequence of GYSVd-1. However, the A residue found at position 87 of the GYSVd-2

sequence and the U residue found at position 116 of GYSVd-2 sequence has been altered in GYSVd-1 to a G and a C residue respectively (Koltunow and Rezaian 1988, 1989a). ASSVd has an 8-base inverted repeat sequence flanking the conserved central sequence. The inverted repeat sequence in ASSVd bears little resemblance to the inverted repeat sequence in GYSVd-1 and GYSVd-2 (Koltunow and Rezaian 1988, 1989a; Hashimoto and Koganezawa 1987). The T2 domain of GYSVd-2 generally exhibits little sequence homology with the T2 domains of other viroids. However, there is some sequence conservation among the T2 domains of GYSVd-1, GYSVd-2 and ASSVd. Residues 170 to 185 in the terminal portion of T2 region of GYSVd-2 are repeated in the terminal portion of the T1 region (residues 351 to 6) (Figure 28.1C). The repeat in the T1 region is imperfect because 3 additional residues are present in positions 356, 357 and 5 of the GYSVd-2 sequence. A U-rich region flanks the 3´ end of each repeat sequence. A direct repeat (Figure 28.1C) at the termini of the T regions is not evident in other viroids. There is a block of sequence in the T1 region of GYSVd-2 (Figure 28.1C), which is conserved and found in a similar location in both GYSVd-1 and ASSVd (Koltunow and Rezaian 1989b). Residues 18 to 30 (Figure 28.1C) are also conserved in a number of the PSTVdlike viroids (Koltunow and Rezaian 1988). GYSVd-2 has 49% sequence similarity with Tomato planta macho viroid (TPMVd). The high sequence similarity between TPMVd and GYSVd-2 is primarily because a sequence of 69 nucleotide residues in the left hand end of GYSVd-2 is extremely similar to a corresponding region in the left hand end of TPMVd. In general, the T1 domain of GYSVd-2 has high sequence similarity with the T1 domains of viroids in the Pospiviroid genus (Koltunow and Rezaian 1989a), namely TPMVd, CEVd and PSTVd, providing additional evidence for the importance of RNA recombination in viroid evolution. Genus

GYSVd-2 contains sequences in its central region identical to those found in GYSVd-1 and ASSVd, which form the Apscaviroid genus (Koltunow and Rezaian 1988, 1989a). The CCR of GYSVd-2, like those of GYSVd-1 and ASSVd, is quite distinct from those found in the genus Pospiviroid. This provided further support for the classification of these viroids in the genus Apscaviroid (Koltunow and Rezaian 1988, 1989b). Sequence variation

Sequence heterogeneity was observed among the cDNA clones of GYSVd-2. The changes were A→G at positions 300 and 328, C→U at position 344, U→C at position 360, and the deletion at position 8 (Koltunow and Rezaian 1989a).

199


SYNERGISM OF YELLOW SPECKLE VIROIDS WITH GRAPEVINE FANLEAF VIRUS (GFLV) Vein-banding disease of the grapevine is common in Europe and California. Viroid-free Vitis vinifera cultivars ‘Cabernet Sauvignon’ and ‘Sauvignon Blanc’ were established in controlled field trials to evaluate the relationship between grapevine viroids and Grapevine fanleaf virus (GFLV) for the induction of the vein-banding disease (Szychowski et al. 1995). Vein-banding symptoms were observed on vines, which contained GYSVd-1, GYSVd-2, HSVd together with GFLV. ‘Sauvignon Blanc’ vines, which contained only HSVd and GFLV, were non-symptomatic for vein-banding indicating an absence of correlation between HSVd and this disease. Vein-banding symptoms only occurred when GFLV was combined with either GYSV-1 or GYSV-2. The intensity of vein-banding symptoms was directly correlated with the enhanced titre of GYSVd-1 and GYSVd-2 (Szychowski et al. 1995). Grapevines without disease or expressing either vein-banding or yellow speckle symptoms in Italy all contained both GYSVd-1 and HSVd (Szychowski et al. 1995). However, symptomatic vines displayed a higher titre of GYSVd-1 than non-symptomatic materials and vein-banding symptomatic vines were GFLV-infected. These data demonstrated that expression of vein-banding disease is induced by a synergistic reaction between Grapevine yellow speckle viroids 1 and 2, and a virus, GFLV (Szychowski et al. 1995) confirming the same observations by biological indexing (Krake and Woodham 1983).

Figure 28.2 Sequence relationship between AGVd and other viroids. Boxes denote regions of high similarity. For clarity, the homologous boxes occurring in AGVd and in other viroids are shown with the same shades and identified with the same numbers. The degrees of sequence similarities are given in Table 28.2.

Pospiviroidae

sequence similarity with any previously known viroids (Rezaian 1990). Nevertheless its entire sequence can be divided into regions, each with high sequence similarity with segments from one of CEVd, PSTVd, ASSVd, and GYSV-1 and 2 (See Figure 28.2 and Table 28.2). This suggests that AGVd has originated from extensive RNA recombination involving other viroids. The vegetatively propagated grapevines, which have been exposed to multiple viroid infections during their long history of cultivation, may have allowed such recombination. The nucleotide sequence of AGVd and its proposed secondary structure are shown in Figure 28.1A. AGVd consists of 103 C (27.9%), 111 G (30.1%), 76 A (20.6%) and 79 U (21.4%). The AGVd sequence was analyzed for the presence of putative reading frames in both the plus and minus strand using AUG and GUG as possible initiation codons. The only AUG codon is present on the plus strand (residue 141–143) and is followed by nine nucleotides before a stop codon. The largest GUG initiated reading frames in the plus strand (residue 337–49) and in the minus strand (residues 337–60) can potentially encode peptides of 27 and 31 amino acid residues respectively. Like other viroids, AGVd does not appear to encode any protein.

Genus

Structure

Apscaviroid

The primary sequence of AGVd was arranged to allow for the highest degree of base pairing. The resulting structure is rod-like and typical of viroids. Overall, 69% of the bases are paired and the paired residues consist of 54.7% GC, 29.7% AU and 15.6% GU pairs. Compared to the other viroids of the genus Apscaviroid, to which it belongs (see genus section), AGVd contains a larger number of uninterrupted base pair stretches and presumably a more stable secondary structure.

AUSTRALIAN GRAPEVINE VIROID (AGVd) Family

Original source

Australian grapevine viroid was isolated from grapevines, which also contained GYSVd-1, GYSVd-2, CEVd-g and HSVd-g (Rezaian et al. 1988). It was distinguished from these four viroids by a combination of its electrophoretic properties, ability to replicate in cucumber and in tomato and its lack of hybridisation to other viroid probes. Genome sequence

AGVd was partially sequenced by direct enzymic sequencing of end-labeled RNA fragments obtained by digestion of the viroid with nuclease T1. The sequence of 268 nucleotide residues of AGVd was determined by this procedure. To complete the sequence and to resolve ambiguities in the identification of C and U residues resulting from the direct enzymic sequencing, cDNA clones of AGVd were constructed and sequenced (Rezaian 1990). This 369-nucleotide viroid had less than 50%

200

The CCR of AGVd is the same as that in ASSVd (Rezaian 1990), GYSVd-1 (Koltunow and Rezaian 1989a) and GYSVd2 (Koltunow and Rezaian 1989b). These conserved sequences consist of 16 and 18 residues, respectively, in the lower part and the upper part of the secondary structure of AGVd (boxed in Figure 28.1A). The upper conserved sequence of AGVd and flanking 13 residues on either side (42 residues total) has the potential to base pair with the same sequence of 42 residues present in another AGVd molecule or in another region of a multimeric AGVd molecule to form a palindromic structure. In

GRAPEVINE VIROIDS

Table 28.2

Sequence similarities of different regions of AGVd with other viroids.

Region* number 1 2 3 5 6 7 8 9 10 11

AGVd sequence similarity with ASSVd ASSVd CEVd CEVd PSTVd ASSVd CEVd GYSVd-1 CEVd ASSVd

Position in AGVd

Length of sequence

% similarity

82–123 126–161 162–171 190–227 232–255 262–279 292–34 35–43 46–61 65–81

42 36 10 38 24 18 112 9 16 17

69 52 100 63 79 100 69 100 82 71

*The regions of AGVd are identified in Figure 28.2. The region 4, which consists of only a homologous quadruple, starting at the position 180 of AGVd, is not included.

AGVd, sequences flanking the 16 residue core sequence are similar to those of ASSVd except that there are two five residue insertions in symmetrical positions on either side of the core sequence thus allowing the formation of a longer palindrome than in ASSVd. The 42-residue sequence of AGVd and the corresponding residues in the lower strand can also be folded to a cruciform structure. An A rich sequence of 18 nucleotides (containing 14 A residues) paired with a U rich sequence of 19 nucleotides (containing 15 U residues) is present to this left of the CCR. This sequence resembles the pathogenicity (P) domains of other viroids (Keese et al. 1988). The sequence AAAGAAAA found in the P domains of most viroids of Pospiviroid genus (Keese et al. 1988) is present in AGVd (residues 53–61). Compared to the P domains of other viroids the corresponding region of AGVd is more regularly base paired and contains a single loop. With PSTVd, it has been found that increasing stability of the base pairing of the sequences in the P domain correlates with decreasing virulence (Schnolzer et al. 1985). The left terminal region of AGVd contains a sequence of 17 nucleotides (residues 11–27), which is conserved in identical or similar positions of most other viroids. The role of this terminal conserved region (TCR) in viroid replication and pathogenesis is unknown. Genus

AGVd has the highest overall sequence similarity of 49% with both ASSVd (prototype of this genus) and CEVd (a member of the genus Pospiviroid). It cannot therefore be assigned to a genus on the basis of sequence similarity. However, AGVd contains the entire central conserved region of the genus Apscaviroid and on this basis, it was proposed as a member of this genus (Rezaian 1990).

Sequence variation

A single sequence variation was detected in a cDNA clone in which the residue at position 55 was deleted. It is possible that the passage of AGVd through cucumber for viroid purification has had a filtering effect in removing sequence variants, as two other viroids, GYSVd-1 and GYSVd-2, purified directly from grapevine show frequent sequence variations (Koltunow and Rezaian 1989a, b).

HOP STUNT VIROID (HSVd) Family

Pospiviroidae Genus

Hostuviroid Original source

As early as 1952, hop plants showing a stunting syndrome were observed in Japan (Yamamoto et al. 1970). Sasaki and Shikata (1977) established a biological assay using cucumber as an indicator and provided evidence that hop stunt was a viroid-incited disease. The complete cDNA of HSVd was cloned and HSVd was sequenced (Ohno et al. 1983). A cucumber isolate of HSVd (HSVd-c), previously recognized as Cucumber pale fruit viroid in Holland (van Dorst and Peters 1974), was shown to be biologically indistinguishable from HSVd and its sequence was closely related to HSVd (Sano et al. 1984). An infectious lowmolecular weight RNA was detected in grapevines by molecular hybridization with HSVd-c cDNA (Shikata et al. 1984). The RNA, HSVd-g, produced symptoms on cucumber plants indistinguishable from those infected with HSVd and HSVd-c. HSVd-g was detected in almost all grapevines collected in Japan, including stocks imported from the United States and Europe (Sano et al. 1985).

201


T1

A. CEVd-g

P

20

1

40

CCR 60

80

V

100

T2 140

120

160

180

A A G UG A A A G A A G C A CU G A U U A C U GC G C C G A G A G AA UC G A G C C A U A U U G CG C C U G A C C C GG UCU UCU GGUUC GGU UCACCU CCUG AGGA AAGA AGAGGCGGC GGGG GAAG CUUCAG UCC CCGGG CUGGAG GAAGU GG CGGGGGGG AC GCUU GGCGGC CGGAU UGG CAGCGG GAAA AGGAC U G U CC GGG AGA CCAAG CCG AGUGGG GGGC UCUU UUCU UUUUCGCCG CCCC UUUC GAAGUC AGG GGCCC GGUCUC CUUCG CC GCUCCCCC UG CGAA UCGCUG GCCUA ACC GUCGUC CUUU UCCUG G C A UU UC C C AA A C C G C A C AA AA A C U A A U A G G U G C A C U G G C U U CC C U A U U U A U G GG CG U C C C AU C CA

369

B. HSVd-g

340

320

300

T1

P

20

1 U GGGG

AU A U

U CUCGAG

U

280

A

40

UC AA G A A A AA A A CCGC GGCA GC AGA ACA

AGGCAGG

260

240

60

80

GA U UU U AA A GG AC ACC GAGA GG

200

V

CCR G

220

100

CU A C G A U GCC CCGG GC UCUCA

GAA

U

T2

120 CG C C CCAG AGAGG GUGGAGA GA GGG CG

140

CG

GUG

C

U A UC GGA GUAG G G

C U

CCCC

GAGUUC

GGCG A

U C U U C C

297

280

CCGU

C C U

CG

UCU AG A

UGU UUCGUCC CC UG UGG CUCU C UU AA C CU U

260

240

CC

CGG GGCC CG A A U AG CU

C

AGAGU CUU GGUC UCUUC CAUUUCU CU CCC GC GC CAC AG CUU CGUC C A C C C U U U U UA U C AA C U G U A

220

200

180

160

Figure 28.3 (A) The proposed sequence and secondary structure of CEVd-g. Positions of the five domains are indicated, the P and V domains being boxed. Nucleotide residues differing from the sequences of CEVd-A(_) or CEVd–DE26 (°) are marked. (B) The proposed sequence and secondary structure of HSVd-g. Single nucleotide differing from HSVd is indicated by arrow.

