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Seed-borne Plant Virus Diseases K. Subramanya Sastry Seed-borne Plant Virus Diseases 123 K. Subramanya Sastry Emeritus Professor Department of Virology S.V. University Tirupathi, AP India ISBN 978-81-322-0812-9 ISBN 978-81-322-0813-6 (eBook) DOI 10.1007/978-81-322-0813-6 Springer New Delhi Heidelberg New York Dordrecht London Library of Congress Control Number: 2012945630 © Springer India 2013 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) About the Author Prof. K. Subramanya Sastry, Ph.D., is emeritus professor in the department of virology at S.V. University, Tirupathi – 517502, A.P., India. (India). He obtained his M.Sc. (Botany) and Ph.D. (Botany) with plant virology specialization in 1966 and 1973, respectively, from Sri Venkateswara University, Tirupati (India). He joined Indian Council of Agricultural Research (ICAR) as a scientist (Plant Virology) during the year 1971 and retired in 1999. He has served at Indian Institute of Horticultural Research, Hessaraghatta, Bangalore 560 089, Karnataka, and also at Directorate of Oil Seeds Research, Hyderabad 500 080, Andhra Pradesh. Prof. Sastry’s research has been primarily on epidemiology and management of virus and virus-like diseases of Horticultural and Oil Seed crops. He has done pioneer research on Begomoviruses of tomato and okra. He has research experience in molecular and biotechnological approaches for characterization and control of viral diseases of horticultural crop plants. Further, he has also published over 120 research papers both in national and international journals. He has also published “Compendium of the Plant Virus Research in India (1903–2008)” having 8,652 plant virus references that is considered as one of the rich sources of information for Indian plant virus research. v Foreword I am delighted to write the foreword for a book on seed-borne viruses, since many economically important viral diseases are spread in nature through seeds. Increasing our knowledge on all aspects of seed-borne viruses is the first step towards developing a strategy for their control. This book represents a comprehensive up-to-date treatise for seed-borne viruses, including detection methods, ecology, epidemiology and control. Attention is also placed on the importance of integrated management to reduce losses caused by seedborne viruses. I congratulate Dr. K. S. Sastry for his sincere effort and many years of hard work to assemble existing information on seed-borne viruses and make them available in a well organized manner to a wide audience: research scientists, graduate students, extension workers, progressive farmers as well as individuals who are interested in agricultural production at large. I am confident that this book will serve as an important reference for seed-borne viruses which affect agricultural crops, globally. Legume crops are the main source of protein for the majority of people in developing countries. Around 50% of the viruses which infect legumes are seed-borne, and some of them could lead to a complete crop failure. Improving our knowledge on these viruses can be well translated to improved legume crops production, worldwide. It is hoped that this book will serve as an important resource for all agricultural workers dedicated to improved and stabilized crop production through adoption of environment-friendly practices, including the use of virus-free seeds. Khaled M. Makkouk Advisor for Agriculture and Environment National Council for scientific Research (CNRS) P.O. Box 11-8281 Riad El-Solh 1107 2260, Beirut, LEBANONCNRS, Beirut, Lebanon vii Preface Seed, a highly ordered plant structure, is the basic input in crop production. It possesses the qualities necessary for cell division, morphogenesis and regeneration of species. The study of seed itself is as good as the study of life. Seed is one of the vital inputs in the development of agriculture in any country. To increase agricultural production, viability of quality seed is one of the prerequisites. The seed is also one of the most important source for the perpetuation of fungi, bacteria, nematodes, insects, viruses, etc. and acts as an efficient carrier for their spread to new areas through introduction and/or seed trade which is a global enterprise. Among these plant pathogens, viruses are unique in nature and behaviour. As is the case with any seedtransmitted plant pathogens, virus transmission through seeds of higher plants also result from complex interactions between the genetic systems of the host, pathogen and the environment. There is increasing awareness of seedtransmitted viruses with particular reference to their mode of transmission, survival and management. Till date, more than 231 viruses have been reported to be seed transmitted in different food, fiber, weed and ornamental crops. Virus-free seed has assumed multifold significance in quarantine and seed certification for ensuring initial crop health. Circumstantial evidence shows that several viruses have spread to different geographical regions during the process of liberalized seed exchange of crop plants in recent years. Seeds are instrumental in an effective worldwide spread of a range of diseases through international exchange of seeds. The techniques of identification and management of seed-transmitted viruses are completely different from those of other pathogens like fungi, bacteria and phytoplasmas. The information on reliable techniques for detection of seed-transmitted viruses and their management is of immense use in the present international seed trade. The coverage of seed-transmitted plant viruses is limited to few chapters in books on seed pathology. Considerable information in respect of new seedtransmitted viruses, their detection and identification techniques, transmission and management has been generated in recent years. This publication is an endeavor to compile up-to-date literature available on seed-transmitted viruses in a comprehensive form. It is hoped that this book will have an important role to play in the context of the government agencies on new seed policy for liberal import of the seeds of coarse cereals, oilseeds and pulses. The knowledge of seed-transmitted virus diseases on the isolation and identification of viruses in fresh seed lots and their management not only restricts the entry of virus diseases but also will help to prevent the spread ix x Preface of unrecorded virus diseases in the country. Within the scope of this book, elaborate attempts have been made to present a comprehensive account on identification, mode of transmission, ecology, epidemiology and management of seed-transmitted virus and viroid diseases covered in ten chapters. An up-to-date list of all seed-transmitted viruses and viroids of different host plants is presented in the form of a table for ready reference. The information given on latest molecular techniques for virus detection and management included in this volume will be of immense practical value to researchers and field workers. This work has immensely benefited from critical comments and constructive suggestions made by Prof. M. V. Nayudu, Dr. S. E. Albrechtsen, Dr. P. Sreenivasulu, Dr. G. P. Rao, Dr. R. K. Khetarpal, Dr. D. V. R. Saigopal, Dr. V. C. Chalam and also assistance offered by our scholarly friends and colleagues. I am highly thankful to all the persons, organizations and various publishers for their prompt help in providing information, photographs and consents for reproduction. I also wish to express my sincere gratitude to Mr. C. Nagaraja for secretarial work. I thank my wife Mrs. B. N. K. Kumari for her continuous support during the preparation of this book. I dedicate this book to the memory of my parents late K. Panduranga Sastry and Smt. K. Subadramma who have sacrificed everything to give me the best education possible and for their eternal blessings. I hope this book will be of value and interest to many teachers, students, seed biologists, seed technologists, seed companies and researchers at quarantine stations as a comprehensive, accurate and easily readable reference book on seed-transmitted plant virus and viroid diseases. I shall deem it an honour and reward if readers find this book useful to them. I welcome suggestions and comments for the improvement of this book in future editions. K. Subramanya Sastry Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Seed (Sexual Propagule) . . . . . . . . . . . . . . . . . . . . . . 1.2 Seed Transmission of Viruses . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 History of Seed-Transmitted Plant Virus Research 1.3 Seed Transmission of Partitiviridae . . . . . . . . . . . . . . . . . . . . 1.4 Seed Transmission of Viroids . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.1 Extent of Seed Transmission . . . . . . . . . . . . . . . . . . 1.5 Viruses Erroneously Listed as Seed Transmitted . . . . . . . . . 1.5.1 Seed-Transmitted Plant Virus Names That Appeared Only Once in the Literature . . . . . . . . . . 1.5.2 Establishing Certain Erotic Positive SeedTransmitted Viruses to Be Non-seed Transmissible References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 32 2 Identification and Taxonomic Groups . . . . . . . . . . . . . . . . . . . . . . 2.1 Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Classification of Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Variability in Certain Seed-Transmitted Viruses . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 56 56 62 65 3 Economic Significance of Seed-Transmitted Plant Virus Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Assessment of Crop Losses . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Viruses and Seed Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Factors Affecting Yield Losses . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 67 67 69 69 70 Virus Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Vector Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1 Aphids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2 Beetles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3 Thrips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4 Whiteflies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.5 Mites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.6 Nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.7 Bumblebees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 75 76 77 77 78 78 78 79 4 1 1 2 2 4 5 5 6 7 8 xi xii Contents 4.1.8 Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.9 Mealybug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Nonvector Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Mechanical Spread . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2 Wind . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3 Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4 Obligate Symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 79 80 80 80 80 80 81 81 5 Mechanism of Seed Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Embryology and Development of Seed Structures . . . . . . . . 5.2 Distribution of Virus in the Seed . . . . . . . . . . . . . . . . . . . . . . . 5.3 Virus Longevity in Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Genetics of Seed Transmission . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Factors Influencing Rate of Seed Transmission . . . . . . . . . . . 5.5.1 Number of Infection Sources . . . . . . . . . . . . . . . . . . 5.5.2 Virus Strain/Isolate . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.3 Mixed Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.4 Host Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.5 Stage of Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.6 Environmental Factors . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Reasons for Failure of Seed Transmission . . . . . . . . . . . . . . . 5.6.1 Inability to Infect Embryos . . . . . . . . . . . . . . . . . . . . 5.6.2 Inability of Virus Survival in the Embryos . . . . . . . 5.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 85 87 88 88 88 90 90 90 90 92 92 93 93 94 95 95 6 Detection of Plant Viruses in Seeds . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1 Seed Health Testing . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2 Low Seed Transmission/ Symptomless Carriers . . . 6.2 Biological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1 Visual Examination . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2 Grow-Out Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.3 Indicator Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.4 Biological Properties . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Physical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.1 Inclusion Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2 Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Serological Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.1 Monoclonal and Polyclonal Antibodies . . . . . . . . . . 6.4.2 Immunodiffusion Tests . . . . . . . . . . . . . . . . . . . . . . . 6.4.3 Labelled Antibody Techniques . . . . . . . . . . . . . . . . . 6.4.4 Dot-Immunobinding Assay (DIBA or DIA) . . . . . . 6.4.5 Disperse Dye Immunoassay (DIA) . . . . . . . . . . . . . . 6.4.6 Rapid Immunofilter Paper Assay (RIPA) . . . . . . . . . 6.4.7 Immunosorbent Electron Microscopy (ISEM) . . . . 101 101 102 103 105 105 107 108 109 109 109 111 111 112 114 116 126 128 128 129 Contents xiii 6.5 7 8 Biotechnology/Molecular Biology-Based Virus Diagnosis . 6.5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.2 Molecular Hybridisation . . . . . . . . . . . . . . . . . . . . . . 6.5.3 Double-Stranded RNA (dsRNA) Analysis . . . . . . . 6.5.4 Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.5 Nucleic Acid-Specific Hybridisation . . . . . . . . . . . . 6.5.6 Array Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.7 Polymerase Chain Reaction (PCR)-Based Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.8 Real-Time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 Molecular Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 131 132 133 134 135 137 Ecology and Epidemiology of Seed-Transmitted Viruses . . . . . 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1 Primary Inoculum Source . . . . . . . . . . . . . . . . . . . . . 7.2 Host Plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.1 Factors Influencing Vector Movement . . . . . . . . . . 7.4 Pollen Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.1 Epidemiological Role of Pollen-Transmitted Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 166 166 167 170 170 172 Methods of Combating Seed-Transmitted Virus Diseases . . . . . 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Avoidance of Virus Inoculum from Infected Seeds . . . . . . . 8.2.1 Removal of Infected Seeds . . . . . . . . . . . . . . . . . . . . 8.2.2 Chemical Seed Disinfection . . . . . . . . . . . . . . . . . . . 8.2.3 Seed Disinfection by Heat . . . . . . . . . . . . . . . . . . . . 8.2.4 Storage Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.5 Irradiation Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Reducing the Rate of Virus Spread Through Vector Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1 Avoiding of Continuous Cropping . . . . . . . . . . . . . . 8.3.2 Elimination of Weed, Volunteer and Wild Hosts . . 8.3.3 Roguing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.4 Crop Rotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.5 Planting Dates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.6 Plant Density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.7 Barrier and Cover Crops . . . . . . . . . . . . . . . . . . . . . . 8.4 Integrated Cultural Practices for Seed-Transmitted Virus Disease Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 Crops Hygiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.1 Raising Transplants . . . . . . . . . . . . . . . . . . . . . . . . . . 185 185 186 186 186 187 189 189 138 143 144 145 145 173 177 178 179 189 189 190 190 191 191 191 192 192 193 194 xiv Contents 8.6 8.7 8.8 8.9 8.10 8.11 8.12 8.13 8.14 8.15 8.16 8.17 8.18 8.19 8.20 8.21 Control of the Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.1 Insecticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.2 Mineral Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.3 Repelling Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . Virus Avoidance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7.1 Exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.1 Host Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.2 Sources of Resistance . . . . . . . . . . . . . . . . . . . . . . . . 8.8.3 Conventional Breeding of Natural Resistance Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.4 Cultivars with Low Seed Transmission . . . . . . . . . . 8.8.5 Vector-Resistant Cultivars . . . . . . . . . . . . . . . . . . . . . Immunisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Approved Seed Certification Standards . . . . . . . . . . . . . . . . . Stages of Seed Multiplication . . . . . . . . . . . . . . . . . . . . . . . . . Inoculum Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . International Seed Testing Association (ISTA) . . . . . . . . . . . 8.13.1 Objectives of ISTA . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13.2 ISTA Certificates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13.3 Conditions for Issuance of ISTA Certificates . . . . . 8.13.4 Accredited Laboratory . . . . . . . . . . . . . . . . . . . . . . . . Seed Certification Against Plant Virus Diseases . . . . . . . . . . 8.14.1 The Quality Control by ELISA . . . . . . . . . . . . . . . . . 8.14.2 Certification Schemes Against Crops . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quality Control of Bulk Seed Lots . . . . . . . . . . . . . . . . . . . . . 8.16.1 Determination of Seed Transmission Rate . . . . . . . . 8.16.2 Infection Status of a Bulk Seed Lot with Respect to a Tolerance Limit . . . . . . . . . . . . . . . . . . . . . . . . . . 8.16.3 Infection Status of a Bulk Seed Lot with Respect to a Virus Not Known in the Importing Country . . . World Trade Organization (WTO) Regime and Its Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.17.1 Examples of Introduced Plant Viruses Through Seed Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of Plant Biosecurity in Preventing and Controlling Emerging Plant Virus Disease Epidemics . . Pest Risk Analysis (PRA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.19.1 Pest and Pathogen Risk Analysis . . . . . . . . . . . . . . . 8.19.2 Pest Risk Analysis for Viral Diseases of Tropical Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.20.1 Biosafety Regulations . . . . . . . . . . . . . . . . . . . . . . . . 8.20.2 History of Biosafety Protocol and Regulations . . . . 8.20.3 Biosafety Regulations of Asia-Pacific Countries . . Risks Associated with Genetically Modified Crops . . . . . . . 195 195 196 196 197 197 199 201 201 203 205 206 207 209 210 210 213 214 214 214 215 215 216 217 220 220 220 222 222 222 223 223 225 225 225 227 227 227 228 229 Contents xv 8.22 Quarantines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.22.1 Plant Quarantine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.22.2 Plant Quarantine Measures . . . . . . . . . . . . . . . . . . . . 8.22.3 Functions of Plant Quarantine . . . . . . . . . . . . . . . . . 8.22.4 Pathways of Spread of Pests and Pathogens . . . . . . 8.22.5 Quarantine Status of Plant Importations . . . . . . . . . 8.22.6 Types of Materials Received . . . . . . . . . . . . . . . . . . 8.23 Role of FAO/IBPGR in Germplasm Exchange . . . . . . . . . . . 8.23.1 Conceptual Guidelines for Exchange of Legume Germplasm, Breeding Lines and Commercial Seed Lots as Follows . . . . . . . . . . . . . . . . . . . . . . . . . 8.23.2 The Technical Guidelines for Exchange of Germplasm and Breeding Lines . . . . . . . . . . . . . 8.23.3 Movement of Germplasm . . . . . . . . . . . . . . . . . . . . . 8.24 Steps in Technical Recommendations for Seed Germplasm Exchange in the Country of Origin or Destination . . . . . . . . 8.25 Part of the Planting Material to Be Tested and Post-Entry Quarantine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.26 Exclusion of Exotic Plant Viruses Through Quarantine . . . . 8.26.1 International Scenario . . . . . . . . . . . . . . . . . . . . . . . . 8.26.2 National Scenario . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.27 Challenges in Diagnosis of Pests in Quarantine . . . . . . . . . . 8.28 Quarantine for Germplasm and Breeding Material . . . . . . . . 8.29 Role of IPGRI and NBPGR in Germplasm Maintenance and Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.29.1 Objectives of NBPGR . . . . . . . . . . . . . . . . . . . . . . . . 8.29.2 Methods of Testing at Quarantine Stations . . . . . . . 8.30 Important Cases of Introduction . . . . . . . . . . . . . . . . . . . . . . . 8.31 Important Diseases Restricted to Some Countries . . . . . . . . 8.32 Effective Methods of Plant Importations . . . . . . . . . . . . . . . . 8.32.1 Phytosanitary Certificates . . . . . . . . . . . . . . . . . . . . . 8.32.2 Closed Quarantines . . . . . . . . . . . . . . . . . . . . . . . . . . 8.32.3 Quantity of Plant Materials . . . . . . . . . . . . . . . . . . . 8.32.4 Open Quarantine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.32.5 Examination of Exportable Crops During Active Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.32.6 The Intermediate Quarantine . . . . . . . . . . . . . . . . . . 8.32.7 Aseptic Plantlet Culture . . . . . . . . . . . . . . . . . . . . . . 8.32.8 Embryo Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.32.9 Use of Shoot Tip Grafting or Micrografting . . . . . . 8.33 General Principles for the Overall Effectiveness of Quarantines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.34 Quarantine Facilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.35 Need for Networking for the Developing Countries . . . . . . . 8.36 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.37 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.38 Biotechnology and Virus-Derived Resistance . . . . . . . . . . . . 8.38.1 Capsid Protein-Mediated Resistance . . . . . . . . . . . . 229 230 230 231 232 233 233 234 234 235 235 236 237 238 238 239 241 241 243 243 244 246 248 249 249 249 250 250 250 251 251 252 253 253 254 254 255 256 256 257 xvi Contents 8.39 Molecular Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.39.1 Molecular Interactions of Seed-Transmitted Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.39.2 Transgenic Approach . . . . . . . . . . . . . . . . . . . . . . . . . 8.40 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Plant Virus Transmission Through Vegetative Propagules (Asexual Reproduction) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1 Different Vegetative Propagative Plant Materials . . . . . . . . . 9.2 Role of Vegetatively Propagated Plant Materials in Virus Spread . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Different Virus, Phytoplasma and Viroid Diseases . . . . . . . . 9.4 Virus Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Yield Losses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.6 Virus Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.7 Vegetatively Transmitted Plant Virus and Virus-Like Disease Management by Certification Schemes . . . . . . . . . . 9.7.1 Success Stories of Production of Virus-Free Plant Propagules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.7.2 Certification Schemes . . . . . . . . . . . . . . . . . . . . . . . . 9.8 IPGRI’S Role in Controlling Virus Diseases in Fruit Germplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Future Strategies and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Plant Virus Management by Integrated Approach . . . . . . . . . 10.3 Challenges for the Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 257 258 266 266 285 286 286 286 286 288 289 291 291 295 296 296 296 307 308 310 313 315 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 List of Standard Acronyms of Plant Virus and Viroids Virus and viroid names Abaca mosaic virus African cassava mosaic virus Alfalfa cryptic virus Alfalfa mosaic virus Andean potato latent tymovirus Apple chlorotic leaf spot virus Apple mosaic virus Apple scar skin viroid Apple stem grooving virus Arabis mosaic virus Arracacha virus A Artichoke Italian latent virus Artichoke latent virus Artichoke yellow ringspot virus Asparagus virus I Asparagus virus II Avocado sunblotch viroid Bamboo mosaic virus Banana bract mosaic potyvirus Banana bunchy top virus Banana streak badnavirus Barley stripe mosaic virus Barley yellow dwarf virus Bean common mosaic virus Bean common mosaic necrosis virus Bean pod mottle virus Bean southern mosaic virus Bean yellow mosaic virus Beet 1 alpha cryptovirus Beet 2 alpha cryptovirus Beet 3 alpha cryptovirus Beet mild yellowing virus Blackeye cowpea mosaic virus Blackgram mild mottle virus Blackgram mottle virus Bramble yellow mosaic virus Broad bean mottle virus Broad bean stain virus Broad bean true mosaic Broad bean wilt virus Brome mosaic virus Carnation necrotic fleck virus Carrot red leaf virus Acronym AbMV ACMV ACV AMV APLV ACLSV ApMV ASSVd ASGV ArMV AVA AILV ALV AYRSV AV1 AV2 ASBV BaMV BBMV BBTV BSV BSMV BYDV BCMV BCMNV BPMV BSMV BYMV BCV-1 BCV-2 BCV-3 BMYV BICMV BMMV BgMV BrmYMV BBMV BBSV BBTMV BBWV BMV CNFV CtRLV (continued) xvii xviii List of Standard Acronyms of Plant Virus and Viroids (continued) Virus name Cassava brown streak virus Cassava common mosaic virus Cauliflower mosaic virus Cherry leaf roll virus Cherry necrotic rusty mottle virus Cherry rasp leaf virus Chicory yellow mottle virus Chrysanthemum stunt viroid Citrus exocortis viroid Citrus mosaic virus Citrus psorosis virus Citrus ringspot virus Citrus tristera virus Citrus variegation virus Citrus yellow mosaic virus Clover yellow mosaic virus Clover yellow vein virus Cocao necrosis virus Cocao swollen shoot virus Cocao yellow mosaic virus Coconut cadang cadang viroid Coffee ringspot virus Coleus blumei viroid Cow parsnip mosaic virus Cowpea aphid-borne mosaic virus Cowpea banding mosaic virus Cowpea chlorotic mottle virus Cowpea chlorotic spot virus Cowpea green vein-banding virus Cowpea mild mottle virus Cowpea Moroccan aphid-borne mosaic Cowpea mosaic virus Cowpea mottle virus Cowpea severe mosaic virus Crimson clover latent virus Cucumber cryptic virus Cucumber pale fruit viroid Cucumber green mottle mosaic virus Cucumber leaf spot virus Cucumber mosaic virus Cymbidium ringspot virus Dahlia mosaic virus Dapple apple viroid Dasheen mosaic virus Desmodium mosaic virus Dioscorea bacilliform virus Echtes Ackerbohnen mosaik viruses Eggplant mosaic virus Elm mosaic virus Elm mottle Euonymus mosaic Fescue cryptic virus Fig latent virus 1 Garland chrysanthemum temperate Garlic common latent virus Grapevine Bulgarian latent virus Acronym CBSV CsCMV CaMV CLRV CNRMV CRLV CYMV CSV CEVd CiMV CPsV CRSV CTV CVV CYMV ClYMV CYVV CoNV CSSV CYMV CCCV CoRSV CbVd CpAMV CABMV CpBMV CCMV CpCSV CGVBV CMMV CABMV CPMV CPMoV CpSMV CCLV CuCV CPFVd CGMMV CLSV CMV CyRSV DMV DAV DsMV DesMV DBV EAMV EMV EIMV EMoV EuoMV FCV FLV-1 GCTV GCLV GBLV (continued) List of Standard Acronyms of Plant Virus and Viroids xix (continued) Virus name Grapevine fanleaf virus Grapevine yellow speckle viroid Groundnut bud necrosis virus Guar symptomless virus Hibiscus latent ringspot virus High plains virus Hop stunt viroid Hop trefoil cryptic 1 Hop trefoil cryptic 2 Hop trefoil cryptic 3 Hosta virus x Humulus japonicas virus Hydrangea mosaic virus Indian cassava mosaic virus Indian citrus ringspot virus Indian peanut clump virus Iris mild mosaic virus Iris yellow spot virus Kalanchoe top-spotting Leek yellow stripe virus Lettuce mosaic virus Lilac ring mottle virus Lucerne Australian latent virus Lucerne (Australian) symptomless Lucerne transient streak virus Lychnis ringspot virus Maize chlorotic mottle virus Maize dwarf mosaic virus Maize mosaic virus Maize streak virus Melon necrotic spot carmovirus Melon rugose mosaic virus Mibuna temperate virus Mulberry ringspot virus Muskmelon mosaic virus Muskmelon necrotic spot virus Nicotiana velutina mosaic virus Oat mosaic virus Olive latent virus Onion yellow dwarf virus Panicum mosaic virus Papaya ringspot virus Paprika mild mottle tobamovirus Parsley latent virus Passionfruit woodiness virus Peach rosette mosaic virus Pea early browning virus Pea enation mosaic virus Pea mild mosaic virus Pea mosaic virus Peanut clump virus Peanut mottle virus Pea streak virus Peanut bud necrosis virus Peanut stripe virus Peanut stunt virus Acronym GFLV GYSV GBNV GSLV HLRSV HPV HsVd HTCV1 HTCV2 HTCV3 HSVX HJV HdMV ICMV ICRSV IPCV IMMV IYSV KTSV LYSV LMV LRMV LALV LASV LTSV LRSV MCMV MDMV MMV MSV MNSV MRMV MTV MRSV MuMV MNSV NVMV OMV OLV OYDV PMV PRSV PaMMV PLV PWV PRMV PEBV PEMV PMiMV PMV PCV PeMoV PeSV PBNV PStV PSV (continued) xx List of Standard Acronyms of Plant Virus and Viroids (continued) Virus name Pea seed-borne mosaic virus Pelargonium zonate spot virus Pepino mosaic virus Pepper mild mosaic virus Pepper mild mottle virus Piper yellow mottle virus Plum pox virus Potato leaf roll virus Potato spindle tuber viroid Potato virus M Potato virus S Potato virus T Potato virus U Potato virus X Potato virus Y Prune dwarf virus Prunus necrotic ringspot virus Radish yellow edge virus Raspberry bushy dwarf virus Raspberry ringspot virus Red clover cryptic virus Red clover mottle virus Red clover vein mosaic virus Red pepper cryptic–1 Red pepper cryptic–2 Rhubarb temperate virus Rice yellow mottle virus Rubus Chinese seed-borne nepovirus Rye grass cryptic virus Santosai temperate virus Satsuma dwarf virus Soil-borne wheat mosaic virus Southern bean mosaic virus Sowbane mosaic virus Soybean mild mosaic virus Soybean mosaic virus Spinach latent virus Spinach temperate virus Squash mosaic virus Strawberry latent ringspot virus Subterranean clover mottle virus Sugarcane mosaic virus Sunflower mosaic potyvirus Sunflower rugose mosaic virus Sunn-hemp mosaic virus Sweet potato feathery mottle virus Sweet potato ringspot virus Tobacco mosaic virus Tobacco necrosis virus Tobacco rattle virus Tobacco ringspot virus Tobacco streak virus Tomato apical stunt viroid Tomato aspermy virus Acronym PSbMV PZSV PepMV PMMV PMMoV PYMoV PPV PLRV PSTVd PVM PVS PVT PVU PVX PVY PDV PNRSV RYEV RBDV RRV RCCV RCMV RCVMV RPCV1 RPCV2 RTV RYMV RCSV RGCV STV SDV SBWMV SBMV SoMV SMMV SMV SpLV SpTV SqMV SLRSV SCMoV SCMV SuMV SRMV SHMV SPFMV SPRSV TMV TNV TRV TRSV TSV TASVd TAV (continued) List of Standard Acronyms of Plant Virus and Viroids xxi (continued) Virus name Tomato black ring virus Tomato bushy stunt virus Tomato chlorotic dwarf viroid Tomato mosaic virus Tomato ringspot virus Tomato spotted wilt virus Tomato streak virus Turnip mosaic virus Turnip yellow mosaic virus Urdbean leaf crinkle virus Vicia cryptic virus Watermelon mosaic virus Wheat soil borne mosaic virus Wheat streak mosaic virus White clover cryptic virus White clover mosaic virus Zucchini yellow mosaic virus Acronym TBRV TBSV TCDV ToMV ToRSV TSWV TSV TuMV TYMV ULCV VCV WMV WSBMV WSMV WCCV WClMV ZYMV 1 Introduction Abstract Good quality seeds must be genetically and physically pure, healthy vigorous and high in germination percentage. The term ‘seed transmission’ refers to the passage of pathogen from seeds to seedlings and plants. Besides fungal and bacterial pathogens, the viruses are also established to be seed transmitted in a number of crops. The rate of seed transmission depends on the host, virus, environment, vectors and their interactions. The spread of seed-transmitted virus diseases will be rapid and irreversible if initial inoculum is high and vector flight activity is great. Seed-transmitted plant viruses are also small, submicroscopic infectious particles composed of a protein coat and a nucleic acid. They are prevalent in vegetable, fruit, cereal and ornamental crops and are great concern to successful crop production. The up-to-date list of seedtransmitted plant virus and viroid diseases is presented in the form of tabular form in this chapter. There are nearly 231 plant virus and viroid diseases which are reported to be seed transmitted from different parts of the world. Seed transmission of nearly 68 viruses is more common in leguminous species than in any other species of crop plants. Seed transmission of plant viruses plays a pivotal role in the spread and survival of a number of important plant viral and viroid diseases. Viroid diseases are effectively transmitted vertically through pollen and ovule to the seed and seedlings. Attempts are also made to list out the virus diseases, which are erroneously listed as seed-transmitted viruses in the earlier days, which in the future are not to be considered as seedtransmitted viruses. 1.1 Introduction The agricultural scenario as a basis for socioeconomic and physical development of a country is undergoing rapid changes all over the world. Satisfying the basic hunger and nutritional needs of the growing population of the world constitutes the first priority in the agricultural production– demand equation. There are 800 million undernourished people in the world, and with the present rate of population growth, this number is expected to reach 1,000 million and result into K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 1, © Springer India 2013 1 2 1 starvation deaths due to food shortage. At present nearly 1.3 billion population live on less than $1 a day. According to the FAO, food prices have increased by over 75% since 2000 and alarming increase in prices has been witnessed especially since 2006. In 2008, internationally the prices of rice increased by 74% and wheat by more than double of the previous year. As a result of this sharp increase in food prices, widespread protests and clashes were reported in several parts of Latin America, Africa and South Asia. According to FAO, about 14% of the 6.5 billion world population is affected by hunger. Twentynine countries including India have ‘alarming’ or ‘extremely alarming’ levels of hunger. The situation is likely to be aggravated by 2050, as the global population is expected to increase by 40%. This desperate overpopulation situation cannot be reduced. On the other hand, there should be rapid agricultural and industrial development, and out of the two, intensive agriculture will play major role. Expansion of cultivated land can increase agriculture production to some extent, but high-yielding varieties and hybrids along with advanced agro-production technologies will play a major role. The tremendous increase in population in recent years has given an impetus to man’s urgent quest for rapid means of increasing agricultural production. This will mean multiple cropping and increasing yields per unit land. Among the major factors which influence agricultural productivity, seed has a place of prime importance. 1.1.1 Seed (Sexual Propagule) Seed is a productive propagule to perpetuate the species that germinates to produce a new plant. It is a fertilised mature ovule that possesses an embryonic plant, stored material (sometimes absent) and a protective coat or coats. Most species are adapted to environments that allow the seed to desiccate and survive adverse environmental conditions in which the species normally cannot grow. When the environmental conditions are congenial for plant growth, the seeds germinate, differentiate, grow and set flower, followed by Introduction seed that starts the cyclic process all over again. When man learnt cultivation of plants, he also realised that he had to save part of the seed produced for sowing in the subsequent year. About 90% of food crops grown are propagated through seed (Maude 1996). Since seed is the carrier of the genetic potential for higher crop production, improved varieties of seed have been produced by modern selection and breeding techniques to help in increasing the yield per unit area and in turn to boost agricultural production leading to green revolution. Asexually or vegetatively clonally propagated crops in which no true seeds are involved are also covered in this book. 1.2 Seed Transmission of Viruses Seeds, from the time of their inception at flowering of the parent plants till their germination and development into seedlings, are prone to microbial attacks. They are known to be the most efficient vehicles of transport for a number of plant pathogens comprising fungi, bacteria and viruses and cause catastrophic yield and economic losses. Worldwide, plant pathogens cause about 12% of crop losses, weeds about 13% and insect pests about 15%. The value of crop losses due to pests is estimated to be more than $1 1012 per year. Probably developing countries suffer the most from crop attacks by plant pathogens. It is reported that plant pathogens cause several billions of dollars in crop losses each year (http://apsnet. org). In intensive agricultural systems, constant coordinated efforts are required to control the diseases to ensure higher food production and provide raw material to agro-based industries. Among the plant diseases, virus diseases are prevalent in cultivated plants in highly intensive agricultural systems as well as under more traditional farming conditions. The most important effects of seed transmission of plant viruses are as follows: (1) direct and/or indirect injury as even a low incidence of infected seeds sown results in numerous randomly scattered foci of inoculum, facilitating early secondary spread in the crop through insect vectors; (2) survival of viral inoculum from one crop season to the next; and 1.2 Seed Transmission of Viruses 3 Fig. 1.1 Symptomatology of some seed-borne virus diseases (3) several viruses and viroid diseases have been and undoubtedly still are disseminated worldwide through exchange of seeds having undetected infection (Fig. 1.1). Viruses are defined as ultramicroscopic, filterable and pathogenic entities which multiply in living cells, using host components and induce diseases in higher animals, plants, bacteria, phytoplasmas, spiroplasmas, insects, fungi and algal organisms (Agrios 2005; Khan and Dijkstra 2002; Nayudu 2008). They have ss or ds RNA or DNA not both, enclosed in a protein coat which may be enveloped in a few cases by a lipid envelope as in Tomato spotted wilt virus (TSWV). The genome may be a single- or double-stranded or a segmented form enclosed in a single coat protein or occur in two or three particles. They may be rigid rod-shaped or flexuous filamentous particles or icosahedral particles. Seed transmission plays a pivotal role in the spread and survival of a number of important plant viral and viroid diseases. Infected seed is probably the most important source of viruses and subviral pathogens in commercial plantings. 4 1 In fields, the seedlings raised from the randomly dispersed infected seeds serve as initial sources of virus inoculum or foci of infection from which secondary spread occurs within and outside the field by suitable vectors. Certain viruses like Lettuce mosaic (LMV) and Bean common mosaic (BCMV) have the least number of alternate hosts, their principal crop hosts do not overwinter and seed transmission is one of the chief ways of virus survival from one season to another. Besides being a source of inoculum, the seed also helps in perpetuation of the virus over long periods. For example, BCMV in French bean seed and Prunus necrotic ring spot virus in Prunus pensylvanica seed persists for about 38 and 6 years, respectively (Walters 1962a; Fulton 1964). Many countries import plant germplasm to diversify the genetic base of crop to improve yields and raise the levels of disease resistance and other economic and agronomic characteristics. But due to indiscriminate international exchange of germplasm, areas hitherto free of certain pathogens now have new virus and virus-like diseases. There are number of established reports from many countries that some new virus and virus strains are being introduced along with the germplasm/food grains when imported for research/consumption purposes, and the examples are fruit tree, peanut, pea and bean viruses (Chalam and Khetarpal 2008). 1.2.1 History of Seed-Transmitted Plant Virus Research The first seed transmission of Tobacco mosaic virus through tomato seed was suspected by Westerdijk in 1910 and later by Allard (1914). As early as 1915, Soybean mosaic virus was reported in the annual report of Connecticut Agricultural Experiment Station (Clinton 1916). Subsequently seed transmission of virus disease of Lima bean mosaic was studied by Mc Clintock (1917). Further, instances of transmission of Bean common mosaic virus (BCMV) through bean seed were reported (Stewart and Reddick 1917; Reddick and Stewart 1918, 1919). Similarly, Introduction seed transmission of Cucumber mosaic virus (CMV) in wild cucumber (Echinocystis lobata) was reported by Doolittle and Gilbert (1919), Lettuce mosaic virus in lettuce by Newhall (1923) and Soybean mosaic virus in soybean by Kendrick and Gardner (1924). Later the BCMV transmission through seed was reported by several workers (Burkholder and Muller 1926; Merkel 1929; Pierce and Hungerford 1929). Regarding listing of seed-transmitted plant viruses as early as 1951, eight seedtransmitted viruses were reported by Smith (1951), and 6 years later around 20 seedtransmitted viruses were reported in 40 species of various plants by Crowley (1957a, b). Thirtysix seed-transmitted viruses in 63 plant species were recorded by Fulton (1964), later Bennett (1969) reported 47 and Phatak (1974) listed 85. Agarwal and Sinclair (1988) listed 156 viruses to be transmitted through seed of several plant species. Neergaard’s (1977) and Agarwal and Sinclair’s (1987) voluminous compilations and other reviews by Crowley (1957a), Fulton (1964), Baker and Smith (1966), Kunze (1968), Bennett (1969), Baker (1972), Shepherd (1972), Phatak (1974, 1980), Bos (1977), Richardson (1979, 1981, 1983), Mandahar (1981, 1985), Quiot et al. (1982), Mishra et al. (1984), Stace-Smith and Hamilton (1988), Bos et al. (1988), Frison et al. (1990), Rishi and Nain (1992), Sastry et al. (1992), Mink (1993), Johansen et al. (1994), Gaur et al. (1996), Maude (1996), Maule and Wang (1996), Sutic et al. (1999), Hull (2002), Power and Flecker (2003) and Albrechtsen (2006) provided a great deal of information on seedtransmitted viruses in terms of identification, ecology, epidemiology and management aspects. During 1988, Stace-Smith and Hamilton have stated that about 18% of the described plant viruses are seed transmitted in one or more hosts. Later Mink (1993) has listed 108 viruses (excluding Partitiviridae) and 7 viroid diseases to be seed transmitted in one or more hosts. Hull (2002) reported that one seventh of the known plant viruses are transmitted through the seed. During 2006, Albrechtsen from Danish seed health centre for developing countries, Denmark, has listed seed-transmitted 113 conventional viruses, 1.4 Seed Transmission of Viroids 31 cryptoviruses and 12 viroids. During 2003, Power and Flecker have reported 131 viruses to be carried through seed. Since then, the number of seed-transmitted viruses has phenomenally increased year after year, and presently more than 231 viruses are reported to be transmitted through seed in different cultivated and weed hosts. An updated list of seed-transmitted virus diseases is furnished in Table 1.2. Descriptions and lists of VIDE database edited by Brunt et al. (1990, 1996) and CMI/AAB descriptions and also the virus diseases of plants on CD by Barnett and Sherwood (2009) will provide more information on seed-transmitted plant viruses. However, an attempt is made in this book by collecting the information on seed-transmitted viruses from Review of Plant Pathology and CAB abstracts and other information sources including VIDE/ICTV, and the list is prepared and presented in the form of Table 1.2. Approximately, out of the total 1,500 plant viruses reported on different crops and weed plants, nearly 231 virus and viroid diseases are found to be seed transmitted. In Table 1.2, some synonyms or strains of seed-transmitted viruses are also listed as the author has included all the publications on seedtransmitted plant viruses without having any discrimination. Future studies by taking many characters including molecular detection techniques may reveal authentic data on seed-transmitted viruses. 1.3 Seed Transmission of Partitiviridae Partitiviridae (cryptoviruses) are unique in nature and do not induce any type of symptoms in infected plants. Cryptoviruses are transmitted only through seed and pollen and consist of a doublestranded RNA genome encapsidated in protein, without lipid envelope (Fraser 1989). The virions are small and isometric and no vectors were identified. Cryptoviruses as per ICTV report classified in the family Partitiviridae (Boccardo et al. 1983, 1987; Milne and Natsuaki 1995). The examples are Alfalfa cryptic virus 1 and 2; Beet cryptic virus 1, 2 and 3; Carnation cryptic virus 1 and 2; 5 Carrot temperate virus 1, 2, 3 and 4; Cucumber cryptic virus; Fescue cryptic virus; Hop trefoil cryptic 1, 2 and 3; Red clover cryptic virus 2; Spinach temperate cryptic virus; Vicia cryptic virus; White clover cryptic virus 1, 2 and 3; and Ryegrass cryptic virus (Boccardo et al. 1983, 1987; Milne and Natsuaki 1995; Albrechtsen 2006). 1.4 Seed Transmission of Viroids Viroids are independently replicating circular RNAs capable of causing diseases in infected plants. They consist of naked RNA which does not code for any protein nor is protein associated with it and replicate independent of any associated plant viruses. The two families of viroids are the Pospiviroidae and Avsunviroidae with five and two genera, respectively. Viroid diseases are effectively transmitted through seed and pollen (Mink 1993; Diener 1999; Hadidi et al. 2003; Flores et al. 2005; Hammond and Owens 2006). For the first time, Diener (1971) reported that potato spindle tuber disease was induced by small, unencapsidated molecules of autonomously replicating circular RNA, and the term viroid has been adopted. Some of the properties of viroids that differentiates them from the viruses are as follows: (1) the pathogen exists in vivo as an unencapsidated RNA, that is, no virion-like particles are detectable in infected tissue; (2) the infectious RNA is of low molecular weight; (3) despite its small size, the infectious RNA is replicated autonomously in susceptible cells, that is, no helper virus is required; and (4) the infectious RNA consists of one molecule only. Viroid species are clustered into the families Pospiviroidae and Avsunviroidae, whose members replicate (and accumulate) in the nucleus and chloroplast, respectively. Out of the 28 viroid diseases known, 10 are reported to be seed transmitted. For more details about viroid diseases, refer to Hadidi et al. (2003). Viroid diseases are effectively transmitted vertically through pollen and ovule to the seed and seedling. Some of the viroid diseases have now been shown to be transmitted through seed, 6 1 namely, Apple scar skin viroid, Apple dapple viroid, Chrysanthemum stunt viroid, Coleus viroid, Grapevine viroid, Hop stunt viroid and Potato spindle tuber viroid. High degree of seed transmission is reported from viroid diseases of Potato spindle tuber, Cucumber pale fruit, Avocado sunblotch and Chrysanthemum stunt. 1.4.1 Introduction and Holdeman 1965) and as high as 80–100% with Pea seed-borne mosaic (PSbMV), Soybean stunt, Cucumber mosaic (CMV), BCMV and Red clover vein mosaic viruses (RCVMV) in certain legume cultivars (Sander 1959; Medina and Grogan 1961; Stevenson and Hagedorn 1973; Brunt and Kenten 1973; Iizuka 1973; Takahashi et al. 1980; Truol et al. 1987; Morales and Castano 1987; Khetarpal 1989). Extent of Seed Transmission 1.4.1.1 Viruses The extent of seed transmission depends on particular host–virus combination. Variable estimates for seed transmission in a majority of cases have been due to the use of different cultivars of the same host species or different strains of the same virus. Complete absence of seed transmission is virtually impossible to demonstrate with certainty in some virus–host combinations. Lack of seed transmission in a small sample does not preclude its possible transmission. Hence, negative results can at best be tentative or inconclusive. Seed transmission of viruses predominates in some plant families and is rare in others. Seed transmission of nearly 68 viruses is more common in leguminous species than in any other species of crop plants (Table 1.2). Some viruses with nonlegume principal hosts are also seed transmitted in legumes. In Rosaceae family, there is an affinity between a group of hosts and seed-transmitted viruses. In Prunus avium, P. cerasus, P. mahaleb, P. pensylvanica and P. persica, the seed transmission of Prunus necrotic ring spot (PNRSV) was most common (Millikan 1959; Wagnon et al. 1960; Megahed and Moore 1967). As a group, greater seed transmission of viruses is seen with nepoviruses which may show up to 100% (Athow and Bancroft 1959; Murant and Lister 1967; Allen et al. 1970; Hansen et al. 1974). Tobraviruses in general have a low rate of transmission (1–6%) except Dutch isolate of Pea early browning virus, PEBV (Fiedorow 1983). Among the aphid-borne seed-transmitted viruses, it is as low as 0.1–0.4% in corn infected with Sugarcane mosaic virus (SCMV) (Shepherd 1.4.1.2 Viroids High degree of seed transmission is noticed in certain viroid diseases, namely, Australian grapevine viroid; Avocado sunblotch viroid; Chrysanthemum stunt viroid; Citrus exocortis viroid; Coconut cadang-cadang viroid; Coleus blumei viroid 1,2 and 3; Grapevine yellow speckle viroid 1 and 2; Hop stunt viroid; Potato spindle tuber viroid; Apple scar skin viroid; Apple dapple viroid; and Tomato apical stunt viroid (Mink 1993; Pacumbaba et al. 1994; Hadidi et al. 2003; Albrechtsen 2006; Antignus et al. 2007), and various aspects of seed-transmitted viroid diseases were discussed in the relevant chapters. 1.4.1.3 Suspected Seed Transmission of Phytoplasma Diseases During Khan et al. (2002), have reported the transmission of phytoplasma in both seed and seedling progeny of alfalfa plants affected by witches’ broom disease, indicating the seed transmission in certain plant host–phytoplasma pathosystem. Necas et al. (2008) have reported the preliminary information of possibility of European stone fruit yellows phytoplasma (ESFY) transmission through apricot seeds. Seeds from ESFY-infected trees showed very low viability (21.6%) and practically no germination activity. Earlier Cordova et al. (2003) have observed the presence of phytoplasma DNA in coconut embryos by PCR studies, raising the possibility of seed transmission of lethal yellowing disease of coconut palms. These research reports warrant further research on the role of seed transmission in the epidemiology of phytoplasma diseases, and the observations cited here requires further study. 1.5 Viruses Erroneously Listed as Seed Transmitted Bove et al. (1988) stated in their review article that the occurrence of stubborn symptoms on citrus seedling trees or plants was taken as evidence for natural spread of the disease because Spiroplasma citri has never been found to be transmitted vertically through seeds, even though the seed coats from infected citrus trees can carry S. citri. The aetiological agent, Spiroplasma, in citrus stubborn and corn stunt diseases is suspected to be seed transmitted, but confirmation is warranted. 1.4.1.4 Lack of Evidence for Transmission of Citrus Huanglongbing Lot of controversy existed regarding the aetiology of citrus greening disease. As early as Ghosh et al. (1971) have reported the association of phytoplasma with citrus greening. In the subsequent researches based on electron microscopy (Garnier and Bove 1983) and PCR studies (Jagoueix et al. 1994), the presence of bacterium Candidatus Liberibacter asiaticus (Ca. L. asiaticus) was observed and reported to be the aetiological agent of Citrus greening which was later named as huanglongbing (HLB). Transmission of ‘Ca. L. asiaticus’ through infected citrus seed has been reported (Tirtawidjaja 1981) based on the rapid appearance of HLB-like symptoms in seedlings when apparently healthy seed harvested from symptomatic fruit was sown. ‘Ca. L. asiaticus’ was also readily detected throughout HLB-affected fruit by quantitative qPCR (Tateneni et al. 2008; Li et al. 2009) and in the seed coat but not the embryos of limited numbers of seed collected from HLB-affected fruit (Tateneni et al. 2008). Citrus germplasm is often exchanged as seed, and thus, a pathway for the potential introduction of huanglongbing (HLB) into previously HLBfree regions would exist, if the pathogen is seed transmitted. Hence, intensive researches were carried out by Hartung et al. (2010) to provide evidence as to whether or not ‘Ca. L. asiaticus’ can be transmitted through seed. They have grown various citrus species from the seed collected from symptomatic ‘Ca. L. asiaticus’, and the seedlings were tested by PCR 7 for number of times over periods of up to 3 years. No symptoms typical of huanglongbing, such as blotchy leaf mottle, chlorotic shoots or dieback of branches, were observed in these seedlings, and none of these 723 seedlings tested positive for the presence of ‘Ca. L. asiaticus’, even after repeated testing by sensitive quantitative PCR assays. Some sour orange seedlings did have quite pronounced and atypical growth, including stunting and mild to severe leaf malformation. These atypical growth habits were limited to seedlings that arose from zygotic embryos as determined by expressed sequence tag simple sequence repeat analyses. Thus, no evidence of transmission of ‘Ca. L. asiaticus’ through citrus seed was obtained by Hartung et al. (2010), and an earlier report of transmission of the pathogen through seed was not confirmed. 