Genome sequence

Occurrence

The HSVd-g RNA consists of 297 nucleotides (Sano et al. 1985), and differs from the previously published HSVd sequence (Ohno et al. 1983) by only one nucleotide at position 54 (A→G). The single nucleotide change was confirmed in an independent isolation of HSVd-g from grapevines in Australia (Rezaian et al, 1988). HSVd-g is 6 nucleotides smaller than HSVd-c and 15 nucleotides different. The sequence of HSVdg and its proposed secondary structure is presented in Figure 28.3B.

The Japanese commercial hop was first introduced in 1875 from the United States and Germany. Since then, most of the hop varieties grown in Japan were obtained from the United States or European countries. Hop stunt disease, however, has not been reported to occur in these countries, despite the presence of HSVd and the cucumber isolate of HSVd (Sano et al. 1985; van Dorst and Peters 1974). Thus the disease appears to be specific to Japan. HSVd-g has been reported in grapevine cultivars and rootstocks in Australia, the United States and Europe (Koltunow et al. 1988; Semancik et al. 1987; Sano et al. 1986). This suggests that HSVd-g is probably distributed worldwide in grapevines. The significance of HSVd-g infection in grapevines may lie in the potential for the viroid to infect hops.

Structure

A possible secondary structure model for the HSVd-g was proposed (Sano et al. 1985). It provides maximum sequence and structural homology with the proposed secondary structures of other viroids. In total, 67% of the nucleotides are base-paired resulting in a rod-like native structure. The bases paired, expressed as a percentage of the total, include 29% A:U, 64% G:C and 7% G:U base pairs. HSVd-g shares essentially all structural features of HSV (Ohno et al. 1983) including the presence of a PSTVd CCR, a U1a RNA related sequence and an apparent lack of viroid-encoded translation products either in vitro or in vivo (Ohno et al. 1983). Disease symptoms

Infected hop plants have a stunted appearance because of internode shortening in the main and lateral shoots. The disease symptoms also include a downward curling of the upper leaves, a decrease in laminae size and yellowing of the leaves (Yamamoto et al. 1973). No disease symptoms have been observed in grapevines as a result of HSVd-g infection.

202

CITRUS EXOCORTIS VIROID (CEVd) Family

Pospiviroidae Genus

Pospiviroid Original source

Fawcett and Klotz (1948) first described citrus exocortis symptoms. Subsequent indexing for exocortis was based on trunk symptoms on trees grown in the field. The severe isolates were used for transmission to herbaceous species and Gynura aurantiaca was found as an indicator host (Weathers et al. 1967). The causal agent of the disease was isolated and characterized as a low molecular weight infectious RNA and named CEVd (Semancik and Weathers 1972). CEVd infects many citrus species and

GRAPEVINE VIROIDS

cultivars and some citrus relatives. Flores et al. (1985) isolated a viroid from a symptomless grapevine in Spain, which was shown to have 89% to 92% sequence homology with CEVd (GarcíaArenal et al. 1987). CEVd-g has also been isolated from symptomless grapevines in Australia (Rezaian et al. 1988).

82(U/A), 108 (G/C), 317 (U/A) and 319-321 (ACU/GUA). The U at position 317 occurs together with ACU at positions 319–321, while the A at position 317 occurs with GUA at 319–321. Occurrence

Genome sequence

The complete nucleotide sequence of CEVd-g has been determined by the partial RNA digestion method (García-Arenal et al. 1987). The CEVd-g molecule is a circular RNA consisting of 369 nucleotides. The sequence of CEVd-g and its proposed secondary structure is presented in Figure 28.3A. In total, 66% of the nucleotides are base-paired. The bases paired, expressed as a percentage of the total, include 18% A:U, 73% G:C and 9% G:U base pairs.

CEVd is present in most, if not all, citrus-growing countries. Its incidence in countries such as the United States, Australia, South America, and the Mediterranean region has been high, but its impact has diminished in countries with established pathogen testing schemes. CEVd-g has only been reported from grapevines in Spain (García-Arenal et al. 1987), Australia (Rezaian et al. 1988) and in California (Semancik and Szychowski 1992).

TRANSMISSION AND SPREAD OF GRAPEVINE VIROIDS Structure

A secondary structure model of CEVd-g has been proposed (García-Arenal et al. 1987) and adjusted to provide maximum sequence and structural homology with the one proposed for CEVd-A (Visvader et al. 1982). The percentage of base-paired residues for CEVd-g and CEVd-A is 66% and 67%, respectively. In the secondary structure of CEVd-g (Figure 28.3A) the pathogenicity (P) and variable (V) domains are boxed as defined by Keese and Symons (1985), and residues differing from severe isolate CEVd-A and mild isolate CEVd-DE26 have been marked. Differences with respect to both CEVd type sequences occur throughout the molecule and although many of these are concentrated in the P and V domains, differences in the T1 and T2 left and right terminal regions also occur. No AUG starting open reading frame (ORF) is found in the CEVd-g plus strand, but there is one in the minus strand coding potentially for a 41 amino acid peptide (García-Arenal et al. 1987). Five GUG initiation codons exist in each of the plus and minus strand. An ORF starting with GUG at residue 337 and ending with a UGA at residue 13 of the plus strand is also found in all the sequenced CEVd isolates (Visvader and Symons 1985). Genus

CEVd-g shows extensive homology with viroids in the genus Pospiviroid: 62% with PSTVd, 63% with Chrysanthemum stunt viroid, 65% with Tomato planta macho viroid and 74% with Tomato apical stunt viroid (García-Arenal et al. 1987). The homology with Class B sequences of CEVd found in mild isolates (CEVd-DE26) is 89% (Visvader and Symons 1983) and with Class A sequences typical of isolates inducing severe symptoms in tomato (CEVd-A) is 92% (Visvader et al. 1982). Sequence variation

As has been found with other viroids, the CEVd-g isolate was found to consist of a number of sequence variants (GarcíaArenal et al. 1987). The heterogeneity is found at residues

Two possible sources responsible for the ubiquitous occurrence and distribution of viroids in grapevines have been identified (Szychowski et al. 1988). Mechanical inoculation among vines by contaminated cutting tools during the processes of vine management and propagation may play a significant role in the field spread of viroids (Szychowski et al. 1988). Viroid spread in other woody plant species has been observed and it has been demonstrated that sodium hypochlorite or formaldehyde can be used for effective decontamination of pruning tools (Garnsey and Jones 1967; Roistacher et al. 1969; Wutscher and Schull 1975). Systemic transmission between grafted rootstocks and scion varieties containing grapevine viroids has also been demonstrated (Szychowski et al. 1988). Staub et al. (1995) looked at viroid distribution patterns within a vineyard and in different rootstock clones, which are routinely used for grafting in Germany, allowing an insight into the mechanisms of viroid spread in the field. They concluded that mechanical transmission observed by Szychowski et al. (1988) through pruning tools may occur, however, this method of viroid spread plays a minor role in the field (Staub et al. 1995) and viroid spread in grapevines mainly occurrs through systemic transmission upon grafting. Until recently, grape seeds have generally been considered as viroid-free germplasm, with grape seedlings derived from viroid-infected sources reported to be free from infection (Taylor and Woodham 1972; Semancik et al. 1987; Koltunow and Rezaian 1988; Koltunow et al. 1988; Minafra et al. 1990). However, viroids have previously been found in grape seedlings (Shanmuganathan and Fletcher 1980; Koltunow et al. 1988), but their presence was interpreted as evidence for the spread of viroids in the field. Recently, four grapevine viroids have been found in two ‘Emperor’ table grape seedlings by the more sensitive reverse transcription polymerase chain reaction (RTPCR) (Wan Chow Wah and Symons 1997). These were GYSVd-1, GYSVd-2, CEVd-g and AGVd. The transmission of GYSVd-1 and HSVd-g via seeds was later confirmed in eleven

203


seedlings of eight grapevine varieties using a combination of RT-PCR, dot-blot and northern-blot hybridization (Wan Chow Wah and Symons 1999). The viroid profiles found in nine seedlings indicated a differential reduction of viroids transmitted via seeds similar to the differential reduction of viroids observed in vines regenerated after shoot apical meristem culture (Wan Chow Wah and Symons 1997, 1999). Given that viroids are widespread in all grapevine rootstocks and varieties tested, evaluation of the effect of viroids on the growth and productivity of grapevines, as well as on grape quality, relies on the availability of viroid-free grapevines. Tissue culture techniques have been used to provide viroid-free grapevines. Grapevine shoot tips (0.1–0.2 mm in length) from a viroid-infected plant containing one or two leaf primordia were cultured in vitro (Duran-Vila et al. 1988). They developed into multipleshoots. These shoots were rooted easily and produced whole plantlets that were successfully transplanted to soil. Analysis of nucleic acid by sequential polyacrylamide gel electrophoresis showed that all shoot tip recovered plants were viroid-free, in contrast with the mother plant, which contained two viroid-like RNAs. The micro-propagation system ensured the availability of large numbers of viroid-free grapevines when needed (DuranVila et al. 1988). The efficacy of viroid removal by tissue culture techniques was also evaluated by Wan Chow Wah and Symons (1997). They optimized the viroid extraction and diagnostic methods to detect viroids in low copy number. The level of viroids in vines regenerated by shoot apical meristem culture and fragmented shoot apex culture were tested. The data indicated a differential reduction of viroids, rather than viroid elimination, in the regenerated vines (Wan Chow Wah and Symons 1997).

VIROID INFECTIVITY Infectious viroid cDNA clones (Cress et al. 1983) have been widely used to test infectivity of viroids (Owens and Hammond 1987). The infectivity of DNA clones and their respective transcripts depends generally on the presence of longer than unit length viroid sequence in the DNA constructs (Tabler and Sänger 1984; Visvader et al. 1985) although certain monomeric clones are also infectious (Tabler and Sänger 1984; Visvader et al. 1985). Given the common occurrence of viroid mixed infections in grapevines, it is impractical to reliably isolate individual viroids in pure form by analytical means. Using in vitro synthesis it is possible to produce pure viroid samples without the risk of contamination from other viroids. This technique has been applied to grapevine viroids GYSVd-1 and GYSVd-2 (Koltunow et al. 1989a). A full-length cDNA of each viroid cloned downstream of a SP6 promoter and upstream of the T7 promoter, allowed positive and negative sense RNA transcripts to be synthesized using SP6 and T7 RNA polymerase, respectively. The plus and minus sense dimeric transcripts were used for

204

infectivity studies and allowed Koltunow et al. (1989) to prove Koch’s postulates for GYSVd-1 and GYSVd-2 inducing yellow speckle disease symptoms. A simpler method reported by Rigden and Rezaian (1992), describes in vitro synthesis of infectious viroids without resorting to cloning procedures. This procedure involves the synthesis and amplification of double stranded cDNA fused with a T7 RNA polymerase promoter. This is followed by transcription of the DNA resulting in the production of an infectious linear viroid monomer. This procedure also revealed that a 2´, 3´cyclic phosphate terminus is not a prerequisite for viroid infectivity as previously suggested (Hashimoto et al. 1985). The linear RNA could be circularized using the T4 RNA ligase to produce an authentic viroid molecule. This procedure has been used to produce infectious GYSVd-1 sequence variants (Rigden and Rezaian 1993). The use of this in vitro system combined with viroid-free grapevine and highly sensitive diagnostic methods would allow future examination of grapevine viroid pathogenesis involving variants and viroid combinations. Acknowledgements

We thank Anna Koltunow, Akbar Behjatnia and Nuredin Habili for reviewing the manuscript and Les Krake for the picture of vein banding symptoms. References Bovey, R., and Martelli, G.P. (1992). Directory of major virus and virus-like diseases of grapevines. Editions Payot: Lausanne. Cress, D., Kiefer, M.C., and Owens, R.A. (1983). Construction of infectious potato spindle tuber viroid cDNA clones. Nucleic Acids Res. 11, 6821-6835. Diener, T.O. (1986). Viroid processing: a model involving the central conserved region and hairpin I. Proc. Natl. Acad. Sci. USA 83, 58-62. Duran-Vila, N., Juárez, J., and Arregui, J.M. (1988). Production of viroidfree grapevines by shoot tip culture. Am. J. Enol. Viticult. 39, 217-220. Fawcett, H.S., and Klotz, L.J. (1948). Exocortis on trifoliate orange. Citrus Leaves 28, 8. Flores, R., Duran-Vila, N., Pállas, V., and Semancik, J.S. (1985). Detection of viroid and viroid-like RNAs from grapevines. J. Gen. Virol. 66, 2095-2102. Garciá-Arenal, F., Pallás, V., and Flores, R. (1987). The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid. Nucleic Acids Res. 15, 4203-4210. Garnsey, S.M., and Jones, J.W. (1967). Mechanical transmission of exocortis virus with contaminated budding tools. Plant Dis. Rep. 51, 410-413. Gubler, U., and Hoffman, B.J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. Hashimoto, J., Suzuki, K., and Uchida, T. (1985). Infectivity of artificially nicked viroid molecules. J. Gen. Virol. 66, 1545-1551. Hashimoto, J., and Koganezawa, H. (1987). Nucleotide sequence and secondary structure of apple scar skin viroid. Nucleic Acids Res. 15, 7045-7052.

GRAPEVINE VIROIDS

Keese, P., and Symons, R.H. (1985). Domains in viroids: Evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proc. Natl. Acad. Sci. USA 82, 4582-4586. Keese, P., and Symons, R.H. (1987). Physical-chemical properties: molecular structure (primary and secondary). Pages 37-62 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Keese, P., Osorio-Keese, M.E., and Symons, R.H. (1988). Coconut tinangaja viroid: Sequence homology with coconut cadang-cadang viroid and other potato spindle tuber viroid related RNAs. Virology 162, 508-510. Koltunow, A.M., and Rezaian, M.A. (1988). Grapevine yellow speckle viroid: structural features of a new viroid group. Nucleic Acids Res. 16, 849-864. Koltunow, A.M., and Rezaian, M.A. (1989a). Grapevine viroid 1B, a new member of the apple scar skin viroid group contains the left terminal region of tomato planto macho viroid. Virology 170, 575578. Koltunow, A.M., and Rezaian, M.A. (1989b). A scheme for viroid classification. Intervirology 30, 194-204. Koltunow, A.M., Krake, L.R., and Rezaian, M.A. (1988). Hop stunt viroid in Australian grapevine cultivars: potential for hop infection. Aust. Plant Pathol. 17, 7-10. Koltunow, A.M., Krake, L.R., Johnson, S.D., and Rezaian, M.A. (1989). Two related viroids cause grapevine yellow speckle disease independently. J. Gen. Virol. 70, 3411-3419. Krake, L.R., and Woodham, R.C. (1983). Grapevine yellow speckle agent implicated in the aetiology of vein banding disease. Vitis 22, 40-50. Minafra, A., Martelli, G.P., and Savino, V. (1990). Viroids of grapevine in Italy. Vitis 29, 173-182. Ohno, T., Takamatsu, N., Meshi, T., and Okada, Y. (1983). Hop stunt viroid: Molecular cloning and nucleotide sequence of the complete cDNA copy. Nucleic Acids Res. 11, 6185-6197. Owens, R.A., and Hammond, R.W. (1987). Molecular biology of viroid-host interactions. Pages 167-188 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Polivka, H., Staub, U., and Gross, H.J. (1996). Variation of viroid profiles in individual grapevine plants: Novel grapevine yellow speckle viroid 1 mutants show alterations in hairpin I. J. Gen. Virol. 77, 155-161. Rezaian, M.A. (1990). Australian grapevine viroid – evidence for extensive recombination between viroids. Nucleic Acids Res. 10, 5587-5598. Rezaian, M.A., Koltunow, A.M., and Krake, L.R. (1988). Isolation of three viroids and a circular RNA from grapevines. J. Gen. Virol. 69, 413-422. Rigden, J.E., and Rezaian, M.A. (1992). In vitro synthesis of an infectious viroid: Analysis of the infectivity of monomeric linear CEV. Virology 186, 201-206. Rigden, J.E., and Rezaian, M.A. (1993). Analysis of sequence variation in grapevine yellow speckle viroid 1 reveals two distinct alternative structures for the pathogenic domain. Virology 193, 474-477. Roistacher, C.N., Calavan, E.C., and Blue, R.L. (1969). Citrus exocortis virus-chemical inactivation on tools, tolerance to heat and separation of isolates. Plant Dis. Rep. 53, 333-336. Sano, T., Ohshima, K., Hataya, T., Uyeda, I., Shikata, E., Chou, T., Meshi, T., and Okada, Y. (1986). A viroid resembling hop stunt viroid in

grapevines from Europe, the United States and Japan. J. Gen. Virol. 67, 1673-1678. Sano, T., Ohsima, K., Hataya, T., Uyeda, I., Shikata, E., Chou, T., Meshi, T., and Okada, Y. (1985). A viroid-like RNA isolated from grapevine has high sequence homology with hop stunt viroid. J. Gen. Virol. 66, 333-338. Sano, T., Uyeda, I., Shikata, E., Ohno, T., and Okada Y. (1984). Nucleotide sequence of cucumber pale fruit viroid: Homology to hop stunt viroid. Nucleic Acids Res. 12, 3427-3434. Sasaki, M., and Shikata, E. (1977). Studies on the host range of hop stunt disease in Japan. Proc. Jpn. Acad. Ser. B 53, 103-108. Schnolzer, M., Haas, B., and Ramm, K. (1985). Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV). EMBO J. 4, 2181-2190. Semancik, J.S., and Weathers, L.G. (1972). Exocortis disease: evidence for a new species of ‘infectious’ low molecular weight RNA in plants. Nature 237, 242-244. Semancik, J.S., Rivera-Bustamante, R., and Goheen, A.C. (1987). Widespread occurrence of viroid-like RNAs in grapevines. Am. J. Enol. Viticult. 38, 35-40. Semancik, J.S., and Szychowski, J.A. (1992). Relationships among the viroids derived from grapevines. J. Gen. Virol. 73, 1465-1469. Shanmuganathan, N., and Fletcher, G. (1980). The incidence of grapevine yellow speckle disease in Australian grapevines and the influence of inoculum on symptom expression. Aust. J. Agric. Res. 31, 327-333. Shikata, E., Sano, T., and Uyeda, I. (1984). An infectious low molecular weight RNA was detected in grapevines by molecular hybridisation with hop stunt viroid cDNA. Proc. Jpn. Acad. Ser. B 60, 202. Staub, U., Polivka, H. Herrmann, J.V., and Gross, H.J. (1995). Transmission of grapevine viroids is not likely to occur mechanically by regular pruning. Vitis 34, 119-123. Symons, R.H. (1981). Avocado sunblotch viroid: Primary sequence and secondary structure. Nucleic Acids Res. 9, 6527-6537. Szychowski, J.A., Credi, R., Reanwarakorn, K., and Semancik, J.S. (1998). Population diversity in grapevine yellow speckle viroid 1 and the relationship to disease expression. Virology 248, 432-444. Szychowski, J.A., Goheen, A.C., and Semancik, J.S. (1988). Mechanical transmission and rootstock reservoirs as factors in the widespread distribution of viroids in grapevines. Am. J. Enol. Viticult. 39, 213-216. Szychowski, J.A., McKenry, M.V., Walker, M.A., Wolpert, J.A., Credi, R., and Semancik, J.S. (1995). The vein-banding disease syndrome: A synergistic reaction between grapevine viroids and fanleaf virus. Vitis 34, 229-232. Tabler, M., and Sänger, H.L. (1984). Cloned single and double stranded cDNA copies of potato spindle tuber viroid (PSTV) RNA and coinoculated subgenomic DNA fragments are infectious. EMBO J. 3, 3055-3062. Taylor, R.H., and Woodham, R.C. (1972). Grapevine yellow speckle – a newly recognized graft-transmissible disease of Vitis. Aust. J. Agric. Res. 23, 447-452. van Dorst, H. J. M., and Peters, D. (1974). Some biological observations on pale fruit, a viroid-incited disease of cucumber. Neth. J. Plant Pathol. 80, 85. Visvader, J.E., and Symons, R.H. (1985). Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity. Nucleic Acids Res. 13, 2907-2920.

205


Visvader, J.E., and Symons, R.H. (1983). Comparative sequence and structure of different isolates of citrus exocortis viroid. Virology 130, 232-237. Visvader, J.E., Forster, A.C., and Symons, R.H. (1985). Infectivity and in vitro mutagenesis of monomeric cDNA clones of citrus exocortis viroid indicates the site of processing of viroid precursors. Nucleic Acids Res. 13, 5843-5856. Visvader, J.E., Gould, A.R., Bruening, G.E and Symons, R.H. (1982). Citrus exocortis viroid: nucleotide sequence and secondary structure of an Australian isolate. FEBS Lett. 137, 288-292. Wan Chow Wah, Y.F., and Symons, R.H. (1997). A high sensitivity RTPCR assay for the diagnosis of viroids in grapevines in the field and in tissue culture. J. Virol. Methods 63, 57-69. Wan Chow Wah, Y.F., and Symons, R.H. (1999). Transmission of viroids via grape seeds. J. Phytopathology 147, 285-291.

206

Weathers, L.G., Greer, F.C., JR and Harjung, M.K. (1967). Transmission of exocortis virus of citrus to herbaceous plants. Plant Dis. Rep. 51, 868-871. Wolpert, J.A., Szychowski, J.A., and Semancik, J.S. (1996). Effect of viroids on growth, yield and maturity indices of Cabernet Sauvignon grapevines. Am. J. Enol. Viticult. 47, 21-24. Wutscher, H.K., and Schull, A.V. (1975). Machine hedging of citrus trees and transmission of exocortis and xyloporosis viruses. Plant Dis. Rep. 59, 368-369. Yamamoto, H., Kagami, Y., Kurokawa, M., Nishimura, S., and Kubo, S. (1973). Studies on hop stunt disease in Japan. Rep. Res. Lab. Kirin Brew. Co. 16, 49. Yamamoto, H., Kagami, Y., Kurokawa, M., Nishimura, S., Kubo, S., Inoue, S., and Murayama, D. (1970). Studies on hop stunt disease. I. Mem. Fac. Agric. Hokkaido Univ. 7, 491.

PART IV

HOP VIROIDS HOP STUNT VIROID T. Sano

....................................................................................................

CHAPTER 29

.................................................................................................................................................................................................................................................................

The acreage of hop cultivation in Japan is now decreasing, its peak having been from the mid-1960s to the beginning of the 1970s. In 1999 hop farmers numbered 550, the gross cultivation acreage was 341 ha, and the gross cone yield was 721 t (Annual Report 2000, The Federation of Hop Agricultural Cooperative Association of Japan). The hop cultivation area has now been restricted to Hokkaido Island and five prefectures in the Tohoku district of northern Honshu. Why has hop cultivation in Japan decreased? Even though the consumption of hops as a raw material appears to have been on the increase since the 1970s, it seems there has been a decline in the demand for domestically grown hops. The self-sufficiency rate, which exceeded 70% in the 1960s, decreased to 8.7% in 1998. Weakening of international competitiveness due to the local production cost is probably the main reason for this lowering of the self-sufficiency rate. The contract price of domestic hops now reaches double the import price. However, it is not only the price but also the quality of factors such as bitterness and perfume compounds of the domestic hops, elements related to consumer preference, that might have weakened the demand for the local product.

In addition to the deterioration of the hop growing environment described above, it should be emphasized that the emergence of a new infectious disease, now known as hop stunt disease has also caused considerable negative effects upon hop production in Japan. And hop stunt disease caused serious damage just when it was the golden age of hop production in Japan. Hop stunt disease spread rapidly with the expansion of the hop cultivation area in the Tohoku district. For example, on the farm contracted to supply hops to Kirin Brewery Co. Ltd., the disease was prevalent in 19% of the total hop cultivating areas in 1977; the year when the causal agent of hop stunt disease was discovered (Sasaki et al. 1989). Full-scale control measures were then conducted for 10 years; the diseased hops were surveyed, removed and replanted with healthy plants. As a result of these energetic efforts, the disease was almost completely eliminated from the hop gardens associated with the Kirin Company (Sasaki et al. 1989). However, complete eradication has not yet been accomplished. Hop stunt is still one of the most feared diseases in the hop production areas of Japan. Once infected stock is found, several nearby plants including the infected individuals or all the stocks are replanted. Measures to counter the outbreak

207

T. Sano

of hop stunt disease need to be enforced as long as hop cultivation continues in Japan.

Although the disease is under control, it still poses a threat for renewed outbreaks.

HISTORY AND GEOGRAPHICAL DISTRIBUTION

ECONOMIC IMPACT AND SYMPTOMATOLOGY

Hop stunt is a disease endemic to hops cultivated in Japan and is not observed in any other region of the world. The disease had already been sparsely distributed in Fukushima prefecture in 1952 and was prevalent in the 1960s due to the expansion of hop cultivation throughout the Tohoku district. The affected hop was called ‘dwarf hop’ or ‘cedar-shaped hop’ (Yamamoto et al. 1973). A similar mysterious hop disorder was also recognized in Nagano prefecture from the 1940s and was called ‘Tozama’ disease, taking its name from the place where the disease first emerged (Mori 1995).

Stunting of bines (shoots): Hop stunt is a disease which was first recognized through the deterioration in growth of infected hop bines. However, our field experiment using hops artificially inoculated with HSVd clearly showed that ‘stunt’ was not the most typical symptom of the disease (Sano et al. 1999).

Hop (Humulus lupulus) is a dioecious perennial herb and is assumed to have originated from the Mediterranean sea coast, in the Caucasus region. Hop cultivation began in Germany in the mid-eighth century, and has a long history mainly in the central European countries (Hamaguchi 1979; Mori 1995). Hop cultivation started in Japan in the late nineteenth century with experimental cultivation in 1876 and more large-scale cultivation started soon after. Programs for selection and breeding to produce original hop varieties in Japan started in 1910 with foreign varieties imported mainly from Germany and the US (Hamaguchi 1979; Mori 1995). Therefore, most of the hop varieties now cultivated in Japan are progenies which were propagated vegetatively from selected mother stocks originating from Germany or the US. Four viruses and a viroid being isolated from hop in Japan are all known to have spread widely among hops around the world. In particular, Hop latent virus, Apple mosaic virus and Hop latent viroid are considered to have been introduced from foreign countries with some infected hop scions which were introduced for the purpose of breeding, because infection rates were extremely high (almost 100%) in the commercial hops established before virus-free hop scions were available (Sano et al. 1985; Sano and Shikata 1989; Kanno et al. 1993; Hataya et al. 1992). It is also apparent that neither Hop mosaic virus nor Prunus necrotic ringspot virus are of Japanese hop origin, because they were isolated only from limited numbers of foreign varieties introduced for breeding (Sano and Shikata 1989; Kanno et al. 1994), they have never been isolated from Japanese commercial hop gardens. On the other hand, Hop stunt viroid (HSVd), the causal agent of hop stunt disease, is the exception. Hop stunt disease and HSVd from hop, have been reported only from Japan and Korea. It is apparent that imports of infected scion stocks from Japan are the cause of the outbreak in Korea (Lee et al. 1988). In conclusion, hop stunt disease emerged in Japan and was endemic during the 1960s–1970s in the Tohoku district.