1.5 Viruses Erroneously Listed as Seed Transmitted Nearly 13 review articles with comprehensive lists of seed-transmitted plant virus and viroid diseases were published since 1951, and each reviewer had added a substantial number of names to the lists of their predecessors. The book entitled Testing methods for seed-transmitted viruses: Principles and Protocols by Albrechtsen is the latest and has covered various aspects of seed-transmitted viruses. Perusal of the review articles of Mandahar (1981), Agarwal and Sinclair (1987), Mink (1993), Niazi et al. 2007 and others reveal that at least 14 virus or virus disease names are erroneously listed as seed transmitted and has been furnished in Table 1.1. Mink (1993) has furnished details of the 14 erroneously listed seed-transmitted viruses in his review article. Of the 14 virus disease names considered by Mink (1993), four have been perpetuated in the literature as seed transmitted on the basis of erroneous reports or erroneously cited reports: ACLSV, Barley mottle mosaic, CaMV and TuMV. These should be eliminated from future lists of seed-transmitted viruses. Four other viruses should also be removed from lists of seed- 8 1 Introduction Table 1.1 Virus or disease names erroneously listed as seed transmitted in earlier reviews Disease name and virus Abutilon mosaic Apple chlorotic leaf spot Barley mottle mosaic Barley yellow dwarf Bean yellow dwarf Beet western yellows Carrot motley dwarf Carrot red leaf Cauliflower mosaic Cherry necrotic rusty mottle Citrus psorosis Potato leaf roll Sugarcane mosaic Turnip mosaic Virus listed as Seed transmitted Yes Yes Yes Yes No No No Yes Yes Yes Yes No Yes Yes Seed borne Yes Yes No No Yes No Yes Yes No Yes Yes Yes Yes Yes Comment Geminivirus Misidentification No such name listed Luteovirus Luteovirus Luteovirus Not seed transmitted Luteovirus Not seed transmitted Not seed transmitted Misidentification Luteovirus Not seed transmitted Not seed transmitted Source: Mink (1993) transmitted viruses because of nomenclatural problems: Abutilon mosaic virus, Beet curly top virus, Citrus psorosis virus and Sugarcane mosaic virus. Six of the names in Table 1.1 are either luteoviruses or associated with a luteovirus. In each case (except bean yellow dwarf), reports claim transmission through seed based on the occurrence of symptomatic seedlings. These reports have not been taken seriously, either by many reviewers or the ICTV. Credibility of any future reports of luteovirus seed transmission will be enhanced by a demonstration that virus can be transmitted from seedlings (whether symptomatic or not) using aphid vectors. The occurrence of symptomatic but apparently noninfected seedlings for several luteoviruses, CNRMV and the causal agent of Abutilon variegation represents a phenomenon that deserves additional study. 1.5.1 Seed-Transmitted Plant Virus Names That Appeared Only Once in the Literature Excellent exercise has been done by Mink (1993) in his review article while listing the seed-transmitted plant viruses. Forty-six disease and virus names were found among the lists of seed-transmitted viruses in previous reviews that occurred only once, or very few times, in the literature. Plant virus literature searches have failed to provide much additional information regarding most of the causal agents. Usually, transmission through seed was demonstrated by the occurrence of symptomatic seedlings. In some cases, the causal agents were demonstrated to be viruses, which were at least partially characterised in the original reports although their relationships to other viruses were not clearly established (Mink 1993). Forty-one of the 46 names were reported prior to 1980 and some of these names may represent newly recognised seed-transmitted viruses that have received little attention since the initial report. However, most were more likely applied to diseases that were eventually found to be caused by commonly recognised viruses, and the disease names were well documented in the review article of Mink (1993). Although many agents are indubitably seed transmitted, Mink (1993) has listed their names in a separate table in his article to call attention to their plight. Specialists working with one or more of these agents may be able to provide up-to-date information regarding their identity. Mink (1993) and also other reviewers of seedtransmitted viruses have complied the available 1.5 Viruses Erroneously Listed as Seed Transmitted literature in the form of tables. Primarily Mink (1993) has observed many names of seed – transmitted viruses that appeared on earlier lists are synonyms of other viruses. Because of these synonyms, a list of 50 such names that have often been cited as distinct seed-transmitted viruses has been discussed thoroughly. In some cases, several synonyms of a single virus have been used. The five synonyms for Prunus necrotic ring spot virus are an extreme example: cherry necrotic ring spot, peach latent, peach necrotic leaf spot, peach ring spot and stone fruit ring spot. These disease names convey no unique strain identity (Mink 1991). Other synonym names identified by Mink (1993) are also not strain specific, and to include multiple synonyms on lists of seed-transmitted viruses creates redundancy. 1.5.2 Establishing Certain Erotic Positive Seed-Transmitted Viruses to Be Non-seed Transmissible In recent years, ilar- and tospoviruses which cause enormous yield losses in majority of economically important crops of both positive and negative seed transmission in different crops are reported. Positive seed transmission reports of ilarviruses (Tobacco streak virus) are reported in soybean by Ghanekar and Schwenk (1974), Kaiser et al. (1982), Truol et al. (1987) and Jain et al. 2006; in tomato by Sdoodee and Teakle (1988); in beans by Kaiser et al. (1991); and in Parthenium hysterophorus by Sharman et al. (2009). Extensive seed transmission studies by Prasada Rao et al. (2003, 2009), Reddy et al. (2007) and Vemana and Jain (2010) have established the negative seed transmission of TSV in groundnut and sunflower. It was further established by Vemana and Jain (2010) that TSV is not true seed transmitted in V. mungo, V. radiata, G. max. and Dolichos lablab. So far TSV seed transmission was not proved from any crop or weed hosts in India. Hence, it is concluded that non-seed-transmissible strain of TSV may be existing in India. Moreover, complete genome sequence is lacking for Indian 9 TSV isolate, and comparison has to be made with seed-transmissible strain of TSV from different countries as Walter et al. (1995) observed extra minor RNAs in non-seed-transmissible strain (MelF) of TSV. So, differentiation has to be made between the seed-transmissible strains of TSV with respect to their host range, basic chemical properties and genetic diversity. Similarly, seed transmission of Tomato spotted wilt virus in tomato and Senecio cruentus were reported by Jones (1944). Reports exist regarding seed transmission of Groundnut bud necrosis virus (GBNV) in peanut; however, the recent studies carried out at NBPGR and ICRISAT, Hyderabad, and elsewhere have proved that GBNV is not seed transmitted in peanut and other hosts. Rice yellow mottle virus (RYMV), genus Sobemovirus, causes severe yield losses ranging from 25 to 100% in most countries of Africa. Studies carried out by Konate et al. (2001) and Allarangaye et al. (2006) have established the evidence of non-transmission of RYMV through seeds of rice and wild rice species. RYMV was infectious in freshly harvested seed extracts, whatever the plant species or the virus isolate. However, most infectivity was lost in dried seeds, possibly due to virus inactivation following dehydration of the seeds. Therefore virus dissemination or epidemics of rice yellow mottle do not originate from infected seeds. Similarly, Beet curly top virus transmitted by Cicadellidae vector Circulifer tenellus was seed transmitted in Beta vulgaris to the extent of 11–25% as reported by Abdel-Salam and Amin (1990) requires confirmation. Although plant virus and viroid diseases infect number of hosts belonging to different families, either in natural or controlled conditions, seed transmission is noticed only in certain virus–host combinations (Table 1.2). The mere presence of virus in/on the seed does not always lead to seedling infection. Some viruses may be confined only on seed coat or in cotyledons or in embryos. Some viruses are present before seed maturation and drying, whereas certain viruses will get eliminated with seed maturation and drying. The reasons for positive or negative seed transmission are discussed in Chap. 5. 10 1 Introduction Table 1.2 Per cent transmission of virus and viroid diseases through seeds in different hosts Virus/viroid Alfalfa mosaic (Syn. Berseem mosaic) Host Amaranthus albus Capsicum annuum Cicer arietinum Lathyrus cicera Lathyrus sativus Lens culinaris Per cent 1.9–15.5 1–5 – 2 0.9–4 – Lupinus angustifolius Medicago lupulina Medicago polymorpha Medicago sativa M. sativa M. sativa M. sativa M. sativa 0.8 – 80–100 6 55 1–4 0.2–6 0.6–17 M. sativa M. sativa M. sativa 4 10.6 0.3–74 M. sativa Medicago scutellata Nicandra physalodes Petunia violacea Phaseolus vulgaris Trifolium alexandrinum 3.5–6 – 23 30.3 0.7–4.9 60–70 Trifolium michelianum Trigonella balansae Vicia sativa 0.05 7 0.04–0.7 Alfalfa cryptic Medicago sativa High Alfalfa temperate Apple chlorotic leaf spot Apple dapple viroid Apple mosaic M. sativa Rubus spp. Prunus americana Prunus amygdalus Vigna unguiculata Prunus americana Prunus americana P. avium P. domestica P. serrulata Beta vulgaris Capsella bursa-pastoris Chenopodium album Fragaria x ananassa Glycine max Humulus spp. Lactuca sativa Lamium amplexicaule Lycopersicon esculentum High 30–40 – – 2 – – 15 – – 13 6–33 80 6.9 6.3 10 60–100 1.2–25 1.8 Apple scar skin viroid Apricot gummosis Arabis mosaic References Kaiser and Hannan (1983) Sutic (1959) Jones and Coutts (1996) Latham and Jones (2001a) Latham and Jones (2001a) Jones and Coutts (1996); Latham et al. (2004) Jones et al. (2008) Paliwal (1982) Jones and Nicholas (1992) Belli (1962) Zschau and Janke (1962) Frosheiser (1964); Jones (2004a, b) Frosheiser (1970) Beczner and Manninger (1975); Tosic and Pesic (1975); Hemmati and McLean (1977) Ekbote and Mali (1978) Pesic and Hiruki (1986) Jones and Pathipanawat (1989); Pathipanawat et al. (1995, 1997); Jones (2004a, b) Avegelis and Katis (1989a, b) Paliwal (1982) Gallo and Ciampor (1977) Phatak (1974) Kaiser and Hannan (1983) Mishra et al. (1980); Fugro and Mishra (1996) Latham and Jones (2001b) Latham and Jones (2001b) Latham and Jones (2001b); Latham et al. (2004) Boccardo et al. (1983); Natsuaki et al. (1986) Natsuaki et al. (1984, 1986) Cadman (1965); Converse (1967) Hadidi et al. (1991) Barba (1986) Gottlieb and Berbee (1973) Hadidi et al. (1991) Fridlund (1966) Fridlund (1966) Traylor et al. (1963) Fridlund (1966) Lister and Murant (1967) Lister and Murant 1967 Lister and Murant 1967 Lister and Murant (1967) Lister (1960) Kriz (1959) Walkey (1967) Lister and Murant (1967) Lister and Murant (1967) (continued) Table 1.2 (continued) Virus/viroid Host Myosotis arvensis Nicotiana clevelandii Petunia hybrida P. violacea Plantago major Poa annua Polygonum persicaria Rheum rhaponticum Rosa rugosa Senecio vulgaris Stellaria media Nicotiana clevelandii Solanum tuberosum C. quinoa Per cent 19–95 35 20 20–37 5.4–28 4 21–100 10–24 – 2.2 57 – 4–12 4–12 Artichoke Italian latent Cynara scolymus – Artichoke latent Artichoke yellow ring spot Cynara scolymus Cynara scolymus Vicia faba Vigna sesquipedalis Asparagus officinalis A. officinalis 5–10 High 9–33 35 65 0.5 A. officinalis Petunia hybrida Zinnia elegans A. officinalis Medicago sativa Persea americana P. americana P. americana Musa spp. Musa acuminate M. balbisiana Hordeum vulgare Aegilops spp. Agropyron elongatum Avena fatua A. sativa Avena spp. Bromus inermis Bromus spp. Commelina communis Hordeum depressum H. glaucum H. glaucum H. glaucum H. glaucum H. glaucum H. glaucum H. glaucum H. vulgare H. vulgare 3–27 – – 18–53 – 76 86–100 High – High High 2–45 – – 22 0–9.5 – 8 – 4 3 2 58 Up to 90 50–100 4–64 38–86 3–53 55–75 38 Arracacha virus A Arracacha virus B Asparagus bean mosaic Asparagus latent (Syn. Asparagus virus II) Asparagus virus I Australian Lucerne latent Avocado sunblotch Avocado viruses 1,2,3 Banana streak Banana viruses Barley mottle mosaic Barley stripe mosaic (Syn. Barley false stripe) References Lister and Murant (1967) Tomlinson and Walkey (1967) Lister (1960) Phatak (1974) Lister and Murant (1967) Taylor and Thomas (1968) Lister and Murant (1967) Tomlinson and Walkey (1967) Thomas (1981) Lister and Murant (1967) Lister and Murant (1967) Jones and Kenten (1980) Jones (1982); Jones and Kenten (1981) Kenten and Jones (1979); Jones and Kenten (1981); Jones (1982) Vide (1996); Bottalico et al. (2002); Albrechtsen (2006) Bottalico et al. (2002) Jones (1979); Kyriakopoulou et al. (1985) Avegelis et al. (1992) Snyder (1942) Paludan (1964) Uyeda and Mink (1981); Fujisawa et al. (1983); Falloon et al. (1986) Bertaccini et al. (1990) Fujisawa et al. (1983) Fujisawa et al. (1983) Bertaccini et al. (1990) Jones et al. (1979) Wallace and Drake (1953), (1962) Thomas and Mohamed (1979) Jordan et al. (1983) Daniells et al. (1995) Gold (1972) Gold (1972) Dhanraj and Raychaudhuri (1969) Nitzany and Gerechter (1962) Inouye (1962) Chiko (1975) Mckinney and Greely (1965) Nitzany and Gerechter (1962) Inouye (1962) Nitzany and Gerechter (1962) Inouye (1962) Inouye (1962) Inouye (1962) McKinney (1951a, b) McKinney (1953) Gold et al. (1954) Eslick and Afanasiev (1955) Inouye (1962) Mckinney and Greely (1965) Phatak and Summannwar (1967) Catherall (1972) (continued) 12 1 Introduction Table 1.2 (continued) Virus/viroid Bean common mosaic (Syn. Bean western mosaic, Syn. Azuki bean mosaic) Host H. vulgare H. vulgare H. vulgare H. vulgare H. vulgare Lolium spp. Poa exilis Triticum aestivum T. aestivum T. aestivum Cyamopsis tetragonoloba Lupinus luteus Macroptilium lathyroides Per cent 5–60 38–45 61–70 52 45 3–8 – 71 6.7–80 70 94 16 5–33 Phaseolus aborigineus Phaseolus acutifolius var. latifolius P. acutifolius var. latifolius P. angustifolius P. mungo 6.7 7–34 7–20 1.0 – P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris 2–3 50 43 10–25 50 21–51 10–30 20–60 2–66 10–86 2–3 5–33 7–20 20–80 P. vulgaris P. vulgaris P. vulgaris P. vulgaris P. vulgaris Phaseolus vulgaris P. vulgaris P. vulgaris – 12–66 1.3 93 39.7–54.4 1–18 12.4 39 Vigna angularis – Vigna mungo Vigna mungo Vigna mungo Vigna radiata Vigna radiata Vigna radiata Vigna radiata Vigna radiata Vigna sesquipedalis V. sinensis Vigna unguiculata 2–10 20–48 67 25 4.1–7.2 8–32 1–4.9 78 37 25–40 0.8–12.4 References Phatak (1974) Slack et al. (1975) Carroll and Mayhew (1976a, b) Lange et al. (1983) Makkouk et al. (1992) Inouye (1962) Nitzany and Gerechter (1962) Hagborg (1954) McNeal and Afanasiev (1955) Lange et al. (1983) Gillaspie et al. (1998a, b) Frencel and Pospieszny (1979) Kaiser and Mossahebi (1974); Provvidenti and Braverman (1976) Klein et al. (1988) Lockhart and Fischer (1974) Provvidenti and Cobb (1975) Klein et al. (1988) Nene (1972); Agarwal et al. (1977, 1979) Scotland and Burke (1961) Reddick and Stewart (1919) Archibald (1921) Kendrick and Gardner (1924) Burkholder and Muller (1926) Merkel (1929) Fazardo (1930) Harrison (1935) Smith and Hewitt (1938) Medina and Grogan (1961) Skotland and Burke (1961) Ordosgoitty (1972) Phatak (1974) Muniyappa (1976); Drijfhout and Bos (1977) Uyemoto and Grogan (1977) Capoor et al. (1986) Tsuchizaki et al. (1986a, b) Edwardson and Christie (1986) Morales and Castano (1987) Puttaraju et al. (1999) Njau and Lyimo (2000) Nalini et al. (2004, 2006a, b); Suteri (2007) Tsuchizaki et al. (1970a, b); Hampton et al. (1978) Agarwal et al. (1979) Provvidenti (1986) Dinesh et al. (2007) Kaiser et al. (1968) Tsuchizaki et al. (1986a, b) Kaiser and Mossahebi (1974) Choi et al. (2006) Dinesh et al. (2007) Snyder (1942) Sachchidananda et al. (1973) Hao et al. (2003) (continued) Table 1.2 (continued) Virus/viroid Bean common mosaic necrosis Bean pod mottle Bean southern mosaic (Syn. Southern bean mosaic) Host Phaseolus vulgaris Glycine max Phaseolus vulgaris Per cent 36.6 0.1 1–30 Vigna sinensis 1–40 V. unguiculata 1–3 Lens culinaris Lens culinaris – – Lens esculenta Lupinus albus L. luteus – – 5–6.2 Beet 1 alpha crypto (Syn. Beet temperate) Beet 2 alpha crypto L. luteus Melilotus alba Phaseolus vulgaris Phaseolus radiata P. vulgaris Pisum sativum Trifolium pratense Vicia faba V. faba V. faba V. faba V. faba V. faba V. radiata Vigna sinensis Beta vulgaris Beta vulgaris 7–21 3–5 7 – – 10–30 12–15 Low 0.1–2.4 0.1–0.2 1.8 2.6 9.8 – 1–37 100 100 Beet 3 alpha crypto Beet mild yellowing Beet 41 yellows Blackgram mild mottle Blackgram mottle Beta vulgaris Beta vulgaris Beta vulgaris Vigna mungo Vigna radiata Low – 47 4.9–14.9 8 Blackeye cowpea mosaic Vigna radiata Vigna mungo Vigna mungo Vigna mungo Vigna sinensis 1–1.5 5.3–16.70 1.3–15.9 5–10 30.9 V. sinensis 1.8 Vigna mungo Vigna sinensis 14 1.2–30.9 Bean yellow mosaic References Njau and Lyimo (2000) Lin and Hill (1983) Crowley (1959); Smith (1972); Jayasinghe (1982); Morales and Castano (1985) Shepherd and Fulton (1962); Givord (1981); O’Hair et al. (1981) Shepherd and Fulton (1962); Lamptey and Hamilton (1974) Bayaa et al. (1998) Kumari et al. (1994); Kumari and Makkouk (1995) Erdiller and Akbas (1996) Blaszczak (1963, 1965) Mastenbroek (1942); Corbett (1958); Zschau (1962) Porembskaya (1964) Phatak (1974) Crowley (1957a, b) Benigno and Favali-Hedayat (1977) Hino (1962) Inouye (1967) Hampton (1967) Quantz (1954a, b); Bos (1970) Kaiser (1972a, b, 1973); Evans (1973) Fiedorow (1980) Eppler and Kheder (1988) Aftab et al. (1989) El-Dougdoug et al. (1999) Benigno and Favali-Hedayat (1977) Snyder (1942) Natsuaki et al. (1983a, b, 1986) Kassanis et al. (1977, 1978); White and Woods (1978); Accotto and Boccardo (1986); Natsuaki et al. (1986); Osmond et al. (1988) VIDE (1996) Fritzsche et al. (1986) Clinch et al. (1948) Krishnareddy (1989) Phatak (1974, 1983); Scott and Phatak (1979) Saleh et al. (1986) Krishnareddy (1989) Varma et al. (1992) Dinesh Chand et al. (2004) Anderson (1957); Zettler and Evans (1972) Lin et al. (1981a, b); Tsuchizaki et al. (1984) Provvidenti (1986) Edwardson and Christie (1986) (continued) 14 1 Introduction Table 1.2 (continued) Virus/viroid Host Vigna sinensis Vigna unguiculata Per cent 2.3–38.4 6–41.6 Black raspberry latent Rubus occidentalis Chenopodium quinoa Vaccinium corymbosum Vitis labrusca Rubus rigidus S. melongena S. melongena Vicia faba Vicia faba 10 12 29 5 – 6.5 10.1 – 0–28 Cicer spp. Phaseolus vulgaris Vicia faba Lens culinaris Lens esculenta – 7 1.37 2.1 – Pisum sativum Vicia faba V. faba V. faba V. faba V. faba V. faba V. faba V. faba (minor) V. faba V. faba V. faba V. faba Vicia palastina Vicia faba Vicia faba – 1 1 10 5 7.3 4–16 3.2 0.06–2.7 6.7 16.0 3–20 6.7–15.4 – 28 0.6 Triticum aestivum Phaseolus vulgaris Phaseolus lunatus Glycine max Theobroma cacao Arachis hypogaea Daucus carota Daucus carota Daucus carota Daucus carota Daucus carota Daucus carota Cassia occidentalis > 50 1–24 – – 34–54 Low – 25 – – – – – Bramble yellow mosaic Brinjal ring mosaic Brinjal severe mosaic Broad bean mild mosaic Broad bean true mosaic Broad bean mottle Broad bean stain Broad bean true mosaic Broad bean wilt Brome mosaic Cacao necrosis Cacao swollen shoot Carlavirus Carrot motley leaf Carrot red leaf Carrot temperate 1 Carrot temperate 2 Carrot temperate 3 Carrot temperate 4 Cassia yellow spot (poty) References Sumana and Keshava Murthy (1992) Pio-Ribeiro et al. (1978); Mali and Kulthe (1980) Mali et al. (1987, 1988, 1989) Bashir and Hampton (1996a, b); Puttaraju et al. (2000), (2004a, b); Shilpashree (2006) Lister and Converse (1972) Uyemoto et al. (1977) Ramsdell and Stace-Smith (1983) Uyemoto et al. (1977) Engelbrecht (1963); Vide (1996) Sharma (1969) Sharma (1969) Devergne and Cousin (1966) Brunt (1970); Neergaard (1977); Mali et al. (2003) Erdiller and Akbas (1996) Phatak (1974) Makkouk et al. (1988) Al–Mabrouk and Mansour (1998) Erdiller and Akbas (1996); Bayaa et al. (1998) Kowalska and Beczner (1980) Lloyd et al. (1965) Varma and Gibbs (1967) Gibbs and Smith (1970) Moghal and Francki (1974) Cockbain et al. (1976) Vorra-urai and Cockbain (1977) Vorra-urai and Cockbain (1977) Jones (1978) Eppler and Kheder (1988) El-Dougdoug et al. (1999) Mali et al. (2003) El-Kewey et al. (2007) Makkouk et al. (1987) Mali et al. (2003) Putz and Kuszala (1973); Makkouk et al. (1990) Von Wechmar et al. (1984) Kenten (1972) Kenten (1977); Vide (1996) Kenten (1977); Vide (1996) Quainoo et al. (2008) Sivaprasad et al. (1990) Scott (1972) Watson and Serjeant (1962) Natsuaki et al. (1983a, b) Natsuaki et al. (1983a, b); VIDE (1996) Natsuaki et al. (1983a, b); VIDE (1996) Natsuaki et al. (1983a, b); VIDE (1996) Souto (1990); VIDE (1996) (continued) Table 1.2 (continued) Virus/viroid Cauliflower mosaic Host Capsella bursa-pastoris Raphanus raphanistrum Apium graveolens Amaranthus caudatus Amaranthus hybridus Per cent – – 34 – 0.3 Chenopodium quinoa 89 Trigonella foenumgraecum Arabidopsis thaliana Betula pendula 0.5 – 4–22 Chenopodium amaranticolor Glycine max Juglans regia 100 100 4–32 Cherry ring spot Chicory yellow mottle Chrysanthemum stunt viroid Juglans regia J. regia Nicotiana clevelandii Nicotiana megalosiphon Nicotiana tabacum Olive spp. Phaseolus vulgaris Prunus serotina Rheum rhaponticum Sambucus racemosa Solanum acaule Ulmus americana Viola tricolor Prunus avium P. cerasus Chenopodium amaranticolor Prunus spp. Taraxacum officinale Prunus avium Cichorium intybus Chrysanthemum morifolium 52.3 6 – – 1 41–90 12–48 0.5–0.8 72 13–44 – 1–48 1.2–6.1 – – 4–25 – 30 5–56 3 0–75.7 Cineraria mosaic Citrus exocortis viroid Lycopersicon esculentum Senecio cruentus Impatiens walleriana – – 66 Verbena x hybrida 28 Citrus spp. Citrus sinensis Citrus sinensis x Poncirus trifoliata Citrus spp. Trifoliate orange Chenopodium quinoa Citrus spp. Citrus spp. Citrus aurantifolia – – 19 Trace 1–10 – 1.2 70 66 Celery latent Cherry leaf roll Cherry necrotic rusty mottle Cherry rasp leaf Citrus leaf blotch Citrus mosaic Citrus psorosis Citrus tatter leaf Citrus yellow mosaic Citrus veinal chlorosis Citrus xyloporosis References Tomilson and Walker (1973) Tomilson and Walker (1973) Bos (1973); Bos et al. (1978) Bos et al. (1978) Luisoni and Lisa (1969); Bos et al. (1978) Luisoni and Lisa (1969); Bos et al. (1978) Luisoni and Lisa (1969) Artemis Rumbou et al. (2009) Cooper (1976); Schimanski et al. (1980) Lister and Murant (1967) Lister and Murant (1967) Quacquarelli and Savino (1977); Mircetich et al. (1980) Kolber et al. (1982) Cooper and Edwards (1980) Hansen and Stace-Smith (1971) Hansen and Stace-Smith (1971) Schmelzer (1965, 1966) Saponari et al. (2002) Lister and Murant (1967) Schimanski et al. (1976) Tomlinson and Walkey (1967) Schimanski and Schmelzer (1972) Crosslin et al. (2010) Bretz (1950); Callahan (1957) Lister and Murant (1967) Nyland (1962) Nyland (1962) Hansen et al. (1974) Hansen et al. (1974) Hansen et al. (1974) Cochran (1946); Cation (1949, 1952) Vovlas (1973) Monsion et al. (1973); Chung and Pak (2008) Kryczynski et al. (1988) Jones (1944) Singh and Baranwal (2008); Singh et al. (2009) Singh and Baranwal (2008); Singh et al. (2009) Guerri et al. (2004) Murthy and Subbaiah (1981) Childs and Johnson (1966) Wallace (1957) Campiglia et al. (1976) Inouye et al. (1979) Reddy et al. (1996) Sawant et al. (1983) Childs (1956) (continued) 16 1 Introduction Table 1.2 (continued) Virus/viroid Clover (red) vein mosaic Clover (red) mosaic Clover (white) mosaic Clover yellow mosaic Cocksfoot alphacryptovirus Coconut cadang-cadang viroid Coffee ring spot Coleus blumei viroid 1 Coleus blumei viroid 2 Coleus blumei viroid 3 Cowpea aphid-borne mosaic Cowpea green vein banding Cowpea chlorotic spot virus Cowpea mild mottle Cowpea mosaic Cowpea mottle Host Trifolium pratense Vicia faba Trifolium pratense Vicia faba Trifolium pratense Per cent – 100 12–18 – 1–12 T. pratense – Cocos nucifera Coffea excelsa Coleus scutellarioides Coleus scutellarioides Coleus scutellarioides Arachis hypogaea Arachis hypogaea Phaseolus angularis Vigna sesquipedalis V. sinensis V. sinensis V. sinensis V. sinensis 7.6 – – 8.1 16–68 71.4 – 0.15 – – – 23 0.3–1.6 5–16 35 V. unguiculata V. unguiculata V. unguiculata V. unguiculata V. unguiculata 27 0–30 0–2 3–19 1.1–39.8 V. unguiculata V. unguiculata up to 20.9 6.3–18.3 V. unguiculata V. unguiculata V. unguiculata V. unguiculata V. unguiculata V. sinensis V. sinensis Glycine max G. max G. max Phaseolus vulgaris V. unguiculata Vigna catjang V. sinensis V. sinensis V. sinensis V. sinensis V. sinensis Vigna unguiculata Phaseolus vulgaris V. unguiculata V. unguiculata Voandzeia subterranea 4.7 5.0–20 5.9 3.8–5.4 0.67–13.49 – 3–18 90 0.9 0.5 6 90 17 17.5 23 0–55 – 1–5 75–84 – 3–10.3 0.4 2 References Matsulevich (1957) Sander (1959) Hampton (1967) Sander (1959) Hampton (1963); Hampton and Hanson (1968) Hampton (1963) VIDE (1996) Pacumbaba et al. (1994) Reyes (1961) Singh et al. (1991a) VIDE (1996) VIDE (1996) Gillaspie et al. (2001) Pio–Ribeiro et al. (2000a, b) Tsuchizaki et al. (1970a, b) Chang and Kno (1983) Capoor and Varma (1956) Lovisolo and Conti (1966) Chenulu et al. (1968) Phatak (1974); Ramachandran and Sunmanwar (1982) Snyder (1942) Kaiser et al. (1968); Phatak (1974) Bock (1973) Phatak (1974) Kaiser and Mossahebi (1975); Ndiaye et al. (1993) Ladipo (1977) Ata et al. (1982); Bashir and Hampton (1996a, b) Chang and Kno (1983) Mali et al. (1987, 1988, 1989) Pio–Ribeiro et al. (2000a, b) El-Kewey et al. (2007) Udayashankar et al. (2009) VIDE (1996) Sharma and Varma (1975a, b, 1986) Brunt and Kenten (1973) Iwaki et al. (1982) Thouvenel et al. (1982) Brunt and Kenten (1973) Brunt and Kenten (1973) Capoor and Varma (1956) Diwakar and Mali (1977) Capoor and Varma (1956) Anderson (1957); Haque and Chenulu (1972) Khatri and Chohan (1972) Gilmer et al. (1974) Mahalakshmi et al. (2008) Shoyinka et al. (1978) Shoyinka et al. (1978) Allen et al. (1982) Bird and Corbett (1988); Robertson (1966) (continued) Table 1.2 (continued) Virus/viroid Cowpea Moroccan aphid-borne mosaic Cowpea severe mosaic Cowpea severe mottle Crimson clover latent Cucumber green mottle mosaic Cucumber leaf spot carmovirus Cucumber mosaic (Syn. Cowpea banding mosaic, Syn. Cowpea ring spot, Syn. Soybean stunt) Host Vigna unguiculata Per cent – References VIDE (1996) Vigna sesquipedalis Vigna sinensis Vigna unguiculata Vigna sinensis Trifolium incarnatum Citrullus vulgaris Citrullus vulgaris Cucumis sativus C. sativus 8 3.3–5.8 10 0.7 97 5 – 44 4.2 Lagenaria siceraria – Cucumis melo Arachis hypogaea Benincasa hispida Capsicum annuum Capsicum frutescens Cerastium holosteoides Cicer arietinum 10–40 1.3 1 1 53–83 2 – Cucumis melo C. melo C. melo C. sativus Cucurbita moschata C. pepo 2.1 16 11.37–23.07 1.4 0.7 0.07 Echinocystis lobata E. lobata E. lobata G. max G. max G. max G. max Lamium purpureum Lens culinaris 9.1 55 15 50 95 – > 70 4 7.0–64.2 Lupinus angustifolius Lupinus luteus L. luteus Lycopersicon esculentum Medicago sativa Phaseolus vulgaris P. vulgaris P. vulgaris P. vulgaris Pisum sativum Solanum melongena Spergula arvensis Stellaria media S. media 12–18 – 14 0.2 0.1–0.3 41 30 0–49 30–100 1 – 2 1–30 5–8 Dale (1949) Haque and Persad (1975) Shepherd (1964); VIDE (1996) Dos (1987) Kenten et al. (1980a, b) Komuro et al. (1971) Lee et al. (1990) Yakovleva (1965) Kawai et al. (1985); Rama Murthy et al. (2008) Komuro et al. (1971); Sharma and Chohan (1973) VIDE (1996) Xu and Barnett (1984) Sharma and Chohan (1973) Glaeser (1976) Akhtar Ali and Kobayashi (2010) Tomlinson and Carter (1970a, b) Jones and Coutts (1996); Makkouk et al. (2001) Kendrick (1934) Mahoney (1935) Sandhu and Kang (2007) Doolittle (1920) Sharma and Chohan (1973) Reddy and Nariani (1963); Sharma and Chohan (1973) Doolittle and Gilbert (1919) Doolittle and Walker (1925) Lindberg et al. (1956) Koshimizu and Iizuka (1963) Iizuka (1973) Shen et al. (1984) Honda et al. (1988) Tomlinson and Carter (1970a, b) Jones and Coutts (1996); Fletcher et al. (1997); Makkouk et al. (2001, 2003); Jones (2004a, b) Alberts et al. (1985); Jones (1988) Troll (1957) Porembskaya (1964) Van Koot (1949) Jones (2004a, b) Bos and Maat (1974) Marchoux et al. (1977) Davis and Hampton (1986) Bhattiprolu (1991) Latham and Jones (2001a) Sriram and Doraiswamy (2001) Tomlinson and Carter (1970a, b) Hani et al. (1970); Hani (1971) Tomlinson and Carter (1970a, b) (continued) 18 1 Introduction Table 1.2 (continued) Virus/viroid Cucumis sativus cryptic Cucumber cryptic Cycas necrotic stunt Dahlia mosaic Desmodium mosaic Desmodium trifolium mottle Dodder latent mosaic Dulcamara mottle Echtes Ackerbohnen mosaic (Syn. Broad bean true mosaic) Eggplant mosaic (Syn. Andean potato latent) Elm mosaic Elm mottle Host S. media S. media Spinacia oleracea Trifolium subterraneum Per cent 3–40 1–4 15 8.8 References Tomlinson and Carter (1970a, b) Tomilson and Walker (1973) Yang et al. (1997) Jones and Mc Kirdy (1990); Jones (1991a, b) Vicia faba – Latham and Jones (2001a) Vigna radiata 8–32 Kaiser et al. (1968); Kaiser and Mossahebi (1974) V. radiata 5 Phatak (1974) V. radiata 0.61 Purivirojkul et al. (1978) V. radiata 11 Iwaki (1978) V. sesquipedalis 4–28 Anderson (1957) V. sinensis 30 Meiners et al. (1977) V. sinensis 10 Iwaki (1978) V. sinensis 15–31 Sharma and Varma (1975a, b, 1984); Prakash and Joshi (1980) V. unguiculata 4–28 Anderson (1957) Vigna unguiculata 5 Iizuka (1973) V. unguiculata 10–30 Phatak (1974) V. unguiculata 15–20 Phatak et al. (1976) V. unguiculata 26 Fischer and Lockhart (1976) V. unguiculata 3–10 Pio-Ribeiro et al. (1978) V. unguiculata 4–18 Mali et al. (1987) V. unguiculata 1.2–2 Dos (1987); Bashir and Hampton (1996a, b) V. unguiculata 1.5–37 Gillaspie et al. (1998a, b) V. unguiculata 1.3–25.8 Mali et al. (1989) V. unguiculata 1.5–37 Gillaspie et al. (1998a, b) V. unguiculata 10–30 Abdullahi et al. (2001) Cucumis sativus – Jelkmann et al. (1988) Cucumis sativus – Boccardo et al. (1983, 1987) Chenopodium amaranticolor 30 VIDE (1996) C. serotinum 80 VIDE (1996) Dahlia pinnata – Pahalawatta et al. (2007) Desmodium canum 8 Edwardson et al. (1970) Desmodium trifolium – Suteri and Joshi (1978) Cuscuta californica 2.4 Bennett (1944) C. campestris 4.9 Bennett (1944) Solanum dulcamara 23 Gibbs et al. (1966) Solanum tuberosum <1 Jones and Fribourg (1977) Vicia faba 1–3 Quantz (1953) V. faba 15 Gibbs and Smith (1970); Gibbs and Paul (1970) V. faba 0.8 Cockbain et al. (1976) V. faba 5 Vorra-urai and Cockbain (1977) V. faba 4.8 Eppler and Kheder (1988) Andigena clone 1 Jones and Fribourg (1977) Nicotiana clevelandii – Jones (1982) Petunia hybrida – Gibbs et al. (1966) Solanum tuberosum <1 Jones and Fribourg (1977) Ulmus americana 1–3.5 Bretz (1950) Ulmus americana 48 Callahan (1957) Ulmus glabra – Jones and Mayo (1973) (continued) Table 1.2 (continued) Virus/viroid Eucharis mottle nepovirus Euonymus mosaic Fescue cryptic Fig latent virus 1 Foxtail mosaic potexvirus Fragaria chiloensis ilarvirus French bean mosaic Garland chrysanthemum temperate Grape decline Grapevine Bulgarian latent Grapevine fanleaf Grapevine yellow mosaic Grapevine yellow speckle Guar symptomless Hibiscus latent ring spot Hemp streak Hippeastrum mosaic High plains virus Hop chlorosis Hop stunt viroid (Syn. Cucumber pale fruit, Syn. Grapevine viroid) Hop trefoil cryptic1 Hop trefoil cryptic 2 Hop trefoil cryptic 3 Hosta virus x Humulus japonicus Hydrangea mosaic Kalanchoe top-spotting Leek yellow stripe Lettuce mosaic Host Eucharis candida Euonymus europaeus Festuca pratensis Ficus carica Setaria italica Fragaria chiloensis Phaseolus vulgaris Chrysanthemum coronarium Vitis vinifera Vitis vinifera Chenopodium quinoa C. amaranticolor C. quinoa Per cent – – – – – 30–50 12–66 – – 4.54 12.50 1.3 3 Glycine max Vitis vinifera Chenopodium amaranticolor Vitis vinifera Cyamopsis tetragonoloba 59 0–66 0.7 – 12–28 Hibiscus cannabinus C. amaranticolor C. quinoa Cannabis sativa Hippeastrum hybridum Zea mays Humulus lupulus Lycopersicon esculentum Vitis vinifera Medicago lupulina Medicago lupulina Medicago lupulina Hosta longipes Humulus japonicus Chenopodium quinoa Kalanchoe blossfeldiana 26 11 1 – – Low 27 – – High High High 7.5 – 82.3 – Allium cepa Chenopodium quinoa Lactuca sativa L. sativa L. sativa L. sativa L. sativa – 0–1 3.1 10 2–8 6–15 1–8 L. sativa L. sativa L. sativa L. sativa L. scariola Senecio vulgaris 3–10 11 13 5 0.2–6.2 2.3 References VIDE (1996) Bojannsky and Koslarova (1968) Boccardo et al. (1983) Castellano et al. (2009) VIDE (1996) VIDE (1996) Capoor et al. (1986) Natsuaki et al. (1983a, b) Dias and Cation (1976) Uyemoto et al. (1977) Uyemoto et al. (1977) Dias (1963) Bruckbauer and Rudel (1961); Cory and Hewitt (1968) Cory and Hewitt (1968) Cory and Hewitt (1968) Dias (1963) Wan chow wah and Symons (1999) Hansen and Leseman (1978); Behncken (1983) Rubies-Autonell (1997) Rubies-Autonell (1997) Rubies-Autonell (1997) Brierly (1944) Brants and Vanden Heuvel (1965) Forster et al. (2001) Salmon and Ware (1935) Kryczynski et al. (1988) Wah and Symons (1999); Mink (1993) Boccardo et al. (1983) Boccardo et al. (1983, 1987) Boccardo et al. (1983, 1987) Ryu et al. (2006) VIDE (1996) Thomas et al. (1984) Hearon and Locke (1984); Brunt (1992) Vishnichenko et al. (1990) Phatak (1974) Newhall (1923) Ogilvie et al. (1935) Ainsworth and Ogilvie (1939) Kramer et al. (1945) Grogan and Bardin (1950); Grogan et al. (1952) Couch (1955) Herold (1956) Rohloff (1962) Ryder (1964) van Hoof (1959) Phatak (1974) (continued) 20 1 Introduction Table 1.2 (continued) Virus/viroid Lettuce yellow mosaic Lilac ring mottle Lima bean mosaic Lucerne (Australian) symptomless Lucerne Australian latent Lucerne transient streak Lychnis ring spot Maize chlorotic mottle machlomovirus Maize dwarf mosaic Maize leaf spot Maize mosaic Melon rugose mosaic Melon necrotic spot Mibuna temperate Mung bean isometric yellow mosaic Mung bean mosaic potyvirus Mulberry ring spot Muskmelon mosaic (Syn. Squash mosaic) Host L. sativa C. amaranticolor C. quinoa Celosia argentea Phaseolus limensis P. limensis P. lunatus Chenopodium quinoa Medicago sativa Per cent 30 13 91 24 25 2.2 0.3 6 8 C. amaranticolor – C. quinoa 9 Melilotus albus Beta vulgaris Capsella bursa-pastoris Cerastium viscosum Lychnis divaricata Silene gallica S. nodiflora Zea mays Zea mays 2.5 9.5 9.4 27 58 28 41 – 0.02–1.65 Zea mays Z. mays Cucumis melo Cucumis melo var. flexuosus Cucumis melo – – 0.9 3.8 Brassica rapa var. laciniifolia Vigna radiata – – – Glycine max Citrullus vulgaris Cucumis melo Cucumis melo – 10 1.5 1–27 12–93 C. melo 12–93 C. melo C. melo C. melo 7–21 9–10 3 20–22.5 References Mandahar (1978) Van der Meer et al. (1976) Van der Meer et al. (1976) Van der Meer et al. (1976) Mandahar (1978) Sawant and Capoor (1983) Gay (1972) Remah et al. (1986) Taylor and Smith (1971); Blackstock (1978); Jones et al. (1979) Taylor and Smith (1971); Blackstock (1978); Jones et al. (1979) Taylor and Smith (1971); Blackstock (1978); Jones et al. (1979) Paliwal (1983) Bennett (1959) Bennett (1959) Bennett (1959) Bennett (1959) Bennett (1959) Bennett (1959) VIDE (1996) Williams et al. (1968); Hill et al. (1974); Tosic and Sutic (1977) Tsvet kov (1967) Tosic and Sutic (1977) Mahgoub et al. (1997) Mahgoub et al. (1997) Kishi (1966) ; Gonzalez-Garza et al. (1979); Avegelis (1985); Campbell et al. (1996) Natsuaki et al. (1983a, b) Benigno and Favali-Hedayat (1977) Mink (1993) Tsuchizaki et al. (1971) Nelson and Knuhtsen (1969) Mahoney (1935) Rader et al. (1947); Seth et al. (1980) Rader et al. (1947); Mukhayyish and Makkouk (1983) Grogan et al. (1959) Kemp et al. (1972) Nelson and Knuhtsen (1973) (continued) Table 1.2 (continued) Virus/viroid Muskmelon necrotic spot Nicotiana velutina mosaic Oat mosaic Olive latent virus-1 Onion mosaic Onion yellow dwarf Panicum mosaic sobemo Papaya ring spot Paprika mild mottle tobamovirus Parsley latent Pea early browning Pea enation mosaic Pea false leaf roll Pea mild mosaic Pea mosaic (Syn. Bean yellow mosaic) Host C. melo C. melo C. melo C. melo Per cent 4 0–34.6 30 – Cucurbita flexuous C. maxima Cucumis melo C. melo C. mixta C. moschata – 0.2–1.5 6.6–20 3 0.3 – C. pepo C. pepo C. pepo 2.2 – 5 Chenopodium murale C. quinoa Cucumis melo 23 20 – C. melo Sechium edule Nicotiana glutinosa Nicotiana spp. Avena sativa Olive Allium cepa A. cepa Panicum spp. Carica papaya Capsicum spp. Petroselinum crispum Pisum sativum P. sativum P. sativum P. sativum P. sativum Vicia faba V. faba V. faba Lathyrus ochrus P. sativum P. sativum P. sativum P. sativum 22.5 – <72 – – 35–82 – 6–29 – 0.15 – – 37 1–2 30 61 25 5 0.3–8 9–45 – 1.5 – 4–5 40 P. sativum P. sativum Lathyrus Trifolium hybridum T. pratense 15 – – 0.7 47 References Phatak (1974) Alvarez and Campbell (1978) Lange et al. (1983) Izadpanah (1987); Avegelis and Katis (1989a, b) Rader et al. (1947) Grogan et al. (1959) Grogan et al. (1959) Nelson and Knuhtsen (1973) Grogan et al. (1959) Rader et al. (1947); Campbell (1971) Middleton (1944) Rader et al. (1947) Grogan et al. (1959); Knuhtsen and Nelson (1968) Lockhart et al. (1985) Lockhart et al. (1985) Gonzalez-Garza et al. (1979); Avegelis (1985) Avegelis (1985) Singh (1981) Randles et al. (1976) Randles et al. (1976) Carr (1971) Saponari et al. (2002) Cheremuskina (1977) Hardtl (1964, 1972) VIDE (1996) Bayot et al. (1990) VIDE (1996) Bos et al. (1979) Bos and van der Want (1962) Harrison (1973) Lockhart and Fischer (1976) Fiedorow (1983) Pospieszny and Frencel (1985) Cockbain et al. (1983) Fiedorow (1983) Mahir et al. (1992) Kovachevski (1978) Blattny (1956) Kovachevski (1978) Kheder and Eppler (1988) Thottappilly and Schmutterer (1968) Clark (1972) Quantz (1958) Mandahar (1978) Mandahar (1978) Mandahar (1978) (continued) 22 1 Introduction Table 1.2 (continued) Virus/viroid Pea seed-borne mosaic (Syn. Pea fizzle top and Pea leaf rolling virus) Pea stem necrosis Pea streak Peach latent Peach rosette mosaic Peanut clump (African) Peanut clump (Indian) Host Cicer arietinum Lathyrus clymenum Lens culinaris Per cent – 5 0.8 Lens culinaris 32–44 L. culinaris 0.5–5 L. culinaris – L. culinaris Pisum arvense Pisum sativum P. sativum 6 9 8–30 0–88 P. sativum P. sativum 20–80 65–90 P. sativum P. sativum P. sativum P. sativum 2–55 4–32 0.5–5.8 30–60 P. sativum 23–24 P. sativum P. sativum P. sativum 10 58 10 P. sativum 1–18 P. sativum P. sativum Vicia spp. 1.9–32.7 5–30 0.11–3 Vicia faba 0.2–2 Pisum sativum Pisum sativum Prunus sp. Chenopodium quinoa Taraxacum officinale Vitis vinifera A. hypogaea Eleusine coracana Pennisetum glaucum A. hypogaea A. hypogaea Eleusine coracana Pennisetum glauca Setaria italica Triticum aestivum – 1.7 2.4 90 3.6 9.5 24–48 5.2 0.9 3.5–17 9–11 5.2–6.5 0.93 9.7–10.2 0.5–1.3 References Makkouk et al. (2001) Latham and Jones (2001a) Al–Mabrouk and Mansour (1998) Hampton and Muehlbauer (1977); Eppler et al. (1988) Hampton (1982); Goodell and Hampton (1984); Bayaa et al. (1998) Erdiller and Akbas (1996); Makkouk et al. (2001) Coutts et al. (2008) Zimmer and Ali-Khan (1976) Inouye (1967) Stevenson and Hagedorn (1969, 1973) Hampton (1969) Mink et al. (1969); Cockbain (1988); Alconero and Hoch (1989) Musil (1970) Hampton (1972) Chiko and Zimmer (1978) Thakur et al. (1984); Rishi and Singh (1987) Kheder and Eppler (1988); Johansen et al. (1996) Kumar et al. (1991) McKeown and Biddle (1991) Zimmer and Lamb (1993); Sontakke and Chavan (2007) Latham and Jones (2001a); Deepti Anand et al. (2006) Gallo and Jurik (1995) Coutts et al. (2008) Hampton and Mink (1975); Musil (1980); Boulton et al. (1996) Latham and Jones (2001b); Coutts et al. (2008) VIDE (1996) Kheder and Eppler (1988) Mandahar (1978) Dias and Cation (1976) Ramsdell and Myers (1978) Ramsdell and Myers (1978) Thouvenel et al. (1978) Dieryck et al. (2009) Dieryck et al. (2009) Reddy et al. (1998a, b) Reddy et al. (1988, 1989) Reddy et al. (1989, 1998a, b) Reddy et al. (1989, 1998a, b) Reddy et al. (1989, 1998a, b) Delfosse et al. (1999) (continued) Table 1.2 (continued) Virus/viroid Peanut marginal chlorosis Peanut mottle Host A. hypogaea A. hypogaea Per cent 30–100 0.02–2 A. hypogaea A. hypogaea A. hypogaea A. hypogaea 20 3.7 0–8.5 1.3 A. hypogaea Glycine max Lupinus albus (white lupin) Phaseolus vulgaris Vigna unguiculata A. hypogaea Glycine max 1–7 0.22 0.37 1.0 0.8 3–4 0.2 Pelargonium zonate spot Pepino mosaic Pepper chat fruit viroid Piper yellow mottle Plum pox virus A. hypogaea A. hypogaea A. hypogaea A. hypogaea A. hypogaea A. hypogaea A. hypogaea Glycine max Glycine max Vigna unguiculata Nicotiana glutinosa Lycopersicon esculentum Capsicum annuum Piper nigrum Apricot 5–20 1.3–4.8 19.3–37.6 28.8 43 12.5 60.0 28.0 3 17.8 5 1.84 19 30 24–92 Potato Andean latent tymovirus Peach Plum Solanum tuberosum 15–84 64 <1 Potato spindle tuber Lycopersicon esculentum 2–11 Physalis peruviana Scopolia sinensis Solanum incanum S. tuberosum 29 71 53 6–66 S. tuberosum 87–100 Datura stramonium Nicandra physalodes Solanum demissum-A Solanum tuberosum cv. Cara Solanum tuberosum cv. D 42/8 5–72 28 39 33–59 0–2 Peanut stunt Peanut stripe (Syn. Peanut mild mottle) Potato virus T References van Velsen (1961) Kuhn (1965); Demski et al. (1983) Bock (1973) Paguio and Kuhn (1974) Adams and Kuhn (1977) Bharatan et al. (1984); Iizuka and Reddy (1986) Puttaraju et al. (2001) Iwaki et al. (1986) Demski et al. (1983) Demski et al. (1983) Demski et al. (1983) Iizuka and Yunoki (1974) Troutman et al. (1967); Kuhn (1969) Xu et al. (1991) Xu et al. (1983) Demski et al. (1984a, b) Prasada Rao et al. (1988) Okhi et al. (1989) Chang et al. (1990) Matsumoto et al. (1991) Warwick and Demski (1988) Vetten et al. (1992) Vetten et al. (1992) Gallitelli (1982) Cordoba-Selles et al. (2007) Verhoeven et al. (2009) Hareesh and Bhat (2010) Nemeth and Kolber (1982); Pasquini et al. (1998) Nemeth and Kolber (1983) Nemeth and Kolber (1983) VIDE (1996); Jones and Fribourg (1977) Singh (1970); Kryczynski et al. (1988) McClean (1948) Singh and Finnie (1973) McClean (1948) Singh (1970); Singh et al. (1992a, b) Hunter et al. (1969); Fernow et al. (1970); Grasmick and Slack (1986) Salazar and Harrison (1978) Salazar and Harrison (1978) Salazar and Harrison (1978) Jones (1982) Jones (1982) (continued) 24 1 Introduction Table 1.2 (continued) Virus/viroid Potato virus U Potato virus X Potato virus Y (Syn. Brinjal mosaic) Potato yellowing Prune dwarf (Syn. Cherry ring mottle, Syn. Cherry yellows) Prunus necrotic ring spot (Syn. Peach necrotic leaf spot, Syn. Peach ring spot) Radish yellow edge Loganberry degeneration Raspberry bushy dwarf (Syn. Loganberry degeneration) Raspberry latent (Black raspberry latent) Raspberry ring spot Red clover cryptic Host Chenopodium quinoa C. amaranticolor Nicotiana debneyi N. tabacum cv. Xanthi Solanum tuberosum S. tuberosum Solanum nigrum Solanum melongena Solanum brevidens Prunus spp. Prunus spp. Prunus avium Prunus cerasus P. mahaleb P. mahaleb P. persica P. persica Cucurbita maxima Prunus americana Per cent – – – – 0.6–2.3 14–16 – 9–41 20 33.3 17–77.7 0–58 3–30 9 1.5–21.4 – 10 2.7 1.1 P. amygdalus P. avium P. avium P. cerasus P. cerasus P. cerasus P. mahaleb P. mahaleb P. pensylvanica P. persica P. persica P. persica P. persica P. persica Rubus idaeus Brassica vulgaris Raphanus sativus Rubus longanobaccus Rubus idaeus Rubus longanobaccus Rubus idaeus Stellaria media Beta vulgaris Capsella bursa-pastoris Fragaria x ananassa Fragaria spp. Glycine max G. soja Petunia violacea Rubus idaeus Stellaria media Trifolium pratense – 6.0 37 20–56 91 – 10 70 37 3–9 16 1.1–11.7 16 17 40–50 – 80–100 – 22–60 – 10 8–26 50–55 2–10 References Jones et al. (1983) Jones et al. (1983) Jones et al. (1983) Jones et al. (1983) Darozhkin and Chykava (1974) Mandahar (1978) Eskarous et al. (1983) Mayee and Khatri (1975) Valkonen et al. (1992) Ramaswamy and Posnette (1971) Mandahar (1978) Mink and Aichele (1984) Cation (1952); Gilmer and Way (1960) Cation (1949, 1952) Schimanski and Schade (1974) Cochran (1950) Mink and Aichele (1984) Das et al. (1961) Hobart (1956); Schimanski and Fuchs (1984) Williams et al. (1970); Barba (1986) Cochran (1946) Megahed and Moore (1967) Cation (1949, 1952) Megahed and Moore (1967) Davidson (1976) Cation (1949, 1952) Megahed and Moore (1967) Megahed and Moore (1967) Cochran (1950) Millikan (1959); Wagnon et al. (1960) Wagnon et al. (1960); Mandahar (1978) Millikan (1959); Wagnon et al. (1960) Mink and Aichele (1984) Cadman (1965) Natsuaki et al. (1979) Natsuaki et al. (1979, 1983a, b) Ormerod (1970) Cadman (1965); Converse (1973) Ormerod (1970) Converse and Lister (1969) Murant et al. (1968) Lister and Murant (1967) Lister and Murant (1967) 35–49 35 7.2 20 16.5 18 29 – Lister (1960); Lister and Murant (1967) Lister and Murant (1967) Lister (1960); Lister and Murant (1967) Mandahar (1978) Phatak (1974) Lister and Murant (1967) Lister and Murant (1967) Boccardo et al. (1983) (continued) Table 1.2 (continued) Virus/viroid Red clover mottle comovirus Red clover vein mosaic carlavirus Red pepper cryptic-1 Host Trifolium spp. Trifolium pratense Vicia faba Capsicum spp. Per cent – – – – Red pepper cryptic-2 Capsicum spp. – Rhubarb temperate Rubus Chinese seed-borne nepovirus Runner bean mosaic Rye grass cryptic Rheum rhaponticum Rubus spp. Phaseolus coccineus Lolium multiflorum – – 42 82 Safflower mosaic Santosai temperate Carthamus tinctorius Brassica rapa var. amplexicaulis sub var. dentata Phaseolus vulgaris Pachyrhizus erosus Atriplex pacifica Chenopodium album C. amaranticolor 2.2–5.0 – C. murale C. quinoa 45–70 2–46 Glycine max Glycine max 22–70 0–68 G. max G. max G. max G. max G. max G. max G. max G. max G. max G. max G. max G. max G. max G. max G. soja Lupinus albus Phaseolus vulgaris Glycine max Celosia cristata Chenopodium quinoa C. quinoa Nicotiana clevelandii 40 1–18 1–24 34 55.9 10.6–29.1 20.5–29.5 64 10 11.2–41.1 25.7–91.7 43 32.9 5.3 10–25 1.2 1.6 95 53 90 60 90 Satsuma dwarf Sincomas mosaic Sowbane mosaic Soybean mild mosaic Soybean mosaic Soybean streak Spinach latent virus 8.6 40–80 21 30 14–62 References Mink (1993) VIDE (1996) VIDE (1996) Boccardo et al. (1985); Natsuaki et al. (1987); Vide (1996) Boccardo et al. (1985); Natsuaki et al. (1987); VIDE (1996) Natsuaki et al. (1983a, b) VIDE (1996) Vashisth and Nagaich (1965) Plumb (1973); Plumb and Misari (1974); Boccardo et al. (1983) Chauhan and Singh (1979) Natsuaki et al. (1983a, b) Kishi (1967) Fajardo and Maranon (1932) Bennett and Costa (1961) Bennett and Costa (1961) Kado (1967); Dias and Waterworth (1967) Bennett and Costa (1961) Bancroft and Tolin (1967); Dias and Waterworth (1967) Takahashi et al. (1974, 1980) Kendrick and Gardner (1924); Ganesha Naik and Keshava Murthy (1997) Heinze and Kohler (1941) Ross (1963) Kennedy and Cooper (1967) Iizuka (1973) Phatak (1974) Suteri (1981) Kim and Lee (1986) Edwardson and Christie (1986) Nakano et al. (1988) Tu (1989) Pacumbaba (1995) Domier et al. (2007) Patil and Byadgi (2005) Golnaraghi et al. (2004) Mandahar (1978) Vroon et al. (1988) Castano and Morales (1983) Iizuka (1973) Bos et al. (1980) Bos et al. (1980) Stefanac and Wrischer (1983) Stefanac and Wrischer (1983) (continued) 26 1 Introduction Table 1.2 (continued) Virus/viroid Spinach temperate crypto Stone fruit ring spot Strawberry latent ring spot Strawberry pallidosis Subterranean clover mottle Sugarcane mosaic Sunflower mosaic potyvirus Sunflower rugose mosaic Sunflower ring spot ilarvirus Sunn-hemp mosaic (Syn. Sunn-hemp rosette) Sweet potato ring spot Telfairia mosaic Tobacco mosaic Host N. megalosiphon Nicotiana rustica N. tabacum white Burley N. tabacum Xanthi Spinacia oleracea S. oleracea Spinacia oleracea Cucurbita pepo Amaranthus viridis Apium graveolens Capsella bursa-pastoris Per cent 95 30 90 94 50 56 – – 96 98–100 4 Chenopodium quinoa C. quinoa Lamium amplexicaule Mentha arvensis Pastinaca sativa Petroselinum crispum var. neapolitanum Petroselinum hortense Rosa multiflora Rosa rugosa Rubus idaeus Senecio vulgaris 63–100 74 – 6 22.4 6.6 Solanum nigrum Stellaria media Stellaria media Fragaria vesca Trifolium subterraneum Zea mays 0.1 < 60 97 – 0.5–3 0.1–0.4 Zea mays Helianthus annuus Helianthus annuus Helianthus annuus Vigna unguiculata Crotalaria juncea Ipomoea batatas Telfairia occientatis Arabidopsis thaliana Capsicum annuum Capsicum annuum 4.81 – 5.6 ?? 2.5–17.5 10–20 – 6–20 High 45 13.5–29.6 Capsicum frutescens 22 – – – 75 20 References Stefanac and Wrischer (1983) Bos et al. (1980) Bos et al. (1980) Bos et al. (1980) Bos et al. (1980) Stefanac and Wrischer (1983) Natsuaki et al. (1983a, b); VIDE (1996) Mandahar (1978) Van Hoof (1976) Walkey and Whittingham-Jones (1970) Schmelzer (1969); Allen et al. (1970); Van Hoof (1976) Schmelzer (1969); Allen et al. (1970) Hanson and Campbell (1979) Murant and Goold (1969) Taylor and Thomas (1968) Hicks et al. (1986) Hanson and Campbell (1979) Bellardi and Bertaccini (1991, 1992) Thomas (1981) Thomas (1981) Anon (1968); Murant and Goold (1969) Murant and Goold (1969); Van Hoof (1976) Van Hoof (1976) Van Hoof (1976) Murant and Goold (1969) Yoshikawa and Converse (1990) Francki et al. (1988); Njeru et al. (1997) Shepherd and Holdeman (1965); Williams et al. (1968); Baudin (1969); Mikel et al. (1984); Von Wechmar et al. (1984) Li et al. (2007) Mink (1993); VIDE (1996) Singh (1979) VIDE (1996) Mali et al. (1989) Verma and Awasthi (1978) VIDE (1996) Anno-Nyako (1988) de Assis Filho and Sherwood (2000) Glaeser (1976); Demski (1981) Tosic et al. (1980); Chitra et al. (1998, 2002) McKinney (1952); Sakamoto and Matsuo (1972) (continued) Table 1.2 (continued) Virus/viroid Tobacco necrosis Tobacco rattle Tobacco ring spot Host Capsicum frutescens Carthamus tinctorius Lycopersicon esculentum Per cent 18.4 – 2–6 Lycopersicon esculentum Malus platycarpa Malus pumila M. sylvestris Plantago major Pyrus communis Vigna unguiculata V. unguiculata Vitis vinifera Zea mays Beta vulgaris Capsella bursa-pastoris Lamium amplexicaule Myosotis arvensis Papaver rhoeas Senecio vulgaris Viola arvensis 98.1 38 21 3–37 – 35 1–4 14.6–22.6 20 – 15–20 1.9 2.2 6.0 1.1 1 3 Amaranthus hybridus Cucumis melo Cucumis sativus Gladiolus spp. Glycine max G. max G. max G. max G. max G. max G. max 14–21 3–7 – 4 54–78 78–82 100 40 100 100 94–97 G. max Gomphrena globosa G. globosa Lactuca sativa L. sativa Nicotiana glutinosa N. tabacum Pelargonium hortorum Petunia violacea Phaseolus aureus Senecio vulgaris Solanum melongena Solanum tuberosum Taraxacum officinale Vigna sinensis Zinnia elegans 2.1 25–50 46 3 21 – 4.9–17 4–6 20 68–91 52 3.2–9.8 2–9 9–36 82 5 References Cicek and Yorganci (1991) Lockhart and Goethals (1977) Doolittle and Beecher (1937); Taylor et al. (1961); Chitra et al. (1998, 1999, 2002) Cicek and Yorganci (1991) Gilmer and Wilks (1967) Allen (1969) Gilmer and Wilks (1967) Prochazkova (1977) Gilmer and Wilks (1967) Phatak (1974) Mali et al. (1987) Gilmer and Kelts (1968) Von Wechmar et al. (1992) Dikova (2005) Lister and Murant (1967) Lister and Murant (1967) Lister and Murant (1967) Lister and Murant (1967) Cooper and Harrison (1973) Lister and Murant (1967); Cooper and Harrison (1973) Sammons and Barnett (1987) McLean (1962) Stace-Smith (1970) Sushak (1976) Desjardins et al. (1954) Kahn (1956) Athow and Bancroft (1959) Kahn et al. (1962) Owusu et al. (1968) Iizuka (1973) Yang and Hamilton (1974); Hamilton (1985a, b) Golnaraghi et al. (2004) Kahn et al. (1962) Iizuka (1973) Grogan and Schnathorst (1955) Iizuka (1973) Stace-Smith (1970) Valleau (1941) Scarborough and Smith (1975) Henderson (1931) Shivanathan (1977) Tomilson and Carter (1971) Sastry and Nayudu (1976) Jones (1982) Tuite (1960) Kahn (1956) Iizuka (1973) (continued) 28 1 Introduction Table 1.2 (continued) Virus/viroid Tobacco streak (Syn. Asparagus stunt, Syn. Bean red node, Syn. Datura quercina) Tomato apical stunt viroid Tomato aspermy Tomato black ring Host A. officinalis Chenopodium quinoa Datura stramonium D. stramonium Fragaria vesca Glycine max G. max G. max G. max Lycopersicon esculentum Melilotus albus Parthenium hysterophorus Phaseolus vulgaris Per cent – – 79 94 0–35 2.6–30 90 30–80 2.3 40–76 – 6.8–48 1–26 Phaseolus vulgaris Vigna unguiculata 27 1 Lycopersicon esculentum Phaseolus vulgaris Stellaria media Beta vulgaris B. vulgaris Capsella bursa-pastoris Cerastium vulgatum Chenopodium album C. quinoa Fragaria x ananassa Fumaria officinalis Geranium dissectum Glycine max Lactuca sativa Lamium amplexicaule Ligustrum vulgare Lycopersicon esculentum Myosotis arvensis Nicotiana clevelandii N. rustica N. tabacum Petunia violacea Poa annua Polygonum aviculare P. convolvulus P. persicaria Rubus idaeus Rubus spp. Sambucus nigra Senecio vulgaris Spergula arvensis Stellaria media Vigna sinensis 80 18.7 – 3–27 56 90 33–100 84 78 40 100 – 83 3 10–48 5.7–8.3 19 100 – 4.4–8.8 6 29.1 2.7 – – 21–100 1–6 5–19 0.1–1 3–40 63 65 23 References Van Hoof (1970) Brunt (1968); Shukla and Gough (1983) Blakeslee (1921); Brunt (1968) Edwardson and Purcifull (1974) Johnson et al. (1984) Ghanekar and Schwenk (1974) Kaiser et al. (1982) Truol et al. (1987) Golnaraghi et al. (2004) Sdoodee and Teakle (1988) Kaiser et al. (1982) Sharman et al. (2009) Thomas and Graham (1951); Kaiser et al. (1991) Thomas and Graham (1951) Kaiser et al. (1982); Shukla and Gough (1983) Antignus et al. (2007) Wang (1982) Noordam et al. (1965) Gibbs and Harrison (1964) Lister and Murant (1967) Lister and Murant (1967) Lister and Murant (1967) Lister and Murant (1967) Hanada and Harrison (1977) Lister (1960) Lister and Murant (1967) Murant and Lister (1967) Lister (1960) Morand and Poutier (1978) Lister and Murant (1967) Lister and Murant (1967) Lister and Murant (1967) Lister and Murant (1967) Hanada and Harrison (1977) Lister and Murant (1967) Hanada and Harrison (1977) Phatak (1974) Lister and Murant (1967) Murant and Lister (1967) Murant and Lister (1967) Lister and Murant (1967) Lister and Murant (1967) Lister and Murant (1967) Schimanski (1987) Lister (1960); Lister and Murant (1967) Lister and Murant (1967) Lister and Murant (1967) Lister (1960) (continued) Table 1.2 (continued) Virus/viroid Tomato bushy stunt Tomato chlorotic dwarf viroid Tomato mosaic Host Lycopersicon esculentum Malus pumila M. pumila Prunus avium Lycopersicon esculentum Lycopersicon esculentum Per cent 50–65 17 1.7–6.9 High 85.5–94.4 16.7 Tomato planta macho viroid Tomato ring spot (Syn. Grapevine yellow vein) Lycopersicon esculentum Chenopodium amaranticolor Fragaria vesca – 57 68 Gladiolus spp. 7 Fragaria x ananassa Glycine max 26 76–80 Glycine max G. max Gomphrena globosa Lycopersicon esculentum Nicotiana tabacum Pelargonium hortorum Rubus idaeus Sambucus spp. Taraxacum officinale Trifolium pratense Lycopersicon esculentum Senecio cruentus Lycopersicon esculentum Raphanus raphanistrum Arabidopsis thaliana Alliaria petiolata Brassica chinensis var. parachinensis Brassica napus var. silvestris Camelina sativa Crambe hispanica Vigna mungo 58 1.4 76 3 11 11 2 11 23.8 3–7 – 96 66 4 72.6 2.3 9.5 Tomato spotted wilt Tomato streak Turnip mosaic Turnip yellow mosaic Urd bean leaf crinkle (Syn. Blackgram leaf crinkle, Syn. Bean urd leaf crinkle virus) V. mungo V. mungo V. mungo V. mungo V. mungo Vigna unguiculata References Tomilson and Faithfull (1984) Allen (1969) Kegler and Schimanski (1982) Allen and Davidson (1967) Singh and Dilworth (2009) Chitra et al. (2002); Ismaeil et al. (2011); Hadas et al. (2004) Galindo et al. (1982) Cory and Hewitt (1968) Kahn (1956); Mellor and Stace-Smith (1963) Mellor and Stace-Smith (1963); Sushak (1976) Mellor and Stace-Smith (1963) Kahn (1956); Lister and Murant (1967) Cory and Hewitt (1968) Golnaraghi et al. (2004) Keplinger and Braun (1973) Hollings et al. (1972) Hollings et al. (1972) Hollings et al. (1972) Braun and Keplinger (1973) Uyemoto et al. (1971) Mountain et al. (1983) Hampton (1967) Jones (1944) Jones (1944) Berkeley and Madden (1932) Tomilson and Walker (1973) de Assis Filho and Sherwood (2000) Pelikanova (1990) Benetti and Kaswalder (1982/83) 1.6–8.3 20 2.7 18.3 Spak et al. (1993) Hein (1984) Benetti and Kaswalder (1982/83) Kolte and Nene (1972); Beniwal and Chaubey (1984); Beniwal et al. (1984); Makwana et al. (2001) 20.3–41.8 Narayanaswamy and Jaganathan (1975); Patel et al. (1999); Mahajan and Joi (1999); Prasad et al. (1998) 17.6 Dubey and Sharma (1985) 8.6 Ravinder and Jeyarajan (1989); Ravinder Reddy et al. (2005a, b) 1–83 Pushpalatha et al. (1999) 2.2–28.7 Sharma et al. (2007) 6–15 Beniwal et al. (1980) (continued) 30 1 Introduction Table 1.2 (continued) Virus/viroid Vicia cryptic Host Vicia faba Per cent High Watermelon mosaic Cucumis pepo Echinocystis lobata Triticum spp. Zea mays Secale cereale Triticum aestivum Zea mays – 2 0.01 0.03 3 0.22–0.4 0.01–0.1 Wheat striate mosaic White clover cryptic 1 Z. mays Triticum aestivum Trifolium repens 0.2–1.5 – High White clover cryptic 2 T. repens High White clover cryptic 3 T. repens Low White clover mosaic White clover temperate Winged bean ring spot Zucchini yellow mosaic T. pretense T. repens Psophocarpus tetragonolobus Cucumis pepo 6 High – 1.4–18.95 Cucurbita pepo 1.6 Wheat mosaic Wheat soil-borne mosaic Wheat streak mosaic References Kenten et al. (1978, 1979, 1980a, b, 1981); Abou-Elnasr et al. (1985) Bhargava and Joshi (1960) Lindberg et al. (1956) Panarin and Zabavina (1978) Panarin and Zabavina (1978) Jezewska (1995) Lanoiselet et al. (2008) Hill et al. (1974); Panarin and Zabavina (1978); Jones et al. (2005) Jones et al. (2005) Abdel Hak et al. (1977) Boccardo et al. (1985); Natsuaki et al. (1986) Boccardo et al. (1985); Natsuaki et al. (1986) Boccardo et al. (1985); Natsuaki et al. (1986) Hampton (1963); Lee et al. (2004) Natsuaki et al. (1986) Fauquet et al. (1979) Davis and Mizuki (1986); Schrijnwerkers et al. (1991); Riedle–Bauer et al. (2002); Tobias et al. (2008) Simmons et al. (2011) Note: Per cent seed transmission has not been given or doubtful in some cases because of inadequate studies or has not been reported or due to non-availability of original article. Fig. 1.2 Symptoms on foliage and seeds due to some seed-borne viruses 32 References Abdel Hak T, Ghobrial E, Kamel AH (1977) Studies on striate mosaic of wheat in Egypt. 