208

Figure 29.1 shows the annual change of the main bine length of infected hops from 3 to 7 years post-infection. The average length of 10 main bines of the infected plants was indicated by an index relative to healthy bines. Stunting of the bines was not recognized up until the fourth growing season post-infection. About 17% of stunting was first recognized at the end of the fifth growing season, however, the average length was still nearly 8 m which was much higher than the 4.5 m high shelf. The infected bines were 65% of the height of the healthy bines at the end of the seventh growing season, but were still higher than the shelf. Because the stunting level observed during the first seven years was so slight as to be not recognized visually in experimental field plots, it should be emphasized that the early stage of hop stunt, at least during the several years post-infection, easily escapes recognition in commercial hop gardens. Since the severity of stunting, however, increases year by year, the infected hop eventually exhibits typical stunting after several years. Hop is a climbing vine and is usually cultivated in Japan by winding the bines on strings which are hanging from the shelf up to a height of 4.5–6 m. Since the main bine of the major Japanese cultivars grows more than 10 m in a growing season, pulling down the bines or cutting off the tips is essential to adjust the length of bines to the shelf and get maximum cone yields. Such a cultivation style, in addition to the congenital slow stunting progress, makes stunting of the infected hop plants more unrecognizable. The temperature in the growing season is also another key factor that determines the severity of stunting; with stunting milder in the northern (i.e. cooler) prefectures in Japan. Consequently, in spite of the name, the stunting progresses slowly year by year and may take up to 10 years, depending on the temperature etc., to be noticed. Taking these points into consideration, when hop stunt disease first prevailed in the 1960s and 1970s, the stunted hops observed by farmers were those most seriously affected cases which had been infected presumably more than 10 years earlier. Reduction of alpha acid content in cone

On the other hand, the alpha acid content in the cone decreases immediately after infection of HSVd (Sano et al. 1999). The annual change of average alpha acid content (dry material %) in cones produced by 10 infected hops from 3 to 7 years post-

HOP STUNT VIROID

sequences of HSVd and CPFVd were determined (Ohno et al. 1983, Sano et al. 1984). They shared 95% sequence identity and are now considered to be variants of the same species. Furthermore, a large number of HSVd sequence variants have been isolated from several woody hosts: grapevine, citrus, plum, peach, pear, apricot, almond, and Punica granatum (Sano et al. 1985, 1986, 1989; Shikata 1990, Astruc et al. 1996). The variants in plum and peach cause dapple fruit disease, but those in grapevine, apricot, almond, and Punica granatum occur as symptomless infections. Most of the variants in citrus are also symptomless, however, a certain variant was proved to cause citrus cachexia disease (Reanwarakorn and Semancik 1999). It is now known that variants of HSVd are distributed widely among several fruit tree species around the world, and that HSVd is a very widely distributed viroid which is frequently pathogenic. Figure 29.1 Length of the main bine and alpha acids content of HSVdinfected hops from three to seven years post-infection, relative to healthy hop plants.

infection is presented in Figure 29.1 by an index relative to healthy plants. It is apparent that the alpha acid content in the infected cones decreased to 50% of that of the healthy cones by the third year post-inoculation. Surprisingly, the same reduction had already been recognized at the first fall season of the infected year. In fact, the dramatic decrease of alpha acid content was considered to be the most serious problem since hop stunt disease was first recognized. In severe infections, dwarfing of the whole plant led to a reduction of the number and size of cones. For example, in the epidemic of the 1960s–1970s, the average length of a severely infected bine was ca. 66% that of a healthy one, though the number of internodes in a main bine was normal. The total number of cones in the infected bines was 30–50% of a healthy bine. The average weight of an infected cone was ca. 66% of a healthy one. Finally, the alpha acid content in an infected cone was 42% of a healthy cone (Yamamoto et al. 1973). Combining these data, the yield of alpha acids obtained from a severely infected hop would be decreased to 8–14% of a healthy one.

TAXONOMIC POSITION AND NUCLEOTIDE

Since the complete nucleotide sequence of HSVd isolated from hop (the type HSVd-hop) was determined (Ohno et al. 1983), the sequences of HSVd variants isolated from grapevine, citrus, plum, peach, apricot, almond and Punica granatum have been reported (Sano et al. 1986, 1988, 1989; Kofalvi et al. 1997), and more than 40 HSVd sequence variants are now deposited in the Viroid Sequence Database (Lafontaine et al. 1999). In addition to the type HSVd-hop, nine isolates of HSVd-hop were recovered for sequencing from leaves collected between 1994 to 1999 from symptomatic hop plants growing in commercial hop gardens in the Tohoku district. All the isolates exhibited minor sequence variations. At least six different consensus and one sub-consensus sequence as well as 12 sequence variants were detected in the nine isolates (Sano et al. 2000). In other words, the sequence of HSVd-hop isolated from the area where hop stunt disease is prevalent was remarkably variable. A neighbor-joining analysis on the nine new HSVd-hop consensus sequence, one sub-consensus, and two minor sequence varinats, together with 44 previously described variants of HSVd isolated from hop and other species revealed that HSVd isolates can be divided into five clusters depending on original host species: i

a plum-peach-almond-apricot cluster (Sano et al. 1989; Kofalvi et al. 1997);

ii

a German grapevine cluster (Polivka et al. 1996; Puchta et al. 1988a, 1989);

iii

a general grapevine-hop cluster (Sano et al. 1986; Polivka et al. 1996; Sano et al. 2000);

iv

a US citrus cachexia cluster (Reanwarakorn and Semancik 1999); and

v

a general citrus-cucumber cluster (Sano et al. 1988; Puchta et al. 1988b; Reanwarakorn and Semancik 1999).

SEQUENCE

The causal agent of hop stunt disease — HSVd — was first discovered in 1977 (Sasaki and Shikata 1977). The agent was successfully transmitted by mechanical inoculation on cucumber (Cucumis sativus), and shown to be a viroid. At the beginning, the viroid was believed to occur only in certain hop varieties growing in Japan, however, it was soon noted that the symptoms of cucumber were quite similar to those incited by Cucumber pale fruit viroid (CPFVd) reported in the Netherlands (Van Dorst and Peters 1974; Sano et al. 1981). The similarity of the two viroids was confirmed when the complete nucleotide

209

T. Sano

It is noteworthy that all the HSVd-hop isolates group together with the grapevine isolates (HSVd-g).

POSSIBLE ORIGIN Since the discovery of hop stunt disease, it has long been a mystery why this type of new disease has emerged only in Japan. As described earlier, hops were initially introduced into Japan from Europe and the US at the end of the nineteenth century (Hamaguchi 1979, Mori 1995). Present day Japanese cultivars have been developed from these parental materials by crossing and selection (Mori 1995). Hop stunt disease was first recognized in Nagano and Fukushima prefectures in the 1940s and 1950s (Yamamoto et al. 1973). Thus far, the disease has not been observed in Europe or the US. By taking account of this circumstantial evidence, it would appear that the causal agent of the disease was introduced into hop from some other hosts harboring the pathogen approximately 40–50 years after the introduction of hops into Japan. Considering that HSVd can be recovered from grapevine, citrus, and plums growing throughout the world, these hosts would seem to have encountered HSVd much earlier than did the cultivated hop. As shown by phylogenetic analysis, all the 12 HSVd-hop sequences recovered from hops cultivated in the Tohoku district of Japan as well as the type isolate and two Korean isolates form a cluster with the HSVd isolate commonly recovered from grapevine. This suggests that the causal agent of hop stunt disease may have come from grapevines. In addition, since phylogenetic analysis indicated that most HSVd-hop sequences form a single clade, the putative movement of HSVd-g from grapevine to hop must have been a unique (or at least an extremely rare) event. Presumably, a transfer of HSVd-g from grapevine to hop occurred more than 50–60 years ago somewhere around the Nagano and/or Fukushima prefectures. In fact, as both prefectures are popular fruit production centers, it is not uncommon to find a hop garden adjoining a vineyard. Then the vegetative propagation of the viroid-contaminated hops could have led to the spread of hop stunt disease throughout the hop-growing areas in Japan (Yamamoto et al. 1973). Once established in hops, the viroid was easily transmitted mechanically from hop to hop through contaminated implements (Sasaki et al. 1989) because the viroid titer in hops is more than 10 times higher than that in grapevine (Li et al. 1995). Finally, the increased numbers of infected hops would have appeared as an epidemic (Sano et al. 2000).

DIAGNOSIS Diagnosis of hop stunt disease can be done by observation of symptoms, analysis of alpha- and beta-acids ratio in cones, bioassay using cucumber plants, polyacrylamide gel electrophoresis,

210

nucleic acids hybridization and by reverse transcription–polymerase chain reaction. Before the discovery of HSVd, the disease was detected through observing symptoms in the field, in which the stunting of bines or drooping of leaf stalks were diagnostic criteria (see Plate 12). Analysis of alpha- and beta-acids ratio in cones has also been used as a practical diagnosis, because only the alpha acids content of the resin components in the infected cones is reduced to half that of the healthy cones. A bioassay called ‘cucumber assay’ was used as the diagnostic tool during epidemics in the late 1970s–1980s. Sasaki and Shikata (1977) found that the causal agent of the disease can be transmitted mechanically to cucumber, which led them to the discovery of HSVd. At the same time, they devised a new diagnostic method by using cucumber as an indicator plant. They examined several cucumber cultivars and found that cv. ‘Suyo’ showed the highest sensitivity to HSVd infection. The assay was performed as follows (Sasaki et al. 1989): hop leaves dried in silica gel or frozen were homogenized in 3 volumes (weight/ volume) of 0.1 M phosphate buffer, pH 7.5. The crude extract, or the supernatant obtained by low speed centrifugation (800 × g, 5 min.) was mechanically inoculated onto cucumber cotyledons dusted with carborundum (600 mesh). The plants were kept in a greenhouse at 30°C for 40 days. The infected plants showed symptoms of leaf curling, vein clearing and finally stunting 3–4 weeks post-inoculation. The assay was sensitive enough to detect HSVd in a bulked sample even when only one positive hop leaf disc was mixed with 200 healthy ones. Based on the data, inoculum made from bulked samples of 50 hop plants were used for routine diagnosis. The assay was sensitive enough to detect HSVd from symptomless hop plants. The result of the cucumber bioassay followed by a replanting program using HSVd-free hops led to the almost complete elimination of hop stunt disease in hop gardens associated with the Kirin Company by 1987. Although the cucumber bioassay was such a good diagnostic tool and played an important role in the cessation of hop stunt disease epidemics, it does require 1–2 months to complete. The assay has now been replaced by nucleic acid hybridization which can be done more quickly without reducing sensitivity. Li et al. (1995) examined return-polyacrylamide gel electrophoresis and nucleic acid hybridization for the practical diagnosis of HSVd. The return-PAGE followed by silver staining was sensitive enough to detect HSVd in hops, however, the frequent appearance of double or triple bands which was caused by the mixed infection of Hop latent viroid (HLVd) may result in some confusion. This is in fact the situation in a major Japanese hop cultivar, because the concentration of HLVd was several times higher than that of HSVd. Nucleic acid hybridization using a digoxigenin- (DIG) labeled cRNA probe was more reliable for

HOP STUNT VIROID

the practical HSVd diagnosis, and sensitive enough to detect HSVd in 2–10 mg of infected hop leaves. Our field experiment using artificially infected hop plants indicated that HSVd was easily detected by nucleic acid hybridization at the end of the growing season, even when the plants were inoculated in the spring and no discernible disease symptoms would be expected for several years. The method is sensitive enough to detect HSVd in symptomless carriers during the incubation period. Furthermore, tissue blot hybridization is also effective, although the sensitivity is lower than dot-blot hybridization especially when leaf material collected early in the growing season is used for diagnosis. By using this method, HSVd diagnosis can be performed within 2 days.

TRANSMISSION AND CONTROL In the initial stage of the epidemics, distribution of the infected cuttings among farmers played the most important role for the spreading of the disease. Since farmers are supplied with cuttings which have been propagated vegetatively, the distribution of contaminated cuttings could cause a broad and long distance spread of the disease. Once the disease has been established in a garden, mechanical transmission from the infected stocks to the adjacent plants by production practices becomes more important in the spread of the disease. This type of spread is in general restricted to a relatively small area of farm land; i.e. within one or several gardens belonging to the same owner. At present, spread via cuttings is not serious, because associations of hop growers now supply farmers with HSVd-free hop cuttings propagated vegetatively from mother plants free from viruses and viroids. On the other hand, it is not easy to prevent spread between vines. HSVd is easily transmitted mechanically through contaminated implements by cultural practices such as dressing of stocks, rubbing of young buds or pulling to remove excess sprouts which are all concentrated early in the spring. Investigations on spread of the disease in some hop gardens clearly indicated that the number of infected plants increased each year along the ridge next to the previously infected plants (Yamamoto et al. 1973; Sasaki et al. 1989). In order to prevent infection of hops with HSVd, farmers are instructed to cultivate and harvest HSVd-free areas prior to HSVd-contaminated areas, and to change and/or disinfect the implements such as sickles, scissors, etc., at every garden and for every plant by dipping them into a disinfectant such as formalin and caustic soda (Takahashi 1979; Sasaki et al. 1989). If infected plants are found, the bines are cut off and the stocks are removed from the garden to be burned after drying. In the days of the epidemics, ten plants on either side in the same ridge and three plants in the adjacent ridges on either side of the infected plants were also removed simultaneously, because they were sus-

pected to have been infected even if they did not show any detectable disease symptoms. Currently, once infected plants are detected, farmers are encouraged to replant all the plants in the garden with virus/viroid-free cuttings, because symptomless carriers may possibly remain in the garden. Consequently, it is necessary for successful disease control to continuously promote the following measures: i

the periodic surveillance of the infected plants in the fields for symptoms;

ii

early diagnosis of HSVd in the laboratory by hybridization or alpha acids test; and

iii

the establishment of a replanting program using virus/ viroid-free cuttings.

References Astruc, N., Marcos, J.F., Macquaire, G., Candresse, T., and Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. Eur. J. Pl. Pathol. 102, 837-846. Hamaguchi, N. (1979). Hop. 116pp. Tokusan Series 15, Nou-san-gyoson Bunka Kyokai (in Japanese). Hataya, T., Hikage, K., Suda, N., Nagata, T., Li, S., Itoga, Y., Tanikoshi, T., and Shikata, E. (1992). Detection of hop latent viroid (HLVd) using reverse transcription and polymerase chain reaction (RT-PCR). Ann. Phytopathol. Soc. Jpn. 58, 677-684. Kanno, Y., Iida, H., Yoshikawa, N., and Takahashi, T. (1994). Some properties of hop mosaic virus isolated in Japan. Ann. Phytopathol. Soc. Jpn. 60, 675-680. Kanno, Y., Yoshikawa, N., and Takahashi, T. (1993). Some properties of hop latent and apple mosaic virus isolated from hop plants and their distributions in Japan. Ann. Phytopathol. Soc. Jpn. 59, 651-658. Kofalvi, S. A., Marcos, J. F., Cañizares, M. C., Pallás, V., and Candresse, T. (1997). Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. J. Gen. Virol. 78, 3177-3186. Lafontaine, D. A., Deschenes, P., Bussiere, F., Poisson, V., and Perreault, J-P. (1999). The viroid and viroid-like RNA database. Nucleic Acids Res. 27, 186-187. Lee, J. Y., Puchta, H., Ramm, K., and Sänger, H.L. (1988). Nucleotide sequence of the Korean strain of hop stunt viroid (HSV). Nucleic Acids Res. 16, 8708. Li, S., Onodera, S., Sano, T., Yoshida, K., Wang, G., and Shikata, E. (1995). Gene diagnosis of viroids: comparison of return-PAGE and hybridization using DIG-labeled DNA and RNA probes for practical diagnosis of hop stunt, citrus exocortis and apple scar skin viroids in their natural host plants. Ann. Phytopathol. Soc. Jpn. 61, 93-102. Mori, Y. (1995). Hop. 520pp. Hokkaido Univ. Co-op Press: Sapporo, Japan. (in Japanese). Ohno, T., Takamatsu, N., Meshi, T., and Okada, Y. (1983). Hop stunt viroid: molecular cloning and nucleotide sequence of the complete cDNA copy. Nucleic Acids Res. 11, 6185-6197.

211

T. Sano

Polivka, H., Staub, U., and Gross, H.J. (1996). Variation of viroid profiles in individual grapevine plants: novel grapevine yellow speckle viroid 1 mutants show alterations of hairpin I. J. Gen. Virol. 77, 155-161. Puchta, H., Ramm, K., and Sänger, H. L. (1988a). Nucleotide sequence of a hop stunt viroid isolate from the German grapevine cultivar ‘Riesling’. Nucleic Acids Res. 16, 2730. Puchta, H., Ramm, K., and Sänger, H. L. (1988b). Molecular and biological properties of a cloned and infectious new sequence variant of cucumber pale fruit viroid (CPFV). Nucleic Acids Res. 16, 8171. Puchta, H., Ramm, K., Luckinger, R., Freimuller, K., and Sänger, H.L. (1989). Nucleotide sequence of a hop stunt viroid (HSVd) isolate from the German grapevine root stock 5BB as determined by PCR-mediated sequence analysis. Nucleic Acids Res. 17, 5841. Reanwarakorn, K., and Semancik, J. S. (1999). Correlation of hop stunt viroid variants to Cachexia and Xyloporosis diseases of citrus. Phytopathology 89, 568-574. Sano, T., Hataya, T., Sasaki, A., and Shikata, E. (1986b). Etrog citron is latently infected with hop stunt viroid-like RNA. Proc. Japan Acad. 62(B), 325-328. Sano, T., Hataya, T., and Shikata, E. (1988). Complete nucleotide sequence of a viroid isolated from Etrog citron, a new member of hop stunt viroid group. Nucleic Acids Res. 16, 347. Sano, T., Hataya, T., Terai, Y., and Shikata, E. (1989). Hop stunt viroid strains from dapple fruit disease of plum peach in Japan. J. Gen. Virol. 70, 1311-1319. Sano, T., Ito, S., Narita, M., Murakami, A., and Shikata, E. (1999). Assessment of potential risks of hop stunt viroid isolates harboring in grapevine, plum, citrus and hop. In Abstract of XIth International Congress of Virology, August 9-13, Sydney, Australia. Sano, T., Mimura, R., and Ohshima, K. (2000). Phylogenetic analysis of hop stunt viroid supports a grapevine origin for hop stunt disease. Virus Genes 22, 53-59. Sano, T., Ohshima, K., Hataya, T., Uyeda, I., Shikata, E., Chou, T. G., Meshi, T., and Okada, Y. (1986). A viroid resembling hop stunt

212

viroid in grapevines from Europe, the United States and Japan. J. Gen. Virol. 67, 1673-1678. Sano, T., Sasaki, M., and Shikata, E. (1981). Comparative studies on hop stunt viroid, cucumber pale fruit viroid and potato spindle tuber viroid. Ann. Phytopathol. Soc. Jpn. 47, 599-605. Sano, T., Sasaki, M., and Shikata, E. (1985). Apple mosaic virus isolated from hop plants in Japan. Ann. Appl. Biol. 106, 305-312. Sano, T., and Shikata, E. (1989). Hop virus diseases in Japan. Pages 312 in: Proceedings of Int. Workshop on Hop Virus Diseases. A. Eppler, ed. Deutsche Phytomedizinische Gesellschaft: Ulmer (Germany). Sano, T., Uyeda, I., Shikata, E., Meshi, T., Ohno, T., and Okada, Y. (1985). A viroid-like RNA isolated from grapevines has high sequence homology with hop stunt viroid. J. Gen. Virol. 66, 333-338. Sano, T., Uyeda, I., Shikata, E., Ohno, T., and Okada, Y. (1984). Nucleotide sequence of cucumber pale fruit viroid: homology to hop stunt viroid. Nucleic Acids Res. 12, 3427-3434. Sasaki, M., and Shikata, E. (1977). On some properties of hop stunt disease agent, a viroid. Proc. Jpn. Acad. Ser.B. 53, 109-112. Sasaki, M., Fukamizu, K., Yamamoto, T., Ozawa, M., Kurokawa, M., and Kagami, Y. (1989). Epidemiology and control of hop stunt disease. Pages 165-178 in: Proceedings of Int. Workshop on Hop Virus Diseases. A. Eppler, ed. Deutsche Phytomedizinische Gesellschaft: Ulmer (Germany). Shikata, E. (1990). New viroids from Japan. Semin. Virology 1, 107-116. Takahashi, T. (1979). Diagnosis and control of hop stunt disease. Agriculture and Horticulture 54, 1031-1034. Van Dorst, H.J.M., and Peters, D. (1974). Some biological observations on pale fruit, a viroid-incited disease of cucumber. Neth. J. Plant Pathol. 80, 85-96. Yamamoto, H., Kagami, Y., Kurokawa, M., Nishimura, S., and Kubo, S. (1973). Studies on hop stunt disease in Japan. Rep. Res. Lab. Kirin Brew Co. Ltd. 16, 49-62.

PART IV

CHAPTER 30

HOP LATENT VIROID ....................................................................................................

D. J. Barbara and A.N. Adams

.................................................................................................................................................................................................................................................................

Two viroids are known to occur in hop. Hop stunt, which is of limited geographic distribution in this host, is described in Chapter 29 in this volume. Hop latent viroid (HLVd) is much more widespread. HLVd was first described in detail and named by Puchta et al. (1988) but slightly earlier a viroid-like RNA from hops in Spain was described by Pállas et al. (1987). This latter moiety (which they referred to as ‘hop viroid-like RNA fast’ or HV-f) was of a similar size to HLVd and almost certainly represents the first tentative description of HLVd. The hop (Humulus lupulus L., family Cannabinaceae) is a climbing, perennial, dioecious plant that is cultivated for the resins contained in the dried female cones. The resins are used by the brewing industry mainly to impart the bitter flavor to beer but historically they also acted as a preservative. The most important resin compounds are the alpha-acids and for most varieties the quality and selling price of the harvest is assessed by the alphaacids content. The plant is native to the northern temperate region and is cultivated commercially in many parts of Europe, in the US, Canada, China, Japan, South Korea, South Africa, Australia and New Zealand (Barth et al. 1994). The shoots (also known as bines) are usually supported on a framework of poles and wires 4.5–8 m high. Recently, however, cultivation of traditional varieties has been modified for growth on low wirework

about 3 m high. Also dwarf hops which are grown on 2.4 m trellises have been introduced in the UK. Tall hops are harvested by cutting off the bines at 1–2 m above the soil and transporting them to a large static machine to strip off the cones. The remains of the shoots are then trimmed to soil level in the winter. The cones are removed by machine in the field from plants growing on low wirework and dwarf wirework systems. In the spring many shoots grow from the substantial rootstock which can remain in production for several decades. Usually 8–12 shoots per plant are trained up supporting strings or wires and when these selected shoots have reached about 2 m the remainder are removed by hand-pulling, strimming, cutting or chemically. Hops are propagated vegetatively to preserve varietal characteristics, usually by rooting stem cuttings in a mist bench. It is important to monitor the source material for viruses and viroids (Anonymous 1998) but it has only been possible to do this for HLVd since its description in 1988 (Puchta et al. 1988).

ECONOMIC IMPACT AND SYMPTOMOLOGY Overt symptoms of infection with HLVd have only been recorded in one commercial hop cultivar, ‘Omega’. In this

213


Table 30.1

Cone analysis and net yield of alpha-acids per plant in three cultivars of hop grown in the UK: ‘Omega’, ‘Wye Northdown’ and ‘Wye Challenger’.

Cone analysis

Omega

Northdown

Challenger

Cone weight

63*

92

89

Alpha-acids

69

85

89

Beta-acids

130

119

108

Net alpha-acids per plant

43

78

79

*Infected as a percentage of uninfected.

variety diseased plants are paler, grow more slowly, and produce fewer laterals than uninfected plants (Barbara et al. 1990a). The symptoms can be obvious in plantings containing adjacent healthy and infected plants but can be difficult to recognize without healthy plants for comparison. The effects of HLVd on productivity and quality were examined in plants growing in commercial plantations by Barbara et al. (1990a) and Adams et al. (1991). The cultivar ‘Omega’ was severely affected with cone yield reduced by 27% and alphaacids content by 31%, equivalent to an overall reduction in the yield of alpha-acids per plant of over 50%. The reduction in cone yield was due mainly to the cones on infected plants being smaller. Chemical analysis of the hops showed that the betaacids and oil content in cones from infected plants was higher than in cones from healthy plants, suggesting that viroid infection may advance the maturation of the cones. The effects of HLVd infection were less dramatic in two other cultivars, ‘Wye Northdown’ and ‘Wye Challenger’, but the individual cone constituents were affected in a similar way with a net reduction of alpha-acids production of approximately 20% (see Table 30.1). These yield effects were sufficiently serious to stimulate a program in the UK for the replacement of all hop planting material with propagules from viroid-tested Nuclear Stock plants (Adams and Barbara 1990). The rooting and establishment of plants infected with HLVd is poorer than that of uninfected plants (Darby, 1999).

HOST RANGE HLVd infects Humulus lupulus (the commercial hop) and H. japonicus Sieb. & Zucc., an annual found in the Orient. There is no information on the susceptibility of the third member of the genus, Humulus yunnanensis, which is rare and is mainly known mainly from a few herbarium specimens. The viroid has also been found naturally infecting Urtica dioica L. (stinging nettle) in Germany (Knabel et al. 1999).

TRANSMISSION HLVd is easily transmitted by mechanical methods and this is probably the main means of spread within commercial plant214

ings (Adams et al. 1992, 1996). There is evidence for transmission of some viroids by aphids under certain circumstances. For example, Potato spindle tuber viroid was transmitted from potato plants that were infected with potato leafroll virus in addition to the viroid; this was thought to be due to transencapsidation (Salazar et al. 1995; Syller et al. 1997; Querci et al. 1997). However, no transmission of HLVd by the damson hop aphid, Phorodon humuli (Schrank), was detected from hop plants doubly that were infected with Hop mosaic and/or Hop latent carlaviruses (the two most widespread aphid-borne viruses in hop) and HLVd (Adams et al. 1996). No infection was found in seedlings derived from infected male or female plants by Darby (1999), suggesting that there is little or no transmission by pollen or through the seed.

GEOGRAPHICAL DISTRIBUTION Early tests (Puchta et al. 1988) on dried cone samples suggested very high levels of infection (73 of 80 hop varieties tested were found to be infected). In most countries: (Europe: Belgium, Czech Republic, Slovania, France, Germany, Hungary, Poland, Portugal, Russia, Spain, United Kingdom, Yugoslavia; Asia: Japan, South Korea, China; Americas: USA) 85–100% of varieties were apparently infected. These same tests suggested a lower level of infection in South Africa (50%, but only two varieties were tested) and that HLVd was absent from Australia and New Zealand (only one variety tested for each). Some (but not all) later studies suggested that infection levels were lower in some of these countries and it appears that at least some of the samples used in the early study were from a hop variety collection being grown in Germany and therefore they were probably not truly representative. HLVd has been detected in hop samples known to be from the following countries: UK (Barbara et al. 1990b), Germany (Puchta et al. 1988; Knabel et al. 1999), Slovenia (Knapic and Javornik 1998), Japan (Hataya et al. 1992), Poland (Solarska et al. 1995), Korea (Lee et al. 1990), Czech Republic (Matousek et al. 1994) New Zealand (F. Hay, personal communication) and in seven hop varieties introduced to Brazil from the US (Fonseca et al. 1993).