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Can J Plant Pathol Rev Canadienne De Phytopathol 15:17–22 Zschau K (1962) Versuche and Beobachtungen Zur Samenubentragurg der Mosaikkrankheit der lupinen, insbesondere der Gelblupine. Nachr Bl.dt Pflschutzdienst 16:1–7 Zschau K, Janke C (1962) Samenubertragung des Luzernemosaik Virus and Luzerne. NachBl dt Pfschutzdienst 16:94–96 2 Identification and Taxonomic Groups Abstract In the early period of plant virus research work, inadequate identification has led to lot of confusion due to lack of an internationally accepted classification of plant viruses. The discriminatory criteria are established by ICTV study groups, who have followed the rules laid out in the International Code of Virus Classification and Nomenclature (ICVCN). As new types of viruses continue to be discovered, new names must be created for taxa; several rules in the ICVCN govern the construction of these names. Till now, nearly 231 virus and viroid diseases are reported to be seed transmitted in different crop plants. As per the latest ICTV classification by King et al. (Virus Taxonomy: 9th report of the international committee on taxonomy of viruses. Elsevier, San Diego, 2011), the seed-transmitted viruses were concerned to 231 virus families/plant virus groups. Among these plant virus groups, seed-transmitted viruses are distributed in 24 virus groups including Alfamovirus, Bromovirus, Capillovirus, Carlavirus, Carmovirus, Caulimovirus, Comovirus, Cryptovirus, Cucumovirus, Enamovirus, Fabavirus, Furovirus, Hordeivirus, Ilarvirus, Necrovirus, Nepovirus, Potexvirus, Potyvirus, Sobemovirus, Tobamovirus, Tobravirus, Tombusvirus, Tospovirus and Tymovirus groups. Greater number of seed-transmitted viruses are found in Poty (35), Nepo (28), Crypto (28), Ilar (14), Tobamo (7), Potex (7), Como (6), Carla (5), Carmo (5), Cucumo (5), Sobemo (5), Furo (4), Bromo (3) and Tymo (3) virus groups. On the other hand, in some groups such as Alfamo, Capillo, Caulimo, Enamo, Faba, Hordei, Necro, Tobra and Tombus groups, the seed-transmitted viruses recorded were very few. No seed transmission was noticed in Clostero, Diantho, Gemini, Luteo, Marafi, Parsnip yellow fleck, Reo, Rhabdo, Tenui and Waika groups. K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 2, © Springer India 2013 55 56 2.1 2 Identification In order to detect and identify a seed-transmitted virus, it is imperative to understand the characteristics of seed-transmitted viruses for comparison with the available information on previously described viruses. Diagnosis of a virus disease is never unequivocal unless the virus is isolated, studied outside its host and demonstrated by Koch’s postulates. By following the ‘ten commandments of the plant viruses’ proposed by Bos (Bos 1976a), a reasonable diagnosis of the viral disease could be made. The diagnosis begins with particulars of the diseased plant and then deals with the virus itself, indicating a gradual shift from clinical observations to etiological diagnosis. The pragmatic approach depends on the fact that each virus has a definite host range that is often confined to a wide or limited number of host plants. The natural hosts of the virus are first identified and a comparison is then made with previously isolated species or their close relatives, and their properties are also compared with those of the unknown virus. Based on the morphology of the virus involved, they can easily be differentiated by their shape and size. Serology and nucleic acid hybridisation tests are being widely used for the identification of different plant viruses and their strains. At present the virus identification is being done in scientific laboratories through specialised techniques. Different steps involve are seed morphology, indicator hosts, transmission, electron microscopy, serology, molecular methods, etc., which can help in detection and identification of seed-transmitted viruses. The total list of conventional viruses, cryptic viruses and viroids which are seed transmitted and reported from different parts of the world is presented in Table 1.2. 2.2 Classification of Viruses In the early period of plant virus research work, inadequate identification has lead to lot of confusion due to lack of an internationally accepted classification of plant viruses. Looking into this Identification and Taxonomic Groups problem, Bos (1964) suggested the use of standardised vernacular names while listing out the viruses from legume crops. Subsequent schemes of nomenclature (Gibbs and Harrison (1976); Matthews (1979, 1982); Fauquet et al. (2005); King et al. (2011) and grouping Harrison et al. (1971); Francki (1981)) of viruses and the recent CMI/AAB descriptions have contributed greatly to the knowledge of plant viruses. During 1996, Brunt and his associates’ compiled publication is one of the most important sources of descriptions of viruses of tropical plants. Information on the identification of virus diseases in the form of Virus Information Data Exchange (VIDE) is published by Boswell and Gibbs 1986 and Gibbs 1989. A system called ‘DELTA’ which is specifically designed to handle all forms of taxonomic information has been used to store the information in computers (Brunt et al. 1996). The taxonomic approach assesses the group to which a virus belongs by determining some of its group-specific characteristics such as the shape and size of its particles. Then, more specific tests including host range are used to check whether the unknown virus is an already described member of the likely group or not. This approach requires some understanding in virus classification. Modern taxonomic classification is also taken into the consideration of molecular characterisation of viruses which has become reliable and authentic (Van Regenmortal et al. 2000; Fauquet et al. 2005; King et al. 2011). The ICTV 9th report of 2009 which was published by King et al. (2011) has 6 orders, 87 families, 19 subfamilies, 349 genera and 2,284 species. Based on a set of characteristics, more than 975 plant viruses have been described and classified by International Committee on Taxonomy of Viruses (ICTV) into 34 well-defined groups and a few less well-defined groups or subgroups (Hamilton et al. 1981; Brown 1989; Martelli 1992; Brunt et al. 1996; Fauquet et al. 2005; Van Regenmortel et al. 2005; King et al. 2011). In the Table 1.2, available information on seed-transmitted virus diseases of fruits, vegetable and ornamental crops, including in collateral hosts, was listed along with percentage of seed transmission. In Table 2.1 taxonomic position of seed-transmitted plant viruses is 2.2 Classification of Viruses 57 Table 2.1 Taxonomic position of seed-transmitted viruses S. no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Species (virus/viroid) Alfalfa mosaic (Syn.) Berseem mosaic Potato Yellowing Alfalfa cryptic Alfalfa temperate Avocado viruses 1,2,3 Beet 1 alpha crypto (Syn.) Beet temperate Beet 2 alpha crypto Beet 3 alpha crypto Carrot temperate 1 Carrot temperate 3 Carrot temperate 4 Fescue cryptic Garland chrysanthemum temperate Hop trefoil cryptic 1 Hop trefoil cryptic 3 Mibuna temperate Radish yellow edge Red clover cryptic Red pepper cryptic-1 Red pepper cryptic-2 Rhubarb temperate Rye grass cryptic Santosai temperate Spinach temperate crypto Vicia cryptic White clover cryptic 1 White clover cryptic 3 Pelargonium zonate spot Apple dapple viroid (Syn.) Apple scar skin viroid Grapevine yellow speckle Cucumber leaf spot carmovirus Avocado sunblotch Banana streak Cacao swollen shoot Citrus mosaic Citrus yellow mosaic Kalanchoe top-spotting Piper yellow mottle Abutilon mosaic Carrot temperate 2 Hop trefoil cryptic 2 White clover cryptic 2 Broad bean mottle Brome mosaic Cowpea chlorotic spot virus Oat mosaic Citrus tatter leaf Order – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – Family Bromoviridae Bromoviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Bromoviridae Pospiviroidae Pospiviroidae Tombusviridae Avsunviroidae Caulimoviridae Caulimoviridae Caulimoviridae Caulimoviridae Caulimoviridae Caulimoviridae Geminiviridae Partitiviridae Partitiviridae Partitiviridae Bromoviridae Bromoviridae Bromoviridae Potyviridae Flexiviridae Genus Alfamovirus Alfamovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Alphacryptovirus Anulavirus Apscaviroid Apscaviroid Aureusvirus Avsunviroid Badnavirus Badnavirus Badnavirus Badnavirus Badnavirus Badnavirus Begomovirus Betacryptovirus Betacryptovirus Betacryptovirus Bromovirus Bromovirus Bromovirus Bymovirus Capillovirus (continued) 58 2 Identification and Taxonomic Groups Table 2.1 (continued) S. no 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 Species (virus/viroid) Carlavirus Clover (red) vein mosaic Cowpea mild mottle Pea streak Red clover vein mosaic Blackgram mottle Cowpea mottle Melon necrotic spot Muskmelon necrotic spot Pea stem necrosis Cauliflower mosaic Dahlia mosaic Citrus leaf blotch Coconut cadang-cadang viroid Coleus blumei viroid 1 Coleus blumei viroid 2 Coleus blumei viroid 3 Bean pod mottle Broad bean true mosaic Broad bean stain Cowpea mosaic Cowpea severe mosaic Echtes Ackerbohnen mosaic (Syn. Broad bean true mosaic) Muskmelon mosaic (Syn.) Squash mosaic Pea mild mosaic Red clover mottle comovirus Strawberry pallidosis Banana viruses Cucumber mosaic (Syn.) Cowpea banding mosaic (Syn.) Cowpea ring spot (Syn.) Soybean stunt Peanut stunt Tomato aspermy Winged bean ring spot Beet curly top Pea enation mosaic Broad bean wilt Nicotiana velutina mosaic Peanut clump (Indian) Wheat mosaic Wheat soil-borne mosaic Barley stripe mosaic(Syn. Barley false stripe) Lychnis ring spot Order Tymovirales Tymovirales Tymovirales Tymovirales Tymovirales – – – – – – – Tymovirales – – – – Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Family Betaflexiviridae Betaflexiviridae Betaflexiviridae Betaflexiviridae Betaflexiviridae Tombusviridae Tombusviridae Tombusviridae Tombusviridae Tombusviridae Caulimoviridae Caulimoviridae Betaflexiviridae Pospiviroidae Pospiviroidae Pospiviroidae Pospiviroidae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Genus Carlavirus Carlavirus Carlavirus Carlavirus Carlavirus Carmovirus Carmovirus Carmovirus Carmovirus Carmovirus Caulimovirus Caulimovirus Citrivirus Cocadviroid Coleviroid Coleviroid Coleviroid Comovirus Comovirus Comovirus Comovirus Comovirus Comovirus Picornavirales Secoviridae Comovirus Picornavirales Picornavirales – – – Secoviridae Secoviridae Closteroviridae Bromoviridae Bromoviridae Comovirus Comovirus Crinivirus Cucumovirus Cucumovirus – – – – – Picornavirales – – – – – Bromoviridae Bromoviridae Bromoviridae Geminiviridae Luteoviridae Secoviridae Virgaviridae Virgaviridae Virgaviridae Virgaviridae Virgaviridae Cucumovirus Cucumovirus Cucumovirus Curtovirus Enamovirus Fabavirus Furovirus Furovirus Furovirus Furovirus Hordeivirus – Virgaviridae Hordeivirus (continued) 2.2 Classification of Viruses 59 Table 2.1 (continued) S. no 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 Species (virus/viroid) Hop stunt viroid (Syn.) Cucumber pale fruit viroid (Syn.) Grapevine viroid Raspberry bushy dwarf (Syn.) Loganberry degeneration Apple mosaic Asparagus latent (Syn.) Asparagus virus II Black raspberry latent Elm mottle Fragaria Chiloensis Ilarvirus Humulus Japonicas Hydrangea mosaic Lilac ring mottle Prune dwarf (Syn.) Cherry ring mottle (Syn.) Cherry yellows Prunus necrotic ring spot (Syn.) Peach necrotic leaf spot (Syn.) Peach ring spot Raspberry latent (Black raspberry latent) Spinach latent virus Sunflower ring spot Tobacco streak (Syn.) Asparagus stunt (Syn.) Bean red node (Syn.) Datura quercina Maize chlorotic mottle machlomovirus Olive latent virus-1 Tobacco necrosis Arabis mosaic Arracacha virus A Arracacha virus B Artichoke Italian Latent Artichoke yellow ring spot Australian Lucerne latent Cacao necrosis Cherry leaf roll Cherry rasp leaf Chicory yellow mottle Crimson clover latent Cycas necrotic stunt Eucharis mottle Grapevine Bulgarian latent Grapevine fanleaf Hibiscus latent ring spot Lucerne (Australian) symptomless Order – Family Pospiviroidae Genus Hostuviroid – – Idaeovirus – – Bromoviridae Bromoviridae Ilarvirus Ilarvirus – – – – – – – Bromoviridae Bromoviridae Bromoviridae Bromoviridae Bromoviridae Bromoviridae Bromoviridae Ilarvirus Ilarvirus Ilarvirus Ilarvirus Ilarvirus Ilarvirus Ilarvirus – Bromoviridae Ilarvirus – Bromoviridae Ilarvirus – – – Bromoviridae Bromoviridae Bromoviridae Ilarvirus Ilarvirus Ilarvirus – Tombusviridae Machlomovirus – – Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales – Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Tombusviridae Tombusviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Necrovirus Necrovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus (continued) 60 2 Identification and Taxonomic Groups Table 2.1 (continued) S. no 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 Species (virus/viroid) Lucerne Australian latent Mulberry ring spot Peach rosette mosaic Potato virus U Raspberry ring spot Rubus Chinese seed-borne Satsuma dwarf Sweet potato ring spot Tobacco ring spot Tomato black ring Tomato ring spot (Syn.) Grapevine yellow vein Clover (red) mosaic Coffee ring spot Maize mosaic Wheat striate mosaic Citrus psorosis Peach latent Beet mild yellowing Carrot red leaf Chrysanthemum stunt viroid Citrus exocortis viroid Pepper chat fruit viroid Potato spindle tuber Tomato apical stunt viroid Tomato chlorotic dwarf viroid Tomato planta macho viroid Clover (white) mosaic Clover yellow mosaic Foxtail mosaic potexvirus Hosta virus x Pepino mosaic Potato virus X White clover mosaic Mung bean mosaic Artichoke latent Asparagus virus I Bean common mosaic (Syn.) Bean western mosaic (Syn.) Azuki bean mosaic Bean common mosaic necrosis Bean yellow mosaic Blackeye cowpea mosaic Bramble yellow mosaic Broad bean mild mosaic Cassia yellow spot (poty) Celery latent Cowpea aphid-borne mosaic Order Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Picornavirales Family Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Secoviridae Genus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Nepovirus Mononegavirales Mononegavirales Mononegavirales Mononegavirales – – – – – – – – – – – Tymovirales Tymovirales Tymovirales Tymovirales Tymovirales Tymovirales Tymovirales – – – – Rhabdoviridae Rhabdoviridae Rhabdoviridae Rhabdoviridae Ophioviridae Avsunviroidae Luteoviridae Luteoviridae Pospiviroidae Pospiviroidae Pospiviroidae Pospiviroidae Pospiviroidae Pospiviroidae Pospiviroidae Alphaflexiviridae Alphaflexiviridae Alphaflexiviridae Alphaflexiviridae Alphaflexiviridae Alphaflexiviridae Alphaflexiviridae Potyviridae Potyviridae Potyviridae Potyviridae Nucleorhabdovirus Nucleorhabdovirus Nucleorhabdovirus Nucleorhabdovirus Ophiovirus Pelamoviroid Polerovirus Polerovirus Pospiviroid Pospiviroid Pospiviroid Pospiviroid Pospiviroid Pospiviroid Pospiviroid Potexvirus Potexvirus Potexvirus Potexvirus Potexvirus Potexvirus Potexvirus Poty virus Potyvirus Potyvirus Potyvirus – – – – – – – – Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus (continued) 2.2 Classification of Viruses 61 Table 2.1 (continued) S. no 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 Species (virus/viroid) Cowpea green vein-banding Cowpea moroccan aphid-borne mosaic Desmodium mosaic Guar symptomless Hippeastrum mosaic Leek yellow stripe Lettuce mosaic Maize dwarf mosaic Onion yellow dwarf Papaya ring spot Pea mosaic (Syn. Bean yellow mosaic) Pea seed-borne mosaic (syn. pea fizzle top and Pea leaf rolling virus) Peanut mottle Peanut stripe (Syn. Peanut mild mottle) Plum pox virus Potato virus Y (Syn.) Brinjal mosaic Soybean mosaic Sugarcane mosaic Sunflower mosaic potyvirus Telfairia mosaic Turnip mosaic Watermelon mosaic Zucchini yellow mosaic Bean southern mosaic (Syn. Southern bean mosaic) Lucerne transient streak Panicum mosaic Sowbane mosaic Subterranean clover mottle Cucumber green mottle mosaic Paprika mild mottle tobamovirus Pepper mild mottle Pepper mild mottle tobamovirus Sunn-hemp mosaic (Syn.) Sunn-hemp rosette Tobacco mosaic Tomato mosaic Pea early browning Tobacco rattle Tomato bushy stunt Tomato spotted wilt Apple chlorotic leaf spot Wheat streak mosaic Dulcamara mottle Eggplant mosaic (Syn. Andean potato latent) Order – – – – – – – – – – – – Family Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Genus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus – – – – – – – – – – – – Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae Potyviridae – Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Potyvirus Sobemovirus – – – – – – – – – – – – – Virgaviridae Virgaviridae Virgaviridae Virgaviridae Virgaviridae Sobemovirus Sobemovirus Sobemovirus Sobemovirus Tobamovirus Tobamovirus Tobamovirus Tobamovirus Tobamovirus – – – – – – Tymovirales – Tymovirales Tymovirales Virgaviridae Virgaviridae Virgaviridae Virgaviridae Tombusviridae Bunyaviridae Betaflexiviridae Potyviridae Tymoviridae Tymoviridae Tobamovirus Tobamovirus Tobravirus Tobravirus Tombusvirus Tospovirus Trichovirus Tritimovirus Tymovirus Tymovirus (continued) 62 2 Identification and Taxonomic Groups Table 2.1 (continued) S. no 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 Species (virus/viroid) Melon rugose mosaic Potato Andean latent Turnip yellow mosaic Sunflower rugose mosaic Cherry necrotic rusty mottle Potato virus T Strawberry latent ring spot High plains virus Urdbean leaf crinkle (Syn.) Black gram leaf crinkle (Syn.) bean urd leaf crinkle virus Barley mottle mosaic Brinjal ring mosaic Brinjal severe mosaic Carrot motley leaf Cherry ring spot Cineraria mosaic Mung bean isometric yellow mosaic Parsley latent Peanut marginal chlorosis Soybean mild mosaic Order Tymovirales Tymovirales Tymovirales – Tymovirales Tymovirales Picornavirales – – Family Tymoviridae Tymoviridae Tymoviridae Luteoviridae Betaflexiviridae Betaflexiviridae Secoviridae – – Genus Tymovirus Tymovirus Tymovirus Umbravirus Unassigned Unassigned Unassigned Unassigned Unassigned – – – – – – – – – – – – – – Unassigned Unassigned Unassigned Unassigned Unassigned Unassigned Unassigned – – – – – – Unassigned Unassigned Unassigned presented by taking the information from Fauquet et al. (2005) and King et al. (2011) classification tables. The seed-transmitted viruses are confined to 34 virus groups, which are in Table 2.2. To have more clarity, the information on a particle morphology and genome and vector is also provided. Among these plant virus groups, 231 wellcharacterised seed-transmitted viruses are distributed in 24 virus groups including Alfamovirus, Bromovirus, Capillovirus, Carlavirus, Carmovirus, Caulimovirus, Comovirus, Cryptovirus, Cucumovirus, Enamovirus, Fabavirus, Furovirus, Hordeivirus, Ilarvirus, Necrovirus, Nepovirus, Potexvirus, Potyvirus, Sobemovirus, Tobamovirus, Tobravirus, Tombusvirus, Tospovirus and Tymovirus groups. Greater number of seed-transmitted viruses are found in Potyvirus (35), Nepovirus(28), Cryptovirus (28), Ilarvirus (14), Tobamovirus (7), Potexvirus (7), Comovirus (6), Carlavirus (5), Carmovirus (5), Cucumovirus (5), Sobemovirus (5), Furovirus (4), Bromovirus (3) and Tymovirus (3) groups (Tables 2.1 and 2.2). On the other hand, in some groups such as Alfamovirus, Capillovirus, Caulimovirus, Enamovirus, Fabavirus, Hordeivirus, Necrovirus, Tobravirus, Tombusvirus and Tospovirus groups, the seedtransmitted viruses recorded were very few. No seed transmission was noticed in Closterovirus, Dianthovirus, Geminivirus, Luteovirus, Parsnip yellow fleck virus, Reovirus, Rhabdovirus, Tenuivirus and Waikavirus groups. 2.3 Variability in Certain Seed-Transmitted Viruses From the above discussed aspects on plant virus taxonomy, it is clear that the viruses resembling each other in genome properties and virion morphology are grouped into genera and given genus names. The phylogenic analysis of some of the viruses indicates that recently described viruses are established based on molecular studies as strains of the earlier described viruses. More Geminivirus Group A mastro Group B begomo Hordeivirus Ilarvirus Luteovirus Marafivirus Necrovirus 15 16 17 18 19 20 10 11 12 13 14 Genus/group Alfamo virus Bromovirus Capillovirus Carlavirus Carmovirus Caulimovirus Closterovirus Comovirus Cryptovirus Subgroup A Subgroup B Cucumovirus Dianthovirus Enamo virus Fabavirus Furovirus Sl. no. 1 2 3 4 5 6 7 8 9 Geminate Geminate Rigid rods Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Rigid rods White clover cryptic virus-1 White clover cryptic virus-2 Cucumber mosaic virus Carnation ring spot virus Pea enation mosaic virus Broad bean wilt virus Soil-borne wheat mosaic Maize streak virus African cassava mosaic Barley stripe mosaic virus Tobacco streak virus Barley yellow dwarf virus Maize rayadofino virus Tobacco necrosis virus Particle morphology Bacilliform Isometric Flexuous Flexuous Isometric Isometric Flexuous rods Isometric Type member Alfalfa mosaic Brome mosaic Apple stem grooving virus Carnation latent virus Carnation mottle virus Cauliflower mosaic virus Beet yellows virus Cowpea mosaic virus 18 30 18 30 100–150 20 26–35 25 31 28 30 38 28 31–34 28 30 280–330 20 92–160 20 Particle size (nm) 28–58 18 26 640 12 620–700 11 28–30 50 600–2,000 10 28 Table 2.2 Seed-transmitted viruses in different taxonomic groups of plant viruses and their characteristic features ssRNA ssRNA ssRNA ssRNA ssRNA ssRNA ssRNA ssRNA dsRNA dsRNA ssRNA ssRNA ssRNA ssRNA Genome ssRNA ssRNA ssRNA ssRNA ssRNA dsDNA ssRNA ssRNA Leaf- hopper Whitefly – Thrips Aphid Leaf- hopper Fungus – – Aphid – Aphid Beetle Fungus Vector Aphid Beetle – Aphid, whitefly Beetle Aphid Aphid Beetle 0 2 14 0 0 2 – – 25 3 5 0 1 1 4 (continued) No. of Seedtransmitted viruses 2 3 1 5 5 2 0 6 2.3 Variability in Certain Seed-Transmitted Viruses 63 Sobemovirus Tenuivirus Tobamovirus Tobravirus Tombusvirus Tospovirus Tymovirus Waikavirus 27 28 29 30 31 32 33 34 26 23 24 25 Genus/group Nepovirus Parsnip yellow fleck virus Potexvirus Potyvirus Reovirus (a) Phytoreo (b) Fijivirus Rhabdovirus Sl. no. 21 22 Table 2.2 (continued) ssRNA ssRNA ssRNA ssRNA ssRNA ssRNA ssRNA ssRNA dsRNA dsRNA ssRNA ssRNA ssRNA 470–580 13 680–900 11 70 71 160–380 50–95 14–114 22 28–30 >400 8 300 18 160–215 22 14–114 22 30 85 29 25 Genome ssRNA ssRNA Particle size (nm) 28 30 – Thrips Beetle Leaf- hopper Beetle Plant- hopper – Nematode Leaf- hopper Plant- hopper Aphid, Leafhopper – Aphid, whitefly, mite Vector Nematode Aphid 1 1 3 0 5 0 7 2 0 0 0 7 35 No. of Seedtransmitted viruses 28 0 2 Tomato bushy stunt virus Tomato spotted wilt virus Turnip yellow mosaic virus Maize chlorotic dwarf Isometric Flexuous Rigid rods Short & long rigid rods Isometric Isometric Isometric Isometric Isometric Isometric Bacilliform Wound tumour virus Fiji disease virus Lettuce necrotic yellows virus Southern bean mosaic virus Rice stripe virus Tobacco mosaic virus Tobacco rattle virus Flexuous rods Flexuous rods Particle morphology Isometric Isometric Potato virus X Potato virus Y Type member Tobacco ring spot virus Parsnip yellow fleck virus 64 Identification and Taxonomic Groups References examples one can find are in potyvirus group. Viruses like Blackeye cowpea mosaic and Peanut stripe virus are nothing but strains of the Bean common mosaic virus with little variation. Variability in viruses is noticed in almost all described virus groups. The molecular structure of viruses has the capacity of transferring its characters to its duplicates produced in the host cells. (a) Hybridisation: This is one of the means by which new virus strains are formed. If two strains of virus are inoculated into the same host plant, one or more new virus strains may be recovered with properties (virulence, symptomatology, etc.) different from those of either of the original strains introduced into the host. These new strains are probably hybrids (RNA or DNA recombinants). Albersio et al. (1975) reported variability in Squash mosaic virus (SqMV) by hybridisation between two strains of virus. They had crossed strain 1 H and II A of SqMV in pumpkin (Cucurbita pepo) and cantaloupe (Cucumis melo) plants and observed the interaction between them. (b) Mutations: The evolution of new strains of viruses may also be due to mutation. These may be a heritable change in the genetic material (RNA or DNA). The production of mutants differing in virulence has also been reported in several viruses, especially TMV, although they seem to vary mostly in the type of symptoms and severity of disease they produce rather than in their ability to infect different host plant varieties. The previous reports when the advanced identification techniques were not available have reported very negligible (<1%) percentage of seed transmission in certain virus–host combinations, which the later research workers have disproved. Without any discrimination based on the available literature all the seed-transmitted viruses, Cryptoviruses and viroid diseases were reported in the Tables 1.2, 2.1 and 2.2. In the earlier days, when the computer knowledge was not available, the students, research workers and others used to depend on Review of Plant Pathology, review articles 65 and other abstract journals for locating subject literature for use in their research work. Since 1990, lot of databases and websites are available which are quite useful for day-to-day plant virus research work, where even the poor library facilities existed and such problems were solved by certain international organisations. For example, the ‘Crop Protection Compendium’ (CPC), which was available on CD-ROM, was published by CAB International, which is being updated annually on http://www. cabicompendium.org/cpc. Another valuable information source for viruses is of AAB descriptions on plant viruses and can be accessed on http://www.dpvweb.net. Even the descriptions and lists from VIDE Database of the International Committee on Taxonomy of Viruses are available on http://www.ncbi.nlm.nih.gov/ICTVdb/index. htm. ‘The Plant Pathology Internet Guide Book’ (http://www.pk.uni-bonn.de/ppigb/ppigb.htm) is another source of information for many virology topics. Because of the availability of databases of plant viruses on internet, it has become much easier for the virus research workers to know the latest progress that is happening in any corners of the world (http://www.virology.net). References Albersio J, Lima A, Nelson MR (1975) Squash mosaic virus variability: nonreciprocal cross-protection between strains. Phytopathology 65:837–840 Bos L (1964) Tentative list of viruses reported from naturally infected leguminous plants. Neth J Plant Pathol 70:161–174 Bos L (1976a) Problems and prospects in plant virus identification. EPPO Bull 6:63–90 Bos L (1976b) Research on plant virus diseases in the developing countries: possible ways for improvement. FAO Plant Prot Bull 24(4):109–118 Boswell K, Gibbs A (1986) The VIDE data bank for plant viruses. In: Jones RAC, Torrance L (eds) Developments and applications in virus testing. Association of Applied Biologists, Warwickshire, pp 283–287 Brown F (1989) The classification and nomenclature of viruses. Summary of results of meetings of International Committee on Taxonomy of Viruses. Edmonton, Canada 1987. Intervirology 30:181–186 66 Brunt AA, Crabtree K, Dallwitz MJ, Gibbs AJ, Watson L (eds) (1996) Viruses of plants. Description and lists from VIDE data base. CAB International, Wallingford, p 1484 Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (2005) Virus taxonomy, VIIIth report of the ICTV. Elsevier Academic Press, London, pp 751–756 Francki RIB (1981) Plant virus taxonomy. In: Kurstak E (ed) Handbook of plant virus infection and comparative diagnosis. Elsevier, Amsterdam, 3 Gibbs AJ (1989) A virus database for aiding plant pathologists. In: Proceedings of the 2nd meeting of peanut stripe virus coordinators, held at ICRISAT, Patancheru, India Gibbs AJ, Harrison BD (1976) Plant virology: the principles. Edward Arnold, London, p 292 Hamilton RI, Edwardson JR, Francki RIB, Hsu HT, Hull R, Koenig R, Milne RG (1981) Guidelines for identification and characterization of plant viruses. J Gen Virol 54:223–241 Harrison BD, Finch JT, Gibbs AJ, Hollings M, Shepherd RJ, Valenta V, Wetter C (1971) Sixteen groups of plant viruses. Virology 45:356–363 2 Identification and Taxonomic Groups King AMQ, Lefkowitz E, Adams MJ, Carstens EB (2011) Virus taxonomy: 9th report of the international committee on taxonomy of viruses. Elsevier, San Diego Martelli GP (1992) Classification and nomenclature of plant viruses: state of the art. Plant Dis 76: 436–442 Matthews REF (1979) Classification and nomenclature of viruses. Third report of the International Committee on the Taxonomy of Viruses. Intervirology 12: 131–296 Matthews REF (1982) Classification and nomenclature of viruses. Fourth report of the International Committee on the Taxonomy of Viruses. Intervirology 17:1–199 Van Regenmortal MHV, Fauquet CM, Bishop DHL, Carstens E, Estes M, Lemon S, Maniloff J, Mayo M, Mc Geoch D, Pringle C, Wickner R (2000) Virus taxonomy: Seventh report of the International Committee on Taxonomy of Viruses. Academic Press, New York/San Diego Van Regenmortel HR, Bishop DHL, Van Regenmortel MH, Fauquet C (2005) Virus taxonomy: Eighth report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego 3 Economic Significance of Seed-Transmitted Plant Virus Diseases Abstract The assessment of yield losses due to virus diseases is particularly difficult to estimate accurately both qualitatively and quantitatively. Yield losses are significantly greater when the plants are infected at early stages. Attempts were made to compile the available yield loss data due to seedtransmitted virus diseases in different crop plants. Increased yield losses would also result due to mixed infection of two or more viruses which exhibit synergistic effect. The extent of yield loss varies with the cultivar, stage of infection, virus strain and environmental factors. The yield loss estimates give the necessity of framing suitable virus management measures under field conditions and also stresses to develop resistant cultivars. 3.1 Introduction Some of the seed-transmitted viral diseases are cosmopolitan in distribution. They greatly influence man’s economic and social facets of life by reducing the yield and quality of plant products. The extent of crop loss depends mostly on the disease intensity and its distribution. The losses caused by the viral and viroid diseases are difficult to estimate unless crops are completely destroyed. Farmers and government agencies are still faced with questions like (1) how damaging the viruses are, (2) which areas are the most damaging, (3) how yield reductions can be assessed on a farm, in a district, a country or a region and (4) how such losses can be predicted. Precise answers are not available for virus diseases, and plant virology confronts with the lacunae in the appraisal of losses. The information available on crop losses varies considerably in precision as the severity of the disease varies greatly with factors like crop variety, virus strain, locality, the activity of the vectors, nutritional status of the infected crop, etc. 3.2 Assessment of Crop Losses The assessment of losses due to viral diseases is particularly difficult because it is extremely hard to prevent contamination of healthy control plants. Similarly, inoculation under vector-proof conditions in mesh/glass houses may not reflect the true picture of natural field conditions. Generally, yield losses are significantly greater when the plants are infected at early stages. Virus infection causes not only quantitative loss of the harvested product in terms of weight and K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 3, © Springer India 2013 67 68 3 Economic Significance of Seed-Transmitted Plant Virus Diseases number of units but qualitatively for taste and nutritive composition. Estimation of losses based on yield comparisons between plots of inoculated and uninoculated plants mostly represents only the maximum loss caused by the virus, but rarely 100% infection takes place under natural conditions (Irwin and Schultz 1981; Irwin et al. 2000; Ruesink and Irwin 2006). Even though in this chapter several examples of yield losses in different virus–host combinations are furnished, two major crops like barley and lettuce have faced very significant enormous yield losses due to Barley stripe mosaic virus in Montana (USA) and Lettuce mosaic virus in California (USA), respectively (Grogan 1980; Carroll 1983). Some outstanding examples of crop loss estimates due to viruses have been reported by Bos (1981, 1982) and Waterworth and Hadidi (1998) in their review articles. To quote few examples of crop loss estimates: A 0.1% LMV seed infection resulted in total crop loss of lettuce (Broadbent et al. 1951; Grogan et al. 1952; Zink et al. 1956). In the USA an estimated annual loss of 4% was caused by the same virus during 1951–1960 (USDA 1965). On the other hand, yield loss of 50–68% due to BCMV in bean crop has been reported by a number of workers (Lockhart and Fischer 1974; Hampton 1975). Natural aphid transmission of Soybean mosaic virus (SMV) caused up to 35% loss in seed yield in susceptible soybean cultivars (Ross 1977), while mechanical inoculation of soybean seedlings resulted in yield reductions up to 86% (Goodman and Oard 1980). From the USA, yield losses in certain soybean cultivars were between 8 and 25% due to SMV strains (Ross 1969a). The Peanut mottle virus (PMV) reduced the peanut seed yield from 31 to 47% (Kuhn et al. 1978), and in Georgia (USA) yield losses exceeded around $10 million per annum (Paguio and Kuhn 1974; Kuhn et al. 1978). The losses due to Peanut stripe virus (PStV) were 23 and 21%, respectively, in peanut cultivars Argentine and Florunner (Demski et al. 1984). However, in Virginia (USA), Peanut stunt virus caused 80% loss of matured nuts in three commercial peanut fields (Culp and Troutman 1967). In cowpea, a yield loss of 13–87% and 64–75% due to Cowpea mosaic virus was recorded by Kaiser and Mossahebi (1975) and Suarez and Gonzalez (1983), respectively. While another seed-transmitted virus of cowpea, Cowpea banding mosaic virus (CpBMV) reduced the seed yield to 11.5–43.5% (Sharma and Varma 1981a). In Montana, an incidence of 90% Barley stripe mosaic virus (BSMV) has reduced barley yields by 35–40% (Eslick 1953). Catherall (1972) has reported 62% grain yield loss in barley cultivar Akka. The same virus has caused yield reduction in winter wheat by 19% (Fitzgerald and Timian 1960). In currency the estimated total loss in barley due to BSMV exceeded $30 million during 1953–1970 (Carroll 1980). Similarly, from Western Australia, Coutts et al. (2008) have reported yield losses in lentil cv. Nugget, due to PSbMV; the shoot dry weight decrease was 23% and seed yield by 96% and individual seed weight by 58%. The crop loss estimates are also available for lucerne (Medicago sativa) infected with Alfalfa mosaic virus (AMV) (Hemmati and McLean 1977; Bailiss and Ollennu 1986; Jones and Pathipanawat 1989) and Vigna angularis and Broad bean (Latham et al. 2004); asparagus bean infected with Cowpea aphid-borne mosaic (Chang and Kno 1983; Giesler et al. 2002; Latham et al. 2004); soybean with Bean pod mottle virus (Hopkins and Mueller 1984; Ross 1986; Giesler et al. 2002) and SMV (Irwin and Schultz 1981; Hill et al. 1987; Tu 1989); wheat with Indian peanut clump virus (Delfosse et al. 1999); barley with BSMV (Nutter et al. 1984); French bean with Southern bean mosaic virus (Morales and Castano 1985) and BCMV (Hampton 1975; Omar et al. 1978; Sastry et al. 1981; Nakano et al. 1988; Rao et al. 1990; Vishwadhar and Gupta 1990); maize with Maize dwarf mosaic virus (Scott et al. 1988); peanut with Peanut mild mottle virus (PMMV); Peanut mottle virus (Kuhn 1965; Idris and Ahmed 1981; Puttaraju et al. 2001); Cucumber mosaic virus (CMV) (Xu and Zhang 1986); pea with PSbMV (Kraft and Hampton 1980; Ovenden and Ashby 1981; Khetrapal et al. 1988; Ali and Randles 1998; Coutts 3.4 Factors Affecting Yield Losses et al. 2009) and lentil with PSbMV (Coutts et al. 2008); urd bean with Urdbean leaf crinkle virus (ULCV) (Nene 1972; Beniwal et al. 1979; Kadian 1982; Indu Sharma and Dubey 1984); urd bean with BCMV (Agarwal et al. 1976); pepper with TMV (Feldman et al. 1969); lettuce with LMV (Ryder and Duffus 1966); subterranean clover with CMV (Jones and Mc Kirdy 1990; Jones 1991); asparagus lettuce with a strain of Lettuce mosaic virus (Xia et al. 1986); Peanut clump virus in wheat (Delfosse et al. 1999); sorghum with Sugarcane mosaic virus (Mock et al. 1985); urd and mung bean with Leaf crinkle disease (Beniwal and Bharathan 1979; Chattopadhyay et al. 1986; Manadhare et al. 1999; Negi and Vishunavat 2004, 2006; Ravinder Reddy et al. 2005b; Sharma et al. 2007); lentil with Bean yellow mosaic virus (Kumari et al. 1994), Broad bean stain virus (Makkouk and Kumari 1990), Alfalfa mosaic virus (Latham and Jones 2001a), PSbMV (Kumari and Makkouk 1995; Mabrouk and Mansour 1998) and Broad bean stain virus (Mabrouk and Mansour 1998); cowpea with Blackeye cowpea mosaic (Puttaraju et al. 2002; 2004a); lupin with CMV (Bwye et al. 1994); chickpea with Tobacco streak virus (Kaiser et al. 1991; Kumari et al. 1996); and raspberry with Raspberry bushy dwarf disease (Jones 1979). It is very difficult to get data on exact yield losses in different host and virus infections. There are several obvious reasons for not having accurate yield loss data which will probably remain the foreseeable future. Among them are variations in losses by a particular virus in a particular crop from year to year, different degrees of loss among regions within the same year, differences in loss assessment methodologies and on different agronomical practices followed, etc. 3.3 Viruses and Seed Viability Some viruses directly affect the viability of seed. A mild strain of seed-transmitted hop infecting virus (Prunus necrotic ring spot virus) caused a reduction of 20% in germination, while a severe strain reduced germination by not less than 90% 69 (Blattny and Osvald 1954). Seeds of spurrey, Spergula arvensis, infected by Tomato black ring virus germinated more slowly than healthy seeds, but there was little or no effect on seedlings growth (Lister and Murant 1967). 3.4 Factors Affecting Yield Losses Usually, virus infection at early stages of the crop causes greater loss in yield. For example, Fletcher et al. (1969) reported 15% yield loss in cucumbers with Cucumber green mottle mosaic virus (CGMV) when infected at the very early stage. Similarly in soybean, Tobacco ring spot virus (TRSV) infection caused a yield reduction of 60% in 80% infected plants, while early infection resulted in yield loss of 79% (Crittenden et al. 1966). Puttaraju et al. (2004a, b) observed that early infection in cowpea with BCMV–ICM has led to severe yield losses, while infection at 50 days did not differ much from those of healthy cowpea plants. Similarly, Bean yellow mosaic virus infection of lentil crop at pre-flowering stage and flowering stage caused yield losses of 96 and 34%, respectively (Kumari et al. 1994). Increased yield losses would also result due to mixed infection of two or more viruses which exhibit synergistic effect and lead to greater yield losses than yield loss from either virus alone. Demski and Jellum (1975) recorded average yield losses of soybean to be 18, 31, 66 and 76% for single infection by PMV, Cowpea chlorotic mottle virus (CCMV), SMV and TRSV, respectively. However, in doubly infected plants, the yield loss percentage was 46 (PMV– CCMV), 78 (PMV–SMV), 80 (PMV–TRSV), 82 (CCMV–TRSV), 87 (CCMV–SMV) and 98 (SMV–TRSV) along with decrease in oil content. Similar synergistic reaction and increased yield losses were recorded with Bean pod mottle and Soybean mosaic viruses in soybean (Ross 1968) and Pea enation mosaic virus and Pea seed-borne mosaic virus in pea (Bossennec et al. 2000). Even in the case of cowpea stunt (which is a resultant of synergism between Blackeye cowpea mosaic and CMV) in cowpea cv. California, the yield 70 3 Economic Significance of Seed-Transmitted Plant Virus Diseases loss was 86.4%, while for the individual viruses, it was 2.5 and 14.2%, respectively (Pio-Ribeiro et al. 1978). Losses caused by virus and its vector sometimes may cause huge yield losses. For example, the yield reduction of broad bean seeds was 6% due to infection with Pea enation mosaic alone, 50% by the infestation of Aphis fabae as pest alone, but 93% due to both virus and aphid vector (Hinz and Daebeler 1979). Similar type of studies has to be carried out in some more host–virus– vector combinations to get clear understanding on these aspects. The yield loss estimates varied depending on the cultivar, the virus strain and the stage of infection. For example, at Georgia (USA), Peanut mottle virus (PMV) strain-N in peanuts reduced seed yield by 68% in the cv. Starr and 47% in PI-261945, but no losses were reported in PI261946. The PMV strain-M2 caused 31% loss in seed yield in the cv. Starr and 20% loss in the above two introductions (Kuhn et al. 1978). Tu (1989) studied the impact of 7 strains of SMV on seed yield in 8 soybean cultivars including Oxley 615, which is a resistant line. Similarly, the extent of yield losses varies with the age of the crop infected. From the examples cited, it is clear that loss estimates vary and are influenced by a number of factors. Practical quantitative assessment methods and the prediction of crop losses caused by seed-transmitted viruses are still in their infancy. References Agarwal VK, Nene YL, Beniwal SPS (1976) Influence of bean common mosaic virus infection on the flower organelles, seed characters and yield of urd bean. Indian Phytopathol 29:444–446 Ali A, Randles JW (1998) The effects of two pathotypes of pea seed-borne mosaic virus on the morphology and yield of pea. Aust Plant Pathol 27:226–233 Bailiss KW, Ollennu LLA (1986) Effect of alfalfa mosaic virus isolates on forage yield of lucerne (Medicago sativa) in Britain. Plant Pathol 35(2):162–168 Beniwal SPS, Bharathan N (1979) Urd bean leaf crinkle virus: effect on yield contributing factors, total yield and seed characters of Urd bean. Seed Res 7:175–181 Beniwal SPS, Kolte SJ, Nene YL (1979) Nature and rate of spread of urd bean leaf crinkle diseases under field conditions. Indian J Mycol Plant Pathol 9:188–192 Blattny C, Osvald V (1954) Pfenos viros chmele (Humulus lupulus L.) na potomsto semenem. Preslia, (Prague) 26:1–26 Bos L (1981) Wild plants in the ecology of virus diseases. In: Maramorosch K, Harris KF (eds) Plant diseases and vectors: ecology and epidemiology. Academic, New York, pp 1–33 Bos L (1982) Crop losses caused by viruses. Crop Prot 1:263–282 Bossennec JM, David O, Demange N, Faivre B, Letarnec B, Palluault M, Taupin P, Maury Y (2000) Incidence of viruses on yield of field pea. Phytoma 526:21–24 Broadbent L, Tinsley TW, Buddin W, Roberts ET (1951) The spread of lettuce mosaic in the field. Ann Appl Biol 38:689–706 Bwye AM, Jones RAC, Proudlove W (1994) Effects of sowing seed with different levels of infection, plant density and he growth stage at which plant first develop symptoms on cucumber mosaic virus infection of narrow leafed lupins lupines angustifolius). Aust Gen Agric Res 45:1395–1412 Carroll TW (1980) Barley stripe mosaic virus: its economic importance and control in Montana. Plant Dis 64:135–140 Carroll TW (1983) Certification schemes against barley stripe mosaic. Seed Sci Technol 11:1033–1042 Catherall PL (1972) Barley stripe mosaic virus. Rept. Welsh Plant Breed Stn 1971:62 Chang CA, Kno YJ (1983) Cowpea aphid borne mosaic virus and its effect on the yield and quality of asparagus bean. J Agric Res China 32(3):270–278 Chattopadhyay AK, Bhatttacharya S, Dutta SK (1986) Assessment of loss in mungbean cultivars due to leaf crinkle disease. 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American Phytopathological Society, Saint Paul, pp 1–13 Xia JQ, Wang QH, Yan DY, Zuh HC, Zheng JF (1986) On the mosaic disease of asparagus lettuce II. The distribution, loss, hosts and transmission of asparagus lettuce mosaic virus. Acta Phytopathol Sin 16(1):37–40 Xu Z, Zhang Z (1986) Effect of infection by three major peanut viruses on the growth and yields of peanut. Agric Sci China 4:51–56 Zink FW, Grogan RG, Welch JE (1956) The effect of the percentage of seed transmission upon subsequent spread of lettuce mosaic virus. Phytopathology 46:662–664 4 Virus Transmission Abstract The secondary spread of the seed-transmitted virus diseases both in the field and glass houses takes place through different vectors. Under field conditions, the infected seedlings raised from virus-infected seeds act as primary foci of infection and further spread takes place through insect vectors. Transmission through mite and insects is a natural and main method of virus spread, and the major vectors of viruses are from the phylum Arthropoda (94%) and other important vectors (about 6%) are nematodes (Phylum: Nematoda). Nearly 35 seed-transmitted viruses are from Potyvirus group, which have aphid vectors. The next highest seedtransmitted viruses (28) are found in Nepovirus group, in which nematodes are the vectors. Even beetle-transmitted seed-borne viruses are found in Fabavirus group (3) and Sobemovirus group (5). Soil-borne fungal vectors have also transmitted 4 and 2 viruses in Furovirus and Necrovirus groups. While dealing with the insect vector transmission, the virus–vector relationship of some of the seed-transmitted viruses was also discussed. Doubtful reports of seed transmission of certain viruses by whiteflies and mites are to be confirmed by further studies. Information on role of pollen in seed transmission is discussed. Potexvirus and Tobamovirus groups, which are externally seed-borne in certain solanaceous crops, were also presented. 4.1 Vector Transmission All seed-transmitted viruses are vertically seed transmitted and the movement of the virus is temporal from generation to generation down the pedigree line. In the present context, the vertical transmission means the movement of a virus from a male and/or female parent via pollen and/or ovule leading to the formation of the next generation infected seeds. More than 231 seed-transmitted viruses are known to be vertically transmitted. The secondary spread of the seed-transmitted virus diseases both in the field and glasshouse takes place through different vectors as reviewed. Under field conditions, the diseased plant raised from infected seed serves as focus of infection for further secondary spread either mechanically or by the vectors, and hence K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 4, © Springer India 2013 75 76 seed-transmitted virus diseases are compound interest diseases. Further, horizontal spread of the virus to the contemporary plants in the field from the initial foci of infection takes place mechanically or through pollen or vectors resulting in heavy infections. The word ‘vector’ is derived from the Latin word ‘vectus’ as past participle of vehere meaning ‘to carry’, and in a biological sense, vector is an organism carrying pathogenic agents. Barring a few viruses, majority of the seed-transmitted plant viruses spread through arthropod vectors like aphids, whiteflies, beetles, mites and thrips. Only about three decades ago, certain nematodes and fungi have joined the vector group for some seedtransmitted viruses. The insect vectors transmit plant viruses by four major transmission modes and a number of virus and insect proteins have been found to control virus–vector association. Majority of the seed-borne plant viruses are transmitted by aphids in nonpersistent manner. Most vectors are piercing–sucking insects that transmit plant viruses in either the circulative virus (CV) or noncirculative virus (NCV). NCVs are carried on the lining cuticle of vector stylets. Circulative viruses (CVs) cross the vector’s gut, move internally to the salivary glands (SG) and cross the SG membranes to be ejected upon feeding. Transmissibility of NCVs depends on motifs of coat protein and for Potyviruses and Caulimoviruses also on helper proteins (encoded by the virus). NCV proteins were found to associate with vector’s cuticle proteins. Transmissibility of CVs depends on proteins comprising the virus capsid (the coat protein and the read-through protein) and on symbionin (produced by vectors symbionins). Passage of CV through the gut has been also associated with vector’s proteins. Raccah and Fereres (2009) have furnished more details on vector transmission. The arthropod vectors have a diverse assemblage of mouth parts with great differences between the various groups in life cycle, behaviour and activity. The viruliferous winged adult aphids and thrips are blown by wind to distant places where they initiate infection. For example, Johnson (1967) recorded that a nonpersistent virus 4 Virus Transmission was carried up to 60 km or even more in a nonstop flight. Certain vectors like the mealybugs and mites also spread viruses to nearby plants by crawling or walking. No reports are available on the transmission of any seed-transmitted virus either by mealybug or by leaf and plant hoppers. Although the mite transmission of Wheat streak mosaic virus is reported to be seed transmitted in corn (Hill et al. 1974), it requires confirmation. 4.1.1 Aphids Aphids constitute the most important group of virus vectors and transmit more plant virus diseases than any other group of vectors. About 50% of seed-transmitted viruses are aphid transmitted and belong to Caulimovirus, Carlavirus, Cucumovirus, Alfamovirus, and Potyvirus groups. It is a nonpersistent relationship in which both the acquisition and inoculation probes of 15–60 s each are optimal for transmission. Both winged and wingless single aphids can transmit the virus. High percent of transmission is achieved by the preliminary fasting period with optimal aphid numbers; however, the inoculative capacity of the aphid decreases as the period between acquisition and inoculation of the virus increases. Aphids almost always probe in the anticlinal grooves of adjacent epidermal cells, which they locate via receptors-mechano pegs present on the labial tip. Aphids always secrete saliva at the start of the probe as well as during stylet penetration forming a sheath. Stylets move fairly rapidly within this sheath but subsequently extend beyond the sheath for ingestion of food material from host cells. During the process of feeding, the viruses present in the sap usually adhere to their mouth parts and are introduced subsequently into another plant when the viruliferous aphids feed, which is termed as mechanical contamination hypothesis. Forbes (1977) has extensively reviewed the feeding mechanism of aphids. In general, the nonpersistent seed-transmitted viruses have low level of vector specificity because they are transmitted by several aphid species, such as AMV by 14 aphid species (Crill et al. 1970), CMV 4.1 Vector Transmission by 60 aphid species (Kennedy et al. 1962) and SMV by 31 aphid species (Irwin and Goodman 1981). Domier et al. (2007) have studied eight SMV strains in soybean and showed that there was direct correlation with transmission through aphid vector, and seed coat mottling caused by SMV virus isolate. The poor seed transmission showed even poor transmission by the soybean aphid, Aphis glycines. The loss of aphid and seed transmissibility by repeated mechanical transmission suggests that constant selection pressure is needed to maintain the regions of the SMV genome controlling the two phenotypes from genetic drift and loss of function. 4.1.2 Beetles Another active vector of seed-transmitted viruses is beetle and five members from Comovirus, three from Carmovirus, two from Tymovirus, two from Sobemovirus and one each from Bromovirus and Fabavirus groups are beetle transmitted (Fulton et al. 1980, 1987). There is a high degree of specificity between the beetle vectors and the viruses they transmit. Squash mosaic and Southern bean mosaic viruses are seed transmitted in a number of cucurbitaceous and leguminous hosts, respectively. As low as 0.1% seed transmission has been recorded in different virus–host combinations, namely, Bean pod mottle virus in soybean which is transmitted by bean leaf beetle (Cerotoma trifurcata) (Lin and Hill 1983; Giesler et al. 2002; Krell et al. 2003), while as high as 93% seed transmission in C. melo with Squash mosaic virus was recorded (Rader et al. 1947). There are certain controversial reports of seed transmission of viruses having beetles as vectors. Seed transmission (1–5%) of beetle-transmitted Cowpea mosaic virus was reported by Gilmer et al. (1973), but Thottappilly and Rossel (1987) could not confirm the seed transmission. Urdbean leaf crinkle virus in Vigna mungo is also seed transmitted up to 83% (Pushpalatha et al. 1999) and reported to be transmitted by leaf-feeding beetle Henosepilachna dodecastigma (Beniwal and Bharathan 1980), and this vector has to be 77 confirmed. Cowpea mottle virus in cowpea has beetle vector, namely, Ootheca mutabilis, which acquired virus in 10 min, transmitted within 1 h and remained viruliferous for 5 days (Shoyinka et al. 1978). The beetle vectors are confined to the families of Chrysomelidae, Coccinellidae, Curculionidae and Meloidae. They have biting type of mouth parts and transmit viruses mechanically by carrying them on the mouth parts. These vectors acquire the virus during acquisition feeding periods ranging from a few minutes to 24 h and transmit them immediately. The length of time a beetle retains and transmits the virus primarily depends on the type of virus, its host and environmental conditions. Both Walters (1969) and Selman (1973) suggested that virus retention time by the vector is characteristic of a particular virus. The viruses transmitted for 1 or 2 days fall into one group, while that of longer periods into the other group. The Mexican bean beetle retains different viruses and transmits only for short periods of time, whereas the bean leaf beetle retains the same viruses for extended periods. Beetles can retain viruses up to a period of 8 days and can be detected in haemolymph, but apparently do not multiply in their beetle vectors. Virus–vector relationship appears to be similar to semi-persistent type. As early as 1949, Markham and Smith pointed out that beetles regurgitate during feeding which contain virus over several days. The viruses are also detected in the faecal matter of beetles which have fed on infected plants. Some viruses like Broad bean stain and Broad bean true mosaic (BBTMV) are transmitted by weevils, Apion vorax (Cockbain et al. 1975; Jones 1978). BBTMV is transmitted through seed of faba bean up to 17% (Cockbain et al. 1976) and 2.7–10% by Broad bean stain virus in the same host (Gibbs and Smith 1970). 