DETECTION Despite being mechanically transmissible, the limited host range and lack of obvious symptoms mean that there are no biological indicators for HLVd and practical diagnosis is entirely based on three molecular methods. Dot-blot hybridization using RNA transcripts from a dimeric HLVd clone was first applied to HLVd by Puchta et al. (1988) and was the principal detection method used in the most comprehensive studies of the disease aspects of HLVd published to date (see Barbara et al. 1990b et seqq.). Both radioisotopic labels and non-isotopic labeling of the probe has been used. Whilst this procedure is not particularly sensitive, it is robust and, as sample preparation is simple, applicable to large-scale testing for

HOP LATENT VIROID

epidemiological and similar studies. Although at low stringencies the probe will also hybridize to Hop stunt viroid, at moderate to high stringency it is specific to HLVd. More recently several reverse transcriptase-PCR assays for HLVd have been developed (Hataya et al. 1992; Cajza et al. 1996; Nakahara et al. 1999; Knabel et al. 1999). The sensitivities of these methods for detecting HLVd in plants will vary according to the precise protocols used. However, in one laboratory RT-PCR has been shown to be approximately 1000-fold more sensitive than dot-blot hybridization when agarose gels were used to detect the amplicon and 10,000-fold more sensitive when using hybridization or a serological method to detect the amplicon (Knabel et al. 1999; Seigner 2000). In general, sample preparation for RT-PCR is more laborious than for dotblot hybridization and, especially for large scale studies, PCRbased tests may only be justified where maximum sensitivity is desirable (e.g. quarantine testing). HLVd was first detected and differentiated from Hop stunt viroid by bi-directional (‘return’) electro-phoresis (Puchta et al. 1988; Pállas et al. 1987) and this remains a valuable technique for broad spectrum detection of viroids, possibly as a preliminary screen (e.g. Fonseca et al. 1993). However, sample throughput is limited and for routine studies of HLVd either dot-blot hybridization or RT-PCR are generally to be preferred. In the UK, titers of HLVd were found to vary in the aerial parts of the plants, especially early in the season (Morton et al. 1993), being relatively high near the bases of the plants but apparently absent near the tops of the bines. Viroid levels generally increased as the season progressed and HLVd only became fully systemic by about mid-summer. Similarly in the Czech Republic, in May/June, Matousek et al. (1994) found c. 5 pg mg-1 HLVd in leaves taken at 1 m from the ground in field grown plants but apparently lower levels (down to 0.5 pg mg-1) in samples taken in April. Relative to these Czech results, Pállas et al. (1987) found higher levels of their HV-f RNA in Spanish hop samples collected in ‘early summer’ (150–250 pg mg-1), which (assuming that HV-f was HLVd) may reflect either host variety or, perhaps more likely, a climatic difference. Whilst there may be higher levels of HLVd in roots than in aerial parts (Matousek et al. 1994) these are difficult to sample. Knabel et al. (1999) suggested that in Germany there was a slight decrease in HLVd concentration in lower leaves in late summer, but this decrease was not sufficient to compromize detection. In practice, for routine testing there appears to be adequate levels of HLVd in all types of tissue from leaves through to dried mature cones but in cooler climates sampling date is an important variable; sampling too early may lead to infections being missed. The earliest reliable sampling times should preferably be determined experimentally but, based on UK experience, adequate levels of pathogen are present in leaves collected at 1 m from the ground from late spring onwards.

EPIDEMIOLOGY AND CONTROL It is probable that the main route for the introduction of HLVd into new hop gardens (other than where they are planted adjacent to existing already infected hops) is in infected planting material. The best means to control dissemination by this route is by the provision of viroid-tested planting material through a certification scheme such as that outlined for use in Europe (OEPP/EPPO 1997). Such schemes operate in many countries but they are generally vulnerable to undescribed pathogens. In the UK it is thought that material infected with HLVd was introduced to the Nuclear Stock house (i.e. the start of the propagation chain) in the late 1970s (Barbara et al. 1990b). As infection is latent in most varieties it appears to have passed without notice, as no diagnostic tests were available until the late 1980s. Even in the sensitive cultivar ‘Omega’, infection would have been inconspicuous in the unnatural growth conditions under glass, particularly if all the plants became infected at about the same time (in this case the four or five plants of each variety held in the Nuclear Stock house). The crowded conditions would also have been conducive to rapid spread of the pathogen. By the time the viroid was detected in the UK (Barbara et al. 1990b), one year after the original description (Puchta et al. 1988), it was present in a high proportion of the Nuclear Stock mother plants and in 17% of samples from 476 commercial hop plantings. Infected weeds are a possible source of HLVd inoculum but only one perennial weed host is known, Urtica dioica; this was found in a hop garden containing many infected hop plants and its role in the epidemiology of HLVd is obscure (Knabel et al. 1999). The distribution and spread of the viroid was studied in two commercial plantings in the UK (Adams et al. 1992). One planting was of cv. ‘Omega’ and about 75% of plants were infected at the start of the investigation. Spread within this variety was rapid in the two subsequent years with 29 and 75%, respectively, of the remaining healthy plants, becoming infected. The planting was contiguous with a block of cv. ‘Wye Challenger’ in which few plants were infected at the start of the study. There was no gradient of infection from the highly infected cv. ‘Omega’ into the ‘Wye Challenger’, suggesting that contact between adjacent plants was not an important means of spread. However, the interface between varieties was between rows, not within them, so that during mechanical cultivation (during which spread may occur) plants of the two varieties would not have been cultivated consecutively. Within the planting of cv. ‘Wye Challenger’ there was evidence of spread between adjacent plants but this did not account for all new infections; the occurrence of some new infections in plants remote from infection sources suggested the possibility of a mobile vector but no such vector has been identified. Tests in a commercial hop garden, of hops on high wirework, and in a greenhouse, showed that serial cutting of hop plants 215


Table 30.2

Mechanical inoculation of HLVd between field-grown plants cv. ‘Wye Challenger’ by serial cutting with a razor blade or rubbing together of the stems (plants infected the year following inoculation/plants inoculated) (from Adams et al. 1996).

Year/ type of inoculation

Apr

May

Jun

Jul

Aug

Sep

1992/cut

-

8/10

1/6

0/8

0/10

0/10

1992/rub

-

2/9

0/6

0/8

0/10

0/10

1993/cut

2/14

19/28

2/13

1/14

0/14

-

was a more efficient means of transmitting HLVd between hop plants than rubbing bines together, to mimic plant to plant contact during normal growth (Adams et al. 1996). Furthermore, plants that were infected in the field could only be detected in the year after inoculation and then only if they were inoculated early in the growing season (see Table 30.2). This could partly be due to slow replication and movement of the viroid. The above ground part of the plant is removed at harvest or when tidying the hop garden during the early winter. For inoculations late in the season, therefore, infected tissue may have been removed before the viroid reached the roots. These results agree with the observations on ‘natural’ spread above, which indicated that contact between the bines of plants in the field was not important in spreading the viroid. Also, in the UK, plants are traditionally ‘trained’ in late April and early May, when transmission by cutting is most efficient. The removal of excess growth by hand pulling, which is probably not conducive to spread as infected material is discarded, was the most common practice in the UK in the 1980s and early 1990s and this probably served to limit spread. Most growers in the UK now use chemical dessication of excess shoots and the influence of this procedure on spread is unknown. In greenhouse experiments, mechanical inoculation was not only more efficient but could also occur later in the season (Adams et al. 1996). This was probably due to the higher average temperatures favoring faster replication and movement within the plants and suggests that rates of spread may be higher in warm countries. In the UK, cultural practices are now very different in many hop gardens. Dwarf varieties are widely grown, and the old bines are not cut at harvest or removed during the winter. There are also significant plantings in some countries on low (2–3 m high) trellises. The results of Adams et al. (1996) were obtained in the UK, with varieties growing on traditional high (5 m) wirework. Transmission rates are likely to vary with different cultural practices, different climates and varieties. As mentioned above, Morton et al. (1993) found that HLVd titers were relatively high near the bases of the bines but apparently absent higher in the plant early in the UK growing season. In Germany, HLVd was detected in the shoot tips in May (Knabel et al. 1999) and this may reflect the effects of a warmer climate, earlier growing season and a more sensitive detection method 216

than that used by Morton et al. (1993). As the growing season progresses, HLVd becomes fully systemic. The timing of cultural operations is therefore probably pivotal to the efficiency of transmission as well as the precise method used. Any operation that wounds the plant might lead to transmission and some unexpected routes of transmission may be found (e.g. sheep are allowed to graze the lower leaves of hop plants in Australia and New Zealand (Neve 1991) and might act as vectors). Establishing healthy stocks is clearly an essential prerequisite to any stock replacement scheme. Selection of uninfected material is normally the most effective approach but it may not be possible to find plants of some varieties that are not infected with HLVd. Therapy may therefore be required to provide viroidfree planting stock. Hataya et al. (1992) apparently grew viroidfree plants of two hop cultivars from 0.2 mm explants taken from HLVd-infected mother plants. Moreton et al. (1993) grew plants from shoot tips cut from viroid-infected plants but although HLVd could not be detected by dot-blot or in situ hybridization in 1 mm shoot tips cut from plants grown at 10 or 15°C, none of the 0.5 mm explants cut from such plants were free from HLVd. However, HLVd-free plants were successfully grown from explants of 300 nt)

CEVd minus-strand

tomato

Progeny accumulation slightly suppressed

Not tested

Atkins et al. (1995)

anti-sense RNA + ribozyme

CEVd minus-strand (3 GUC trinucleotides)

tomato

Progeny accumulation slightly suppressed

Not tested

“

anti-sense RNA (>300 nt)

CEVd plus-strand

tomato

Progeny accumulation enhanced

Not tested

anti-sense RNA + ribozyme

CEVd plus-strand (3 GUC trinucleotides)

tomato

Progeny accumulation enhanced

Not tested

anti-sense (9–11 nt) + ribozyme

PSTVd minus-strand (GUC trinucleotide)

potato

23/34 lines resistant Progeny accumulation suppressed

Not tested

anti-sense (9–11 nt) + ribozyme

PSTVd plus-strand (GUC trinucleotide)

potato

2/6 lines resistant

Not tested

pac1 dsRNase

replicative intermediates (PSTVd ?)

potato

33–90% plants resistant (5 transgenic lines)

Resistance stably maintained

Progeny accumulation suppressed

346

Yang et al. (1997)

Sano et al. (1997)

BIOTECHNOLOGICAL APPROACHES FOR CONTROLLING VIROID DISEASES

ribonuclease specific for dsRNA can inhibit viroid replication, sometimes quite dramatically. Figure 55.1 schematically illustrates the various anti-viroid resistance strategies tested to date, and Table 55.1 summarizes the results achieved. Whether a single strategy can confer durable resistance in agricultural applications has yet to be determined. As suggested by Hammond (1997), a combined strategy (i.e. expression of a dsRNA-specific ribonuclease to attack invading viroid plus-strands in the cytoplasm and the concurrent expression of ribozymes targeting the minus-strand RNAs used as template for progeny synthesis in the nucleus) could produce more effective resistance than either strategy alone. RNA silencing effectively inhibits the replication of many different plant viruses (Ratcliff et al. 1997; Al-Kaff et al. 1998). Most plant viruses replicate in the cytoplasm, and even for those DNA-containing viruses that replicate in the nucleus, viral mRNAs must be transported to the cytoplasm for translation. If the RNA degradation triggered by RNA silencing occurs in the cytoplasm, members of Pospiviroidae may be susceptible to RNA silencing only during the brief period of transport to and from the nucleus. ASBVd and Peach latent mosaic viroid replicate and accumulate in the chloroplast. As described by Desjardins (1987), ASBVdinfected trees may undergo a spontaneous recovery process that is strikingly similar to the disappearance of viral symptoms associated with RNA silencing. All typical disease symptoms disappear, the rate of ASBVd seed transmission increases from ≤5% to 90–100%, and the resulting ‘symptomless carrier’ phenotype is stably inherited. Conceptually, it would then appear that highly structured viroid RNAs are potentially capable of inducing silencing. Because viroids encode no protein suppressors of the silencing process (Voinnet et al. 1999; Carrington et al. 2001), RNA silencing might be expected to inhibit viroid infections even more effectively than those caused by viruses. Members of Pospiviroidae may be inherently weak inducers of RNA silencing; if so, simultaneous expression of both sense and antisense RNAs may yield more effective resistance (Waterhouse et al. 1998). Alternatively, their rod-like, highly base-paired secondary structure could inhibit interaction with the small 21–23 nt RNA molecules responsible for conferring sequence specificity on the RNase that degrades target sequences. To date, efforts to create resistance to viroid diseases have targeted the replication process, attempting to inactivate incoming infectious plus-strand RNAs or disrupt the formation of double-stranded RNA replicative intermediates. Interfering with a viroid’s ability to move — from the cytoplasm to the nucleus prior to replication, from cell to cell via the plasmodesmata, or long distances via the phloem — may also lead to resistance. Mutations in the right terminal loop have been shown to interfere with normal intercellular movement of PSTVd (Hammond

1994) as well as its ability to interact with tomato viroid-binding protein X1 (Sägesser et al. 1997; Martinez de Alba et al. 2000). Likewise, nuclear import of PSTVd is a specific, carrier-mediated process that is dependent on specific signals not present in viroids that replicate in the chloroplast (Woo et al. 1999; Zhao et al. 2001). PSTVd also contains signals that allow it to move from cell to cell (Ding et al. 1997). Fundamental studies are currently under way to identify these signals. PSTVd (and presumably other viroids) move long distances in the photoassimilate stream (Palukaitis 1987; Zhu et al. 2001). Results from in situ hybridization analyses suggest that its ability to enter and exit different cell types in the phloem is under strict developmental control. While in the phloem, viroids may bind to phloem protein 2 (PP2), a dimeric lectin and most abundant component of phloem exudate (Owens et al. 2001; Goméz and Pallás 2001). Phloem protein 2 moves from cell to cell via plasmodesmata as well as long distances in the phloem; thus, the ability to bind to phloem protein 2 may facilitate the systemic movement of viroids (and possibly other RNAs) in vivo. Efforts to block viroid movement by inhibiting the interaction with phloem protein 2 are complicated by the large amounts of this protein in the sieve elements. In addition to inhibiting viroid replication, one might also consider trying to block disease development. A variety of evidence suggests that protein phosphorylation plays an important role in viroid pathogenicity (Hiddinga et al. 1988; Diener et al. 1993), and Hammond and Zhao (2000) have recently characterized a tomato protein kinase gene [i.e. pkv(iroid)] that is transcriptionally activated by PSTVd infection. Expression of pkv antisense RNA using a viral-based vector results in a marked suppression of the stunting and epinasty characteristic of viroid infection (Zhao and Hammond, unpublished). Rather than decreasing or eliminating viroid infection, widespread cultivation of varieties in which infection is latent would favor an increase in viroid prevalence. To date, only a few of many possible strategies to use biotechnology to create viroid-resistant crop plants have been tested. This brief review has highlighted some of the challenges and opportunities involved in solving problems that have proven intractable by conventional means. References Al-Kaff, N.S., Covey, S.N., Kreike, M.M., Page, A.M., Pinder, R., and Dale, P.J. (1998). Transcriptional and post transcriptional plant gene silencing in response to a pathogen. Science 279, 2113-2115. Atkins, D., Young, M., Uzzell, S., Kelly, L., Fillatti, J., and Gerlach, W. L. (1995). The expression of antisense and ribozyme genes targeting exocortis viroid in transgenic plants. J. Gen. Virol. 76, 1781-1790. Branch, A. D., and Robertson, H. D. (1984). A replication cycle for viroids and other small infectious RNAs. Science 223, 450-455.

347

T. Sano et al.

Carrington, J. C., Kasschau, K. D., and Johansen, L. K. (2001). Activation and suppression of RNA silencing by plant viruses. Virology 281, 1-5. Desjardins, P. R. (1987). Avocado sunblotch. Pages 299-313 in: The viroids. T.O. Diener, ed. Plenum Press: New York. Diener, T. O., Hammond, R. W., Black, T., and Katze, M. G. (1993). Mechanism of viroid pathogenesis: differential activation of the interferon-induced, double-stranded RNA-activated, M r 68 000 protein kinase by viroid strains of varying pathogenicity. Biochemie 75, 533-538. Ding, B., Kwon, M-O., Hammond, R.W., and Owens, R. (1997). Cell-to-cell movement of potato spindle tuber viroid. Plant J. 12, 931-936. Dougherty, W. G., Lindbo, J. A., Smith, H.A., Parks, T. D., Swaney, S., and Proebsting, W. M. (1994). RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation. Mol. Plant-Microbe Interact. 7, 544-52 Gómez, G., and Pallás, V. (2001). Identification of an in vitro ribonucleoprotein complex between a viroid RNA and a phloem protein from cucumber plants. Mol. Plant-Microbe Inter. 14, 910-913. Hadidi, A., Hashimoto, J., and Diener, T.O. (1982). Potato spindle tuber viroid-specific double-stranded RNA in extracts from infected leaves. Ann. Virol. 133E, 15-31. Hammond, J. (1997). Repelling plant pathogens with ribonuclease. Nature Biotechnology 15, 1247. Hammond, J., Lecoq, H., and Raccah, B. (1999). Epidemiological risks from mixed virus infections and transgenic plants expressing viral genes. Advances in Virus Research 54, 189-314. Hammond, R. W. (1994). Agrobacterium-mediated inoculation of PSTVd cDNAs onto tomato reveals the biological effect of apparently lethal mutations. Virology 201, 36-45. Hammond, R. W., and Zhao, Y. (2000). Characterization of a tomato protein kinase gene induced by infection by Potato spindle tuber viroid. Mol. Plant-Microbe Inter. 13, 903-910. Hammond-Kosack, K. E., and Jones, J. (1996). Resistance gene-dependent plant defense responses. The Plant Cell 8, 1773-1791. Harders, J. Lukács, N., Robert-Nicoud, M., Jovin, T. M., and Riesner, D. (1989). Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy. EMBO J. 8, 3941-3949. Hiddinga, H. J., Crum, C. J., and Roth, D. A. (1998). Viroid-induced phosphorylation of a host protein related to a dsRNA-dependent protein kinase. Science 241, 451-453. Hilleren, P., and Parker, R. (1999). Mechanisms of mRNA surveillance in eukaryotes. Annu. Rev. Genet. 33, 229-260. Itaya, A., Folimonov, A., Matsuda, Y., Nelson, R.S., and Ding, B. (2001). Potato spindle tuber viroid as inducer of RNA silencing in infected tomato. Mol. Plant-Microbe Inter. 14, 1332-1334. Jones, A. L. Thomas, C. L., and Maule, A. J. (1998). De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus. EMBO J. 17, 6835-6393. Keese, P., and Symons, R.H. (1985). Domains in viroids: Evidence of intermolecular RNA rearrangement and their contribution to viroid evolution. Proc. Natl. Acad. Sci. USA 82, 4582-4586. Lima, M. I., Fonseca, M. E. N., Flores, R., and Kitajima, E. W. (1994). Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization. Arch. Virol. 138, 385-390.

348

Lindbo, J. A., and Dougherty, W. G. (1992). Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts. Virology 189, 725-33. Loss, P., Schmitz, M., Steger, G., and Riesner, D. (1991). Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA. EMBO J. 10, 719-727. Martinez de Alba, A.E. Maniataki, E., Denti, M.A., Sägesser, R., Tabler, M., and Tsagris, M. (2000). Characterization of the interaction of the viroid binding protein X1 with potato spindle tuber viroid (PSTVd). Page 12 in: Plant virus invasion and host defence (EMBO Workshop, Kolymbari, Greece). Matousek, J., Schröder, A. R. W., Trnená, L., Reimers, M., Baumstark, T., Dêdiˆc, P., Vlasàk, J., Becker, I., Kreuzaler, F., Fladung, M., and Riesner, D. (1994). Inhibition of viroid infection by antisense RNA expression in transgenic plants. Biol. Chem. Hoppe-Seyler 375, 765-77. Niblett, C. L., Dickson, E., Fernow, K. H., Horst, R. K., and Zaitlin, M. (1978). Cross protection among four viroids. Virology 91, 198-203. Ogawa,T., Hori, T., Tsukahara, M., Yoshioka, M., Ishida, K., Kakitani, M., and Toguri, T. (1998). Transgenic chrysanthemum expressing a double stranded RNA specific ribonuclease is resistant to chrysanthemum stunt viroid. Ann. Phytopathol. Soc. Japan 64, 425. Owens, R. A., Blackburn, M., and Ding, B. (2001). Possible involvement of the phloem lectin in long-distance viroid movement. Mol. PlantMicrobe Inter. 14, 905-909. Palukaitis, P. (1987). Potato spindle tuber viroid: Investigation of the long-distance, intra-plant transport route. Virology 158, 239-241. Papaefthimiou, I., Hamilton, A.J., Denti, M.A., Baulcombe, D.C., Tsagris, M., and Tabler, M. (2001). Replicating potato spindle tuber viroid RNA is accompanied by short RNA fragments that are characterisitic of post-transcriptional gene silencing. Nucleic Acids. Res. 29, 2395-2400. Pélissier, T., and Wassenegger, M. (2000). A DNA target of 30 bp is sufficient for RNA-directed DNA methylation. RNA 6, 55-65. Ratcliff, F., Harrison, B. D., and Baulcombe, D. C. (1997). A similarity between viral defense and gene silencing plants. Science 276, 1558-1560. Sanford, J. C., and Johnston, S. A. (1985). The concept of parasitederived resistance – deriving resistance genes from the parasite’s own genome. J. Theor. Biol. 113, 395-405. Sägesser, R., Martinez, E., Tsagris, M., and Tabler, M. (1997). Detection and isolation of RNA-binding proteins by RNA-ligand screening of a cDNA expression library. Nucleic Acids Res. 25, 3816-22. Sano, T., Nagayama, A., Ogawa, T., Ishida, I., and Okada, Y. (1997). Transgenic potato expressing a double-stranded RNA-specific ribonuclease is resistant to potato spindle viroid. Nature Biotechnology 15, 1290-1294. Tabler, M., Tsagris, M., and Hammond, J. (1998). Antisense RNA and ribozyme-mediated resistance to plant viruses. Pages 79-83 in: Plant virus disease control. A. Hadidi, R. K. Khetarpal, and H. Koganezawa, eds. APS Press: St. Paul, MN. Vaucheret, H., and Fagard, M. (2001). Transcriptional gene silencing in plants: targets, inducers, and regulators. Trends in Genetics 17, 29-35.

BIOTECHNOLOGICAL APPROACHES FOR CONTROLLING VIROID DISEASES

Voinnet, O., Pinto, Y. M., and Baulcombe, D. C. (1999). Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants. Proc. Natl. Acad. Sci. USA 96, 14147-14152. Wang, M-B., Wesley, S. V., Finnegan, E. J., Smith, N. A., and Waterhouse, P. M. (2001). Replicating satellite RNA induces sequencespecific DNA methylation and truncated transcripts in plants. RNA 7, 16-28. Wassenegger, M., Heimes, S., Riedel, L., and Sänger, H. L. (1994). RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567-576. Wassenegger, M., and Pélissier, T. (1999). Signalling in gene silencing. Trends Plant Sci. 4, 207-209. Watanabe, Y., Ogawa, T., Takahashi, H., Ishida, I., Takeuchi, Y., Yamamoto, M., and Okada, Y. (1995). Resistance against multiple plant viruses in plants mediated by a double stranded-RNA specific ribonuclease. FEBS Letters 372, 165-168. Waterhouse, P. M., Graham, M. W., and Wang, M-B. (1998). Virus resistance and gene silencing in plants can be induced by simulta-

neous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. USA 95, 13959-13964. Woo, Y-M., Itaya, A., Owens, R.A., Tang, L., Hammond, R.W., Chou, H-C., Lai, M., and Ding, B. (1999). Characterization of nuclear export of potato spindle tuber viroid RNA in permeabilized protoplasts. Plant J. 17, 627-635. Yang, X., Yie, Y., Shu, F., Liu, Y., Kang, L., Wang, X., and Tien, P. (1997). Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes. Proc. Natl. Acad. Sci. USA 94, 4861-4865. Zhao, Y., Owens, R. A., and Hammond, R. W. (2001). Use of a vector based on Potato virus X in a whole plant assay to demonstrate nuclear targeting of Potato spindle tuber viroid. J. Gen. Virol. 82, 1491-1497 Zhu, Y., Green, L., Woo, Y-M., Owens, R. A., and Ding, B. (2001). Cellular basis of potato spindle tuber viroid systemic movement. Virology 279, 69-77.

349

PART VIII

CHAPTER 56

RIBOZYME REACTIONS OF VIROIDS ....................................................................................................

F. Côté, M. De la Peña, R. Flores, and J.P. Perreault

.................................................................................................................................................................................................................................................................

Viroids replicate through a DNA-independent rolling-circle mechanism involving the synthesis of multimeric strands of both polarities and their subsequent cleavage into monomeric fragments, which are then circularized to produce the progeny (Branch and Robertson 1984). Depending on whether or not the minus multimeric strands are cleaved and ligated to unitlength circular strands, which are then used as templates for the second half of the cycle, the viroid RNA is considered to replicate by either a symmetric or asymmetric mode (see Chapter 5 ‘Replication’). While processing of the multimeric plus RNA intermediates is generally believed to require a host ribonuclease for the members of the family Pospiviroidae (formerly known as group B viroids), this step is autocatalytic and mediated by hammerhead ribozymes in members of the family Avsunviroidae (formerly known as group A viroids) (reviewed in Symons 1989; Flores et al. 1998; see Chapter 8 ‘Classification’). However, the possibility has been raised that the processing step is RNA-catalyzed in all cases (reviewed in Symons 1997). The three viroid species within the family Avsunviroidae known to date, Avocado sunblotch viroid, ASBVd (Symons 1981; Hutchins et al. 1986), Peach latent mosaic viroid, PLMVd (Hernández and Flores 1992; Shamloul et al. 1995) and Chrysanthemum chlorotic mottle viroid, CChMVd (Navarro and Flores 1997), can adopt ham-

350

merhead structures on their plus and minus polarity strands and, as a consequence, they are presumed to replicate according to the symmetric rolling-circle mechanism. The hammerhead structures appear as the only ‘homologous molecular characters’ shared by these viroids. Briefly, the hammerhead structure is a small RNA motif consisting of three sequence non-specific helices bordering a catalytic core of 11 conserved residues which form a complex array of non-canonical interactions (Prody et al. 1986; Hutchins et al. 1986; Forster and Symons 1987; Pley et al. 1994) (see Figure 56.1). The adoption of this structure in the presence of a divalent cation, usually magnesium, results in the self-cleavage of the RNA chain at a specific phosphodiester bond creating 2',3'-cyclic phosphate and 5'-hydroxyl termini. Biochemical knowledge in respect to both the detailed structural features and a molecular mechanism of the hammerhead structures has been reviewed recently (Flores et al. 2000; StageZimmermann and Uhlenbeck 1998) and, therefore, will not be the main focus of this chapter that primarily aims to present the hammerhead structure as an essential molecular feature of the Avsunviroidae members and, particularly, of their replication cycle. We will also consider the potential of this self-cleaving motif to act in trans targeting cellular RNA and, more specifically, to contribute to viroid pathogenesis.