4.1.3 Thrips The transmission of seed-transmitted viruses by thrips vectors has been reported for Ilarvirus and Tomato spotted wilt virus groups. Tobacco 78 4 streak virus (TSV), seed transmitted in hosts like Datura stramonium, Glycine max and Phaseolus vulgaris, was transmitted by Frankliniella sp. and Thrips tabaci (Costa and Lima Neto 1976; Sdoodee and Teakle 1987). During 2010, Vemana and Jain have detected TSV both in groundnut pod shell and seed coat (testa) and no virus was detected either in cotyledons or embryos obtained from infected seeds, indicating absence of true seed transmission in groundnut and also in other leguminous hosts. Similarly, Tomato spotted wilt virus (TSWV) was also seed transmitted in Senecio sp., but not in peanut. Extensive serological tests carried out at ICRISAT, Hyderabad (India), have clearly indicated the non-seed-transmitted nature of GBNV in mature dried peanuts (Reddy et al. 1983; Prasada Rao et al. 2009). 4.1.4 Whiteflies The whiteflies are small, piercing and sucking insects belonging to the family Aleyrodidae in the order Homoptera. Seed transmission of whiteflytransmitted virus diseases has been reported in Begomovirus and Carlavirus groups for which Bemisia tabaci is the vector and further seed transmission researches are required with different whitefly-transmitted viruses. For example, Keur (1933, 1934) has reported Abutilon mosaic begomovirus to be seed transmitted and subsequent studies have not confirmed the seed transmission. Abutilon mosaic virus belonging to Begomovirus group is transmitted by whitefly vector in a semi-persistent manner, and the Cowpea mild mottle virus (CMMV) of Carlavirus group in a nonpersistent manner. Muniyappa and Reddy (1983); Jeyanandarajah and Brunt (1993) have reported that CMMV was transmitted even by a single whitefly and the adult whitefly acquired the CMMV in an access period of 10 min and transmitted in a 2 min inoculation period in serial 5 min transfers, the virus was retained for a maximum period of 20 min. However, according to Briddon (2003), Begomoviruses belonging to family Geminiviridae has 102 viruses, which are not seed transmitted. Horn et al. (1991) could Virus Transmission not observe the seed transmission of CMMV in soybean and groundnut. Even in extensive seed transmission tests conducted by Rossel and Thottappilly (1993) with two isolates of Cowpea mild mottle virus in 25 soybean accession numbers, seed transmission was not noticed both in grow out tests and by ELISA. 4.1.5 Mites The Eriophyid mite vector is elongated and tiny, about 0.2 mm long, and virus–vector relationship is of noncirculative type. For the first time, Khetarpal (1989) and Khetarpal and Maury (1989) have reported the transmission of the Pea seed-borne mosaic virus in pea by the mite, Tetranychus urticae under glasshouse conditions and requires confirmation. Another seed-transmitted virus, Wheat streak mosaic virus, in wheat is transmitted by mite vector Aceria tosichella (Jones et al. 2005), and this virus is reported to be seed transmitted in wheat as low as 0.22% (Lanoiselet et al. 2008). 4.1.6 Nematodes The soil-inhabiting nematodes are the vectors responsible for transmission of members of Tobravirus and Nepovirus groups which are also seed transmitted (Table 1.2), and this has been reviewed exhaustively (Taylor 1980; Brown et al. 1995). Four nematode genera, namely, Xiphinema, Longidorus, Paratrichodorus and Trichodorus, are known vectors which measure 2–12 mm long and are migratory ectoparasites feeding mainly on root tips. Regarding their habitat, X. diversicaudatum is generally associated with moist and shady sites in the vicinity of water or medium soils with high moisture levels (Fritzsche et al. 1972). L. elongatus is found in light medium soils and L. attenuatus in light sandy soils (Harrison 1977). T. pachydermatus usually occurs in light rather than heavy soils (Van Hoof 1962). Both adult and larval stages can transmit the viruses with equal efficiency. L. elongatus retains the virus for 8–9 weeks and 4.1 Vector Transmission the Xiphinema sp. for many months, and there is no evidence that viruses multiply inside the nematode vector. Based on the particle morphology of the nematode-transmitted viruses, Cadman (1963) and Harrison (1964) categorised them into Nepovirus and Tobravirus groups having polyhedral and tubular morphology, respectively. The spread of these viruses is very slow since the vector nematodes move over short distances of approximately 50 cm in a year. Transport of vector nematodes over longer distances in soil is probably less feasible since they are susceptible to desiccation, but movement of moist soil on agricultural implements, plant roots and the feet of animals and birds may play some role in nematode vector distribution. 4.1.7 Bumblebees Antignus et al. (2007) have confirmed that bumblebees (Bombus terrestris) transmit the Tomato apical stunt viroid from infected to healthy tomato plants in the form of secondary spread. Okada et al. (2000) have demonstrated that TMV was transmitted to tomato plants grown in glasshouses by the bumblebees. The virus transmission by bumblebees may result from the wounding of flowers during visits of the insects as was reported for Pepino mosaic virus or from the introduction of infected pollen to the stigma, from which the viroid could enter the embryo after the fusion of the sperm cells with the ovules. Lacasa et al. (2003) have implicated the involvement of bumblebees on Pepino mosaic virus spread in tomato crop. In Japan, Tomato chlorotic dwarf viroid is also transmitted from infected tomato plants to neighbouring healthy plants through bumblebees (Bombus ignites) during pollination activities (Matsuura et al. 2010). 4.1.8 Fungi Certain fungi like Polymyxa sp., Olpidium sp. and Synchytrium sp. are also implicated as vectors of seed-transmitted viruses belonging to Necrovirus 79 and Furovirus groups. Peanut clump virus (Pecluvirus) occurring to a limited extent in sandy soils of Punjab, Rajasthan and Andhra Pradesh states in India is transmitted both by Polymyxa graminis and also through seed (Thouvenel and Fauquet 1981a; Reddy et al. 1983; Delfosse et al. 1999, 2002; Dieryck et al. 2009). The life cycle of the fungus vector in its graminaceous hosts has been studied by Ratna et al. (1991). Similarly, Pea stem necrosis virus, transmitted by Olpidium sp., is seed transmitted (Brunt et al. 1996). Soil-borne wheat mosaic virus (SBWMV) transmitted by Polymyxa graminis is seed transmitted in rye and wheat crops (Delfosse et al. 1999). Another soil and seed-transmitted virus, Tomato bushy stunt virus of Tombusvirus group, has been suspected to have a fungus vector, but attempts to implicate a specific vector have not been successful. Potato virus X, transmitted by the fungus Synchytrium endobioticum, is seed transmitted in potato to an extent of 0.6–2.3% and requires confirmation (Darozhkin and Chykava 1974). Different aspects of fungus transmission of plant viruses have been reviewed by Teakle (1983). Campbell et al. (1996) have tested the hypothesis of vectorassisted seed transmission (VAST) while working with seed-transmitted Melon necrotic spot carmo virus (MNSV) infection in melons (Cucumis melo) which is transmitted by Olpidium radicale. Seed-transmitted virus rarely infected melon seedlings unless the vector was present, confirming VAST as a novel means of seed transmission. 4.1.9 Mealybug Among the mealybug-transmitted viruses is Cocoa swollen shoot virus for which the vector is Pseudococcus njalensis and also transmitted by seed to the extent of 34–54% (Quainoo et al. 2008). This virus is located in testa, cotyledon and embryo and virus is pollen-borne. Another seed-transmitted virus with mealybug Planococcus citri vector is Piper yellow mottle virus of black pepper, and rate of seed transmission in black pepper seeds is up to 30% (Bhat et al. 2005). 80 4 4.2 Nonvector Transmission 4.2.1 Mechanical Spread The limited persistence of certain viruses and viroids outside living cells restricts the opportunities for their transmission by direct contact. However, majority of the viruses which are spreading by mechanical contact have no known vectors. Viruses like TMV, PVX, BSMV, PepMV and PStV spread by handling, by pruning or by contact with infected plants, debris or contaminated implements. With BSMV being seed transmitted to the tune of 90%, mechanical spread is an efficient source for its introduction in uninfected barley growing areas. No other weed host acts as a significant reservoir of the virus except wild oats. The secondary spread of this virus inside the field takes place mechanically by leaf contact of infected plant with healthy plants. Even Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV) and Pepino mosaic virus (PepMV) strains in most of the solanaceous crops like tobacco, tomato and chillies are spreading through contact, contaminated tools, workers’ hands and clothing. Wind currents and walking of animals and workers in the field promote increasing spread. Besides this, TMV infection persists in the soil even 20 months after removal of the diseased plants and the virus also survives in dried debris. TMV spreads through irrigated water or soil as a contaminant and poses a problem in glass houses. Mechanical spread of Rice yellow mottle virus by cows, donkeys and grass rats in irrigated rice crops was reported by Sarra and Peters (2003). In almost all the viroid diseases, the secondary spread takes place via workers’ infested hands and tools (Singh et al. 1998a, b; Ramachandran et al. 1996). 4.2.2 Virus Transmission vulgaris) and other Compositae members, viruses like Arabis mosaic, Tomato black ring and Strawberry latent ring spot are seed transmitted and the infected seed is carried through wind. Seeds of S. vulgaris also carry LMV (Costa and Duffus 1958). Wind-mediated spread of Rice yellow mottle virus (RYMV) in irrigated rice crops was reported by Sarra et al. (2004). 4.2.3 Water Viruses present in water can infect plant through the root system and cause the appearance of disease symptoms. They can also be released from infected plants into drainage and then spread to other plants (Koenig 1986). In a Hungarian study, the presence of 26 plant viruses in 47 environmental water samples was detected (Horvath et al. 1999). Yakovleva (1965) reported that Cucumber green mottle mosaic virus (CGMMV) is being seed transmitted in Cucumis sativus to an extent of 44% and spreads with surface water used for watering. In the Netherlands, in areas where CGMMV prevails, it has been demonstrated that the virus is present in high concentrations in the surface water contaminated from waste heaps at dumping sites. Under glasshouse or field conditions, the virus may be released from dying roots and end up in drainage water. It has also been observed that a high percentage of the healthy plants are infected due to the sprinkling of contaminated water (Van Dorst 1988). In India, CGMMV was detected in Jamuna river waters flowing near cucurbit cultivated areas (Vani and Varma 1988). Similarly, CMV and TMV were also detected in some rivers of Southern Italy (Piazzolla et al. 1986). During 2007, Boben et al. have done detection and quantification of Tomato mosaic virus in irrigation waters in Slovenia by using RT–PCR technique. Wind Dispersal of the infected seed over long distance through wind has been observed among seedtransmitted viruses. In wild hosts such as Betula spp. and in weeds such as groundsel (Senecio 4.2.4 Obligate Symbiosis Obligate symbiosis is a mutual molecular beneficial mechanism of sharing of genetic References material of two different viral strains at the cellular system. Majority of the plant viruses are independent of their genetic material replication in the in vivo system, but very few virus strains depend on their replication mutually, for example, Pea enation mosaic virus (PEMV) is caused by a complex of two viruses, PEMV-1 is a Luteoviridae member and PEMV-2 is an Umbravirus. Two sizes of isometric particles are found, that of PEMV-1 (28 nm diameter) and that of PEMV-2 (25 nm diameter) having icosahedral and quasi-icosahedral symmetry, respectively. Both types of particles are composed of coat protein encoded by PEMV-1. PEMV-1 can infect protoplasts but does not spread in plants unless PEMV-2 is present. Unlike other members of the Luteoviridae, Pea enation mosaic virus is transmissible mechanically, as well as by aphids, and spreads to mesophyll tissues; these two properties are conferred by PEMV-2. Virions of some strains of PEMV-1 plus PEMV-2 also contain a satellite RNA. Aphid transmission of PEMV-1 plus PEMV-2 is conferred. Aphid transmissibility can be lost after multiple passages of mechanical transmission. Differences have been found in the electrophoretic profiles of the particles of aphid-transmissible and non-transmissible isolates, but the cause of these differences has not been fully resolved (Hull 2002). 4.3 Conclusions Cryptic viruses which induce little or no disease symptoms are not transmitted mechanically or by any other vector, but are transmitted through the seed; 100% of progeny is infected when both parents are carriers of the virus. Future studies are very much essential to identify the factors involved in seed infection or the lack of it: the prevalence of seed transmissible virus isolates, potential source of virus, occurrence and behaviour of vectors of such viruses before and after virus acquisition and their interactions with the environment and persistence of virus in seed and pollen in nature and development of effective novel control measures. 81 References Antignus Y, Lachman O, Pearlsman M (2007) The spread of Tomato apical stunt viroid (TAS Vd) in green house tomato crops is associated with seed transmission and bumble bee activity. Plant Dis 91:47–50 Beniwal SPS, Bharathan N (1980) Beetle transmission of Urdbean leaf crinkle virus. Indian Phytopathol 33(4):600–601 Bhat AI, Devasahayam S, Hareesh PS, Preethi N, Thomas T (2005) Planococcus citri (Risso)–an additional mealybug vector of Badnavirus infecting black pepper (Piper nigrum L.) in India. 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The higher the embryonic seed infection, the greater will be the size and number of cytoplasmic connections. Since legumes develop cytoplasmic connections more frequently than cereals, the percentage of virus transmission through seeds in legumes is higher than in cereals. The information on the distribution of virus in the seed is also presented. Survival of the virus in seed varies with virus strain, the cultivar and storage conditions. Some aspects of genetics of seed transmission are also presented. Factors like environmental aspects, stage of infection, host cultivar and virus strain/isolate play major role in seed transmission in different virus–host combinations. The information on reasons for failure of seed transmission is also presented. 5.1 Embryology and Development of Seed Structures A typical angiosperm flower consists of four types of floral organs, sepals, petals, stamens and pistil(s), and only the last two are directly concerned in seed production. Stamens form pollen grains which produce male gametes. The pistil consists of ovary which contains the ovules, style and stigma. A simple ovary consists of one carpel with one locule or cavity; a compound ovary is made up of more than one carpel and may contain one or more locules, each of which contains one or more ovules. Thus, a large number of ovules are formed in the ovary which after fertilisation develop into seeds. The developing ovule generally is attached to the placenta by funiculus. The outer layers of the ovule are the outer integument which is joined with funiculus, and within this is the inner integument. These integuments do not completely enclose the inner parts; at one end is a small opening called micropyle. The central part of the ovule is the nucellus with a central single large megaspore mother K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 5, © Springer India 2013 85 86 cell (2n) which gives rise to an oval or elongate embryo sac. During the development of the embryo sac, the megaspore nucleus divides three times by mitosis to form eight genetically identical nuclei. Three of the nuclei become located at the micropylar end of the sac and three at the opposite end. The two polar nuclei move towards the central area of the polar embryo sac and fuse, forming the secondary nucleus. The three cells at the micropylar end constitute the egg cell and the two synergids. The three cells at the opposite end of the embryo sac are the antipodals. The ovule and the organised embryo sac may be oriented in different ways, depending on the positions of the hilum and micropyle. The pollen grains are transferred to stigma by wind currents or through insects or by rain water. The transfer of pollen either to the stigma of the same flower or another flower in the same plant is known as ‘self-pollination’ and that to the stigma of flowers on other plants is termed ‘cross-pollination’. Mature pollen consists of a spore wall, a tube cell nucleus and a generative nucleus. The pollen germinates on the stigma, and the pollen tube grows through the style and ovary, reaches the tip of an ovule. Fertilisation is of three types depending on how the pollen tube enters into the ovule. The pollen tube enters through the micropyle which is the most common form of fertilisation and is known as porogamy. The second type is called chalazogamy, wherein the pollen tube enters the ovule at the chalazal end as in Casuarina. The third type is mesogamy where the pollen tube enters through the funiculus or through the integuments as in Cucurbita. The tip of the pollen tube passes through the nucellus and enters the embryo sac where it bursts discharging the two male germ cells called sperms. Out of the two sperm cells, one fuses with the egg and forms a zygote. The second fuses with the polar nuclei and results in primary triploid endosperm nucleus. The process of fertilisation where both the sperm cells on fusion form a zygote and an endosperm is known as double fertilisation. The fertilised egg or zygote develops into an embryo, and the primary endosperm nucleus divides a number of times to form many nuclei. These nuclei are separated by cell walls to 5 Mechanism of Seed Transmission form the endosperm. The seed coat results from integuments around the ovule. Many embryo-borne viruses are transmitted through pollen. The pollen grains when released from infected plants, may carry the virus either on exine or intine. When such pollen grains germinate on the stigma, pollen tubes grow through the style and reach the embryo sac, into which they release two male gametes. The infected gamete unites with the egg cell, and the resulting embryo may be infected. However, union of other gamete with polar nuclei may lead to infected endosperm. Cytoplasmic connections between the infected mother plant, flower and developing seeds may influence seed infection. The higher the embryonic seed infection, the greater will be the size or number of cytoplasmic connections. Since legumes develop cytoplasmic connections more frequently than cereals, the percentage of virus seed transmission in legumes is higher than in cereals. Ovule infection occurs with a number of viruses which may move into different parts of the seed including the embryo sac. Similarly, cytoplasmic connections between the developing pollen grains and the infected mother plant result in virus-infected pollen grains. The viruses may enter the embryo sac from the nucellus until cytoplasmic connections exist. When cytoplasmic connections are broken, the membrane of the egg cell is formed which prevents the movement of the virus. The capability of viruses to infect either the ovule or pollen grains systematically prior to fertilisation assists in seed transmission. Seed transmission is achieved either by direct invasion of the embryo via the ovule or by indirect invasion of the embryo, mediated by infected gametes. For some viruses in certain hosts (e.g. BSMV in barley), both processes operate simultaneously, although the relative contribution of the two processes will vary depending upon a large number of factors (Mandahar 1981). For direct embryo invasion, there is currently no explanation for how the virus is able to cross the boundary between the parental and progeny generations in the ovule, and no routes have been identified that lead to the establishment of the virus in the tissues of the developing embryo. Furthermore, genetic and cell biological studies 5.2 Distribution of Virus in the Seed have never been combined to obtain an overall understanding of the principles involved in seed transmission. 5.2 Distribution of Virus in the Seed The location of the virus in seed determines transmissibility of virus through seed. The virus is considered to be externally seed transmitted when it is outside the functional seed and internally seed transmitted when it is within the tissue of the seed, respectively. When externally seed transmitted, the virus is confined to the testa as a contaminant. In the case of seeds from fleshy fruits like tomato, cucumber, watermelon and apple, viruses such as TMV, ToMV, PVX, CGMMV and Tomato bushy stunt, respectively, adhere to the seed coat. During germination, the virus infection takes place through the tiny abrasions caused by small soil particles. It is suggested that the contact points between the testa and suspensor as a route of entry. They further suggested that the virus may be able to pass through the cell wall between the testa and suspensor by an as yet unidentified mechanism that does not require plasmodesmata, or it may be able to induce formation of new plasmodesmata, thus allowing direct invasion of the embryo. There are a large number of research articles which support the view of transmission of Tomato mosaic virus, a strain of TMV which is carried as externally seed transmitted primarily in tomato and capsicum in number of countries and has been confirmed by extensive serological tests. Earlier, Huttings and Rast (1995) have reported that tomato mosaic virus in tomato seed is localised on seed coat and sometimes in the endosperm. During Pradhanang et al. (2005) have expressed the doubt of Tomato mosaic virus transmission through tomato seeds, and negative results were also obtained in DAS ELISA and grow out tests (Pradhanang 2005). Now it is clearly understood that there are two ways to infect the developing embryo: by either indirect invasion through infection of the gametes before fertilisation or by direct embryo 87 invasion after fertilisation. For many virus–host interactions, both modes of embryo infection may result in maximal seed transmission (Johansen et al. 1994). The seed-transmitted viruses of the direct virus invasion after fertilisation are found in different seed tissues and may be confined to the embryo or endosperm tissues. In such cases, the infection originates at the female gamete production. Some examples of internally seedtransmitted viruses are BCMV, PSbMV, TRSV, SBMV, LMV, BSMV, PMV, PEBV, CMV, Urd leaf crinkle virus and CpAMV. Each virus has been detected internally in cotyledons and embryos of their respective hosts (Ekpo and Saettler 1974; Ladipo 1977; Adams and Kuhn 1977; Uyemoto and Grogan 1977; Hoch and Provvidenti 1978; Datta Gupta and Summanwar 1980; Beniwal et al. 1980; Beniwal and Chaubey 1984; Bharatan et al. 1984; Patil and Gupta 1992; Varma et al. 1992; Yang et al. 1997; Wang et al. 1997; De Assis Filho and Sherwood 2000; Roberts et al. 2003; Ali and Kobayashi 2010). Some viruses like BSMV and TMV infect the endosperm (Carroll 1972). Hoch and Provvidenti (1978) provided electron micrographic evidence for localisation of BCMV particles and virus inclusion bodies in mature embryos of dormant and germinating bean seeds. They have also recorded virus in thin sections of embryo and endosperm of infected soybean seeds (Tu 1975). The embryo invasion with Turnip yellow mosaic virus (TYMV) in Arabidopsis thaliana was reported by De Assis Filho and Sherwood (2000). The distribution of viruses within the seed could be studied by bioassay/serology and electron microscopy. Large number of viruses are located in embryo and hence high percentage of seed transmission. Some of the embryo-borne viruses are PSbMV in peas (Wang and Maule 1992, 1994; Roberts et al. 2003), BCMV in beans (Hoch and Provvidenti 1978), SMV in soybean (Bowers and Goodman 1979; Porto and Hagedorn 1975) and CMV in lupin (Lupinus angustifolius) (O’keefe et al. 2007) and Pepper (Ali and Kobayashi 2010). Thus, viral access to embryo is gained either indirectly by infection of reproductive tissues before embryogenesis, that is, ovule, megaspore 88 5 Mechanism of Seed Transmission mother cell and pollen mother cells, or directly by invasion of embryo during stages of embryogenesis (Carroll 1981). The ultimate levels of seed transmission in some virus–host combinations may in fact result from a combination of both routes of infection. 5.3 Virus Longevity in Seeds Stability of the virus in the seed depends on its type, location and the host. In a majority of cases, the viruses infecting embryo survive for longer periods, sometimes even as long as the seeds (Erkan 1998). For example, BCMV in bean seed remained viable for 30 years, sowbane mosaic virus in Chenopodium murale for 14 years, PNRSV in Prunus pensylvanica for 6 years and Squash mosaic (SqMV) in Cucurbita pepo and TRSV in soybean for over 5 years. Many potyviruses like SMV and CpAMV survive for 2– 3 years in their respective legume host seeds. TMV in tomato seed has been known to be active for 9 years. In true potato seed, the Potato spindle tuber viroid survives for 21 years (Singh et al. 1991). Longevity of important seed-transmitted viruses in crops is furnished in Table 5.1. transmission of two pathotypes of TSV, which differ in physico–chemical properties and RNA secondary structure. Their results indicated that infectivity of TSV Mel 40 and Mel F isolates appears to be dependent on homologous RNAs. BSMV contains three RNA species: RNA ’, RNA “ and RNA ”. Major seed determinants are located in the 50 untranslated region (UTR) and a 369 nt repeat present in the ”a and ”b genes. Mutations in these determinants significantly influence replication and translation of the virus. It was shown that seed transmission is completely blocked when the repeat sequence is present. This sequence is suggested to enhance the replication and movement of BSMV, while its mutation affects both the transmission and symptom expression in barley plant. It is worth mentioning that the 12 K gene of tobraviruses, HC-Pro of potyviruses and the ”b gene of hordeiviruses share cysteine-rich domains that regulate the transmission process by affecting the replication and movement of the virus in the reproductive tissues of respective hosts and thus influences the infection of the embryo (Johansen et al. 1994). 5.5 5.4 Genetics of Seed Transmission Various researchers have expressed different opinions regarding the role of genetics in transmission of viruses through seed. For example, Keller et al. (1998) have reported that Potyvirus genome-linked protein (VPg) determines the Pea seed-borne mosaic virus pathotype. On the other hand, Johansen et al. (1996) found that CP and HC-Pro coding regions of the Pea seed-borne mosaic virus genome were required for efficient transmission through seed of Pisum sativum. The ”b protein of Barley stripe mosaic virus has been shown to the involved in seed transmission (Edwards and Steffenson 1996). Walter et al. (1995) have studied the viral genetic basis of symptom severity and seed Factors Influencing Rate of Seed Transmission Some of the seed-transmitted viruses become inactive within a short span of time, while others remain active as long as the seeds remain viable. The active period of the virus generally depends on the seed longevity and location in the seed. It appears to be characteristic of the virus– host combination (Table 5.1). Some nematodetransmitted viruses persist in the seed of certain weed plants and increase the chances of virus perpetuation under field conditions. Some other viruses show high seed transmission since they are effectively translocated into the developing seeds in a passive way along with carbohydrates. But in other cases, certain inhibitors may impede translocation. Considerable variation in seed transmission is influenced by one or more factors such as host variety, virus strain, the time of infection of the host and the prevailing temperature 5.5 Factors Influencing Rate of Seed Transmission 89 Table 5.1 Longevity of some important seed-transmitted viruses in different crops Virus Alfalfa mosaic Barley stripe mosaic Bean common mosaic Bean southern mosaic Bean western mosaic Broad bean stain Cowpea aphid-borne mosaic Cucumber green mottle mosaic Cucumber mosaic Dodder latent mosaic Eggplant mosaic Hop mosaic Lychnis ring spot Muskmelon mosaic Prune dwarf Prunus necrotic ring spot Raspberry ring spot Sowbane mosaic Soybean mosaic Squash mosaic Tobacco mosaic Tobacco ring spot Tomato black ring Source: Agarwal and Sinclair (1987) Crop Medicago sativa M. sativa M. sativa Hordeum vulgare H. vulgare Triticum aestivum Phaseolus vulgaris P. vulgaris P. vulgaris P. vulgaris P. angularis P. vulgaris P. vulgaris Vicia faba Vicia faba Vigna unguiculata Cucumis sativus Cucumis sativus P. vulgaris P. vulgaris C. sativus Stellaria media Cuscuta campestris Solanum melongena Humulus lupulus Lychnis divaricata Silene noctiflora Cucumis melo C. melo Prunus sp. P. pensylvanica Capsella bursa-pastoris Stellaria media Chenopodium murale C. murale Glycine max Viability (years) 3 5 7 3 19 6:5 30 3 38 6 4 0:6 3 1 4 2 0:5 3 2:25 0:5 1 1:75 1 0:6 2 3:3 2:15 3 5 3:5 6 6 6 6:5 14 2 G. max Cucurbita pepo C. pepo Lycopersicon esculentum L. esculentum Petunia violacea Glycine max G. max Nicotiana sp. C. bursa-pastoris S. media 1 3 5 3 9 0:6 5 0:75 5:5 6 6 Reference Frosheiser (1970) Frosheiser (1974) Babovic (1976) McNeal et al. (1961) McNeal et al. (1976) Scott (1961) Pierce and Hungerford (1929) Nelson (1932) Walters (Walters 1962a, b) Polak and Chod (1967) Tsuchizaki et al. (1970) Zaumeyer and Harter (1943) Scotland and Burke (1961) Cockbain et al. (1973) Vorra-urai and Cockbain (1977) Khatri and Chohan (1972) Van Koot and Van Dorst (1959) Yakovleva (1965) Bos and Maat (1974) Meiners et al. (1977) Meiners et al. (1977) Tomlinson and Walker (1973) Bennett (1944) Mayee (1977) Blattny and Osvald (1954) Bennett (1959) Bennett (1959) Rader et al. (1947) Middleton and Bohn (1953) Gilmer (1964) Fulton (1964) Lister and Murant (1967) Lister and Murant (1967) Bennett and Costa (1961) Bennett (1969) Kendrick and Gardner (1924), Sinclair and Backman (1993) Provvidenti et al. (1982) Middleton (1944) Middleton and Bohn (1953) Alexander (1960) Broadbent (1965) Henderson (1931) Laviolette and Athow (1971) Athow and Bancroft (1959) Valleau (1939) Lister and Murant (1967) Lister and Murant (1967) 90 5 Mechanism of Seed Transmission conditions (Pathipanawat et al. 1997; Singh and Mathur 2004). Maule and Wang (1996) suggested four factors that possibly regulate the seed transmission. 1. Host genes control the ability of the virus to invade the gametes or the meristematic tissues as in the case of cryptic viruses (Kassanis et al. 1978). 2. Host genes control the survival of the gametes and the embryo in the presence of the virus. 3. Host factors regulate the multiplication and movement of the virus. 4. Host factors govern the maturation of the embryo that affects the longevity of the virus. 5.5.1 PMV differed in frequency of seed transmission in Starr peanut M1 D 0.3%; M2 D 0; M3 D 8.5 and N D 0 (Adams and Kuhn 1977). The impact of virus strains on seed transmission has also been noticed with Bean yellow mosaic virus (Anderson 1957), BSMV (Hamilton 1965), AMV (Frosheiser 1974), BCMV (Morales and Castano 1987), CMV (Davis and Hampton 1986; Sandhu and Kang 2007), SMV (Tu 1989; Domier et al. 2007) and Subterranean clover mottle virus (Wroth and Jones 1992), Broad bean stain virus and Pea seed borne mosaic viruses (Hampton et al. 1981; Kumari and Makkouk 1995; Johansen et al. 1996) and TSV in soybean (Ghanekar and Schwenk 1974). Number of Infection Sources 5.5.3 The effect of number of sources of infection has attracted particular attention where infection originates partially in infected stocks. In England, lettuce crop grown in five commercial seed lots with 2.2–5.3% infection by LMV had 25–96% infection with mosaic at the end of the growing season. But six batches of seed with LMV infection up to 0.1% have yielded crops with infected plants up to 0.5% (Tomlinson 1962). 5.5.2 Virus Strain/Isolate The amount of seed transmission varies greatly with the virus strain/pathotype. At Corvallis (USA)., two isolates of PSbMV (P-1 and P-4) were transmitted through seed at 24 and 0.3%, respectively, in Pisum sativum 549 (Johansen et al. 1996). Earlier, Ghanekar and Schwenk (1974) noticed 2.6–30.6% seed transmission for soybean isolates of Tobacco streak virus, whereas a tobacco isolate of the same virus did not show any transmission. Isolates belonging to serological group I of SqMV were seed transmitted in pumpkin, squash, cantaloupe, honey dew and watermelon, while isolates belonging to group II of SqMV were seed transmitted only in pumpkin and squash (Nelson and Knuhtsen 1973a). Similarly, four isolates of Mixed Infections The rate of seed transmission of a virus may also be influenced by infection with other viruses. Seed transmission of SMV in seeds from soybean plants infected with either SMV alone or in combination with Bean pod mottle virus was 6 and 11%, respectively (Ross 1963). Similarly, Southern bean mosaic virus (SBMV) was 12% in cowpea but increased to 20% in the presence of Cowpea chlorotic mottle virus (CCMV) (Kuhn and Dawson 1973). Seed transmission of Turnip yellow mosaic virus (TYMV) in Arabidopsis thaliana doubly infected with TYMV and TMV was 70% compared to infection of TYMV alone, which was only 31% (De Assis Filho and Sherwood 2000). Mechanism of increases in transmission of a virus in mixed infections is not clearly understood. 5.5.4 Host Species The degree of seed transmission of a virus varies with the host species which was noticed as early as 1935 by Hewitt, while working with BCMV. Bock and Kuhn (1975) reported seed transmission of PMV in peanut, but not through cowpea or soybean. Raspberry ring spot virus (RRV) was seed transmitted at 2–3% in Capsella bursa- 5.5 Factors Influencing Rate of Seed Transmission pastoris but at more than 50% in Beta vulgaris. Tomato black ring virus (TBRV) was transmitted at 5% in Rubus idaeus but reached up to 100% in Cerastium vulgatum, Fumaria officinalis, Myosotis arvensis and Polygonum persicaria (Lister and Murant 1967). Seed transmission rates of Broad bean stain virus (BBSV) varied between 0.2 and 32% in 19 lentil genotypes (Makkouk and Kumari 1990; Kumari et al. 1996). Even, Al-khalaf et al. (2002) reported the variation in seed transmission of BBSV in lentil based on genotype variability and seed size. Considerable differences exist in the magnitude of seed transmission in different virus–host– variety combinations. LMV was very commonly seed transmitted in some but not through the seed of certain lettuce varieties (Couch 1955). Similarly, seed transmission of CMV was dependent in cowpea on the host variety (Anderson 1957). At Hyderabad (India), Reddy et al. (1998a, b) have studied seed transmission of Indian peanut clump virus in 22 peanut genotypes of botanical type of Spanish, Virginia, Valentia and runner, which varied from 3.5 to 17% depending on the genotype. A similar results were observed by Eslick and Afanasiev (1955) and Singh et al. (1960) in some barley varieties infected with BSMV. The PSbMV seed transmission varied from 0 to 87.5% in 148 pea varieties (Stevenson and Hagedorn 1973; Musil et al. 1983) and 1.9– 32.7% in 19 pea varieties with the same virus infection (Gallo and Jurik 1995). While working at France, Khetarpal et al. (1993) recorded varied degrees of seed transmission of PSbMV in different cultivars of pea. With the same virus–host combination, Wang et al. (1993) noticed no seed transmission in pea cultivars like Maro, Princess and Progreta, while it was 74% in the cultivar Vedette. In India, Mali et al. (1987) recorded a lot of variation in seed transmission of CpAMV and CMV in 14 and 10 promising varieties of cowpea, respectively. Seed transmission of Cowpea banding mosaic and Cowpea chlorotic spot virus in 15 cultivars ranged from 0% in P-910 and R-352 lines to 19% in Pusa Phalguni and 0% in R-339 to 18% in Iron New line, respectively (Sharma 91 and Varma 1986). The examples in different virus and cultivars/breeding lines cited here clearly indicate that the variation was primarily due to different environmental conditions, virus strain, host–plant age and host plant constitution physiology. High degree of seed transmission in different host cultivars of cryptic viruses has also been recorded. The incidence of Beet cryptic virus M was between 42 and 90% in seedlings of 8 cultivars of Beta vulgaris (Kassanis et al. 1977; White and Woods 1978), Vicia cryptic virus incidence was from 50 to 75% in 9 cultivars of Vicia faba (Kenten et al. 1978), Radish yellow edge virus was detected in 80–100% of seedlings of 6 cultivars of Raphanus sativus (Natsuaki et al. 1979), Carnation cryptic virus seed transmission ranged from 85 to 100% in 13 cultivars of Mediterranean hybrids of carnation but only 9% in the garden carnation cultivar Chabaud (Lisa et al. 1981) and Ryegrass cryptic virus incidence varied from 0 to 82%y in 21 species and cultivars of Lolium (Plumb and Lennon 1981a, b). A similar variation in seed transmission among different cultivars was reported for other viruses such as SMV (Kendrick and Gardner 1924; Ross 1961; Kennedy and Cooper 1967), LMV (Fegla et al. 1983), BCMV (Agarwal et al. 1979), CpAMV (Ladipo 1977; Ata et al. 1982), PSbMV (Musil et al. 1983), Brome mosaic virus (BMV) (Von Wechmar et al. 1984), CMV (Davis and Hampton 1986), AMV (Frosheiser 1974), White clover cryptic virus (Boccardo et al. 1985; Natsuaki et al. 1986), Alfalfa cryptic virus M (Boccardo et al. 1983; Natsuaki et al. 1984), BgMV (Varma et al. 1992), TYMV (De Assis Filho and Sherwood 2000) and CMV (Gillaspie et al. 1998a, b). The effect of host plant on seed transmission of virus is mostly related with its susceptibility or resistance to specific virus/strain. Therefore, greater disease severity of the mother plant results in highly susceptible varieties than less susceptible/resistant varieties. This close relationship has been explained on the basis of mechanisms of disease resistance. 92 5.5.5 5 Mechanism of Seed Transmission Stage of Infection Voluminous literature is available on the degree of seed transmission in relation to the infection stage of the plant. Infection of plants before flowering leads to embryonic infection resulting in maximum seed transmission (Schippers 1963). For example, in Columbia, the seed transmission of BCMV in the bean cultivar Similac was 40 , 9% and nil and 41.8, 2.8 and 10.1% in cultivar Pubbele Witte, when inoculated at 10, 20 and 30 days after sowing, respectively (Morales and Castano 1987). Similarly, 18–19%y seed transmission of SMV in soybean was recorded when inoculated at 3–4 week and only 3–4%y when inoculated at 9–10 week after planting (Bowers and Goodman 1979; Irwin et al. 2000). In the same crop, TRSV was transmitted up to 100% through seeds from plants infected before flowering, but the percentage decreased when infected prior to or immediately after flowering (Athow and Bancroft 1959). While working with TRSV in Phaseolus aureus, Shivanathan (1977) reported that for effective seed transmission, the mother plant must become infected at least 10 days prior to pollination. Similar results were observed with LMV/lettuce crops, but plants infected after flowering did not transmit the virus through seed (Couch 1955). In India, seed transmission of ULCV infection in urad bean seed (Vigna mungo) was 68% when 10-day-old plants were inoculated. Subsequently, the rate of seed transmission declined to 46, 22 and 10% in 20-, 30- and 40-day-old inoculated plants, respectively. The virus failed to become seed transmitted when 50-day-old plants were inoculated (Dubey and Sharma 1985). Similar trend was noticed with Cowpea banding mosaic (CpBMV) and Cowpea chlorotic spot viruses (CpCSV) when inoculated to 10-day-old cowpea plants under field conditions, with 18–25% and 15–20% seed transmission, respectively. However, plants inoculated after 35 days of sowing had 6–8% and 5–6% seed transmission, respectively, for CpBMV and CpCSV (Sharma and Varma 1986). The rate of infection with BSMV in barley increased when plants were inoculated 10 days before heading than from earlier or later infections. Highly determinant plants which flower over a relatively short period might give no seed transmission from the inoculations after flowering begins (Eslick and Afanasiev 1955). However, indeterminate plants like peanut and late flowering may produce virus transmitting seeds if inoculation occurs early enough. Plants infected after flowering generally do not produce infected seed, and transmission is successful only before cytoplasmic separation of the developing embryo from maternal tissue (Bos 1977). Generally, early virus infection leads to high seed-transmitted virus in number of virus–host combinations. Studies carried out by Chitra et al. (1999) have shown that ToMV and TMV can enter in seeds of tomato and bell pepper, irrespective of growth stage at the time of inoculation. Inoculation even at flowering and fruit-set stage can lead to establishment of virus in seeds. However, the concentration of virus in seeds will be higher, if the plants are infected at an early growth stage. Since ToMV and TMV occur in the seed coat, even late infection of the host leads to establishment of virus particles in the seed. 5.5.6 Environmental Factors The seed transmission also markedly depends on the season and temperatures that prevail during plant growth. High seed transmission of CpBMV and CpCSV was noticed in seeds of rainy season cowpea crop (Sharma and Varma 1986). But in Georgia, Peanut stripe virus (PStV) infection reached 37% and 18% in summer and winter harvest, respectively. While in Spanish cultivar while it was 19 and 11% in a runner type during the two seasons (Demski and Warwick 1986). Barley cultivar seed was more prone to BSMV in spring than in autumn (Slack et al. 1975). Thus, difference in seed transmission in sowings at various seasons was due to factors like occurrence of susceptible crop growth stage at a time when appropriate aphid vector activity was at a peak. Seed transmission of BCMV in bean seeds at ı 20 C varied between 16 and 25%, while it was negative at 16.5–18.5ıC (Crowley 1957a, b). On 5.6 Reasons for Failure of Seed Transmission the other hand, the Southern bean mosaic virus infected 95% of the embryos of the immature seed in bean plants grown at 16–20ıC and only 55% when grown at 28–30ıC (Crowley 1959). Maximum (88%) seed transmission of PSbMV was recorded at 28ı C in peas only after 5 weeks of plant growth but not at 21 nor 32ı C (Khetarpal and Maury 1990). Singh et al. (1960) have noted greater seed transmission of BSMV from barley plants grown at 20–24ıC than at higher or lower temperatures. In Scotland, Stellaria media, an important weed host of Raspberry ring spot virus, is slightly more readily seed transmitted at 14ı C than at 18–22ıC, whereas Strawberry latent ring spot virus is less frequently transmitted at 14–18ıC than at 22ı C (Hanada and Harrison 1977). While working with LMV in lettuce, Ryder (1973) observed high rate of seed transmission from mother plants at lower day temperatures than those at higher temperature. However, the results of environmental factors on seed transmission are often contradictory (Bennett 1969; Shepherd 1972). 5.6 Reasons for Failure of Seed Transmission The presence of virus in a seed (either in seedcoat, cotyledons, embryo) does not always lead to seedling infection. This property distinguishes a seed-transmitted virus that is carried by the seed but does not infect the seedling produced from the seed (Neergaard 1979). Most viruses are not seed transmitted. The reasons for the same are still obscure. However, certain inferences can be made based on the available experimental data. The seed-transmitted viruses are found in different seed tissues and may be located in the embryo, endosperm tissues (embryonic) or, in some cases, only in or on seed coats (non-embryonic). It is generally accepted that for true seed transmission, the virus must enter and survive in the embryo. The ability to invade embryos and get transmitted through seed is determined not solely by either the virus or the host. Genetic and structural analysis of seed-transmitted and non-seed-transmitted Pea seed-borne mosaic virus in certain pea lines 93 has been studied by Wang and Maule (1994). Pea seed-borne mosaic virus (PSbMV), a seedtransmitted virus in pea and other legumes, invades pea embryos early in development. This process has been controlled by maternal genes and, in a cultivar that shows no seed transmission, is prevented through the action of multiple host genes segregating as quantitative trait loci. These genes control the ability of PSbMV to spread into and/or multiply in the nonvascular testa tissues, thereby preventing the virus from crossing the boundary between the maternal and progeny tissues. Immuno-cytochemical and in situ hybridisation studies suggested that the virus uses the embryonic suspensor as the route for the direct invasion of the embryo. The programmed degeneration of the suspensor during embryo development may provide a transient window for embryo invasion by the virus and could explain the inverse relationship between the age of the mother plant for virus infection and the extent of virus seed transmission. It appears that two mechanisms, namely, embryonic and non-embryonic, operate for the virus in seed transmission, based on whatever the virus is unable to enter the embryo or get eliminated after entry. Bennett (1969), Neergaard (1977, 1979a, b), Bos (1977) and Carroll (1981) have reviewed this particular aspect in detail. 5.6.1 Inability to Infect Embryos Maximum seed transmission occurs when the embryo is infected. This is apparently the common mode of virus transmission, and Bennett (1969) terms this as embryonic transmission. During the process of embryo initiation by the union of nuclei in an infected embryo sac, the embryo begins to develop in a virus-free medium. There are no protoplasmic connections between the cells of the embryo and cells of the adjacent tissue. The embryo absorbs food materials from virus-infected areas without becoming infected. Only a few viruses are able to pass through cell walls, and in the absence of penetrating protoplasmic strands, usually the cell wall acts as a formidable barrier to virus passage (Neergaard 1977). Lack of 94 direct vascular/cellular contact through the plasmodesmata with the mother plant inhibits seed transmission (Bennett 1969; Carroll 1972). Absence of such virus pathways explains why embryonic infection exclusively requires mother plant infection before the production of gametes. Many researchers have suggested plasmodesmata as the probable avenue of virus movement in infected plants based on their observations of virus and virus-like particles in plasmodesmata (Esau et al. 1967; Davidson 1969; de Zoeten and Gaard 1969; Kitajima and Lauritis 1969; Roberts and Harrison 1970; Kim and Fulton 1971; Weintraub et al. 1976). The virus can move from cell to cell in the form of complete particles or viral nucleic acid. It has been hypothesised by Caldwell (1934) that marked differences between the growth rate of embryonic and endosperm tissues within developing ovules normally lead to rupture of the protoplasmic bridges between these tissues and the nucellus. This view has been based on the assumption that protoplasmic continuity is necessary to bring about embryonic infection by translocating the virus from the surrounding maternal tissues to the developing ovules. Another possibility is that, generally, most viruses do not multiply in meristematic tissues. Embryo being a site of very intense metabolic activity may be viewed as a particular type of meristematic tissue responsible for the failure of the virus invasion. It is likely that natural cytokinins present in the embryo may make the virus inactive. Caldwell (1962) suggested another reason for failure of seed transmission. Many plant viruses lack the ability to invade meristematic tissue since pathways for virus synthesis cannot compete successfully with cellular systems for high-energy phosphorylated compounds. Resistance of gametes to virus infection has also been implicated as one of the factors. Evidence of such resistance was reported by Medina and Grogan (1961) in the bean cultivars Red Mexican and Idaho Refugee, which were resistant to BCMV. No seed transmission was established when pollinated from infected susceptible cultivars. In resistant embryos, even though the 5 Mechanism of Seed Transmission virus was introduced into ovules, it failed to infect but succeeded in susceptible ones (Neergaard 1977). 5.6.2 Inability of Virus Survival in the Embryos It is now clearly known that a virus-inactivating system operates in the infected seed when it is drying during maturity (Duggar 1930; Cheo 1955). Southern bean mosaic virus infection decreases abruptly or disappears as the seed matures even after heavy infection in the immature seed. In West Africa, Konate et al. (2001) reported that in 17 rice genotypes, Rice yellow mottle virus was detected in all seed parts including glumolla, endosperm and embryo at 65–100%. Nevertheless no seed-transmitted infection was found as the virus decreased throughout the process of seed formation, suggesting inactivation of virus due to seed maturation and desiccation. Even Abo et al. (2004) have also proved the nontransmissibility of RYMV through rice seeds. Allarangaye et al. (2006) have reported nontransmission of RYMV through seeds of wild rice species. RYMV was infectious in freshly harvested seed extracts; however, most infectivity was lost in dried rice seeds, possibly due to virus inactivation following dehydration of the seeds (Konate et al. 2001). A similar phenomenon was observed in pea and cowpea infected with Pea streak and Cowpea chlorotic mottle viruses, respectively (Ford 1966; Gay 1969). Although TSWV was not seed transmitted in peanut, virus has been recorded from the testa of immature and freshly harvested mature seeds. However, it could only be detected serologically in the testa of dried seeds. Freshly harvested mature seeds with testa-containing infective virus failed to transmit TSWV when tested by grow out tests (Reddy et al. 1983a). Seed-transmitted viruses possess a unique property which enables them to invade even reproductive tissues. This property may be related to the glycoprotein portion of the coat protein. The carbohydrate residues in the viral coat may function as recognition sites to attach the References virus to gametophyte cell surfaces. However, the association of the carbohydrate with virus coat protein has not been demonstrated conclusively (Partridge et al. 1974). The abortion of microspores due to virus infection may have a bearing on lack of seed transmission (Valleau 1939). Caldwell (1962) suggested virus-induced meiotic irregularities to be concerned with lack of seed transmission. 5.7 Conclusions Perusal of the Table 1.2 indicates that considerable number of plant viruses of different virus groups are seed transmitted in large number of crop plants. The percentage of virus transmission in different hosts varies due to virus strain, host variety, environment factors, etc. There are certain controversial results regarding seed transmission in certain virus–host combinations. For example, Jain et al. (2006) reported the transmission of Tobacco streak virus through seed of cucumber (Cucumis sativus) and gherkins (Cucumis anguria) to the extent of 3–68%. On the other hand, Prasada Rao et al. 2009 have reported the negative seed transmission of Tobacco streak virus in groundnut and sunflower. Such type of variations in seed transmission exists as it depends on number of factors, and further researches are required to confirm the seed-transmitted nature, which will form the basis in the studies on breeding, biotechnology, etc., disciplines. References Abo ME, Alegbego MD, Sy AA (2004) Evidence of non-transmission of rice yellow mottle virus (RYMV) through rice seed. Tropicultura 22:116–121 Adams DB, Kuhn CW (1977) Seed transmission of peanut mottle virus. Phytopathology 67:1126– 1129 Agarwal VK, Sinclair JB (1987) Principles of seed pathology vol I & II. 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Phytopathology 51:546–550 Bharatan N, Reddy DVR, Rajeswari R, Murthy VK, Rao VR (1984) Screening of peanut germplasm lines by enzyme linked immunosorbent assay for seed transmission of peanut mottle virus. Plant Dis 68:757–758 Blattny C, Osvald V (1954) Pfenos viros chmele (Humulus lupulus L.) na potomsto semenem Preslia, (Prague) 26:1–26 Boccardo G, Lisa V, Milne RG (1983) Cryptic virus in plants. In: Company RW, Bishop DHL (eds) Doublestranded RNA ‘viruses’. Elsevier, Amsterdam, pp 425–430 Boccardo G, Milne RG, Luisoni E, Lisa V, Accotto GP (1985) Three seedborne cryptic viruses containing double stranded RNA isolated from white clover. Virology 147:29–40 Bock KR, Kuhn CW (1975) Peanut mottle virus. No 141. In: Descriptions of plant viruses. Commonwealth Mycological Institute and Association of Application Biologists, Kew, Surrey, England, pp 4 Bos L (1977) Seed-borne viruses. In: Hewitt WB, Chiarappa L (eds) Plant health and quarantine in 96 international transfer of genetic resources. CRC Press, Cleveland, pp 36–39, 346 pp Bos L, Maat HZ (1974) A strain of cucumber mosaic virus seed transmitted in beans. Neth J Plant Pathol 80: 113–123 Bowers GR Jr, Goodman RM (1979) Soybean mosaic virus: infection of soybean seed parts and seed transmission. Phytopathology 69:569–572 Broadbent L (1965) The epidemiology of tomato mosaic. XI. Seed-transmission of TMV. Ann Appl Biol 56:177–205 Caldwell J (1934) The control of virus diseases of the tomato. J Ministry Agric 41:743–749 Caldwell J (1962) Seed transmission of viruses. Nature 193:447–459 Carroll TW (1972) Seed transmissibility of two trains of barley stripe mosaic virus. Virology 48:323–336 Carroll TW (1981) Seed borne viruses: virus-host interactions. In: Maramorosch K (ed) Plant diseases and vectors. Ecology and epidemiology. 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J Agric Res 67:305–327 6 Detection of Plant Viruses in Seeds Abstract The methods for detecting seed-transmitted virus infection should be rapid, reliable and sensitive besides being simple to achieve results. This has been a challenge since the advent of the discipline of plant virology over 100 years ago, and a great variety of methods have been developed since that time. Based on biological, serological and molecular tests, the virus and viroid diseases are diagnosed. In biological tests, a close and careful observation on the seed morphology in certain cases gives a tentative indication of the presence of virus (es). Grow-out test also helps in seed-borne virus diagnosis. Among serological tests Ouchterlony, ELISA and its variants, dot-immunobinding assay, TBIA, and Immunosorbent electron microscopy are widely used for virus detection. Among antibody-based detections, and Polymerase chain reaction (PCR) and its variants, (realtime PCR, RT-PCR, IC-PCR, IC-RT-PCR, multiplex PCR), are extensively practice. Protocols have been implemented in the field using portable realtime PCR machines for same-day, on-site results; cDNA probes which are labelled with radioactive markers or nonradioactive markers are used for diagnosis of seed-borne virus and viroid diseases. Array technology has revolutionised the world of viral diagnosis because of its efficiency in screening a large volume of infected seed samples in a single array plate or reaction. 6.1 Introduction The movement of seed material from one country to another poses a serious problem, as it introduces new diseases to that area. The presence of viruses in true seed is often established by raising seedlings from suspected seed lots and observing symptoms on them. This test is time-consuming and of little use if one requires quick information. Hence, methods for detecting seed-transmitted virus infection should be rapid, reliable and sensitive besides being simple to undertake and give clear-cut results. As some of the devastating virus diseases are seed-transmitted, quick and thorough evaluation of the seed lot is essential at the quarantine stations. K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 6, © Springer India 2013 101 102 6.1.1 6 Seed Health Testing It is well known that about 90% of all the food crops grown are propagated by seed. Seeds are both vehicles and victims of disease. The significance of transmission of plant diseases through seeds was realised long ago. It is a well-known fact that infected or contaminated seed is a primary source of inoculums for a large number of destructive diseases of important food, fodder and fibre crops (Neergaard 1977b). Besides affecting the crop yields, the seed-borne pathogens affect the nutritive quality and value of the seed, leading to trade barriers. In some cases infected seeds are the only source of initial inoculums in the field. One of the most relevant aspects about controlled health quality seed is referred to the detection methods used against different pathogens. International Seed Testing Association (ISTA) took the leading role in studying seed-borne diseases. In 1924, people were interested in seed’s gene purity and germination aspects. Later then, thanks to the efforts made by Dr. L.C. Doyer publishing the first manual for the determination of seed-borne diseases and being the first director of the PDC (Plant Disease Committee), the importance of seed-transmitted diseases was turned into first priority, but detection methods were applied according to the criterion of each laboratory, so their results were not comparative. So, in 1957, PDC established a comparative health testing programme and standardised applied methods. The complexity for comparison of these methods made a considerable step back on the publication of working sheets (protocols). ISTA-approved methods became rules. By 1999, only 64 protocols were being compared, and only 14 of them were accepted as rules. As the solutions proposed by ISTA did not satisfy the needs of international seed industry, this industry, in 1994, organised the International Seed Health Initiative for Vegetables (ISHI-Veg) chartered by the vegetable seed industries in the Netherlands and France. The initiative was soon joined by the seed companies in the United States, Israel and Japan. This group represents the production of Detection of Plant Viruses in Seeds over 75% of the world’s vegetable seed supply (Sheppard 1999; Sheppard and Wesseling 1997). ISHI published many protocols, organised by crop. For horticultural crops, the ISHI-Veg, the Seed Health Testing Methods Reference Manual (van Ettekoven 2002) and this one included 21 different protocols with diverse status ranking. 6.1.1.1 ISHI-Veg Status 1. An ISHI-validated reference method is a method that has been through the ISTA/ISHI comparative testing process and is being published as ISTA working sheet. 2. An ISHI reference method is a method that has been through ISHI comparative testing and has been reviewed by the ISTA/ISHI reviewers for publication as ISTA working sheet. 3. An ISHI-accepted method is a method that is commonly used by the seed industry for determining seed health. The method has been published in a journal, as a working sheet, and/or is publicly available for comparative testing and commonly in use by the seed industry. The method is in comparative testing or aspects of the test are being tested for inclusion or deletion from the method. 4. An ISHI-reviewed method is a method that is publicly available or published, documented and prioritised by the ITG, but not subjected to a comparative test. The method usually is accepted by other agencies or groups. After the second ISTA–PDC symposium, a Joint ISTA/ISHI Guidelines for Comparative Testing of Methods for Detection of Seed Transmitted Pathogens was edited. Through this combined effort of many laboratories, organised under this alignments, the ISTA developed the Manual of Seed Health Testing Methods that has two sections: (1) – validated test methods and (2) – peer-reviewed methods. The transition process from working protocol to ruled method is long and laborious, so working protocols can be considered as adequate methods for seed analysis. Nowadays, it is common that technological advances may become 6.1 Introduction obsolete and research institutions, universities and also individual scientists/researchers have published protocols for the easy access to the research scientists (Duncan and Torrance 1992; Foster and Taylor 1998; Dijkstra and de Jager 1998; Hull 2004; Nayudu 2008; Ahlawat 2010). At most of the germplasm centres, the cultivars of some crops like peas, peanuts, beans and potatoes are generally contaminated with seed-transmitted viruses (Hampton and Braverman 1979; Hampton et al. 1982; Bharathan et al. 1984; Alconero and Hoch 1989). For example, from survey of USDA Phaseolus germplasm, Klein et al. (1988) found BCMV in accessions of 10 Phaseolus species. This is a serious problem in Phaseolus accessions since approximately 60% of the germplasm was contaminated. Even in Canada, more than 14,000 pea germplasm lines maintained at the gene bank of the Crop Improvement Research Centre, University of Saskatchewan found contaminated with PSbMV were discarded (Kartha and Gamborg 1978). In the United States, seed-transmitted CMV was introduced through international exchanges of bean germplasm, despite the fact that the plants grown in beanproducing areas of the Pacific Northwest were virus-free (Davis et al. 1981). Hence, it is imperative that all the incoming seed and plant material from various countries should be subjected to strict quarantine regulations before releasing for use in breeding work or for cultivation. Any negligence in examination of imported seeds and planting stocks may lead to introduction of several unknown viruses into the country. Intensive researches have been made in different parts of the world to evolve simple diagnostic techniques. The Danish Government Institute of Seed Pathology established in 1967 at Copenhagen by the Danish International Development Agency (DANIDA) has been doing laudable service in the field of seed pathology by training plant pathologists from developing countries and assisting in programmes on education in the field of seed health certification and quarantine methods (Albrechtsen 2006). 103 From the earlier Chapter 2, it is clearly known that the economic losses caused by viruses are very high and hampers agricultural yields. In India, several government and nongovernment organisations import germplasm for crop diversification and crop improvement. The government of each country should restrict the entry of the viruses through seed which were not recorded in their respective country. Germplasm of different crops are being exchanged between countries for their breeding programmes and also for distribution to the farmers directly for agriculture with the view of introducing new crop or variety which is high yielding. In this regard National Bureau of Plant Genetic Resources (NBPGR) is the nodal agency in India for quarantine processing of imported germplasm. Similarly, Directorate of Plant Protection, Quarantine and Storage, Ministry of Food and Agriculture, New Delhi (India), is also involved in intercepting the new pests and diseases carried in germplasm received from different countries. Inevitably the large-scale introduction of germplasm involves a risk of accidentally introducing seed-transmitted pathogens. In particular diseases that are often symptomless such as those caused by viruses pose a great risk. In order to avoid this risk, sensitive and reliable testing procedures are required to detect the viruses in seeds to ensure that the introduced seed material is free from viruses of quarantine importance. Large number of virus diseases that were intercepted at NBPGR stations, which were not known in India, were detected (Khetarpal et al. 2001; Prasada Rao et al. 2004; Chalam et al. 2005). Harvest from only virus-free plants are being released to the intenders. 6.1.2 Low Seed Transmission/ Symptomless Carriers In some crops, very low rate of seed transmission has caused enormous crop losses due to secondary spread. To quote a few examples, LMV in lettuce seed even at a reasonably low level of infection caused a high disease incidence 104 due to secondary spread through aphids. If seed transmission exceeds 0.1%, control of this virus will not be satisfactory (Broadbent et al. 1951; Zink et al. 1956). The number of infected lettuce seedlings that emerged per 0.4 ha at 0.1% rate would be 200/0.4 ha or 20,000 infected seedlings/40 ha (Greathead 1966). Similarly, low seed transmission (0.3%) of PMV in peanut was found to be the primary source for secondary spread (Demski et al. 1983). Maize/sweet corn is the most extensively grown crop; low levels of seed transmission of viruses have been documented in several reports. Maize dwarf mosaic virus (MDMV) was transmitted at a low level, with only one seed transmission in 22,189 seedlings in one study (Mikel et al. 1984), one seed transmission in 11,448 seedlings in another study (Hill et al. 1974), and two transmissions in 29,735 seedlings in a third study (Williams et al. 1968). Similarly, Zhu et al. (1982) reported 14 seed transmissions of MDMV-B, now known as Sugarcane mosaic virus-MDB, in 22,925 seedlings, and Shepherd and Holdeman (1965) found somewhat higher transmission of MDMV, with 17 transmissions from 9,485 seeds, but 14 of these came from one lot of 3,163 seeds. Jensen et al. (1996) found 21 seed transmissions of Maize chlorotic mottle virus in over 42,000 seeds tested. During 2001, only 3 plants of sweet corn were infected with High plains virus out of 38,473 seedlings, which is again seed transmitted at low frequency. Some of the viruses and viroid diseases will not be causing characteristic and clear symptoms. The example is of the cryptic viruses in different crop plants (Boccardo et al. 1983, 1987). Another important example is of Avocado sunblotch viroid, which has high percentage of seed transmission. The root stock and scions are symptomless and remain undetected (StaceSmith and Hamilton 1988). In olive plants, latent infection is noticed with Olive latent virus-1 (Necrovirus genus) and Cherry leaf roll virus (Nepovirus genus), which are seed transmitted (Saponari et al. 2002). There is, therefore, an urgent need for developing sensitive, reliable and quick tests for detection of seed-transmitted viruses which may 6 Detection of Plant Viruses in Seeds be present at a low percentage and also the hosts may be symptomless carriers. The tests will be quite helpful at quarantine centres as well as field trials. Appropriate control procedures can only be applied effectively if the virus is correctly identified and distribution in an area or crop is known. In advanced countries, identification of seed-transmitted viruses is well attended by the virologists. However, due to meagre facilities in a majority of the underdeveloped countries, there is a great risk of introducing the virus (es) into the countries. Diagnosis is mostly based on the reactions produced on indicator hosts where only greenhouse facility is available. By serological tests, one can easily identify the virus in a short time involving meagre space. Tremendous improvements have been made in developing new techniques in recent years. However, one should not exclusively depend only on serological tests, since in certain cases even with high antibody titres, false-positive serological reaction may be obtained between different viruses possessing the same antigenic sites of virus particle. For instance the viruses like BCMV, Bean yellow mosaic, CpAMV, SMV and WMV belonging to the Potyvirus genus are serologically interrelated (Martyn 1968a; Matthews 1970). Under such circumstances, one should guess the identity of the causal agent, but confirmatory tests including recently developed molecular assays and electron microscopy tests are essential for precise diagnosis. The success of serology, however, depends on the availability of antisera against a wide range of plant viruses, and one can purchase the antisera from the American Type Culture Collection centre at Rockville, Maryland, and also as gratis samples from persons who have produced the antisera. Logical and systematic 10-step approach suggested by Bos (1976) is of immense use in diagnosing virus diseases. Intensive researches in these directions have led to the development of a number of biological, physical, serological and molecular methods for detecting the presence of virus infections. The value of plant health certificates issued for seed is entirely dependent on the testing procedure applied. 6.2 Biological Methods 6.2 Biological Methods 6.2.1 Visual Examination A close and careful observations on the seed morphology in certain cases gives tentative indication of the presence of virus(es). Seed morphological abnormalities like shrunken shape, shrivelled seed coats, discolouration, size, weight, cracking, and necrotic spots or bands are the common morphological criteria used for detecting the presence of viruses in seeds (Fig. 6.1). The extensive studies carried out on seed coat morphology and virus transmission by Hobbs et al. (2003) revealed that the seed coat mottling in soybean caused by Bean pod mottle virus (BPMV) and Soybean mosaic virus has resulted into infected plants when grown. Even the seeds collected from certain crop plants infected with viruses are small, poorly filled with disfigured seed coats and are lighter in weight. For example, in seeds of cowpea and barley infected with Cowpea mosaic virus and Barley false stripe virus, respectively, maximum virus transmission was associated with small shrivelled seeds (Phatak and Summanwar 1967). Similarly, small-sized peanut seeds showed maximum seed transmission of PMV (Paguio and Kuhn 1974). The available information on the morphological seed abnormalities due to different viruses are furnished in Table 6.1. 105 There are few examples wherein removal of small shrivelled and discoloured seeds minimised the risk of introduction of seed-transmitted viruses in the case of three important leguminous food crops (Jeyanandarajah 1992) and mottled and non-mottled seeds in the case of soybean due to Soybean mosaic virus (Khetarpal et al. 1992; Parakh et al. 1994, 2005; Chalam et al. 2004). It is also known that the external morphology of seed should not be considered alone as a major criterion for identification, since in some virus hosts, it is misleading. For example, Porto and Hagedorn (1975) considered mottling of soybean seeds to be due to hereditary or environmental factors and not due to viral infection. Even Koning et al. (2003) reported that Soybean mosaic virus (SMV) and also Phomopsis spp. (fungus) cause seed coat mottling, and this seed coat mottling was inconsistent across cultivars and years. Pacumbaba (1995), Bazwa and Pacumbaba (1996) and Andayani et al. (2011) have also reported no correlation between mottling of soybean seeds and SMV transmission. Similarly, seed coat cracking in pea seeds, was not due to Pea seed-borne mosaic virus (PSbMV) infection (Stevenson and Hagedorn 1970). Even Chalam et al. (2004), while working with SMV in soybean, have observed that many a time, mottled soybean seeds are found to be free from SMV and healthy looking seeds were found to be infected when tested by ELISA. Thus, seed abnormalities are not necessarily being implicated as seed Fig. 6.1 Morphological abnormalities due to seed-borne viruses 106 6 Detection of Plant Viruses in Seeds Table 6.1 Morphological seed abnormalities correlated with infection of some seed-transmitted viruses Plant species Arachis hypogaea Virus Peanut mottle Cicer arietinum Pea seed-borne mosaic Cucumis melo Cucurbita pepo Glycine max Glycine max Squash mosaic Squash mosaic Bean pod mottle Soybean mosaic Seed abnormality Small size, discoloured seed coat Darkening of the seed coat Poorly filled, deformed Poorly filled, deformed Seed coat mottling Discoloured seed coat Glycine max Soybean stunt Discoloured seed coat Hordeum vulgare Barley stripe mosaic Lactuca sativa Lens culinaris Lupinus luteus Lettuce mosaic Pea seed-borne mosaic Bean yellow mosaic (narrow-leaf lupin strain) Cucumber mosaic Tobacco mosaic Ring spot Mung bean mosaic Small size, shrivelled, lightweight Lightweight Necrotic rings Large or sharp edged Middleton (1944) Middleton (1944) Hobbs et al. (2003) Koshimizu and Iizuka (1963), Ross (1963), Phatak (1974), Almeida (1981), Taraku et al. (1987), Hobbs et al. (2003), Koning et al. (2003), and Domier et al. (2007) Koshimizu and Iizuka (1963), Ross (1963), Van Niekerk and Lombard (1967), Kennedy and Cooper (1967), Phatak (1974), and Laguna et al. (1987) Inouye (1962) and Phatak and Summanwar (1967) Ryder and Johnson (1974) Coutts et al. (2008) Blaszczak (1963) Large size Necrotic, blackened Small, lightweight Wrinkled, greenish grey Troll (1957) Broadbent (1965) Marcelli (1955) Phatak (1974) Wrinkled, green grey seed coat Seed coat splitting, mottling, irregular depressions, discoloration Shrunken, reduced size Small size, lightweight Shrunken seed coat Discoloured and Crinkled seed coat Necrotic rings and line markings on seed coat malformation, reduced size and splitting. Shrunken, shrivelled Mottling of testa Bos and Van der Want (1962) and Stevenson and Hagedorn (1969) Hamptom and Mink (1975), Kumar et al. (1991), and Coutts et al. (2008) Lupinus luteus Lycopersicon esculentum Nicotiana tabacum Phaseolus aureus (syn. Vigna radiata var. radiata) Pisum sativum Pea early browning Pisum sativum Pea seed-borne mosaic Sorghum vulgare Triticum aestivum Vicia faba Vicia faba Sugarcane mosaic Brome mosaic Broad bean stain Bean yellow mosaic Vicia faba Pea seed-borne mosaic Vigna unguiculata Vigna unguiculata Cowpea aphid-borne Blackeye cowpea mosaic infection albeit is an indication. Definitive tests are essential to relate seed abnormalities with infection. Seed collection from a healthy crop by Reference Kuhn (1965) and Paguio and Kuhn (1974) Coutts et al. (2008) Edmunds and Niblett (1973) Von Wechmar et al. (1984a) El-Dougdoug et al. (1999) Kaiser (1973) and El-Dougdoug et al. (1999) Coutts et al. (2008) Phatak and Summanwar (1967) Jeyanandarajah (1992) visual judgement alone does not yield absolute disease-free seed, since certain plants that carry the virus exhibit no symptoms of infection. 6.2 Biological Methods 107 Fig. 6.2 Detection of BCMV–BlCM by growing-on test. Two-leaf stage seedlings showing mosaic and banding symptoms (Source: Udayashankar et al. 2010; H.S. Prakash) 6.2.2 Grow-Out Test It involves germinating the infected seeds in sterile soil under glasshouse conditions either in Petri dishes or in moist paper towels in controlled conditions of light and temperature. Emerging leaves of the seedlings are observed for characteristic visible symptoms develop according to the virus–host combinations. The symptoms could be mild mottle or mosaic on cotyledons or primary leaves. Period of incubation varies from crop to crop and virus to virus. Germinating and growing seed samples in glasshouse for virus detection is also known as sand bench germination test. For instance, LMV in lettuce was detected by this test after 16–21 days when seedlings exhibit clearcut mosaic symptoms (Rohloff 1967). Similarly, Hampton (1972), Naim and Hampton (1979) and Hampton et al. (1981) have detected pea fizzle top (Pea seed-borne mosaic virus) by growing seeds under glasshouse conditions. Alfalfa mosaic virus was detected in 7-day-old alfalfa seedlings and also even in the cotyledonary stage (Tosic and Pesic 1975). Phatak (1974) observed typical symptoms of BSMV in 1-week-old barley seedlings. He also developed a ‘blotter test’ in Petri dishes for nepoviruses in Petunia violacea and was able to detect TRSV infection by growout test as well as by sap inoculation. From Karnataka (India), Puttaraju et al. (1999) have conducted grow-out test for detection of BCMV in French bean seed collections. Similarly from NBPGR, New Delhi, Dinesh Chand et al. (2004) detected Blackgram mottle virus in blackgram by grow-out test. Effectiveness of this test was also observed by Pena and Trujillo (2006) and Udayashankar et al. (2010) while working with cowpea and French bean seeds against different seed-transmitted viruses (Fig. 6.2). Chowdhury and Nath (1983) developed a technique in which 48-h young seedlings of blackgram were inoculated with Blackgram mottle virus. The virus inoculum was prepared in 0.2 M phosphate buffer and young healthy seedlings that were 48 and 72 h are placed in the virus extract with Carborundum and shaken for 40–50 times. Seedlings were washed gently by dipping in water and transplanted in pots. After 21– 28 days, the symptoms will be noticed if the virus transmission has taken place. The seedling inspection method of detecting virus-infected seed lots is unsatisfactory for some virus–host combinations under certain environmental conditions since seedlings from infected 108 seeds fail to exhibit symptoms (McKinney 1954; Afanasiev 1956; Hampton et al. 1957; Mac Withey et al. 1957; Lister 1960; Jeyanandarajah 1992). For example, Lister (1960) observed latent infections in one or more hosts with three seedtransmitted viruses and the growth of seedlings was similar in both healthy and diseased lots. Subsequently, several viruses producing latent or semi-latent infections in their hosts were found transmitted by seed which escaped detection (Frosheiser 1964; Hampton 1962, 1963, 1967). Even in red clover (Trifolium pratense) infected with Clover yellow mosaic or White clover mosaic viruses, detection of virus by assaying on host plants was not possible between July and September (Hampton and Hanson 1968). At Montana (USA), under glasshouse conditions, many plants did not show distinct symptoms, even though serological tests confirmed their infection. Moreover, in some crops physiological spotting of the leaves tend to be confused with symptoms associated with virus infection (Hamilton 1965). Hampton et al. (1957) found light intensity as a critical factor for the symptom expression of Barley stripe mosaic virus and that when plants are grown under 10,000 fc, the diseased plants exhibit clear-cut symptoms. In Sri Lanka, Jeyanandarajah (1992) could not detect Blackeye cowpea mosaic, Cucumber mosaic and Bean common mosaic viruses by grow-out tests in cowpea, winged bean and mung bean hosts. Under such conditions, large number of seedlings need to be tested to derive precise recommendations on seed transmission. The major demerits of grow-out test are as follows: 1. Large space in the form of controlled room or a greenhouse/glasshouse is required. 2. Long time for standardisation to record reproducible symptoms. 3. Positive result may not indicate involvement of virus(es). 4. Negative result may not mean the absence of virus, since the less virulent strains and latent infections do not produce external symptoms. Embryo culture technique is quite useful for quick detection of viruses in seed lots and this technique involves excision of embryo and its 6 Detection of Plant Viruses in Seeds culturing on artificial medium. Besides providing an improved ‘growing-on’ system, culturing embryos on synthetic media allows the virus to develop a certain detectable concentrations not only for the expression of symptoms of newly formed leaves but also for detection by other methods. For the first time, Crowley (1957) employed this technique to study the nature of seed transmission. Examples wherein this technique was successfully employed for seed-transmitted viruses detection are Runner Bean mosaic virus in runner bean (Mishra et al. 1967), BCMV in urd bean (Agarwal et al. 1979), mosaic virus in berseem (Fugro 1980) and LMV in lettuce seeds (Ramachandran and Mishra 1987). More information on embryo culture is provided in a review by Mishra and Ramachandran (1986). 6.2.3 Indicator Hosts In order to increase the reliability of growingon tests and to eliminate symptomless infections especially for quarantine work, infectivity tests are appropriate. In this test, the indicator plants after mechanical inoculation produce characteristic either systemic or local lesion symptoms, which help in identification of the virus(es) and sometimes even the latent or mixed infections. The local lesion hosts are faster to react although they may not be as sensitive as systemic hosts (Green 1991); Albrechtsen (2006) have provided extensive information on indicator hosts. The most commonly used technique is the direct seed test, which includes examination of dry seed and an infectivity assay. In this test, suspected abnormal seeds are soaked in an aqueous medium and then triturated in a mortar with pestle or in a mixer or a Wiley mill. The slurries produced are applied to the abrasive dusted indicator plants and their leaves rinsed with water immediately after inoculation. The inoculated as well as the subsequently developed leaves are observed for symptom development. The symptoms like local lesions (hypersensitive reaction) or systemic reactions such as vein clearing, various types of mosaics, line pattern, chlorosis, necrosis and stunting are recorded on the inoculated test 6.3 Physical Methods plants. Single-seed or composite-seed samples may be processed by the infectivity test. Sometimes viruses like Tomato mosaic virus in tomato can be detected by inoculating the seed washings to indicator hosts like N. glutinosa, N. tabacum var. Xanthi nc. and P. vulgaris var. Scotia (Phatak 1974); hypersensitive cultivars of tobacco, bean and cucumber cotyledons for TMV (Shmyglya et al. 1984); Nicotiana tabacum ‘Xanthi NN’, a local lesion host for detection of tobamoviruses carried on tomato seeds (Grimault et al. 2012); bean for BCMV (Quantz 1957) Chenopodium amaranticolor, C. quinoa, C. murale, Phaseolus vulgaris and Vigna sinensis for majority of the nepoviruses (Lister and Murant 1967); tobacco for Tobacco rattle virus (TRV) (Sol and Seinhorst 1961); pea for Pea early browning virus (Van Hoof 1962); C. amaranticolor and pea cv. ‘Perfection’ for PSbMV (Hampton et al. 1976); Bountiful and Top Crop beans for AMV (Frosheiser 1970, 1974); C. quinoa for LMV (Rohloff 1962; Pelet 1965; Marrou et al. 1967; Phatak 1974; Kimble et al. 1975); and tomato and Solanum berthaultii for PSTVd (Yang and Hooker 1977; Singh 1984; Singh et al. 1991b). These indicators are now routinely used in different countries. Hampton et al. (1978) have listed diagnostic hosts for the identification of mechanically transmitted legume viruses which are now being used in most of the laboratories. Boswell and Gibbs (1986) have assembled virus identification data exchange, comprising the list of infected species and description of virus-associated symptoms. To accommodate more samples and minimise space, detached leaf technique which involves incubation of detached leaves on moist filter paper in Petri dishes at 30–32ıC under artificial illumination of ca.1000 lx for 3–4 days is also used. Quantz (1962a, b) employed this technique for the detection of BCMV by using the bean cultivar ‘Top Crop’. Detached leaves of N. glutinosa and N. tabacum var. Xanthi nc have been used for the detection of Tomato mosaic virus in tomato seed (Phatak 1974). For some viruses, where the titre values are very low in dry seed coats, the inoculum has to be prepared by removing the seed coat (Quantz 1962a, b; Gilmer and Wilks 1967). Both these techniques require modifica- 109 tions in accordance with the seed material as well as the virus strain. Any tentative diagnosis from the reaction of host plants requires confirmation by the serological or other authentic tests. Problems associated with virus indexing based on the symptomatology are many and variable. Some such problems are (1) symptoms induced by a specific virus may be mimicked by another virus; (2) symptoms vary with plant species, crop variety age and physiological condition of the test plant; (3) indexing based on symptomatology is time-consuming and expensive with respect to manpower and plant growth facilities with large number of test samples. 6.2.4 Biological Properties Up to 1975, virologists used to depend on biological properties like thermal inactivation point, dilution end point and ageing in vitro for virus identification besides host range, vector transmission and particle morphology. With technological progress in virology, the biological properties gradually lost their weightage. For instance, Francki (1980) summarised the ranges of the above properties in 20 taxonomic virus groups. His graphical illustrations clearly show that although these data may differ for distinct groups, there is widespread overlap between many of the groups. Therefore, these properties should not be exclusively considered for virus characterisation, identification and classification. 6.3 Physical Methods 6.3.1 Inclusion Bodies The morphology and structure of inclusion bodies are highly characteristic of the infecting viruses in spite of the host variability which greatly aid in diagnosis. The plant virus subcommittee of the International Committee on Taxonomy of Viruses (ICTV) listed ‘types of inclusion bodies’ as one of the criteria to be used in plant virus classification (Harrison et al. 1971; Matthews 1982). The inclusions contain either virus particles and/or other products of 110 6 Detection of Plant Viruses in Seeds Table 6.2 Inclusions in different virus groups of seed-transmitted viruses Virus genus Alfamovirus Bromovirus Carlavirus Closterovirus Cucumovirus Comovirus Hordeivirus Potexvirus Potyvirus Sobemovirus Tobamovirus Tombusvirus Tymovirus Inclusions Hexagonally packed layers as whorl in cytoplasm vacuole Granular and crystalline in cytoplasm and nuclei Some banded, most paracrystalline arrays with cell components Cross-banded masses in phloem Crystals in vacuole but usually no inclusions Crystalline arrays of virus particles Particles in cytoplasm and nuclei Stranded and fibrous masses of LIC Cylindrical, pinwheels Crystalline aggregates, hexagonal Crystalline, monolayer Multi-vesicular body Flask-shaped double membrane viral genome with occasionally modified cell constituents. Most of these inclusions are located in the cytoplasm of mesophyll, epidermal and hair cells and sometimes in vacuoles, nuclei and phloem cells depending on the viruses. The use of these inclusions for the rapid identification of seed-transmitted virus diseases plays an integral role at research and plant quarantine stations where adequate facilities are not available for tentative virus identification. The inclusions can easily be observed by light microscopy in epidermal strips on the underside of the leaves, but easily from main veins, petioles or young herbaceous stems because of larger size and regular shape than those of laminar leaf parts and in leaf hairs. Several selective stains such as trypan blue, azure A, Luxol brilliant green, phloxine and calcomine orange have been used for distinguishing inclusions from normal cell components, even though they may have similar morphology and locations. With trypan blue (0.5% in 0.9% NaCl) staining, the nuclei turn blue quickly and inclusions stain strongly. For some inclusion bodies, aqueous phloxine (1%) is better and stains bright red. The azure A stain imparts a red to magenta colour to inclusions high in ribonucleoprotein and does not stain other type of proteins. The Luxol brilliant green–calcomine (O–G) combination stain imparts green colour to inclusions containing different types of protein. Group characteristic C C C C C C C C C C C C Diagnostic at group level C C C C C C C C C C Phloxine staining proved extremely simple and rapid for fresh tissues (Christie and Edwardson 1977, 1986). Viruses within groups are now known to induce the same or closely similar type of inclusions (Fenner 1976a, b; Martelli and Russo 1977; Christie and Edwardson 1977, 1986; Matthews 1982; Edwardson and Christie 1986). In general, the virus identification should not be dependent exclusively on inclusion morphology, since the viruses of different groups also induce similar type of inclusion bodies, and hence, virus identification needs consideration of other characteristics. Both light microscopy and electron microscopy are being used in studies on inclusion bodies induced by seed-transmitted viruses. Light microscopy with staining of test material helps in differentiating the virus-induced inclusions from each other and from cell organelles. Since the resolving power of light microscope is low, one can study only the location and morphology of the inclusions in the tissues. However, it is possible to study the internal structure and exact shape of the inclusion bodies and also the smaller inclusions which are not seen under light microscope by using electron microscope at higher magnifications. However, the usage of electron microscope for routine detections is costly. The characteristic inclusions found from each virus group are listed in Table 6.2. 6.4 Serological Techniques Based on inclusion body morphology and location, it is possible to identify several seedtransmitted viruses among various virus groups. For instance, when a diagnostician detects banded body inclusions, he/she will know that he/she is dealing with a carlavirus or a closterovirus or a potexvirus or a potyvirus. Similarly, in nepovirus infections the formation of inclusion bodies at early stage of infection, usually adjacent to the nucleus containing elements of endoplasmic reticulum, membranous vesicles and ribosomes are noted in ultrathin sections. Another characteristic feature of the nepovirus infection is the occurrence of virus particles, usually in single files with membrane-walled tubules, present in the cytoplasm of infected cells (Murant 1981a). One of the diagnostic characters of Potyviridae family recognised by ICTV is the formation of cytoplasmic inclusions called pinwheels, laminated aggregates, scrolls, etc. Seed-transmitted viruses of Potyvirus genus like Bean common mosaic, Blackeye cowpea mosaic and Water melon mosaic virus-1 induce cytoplasmic scroll inclusions, while Bean yellow mosaic and Lettuce mosaic viruses induce laminated aggregates. Both scrolls and laminated aggregates are induced by PSbMV, SMV and Turnip mosaic virus (Edwardson 1974). It is also possible to identify virus strains based on the shape of inclusion bodies, that is, hexagonal crystals have been found with TMV vulgare and Tomato mosaic strains; roundedplate inclusions with Sunn-hemp mosaic virus strain, Cucumber green mottle mosaic virus, orchid strain and Holmes rib grass strain; and angled-layer aggregates with aucuba strain and U5 strain (Warmke and Edwardson 1966; Warmke 1968; Milicic et al. 1968; Milicic and Stefanac 1971). Precise comparisons of the information derived from both light and electron microscopic techniques should be related with other virusinduced inclusions. 111 viruses. Depending on the particle morphology and size of the virus in test samples, a tentative virus grouping can be done. Especially, viruses with elongated particles are easy to assign as the size range of most groups of viruses is sufficiently distinct from others (Harrison et al. 1971; Hamiltan et al. 1981; Matthews 1982; Francki et al. 1991). The particle morphology of isometric viruses (25–30 nm in diameter) is also useful in preliminary separation, that is, round and smooth (cucumoviruses and bromoviruses); round and knobby (tombusviruses and tymoviruses); ovoid or imperfectly spherical (ilarviruses); and angular (nepoviruses, comoviruses and fabaviruses). The virus extraction procedure varies with host and routine tests require modification when infected seeds are processed containing inhibitors (Pena and Trujillo 2006). However, limited information is available on the application of this test. Gold et al. (1954) observed virus particles of BSMV in embryo and endosperm of individual seeds of barley and wheat. From Poland, Garbaczewska et al. (1997) have detected Soil-borne wheat mosaic virus particles and inclusion bodies in the tissue of 3-day-old rye seedlings. Similarly, Walkey and Webb (1970) observed virus particles of Cherry leaf roll virus and tubular inclusion bodies in homogenates of mature seeds of N. rustica. Phatak (1974) found virus particles of BSMV, BCMV and SMV in plumule extracts of barley, bean and soybeans, respectively, but failed to find LMV in lettuce seeds. In recent years, electron microscopy is also used successfully in the quarantine division of NBPGR, New Delhi, (India), while processing the imported germplasm of a number of crops from different countries and number of seed-transmitted viruses are also intercepted (Chalam et al. 2009b). The high cost of electron microscope makes limitation to most of the workers. However, if available it requires less time for diagnosis when compared to the germination test. 6.4 6.3.2 Serological Techniques Electron Microscopy An electron microscope is useful in conjunction with grow-out tests for several seed-transmitted Serological techniques are indispensable for the detection and identification of plant viruses. Many of these techniques have been developed 112 for the detection of seed-transmitted viruses and certain tests are highly specific and most promising in the regular diagnosis. The details of different serological techniques are covered in certain annual reviews and laboratory manuals (Van Slogteren and Van Slogteren 1957; Ouchterlony 1968; Ball 1974; Torrance and Jones 1981; Padma and Chenulu 1985a, b; Clark et al. 1986; Hampton et al. 1990; Bashir and Hassan 1998; Bos 1999; Webster et al. 2004; Akinjogunla et al. 2008; Rao and Singh 2008; Koenig et al. 2010). The serological tests involve physicochemical interactions between antisera produced to a plant virus in a warm-blooded animal and plant viruses (antigen). The antigen may be a protein or polysaccharide and capable of inducing an immune response when introduced into the warm-blooded animals. Most of the plant virus proteins are effective antigens and the antibodies to the viruses are produced via their immunological protective system. Antisera are produced by injecting purified virus preparations into rabbit, goat, chicken, mouse or other suitable animals. Several injections are administered intravenously or intramuscularly or a combination of both over a period of 4– 5 weeks. The virus preparations are emulsified with equal volumes of Freund’s incomplete adjuvant before injection. Test bleeds to check the level of antibody are made 3 weeks after the immunisation. Several large bleeds can be made without sacrificing the immunised animal when the antibody level is satisfactory. Such blood samples are allowed to coagulate for an overnight and kept in a refrigerator; then the serum may be decanted and clarified by lowspeed centrifugation. Small aliquots of serum are stored in 1 vol of glycerol/1 vol of antiserum or with 0.02% sodium azide or may be lyophilised. Serological reactions rely on the unique nature of the way in which the antigen and antibody molecules fit together. The combining sites on antibody molecules are complementary to different antigenic determinants on the surfaces of the homologous antigen. When antibody and antigen are mixed in suitable combinations, a visible precipitate results. Such a precipitate probably 6 Detection of Plant Viruses in Seeds results from the linking of antigen molecules by antibody, forming an aggregate and precipitates. 6.4.1 Monoclonal and Polyclonal Antibodies Polyclonal antibodies represent the antibodies from multiple clones or ß-lymphocytes and, therefore, bind to a number of different epitopes. Polyclonal antisera have served us well over the years. For the past 30 years, ELISA involving polyclonal antibodies are still the widely used method for practical plant virus detection. Nalini et al. (2006) and Puttaraju et al. (2004b) have produced polyclonal antibody against Bean common mosaic virus (BCMV) in French and Blackeye cowpea mosaic virus in cowpea and tested their application in seed health. Many commercial plant virus kits are designed based on that principle. The quantity, quality and affinity of antibodies specific for the antigenic determinants of an antigen often vary from animal to animal and even from different bleedings of the same animal. The hybridoma technology introduced by Kohler and Milstein (1975) has provided a revolutionary advances in the method of antibody production that eliminates many of the problems associated with polyclonal antisera. Hybridoma technology has the potential for producing an unlimited quantity of monospecific antibodies (monoclonal antibodies). Monoclonal antibodies (mAbs) potentially eliminate the variation inherent in whole animal systems of serum production, should have maximal usable sensitivity and are currently marketed for routine virus testing of seed stocks. The monoclonal antibodies have become extremely useful for detecting specific virus strains or viruses that are present in low concentrations in infected seed material. However, due to high cost of producing monoclonal antibodies, they are not as widely used as the polyclonal antibodies. Hybridomas are somatic cell hybrids made by the fusion of mouse spleen B-lymphocytes to mouse myeloma cells. The resulting hybrids (hybridomas) acquire the properties of antibodyproducing ability of the spleen lymphocyte and 6.4 Serological Techniques the ability to grow in culture as well and produce tumours of the myeloma cells. Antibodies produced by a single hybridoma clone are identical and are specific for a single antigenic determinant. They are, in essence, a chemically defined immunological reagent (Halk and De Boer 1985). Monoclonal antibodies offer several advantages over conventional polyclonal antiserum: (a) An unlimited quantity of antibody can be produced from a small quantity of antigen. (b) Pure antibodies specific for a single antigenic determinant can be obtained, even when impure antigen or antigen mixtures are used as the immunogen. (c) Hybridomas can be preserved by freezing in liquid nitrogen, thereby assuring a continuous supply of antibody over time. (d) Highly specific mAbs may reveal serological relationships between viruses and antigens that were previously unrecognised with polyclonal sera. (e) The use of monoclonal antibodies eliminates the qualitative and quantitative variability in specific antibody content in different batches of polyclonal serum (Halk and De Boer 1985). (f) It is possible for avoidance of background reaction and nonspecific detection of host proteins. Monoclonal antibody technology has a few disadvantages as well. The production and characterisation of a collection of mAbs for strain and epitope specificity can take over a year. By comparison, the preparation of mAbs is an expensive, complicated and labour-intensive procedure. Some mAbs do not form precipitin bands in double-gel diffusion tests and some classes or subclasses of immunoglobulins do not bind protein A. Some mAbs may be too specific for virus diagnosis, recognising only a rare isolate or a small group of isolates of a particular virus (Oxford 1982). In such cases, a mixture of several mAbs may be needed to detect all strains of a virus. While testing mAbs for specificity, tests should include as broad a range of isolates as possible (Martin 1985). Rishi and Dhawan (1994) have reviewed the production and applications of mAbs in plant virology. mAbs have been produced to more than 40 plant viruses in different taxonomic groups. The greater use of mAbs is to diagnose virus 113 diseases of vegetatively propagated crops such as potato, fruit trees, cassava and bulb crops and of seed-transmitted viruses (Halk et al. 1984; Wang et al. 1984; Huguenot et al. 1996; Bashir and Hampton 1996; Ouizbouben and Fortass 1997; Shang et al. 2011). Monoclonal antibodies to SMV, MDMV and LMV were also incorporated into a competitive radioimmunoassay (RIA) utilising antigencoated plastic beads and tritium-labelled antibody. The sensitivity of these assays for the three viruses was 10–50 ng/ml with purified virus (Hill et al. 1984). The mAbs produced against PVY and PVX performed well in dot-ELISA, reacting with similar virus and strain specific as in the direct or indirect forms of ELISA. Sensitivity of NCM-ELISA was similar to that obtained in the direct or indirect ELISA (Lizarraga and Fernandez-Northcote 1989). Monoclonal antibodies can serve as versatile and powerful immunochemical tools in problems involving viruses and their interactions with host or insect vectors. These antibodies in essence are chemically defined reagents capable of discriminating minute differences on the surface properties of biologically important macromolecules. Diagnostic applications are the use of many mAbs for seed-transmitted virus detection. Encouraging results have already been produced with some viruses, which indicate that mAbs may eventually replace polyclonal serum in large-scale diagnostic progress (Halk and De Boer 1985; Seddas et al. 2000). The mAbs also serve as analytical, functional and structural probes to help in elucidating the biochemical and physiological interaction of gene products and viruses within their host plants. Monoclonal antibodies will become more widely available in the future and permit a high degree of sensitivity in serological assays. (Van Regenmortel 1984, 1986; Martin 1987). Monoclonal and polyclonal antisera for many viruses are available commercially (Agdia, USA, A.C. Diagnostics Inc., USA; BIOREBA, Switzerland; Loewe diagnostics, Germany, and Neogen Europe Ltd., UK) and in individual laboratories. The mAbs are to be used to identify 114 the strainal variation or isolates with single amino acid differences in the viral coat proteins (Chen et al. 1997) and epitope profiling. Before the development of advanced techniques like ELISA, PCR, microarrays and other tests, virologists have tried flocculation tests, which were visible to the naked eye, in liquid media by using test tubes. Later slide agglutination and latex agglutination tests were employed in potato certification schemes (Hardie 1970; Seaby and Caughey 1977). The commonly used serological tests are grouped as follows: 1. Flocculation tests in liquid media 2. Immunodiffusion tests 3. Labelled antibody techniques 4. Immuno-electron microscopy 6 smaller quantities of virus compared to microprecipitin or immunodiffusion tests (Koenig et al. 1979). It can be carried out with lower concentrations of reactants than are required for precipitin tests. The results are expected within 15 min to 1 h. The method can detect one infected seed per 100 seeds or 1 g/ml of virus (Carroll 1979). This test has been routinely employed for largescale testing of potatoes, both in certification schemes and in screening disease resistance (Khan and Slack 1978, 1980). It was used to detect BSMV in germinated barley seedlings (Phatak 1974; Lundsgaard 1976) and SMV in soybean seeds (Phatak 1974). 6.4.2 6.4.1.1 Flocculation Tests in Liquid Media The combination between antigen and specific antibody can be demonstrated in a variety of ways. The most direct method is to observe the formation of an aggregate or a precipitate. In this the size of reacting antigen greatly influences the number of interacting antibody molecules required to produce an aggregation visible to the naked eye. Accordingly, two types of tests are recognised, namely, (a) Precipitation test and (b) agglutination test. (a) Precipitation Tests Precipitation is caused by the formation of a lattice of an antigen and antibody molecules that grow in complex size and become insoluble. This precipitate can be observed in tubes (tube test) or in drops on flat surfaces (microprecipitin test). This test was successfully applied in the early diagnostic period; PSbMV is detected in pea seeds by this method (Kumar et al. 1991). (b) Agglutination Test In this test the size of the reacting antigenic complexes is large and absorbed to larger particles such as red blood cells or latex or bentonite. These tests include slide agglutination and latex agglutination. Even the latex agglutination test is rapid, specific and sensitive. It can detect 100- to 1,000-fold Detection of Plant Viruses in Seeds Immunodiffusion Tests In immunodiffusion tests, the antibody–antigen reactions are carried out in gel instead of liquid. The reactants are allowed to diffuse and precipitation bands form wherever the reactants of suitable concentrations meet. These tests separate the mixtures of antigens and antibodies by their sizes, diffusion coefficient and concentrations. Thus, they are extremely useful for virus detection and identification from seed extract. There are two types of immunodiffusion tests: (1) single or simple immunodiffusion, in which one of the reactants diffuse into the gel, and (2) double immunodiffusion, where both the reactants diffuse into a gel. Diffusion can occur in one or two dimensions depending on whether the reaction takes place in tubes or plates. In immunodiffusion tests, isometric viruses will readily diffuse through the gels and without pretreatment, but the larger rod-shaped viruses have to be degraded into smaller units before they diffuse to give a reaction. They have to be broken down either by treatment with chemicals such as pyridine pyrrolidine, ethanolamine and guanine HCl or by detergents such as sodium dodecyl sulphate (SDS), sodium dibutyl napthalene sulphonate (Leonil SA) and sodium-N-methylN-oleoyl taurate (Igepon T-73) or by physical treatments such as freezing and thawing and ultrasonic vibrations. 6.4 Serological Techniques 6.4.2.1 Single Diffusion in Tubes In this method, one reactant usually antiserum is incorporated in the gel, while the other, the virus (antigen), is allowed to diffuse into it. The position of the leading edge of the precipitin band in the tube is proportional to the square root of time (Oudin 1952). This technique has restricted use in the detection of seed-transmitted viruses. However, this method was employed for testing BSMV in barley seed by Slack and Shepherd (1975) and BMV in wheat by Von Wechmar et al. (1984a). 6.4.2.2 Radial Immunodiffusion Test This technique is based on the principle of single diffusion in two dimensions. It is performed in Petri dishes, with the addition of antibody or antigen to the liquid gel before it sets. A well is then cut in the gel, the antibody or antigen is added and a halo or ring of precipitation is formed around the well if the reaction is positive. This method is also sensitive and rapid. It can detect as little as 1 g/ml of degraded virus. This test has been used for the detection of seedtransmitted Brome mosaic virus (BMV) in wheat seeds (Von Wechmar et al. 1984) and BSMV in barley (Slack and Shepherd 1975; Carroll 1979) and in mass indexing programme for the viruses detected in potato seed stock (Shepard and Secor 1969; Shepherd 1972). The drawback of this test is that it requires large amount of reactants. 6.4.2.3 Gel Double Immunodiffusion (Ouchterlony) Before ELISA invention, this test was the most widely used technique in plant virology research and in quarantine inspections. Thin agar or agarose gel layers are prepared on glass slides or in Petri dishes, and suitably arranged wells are cut to place the reactants. The antibody and antigen (infected seed extract) are added to wells and are allowed to diffuse towards each other. The reactants meet forming a white precipitin band when the optimal proportions of antigens and antibodies diffuse. The number of bands, size, shape and position of the precipitin bands are characteristic to the particular antigen–antibody system. 