RIBOZYME REACTIONS OF VIROIDS

Figure 56.1 Consensus hammerhead structure derived from 23 natural hammerhead sequences, schematically represented as originally proposed with its numbering system and names of helices and loops (Hertel et al. 1992) (left), and according to X-ray crystallography data (Pley et al. 1994) (right). Letters on a dark background refer to absolutely conserved residues in all natural hammerhead structures and N to residues involved in Watson-Crick base pairs. Arrows indicate self-cleavage sites. Watson-Crick base pairs and non-canonical interactions are denoted with continuous and broken lines, respectively.

HAMMERHEAD STRUCTURES OF VIROIDS: MOLECULAR ARCHITECTURE

Figure 56.2 shows the six hammerhead structures described so far in viroids. In ASBVd, the sequences involved in hammerhead structures of both polarities are found in the upper and lower strands of the central domain of the quasi-rod-like secondary structure proposed for this viroid, with the remaining nucleotides of the genome, referred here as ‘extracatalytic’ RNA sequences, flanking the central domain (Figure 56.2A). Therefore, the sequences forming the catalytic core are not contiguous but segregated into two subdomains. In contrast, the sequences involved in the hammerhead structures of PLMVd and CChMVd are contiguous and located in an arm of their proposed branched conformation, with the ‘extracatalytic’ RNA sequences constituting the rest of the genomes (Figure 56.2B and C). There are two classes of viroid hammerhead structures according to their morphology. The monomeric strands of PLMVd and CChMVd can adopt stable hammerhead structures with helices I and II of five-six base pairs closed by short loops 1 and 2 (the CChMVd minus hammerhead structure is an exception in having an unusually long imperfect helix II), and helices III of six-eight base-pairs (Figure 56.2B and C). Conversely, the hammerhead structures that can form the monomeric ASBVd RNAs are thermodynamically unstable, particularly in the plus polarity strand with a stem III of only two base pairs closed by a loop 3 of three residues (Figure 56.2A). This very different architecture of the viroid hammerhead structures has major implications for their in vitro and in vivo self-cleavage efficiency (see section below).

Inspection of natural hammerhead structures shows that they are characterized by a central core with a cluster of strictly conserved nucleotide residues flanked by three double-helix regions (i.e. stems I, II and III) with loose sequence conservation except at positions 15.2 and 16.2, which in most cases form a C-G pair, and positions 10.1 and 11.1, which in most cases form a G-C pair (Figure 56.2). Some viroid hammerhead structures present unusual features. For example, a transition U to C affecting the conserved U4 in the plus hammerhead structure has been observed in a sequence variant of PLMVd (Ambrós and Flores 1998). On the other hand, the common C17 preceding the minus self-cleavage site is A in a sequence variant of ASBVd, and the common pyrimidine residue at position 7 is substituted by an A in the minus hammerhead structure of another ASBVd variant (Rakowski and Symons 1989). An extra A between A9 and G10.1 of the plus hammerhead structure of CChMVd has been also reported (Figure 56.2C). This extra residue, which is compatible with extensive in vitro self-cleavage, could either induce a rearrangement of the junction between helix II and three adjacent non-canonical interactions of the central core, or be accommodated as a bulging residue. These and other sequence variations in the hammerhead structures retrieved in nature from different self-cleaving RNAs have been compiled recently (see Flores et al. 2000). The conservation of the sequences forming the hammerhead structures in the Avsunviroidae members, as well as in most other hammerhead structures known so far, extend beyond the strict requirements for selfcleavage, suggesting that additional selective pressures may act on these sequences. However, the identity of any other selective pressure remains unidentified.

351

F. Côté et al.

Figure 56.2 Nucleotide sequence and alternative secondary structure of the hammerhead self-catalytic motifs of ASBVd (A), PLMVd (B) and CChMVd (C). At the left, delimited by flags, are represented the fragments of the most stable secondary structure proposed for these viroids that contain the sequences involved in forming both polarity hammerhead structures. Extracatalytic sequences are indicated by broken lines, conserved hammerhead residues by bars and self-cleavage sites by arrows. Closed and open symbols refer to plus and minus polarities, respectively. Within the right panels are the plus and minus hammerhead sequences folded into their active secondary structures. Stems I, II and III are shown and the arrowheads indicate self-cleavage sites. Letters on a dark background refer to conserved hammerhead residues. In the case of ASBVd, the double hammerhead structures are also shown. Sequence were retrieved from the viroid and viroid-like database (http://www.callisto.si.usherb.ca/ ~jpperra; Pelchat et al. 2000b). Other details as in Figure 56.1.

CIS-ACTING HAMMERHEAD STRUCTURES OF VIROIDS: IN VITRO AND IN VIVO FUNCTION The similarities found between the plus and minus hammerhead sequences and their genomic organization within each viroid most likely have physical and functional consequences. The PLMVd and CChMVd sequences spanning the two ham-

352

merhead domains are almost complementary and can fold into structures with long double-stranded regions; this is the typical arrangement found in the most stable secondary structures predicted for the two viroids (Figure 56.2B and C). These stable arrangements, formed by the superposition of the hammerhead sequences of both polarities, have the potential to prevent the


adoption of the active hammerhead foldings, which are alternative structures of higher energy (Hernández and Flores 1992; Beaudry et al. 1995; Navarro and Flores 1997). More importantly, self-cleavage inhibition permits the accumulation of certain levels of the viroid monomeric circular forms, which are the templates of the rolling-circle mechanism of replication. In addition, the compact non-self-cleaving structures may contribute positively to the extra- (e.g. during transmission) and intracellular stability of these RNA species. The peculiar organization of the hammerhead sequences may also be informative concerning mechanistic requirements. For example, these RNAs may need to have similar hammerheads in order to perform in vivo self-cleavage to essentially the same extent in both strands, as appears to be the case in PLMVd (Bussière et al. 1999). Interactions with cellular components (e.g. proteins) enhancing self-cleavage may have promoted conservation of similar hammerheads. Therefore, a complex synergy between the stability of the viroid RNA as a whole, and the mechanisms of self-cleavage regulation, has probably contributed to the emergence of the superimposed hammerhead sequences. As already indicated, the ability of viroid RNAs that possess autocatalytic sequences to self-cleave depends on their adoption of a conformation different from the most stable structure (Figure 56.2). Self-cleavage of viroid strands occurs at either single or double hammerhead structures depending on whether or not the sequences can form stable helices surrounding the catalytic core. Whereas the six hammerhead structures of viroids have stable helices I and II, this is not the case for helix III. Both polarity hammerhead structures of PLMVd have stable helices III and selfcleave in vitro, and most likely in vivo, through single hammerhead structures (Hernández and Flores 1992; Beaudry et al. 1995) (Figure 56.2B). This is also probably the case with the two hammerhead structures of CChMVd, which also have stable helices III (Navarro and Flores 1997), although the extended helix II of the minus hammerhead structure might facilitate the adoption of alternative foldings inactive for self-cleavage (Figure 56.2C). In contrast, the single hammerhead structures of ASBVd have unstable helices III closed by short loops, and their self-cleavage is assumed to occur via double hammerhead structures involving longer-than-unit RNAs that allow stabilization of the catalytic core (Forster et al. 1988) (Figure 56.2A). ASBVd plus strands selfcleave through a double hammerhead structure during in vitro transcription and after gel purification, whereas ASBVd minus strands self-cleave via a double hammerhead structure during in vitro transcription, but mostly via a single hammerhead structure after gel purification (Davies et al. 1991). This is most probably the consequence of the different stability of helix III in both hammerhead structures. Direct enzymatic sequencing and primer extension experiments have shown that in vitro self-cleavage of ASBVd, PLMVd and CChMVd occurs at the positions predicted by the hammerhead

structures (Hutchins et al. 1986; Hernández and Flores 1992; Navarro and Flores 1997). The efficiency of the corresponding in vitro self-cleavage reactions can be high; for example, around 50–60% of PLMVd strands self-cleave under standard conditions (Hernández and Flores 1992; Beaudry et al. 1995). However, this efficiency is strongly increased (>95%) when the same RNAs are transcribed under conditions of slow polymerase activity, which favors the adoption of the active hammerhead structures catalyzing self-cleavage reactions (Bussière 1999). The self-cleavage efficiency is also strongly dependent on divalent ions such as Mg2+. There is also solid evidence supporting the involvement of hammerhead structures in the in vivo processing of viroid RNAs with these catalytic domains. For ASBVd (Daròs et al. 1994; Navarro and Flores 2000), CChMVd (Navarro and Flores 1997), and PLMVd (C. Hernández, unpublished data), linear RNAs of one or both polarities with 5’-termini identical to those generated in the corresponding in vitro self-cleavage reactions have been isolated from infected tissues. Moreover, the frequent occurrence in sequence variants of PLMVd (Hernández and Flores 1992; Beaudry et al. 1995; Ambrós et al. 1998) and CChMVd (De la Peña et al. 1999) of compensatory mutations or covariations that preserve the stability of the hammerhead structures, further support their in vivo role, as also does the correlation existing between the infectivity of different PLMVd and CChMVd variants and the extent of their self-cleavage during in vitro transcription (Ambrós et al. 1998; De la Peña et al. 1999). In vivo, self-cleavage of viroid strands should be under regulation, with two different mechanisms appearing to operate for this purpose. In the case of PLMVd and CChMVd, their most stable secondary structures are transiently lost during transcription with the concurrent adoption of the active single hammerhead structures that promote self-cleavage before synthesis is completed and the most stable secondary structures are reformed. In ASBVd self-cleavage of monomeric strands is restricted because the single hammerhead structures are unstable whereas the multimeric replicative intermediates can adopt stable double hammerhead structures and self-cleave to their unit-length strands. Therefore, in both situations the hammerhead ribozymes are active only during replication. Self-cleavage of PLMVd RNAs in vivo appears almost optimal reaching near total processing of the multimeric strands into their corresponding monomeric units (Bussière et al. 1999). For the reasons stated above, this high efficiency may be the result of slow progession of the host RNA polymerase during replication. The situation seems similar in the case of CChMVd, for which the predominant RNAs accumulating in infected cells are also the monomeric linear strands of both polarities (Navarro and Flores 1997). In contrast, the most abundant ASBVd RNA in infected tissue is the plus circular monomer, a clear indication of the low efficiency of the corresponding single hammerhead structure,

353

F. Côté et al.

Figure 56.3 Schematic representation of trans-acting hammerhead ribozymes. A. Two different formats depending on where the separation between the ribozyme itself (black letters) and the substrate (grey letters) is established. B. A hammerhead ribozyme targeting a long mRNA substrate. Letters on a dark background refer to conserved hammerhead residues. Other details as in Figures 56.1 and 56.2.

although decreasing levels of multimeric strands up to octamers in size have been also detected (Bruening et al. 1982).

VIROID HAMMERHEAD STRUCTURES: POTENTIAL FOR TRANS-ACTING FUNCTION In their natural context, the hammerhead structures of viroids operate in cis mediating the self-cleavage of the RNAs in which they are contained. However, active hammerhead structures can also be formed by annealing two different RNA fragments in trans, such that one RNA fragment acts as the ribozyme and the other as the substrate (see Figure 56.3A). If the complementary regions between the two RNAs are short enough, the cleavage products will dissociate from the ribozyme, thus permitting the binding of new substrate molecules. Via successive rounds of binding and cleavage a single ribozyme molecule can therefore cleave many substrate molecules, thereby establishing a classic enzyme/substrate relationship (Uhlenbeck 1987). Furthermore, by changing the complementary sequences between the ribozyme and its substrate, it is possible to create a ribozyme with new substrate specificity. A wide variety of RNAs can be 354

targeted for cleavage by such engineered ribozymes (Figure 56.3B). Because of their ability to interact directly with RNA, ribozymes, particularly those of the hammerhead class, are currently being developed as potential therapeutic agents for a wide range of applications based on the specific cleavage of different RNAs of biological relevance including viroids themselves (see the preceding Chapter ‘Biotechnological approaches for controlling viroid diseases’). In the coming years, altered forms of these versatile molecules will surely emerge as a new class of drugs. Apart from these applications, a detailed description of which falls outside the scope of this chapter, we will consider the possibility that viroid pathogenesis of members of the family Avsunviroidae could result from trans cleavage of host RNAs recognized by the hammerhead ribozymes (Symons 1989). No supporting evidence for such a mechanism has been reported yet. The following discussion is based on experiments performed with PLMVd as a model viroid in an attempt to put this intriguing hypothesis to test (Côté 2000). Minimal artificial hammerhead ribozymes are prefolded into a quasi catalytically


active structure and following the substrate binding, which involves the formation of helixes at both sides of the cleavage site, the break of the scissile bond occurs. Natural hammerhead structures are integral features of viroid RNAs. In the most stable secondary structures of these RNAs, the hammerhead catalytic core is not formed because this is not the most stable structure. For example, active PLMVd hammerhead structures of both polarities are adopted either during the in vitro transcription or by a prior heat denaturation coupled to a snap-cooling treatment, that favor these active structures over others more stable but lacking catalytic activity. Therefore, the knowledge acquired from studies with minimal hammerhead ribozymes can not be simply extended to situations in which this catalytic motif is included in full-length viroid RNAs. In order to compare the cleavage efficiency of a hammerhead catalytic sequence as a model molecule or as part of a viroid, a series of experiments were performed in which four PLMVdderived transcripts, acting as the ribozyme, were tested for their ability to catalyze the cleavage of a short substrate (Côté 2000). As expected, no cleavage products were detected when the ribozyme was a PLMVd 250-nt transcript lacking the hammerhead sequences, whereas most of the substrate (>85%) was cleaved by a ribozyme with the sequences corresponding to the minimal plus hammerhead structure. This efficient cleavage probably results from the absence of extra sequence interfering with the adoption of the catalytically active folding. When the ribozyme was composed of a full-length PLMVd RNA circularized in vitro to have either a 3’,5’- or a 2’,5’-phosphosdiester bond at the self-cleavage site, only a trace amounts of product (

Viroids

Recommend Documents