115 The double diffusion method is generally quite specific and reasonably sensitive. It can detect virus concentrations of 10–25 g/ml and can assay a single seed or parts of a seed. This method has been extensively used for the detection of several seed-transmitted viruses, namely, BSMV in barley embryos (Hamilton 1964; Slack and Shepherd 1975; Carroll et al. 1979a), Blackeye cowpea mosaic virus in cowpea and SMV in soybean hypocotyls (Lima and Purcifull 1980), TMV in tomato seeds (Phatak 1974), Brome mosaic virus (BMV) in wheat seeds (Von Wechmar et al. 1984a) and CMV in French bean (Padma and Chenulu 1985b). Occasionally in gel diffusion method, nonspecific precipitates develop when the antigen was prepared directly from the seed (Phatak 1974; Shrestha 1984). Similar problem was faced in the embryo detection of Broad bean true mosaic and Echtes Ackerbohnen mosaic viruses in broad bean seed (Cockbain et al. 1976). This problem can be overcome by clarifying the seed extract in low-speed centrifugation. Depending on the type of seed material and the virus, double immunodiffusion test was modified by many researchers. Carroll et al. (1979a) successfully modified this technique with filter paper discs serving as sero-reactant depots for the detection of BSMV in barley. The SDS antisera were used successfully by the Montana State Seed Testing Laboratory at Montana State University, Bozeman (USA), for the Montana Seed Growers Association and by plant virology laboratory, and this test has been largely responsible for significant reduction of BSMV in barley at Montana. Hamilton (1965) also modified this technique for rapid detection of BSMV even in a single embryo of barley. In this test, the infected seeds are soaked overnight at room temperature which results in their swelling considerably causing the embryos partially separated from the endosperm. Complete separation is achieved by ‘rolling’ a layer of seeds on a piece of aluminium foil with a metal rolling pin. The seeds are compressed by the force of rolling pins resulting in effective release of the embryos, which are then separated by washing. 116 In this test, quadrant type of Petri dishes is employed. A filter paper disc (Whatman No.1, 6.5 mm dia.) is placed in each of 100 holes, 1 mm deep and of 9 mm diameter in a Lucite ‘embryo crusher’, and individual embryos are transferred to each disc. The discs are sprayed with phosphate-buffered saline and a top plate with 100 pegs of 2 mm length and pressed for crushing embryos. The top plate is removed and the discs supporting the crushed embryos are transferred directly to serological test plates. The immunodiffusion system is incubated for 12–24 h and the number of embryos showing the immunoprecipitates indicates the seedtransmitted infection. This test is extremely useful in large-scale screening of commercial seed lots of barley and possibly other cereal crops too. 6.4.2.4 Direct Immunostaining Assay (DISA) The DISA was developed by Takeuchi et al. (1999) as a technique for detecting tobamoviruses on pepper seeds. In this technique, the seeds are used as the solid phase in an immunoassay by which infected seeds become stained. In the experimentation of DISA, pepper seeds were soaked in a polyclonal antiserum solution for 1 h at room temperature, washed in phosphate-buffered saline with addition of Tween-20 (PBST), incubated with anti-rabbit IgG–AP conjugate for 1 h at room temperature and washed with PBST and distilled water, before being incubated with nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP). After 30–60 min, infected seeds then stained dark purple, while healthy seeds did not. To test whether the detected virus was infective, stained seeds were homogenised and assayed on a hypersensitive tobacco cultivar, Nicotiana tabacum cv ‘Xanthi nc’ and Nicotiana tabacum cv ‘White Burley’ on which tobamoviruses (TMV, ToMV and PMMoV) produced differential symptoms in the form of necrotic local lesions or systemic mosaic (Green et al. 1987; Green and Kim 1991; Albrechtsen 2006). The DISA treatment influenced neither 6 Detection of Plant Viruses in Seeds the biological detectability of the viruses nor the seed viability (Green 1991; Albrechtsen 2006). 6.4.3 Labelled Antibody Techniques The sensitivity of detection of antigen–antibody reactions can be increased by labelling antibody that can readily be noticed or increasing sensitivity or by both. The use of labelled antibodies can help in detecting the virus in minute quantities (Fateanu 1978). Antibodies are generally labelled with ferritin, fluorescent dyes or radioactive iodine or enzymes to locate the viruses in tissue sections by light, fluorescent microscopes, autoradiography and electron microscopy (Ball 1974; Andres et al. 1978; Johnson et al. 1978). These techniques are being applied for virus detection in seeds. The various labelled antibody techniques are: 1. Fluorescent antibody technique (immunofluorescence) 2. Radioimmunoassay (RIA) 3. Enzyme-linked immunosorbent assay ELISA 4. Dot-immunobinding assay (DIBA) 6.4.3.1 Fluorescent Antibody Techniques Immunofluorescence techniques are the most widely used techniques for studying virus location and distribution within the tissues of host plants (Coons et al. 1942; Nagaraj and Black 1961; Tsuchizaki et al. 1978; Thornley and Mumford 1979) as well as in insect vectors (Sinha and Black 1962; Reddy and Black 1972). It is being presently employed for detecting and locating viruses in seed. This technique is perhaps the most specific and versatile among all the histochemical methods, since it provides a means of observing an antigen–antibody reaction by chemically linking a fluorescent dye such as fluorescein isothiocyanate (FITC) or rhodamine B to specific antibody molecules. Such labelled antibodies retain the ability to react specifically with their respective antigens and when viewed under a fluorescent microscope, the reaction site is relatively 6.4 Serological Techniques more common. There are two ways of performing the immunofluorescence techniques, that is, direct and indirect methods. (a) Direct Method: In direct method, antigens are mixed with FITC-labelled specific antibodies. The reaction gives a brilliant yellow-green fluorescence when examined under a microscope with an ultraviolet light source. Blackgram mottle virus was detected by using this method in embryo and germinated seeds of blackgram (Krishna Reddy and Varma 1994). (b) Indirect Method: In indirect method, the antigens are first allowed to react with unlabelled antibodies and then with FITC-labelled sheep antiserum prepared against gamma globulin obtained from animal species (rabbit) in which the virus-specific antiserum was produced. Phatak (1974) used the indirect method of immunofluorescence for the detection of SMV in germinated seeds of soybean. Free-hand sections of seed and squashes of plumule and small shoots are fixed for 5– 10 min in acetone, dehydrated with neutral phosphate-buffered saline and followed by treatment with SMV antiserum and subsequently with the conjugate for 30 min at 37ı C. Sections of virus-infected material under a fluorescent microscope appeared with more bluish-green fluorescence than healthy ones. Similarly, SqMV is detected in infected embryos, seedling protoplasts from cotyledons and microtome sections of dry embryos or seedlings by staining with fluorescein isothiocyanate – labelled and distributed in clusters of cells in epidermal, palisade and spongy mesophyll tissues. The virus is detected only in sections of cotyledons from 6-day-old seedlings (Alvarez and Campbell 1978). Fluorescent Staining of Nucleic Acids Vital stains (fluorochromes) such as acridine orange (A.O.) are gaining importance as compared to the stains used for light microscope because of their differential staining colours for nucleic 117 acids, namely, brick-red fluorescence for RNA and yellowish-green fluorescence for DNA (Hirai and Wildman 1963). Jagadish Chandra and Summanwar (1971) employed A.O. stain for the detection of CPMV in cowpea seeds of cv. P. 378. The seeds are soaked for 2 days. On the third day, the sections are taken from germinating embryos and stained with A.O. The stained tissues are viewed under a fluorescent microscope. Infected seed sections showed aggregation of red fluorescence material. This technique is also found successful for the detection of Wheat mosaic virus (Mayee and Ganguly 1974). 6.4.3.2 Radioimmunoassay (RIA) Antibodies labelled with radioisotopes have been employed in the detection of seed-transmitted viruses. These techniques are very sensitive and well suited to detect and quantify the virus infection in seeds. However, the application of these techniques requires strict safety precautions and highly trained personnel; the conjugate isotopes have a short shelf life and expensive equipment is required to assess the results. The techniques mostly applied for plant virus detection are as follows: (a) Solid-Phase Radioimmunoassay (SPRIA): There are two types of SPRIA. In a competitive type of assay, unlabelled antigen is first incubated in an antibody-coated polystyrene centrifuge tubes, and 125 Ilabelled antigen is added afterwards. The radioisotope-labelled antigen combines specifically with the antibody to give a test with sensitivity in nanogram quantities. An indirect assay can be performed in which known incremental quantities of unlabelled antigen is used. This test is simple, economical and sensitive and requires very small quantity of reactants (Ball 1973). This test was successfully employed to detect SMV in soybean seeds by Hill (1981). Even Diaco et al. (1985) used to detect SMV in seed by using two mAbs that recognised different epitopes on SMV. The assays used mAbs S2 as the capture antibody 118 and mAbs S1 as either the radiolabelled or enzyme-labelled detecting antibody and had a sensitivity of detection of ng/ml of SMV. During 1982, a method based on SPRIA is developed which detects all strains of SMV with equal sensitivity using antibody-coated polystyrene beads (Bryant et al. 1982, 1983). It consists of coating polystyrene beads (solid phase) with antibodies and addition of antigens into the tubes containing beads. The antigens (infected seed extract) bind to the solid-phase antibodies. Radioactive virus antibodies are then added to the antigens. The amount of radioactivity in the tube is directly proportional to the amount of virus antigen. If the virus is absent, there will be no binding sites for the radioactive antibodies and hence are removed during beads washing. The advantage of this method is its ability to detect virus antigen in the presence of extraneous seed material. (b) Radioimmunosorbent Assay (RISA): A simple and highly sensitive method RISA was described by Ghabrial and Shepherd (1980). It is a microplate method based on the principle of double-antibody sandwich (DAS) ELISA and follows essentially the protocol of the enzyme-linked immunosorbent assay (ELISA) with the exception that 125 I-labelled gamma globulin is substituted for the globulin enzyme conjugate. The 125 I-labelled gamma globulin is dissociated by acidification from the double-antibody sandwich, and the released radioactivity is proportional to virus concentration. This test is a valuable tool for viruses in which the ELISA values are too low to be dependable. Another advantage is that it also detects strains of viruses that differ serologically. This technique is effectively used for detection of LMV in very low proportions of lettuce seed lots (Ghabrial et al. 1982). (c) Electro-blot Radioimmunoassay (EBRIA): This technique combines the principles of serology with an analytical technique and is capable of detecting viruses occurring in extremely low concentration in plants or in seed. 6 Detection of Plant Viruses in Seeds This technique consists of sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) of the virus infected seed extract, electrophoretic transfer of protein bands to the activated paper by the electro-blot technique, subsequent probing of the viral coat protein bands by specific antiserum which was prepared against intact virus and detection of immune complex with 125 I-labelled protein A for autoradiographic and scintillation counter detection of virus– antibody immune complexes (O’Donnell et al. 1982; Shukla et al. 1983, 1991). 6.4.3.3 Enzyme-Linked Immunosorbent Assays (ELISAs) Techniques Enzyme immunoassays were introduced in medical diagnostics during the early 1970s and have distinct advantages over radioimmunoassays. As a result, the method known as enzyme-linked immunosorbent assay (ELISA) was adopted to plant viruses by Voller et al. (1976) and Clark and Adams (1977). Use of an enzyme as marker which is linked to the virus-specific antibody augments the detection of a specific reaction in several hundredfold relative to the visibility of an immunoprecipitate. The basic principle of ELISA is that the virus in test sample is selectively trapped and immobilised by specific antibody adsorbed on polystyrene or polyvinyl microtitre plates. Enzyme-labelled antibodies are then complexed with the trapped virus and detected by either colorimetric or spectrophotometric method by adding a suitable substrate. Removal of nonspecific substances is carried out by washing after each step of the procedure, leaving only specific immobilised reactors. Greater use of ELISA in plant virology has been in the large-scale testing and certification of seeds and vegetatively propagated plant materials of crops (Morrison 1999). Visually, it is often possible to distinguish the healthy sample reactions from the infected ones. Now ELISA has been increasingly used for detecting virus in seed due to the following major advantages: 6.4 Serological Techniques (f) Scope for specificity in differentiating serotypes (b) Shorter time for its operation (a) Very much sensitive in detecting low concentrations of virus up to 1–10 ng/ml (c) Direct application to large- or small-scale testing (d) Requires small amounts of antiserum (e) Used for plant or seed extracts as well as purified virus as antigen (h) Amenability to obtain quantitative measurements (i) Involves low cost and long shelf life of the reagents (j) Enables economical and efficient use of antibodies (g) Suitable for both intact and fragmented virion of different sizes as well as morphology Numerous variations of ELISA have been developed, but basically there are two main categories, namely, ‘direct’ and ‘indirect’ ELISA techniques that are widely used in plant virology. 119 enzyme-labelled antibodies, which requires considerably less time for completion of the test (Bar-Joseph et al. 1979; Flegg and Clark 1979; Hamdi and Rizkallah 1997; Chalam et al. 2009a). Besides the above methods, depending on the virus–host combination and convenience, Yolken and Leister (1982), Stobbs and Baker (1985) and Van Vuurde and Maat (1985) have also modified the standard DAS-ELISA technique. Although the method is convenient and effective, it has some disadvantages: 1. It necessitates the preparation of specific antibody conjugates for each virus under test. 2. It is highly strain specific and not suitable for virus detection in disease surveys or for probing a single antigen with several different antisera. Indirect ELISA In indirect ELISA test, immobilised antigen is the target for unconjugated specific antibody. Direct ELISA Trapped antibody is detected by enzyme-labelled In this test, r-globulins prepared for specific virus protein A conjugate or anti-FC-specific or antiare used in coating and the same r-globulins IgG conjugates. Thus, in indirect ELISA, deconjugated with an enzyme are employed for tecting antibodies are not labelled with enzyme. detection. Certain details of some direct ELISA There are several variations among the indirect procedures in common use are as follows: ELISA procedures which are as follows: (a) Double-Antibody Sandwich-ELISA (DAS- (a) Indirect DAS-ELISA: Bar-Joseph and ELISA): This method was first described by Saloman (1980), Koenig (1981), Rybicki and Clark and Adams (1977). As the antigen Von Wechmar (1981) and Van Regenmortel is sandwiched between two r-globulins, the and Burckard (1980) described modified type test is called as double-antibody sandwichof direct ELISA. This method is sometimes ELISA. In this method, the wells of the called as second antibody system. In this test, microtitre plate are first coated with antivirus antigen-specific antiserum is prepared in two globulin and the virus in the test sample is animal species. Gamma globulins from one then trapped by the adsorbed antibody. The species of antiserum is used to coat the plate presence of virus is revealed by an enzymeand trap antigen from the same. Antiserum labelled antivirus conjugate. This method or r-globulin from the other species is is highly strain specific and requires the reacted with the immobilised antigen and preparation of a different enzyme-conjugated the resulting immune complex is detected Ig for each virus to be tested. with an enzyme conjugate specific for the Several modifications have been made to r-globulin of the second animal species. This the standard DAS-ELISA to suit a particular allows the full binding property of specific rvirus–host combination or to reduce the assay globulin to be used, giving greater sensitivity time. One such method is simultaneous incuand overcoming the extreme specificity of bation of virus-containing samples with the DAS-ELISA. 120 (b) (c) (d) (e) 6 This ELISA is particularly suitable for virus detection in disease surveys, testing the presence of viruses in seed and determining serological relationships when specific conjugates cannot be prepared. It is relatively more economical to perform than the DAS form. Indirect F(ab’)2ELISA: Purified IgG is used for coating the plate in common forms of ELISA but Barbara and Clark (1982) used F(ab’)2 portion by cleaving the FC region of the IgG. This technique relies on trapping virus with adsorbed F(ab’)2 fragment and subsequent probing with an unlabelled virusspecific antibody. This immune complex is detected with enzyme-labelled antiglobulin antibody or Staphylococcus protein A (Barbara and Clark 1982). This combines the advantages of an indirect assay with that of DAS-ELISA. Indirect Direct Antigen Coating-ELISA (DAC-ELISA) or Plate-Trapped AntigenELISA (PTA-ELISA): This method is similar to the indirect DAS-ELISA except that the wells are initially coated with antigen. The reaction of antigen attached on solid phase is then carried out as in indirect DAS-ELISA. This method has the advantage of simplicity and convenience of application specially as a general diagnostic procedure where a virus isolate may have to be tested for reaction with several antisera (Mowat and Dawson 1987; Behl et al. 1995). Protein A Sandwich-ELISA (PAS-ELISA or PA-ELISA): Edwards and Cooper (1985) have described a novel form of indirect ELISA, which utilises protein A in two applications to sandwich antibody–antigen–antibody layers. The first applied layer of protein A prepares the plate for coating the antibody layer. The second layer of protein A is conjugated to the enzyme and detects the second antibody layer. Protein A Coating-ELISA (PAC-ELISA): In this method, protein A is only used for initial plate coating and subsequently antigen is added followed by antisera or r-globulins. The resultant protein antigen–antibody complexes are detected by anti-rabbit Detection of Plant Viruses in Seeds FC-specific enzyme-conjugated antibodies. This method is advantageous over the DAS method because of the use of crude antiserum instead of IgG and a single enzyme conjugate with all detection systems (Hobbs et al.1987). Cooper et al. (1986) have detected the cherry leaf roll and Prune dwarf viruses in cherry (Prunus avium) seeds by using PAS-ELISA. This technique is also effectively used in detecting cowpea aphid-borne mosaic virus in cowpea germplasm at IITA, Nigeria (Ojuederie et al. 2009). (f) Clq ELISA: A different indirect method which uses Clq (a component of complement obtained from bovine serum) was developed by Torrance (1980a, b). The plates are first coated with Clq. Since the Clq component of complement binds immune complexes, the virus and its specific antiserum are incubated together in the wells. The resulting virus IgG complexes are trapped by the Clq molecules and detected by an enzyme-labelled antiglobulin conjugate. Choice of Enzyme Labels and Substrates The enzymes most widely used in plant virology are alkaline phosphatase (ALP) and horseradish peroxidase (HRP) (Clark 1981; Clark and BarJoseph 1984). Both of these enzymes have problems associated with their use. These include high cost, limited availability, the potential carcinogenic activity of some of the substrates and the hazardous nature of hydrolyzed products of the substrates. Later urease (Evans et al. 1983) and “-lactamase, generally known as penicillinase (PNC) (Joshi et al. 1978; Yolken et al. 1984; Sudarshana and Reddy 1989), have been introduced in ELISA. Urease has the prospect of becoming routine because it catalyses the release of ammonia from the low-cost substrate urea. The product of this reaction can be conveniently detected by pH indicator such as bromocresol purple which changes colour from yellow to purple. Alkaline phosphatase and penicillinase exhibit the linear reaction kinetics with their substrates p-nitrophenyl phosphate and penicillin, respectively. The reaction kinetics 6.4 Serological Techniques of horseradish peroxidase (HRP) is not linear. Its cost is reasonably low and easily conjugated to immunoglobulins, but the main disadvantage is that many chromogenic substrates used for peroxidase are suspected mutagens. Further, the hydrolysed products are hazardous. Penicillinase has several features that make it a desirable enzyme label for ELISA tests. It has relatively low molecular weight (24 KDa), a high turnover rate, is not too expensive and more readily available in developing countries than other enzymes. The visual result reading from this test is easier than that of the alkaline phosphatase system. Additionally, hydrolysed products in PNC assays are not known to be hazardous (Sudarshana and Reddy 1989). PNC has also been used for labelling biotin and used in conjunction with streptavidin for improving the sensitivity of the ELISA (Yolken et al. 1984). The choice of substrates depends on the enzymes used to make conjugate. For alkaline phosphatase, the substrate most commonly used is p-nitrophenyl phosphate. The fluorogenic substrate, 4-methylumbelliferyl phosphate, has been reported to be more sensitive than the p-nitrophenyl phosphate when ALP was used (Yolken and Stopa 1979; Torrance and Jones 1982). With p-nitrophenyl phosphate, the reaction is stopped by adding excess alkali, and with 4-methylumbelliferyl phosphate, the reaction is stopped by adding K2 HPO4 .KOH (Martin 1985). For HRP, the substrates most commonly used are o-phenylenediamine (OPD) or 2,2 azino-di(3ethylbenzthiazoline-6-sulphonic acid) (ABTS) or 3,30 , 5,500 -tetramethylbenzidine (TMB). OPD is photosensitive; both OPD and ABTS are mutagenic and should be handled accordingly. TMB is neither photosensitive nor mutagenic. With all these substrates, the reaction is stopped by adding H2 SO4 (Clark 1981). For penicillinase, bromothymol blue (BTB), starch iodine complex (SIC) and mixed pH indicator of bromocresol purple (BCP C BTB (2:1) are the substrates used. BTB substrate colour changes from blue to greenish yellow to deep yellow, depending on the amount of penicilloic acid produced and measured at 620 nm (Sudarshana and Reddy 1989). 121 Substrates should ideally be cheap and nontoxic and must provide a sensitive quantitative measure of the bound enzyme. They should be easily prepared, stable and easily soluble for enzyme. Improvements in ELISA Various modifications and improvements have been introduced that give the test a higher degree of sensitivity and better versatility (Koenig and Paul 1982). New developments in the immunoenzymatic diagnosis of plant viruses are (a) biotin– avidin techniques and (b) use of fluorescent substrates. (a) Biotin–avidin System: Biospecific bridges involving biotin–avidin or lectin polysaccharides seem promising alternative strategies in ELISA. This system is based on the very high affinity of avidin for biotin. In the modified ELISA test, the virus is trapped between the coated antibodies and biotinylated antibodies; avidin molecules are used to trap non-specific biotin enzyme conjugate. Though this procedure involves additional steps, the sensitivity is significantly increased over standard ELISA (Kendall et al. 1983; Zrien et al. 1986). This new procedure appears to be very promising for detecting viruses in seed (Maury and Khetarpal 1989). (b) Use of Fluorescent Substrates: The desire to enhance sensitivity of ELISA for detection of antigens led to the development of the enzyme-linked fluorescent assay (ELFA). Recent immunological evidences have suggested that utilisation of fluorescent substrates such as 4-methylumbelliferyl phosphate (MUP) or 4-methylumbelliferyl 1-“-D-galactopyranoside (MUG) in ELISAbased assays enhances detection for several diverse antigens (Shalev et al. 1980; Neurath and Strick 1981; Konijn et al. 1982; Torrance and Jones 1982). The use of fluorescent substrates does not modify or complicate the normal ELISA test. Only the usual substrates are modified. Antibodies conjugated with alkaline phosphatase or galactosidase and a fluorogenic substrate is used. It has been demonstrated that 122 ELFA, using polyclonal antibodies to SMV, can provide sensitive levels of detection (Dolores-Talens et al. 1989), equivalent to biotin–avidin ELISA (Diaco et al. 1985) or radioimmunosorbent assay (RISA) (Ghabrial and Shepherd 1980), and is 10- to 25-fold more sensitive than DAS-ELISA (Hill and Durand 1986; Benner et al. 1990). Enzymelinked fluorescent assay (ELFA) can detect 1 ng/ml of purified virus and has been applied for the detection of SMV in soybean seed using polyclonal antibodies (Hill and Durand 1986) and monoclonal antibodies (Benner et al. 1990) and also for LMV in lettuce seeds (Dolores-Talens et al. 1989). Benner et al. (1990) have also successfully used mAbs of S1 or S2 labelled to biotin and detected SMV in soybean by using enzyme-linked fluorescent assay (ELFA). The indirect ELISA using mAbs is found most sensitive in detecting 2.5 ng/ml of virus and used to screen seed from the PStV-infected peanut cultivars. The mAbs in the indirect ELISA readily detected PStV antigen in peanut cotyledonary tissue and PStV-infected seed part diluted in 32 healthy seed parts (Sherwood et al. 1987; Culver and Sherwood 1988; Culver et al. 1989). (c) Mixed Antisera: To reduce the time and cost, attempts were made by number of workers by mixing the different antisera for the detection of more than one virus at a time. Some of the successful host–virus combinations of detection of viruses by mixed antisera are cowpea and soybean viruses (Joshi and Albrechtsen 1992), nepoviruses (Etienne et al. 1991) and potato viruses (Bantarri and Franc 1982; Grimm and Daniel 1984). Application of ELISA for Detection of Seed-Transmitted Viruses With increased advantages of ELISA, it has been successfully employed for detecting several seedtransmitted viruses (Table 6.3). Further, it has been useful in revealing and detecting virus situation in different seed parts, tolerance limits and detection of latent infections. (a) Virus Detection in Embryos: ELISA is so sensitive that even a single infected embryo 6 Detection of Plant Viruses in Seeds can be detected when diluted up to 2,000 w/v in case of SMV in soybean (Bossennec and Maury 1978), 3,600 w/v for PMV in peanut (Bharathan et al. 1984) or when mixed with 1,400 and 200 healthy seeds in case of LMV in lettuce and BCMV in bean, respectively (Jafarpour et al. 1979). The relative concentration of virus in different embryos has been analysed such as BSMV in barley (Lister et al. 1981), LMV in lettuce (Ghabrial et al. 1982), PMV in peanut (Bharathan et al. 1984), PSbMV in pea (Maury et al. 1987a, b) BCMV in cowpea (Udayashankar et al. 2010) and SqMV in cucurbits (Nolan and Campbell 1984). In barley infected with BSMV, a large variation of virus concentration has been found in different infected embryos from the same seed lot (Miller et al. 1986; Huth 1988). Amount of PMV detected in axis and cotyledon extracts from the same peanut seed were found to be the same (Bharathan et al. 1984), contrary to another isolate of PMV which was not detected in cotyledons (Adams and Kuhn 1977). ELISA also confirms that the concentration of SMV and BgMV was not the same for cotyledons and axis of single embryo, and the virus could even be restricted to either of these two parts (Varma et al. 1992). (b) Correlation Between Embryo Positive in ELISA and Seed Transmission: This test further helps in determining whether the number of infected embryos found positive in ELISA is the same as the infected plants in a progeny. A good correlation has been found for SMV in soybean seed (Maury et al. 1983), PMV in peanut seed (Bharathan et al. 1984), PSbMV in pea seed (Maury et al. 1987b) and LMV in lettuce seed (Falk and Purcifull 1983). On the contrary, for SqMV in cucurbit seed, the number of ELISA-positive embryos largely exceeded the number of virusinfected plants in the ‘grow out’ test (Nolan and Campbell 1984). In this case, when dissected embryos were cut into two halves transversely, the distal half having been tested by ELISA and the germinative half 6.4 Serological Techniques 123 Table 6.3 Application of ELISA techniques in the detection of seed-transmitted plant viruses Virus Alfalfa mosaic Crop Alfalfa Apple mosaic Barley stripe mosaic Annual medics Broad bean Clover Almond Wheat Barley Bean common mosaic French bean Blackgram/green gram Cowpea Bean common mosaic necrosis Bean pod mottle Bean yellow mosaic French bean Cluster bean Mung bean Bean Soybean Broad bean Black eye cowpea mosaic Lentil Lupin Cowpea Blackgram mottle Blackgram mild mottle Blueberry leaf mottle Broad bean stain Broad bean true mosaic Brome mosaic Cherry leaf roll Mung bean Blackgram Blackgram Blueberry Lentil Vetch Broad bean Cowpea aphid-borne Faba bean Wheat Cherry French bean Soybean Walnut Cowpea Cowpea mild mottle Soybean Reference Halk et al. (1982), Lange et al. (1983), and Pesic and Hiruki (1986) Jones and Pathipanwat (1989) Chalam et al. (2009a, b) Bariana et al. (1994) Barba (1986) Lister et al. (1981), Qiu et al. (1982), and Khetarpal et al. (2006b) Qiu et al. (1982), Lange et al. (1983), Mukhayyish and Makkouk (1983), Miller et al. (1986), and Huth (1988) Jafarpour et al. (1979), Lister (1978), Wang et al. (1982), Mukhayyish and Makkouk (1983), Lange and Heide (1986), Dusi et al. (1988), Klein et al. (1992), Khetarpal et al. (1994), Saiz et al. (1994), Puttaraju et al. (1999), Njau and Lyimo (2000), Nalini et al. (2004, 2006), and Chalam et al. (2007) Dinesh chand et al. (2004) Hao et al. (2003), Chalam et al. (2007), and Udayashankar et al. (2010) Puttaraju et al. (1999) Gillaspie et al. (1998b) Jeyanandarajah (1992), Choi et al. (2006) Njau and Lyimo (2000) Krell et al. (2003) and Pedersen et al. (2007) Eppler and Kheder (1988), Raizada et al. (1991), El-Dougdoug et al. (1999), and Chalam et al. (2007) Makkouk et al. (1992) Robertson and Coyne (2009) Jeyanandarajah (1992); Puttaraju et al. (2002, 2003; Puttaraju et al. 2004b) Saleh et al. (1986), Dinesh Chand et al. (2004) Varma et al. (1992) Varma et al. (1992) Childress and Ramsdell (1986) Makkouk and Azzam (1986) Makkouk et al. (1986) Anon (1984), Makkouk et al. (1987), Eppler and Kheder (1988), El-Dougdoug et al. (1999), Khetarpal et al. (2001), Chalam et al. (2007), and Chalam et al. (2009b) Anon (1984) Von Wechmar et al. (1984a) Cooper et al. (1984) Chalam et al. (2005) Chalam et al. (2007) Kolber et al. (1982) Yilmaz and Ozaslan (1989), Hampton et al. (1992), Bashir and Hampton (1993), Ndiaye et al. (1993), Konate and Neya (1996), Khetarpal et al. (2001), Chalam et al. (2007), and Ojuederie et al. (2009) Iwaki (1986), Horn et al. (1991), and Hampton et al. (1992) (continued) 124 6 Detection of Plant Viruses in Seeds Table 6.3 (continued) Virus Cowpea severe mosaic Cucumber mosaic Crop Cowpea Barley Cowpea French bean Lupin Peanut Cucumber Spinach Subterranean clover Lupin Cucumber green mosaic mottle Hibiscus latent ring spot High plains virus Indian peanut clump Iris yellow spot Lettuce mosaic Cucumber Kenaf Sweet corn Peanut Wheat Onion Lettuce Maize chlorotic mottle Maize dwarf mosaic Maize Maize Melon necrotic ring spot Melon rugose mosaic Onion yellow dwarf Pea early browning Melon Melon Garlic/shallot Lettuce Pea Broad bean Lentil Pea Pea seed-borne mosaic Peanut clump virus Peanut mottle Peanut Peanut Peanut mild mottle Peanut stripe Peanut Peanut Peanut stunt Pepino mosaic Peanut Tomato Reference Hampton et al. (1992) and Bashir and Hampton (1993) Von Wechmar et al. (1983) Hampton et al. (1992), Bashir and Hampton (1993), Gillaspie et al. (1998a), Abdullahi et al. (2001), and Ojuederie et al. (2009) Davis and Hampton (1986) and Hampton and Francki (1992) Njeru et al. (1997) and O’keefe et al. (2007) Reddy et al. (1984), Cai et al. (1986), and Demski and Warwick (1986) Ertunc (1992) Yang et al. (1997) Jones and Mckirdy (1990) Wylie et al. (1993), Njeru et al. (1997), and O’Keefe et al. (2007) Kawai et al. (1985) and Shang et al. (2011) Rubies-Autonell and Turina (1997) Froster et al. (2001) Reddy et al. (1988), Reddy et al. (1998a, b) Reddy et al. (1998a, b) and Delfosse et al. (1999) Crowe and Pappu (2005) Jafarpour et al. (1979), Ghabrial et al. (1982), Falk and Purcifull (1983), Van Vuurde and Maat (1983, 1985), Falk and Guzman (1984), Gusenleitner (1985), and Dolores-Talens et al. (1989) Jensen et al. (1991) Hill et al. (1974), Mikel et al. (1984), Khetarpal et al. (2006a, b) Avegelis and Barba (1986) Mahgoub et al. (1997) Delecolle et al. (1985) Van Vuurde and Maat (1985) Van Vuurde and Maat (1985) Chalam et al. (2009a, b) Varma et al. (1991) Hamilton and Nichols (1978), Maury et al. (1987b), Kheder and Eppler (1988), Khetarpal and Maury (1990), Haack (1990), Varma et al. (1991), Phan et al. (1997), Khetarpal et al. (2001), Parakh et al. (2006), and Coutts et al. (2009) Dieryck et al. (2009) Bharathan et al. (1984), Hobbs et al. (1987), Gillaspie et al. (2000), Puttaraju et al. (2001), Prasada Rao et al. (2004), Khetarpal et al. (2006a, b), and Chalam et al. (2007) Cai et al. (1986) Demski and Warwick (1986) Culver and Sherwood (1988), Warwick and Demski (1988), Matsumoto et al. (1991), Xu et al. (1991), Prasada Rao et al. (2004), and Khetarpal et al. (2006a, b) Cai et al. (1986) Cordoba – Selles et al. (2007) (continued) 6.4 Serological Techniques 125 Table 6.3 (continued) Virus Pepper mild mottle Plum pox Prune dwarf Crop Pepper Prunus spp. Sweet cherry Prunus necrotic ring spot Almond Sweet cherry Prunus spp. Ryegrass seed-borne Soybean mosaic Lolium sp. Soybean Southern bean mosaic Squash mosaic Cowpea Cucumber Melon Strawberry latent ring spot Quinoa Parsley Parsnip Maize Tomato Sugarcane mosaic Tobacco mosaic Tobacco ring spot Tobacco streak Tomato black ring Tomato mosaic Tomato ring spot Turnip yellow mosaic Urd bean leaf crinkle Wheat streak mosaic Capsicum Soybean Beans French bean Tomato Soybean Arabidopsis Oil seed rape Winter turnip rape Urd bean Wheat by the grow-out test, none of the ELISAnegative embryos produced a virus-infected plant. This absence of false negatives would suggest that the virus is more homogenously distributed in the embryo of cucurbit seeds than the viruses transmitted through soybean or pea seed. But more individuals were rated positive by ELISA than by the grow-out test; even when derived from half embryos with high positive values, the symptomless seedlings were virus-free when cucurbit seed Reference Svoboda et al. (2006) Thomidis and Karajiannis (2003) Mink (1984), Mink and Aichele (1984), Cooper et al. (1986), and Kelley and Cameron (1986) Mink (1984), Mink and Aichele (1984), Barba (1986), and Kelley and Cameron (1986) Mink (1984), Mink and Aichele (1984), Barba (1986), and Kelley and Cameron (1986) Mink (1984), Mink and Aichele (1984), Barba (1986), and Kelley and Cameron (1986) Chester et al. (1983) Lister (1978), Chen et al. (1982), La et al. (1983), Iwai et al. (1985), Diaco et al. (1985), Hill and Durand (1986), Taraku et al. (1987), Maury et al. (1983, 1985, 1987), Benner et al. (1990), Khetarpal et al. (1992, 2001), Chalam et al. (2004), Golnaraghi et al. (2004), Parakh et al. (2005, 2008), Pedersen et al. (2007), and Andayani et al. (2011) Hampton et al. (1992) and Bashir and Hampton (1993) Nolan and Campbell (1984) Lange et al. (1983), Avegelis and Katis (1989b), and Franken et al. (1990) Hicks et al. (1986) Bellardi and Bertaccini (1991) Hicks et al. (1986) Li et al. (2007) Cicek and Yorganci (1991), Chitra et al. (1999, 2002), and Sevik and Tohumcu (2011) Chitra et al. (1999, 2002) Lister (1978) and Golnaraghi et al. (2004) Walter et al. (1992) Chalam et al. (2005, 2007) Chitra et al. (1999, 2002) and Ismaeil et al. (2011) Golnaraghi et al. (2004), Chalam et al. (2007) de Assis Filho and Sherwood (2000) Spak et al. (1993) Spak et al. (1993) Beniwal et al. (1984) Jones et al. (2005) lots could be related for the frequent false positives by ELISA. The decline with time level of embryonic transmission of SqMV by the cucurbit seed lots could be related for the frequent false positives by ELISA. Similar declines during maturation of seed have also been reported, such as SMV in soybean cv. Merit (Bowers and Goodman 1979). (c) Determination of Percent Seed Transmission by Group Analysis: Where there is low percentage of seed transmission, the number 126 of embryos to be tested to have a good probability of detecting virus in seed would not be economically practical. A method of determining transmission rate consists of dividing a representative sample of the lot (are analysed) into ‘N’ groups of n seeds each. So, ‘N’ tests are then done instead of N x n in single-embryo tests. Some mathematical elements allow estimating the most probable percentage of transmission, with confidence intervals, as a function of the number of ELISA-negative groups (Y) that were given by Maury et al. (1987b). It is the magnitude of confidence intervals for a given level of probability which accounts for precision. The magnitude is determined by four parameters N, n, Y and the level of probability; it keeps small values for Y D N but can be considerable for Y D 0 (all groups positive in ELISA) (Maury and Khetarpal 1989). (d) Determining the Tolerance Limits: After determining the per cent transmission of virus in mature seeds of harvested crops, one requires some information on the subsequent disease spread and degree of yield loss in a given agricultural context. The epidemiological study will help in getting the information by taking into account the source of virus, levels of resistance of the cultivars, the date and intensity of transmission by vectors in relation to climatic conditions and the resulting percentage of infected plants in the field as well as yield reduction. In case of legumes, the per cent seed coats infected in a harvested seed lot gives an estimate of the per cent infected plants. This observation can considerably simplify the studies on the relationship between the seed-transmitted virus and resulting disease spread. However, assessing the per cent infected plants in the field just before flowering is important since early season spread has greater influence on yields, the number of secondary sources and also the virus transmission through seed. The tolerance limit could reach 1% for seed sown in regions that regularly have very low vector intensities early in the season. On the other hand, regions with 6 Detection of Plant Viruses in Seeds high vector intensities would experience major yield losses if more than one seed in 10,000 is infected. In case of LMV in lettuce, a tolerance limit of one infected seed out of 1,000 has restricted the disease satisfactorily in France although it was not determined to such a refinement (Marrou et al. 1967). In California, for the same virus– host combination, the limit chosen was five times lower, a test giving zero-infected seed in 30,000 – for a seed lot a 99.9% probability of having less than 0.022% infection. This limit gave an excellent control of the disease (Grogan 1980). Thus, geographical variability is noted in seed transmission visà-vis the vector prevalence. (e) Detection of Latent Infection: It is also possible to detect the infection in seeds collected from latent infected plants by using the technique. For example, in France some of the pea plants infected with PSbMV under glasshouse conditions remained symptomless and rarely exhibited a very faint leaf rolling, and this weak and subminimal form of infection could be detected by ELISA five weeks after plant growth (Khetarpal and Maury 1990). The ELISA techniques will save time and space. However, virus detection by this method is very strain specific and probably not applicable unless an antiserum with correct antibody is available. The techniques are of little use in situations where numerous serological variants of a virus occur. In developing countries, the availability of specific antisera ”-globulin, enzymeconjugated IgG substrate and ELISA reader are limiting factors for large-scale application of this technique for which regional and international cooperation is very essential. 6.4.4 Dot-Immunobinding Assay (DIBA or DIA) An enzyme immunoassay (EIA) that has been applied to plant viruses is enzymeassisted immunoelectroblotting (IEB) (Towbin 6.4 Serological Techniques et al. 1979; Rybicki and Von Wechmar 1982; Von Wechmar et al. 1984a) and was simplified as dot-immunobinding assay (DIBA) by Hawkes et al. (1982). This technique has been variously described as dot-blot immunobinding assay (DIBA) by (Berger et al. 1984), immunoblot assay (Powell 1984), enzyme-linked immunoblot assay (EIBA) (Wang et al. 1985), NC-ELISA (Bode et al. 1984) and NCM-ELISA (Smith and Banttari 1984; Banttari and Goodwin 1985). However, this technique is popularly referred as dot-immunobinding assay (DIBA) and also as dot-ELISA (Hawkes et al. 1982; Gumpf et al. 1984; Hibi and Saito 1985; Parent et al. 1985). The principle of DIBA is almost the same as that of ELISA, except that antigen or antibody is bound to nitrocellulose membrane instead of polystyrene plate and that the product of the enzyme reaction is insoluble. This technique is useful because the dotted membrane can be stored and processed at convenience or may be sent to other laboratories for processing if required facilities are not available with the worker. DIBA is of two types, direct DIBA and indirect DIBA. The procedure for both the methods is same as that of direct and indirect ELISA. The extract from infected seed is spotted on nitrocellulose membrane (NCM). After incubating and washing the antigen, a blocking agent is added to saturate any unoccupied binding sites on the NCM. Then IgG rabbit antibody specific to the antigen to be detected is incubated with the NCM and unbound antibody is removed by washing. Horseradish peroxidase-conjugated anti-rabbit second IgG is added, incubated and washed to remove unbound antibody. Finally, the substrate is added, that is, hydrolysed to form water-insoluble coloured spots or dots on the membrane. Horseradish peroxidase, the most frequently used enzyme, produces purple spots after addition of substrate. Alkaline phosphatase reacts with naphthol AS-MX phosphate mixed with 5-chloro-2-toluidinediazonium chloride hemizene chloride (fast red TR salt) to produce red dots or with diazotised 4-benzolamino-2,5dimethoxyaniline ZnCl2 (fast blue BBN salt) to produce blue dots on the white background of the NCM. 127 6.4.4.1 Application of DIBA For the first time, this technique was used by Von Wechmar et al. (1984a) for the detection of Brome mosaic virus in seeds and seedlings of wheat. Subsequently, it was also applied for the detection of BCMV in bean (Wang et al. 1985; Lange and Heide 1986; Lange et al. 1989), BSMV in barley (Lange and Heide 1986; Lange et al. 1989), PSbMV in peas (Lange et al. 1989; Ligat et al. 1991; Ali and Randles 1997), Cucumber green mottle mosaic virus (CGMMV) in cucurbitaceous crops (Shang et al. 2011) and Broad bean true mosaic comovirus (BBTMV) in broad beans (Makkouk et al. 1987; Makkouk and Kumari 1996; Khatab Eman et al. 2012). The main advantages of DIBA are (1) rapid, simple and economical; (2) highly sensitive and detects as low as 50–100 picogram of antigen; (3) requires only a single crude-specific antiserum each test virus and also a single generally applicable enzyme conjugate; (4) cost of nitrocellulose is less than that of plastic supports; and (5) part of the seed can be used for testing and the remaining for sowing. A drawback of this technique is that a large volume (50 ml) of relatively concentrated (1 mg/ml) antiserum is required but can also be reused over a period of 6 months to test samples (Abdullahi et al. 2001). 6.4.4.2 Tissue Blot Immunobinding Assay (TBIA) Tissue blotting printing technique is similar to dot-blot immunoassay but involves tissue in printing on nitrocellulose membrane (NCM). This technique does not involve disruption of tissue or extraction of antigen from the seed sample. The fresh material or imbibed seed material can be used as a imprint tissue on NCM (Lin et al. 1990; Hsu and Lawson 1991). In Syria, Makkouk and Attar (2003) have tested the lentil seeds received from ICARDA gene bank through TBIA and found out that CMV infection level was 7.4–35.8% in 2000/2001 and 7.0–64.2% in 2001/2002. When germinating embryo axes of seeds collected from CMVinfected lentil mother plants were tested by 128 TBIA, the CMV infection range was 0.9–9.5% in 2000–2001 and 0.1–1.17% in 2000–2002. Another example of this technique application is with Apple scar skin viroid (ASSVd) which is seed transmitted in apple and pear (Hadidi et al. 1991; Hurtt and Podleckis 1995). They have collected 6–50 ripe fruits from each of 17 cultivars, and tested by chemiluminescent tissue blot hybridisation with a cRNA probe for ASSVd, 93% of the fruits from infected trees were positive. However, none of 100 seedlings grown from the seeds of infected trees showed ASSVd infection in tissue blot hybridisation tests nor was ASSVd detected in any of the seedlings where they were graft inoculated to the bioamplification host Virginia crab (Malus hybrid), and this host was tested for ASSVd by tissue blot hybridisation assay. These data may suggest that ASSVd is not seed transmitted at a high rate in Asian pear even though the pulp of most of the pears on infected trees harbour the viroid and the tissue blotted antigen samples were processed as dot-blot immunoassay. To remove the interference of seed tissues, detergents like Triton-X-100 or sodium hypochlorite can be used. The antigen-blotted membrane can be stored for several weeks at 4ı C and can be used effectively for processing of the bulk samples and also at field level. Makkouk and Kumari (1996) and Makkouk et al. (1997) have studied TBIA application in ten virus–host combinations. Khatab Eman et al. (2012) have tested this technique to detect BBTMV in faba beans. This test was sensitive enough to detect the virus in all parts of the plant and at all growth stages. It is suggested that the test is useful for detecting seed-transmitted viruses after seed germination and is more practical than ELISA. This test was completed in less than 4 h without sacrificing sensitivity and is cheap and does not require sophisticated facilities. This technique is easily applicable to field sampling as tissue printings can be made in the field without the need to collect leaf samples for sap extraction in the laboratory. 6 6.4.5 Detection of Plant Viruses in Seeds Disperse Dye Immunoassay (DIA) This diagnostic method developed by Gribnau et al. (1982) is as sensitive as ELISA and provides an alternative for ELISA for large-scale indexing of seed material. DIA is a solid-phase immunoassay like ELISA wherein the enzyme conjugate is replaced by immediate dissolving of dye molecules with an organic solvent, dimethyl sulphoxide (DMSO). After the addition of DMSO, the plates are shaken for a minute and colour intensity is read at 540 nm; this test was used to detect LMV and PEBV in the seeds of lettuce and pea, respectively (Van Vuurde and Maat 1985). The advantages of DIA are (a) preparation of conjugate is simple and cheaper, (b) eliminates substrate incubation step and (c) possibility of simultaneous detection of two different types of antigens (Gribnau et al. 1983). The disadvantage of the test in comparison with ELISA is that a higher amount of IgG is necessary to prepare the dye solution conjugate. This technique is unsuitable for crude plant extracts. 6.4.6 Rapid Immunofilter Paper Assay (RIPA) RIPA is another simple, rapid, sensitive and virus-specific detection technique which has been adopted for detection of a virus in plant tissues. In this test, at the bottom of the Whatman glass filter paper, latex beads coated with virus antibodies were immobilised as a solid in a line. The bottom end of the paper strip was dipped for 3 min in a mixture of virus-infected leaf/seed extract and dyed latex coated with the virus antibody. A coloured band appears on the line where the white latex has been immobilised and can be detected with the naked eye and the filter paper strips could be measured by chromatoscanner. The filter paper strips coated with antibody can be stored at room temperature for more than 6.4 Serological Techniques 1 year in a desiccator, and the coated strips can be used as easily and simply as pH test papers. The sensitivity of RIPA was demonstrated by Tsuda et al. (1992) with CMV and TMV plant extracts, and attempts should be made to use it for the virus detection in seeds. 6.4.7 Immunosorbent Electron Microscopy (ISEM) The visualisation of immunological reactions on electron microscope grids is one of the most sensitive serological techniques. Two different approaches can be distinguished, depending on whether the viral antigen in suspension is visualised in thin sections of the infected tissue. In recent years, viruses in suspension are visualised directly on the electron microscopic grid. This technique is unique in that it combines direct visualisation of virus particles with the specificity of a serological reaction. Roberts et al. (1982) suggested two general terms for this technique, that is, immuno-electron microscopy (IEM) and electron microscope serology (EMS). However, Derrick (1973) introduced this technique for the first time and termed it serologically specific electron microscopy (SSEM) which has been widely used in the literature (Paliwal 1977; Beier and Shepherd 1978; Hamilton and Nichols 1978; Brlansky and Derrick 1979; Milne and Lesemann 1984; Rao and Singh 2008). However, since the term SSEM would also encompass the technique using ferritin-labelled antibody, it has been argued as an inappropriate term (Roberts et al. 1982). All forms of immuno-electron microscopy are ‘serologically specific’ and prefer to use a special term for the technique in which virus particles are attached to grids previously coated with antiserum. Roberts and Harrison (1979), therefore, introduced the term immunosorbent electron microscopy (ISEM) which is now popular in plant virology. In immunosorbent electron microscopy (ISEM), there are three general methods, namely, clumping, antibody coating or decoration and trapping which are used for virus detection. 129 The main advantage of ISEM is its sensitivity, which equals that of ELISA and local lesion assays (Baier and Shepherd 1978; Hamilton and Nichols 1978) and can be used for quantitative assay in crude seed extracts. It is quick, consuming little antiserum, and also enables the use of relatively low-titre antisera. The grids can also be coated with mixtures of antisera for different viruses, which allows detection of several viruses at a time (Derrick 1973; Thomas 1980). However, the disadvantage of this method is that serologically distantly related strains may not be detected (Nicolaieff and Van Regenmortel 1980). The ISEM technique has been applied successfully in the detection of numerous elongated and isometric plant viruses in seed (Table 6.4) as well as identifying the presence of double-stranded RNA in extracts of tobacco infected with TMV (Derrick 1978). When mixtures of different ratios of infected to healthy seeds were tested, these methods were found sensitive to detect one seed infected with PSbMV or LMV in 100 seeds (Hamilton and Nichols 1978) and one infected with TRSV, SMV or BSMV in 1,000 seeds (Brlansky and Derrick 1979). 6.4.7.1 Clumping of Virus Particles When a virus preparation is mixed with a suitable dilution of specific antiserum, the formation of virus–antibody complexes is visualised in an electron microscope by the appearance of clumps of variable size (Ball and Brakke 1968; Milne and Luisoni 1975; Roberts 1976). This method is simple, faster and suitable for verifying the presence of viruses in seed extracts. The clumping phenomenon is especially valuable when the virus concentration is too low for the particles to be seen directly. However, the disadvantage of this method is that components of plant sap/seed extract and antiserum can interfere with staining and affect the image quality. Also, the attachment of virus– antibody aggregates to the grid is variable and some aggregates may be washed off during the staining process. 130 6 Detection of Plant Viruses in Seeds Table 6.4 Application of immunosorbent electron microscopy (ISEM) for a detection of certain viruses transmitted through seed Virus Alfalfa mosaic Barley stripe mosaic Crop Lucerne Barley Bean common mosaic Beans Black eye cowpea mosaic Blackgram mottle Broad bean stain Broad bean true mosaic Brome mosaic Cacao Cowpea aphid-borne mosaic Cucumber green mottle mosaic Fig latent virus 1 Indian peanut clump Lettuce mosaic Green gram/blackgram Mung bean Cowpea Blackgram Faba bean Faba bean Wheat Swollen shoot Cowpea Cucumber Fig Peanut Lettuce Maize dwarf mosaic Pea seed-borne mosaic Soybean mosaic Squash mosaic Tobacco ring spot Vicia cryptic Sweet corn Pea Soybean Melon Soybean Faba bean 6.4.7.2 Decoration or Antibody Coating Decoration of virus particles in electron microscope grids is similar to the clumping method except that, prior to adding the antibody, the virus is immobilised on the grid by dipping in the antigen or adding the seed extract homogenate to the grid. After the grid is washed, a small drop of serum is added to it. Then it is incubated for 15 min in a water-saturated atmosphere, washed again, stained and examined. Individual virus particles are coated with halo of antibody, if the reaction is positive (Milne and Luisoni 1977; Roberts et al.1982). The decoration can be amplified by using a double decoration procedure (Kerlan et al. 1981). In this method, an antibody specific for decorating antibody is added after initial decoration procedure. Virus particles are then observed at a Reference Lange et al. (1983), Mukhayyish and Makkouk (1983) Brlansky and Derrick (1979), Lister et al. (1981), Lundsgaard (1985), and Lange and Heide (1986) Jafarpour et al. (1979), Russo and Vovlas (1981), Lundsgaard (1983), Hagita and Tamada (1984), Lange and Heide (1986), Raizada et al. (1990), and Nalini et al. (2004) Dinesh chand et al. (2004) Jeyanandarajah (1992) Taiwo et al. (1982) and Jeyanandarajah (1992) Krishnareddy (1989) Anon (1983) Anon (1983) Von Wechmar et al. (1984a) Sagemann et al. (1985) Kositratana et al. (1986) and Raizada et al. (1991) Mukhayyish and Makkouk (1983) Castellano et al. (2009) Reddy et al. (1998a, b) Brlansky and Derrick (1979), Van Vuurde and Maat (1983), and Falk and Purcifull (1983) Mikel et al. (1984) Hamilton and Nichols (1978) Brlansky and Derrick (1979), Maury et al. (1983) Lange et al. (1983) Brlansky and Derrick (1979) Anon (1984) relatively low magnification due to an apparent increase in their diameter (Martin 1985). The efficiency of virus trapping on grids coated with specific antibody or decoration of virus particles is 40–50 times greater than on grids coated with non-specific serum (Derrick 1973). These procedures have been combined for use as a diagnostic tool (Milne and Luisoni 1977; Kerlan et al. 1981). Besides this technique offers additional advantage to detect contaminant and non-specific particles in the preparation. Pares and Whitecross (1982) described goldlabelled antibody decoration (GLAD) with protein A gold complexes using different strains of TMV as an antigen. With this technique, distantly related viruses are distinguished by qualitative analysis of the adsorbed gold particles, but such analyses failed to distinguish between closely related viruses. 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis 6.4.7.3 Trapping in Electron Microscopy The trapping of plant viruses to electron microscope grids coated with specific antiserum was first described and designated by Derrick (1973) as serologically specific electron microscopy (SSEM); later it was renamed as immunosorbent electron microscopy (Roberts and Harrison 1979; Roberts et al. 1982). The method consists of allowing freshly coated microscope grids with a parlodin carbon film to float on 10–50-l drops of diluted antiserum for about 30 min. Excess protein is then removed by floating these grids for several minutes in a buffer solution. The coated grids are drained by a brief contact with filter paper and placed for 1 or 2 h on drops of virus extract. Salts and contaminants are removed by washing with distilled water, and the particles are visualised after negative staining (Van Regenmortel 1982). Use of protein A in ISEM increases twoto sixfold in trapping viruses (Lesemann and Paul 1980) and is specially suitable for low-titre antiserum (Milne and Lesemann 1984). Goldlabelled protein A has been used successfully to label several strains of TMV (Pares and White cross 1982). Protein A binds to the Fc region of most immunoglobulins (IgG). Protein A may also be used to coat grids to provide a more uniform coating of grids with antibody (Shukla and Gough 1979, 1984). The advantages of ISEM is its sensitivity and its lack of strain specificity (Van Regenmortel et al. 1980). It is ideal for small samples if an electron microscope and the specific antisera are available. Direct ISEM of seed extracts may yield results in less than 2 h. The disadvantages of ISEM are that, though sensitive, it is not amenable for large-scale testing programmes where hundreds and thousands of samples are to be tested as in many certification scheme agencies and quarantines (Martin 1985) and lack of availability of the electron microscope facility at many seed testing and quarantine stations due to high cost. 131 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis 6.5.1 Introduction Recent advances in molecular biology and biotechnology are being applied to the development of rapid, specific and sensitive tools for the detection of plant virus and viroid infections in seeds. Immunoassays have reached the point of practical application in agriculture, and the DNA probe technology is established in research laboratories. (Martin et al. 2000; Naidu and Hughes 2003) Immunoassays are being routinely used to detect virus in vegetatively propagated material and seeds for the purpose of quarantine, certification and maintenance. Use of immunoreagents such as monoclonal antibodies in ELISA systems has the potential for enhanced precision and sophistication of detection. When a virus is prone to antigenic variation or occurs without coat protein (Harrison and Robinson 1978) and in viroids, immunological detection systems are unsuitable. Indirect methods for comparing viral nucleic acids are routine and have been used to detect viroids (Owens and Diener 1984) or tobravirus (Harrison et al. 1983). The nucleic acid-based detection systems which make use of cloned DNA or DNA probes in a dot-blot or related assays have the potential to detect even single-nucleotide differences. The ability to make DNA copies (cDNA) of a part or the whole plant viral RNA genome has revealed many new possibilities. The nucleotide sequence of the DNA copy can be determined; it is a time-taking process, but it can be considered as a diagnostic procedure in certain special cases. Fundamentally, four approaches can be used to detect and diagnose the viral nucleic acids: (a) type and molecular size of the viral nucleic acids, (b) cleavage pattern of viral DNA or cDNA, (c) hybridisation between nucleic acids and (d) polymerase chain reaction. 132 6 Morris and Dodds (1979) developed a method for the isolation and detection of dsRNA from virus-infected plants. The properties of dsRNAs associated with RNA viral infections have been used for diagnosis (Dodds 1993). These dsRNAs, which are very resistant to enzymatic degradation, are not normally present in healthy plants. Simplification, together with improved equipment for nucleic acid analysis, has made the technique more practical and attractive to plant virus diagnosis. Application of each of molecular methods as well as dsRNA and monoclonal antibody technologies for the detection and diagnosis of seedtransmitted viruses and viroids are as follows: 6.5.2 Molecular Hybridisation Nucleic acid hybridisation is a powerful technique for the diagnosis of many plant viruses which are not easily detected by serological techniques. It is particularly effective in the detection of viruses occurring in low concentration in plant tissues and also against viruses that are poor immunogens or viroids which lack coat protein. Molecular hybridisation analysis also referred to as the ‘nucleic acid hybridisation’ or ‘spot hybridisation’ or ‘dot-blot’ technique has been shown to be a highly sensitive and specific procedure for identifying RNA or DNA viruses (Abu Samah and Randles 1983; Gould and Symons 1983; Maule et al. 1983) and plant viroids (Palukaitis and Symons 1978; Owens and Diener 1981). Basis of molecular hybridisation of viral nucleic acids was discussed by Hull (1993). The principle of hybridisation analysis for indexing of viroids and viruses is relatively simple. The first requirement is to prepare highly radioactive complementary DNA (cDNA) to the viroid or viral nucleic acid using 32 P or 3 H isotope. The cDNA is prepared enzymatically on the viral or viroid nucleic acid so that its sequence is exactly complementary. When this 32 P or 3 H cDNA is incubated with a nucleic acid extract of a test plant under appropriate conditions, it hybridises to any viral or viroid sequences present to produce a double-strand 32 P cDNA–RNA hybrid in Detection of Plant Viruses in Seeds case of viroid and RNA viruses or 32 P cDNA– DNA hybrid in case of DNA viruses. The extent of hybrid formation is a measure of the concentration of viral or viroid sequences in the plant extract (Symons 1984). Two procedures are available for the molecular hybridisation analysis of viroid or virus infection, similar in principle but different in the way the 32 P cDNA–RNA hybrids are detected. 6.5.2.1 Liquid Hybridisation Assay In this method, nucleic acid hybridisation takes place in solution. Most of basic studies were performed using both the target and probe DNAs in solution, so it is called as liquid–liquid hybridisation; there are also some data available for RNA–RNA and DNA–DNA interaction in solution. Presently most of plant pathogen diagnosis was involved by mixed-phase hybridisation with the target immobilised on a solid matrix; the theory developed for liquid–liquid hybridisation is very relevant (Hull 2002). Liquid– liquid hybridisation has been used to examine the relationship between plant viruses. The main principle of the technique is to hybridise the target nucleic acid with the probe that has been radioactively labelled than to digest away and unhybridised ss probe, precipitate the ds hybrid on a filter membrane and count the radioactivity. It has been extensively used for indexing Avocado sunblotch viroid (ASBV) disease of avocados (Palukaitis et al. 1981; Allen and Dale 1981; Allen et al. 1981) and Coconut cadang-cadang viroid (CCCVd) in extracts of coconut and other palms (Randles and Palukaitis 1979; Randles et al. 1980; Boccardo et al. 1981) and also been used to a very limited extent for detection of Chrysanthemum stunt viroid (CSVd) in extracts of infected plants (Palukaitis and Symons 1979). This method has been used to assess relationships between tombusviruses, and a sensitive nonradioactive procedure has been developed for detection of BaMV and its associated satellite RNA in meristem-tip cultured plants (Hsu et al. 2000). 6.5.2.2 Dot-Blot Hybridisation Assay This method is common for indexing of viroids and viruses and a popular method on large-scale 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis testing. The basic procedures have been described by Thomas (1980) and Owens and Diener (1981) which are extensions and modifications of earlier techniques, which are developed for use in general recombinant DNA technology. The major difference between liquid hybridisation and dotblot assay is that in the latter test nucleic acid is immobilised on a cellulose nitrate sheet for hybridisation rather than being in solution. The preparation of samples in this procedure is quite simple. The crude seed extract samples are simply spotted onto nitrocellulose filters and dried. The filters are subsequently hybridised with radiolabelled DNA probes which are copies of the viral genome. After hybridisation and washing to remove the non-hybridised probe, the presence of virus is detected by autoradiography (Baulcombe et al. 1984). A modification of the dot-blot assay, squash blotting has been used to detect Maize streak virus (MSV) (Boulton and Markham 1986). cDNA probes require the use of radioactive label (32 P) which are not amenable for common use, and very limited success has been achieved in their application. Prehybridisation and the hybridisation steps are carried out in a specialised manner (Dijkstra and de Jager 1998). Salazar et al. (1983) and Bernardy et al. (1987) have used this technique for the detection of PSTVd in mixtures of potato seed extracts equivalent to one infected seed among healthy ones. This technique is being used in routine testing of seeds at International Potato Centre, Peru. Bijaisoradat and Kuhn (1988) used it to detect the presence of PMV and PStV in peanut seeds. Both the viruses have been detected readily in one mg of infected seed tissue and when extracts from seeds have been diluted to 1/62,500 with water. One part of the infected seed can be reliably detected when mixed with 99 parts of healthy seeds. This sensitivity will be 8–10 times greater than that achieved by the use of ELISA. Besides sensitivity, rapidity and simplicity, this test is highly specific. For example, PMV cDNA does not hybridise with PStV from infected peanut seeds and vice versa, even though these two viruses share 60% nucleotide sequence homology when the hybridisation is performed under stringent conditions (Sukorndhaman 133 1987). Hsu et al. (2000) developed dot-blot hybridisation by using nonradioactive method to detect the BaMV and its sat RNA. 6.5.3 Double-Stranded RNA (dsRNA) Analysis Almost 80% of plant viruses have RNA genomes that can be single stranded. During replication of ssRNA viruses in plant cells, high molecular weight double-stranded RNA (dsRNA) is produced as an intermediate product, and this dsRNA is called the replication form (RF) and is consistently present when plant is virus infected. Most of the dsRNAs are associated with plant ssRNA viruses. Double-stranded form of the genome RNA accumulates that is twice the size of the genomic RNA. It is known as replicative form (RF) or replicative intermediate (RI). dsRNAs associated with RNA viral infections have been used for diagnosis (Dodds 1993). dsRNAs are associated with plant reoviruses; cryptoviruses have genome consisting of dsRNA and in tissue infected with ssRNA viruses. Some viruses have dsRNA genomes during replication; others produce dsRNA. Detection of high molecular weight dsRNA has been used to study plant diseases of suspected viral aetiology for which no virus-like particles could be identified (Chu et al. 1983; Dodds and Bar-Joseph 1983; Jordan et al. 1983; Morris and Dodds 1979). Double-stranded RNA (dsRNA), an indicator for the presence of viruses in plants, can be isolated rapidly by simple methods from small tissue weights of seedlings grown out of infected seed and using relatively simple laboratory equipment. The number, size, intensity and complexity of dsRNA detected by gel electrophoresis are distinctive and consistent for different virus groups and also aid in the diagnosis of a virus to a group or even a strain. Detection of dsRNA of appropriate size may provide insight into the cause of diseases of unknown aetiology (i.e. little cherry, lettuce big vein, black currant reversion) (Dodds et al. 1984). Additionally, it was expected that detection of dsRNA molecules would improve awareness of latent infections in 134 apparently healthy plants because any suspect dsRNA species could be labelled directly or after cloning. DNA copies can be multiplied in bacteria, thereby facilitating their routine use in ELISA-based systems or in nucleic acid hybridisation assays (Dodds 1986). The method for extraction of dsRNA from plant tissues, purification and analysis is described by Valverde (2008). Plant virus infections so far tested have been in tobamo, cucumo, potex, carla, poty and clostero virus groups which contain disease-specific dsRNA, molecules of the size expected for the molecular weight of the genomic ssRNA. BarJoseph et al. (1983) showed TMV and CMV could readily be diagnosed in singly or double infected plants by dsRNA analysis. This technique has several advantages over other methods for virus diagnosis. The dsRNA technique overcomes these problems: (1) The technique is simple and relatively inexpensive. (2) dsRNA can be obtained regardless of the host or the RNA virus. (3) Results are obtained in a relatively short time (8–12 h). (4) Interfering host components and instability of the virus or viral RNA are two main problems encountered by plant virologists while using traditional methods. (5) The technique detects mixed infection, which often go undetected with other methods and result in inadequate diagnoses. (6) Unlike most other diagnostic techniques, dsRNA analysis is non-specific. It can be used to distinguish not only different viruses but also strains of the same virus and satellite RNAs (Valverde and Dodds 1986; Valverde et al. 1986). (7) The purified dsRNA could then be used as a reagent for inoculation, probe preparation or molecular cloning (Dodds 1986; Jordan and Dodds 1983; Rosner et al. 1983). The dsRNA analysis has some limitations as only RNA viruses can be detected. The knowledge about the number and size of viral RNAs of different viral groups is required. Some plants contain cryptic viruses and/or cellular dsRNAs that yield dsRNAs similar in size to those associated with ssRNA viruses. Certain viral groups, such as the luteoviruses and most potyviruses, yield very low quantities of dsRNA, making the method impractical for their routine diagnosis (Valverde et al. 1990). 6 Detection of Plant Viruses in Seeds The need for rapid and reliable methods of diagnosing diseases and identifying pathogens is increasing as new technologies become available to researchers and diagnosticians. Despite its limitations, the procedure described by Valverde (2008) could be used in most disease clinics as a primary screening technique and as a complement to other techniques to diagnose viruses (Morris et al. 1983; Dodds 1986; Valverde et al. 1990). Antibodies reacting non-specifically against dsRNAs were found in antisera prepared against phytoreoviruses. Non-specific antisera can be prepared by using synthetic poly (I) poly clonal antigens, but this technique is not well received. mAbs produced against dsRNA have been used in immunoblots to detect this form of RNA in extracts from plants infected with viruses (Lukacs 1994). Polymorphisms in heteroduplex RNAs by adapting this method heteroduplexes of RNA transcripts polymorphism were detected between strains of PNRSV (Rosner et al. 1999). 6.5.4 Gel Electrophoresis Electrophoresis is the migration of charged particles like proteins, nucleic acids or polysaccharides to either cathode or anode under the influence of an electric field. It is usually carried out in gels formed in tubes, slabs or on a flat bed. In electrophoretic units, the gel is mounted between two buffer chambers containing separate electrodes so that the only electrical connection between the two chambers is through the gel. The sample may be run in agarose or polyacrylamide or agarose–acrylamide composite gel. At the end of run, the gels are stained and used for scanning or visual recording of results. The major useful systems for viruses and viroids are (1) agarose gel electrophoresis and (2) polyacrylamide gel electrophoresis (PAGE). 6.5.4.1 Agarose Gel Electrophoresis The standard method used to separate, identify and purify nucleic acid (RNA or DNA) fragments is electrophoresis through agarose gels. This technique is simple and rapid to perform and capable of resolving mixtures of nucleic acid fragments that cannot be separated adequately 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis by other methods, such as density gradient centrifugation. Furthermore, the location of nucleic acids within the gel can be determined directly by staining with low concentration of fluorescent or ethidium bromide dye and subsequent examination of the gel in ultraviolet light. The electrophoretic migration rate of nucleic acids through agarose gels is dependent on (a) molecular size of the nucleic acid, (b) agarose concentration and (c) the applied current. Several different designs of apparatus have been used. Currently agarose gel electrophoresis is being carried out with horizontal slab gels. Several different electrophoresis buffers are available containing tris-acetate, tris-borate or trisphosphate, and among them tris-acetate is the most commonly used buffer. 6.5.4.2 Polyacrylamide Gel Electrophoresis Polyacrylamide gels are used to analyse and prepare fragments of DNA/RNA and proteins. They may be casted in a variety of polyacrylamide concentrations, ranging from 3.5 to 20%, depending on the size of the molecules of interest. Polyacrylamide gels are poured between two glass plates that are held apart by spacers. In this arrangement, most of the acrylamide solution is shielded from exposure to the air so that inhibition of polymerisation by oxygen is confined to a narrow layer at the top of the gel. Polyacrylamide gels can range in length from 10 to 100 cm, depending on the separation required, and are invariably run in the vertical position (Sambrook and Russell 2002). The other major usefulness of PAGE system is determination of the molecular weights of the polypeptides or nucleic acids and detection of cryptic virus and dsRNAs in virusor viroid-infected seed material. 6.5.4.3 Return-Polyacrylamide Gel Electrophoresis (R-PAGE) Return-polyacrylamide gel electrophoresis (RPAGE) has played a pivotal role in the diagnosis of diseases caused by viroids which have low molecular weight RNA (Singh et al. 1992). A modification of PAGE specially designed for the rapid and sensitive detection of circular 135 RNAs and termed return PAGE (R-PAGE) has further facilitated viroid detection in various ways (Schumacher et al. 1986; Singh et al. 1988, 1992, 1993; Singh and Boucher 1987). In R-PAGE assay, nucleic acids are subjected to two electrophoretic runs, one under nondenaturing and the other under denaturing conditions. Because of the denaturation, circular RNAs lose their double-stranded configuration, become single-stranded covalently closed circular forms, migrate much more slowly in the second electrophoresis and thus are well separated from non-circular molecules. The circular RNAs from the lowest bands on the electropherograms render them easily distinguishable from noninfected plant extracts. The procedure takes on a day and requires simple laboratory equipment and nonradioactive chemicals. Viroid concentrations as low as 15– 20 pg can be detected reliably by R-PAGE (Singh 1989). For example, it detects viroids in one infected potato leaf disc mixed with 500 healthy leaf discs and standard nucleic acid extracts diluted to 1:1,024 times. R-PAGE has been successfully utilised to detect viroid from dormant potato tubers or from infected true potato seeds (TPS) singly or mixed with 100 healthy TPS (Singh et al. 1988). It has been used to identify several viroids from various crop plants in developing countries. A unique feature of the R-PAGE has been the separation of viroid strains on the basis of their mobility on the gel, which has greatly aided the studies on cross-protection (Singh 1989). R-PAGE can be used to assess the PSTVd content of various seed lots before planting either from seeds or from in vitro seedlings, when germplasm is valuable and available in small quantities (Singh et al. 1988, 1992). 6.5.5 Nucleic Acid-Specific Hybridisation Nucleic acid-based detection of the plant viruses is an important tool in molecular biology. Four important approaches are being used to detect and diagnose of the plant viruses: (a) type and 136 molecular size of the viral nucleic acid, (b) viral nucleic acid cleavage pattern, (c) hybridisation between nucleic acids and (d) amplification studies of desired viral nucleic acid. Nucleic acid-specific hybridisation techniques are being used for testing plant viruses and it involves the use of labelled complementary DNA (cDNA) or RNA (cRNA) prepared from purified viral nucleic acid as a probe or a recombinant clone of such viral nucleic acid as a probe. These labelled probes are used to detect the presence of plant viral nucleic acid by forming hybrid with them. Presently nucleic acid hybridisation includes the formation of DNA–DNA, DNA–RNA and RNA– RNA complexes (Singh and Dhar 1998). To detect the plant viruses and viroids, three types of ‘molecular hybridisation’ tests are being used: (a) solution hybridisation test, (b) filter hybridisation test or dot-blot hybridisation or nucleic acid spot hybridisation (NASH) and c) in situ hybridisation test. The NASH is one of the widely used techniques to detect the plant viruses and viroids. In this technique the target nucleic acid is immobilised on a membrane (nitrocellulose or nylon) and then hybridised to a labelled specific nucleic acid probe. Crude leaf or seed extracts are applied to the membrane and labelled with specific probe of DNA or RNA. Serological methods (ELISA) give little information on the virus detection, but nucleic acid hybridisation gives more genetic information on the viral or viroids nucleic acids. Due to lack of coat protein in the viroids, the nucleic acid hybridisation techniques are the only detection methods to characterise the genome. The identifying probes were made into two types: (a) radiolabelled probes and (b) non-radio labelled probes. 6.5.5.1 Radiolabelled Probes Several improvements have been made to the application of complementary DNA or RNA probes to detect viral pathogens. Mostly 32 P labelled nucleotides were used to label these nucleic acids radioactively, with specific activities up to 1010 cpm/g, which can detect 1 pg of target sequences. Nucleic acid hybridisation between DNA and RNA was done 6 Detection of Plant Viruses in Seeds in liquid but presently done by blotting the test samples on membrane followed by hybridisation with a radiolabelled (32 P) viral nucleic acid that serves a probe. This technique was more sensitive than ELISA for the detection of Potato virus X (PVX) and Potato leaf roll virus (PLRV) infecting potato plants. The disadvantages of the radiolabelled probes are that they are biohazardous. 6.5.5.2 Non-radiolabelled Probes Due to biohazardous nature of radiolabelled probes, the non-radiolabelled probes are preferred to do the hybridisation studies for the viral nucleic acids. The most used nonradioactive-labelled compounds are biotin vitamin H and hapten digoxigenin (DIG). These two compounds are detected by indirect way, using labelled, specific-binding protein, either streptavidin (biotin) or anti-digoxigenin serum, and later visualised by a chemiluminescent or colorimetric reaction. The biotin–streptavidin system is very sensitive and efficient, but we can get positive or background occurrence. Genome of the various viroids that are 246–375 bases long is being used for the hybridisation studies. Nonradiolabelled hybridisation has been successfully used for detection of PSTVd in true potato seeds (Goldbach et al. 1992; Borkhardt et al. 1994). Advantages of non-radiolabelled probes are stability of probes, no radioactivity, inexpensive and rapid detection, and a disadvantage is possible stearic hindrance during hybridisation because of hapten presence. 6.5.5.3 cDNA Probes cDNA technology utilises the specific recognition between the viral RNA and its complementary DNA (cDNA) (Watson et al. 1983). As majority of the plant viruses have single-stranded RNA (ssRNA) as the genetic material, by using the reverse transcriptase, ssRNA can be copied into cDNA which then by a recombinant DNA technology can be cloned in a suitable cloning vehicle. In this way, cDNA, specific to each virus, can be mass produced. DNA or RNA viruses can be detected in seeds by using cDNA probes which are labelled with 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis radioactive markers (32 P or 3 H) or nonradioactive markers such as biotin. Sensitivity of detection may be related to the probe size and larger probes give more sensitive assays. It is possible to make diagnostic cDNA probes to regions of the certain viral genome where the extent of sequence similarity has been shown to distinguish the viruses and strains (Watson et al. 1983). This technique was also proved to be effective in identifying the seed-transmitted infection of certain viroid diseases. For example, scar skin and dapple apple viroids (Hadidi et al. 1991). 6.5.6 Array Technologies Array technology has revolutionised the world of viral diagnosis because of its efficiency in screening a large volume of field samples in a single array plate. Basic principle of array technology combines the binding of DNA on to a solid support such as membrane filter or array plate and followed by hybridisation technology with a specific probe that will detect the target DNA. This technology was first invented and applied for gene expression studies; later it has been used in various pathogens diagnosis including plant viruses (Boonham et al. 2007). There are mainly two types of arrays: (1) macroarrays and (2) microarrays based on the volume of the sample and the droplet size used for the analysis. 6.5.6.1 Macroarrays Macroarray is a technique used in array technology, where the amount of sample used is higher than microarrays and the droplet size is more than 200-m space. Principle involves simple blotting of oligoprobes of virus-specific sequences either by dot-blot or slot-blot method followed by nucleic acid hybridisation with specific sample in which the virus is to be detected. This technique was first applied in plant virology by Agindotan and Perry (2007) for detection of various RNA viruses and also for detection of 11 potato viruses and a potato-infecting Potato spindle tuber viroid (Agindotan and Perry 2008). 137 6.5.6.2 Microarrays Microarray is a technique used in array technology, where the amount of sample used is much smaller than macroarrays and the droplet size is less than 200-m space. The invention of microarrays was chiefly for the detection of differential gene expression patterns in cells, and this was applied in case of plant viruses by Boonham et al. (2007) and Bystricka et al. (2005). Importance of microarrays in detection of plant viruses was emphasised by Hadidi et al. (2004), Bystricka et al. (2005), Tomlinson and Mumford (2007) and Barba and Hadidi (2008). Microarray or DNA chip technology is used generally for identification of differential gene expression patterns of an organism or tissue. It is chiefly used in functional genomics for understanding the different types of tissues, developmental stages and disease conditions. The principle is mainly based on nucleic acid hybridisation and fluorescent dyes for labelling the hybridisation probes and hence termed as a process of in situ hybridisation. A microarray is a glass slide, to which single-stranded DNA molecules are attached at fixed locations. Generally this slide is either made of glass or nitrocellulose that can bind biomolecules covalently. Each spot will require 0.25–1 nl solution and diameter on the glass will be 100–150 m separated by a space of 200–250 m. The samples are spotted on to the slide or chip in microvolumes in the form of an array. So the technology is defined as microarray technology which is a highly suitable technology for detection of plant viruses on a large scale. High density nucleic acid samples are isolated and purified which can be used as samples or else sometimes cDNA derived from reverse transcription of expressed mRNA or RNA itself and sometimes termed as RNA microarrays. In some cases, oligonucleotides are synthesised directly on to the microarray plates or the glass slides (known as in silico synthesis). To print these samples, the scientists use highthroughput robotic systems. These nucleic acid samples are then immobilised on to the substrate. The next step includes hybridisation of probes. Probes are prepared from many sources, some from cell extracted nucleic acids, some are syn- 138 6 thesised oligonucleotides, some are from PCRamplified products, some include the different cloned inserts, etc. Generally in order to identify the expression patterns in genes, mRNA or cDNA derived from mRNA is used as probes. In case of DNA microarrays, DNA samples or fragments are used as probes. The probes are isolated and purified nucleic acids and they are labelled by two types of fluorescent dyes – one is CY3 that is a green channel excitation fluorophore and the other is CY5 that is a red channel excitation fluorophore. The labelled probes are hybridised to the microarray plate in a hybridiser. After hybridisation, the unhybridised probe will be washed out with appropriate buffers. This microarray plate is ready for scanning. Many microarray scanners are available nowadays which apply briefly two methods: (1) sequential scanning and (2) simultaneous scanning. The image obtained by scanning the microarray plate in a scanner is subjected to analysis by using softwares like ScanAlyze for the expression pattern. Similar principles could be applied in case of plant viruses where the microarray slide could be printed with oligo that are synthesised specific to the plant viruses. At a time, two samples labelled with two different fluorophores could be used for hybridisation, and the presence of specific virus or its genotype could be identified easily. 6.5.7 Polymerase Chain Reaction (PCR)-Based Detection 6.5.7.1 Polymerase Chain Reaction Basics The advent of PCR by Kary B. Mullis in the mid-1980s has revolutionised molecular biology. PCR is a fairly standard procedure now, and its use is extremely wide ranging. At its most basic application, PCR can amplify a small amount of template DNA (or RNA) into large quantities in a few hours. This is performed by mixing the DNA with primers on either side of the DNA (forward and reverse), Taq polymerase (of the species Thermus aquaticus, a thermophile whose polymerase is able to withstand extremely high temperatures), free nucleotides (dNTPs for DNA, NTPs for RNA) and buffer. The temperature is Detection of Plant Viruses in Seeds then alternated between hot and cold to denature and reanneal the DNA, with the polymerase adding new complementary strands each time. In addition to the basic use of PCR, specially designed primers can be made to ligate two different pieces of DNA together or add a restriction site, in addition to many other creative uses. Clearly, PCR is a procedure that is an integral addition to the molecular biologist’s toolbox, and the method has been continually improved upon over the years (Candresse et al. 1998; Dietzen 2001; Albrechtsen 2006). Polymerase chain reaction is a molecular technique for the in vitro amplification of DNA or in general terms known as photocopying of DNA. In this technique, a specific target DNA is amplified into multiple copies by a set of oligonucleotides specific to the target DNA. Before understanding the exact mechanism of how PCR works in amplification of target DNA, let us get into the kinetics of how a DNA molecule replicates in in vivo. In the first step of replication, DNA double helix strand is unwound into two individual strands by the enzyme helicase at the site of replication initiation. The initial primer required for the DNA replication initiation is synthesised by the enzyme primase. Then the DNA polymerase synthesises the DNA strands by utilising the dNTPs present in the cytoplasm, and the synthesis is carried out which leads to the formation of Okazaki fragments in a replication fork and this synthesis is completed by filling the fragments in the replication fork. The basis of invention of PCR is to overcome difficulties in amplification of the desired copy of DNA. Old procedures included cloning and replication of clone for multiplying the copy of desired DNA. The supplements of the DNA replication are provided artificially to replicate a desired copy of DNA in PCR reaction. The most primary problem faced by molecular biologists in in vitro amplification of DNA is the stability of the DNA polymerases. Most of the DNA polymerases are not stable at higher temperatures like 90–95ıC where two strands of DNA get separated by heat denaturation. Studies have shown that certain hot spring living bacteria like Thermus aquaticus have polymerases that are stable at higher temperatures. They are used in 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis PCR as a replacement to conventional DNA polymerases. In the PCR reaction, other requirements are supplemented artificially like Taq DNA buffer (which provides buffered environment for necessary action of Taq DNA polymerase), MgCl2 (essential for activity of Taq DNA polymerase and also aids by binding of dNTPs (dATP, dGTP, dCTP and dTTP) for polymerisation), oligos (short stretch of DNA sequences that are complementary to the target DNA, popularly known as primers that will decide the range of amplification), template DNA from which the amplification of the specific target DNA is achieved and deionised water (to make up the concentration of the components). The PCR reaction is set in polycarbonate tubes or strips or plates designed for the purpose depending on the requirement process. The kinetics of PCR are chiefly dependent on the concentrations of the components like MgCl2 concentration, dNTPs concentration, amount of enzyme used, concentration of primers and amount of the DNA templates added into the reaction mixture. All the components of the reaction mixture are mixed in a PCR tube; the further reactions are carried out on thermal cycler machines specially designed for the PCR. PCR reaction involves three basic steps that include denaturation, annealing and extension. Denaturation step is carried out to separate double helix strands, generally done at approximately 90–95ıC followed by annealing where the temperature of the reaction is lowered to ensure the binding of oligos (primers) to the target. Generally these temperatures are lowered such that the annealing temperature is below the Tm of the primers (temperature of dissociation of primer duplexes). The final step is known as extension, where the temperature of the reaction is adjusted to approximately 72ı C where most of the Taq DNA polymerases have their maximum activity extending the primer by polymerisation of dNTPs towards the 30 OH end thus forming the new copy of the target DNA completely from the template DNA. Additionally, initial denaturation is done for longer time to ensure proper and clear denaturation of template DNA molecules. Also final extension is done to fill the protruded ends of the incomplete fragments. During each cycle, one copy of template DNA is doubled so the total 139 number of copies of the target obtained after the n cycles will be 2n . Generally, these repetitions are done from 25 to 40 cycles depending upon the amount of DNA copies. 6.5.7.2 Variants of PCR and Their Applications PCR is a strong tool in molecular biology; after its invention, it was variantly modified to serve many purposes and these modified types are termed as variants of PCR. The PCR variants are RT-PCR, IC-RT-PCR, nested PCR, multiplex PCR, LAMP, direct binding PCR, IC-PCR, CF-PCR, PCR-RFLP, PCR-ELISA and real-time PCR. Application of these techniques in identifying different seed-transmitted plant virus and viroids are presented in (Table 6.5). The advantages of different PCR variants are discussed here under. 6.5.7.3 RT-PCR: (Reverse Transcription-PCR) During the last two decades PCR and RT-PCR are superb techniques for the isolation of the target DNA or cDNA in a relatively short time, avoiding many of the time-consuming aspects of traditional gene cloning procedures. This technique will help in detecting the virus in picogram levels in infected seed or any other plant tissues or for confirmation of DNA insertion in transgenic plants. The high specificity makes PCR a prime candidate for development of tests that allows not only the detection but also the identification of specific genetic sequences of the pathogens. Sambade et al. (2000) for the first time have developed one-step RT-PCR amplification procedure providing highly specific complementary DNA from the plant virus RNA and being used by a number of researchers. Till recently, more than 200 viruses and viroids are detected using PCR. Using RT-PCR, the ability of specific primer pairs to amplify from individual seed samples were evaluated for Bean common mosaic virus (BCMV), Bean common mosaic necrosis virus in bean, Soybean mosaic virus in soybean and Pea seed-borne mosaic virus (PSbMV) in pea. Potyvirus group – specific degenerate primers which have potential in identifying new or unknown potyviruses were used for their 140 6 Detection of Plant Viruses in Seeds Table 6.5 Application of PCR-based techniques in the diagnosis of seed–transmitted plant viruses and viroids Virus Bean common mosaic virus Bean common mosaic virus (PSt strain) Chrysanthemum stunt viroid Cocoa swollen shoot virus Cowpea aphid-borne mosaic virus Crop Cowpea French bean Mung bean Chrysanthemum Cocoa Cowpea Cowpea mottle virus Cucumber green mottle mosaic virus Cucumber mosaic virus Peanut Cowpea Cucumber Cowpea Dahlia mosaic virus Hosta virus X Lettuce mosaic virus Pea seed-borne mosaic virus Lupin Peanut Pepper Pumpkin Spinach Dahlia Hosta Lettuce Pea Peanut clump virus Peanut mottle virus Peanut stripe virus Peanut stunt virus Pepper chat fruit viroid Piper yellow mottle virus Plum pox virus Subterranean clover mottle virus Subterranean clover red leaf virus Sugarcane mosaic virus Wheat streak mosaic virus White clover mosaic virus Zucchini yellow mosaic virus Peanut Peanut Peanut Peanut Pepper Black pepper Stone fruits Clover Clover Maize Wheat Trifolium Pumpkin detection in RT-PCR. Bariana et al. (1994) have developed RT-PCR assays for the detection of Subterranean clover red leaf luteovirus and Subterranean clover stunt virus, and this system helps in detecting all well-characterised viruses known to infect subterranean clover. The RTPCR assay detected all isolates tested for each virus and was up to five orders of magnitude more sensitive than ELISA. The real-time RTPCR method was also useful in detecting BCMV and PSbMV in single seed. The use of molecular technique has special significance in detecting latent infections and the detection of viruses occurring in very low concentration. Also, Reference Hao et al. (2003) and Udayashankar et al. (2010) Saiz et al. (1994) Choi et al. (2006) Chung and Pak (2008) Quainoo et al. (2008) El-Kewey et al. (2007), Udayashankar et al. (2009), and Salem et al. (2010) Gillaspie et al. (2001)and Salem et al. (2010) Gillaspie et al. (1999, 2000) Hongyun et al. (2008) and Shang et al. (2011) Gillaspie et al. (1998a), Abdullahi et al. (2001), and Salem et al. (2010) Wylie et al. (1993) Dietzgen et al. (2001) Akhtar Ali and Kobayashi (2010) Tobias et al. (2008) Yang et al. (1997) Pahalawatta et al. (2007) Ryu et al. (2006) Revers et al. (1999) and Peypelut et al. (2004) Kohnen et al. (1992), Phan et al. (1997), and Klem and Lund (2006) Lee et al. (2004) Gillaspie et al. (1994, 2001) and Dietzgen et al. (2001) Gillaspie et al. (1994, 2001), and Dietzgen et al. (2001) Dietzgen et al. (2001) Verhoeven et al. (2009) Hareesh and Bhat (2010) Thomidis and Karajiannis (2003) Njeru et al. (1997) Bariana et al. (1994) Li et al. (2007) Jones et al. (2005) Lee et al. (2004) Tobias et al. (2008) and Simmons et al. (2011) by using suitable primer pairs, simultaneous detection of different viruses in seed is possible by multiplex RT-PCR (Chalam et al. 2005). From Australia, Bariana et al. (1994) have detected CMV, CYVV, AMV, BYMV and SCMoV which are seed transmitted in subterranean clover by a cocktail assay which detected all five viruses both individually and collectively. RT-PCR is widely used for detection of RNA viruses in plants. In RT-PCR, PCR is done in two steps: (1) reverse transcription and (2) PCR. Reverse transcription is a process where cDNA is synthesised to an RNA template by involving an enzyme known as reverse transcriptase. This 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis process also requires an important component for cDNA synthesis, that is, a primer possibly an OligodT for the viruses with poly-A tail or a random primer or a reverse primer of the PCR itself. The most commonly used and available reverse transcriptases are from viruses like, Moloney murine leukaemia virus (M-MuLV) and AMV. In this mechanism, the RNA is initially denatured, along with the primer, and snap chilled to unwind the super secondary structures present in RNA and also to aid the primer annealing. Then the other components like dNTPs, RT buffer and RNAse inhibitors are added and the reverse transcription is carried out at a specific temperature, generally 42ı C for most of the RTs, and a final step of termination is held by heating the reaction mix to higher temperatures to inactivate RT. The cDNA can be used as a template in the next step PCR whose details are discussed earlier. In case of viruses where a poly-A tail is not present, either a reverse primer of PCR or a random primer (probably a hexamer) is used for cDNA synthesis. Reports of usage of terminal dinucleotidyl transferases to generate ‘poly’-A tails for cDNA synthesis is also there in case of certain types of ilar- and bromoviruses. This technique was used in detection of Cowpea aphid-borne mosaic virus by Gillaspie et al. (2001), specific detection of Lettuce mosaic virus isolates by Peypelut et al. (2004), differentiation of peanut seed-transmitted potyviruses and cucumoviruses by Dietzgen et al. (2001), detection of Broad bean stain comovirus (BBSV) and Cowpea aphid-borne mosaic potyvirus (CABMV) in faba bean and cowpea plants by EL-Kewey et al. (2007) and detection of ZYMV and CMV in Cucurbita pepo by Tobias et al. (2008). Gillaspie et al. (2001) processed Cowpea aphid-borne mosaic virus-infected peanut seeds by taking 2- to 4-mm seed slices, which included the seed coat as well as cotyledons, and produced more reproducible results by RT-PCR, and this test has also been tested with peanut against Peanut stripe virus in peanut seeds (Pinnow et al. 1990). Individual peanut seeds were subsampled by a modified non-destructive technique in which a slice was removed from each seed distal to the radicle with razor blade and the slices from several seeds were combined and extracted by 141 the Qiagen method (Gillaspie et al. 2001; Pinnow et al. 1990). Earlier this test has been employed against Subterranean clover mottle virus in clover seeds for the routine testing of bulked seed samples (Njeru et al. 1997). Even in fruit crops like plum, the seed transmission of Plum pox virus was confirmed by PCR test (Pasquini and Barba 2006). RT-PCR is applied to detect the viruses in the infected seed samples of the following virus–host combination by different researchers: Arabis mosaic virus, Bean yellow mosaic virus, Cucumber mosaic virus, Clover yellow vein virus, Subterranean clover mottle virus (Bariana et al. 1994) and in certain legume seeds (Bariana et al. 1994; Njeru et al. 1997), Pea seed-borne mosaic virus in pea (Kohnen et al. 1991, 1995), Wheat streak mosaic virus in wheat (Jones et al. 2005), Blackeye cowpea mosaic viruses in cowpea (Udayashankar et al. 2009), Dahlia mosaic virus in Dahlia pinnata (Pahalawatta et al. 2007) and Peanut stripe and Peanut mottle viruses (Gillaspie et al. (1999). As the Cucumber mosaic virus in spinach is carried by the pollen and embryo infection takes place during pollination, for virus detection in pollen grains of CMV-infected spinach, RT-PCR is successfully used (Yang et al. 1997). RT-PCR has been used to detect Potato spindle tuber viroid in true seeds of potato (Shamloul et al. 1997) and Coleus viroid in Coleus scutellarioides (Singh et al. 1991a). During Boben et al. (2007) have applied the RT-PCR for the detection and quantification of Tomato mosaic virus in irrigation water. Application of PCR in detection of various plant virus diseases, both seed-transmitted and non-seed-transmitted is well known after its invention in the mid-1980s. PCR is used for detecting plant DNA viruses like caulimoviruses, geminiviruses, badnaviruses and nanoviruses. 6.5.7.4 Duplex RT-PCR Dietzgen et al. (2001) have developed duplex RTPCR for the detection of two potyviruses, PStV and PeMoV, and the two cucumoviruses, CMV and PSV, that are seed transmitted in peanut. Inclusion of an immunocapture step in the duplex RT-PCR assays may overcome inhibitors of PCR present in peanut seed extracts. 142 6.5.7.5 Nested PCR It is a variant of PCR designed to get amplification of desired fragment specifically. In this technique, the product of the first few cycles (using a first primer set) is used as a template for the second set of nested primers that are designed to amplify the desired fragment. It is designed to increase the specificity of detection and also to enhance the copies of viral nucleic acid in case of low virus titres. This technique was successfully used in Vitivirus and Foveavirus species detection in grape vines (Dovas and Katis 2003) and Beet necrotic yellow vein virus detection in sugar beet (Morris et al. 2001). 6.5.7.6 Multiplex PCR This variant of PCR is targeted for detection of multiple viral pathogens in a single reaction. Multiplex primers are designed specifically for different viruses but with same annealing temperatures and different sized product amplicons. Care is also taken that no secondary structure formations are there between these set of primers. When a PCR reaction is carried out from the template having a mixture of viruses (mixed infections), only the viruses that are present are amplified. When these products are analysed, basing upon the sized fragment obtained, the viruses are identified. This is done in case of many viruses, for example, six citrus viruses (Roy et al. 2005). For the six citrus pathogens that include CMBV, CTV, CLRV, ICRSV, CVV and Citrus exocortis viroid, multiplex primers were designed and used successfully. This technique was used in detection of five seed-transmitted legume viruses by Bariana et al. (1994). 6.5.7.7 CF-PCR It is a modified method of PCR which could be used to differentiate between the different strains of virus where 50 fluorescent dye-labelled oligos are used for amplification. Upon obtaining the amplicon, the dye fluoresces only in a doublestranded hybrid. This technique is used chiefly to differentiate the viruses with divergence in 30 end nucleotide sequences. This was chiefly used to differentiate the multiple strains of the potato viruses (Walsh et al. 2001; Webster et al. 2004). 6 Detection of Plant Viruses in Seeds 6.5.7.8 LAMP (Loop-Mediated Isothermal Amplification) It is a modified, efficient and specific method for the amplification of the DNA templates. The reaction is performed with two sets of primers containing two specific regions of the target sequence for amplification (Notomi et al. 2000). The process starts with annealing of the first primer set for synthesis of the complementary strand that forms a loop out structure for further amplification by second primer set. The amplification is carried out at isothermal conditions (65ı C for 1 h) with Bst DNA polymerase that has strand displacement activity. Target amplification is detected by measuring the turbidity of the reaction mixture. This was used efficiently for detection of TSWV from chrysanthemum (Fukuta et al. 2004) by combining LAMP with IC-RT steps; LAMP was used in diagnosis of plant viruses (Wang et al. 2008). 6.5.7.9 IC-PCR Immunocapture PCR is another variant of PCR that combines the capture of the virus particles by antibodies coated in a PCR tube or micro tube plate. The bound antigen (virus particle) is then lysed in a buffer that will release the total viral nucleic acid content from the virion into the medium, and PCR is carried out by addition of other components to the medium. This method is very sensitive and specific to the virus and is free of contaminants like plant polyphenolic enzymes that inhibit PCR (Varveri 2000). Though, this technique is used for the viruses which had polyclonal or monoclonal antiserum and has been worked out against detection of seed-transmitted viruses like Pea seed-borne mosaic virus in pea seeds (Phan et al. 1997) and also BCMV infection in cowpea seeds (Udayashankar et al. 2010). Gillaspie et al. (1999) used immunocapture reverse transcription-polymerase chain reaction (ICRT-PCR) for large-scale assay of Peanut mottle virus (PeMV) and Peanut stripe virus (PStV) and could be reproducibly detected by RT-PCR from a single seed from 100 seed composites using smaller-sized (approximately 1 mm) seed material. 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis 6.5.7.10 Direct Binding PCR It is a method similar to the IC-PCR, but it involves direct binding of the virus particles from the crude plant sap or seed extract to the PCR tube, washing of the unbound particle and debris, lysis of viral particles in a medium and PCR detection of the target. Although this technique is simple and affordable, rate of success and level of detection is lower than that of IC-PCR for many of the virus hosts with heavy polyphenolics. 6.5.7.11 Reverse Transcription-PCR–DBH During 2009, from Iran, Bagherian et al. have described a new method based on a combination of RT-PCR and dot-blot hybridisation (RT-PCR– DBH); in this method, instead of using nucleic acid extracted directly from the plants, RT-PCR products are subjected to dot-blot hybridisation. By using this method, Hop stunt viroid (HsVd) and Citrus excortis viroid (CEVd) were detected from Washington navel orange plant samples. This RT-PCR–DBH method is over 1,000-folds more sensitive than Southern or Northern blot and over 100-folds more sensitive than electrophoresis of product. High seed transmission is noticed in six viroid diseases of plants (Hadidi et al. 2003). This method which provides a highly sensitive and specific means of diagnostic detection of plant viroid diseases is cost-effective and has been tried (Weissensteiner et al. 2004). 6.5.8 Real-Time PCR It is a modified method of PCR where quantification can be done while the amplification is under process. This is called ‘real-time PCR’ because it allows quantifying the increase in the amount of DNA as it is amplified. Several different types of real-time PCR systems are available having their own advantages and disadvantages. RT-PCR is performed on a real-time machine that has a function of both thermal cycling and also quantitative fluorimetry that will quantify the amount of DNA as it is amplified during the process (Rao and Singh 2008). 143 6.5.8.1 TaqMan Assay In the TaqMan assay system, the real-time PCR is carried out with the help of a set of primers specific for an amplicon and a TaqMan probe that is also complementary to the target amplicon. TaqMan probes depend on the 50 -nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridised to the target amplicon (Rao and Singh 2008). The TaqMan probe consists of two types of fluorophores linked to the 50 and 30 ends of the probe, which are the fluorescent parts of reporter proteins (green fluorescent protein, GFP often-used fluorophore). While the probe is attached or unattached to the template DNA, before the polymerisation starts, the quencher (Q) fluorophore, usually a long-wavelength coloured dye, such as red, reduces the fluorescence from the reporter (R) fluorophore (usually a shortwavelength coloured dye, such as green) by the use of mechanism known as fluorescence resonance energy transfer (FRET), which is the inhibition of one dye caused by another without emission of a proton. The reporter dye is found on the 50 end of the probe and the quencher at the 30 end. First the probe binds to amplicon by following Chargaff’s base pairing rule to the template and the primer set as well. When the amplification is initiated by the Taq DNA polymerase from the primer 30 ends, the TaqMan probe is degraded due to the 50 -nuclease activity of the DNA polymerase thereby releasing the reporter dye and the quencher free. As FRET no longer occurs, the fluorescence increases by each cycle, proportional to the amount of amplicon obtained/probe cleavage (Ha et al. 1996). 6.5.8.2 Molecular Beacons Molecular beacons also use FRET to detect and quantify the PCR amplicons via a fluor coupled to the 50 end and a quench attached to the 30 end of an oligonucleotide. They slightly differ from TaqMan probes as they are designed to remain intact during the amplification reaction and must rebind to target in every cycle for signal measurement. Molecular beacons form a stemloop structure when free in solution. So the fluor 144 and quencher molecule come nearer that prevents the probe from fluorescing. When a molecular beacon is denatured and hybridised to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. Molecular beacons, like TaqMan probes, can be used for multiplex assays by using spectrally separated fluor/quench moieties on each probe. As with TaqMan probes, molecular beacons can be expensive to synthesise, with a separate probe required for each target (Ma et al. 2006; Rao and Singh 2008). 6.5.8.3 Scorpion Probes With scorpion probes, sequence-specific priming and PCR product detection is achieved using a single oligonucleotide. The scorpion probe maintains a stem-loop configuration in the unhybridised state. The fluorophore is attached to the 50 end and is quenched by a moiety coupled to the 30 end. The 30 portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 50 end of a specific primer via a nonamplifiable monomer. After extension of the scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed. The advantage of this configuration is that the unimolecular nature of the primer–probe scorpion probe allows for faster hybridisation kinetics. This may be useful for high-volume screening assays (Ma et al. 2006; Rao and Singh 2008). 6.5.8.4 SYBR Green Dyes SYBR green provides the simplest and most economical source for detecting and quantifying PCR products in real-time PCR reactions. SYBR green binds double-stranded DNA and upon excitation emits fluorescence. Thus, as a PCR product accumulates, fluorescence increases. The advantages of SYBR green are that it is inexpensive, easy to use and sensitive. The disadvantage is that SYBR green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which 6 Detection of Plant Viruses in Seeds results in a wrong quantification of the target concentration. For single PCR product reactions with well-designed primers, SYBR green can work extremely well, with spurious non-specific background only showing up in very late cycles (Ma et al. 2006; Rao and Singh 2008). The same real-time PCR principle can be used to detect and quantify the presence and titre of viruses in plant samples as well as the seed samples in case of seed-transmitted plant viruses. Real-time immunocapture RT-PCR was used for detection of Pepino mosaic virus on tomato seed by Ling (2007). Multiplex real-time fluorescent reverse transcription-polymerase chain reaction was developed for detection of Potato mop top virus and Tobacco mottle virus (Mumford et al. 2000a), Pepino mosaic virus (Ling 2007) and Cherry leaf roll virus (Jalkanen et al. 2007). 6.6 Molecular Markers Molecular markers are any kind of molecule indicating the existence of a chemical or a physical process. Molecular markers include biochemical constituents (e.g. secondary metabolites in plants) and macromolecules (e.g. proteins and deoxyribonucleic acid). Strauss et al. (1992) distinguished the molecular markers into two classes. Biochemical molecular markers derived from the chemical products of gene expression, that is, protein-based markers and molecular genetic markers derived from direct analysis of polymorphism in DNA sequences, that is, DNA-based markers. DNA markers can be used to diagnose the presence of the gene without having to wait for gene effect to be seen. The marker-assisted selection (MAS) permits the breeder to make earlier decisions about the further selections while examining the fewer plants. An added advantage in breeding for disease resistance behaviour is that this could be done in the absence of pathogen including viruses once marker information is available. Many plant viruses are recognised as emerging or re-emerging or new-emerging viruses which have to be studied rapidly and thoroughly for their aetiology, ecology and References epidemiology. Emergence of new strains or new seed-transmitted viruses is a major thrust area of research since Brown’s development of molecular marker to track the movement of viruses in the plant system and attempts were made in this regard to locate genes conferring resistance to certain seed-transmitted viruses. For example, Timmerman et al. (1993) determined the location of Sbm-1 on the Pisum sativum genetic map by linkage analysis with eight synthetic molecular markers. Analysis of the progeny of two crosses confirmed that Sbm-1 is on chromosome six. The inclusion of Fed-1 (encoding ferrodoxin1) and Prx-3 (encoding peroxidase) among the markers facilitated the comparison of this map with the classical genetic map of pea. The Sbm1 gene is most closely linked to RFLP marker GS 185 as a marker for Sbm-1 in breeding programmes. The GS 185 hybridisation pattern and virus resistance phenotypes were compared in a collection of breeding lines and cultivars. In recent years, Smykal et al. (2010) have followed marker-assisted pea breeding against PSbMV and reported that resistance to PSbMV is conferred by a single recessive gene e1F4E marker localised on LG IV (Sbm1 locus). Even in the breeding programmes of soybean, where the Soybean mosaic virus is one of the major problems, the molecular markers of all the three resistance genes have been developed based on fine mapping with several molecular techniques such as restriction fragment length polymorphism (RFLP), random amplified polymorphism DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR) and single-nucleotide polymorphisms (SNP) (Yu et al. 1994; Hayes et al. 2000; Shi et al. 2008; Jeong et al. 2002; Jeong and Saghai Maroof 2004; Hwang et al. 2006; Shi et al. 2008). Molecular markers were also used in identifying the resistance genes for virus diseases in vegetatively propagated crops, as seen in tristeza virus resistance programme in Poncirus trifoliata (Mestre et al. 2007). 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J Virol Methods 13:121–128 7 Ecology and Epidemiology of Seed-Transmitted Viruses Abstract This chapter describes how knowledge of epidemiology helps in understanding the seed-transmitted virus disease spread and in framing suitable management measures. Seed infection is epidemiologically important as this is the primary source of inoculum and forms the starting point for the initiation of the disease. The epidemics of the virus diseases in a particular region are the result of complex interactions between various physical, chemical and biological factors, and major epidemics occur when conditions influencing the virus, host and its vector synchronise. The virus disease epidemics depend on the interaction of four components, namely, the pathogen, the vector, the plant and the environment. Among the seed-transmitted viruses, some have limited host range and some others have wide host range. Either annual or perennial or both are affected with virus diseases. Under field conditions, the weed and wild hosts are important as they are the reservoirs for the virus, vector or both. Survival of the virus in the seed for a long period also plays an integral role in virus perpetuation in off seasons. Seed-transmitted viruses are also vector specific, and aphid or beetle or nematodes or fungi play major role in causing epidemics based on the time of emergence, light, humidity, temperature and wind velocity, and abundance of vector population is a significant factor that determines spread of virus diseases both in time and space. Vectors also may have limited host plants or may be polyphagous. The time and number of vectors visiting the crop varies considerably with species over years. Pollen and seed transmission are closely related factors in virus epidemiology. A number of virus diseases spread horizontally through pollen in a number of fruit and vegetable crops. The epidemiology of virus diseases also depends on different pathogen strains which vary in virulence, host range and transmissibility. If the forecasting system is developed against a particular virus disease, it will help to operate an early warning system or to select growing season or areas for crop growing. Examples of how epidemiological information can be used to develop effective integrated disease management strategies for diverse situations are described. K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 7, © Springer India 2013 165 166 7.1 7 Introduction Epidemiology of seed-transmitted viruses provides a scientific modern theory of epidemic development. Vander Plank (1963, 1982) has provided a unifying concept for its quantitative study and the use of mathematical models to describe, interpret and predict the progress of epidemics insight for effective disease management based on certain principles like environmental factors, sources of infection, vectors and their population dynamics. The value of epidemiological approach lies in its ability to stimulate questions on ‘How many of the viruses are seed transmitted? How are they spreading? How much infection starts from sources other than seeds?’ Vander Plank (1963, 1982) suggested that the relationship between the amount of initial inoculum (Xo), average infection rate (r) and the time (t) over which infection occurs determines the amount of disease (x) that develops. The equation X D Xo ert helps in determining the rate of disease at a given moment of time. Zadoks and Schein (1979), Leonard and Fry (1986) and Khetarpal and Maury (1998) have considered disease control from an epidemiological standpoint and a primer as well as a base in advanced studies. 7.1.1 Primary Inoculum Source Seed infection is epidemiologically important because this is the primary source of inoculum and forms the starting point for the initiation of the disease. It ensures virus association with the planted crop. As the infected seeds are randomly dispersed in the field, the infected dispersed seedlings serve as sources of inoculum for secondary spread. When the infected seedlings are the only virtually source of inoculum, seed transmission plays a critical role in virus epidemiology as the seed-transmitted viruses appear to be the sole and or the primary source of inoculum. In considering the inoculum threshold of seed-transmitted viruses (i.e. the maximum amount of inoculum that can be tolerated), this aspect is by far the most significant. For example, BSMV being seed transmitted to a high degree Ecology and Epidemiology of Seed-Transmitted Viruses has no known vector but still present in most barley-producing areas of the world. No weed or wild grass is a significant reservoir of this virus, but seed transmission in a single plant species, Hordeum vulgare L., is the most important factor for survival from year to year (Timian 1974). In the United Kingdom, for the two beetletransmitted viruses, Broad bean stain and Broad bean true mosaic, which are seed transmitted, the infected seed is the main source of spread for these viruses in spring-sown field bean crop (Cockbain et al. 1975). Seed-transmitted viruses transmitted by aphids are also of great concern as infected seed serves as the primary source of inoculum. Some of the most economically important plant virus diseases of this category are BCMV, LMV, CMV, PMV, PStV, SMV and Black eye cowpea mosaic viruses which are extensively studied at different virus–host combinations (Irwin et al. 2000; Jones 2000; Sharma et al. 2004; Dinesh et al. 2005; Coutts et al. 2009; Udayashankar et al. 2010). The perusal of the Table 1.2 indicates that a number of seed-transmitted virus diseases occur in majority of crop plants cultivated under varied environmental conditions. The epidemics of these virus and viroid diseases in a particular region are the result of complex interactions between various physical, chemical and biological factors, and major epidemics occur when conditions influencing the virus, host and its vector synchronise. Hence, it is imperative to collect information on different aspects like the nature of the virus, the percent seed infection as the primary inoculum and proximity of the host crop to the vector, the number and timing of vector appearance, the diurnal temperature range, the rains, the RH of the air, the dew, the weed hosts of the vector and virus and the duration of the crop and the nature of the produce fruit, seed and tuber. This will help to draw reasonable predictions about disease epidemics and formulate suitable management measures. In a nutshell, the epidemiology of seed-transmitted virus diseases like in any other pathogenic diseases depends on interaction of four components: the pathogen, the vector, the plant and the environment. Extensive and detailed epidemiological studies made 7.2 Host Plant in many crops over long periods have shown the highly complex pattern of interacting factors in the spread of seed-transmitted viruses. More information can be obtained from review articles and textbook chapters (Thresh 1974; Jones 2004; Maramorosch et al. 2006). 7.2 Host Plant New crops have been introduced for the first time in new regions for their high yields and crop uniformity. These introductions will flourish in the new environment, where they will be free for some time from virus diseases that were prevalent in the country of origin. In other instances, catastrophic losses have occurred when exotic crops were soon attacked by indigenous pathogens not previously encountered in the country of origin. For example, when ‘Fasolt’, a hybrid cultivar of Brussel bread from Holland was introduced into England, it was severely affected by Turnip mosaic and Cauliflower mosaic viruses (Tomlinson and Ward 1981). The host plants and the type of vector involved also influence the efficiency of virus transmission as in the case observed by Jansen and Staples (1970) with beetle-transmitted Cowpea severe mosaic virus (CpSMV). Both cowpea and soybean are susceptible when mechanically inoculated. Beetles, however, transmitted this virus from cowpea to cowpea at high levels of efficiency but at very low level to soybean. CpSMV was observed causing a very severe disease in soybeans in the fields at Puerto Rico (Thomgmeearkom et al. 1978) and Brazil (Anjos and Lin 1984). The disease, however, occurred only in few scattered soybean plants immediately adjacent to cowpea planting which was severely infected with CpSMV. Apparently, the inefficiency of transmission of the virus by beetles from cowpea to soybean or soybean to soybean had restricted the disease spread in soybean. The growth habits of the crop plants vary in terms of height and lifetime, that is, annual or perennial. The transmission of seed-transmitted viruses has been recorded both in annual and perennial crops (Table 1.2). Annual crops are 167 highly susceptible to viruses if extensively cultivated with high rate of seed infection, and vector presence results in maximum disease incidence. Among the seed-transmitted viruses, some have limited host range like SMV, and others have a wide host range (Irwin and Goodman 1981). Similarly, BCMV- and LMV-infected seedlings raised from the seed are considered to be the primary source for initiating new infections at the beginning of each season. These viruses spread to other plants through vectors, and plants will subsequently act as secondary sources of infection depending on their susceptibility. In contrast, some seed-transmitted viruses of cucumovirus, tobravirus and nepovirus groups have a very large number of natural and experimental hosts comprising of annuals and perennials. For example, CMV can infect over 470 species of at least 67 families (Kaper and Waterworth 1981). Similarly, Tobacco rattle virus has more than 400 experimental hosts belonging to 50 mono- and dicotyledonous families (Schmelzer 1957). About 237 members of Gramineae have been shown as experimental hosts of various strains of BSMV (Jackson and Lane 1981). Arabis mosaic virus infects 93 out of 136 species in 28 families and Tomato black ring virus has a host range of 76 species in 25 families (Schmelzer 1963). Wherever the virus has a wide host range, the source of virus inoculum was necessarily another crop plant. For example, in Southern USA, peanut infected with PMV not only serves as a source of infection but spreads to nearby soybean crop (Demski and Kuhn 1974) and infected lupins after peanut crop (Demski et al. 1983). Even in beet and potato crops, a number of seedtransmitted viruses are recorded, and the tubers that escape during harvest or grown in waste fields serve as a source for the virus, the vector or both (Wallis 1967). Under field conditions, the weed and wild hosts are important as they are the reservoirs for the virus, vector or both (Duffus 1971; Murant 1981a, b; Sastry 1984a; Bos 1981; Thresh 1981); aid in virus perpetuation during the main or off season; serve as inoculum source; and exert great infection pressure. The survival strategies of 168 7 viruses are just as diverse as their shape, size and physicochemical characteristics. Harrison (1981) distinguished between ‘WILPAD’ viruses from tobravirus and nepovirus groups that seem particularly well adapted for survival in wild plants and ‘CULPAD’ viruses including members of the tobamo and potex groups which seem to thrive best in cultivated plants. A majority of the seed-transmitted viruses infecting economic crops have weed hosts widely which are distributed along irrigation channels, ditches, road sides, orchards, by the side of railway tracks, in fallow lands and in neglected crops. Infected perennial weeds are more dangerous than annuals since they live longer. The chances of outbreak of disease epidemics will be more in areas where infected weeds are abundant and support the multiplication of a particular vector. The importance of source of virus near or especially within a crop has been demonstrated for many virus–crop combinations (Heathcote 1970; Duffus 1971; Bos 1981; Sastry 1984a, b; Jones 2004; Maramorosch et al. 2006). Within plantations, the infected weeds are particularly important since they exert the greatest infection pressure. Biennial or perennial weeds play a significant role in facilitating the survival of viruses that attack annual plants in areas with growing seasons restricted by prolonged drought or cold. Examples of weed hosts as reservoirs of seed-transmitted viruses are many. Tomlinson et al. (1970) reported that in Britain, CMV overwinters in chickweed (Stellaria media), a source of infection for the following season’s lettuce crop. CMV also overwintered on weeds like Echinocystis sp. (9–55%) (Doolittle and Gilbert 1919; Doolittle and Walker 1925), Stellaria sp. (21–40%), Spergula sp. (2%) and Cerastium sp. (2%) (Tomlinson and Carter 1970a, b). In Australia, lupin and clover infected by CMV strain persist between growing seasons in eight alternative host species (McKirdy and Jones 1994). In Europe, overwintering groundsel (Senecio vulgaris) was implicated as the main source of LMV in lettuce, and the infected plants survived in the winter (Kemper 1962). In USA, the leguminous weed Desmodium canum is the alternate host of PMV (Demski et al. 1981). The woody legumi- Ecology and Epidemiology of Seed-Transmitted Viruses nous herb Tephrosia villosa is a common host for CMMV in coastal Kenya (Bock et al. 1981). Bean common mosaic was detected in legume weed Rhynchosia minima (Meiners et al. 1978). At Washington, PSbMV overwintered in hairy vetch (Vicia villosa) and volunteer pea plants under field conditions (Stevenson and Hagedorn 1973). In South Carolina, pigweed (Amaranthus hybridus) formed the source of inoculum for TRSV in squash crop (Sammons and Barnett 1987). In Hungary, the role of weed host on the ecology of CMV in beans (P. vulgaris) has been well studied by Horvath (1983). Hani (1971) demonstrated potentiality of the inoculum due to host and crops. It was noticed that the rates of infection of Stellaria media by CMV were higher in certain areas of Switzerland with continuous cultivation of tobacco than in regions with regular crop rotation. But the infection of tobacco in each year mainly originated from S. media, wherein the virus was also seed transmitted. In fields where tobacco was grown for the first time, the degree of S. media infection rapidly increased during the season. Even for Tomato ring spot virus which is seed transmitted in peach and apple, the weed hosts like Taraxacum officinale, Rumex acetosella and Stellaria sp. proved to be important sources of infections in Indiana, New York and Pennsylvania states in the USA, respectively (Mountain et al. 1983; Powell et al. 1984). In certain cases, the weed hosts not only served as sources of virus infection during the crop season but also for the virus to overwinter and aid in their dispersal to long distances through man, wind and water. A large number of viruses are thus transmitted through the seed of weed hosts which act as a major factor in the ecology of seed-transmitted virus diseases. This factor enhances the chances of virus survival throughout the year and helps in virus spread from crop to crop through the vectors. As early as 1919, Doolittle and Gilbert emphasised the role of wild cucumber (Echinocystis lobata) as a source for the spread of CMV which is also seed transmitted in this host. To cite a few more examples, TRSV was carried in the seed of dandelion weed T. officinale to an extent 7.2 Host Plant of 9–36% and caused severe mosaic diseases of soybean and other crops (Tuite 1960), and the same virus in South Carolina was carried in the seed of pigweed, Amaranthus hybridus, to an extent of 21% (Sammons and Barnett 1987). Murant and Lister (1967) listed several nepoviruses and the hosts in which they were seed transmitted. For example, four viruses were carried by seed of infected Stellaria media plants (Arabis mosaic, Raspberry ring spot, Strawberry ring spot and Tomato black ring viruses), two each by the seed of Capsella bursa-pastoris and of Chenopodium album (Arabis mosaic and Tomato black ring viruses). It was also proved that even tobra group of viruses were carried through the seed of S. media and Myosotis arvensis, but the extent of seed transmission was very low (Murant 1970). As the virus is retained in the seed of certain weeds over long periods, the chances of virus survival are more in the off season and also when the crop is grown intermittently. In UK, the weed, Stellaria media, retained CMV for 21 months in buried seed (Tomlinson and Walker 1973). Hani (1971) reported that the virus was infectious in the same virus–host combination in the seed for 18 months. Even Tomato black ring and Raspberry ring spot viruses were retained in seeds of weeds Capsella bursa-pastoris and S. media for over 6 years (Lister and Murant 1967). However, in the case of nematode-transmitted viruses, they were retained in the vector Longidorus spp. for only 8–9 weeks, and the presence of infected weed seeds was essential for longer survival of some of the nepoviruses. However, viruses transmitted by Xiphinema spp. were retained in the vector for many months, and the role of weed hosts seemed to be less important as the infected woody perennial hosts served as source of inoculum (Murant 1970). Detection and management of seed-transmitted viruses are more difficult when carried in the crop and weed plants without exhibiting any symptoms of the disease. For example, viruses like BSMV in barley and wheat and Avocado pear sunblotch in avocado were often symptomless (Neergaard 1977). Similarly Murant and Lister (1967) detected Tobacco black 169 ring virus in 11 symptomless weed species, and many of these hosts also contained Raspberry ring spot virus. Tuite (1960) recorded TRSV in two symptomless weed hosts. AMV and Strawberry latent ring spot virus were also detected in a number of symptomlessly infected weed species (Taylor and Thomas 1968). A similar situation was recorded for CMV and LMV in a number of weed hosts (Ullrich 1954; Kemper 1962; Tomlinson et al. 1970). Even in wild germplasm, the virus diseases are carried through the off season, and their spread through the exchange and transport from each country has been proven beyond doubt. For example, true seed of Solanum spp. is infected with Andean potato latent virus (Jones and Fribourg 1977), Potato virus T (Salazar and Harrison 1978) and Potato spindle tuber viroid (Diener and Raymer 1971). The reader can refer to the exhaustive compilation made in ‘Plant health and quarantine in international transfer of genetic resources’ (Hewitt and Chiarappa 1977). The risk of spreading viruses even between continents through the exchange of wild plants by botanical gardens, germplasm collectors and through breeding nurseries is very high due to latent infection and also lack of knowledge among the in-charge personnel of botanical collections. Besides acting as a source of virus infection, weeds and wild plants also act as breeding foci for the vectors. In the Pacific Northwest, USA, Myzus persicae overwinters on peach trees in the egg stage and on weeds in drainage ditches as viviparous forms (Wallis 1967). Some of the nematode vectors like Longidorus elongatus multiply in large numbers on some of the weed hosts like S. media, Poa annua and Mentha arvensis (Taylor 1967; Thomas 1969). In India, some of the weed hosts like B. arvensis harbour four to five aphid species like M. persicae, A. gossypii, Brevicoryne brassicae and Rhopalosiphum pseudobrassicae. These are major vectors of mosaic diseases and affect economic crops in which the virus is seed transmitted (Sastry 1984a, b). Similarly, Sorghum arundinaceum was a natural host for both Peanut clump virus and its fungal vector Polymyxa graminis (Thouvenel and Fauquet 1981a, b). 170 7.3 7 Vectors Vector biology and behaviour are of paramount importance, since they have a profound influence on the temporal and spatial spread of virus diseases. The extent of virus spread mostly depends on the density and movements of the vector population. These movements may be of a local nature like between plants in a field or of dispersive type from breeding areas/overwintering sites to crops. Factors responsible for these movements involve biotic factors like life history of the insect, its range and host preference. Aphids are the vectors for a sizeable number of seed-transmitted viruses. Their host range, the time of emergence and abundance of winged forms among the population are the significant factors that determine spread of aphid-transmitted virus diseases both in time and space. Aphid species are mostly specific to one or several closely related host plants, while a few are polyphagous and infest hundreds of plant species. Spread of nonpersistent viruses is greatly affected by the flight behaviour of aphid vectors. The take off of aphid vectors takes place either from the surface of the leaf or by the insect dropping and kicking itself into flight. Take off is influenced by light and temperature and does not occur at wind speed above 3 miles per hour (Kennedy and Booth 1963; Kennedy et al. 1962). The presence of an efficient beetle vector can be sufficient in spread of the virus resulting in serious crop losses, even though seed transmission levels are very low. In Eastern Scotland, seedtransmitted infection of Broad bean stain and Broad bean true mosaic viruses in commercial broad bean fields showed low levels of diseased plants and no detectable further virus spread to healthy beans because of low population of weevil vector, A. vorax (Jones 1978). In Southern England, however, where A. vorax is present, the secondary spread of virus resulted in up to 90% infection of these two viruses (Cockbain et al. 1975). Ecology and Epidemiology of Seed-Transmitted Viruses 7.3.1 Factors Influencing Vector Movement Temperature, humidity, wind velocity and season are some of the factors which influence the vector movement. Generally, increase in relative humidity to a higher level (above 80%) and high temperatures (90ı F) slowed down the flight activity of the aphids, while low temperature also discourages aphid flights. Light intensity between 100 and 1,000 f.c. made little differences in flight, but below 100 f.c., flight activity declined rapidly. Long-distance dispersal of the viruliferous vectors takes place through wind currents. The flights of the winged aphids would be discouraged when wind speed is above 1 m/s (Carter 1962; Eastop 1977). The importance of low level of jet winds in transporting aphids over a long distance has been related to disease outbreaks. Johnson (1967) suggested that certain nonpersistent seed-transmitted viruses can also be transported over relatively long distances of 60 km and that they might be transported three times more distance on nonstop flights. Planting over areas of upward wind rather than downward wind helps in reducing the virus spread. The total vector population and per cent infective individuals in the population also play an important role in the spread of disease. For example, in Eastern Scotland, Apion vorax, an efficient weevil vector of Broad bean stain (BBSV) and Echtes ackerbohnen mosaik viruses (EAMV syn. Broad bean true mosaic), is absent, and hence, no detectable spread of BBSV and EAMV was observed. Therefore, there is a possibility of producing Vicia faba seed free from these two viruses with little effort in this area (Jones 1980). However, the cumulative effect of a large population and high percentage of viruliferous vectors causes catastrophic results. In California, where aphids were numerous, lettuce seed with as low as an infection rate of 0.1% and below resulted in high LMV infection (Zink et al. 1956). Similarly in India, about 15% seed transmission of CpBMV resulted in 100% incidence in kharif and 27% in 7.3 Vectors summer, supporting the role of aphid vectors in two different seasons with the same amount of initial inoculum (Sharma and Varma 1983). In contrast, soil-borne virus diseases tend to spread slowly after their initial appearance due to limited mobility of their vectors, namely, the Dorylaimaida species of nematode Xiphinema, Trichodorus, Paratrichodorus and Longidorus; vectors of tobra and nepo group of viruses were unable to carry the viruses to longer distances. Their migration is about 30 cm per year, and hence, the virus spread takes place either through the infected vegetative planting material or through seed and pollen. Even the soil type also can help in predicting the occurrence of nematode-transmitted viruses like Tobacco rattle virus (TRV) with reasonable accuracy. In soil surveys conducted in Britain, nematode vector, Trichodorus premitivus, occurs predominantly in sandy loams, whereas Paratrichodorus pachydermus was most prevalent in loamy sand soils (Alphey and Boag 1976). In arable soils of Scotland, about 80% of the trichodorid population was found to carry TRV (Cooper 1971). Similarly, Longidorus macrosoma occurs in clay with flint and alluvial soils in Southern England, and Longidorus attenuatus is found in sandy soils mainly in East Anglia (Taylor and Brown 1976). Even the soil temperature and soil moisture have an impact on movement and feeding of nematode vector. At low soil moisture, the nematodes are immobilised, and in dry soils, they are killed due to desiccation, which is more likely to occur in the surface layers of soil than at considerable depths where a large proportion of the nematode population is found (Harrison 1977, 1981). Even against fungal vectors like Polymyxa graminis, the vector of Peanut clump virus (PCV), temperature plays important role in vector transmission; when temperatures are less than 25ı C, only negligible PCV incidence is recorded. The high temperatures that prevail in India and West Africa during the main peanut-growing season (monsoon) are conducive to natural virus transmission. High temperatures during summer in India followed by monsoon rains appear to break the dormancy of resting sporangia of fungal 171 vectors P. graminis. Hence, the solarisation effectively reduces clump disease. Even wellcultivated soils profusely irrigated and covered with two layers of transparent polyethylene sheets for at least 70 days during summer months reduce PCV incidence (Reddy et al. 1988). The timing and number of vectors visiting the crop vary considerably with species over the years. For instance, Irwin and Goodman (1981) and Irwin et al. (2000) while working with SMV in soybean observed higher landing frequency of Rhopalosiphum padi in spring 1976, 1977 and 1978, but still, landing frequency was higher in the fall. Even landing rates of Aphis craccivora were high in the spring of 1976 but low throughout the 1977 and 1978 seasons. In contrast, landing rates of Rhopalosiphum maidis were always higher in the summer and fall and that of M. persicae and M. euphorbia were relatively low and concentrated in the middle of the season. It was also observed that the SMV spread from the source outward was greater in downwind than upwind. Since different species of vectors that transmit the seed-transmitted viruses alight with different frequencies at different times during the growing season, a timing factor must be included to estimate economic losses. In virus epidemiology, accurate measurement of vector numbers and species alighting on plant foliage is most essential. At present, different types of techniques such as use of yellow pan trap, vertical nets, cylindrical sticky thread trap and the Johnson–Taylor suction trap are employed to monitor the vectors, depending on the crop growth and vector behaviour. The traps should be mounted in such a way that they can be maintained at canopy level throughout the crop-growing season. In epidemiological studies, ELISA application is essential for detecting the virus in the vectors. For example, Gera et al. (1978, 1979) reported detection of CMV in single apterous Aphis gossypii by ELISA. Aphids probing on plants infected with a non-transmissible strain of CMV fail to acquire CMV, and short feedings of 1.5 min on healthy plants render viruliferous aphids nonreactive in ELISA tests. These results suggest the use of ELISA for CMV epidemiological studies 172 7 (Gera et al. 1978). However, ELISA application to winged aphids which acquire and carry the virus under natural conditions has been demonstrated in certain virus-host combinations. 7.4 Pollen Transmission Infected pollen plays an integral role in the spread of viruses of some woody plants. This topic has been reviewed by the number of workers, namely, Shepherd (1972), Hardtl (1978), Phatak (1980), Mandahar (1981, 1983, 1985, 1986) and Mink (1993). Transmission of plant viruses through pollen takes place both vertically and horizontally. In case of vertical transmission of viruses, the pollination and fertilisation of healthy ovules by infected pollen result in the formation of infected seeds which on germination produce infected seedlings. For effective horizontal spread through infected pollen, the viruses invade and systematically infect the ovule-bearing mother plant, and large quantities of infected pollen are released to infect the contemporary healthy plants. The virus transmission through pollen is economically important in cross-pollinated woody perennial plants than with annual crops wherein both vertical and horizontal transmission take place. Transmission of certain seed-transmitted plant viruses through pollen is prevalent in bromovirus, alfamovirus, cucumovirus and ilarvirus groups. Virus particles are externally or internally pollen borne and have been observed in electron micrographs of infected pollen or pollen extracts as with BSMV (Gold et al. 1954; Carroll 1974), TRSV (Yang and Hamilton 1974), TMV (Hamilton et al. 1977) and PNRSV (Kelley and Cameron 1986). The entry of the virus from the pollen into the ovules takes place along with the male gametes which move through the pollen tube that grows into the embryo sac. Of the two male gametes infected, one unites with the egg during fertilisation and the other unites with polar nuclei giving rise to endosperm. Even Potato spindle tuber viroid (PSTVd) is pollen transmitted in potato, tomato and Scopolia sinensis (Fernow et al. 1970; Kryczynski et al. 1988). The adverse effects of virus infection on pollen in terms of its Ecology and Epidemiology of Seed-Transmitted Viruses size, morphology, viability and pollen tube size have been recorded (Yang and Hamilton 1974). High level of pollen sterility resulting in poor fertilisation has been observed in certain virus– host combinations (Ryder 1964). Pollen-carrying insects primarily honey bees play a major role in transfer of virus-infected pollen to the stigma of flowers. Mink (1983) indicated the possible role of honey bees for longdistance spread of PNRSV from California to sweet cherry orchards in Washington. Generally, a greater percentage of seedtransmitted virus transmission occurs when the mother plant is infected than when pollen is the sole source of infection. Walter et al. (1992) reported high percentage of seed transmission of Tobacco streak virus in bean plant when anthers from infected plants were used to pollinate healthy plants. In Europe,Rubus spp. particularly R. occidentalis and R. idaeus are infected with Black raspberry latent ilarvirus Syn. Tobacco streak ilarvirus and are pollen and seed transmitted. Virus transmission by pollen from imported Rubus to local plants or propagation of imported Rubus would be the practical means of establishment in Rubus in the EPPO region and is considered as quarantine pest (OEPP/EPPO 1994). In India, Tobacco streak virus (TSV) occurs in high incidence in sunflower, groundnut, mung bean, soybean and some other crop and weed hosts. The intensive seed transmission studies carried out by Prasada Rao et al. (2009) have indicated that seed transmission was not noticed in any of the hosts tested. However, TSV is transmitted in India through pollen assisted by thrips. To quote few more examples of pollen transmission, Vertesey (1976) demonstrated that in Montmorency sour cherry, seed transmission of PNRSV was 28% with pollen, 53% with mother plant infection and 88% with pollen and mother plant-combined infection. Similarly, Timian (1967) observed a high percentage (58%) of seed transmission when both male and female barley parents were infected with BSMV, moderate (46%) with female parent and low (17%) with male parent. This may become more significant if commercial hybrids are developed from male sterile barleys. Similar results were recorded 7.4 Pollen Transmission earlier by Medina and Grogan (1961) while working with BCMV infection in French beans. Pollen and seed transmission are closely related factors in virus epidemiology. Most studies on pollen transmission of viruses indicate that a greater percentage of seed contains virus when the mother plant was infected than when pollen is the sole source of infection (Bennett 1969). In Montmorency sour cherry, PNRSV was seed transmitted to the tune of 28 and 53% when only the pollen and the mother plant were infected individually and 88% with both pollen and the mother plants infected (Vertesy 1976). Similarly, in India, Sharma and Varma (1986), while working with CpBMV in cowpea cv. Pusa Dophasli, recorded seed transmission of 22%, when both parents were diseased, and 19 and 16%, respectively, when the female plant and the pollen-bearing plants were infected. Similar findings were also reported for Raspberry ring spot (Lister and Murant 1967), Prunus necrotic ring spot (Amari et al. 2007, 2009), Apple latent spherical virus (Nakamura et al. 2011), AMV (Frosheiser 1974), BCMV (Medina and Grogan 1961) and Tobacco ring spot virus and Tomato ring spot viruses (Scarborough and Smith 1975) where minimum transmission took place through infected pollen followed by infected ovules and then infected pollen and ovules. Transmission by pollen may be the principal and only method for natural spread of the disease under field conditions for some of the viruses belonging to the ilarvirus group (Davidson 1976). Pollen transmission is probably of little or no ecological significance when the host plant is mainly self-pollinated, as in the case of barley. 7.4.1 Epidemiological Role of Pollen-Transmitted Viruses Some researchers maintain that virus transmission through pollen is of little consequence in nature since the infected pollen cannot compete with healthy pollen during fertilisation (Shepherd 1972; Yang and Hamilton 1974). Others contend that the spread of some viruses through pollen is rapid and widespread (Cameron et al. 1973; Davidson 1976; Daubeny et al. 1978; Mircetich 173 et al. 1980). For the first time, Mandahar and Gill (1984) have brought out a review projecting the epidemiological role of pollen in the spread of viruses. Based on the available literature on the role of pollen in the epidemiology of seed-transmitted viruses, the virus groups were identified and presented in Table 7.1, and the details are discussed. 7.4.1.1 Category A In this category, all externally pollen-borne viruses and viruses causing pollen sterility are included (Table 7.1). No pollen transmission of these viruses was recorded since they are externally pollen borne and are of no epidemiological importance. In case of viruses causing pollen sterility, the seeds are not produced, and the pollen infection becomes a liability to the host plants. Some information is available on the detection of virus particles in pollen homogenates, but the viruses could not be pollen transmitted to seeds, nor any abnormality in morphology or development of anthers and pollen grains was recorded, namely, Echtes Ackerbohnen mosaik virus in Vicia faba, Potato virus T in Datura stramonium and Nicandra physalodes and SqMV in squash (Vorra-urai and Cockbain 1977; Salazar and Harrison 1978; Alvarez and Campbell 1978). It appears that these viruses must be externally pollen borne on exine and should be regarded as such; none of these viruses are pollen transmitted in their respective hosts. 7.4.1.2 Category B The viruses included in this category are detected in the pollen extracts from infected plants, but no experiments have been conducted either in the greenhouse or under field conditions to find out whether cross-pollination of emasculated flowers on healthy plants leads to the formation of infected seeds. Hence, these viruses should not be considered as vertically pollen transmitted unless more definite evidence is present in these category of viruses and are of no epidemiological importance for disease spread in nature. The list of viruses in this category with host plants are cited in Table 7.1. 174 7 Ecology and Epidemiology of Seed-Transmitted Viruses Table 7.1 Grouping of pollen-transmitted viruses into categories based on the importance of pollen transmission of plant viruses in disease spread in nature/greenhouse Virus Category A (a) Externally pollen-borne viruses Apple chlorotic leaf spot Potato virus X Sowbane mosaic Brome mosaic Tobacco mosaic Echtes Ackerbohnen mosaik Potato virus T Host Reference Chenopodium quinoa; C. amaranticolor Petunia hybrida Atriplex coulteri Phaseolus vulgaris Vigna unguiculata Vicia faba Datura stramonium; Nicandra physalodes Cucurbita pepo Cadman (1965) Neergaard (1977) Bennett and Costa (1961) Hamilton et al. (1977) Hamilton et al. (1977) Vorra-urai and Cockbain (1977) Salazar and Harrison (1978) (b) Viruses causing pollen sterility Beet yellows Datura quercina K. Onion mosaic Tobacco ring spot Tomato ring spot Beta vulgaris Datura stramonium Allium cepa Glycine max Pelargonium hortorum Larsen (1981) Blakeslee (1921) Cheremuskina (1977) Yang and Hamilton (1974) Murdock et al. (1976) Category B Bean southern mosaic Bean yellow mosaic Cherry rasp leaf Cucumber mosaic Onion yellow dwarf Pelargonium zonate spot P. vulgaris P. vulgaris Prunus sp. Stellaria media Allium cepa Nicotiana glutinosa Crowley (1959) Frandsen (1952) Williams et al. (1963), Hansen et al. (1974) Tomlinson and Carter (1970a, b) Louie and Lorbeer (1966) Gallitelli (1982) Squash mosaic Category C Arracacha virus B Solanum tuberosum Beet cryptic Beta vulgaris Broad bean stain Vicia faba Cowpea aphid-borne mosaic V. sinensis Cowpea banding mosaic V. sinensis Elm mosaic Sambucus racemosa; Ulmus americana Elm mottle Syringa vulgaris Lettuce mosaic Lactuca sativa Lychnis ring spot Lychnis divaricata; Silene noctiflora Potato virus T Solanum tuberosum Raspberry ring spot Fragaria virginiana; Rubus sp. Tomato black ring Rubus sp. Tomato ring spot Pelargonium hortorum Category C1 Alfalfa mosaic Barley stripe mosaic Bean common mosaic Category D1 Cherry yellows Prune dwarf Medicago sativa Hordeum vulgare Phaseolus vulgaris Prunus cerasus P. cerasus Alvarez and Campbell (1978) Jones (1982) Kassanis et al. (1978) Vorra-urai and Cockbain (1977) Tsuchizaki et al. (1970) Sharma and Varma (1984) Schmelzer (1966), Callahan (1957) Schmelzer (1969) Ryder (1964) Bennett (1959) Jones (1982) Lister and Murant (1967) Lister and Murant (1967) Scarborough and Smith (1975) Frosheiser (1974) Carroll (1974), Carroll and Mayhew (1976a), Hemmati and McLean (1977) Medina and Grogan (1961) Gilmer and Way (1960, 1963), George and Davidson (1963) Gilmer and Way (1960), Ramaswamy and Posnette (1971) (continued) 7.4 Pollen Transmission 175 Table 7.1 (continued) Virus Host Prune necrotic ring spot Cucurbita maxima Tobacco streak (Black raspberry latent strain) Rubus occidentalis Category D2 Cherry leaf roll Prunus necrotic ring spot Raspberry bushy dwarf Juglans regia Prunus cerasus Rubus idaeus R. occidentalis R. ursinus var. loganobaccus Reference Das and Milbrath (1961) Lister and Murant (1967) Mircetich et al. (1980) Gilmer and Way (1960), George and Davidson (1963), Cameron et al. (1973), Davidson (1976) Cadman (1965). Barnett and Murant (1970), Murant et al. (1974), Daubeny et al. (1978), Jones and Wood (1979) Murant (1976) Barnett and Murant (1970), Murant (1976) Source: Mandahar (1978) 7.4.1.3 Category C The viruses included in this category are detected in pollen triturates and/or shown in glasshouse or field experiments on cross-pollination. They are vertically transmitted through pollen and led to the production of infected seed in emasculated healthy female plants. However, their vertical pollen transmission to produce infected seeds has not been detected in nature, and the mother plant does not get back-infected. In this category, there are 13 viruses which are pollen transmitted, and pollen transmissibility of these viruses may not pose any epidemiological threat (Table 7.1). experiments that the viruses from this category are transmitted through pollen and led to infected seed production on emasculated healthy female plants. All these viruses are not only vertically transmitted but also horizontally transmitted through pollen to systemically back-infect the pollinated (by hand or nature) mother plants. Vertical pollen transmission of these viruses is also of little epidemiological significance since their specific hosts are mostly perennial plants. However, based on horizontal pollen transmission, these viruses are further partitioned into two subcategories. 7.4.1.4 Category C1 In this category, the viruses are similar to those in ‘C’ except that their pollen transmissibility appears to be of some limited epidemiological value in disease spread of AMV, BSMV and BCMV; however, these viruses cannot reinfect the female parent. The limited epidemiological value of vertical pollen transmission of these viruses is evidenced by the fact that, usually, pollen vertically transmits the virus to more ovules. In other words, it is somewhat less efficient than ovule transmission but still transmits virus to a considerable number of seeds (Medina and Grogan 1961; Frosheiser 1974; Carroll 1974; Carroll and Mayhew 1976b; Hemmati and McLean 1977). 7.4.1.6 Category D1 Horizontal pollen transmission of viruses like Cherry yellows, Prune dwarf, PNRSV and Tobacco streak was considered of limited epidemiological importance (Das and Milbrath 1961; Gilmer and Way 1963; Lister and Murant 1967; Converse and Lister 1969; Ramaswamy and Posnette 1971). Gilmer and Way (1963) reported that only two sweet cherry and three sour cherry mother plants were back-infected with Cherry yellows virus in the growing season after pollination. They concluded that horizontal pollen transmission was not a highly efficient means of disease spread. Lister and Murant (1967) found that 5 out of the 12 hand-pollinated black raspberry plants got back-infected with Black raspberry latent virus, a strain of Tobacco streak virus, after 1 year of pollination. Later, Converse and Lister (1969) 7.4.1.5 Category D It was shown by embosoming and/or crosspollination tests under greenhouse or field 176 7 concluded that pollen is the major source of horizontal spread of this virus in Black raspberry (Rubus occidentalis) and Blackberry (R. ursinus). Subsequent observations led to revise their earlier opinion and suggested caution for cataloguing this virus among the known pollen-transmitted viruses. Costa and Neto (1976) reported thrips (Frankliniella spp.) as a vector of this virus in Brazil. However, Converse’s own observation is that this virus cannot back-infect the pollinated strawberry plants. More work is needed to resolve this issue. It is therefore tentatively placed in D1 category. 7.4.1.7 Category D2 Horizontal pollen transmission of Cherry leaf roll, PNRSV and Raspberry bushy dwarf virus (RBDV) included in this category is of great epidemiological importance in the spread of these diseases in nature. Cherry leaf roll virus causing the blackline disease of English walnut in the USA spreads horizontally by infected pollen and is a serious threat to the walnut industry in California (Mircetich et al. 1980). During 1975, it was observed that among 361 walnut trees of 10-year age propagated on Northern California Black root stock, only 58 trees showed disease; the number of diseased trees increased to 132 in 1979. This works out to be 5% increase per year. The second orchard, comprising of 1,044 fifteen-year-old walnut trees propagated on paradox root stocks, had 97 infected trees in 1977 but increased to 139 in 1979. This increase was 4% over a 2-year period. Thus, the disease spreads faster as more and more trees were infected as pollen from diseased plants gain ascendancy over the healthy pollen in the environment. Raspberry bushy dwarf virus (RBDV) horizontally spreads naturally only through infected pollen in Red raspberry (Rubus idaeus) orchards in Canada, England, New Zealand, Scotland and USA (Cadman 1965; Murant et al. 1974; Murant 1976; Daubeny et al. 1978; Jones and Woods 1979). In British Columbia (Canada), Daubeny et al. (1978) studied its spread in red raspberry cv. BC 61-6-68 from 1973 to 1977. This selection was propagated in 1973, and 30 healthy plants free of Raspberry bushy dwarf (RBDV) and Ecology and Epidemiology of Seed-Transmitted Viruses Tomato ring spot viruses were established in three randomised blocks of ten plants per block. RBDV was first noticed in 1975 in 6 out of the 30 plants, infected presumably from pollen of the nearby infected cultivars, and increased to 11 and 14 in 1976 and 1977, respectively. Thus, about 50% of the plants became infected over a period of 2– 3 years, which is an alarming rate of horizontal spread. The disease always spreads to adjacent plants – a characteristic of pollen-transmitted viruses. Murant et al. (1974) studied the spread of RBDV in raspberry (R. idaeus) cultivar ‘Lloyd George’ in Scotland and found that after two flowering seasons, it infected 20 out of 24 test plants in field plots containing infector plants but none of the 24 test plants in plots without infectors. However, in plots without infectors, the disease appeared in 1 among 30 plants in 1971, in 16 out of 75 plants in 1972 and increased to 28 in 1973. Thus, the disease spreads to most of the plants in the first two flowering seasons in plots containing internal infection foci. In plots without infector plants, it spreads very rapidly even if a single plant gets infected from outside source. In Canada and USA, the infected pollen is the only means of natural spread of Prunus necrotic ring spot virus (PNRSV) in sour cherry orchards which was always horizontal (Cameron et al. 1973; Davidson 1976). This was the first virus in which pollen transmission was found to reinfect in some of the cross-pollinated mother plants. Way and Gilmer (1958) reported 5 out of 18 cherry seedlings (about 27% pollen transmission) got infected from a cross between an infected male and a healthy female. George and Davidson (1963) found 4 out of 14 emasculated mother plants, fertilised by pollen from infected plants, got systemically infected (about 28% pollen transmission). Subsequently, Cameron et al. (1973) and Davidson (1976) conducted 7-year test for epidemiological efficiency of horizontal pollen transmission of this virus. As many as 73.5 and 87.5% of the naturally flowering sour cherry trees in orchards became infected with Prunus necrotic ring spot virus over the 7-year period in Oregon state of USA and the spread 7.5 Viruses of the same virus at Niagara Peninsula, Ontario, Canada, respectively. The disease progress curve of this virus in sour cherry is a sigmoid curve (Davidson and George 1964), with exponential increase during the 3rd to 9th year when all the trees were infected by the virus. This happened due to infection of more and more plants by infected pollen possibly predominating in the environs of an orchard. This is also the reason why the timescale for the spread of horizontally pollen-transmitted viruses in plantation crops is always in terms of years. The virus particle of PNRSV occurs on the surface of the pollen of infected almond and cherry plants, and infectivity is retained for several days. Cole et al. (1982) have speculated on this basis that foraging bees might mechanically spread and infect the flower parts with the virus through abrasions caused by them. The virus then moves backwards and systemically reinfects the main plant through the plasmodesmata and phloem that connect the various flower parts (except the male and female sporogenous cells) with the sporophyte. In epidemiology of PNRSV-causing cherry rugose mosaic disease from some western states of USA, the mechanical transmission of the virus by honey bees is important. About 50,000 beehives are shifted each year from Washington to California for pollinating various stone fruit orchards and then back to Washington for pollinating sweet cherry (Mink 1983). Bees collect and store huge quantities of PNRSV-infected pollen in their hives in California and continue to store them on re-entry into Washington (Mink 1983). This factor is important epidemiologically in various ways: Even non-viable pollen can act as a virus carrier and transmit virus to healthy plants through abrasion on flowers caused by bees, pollen from even non-compatible species may be equally effective and the disease spreads rapidly over extensive areas. Vertical pollen transmission does not play any part in the spread of viruses which are grouped in A, B and C categories. In category C1 , vertical transmission through pollen seems to play some epidemiological role in their spread. However, these viruses also reach the seed via infected 177 pollen so that their vertical transmission through pollen plays a supplementary role in disease spread by inducing greater production of infected seeds. Thus, vertical virus transmission through pollen per se does not appear to play a major role in disease spread under field conditions. Although Cherry leaf roll virus is present in walnut and gets transmitted vertically, it is the horizontal spread of the disease that threatens the walnut industry in California (Mircetich et al. 1980). Viruses spread horizontally through pollen and invade and systemically reinfect the ovulebearing mother plants which, in turn, lead to the formation and release of larger quantities of infected pollen. This cycle can be repeated during subsequent flowering seasons leading to rapid epidemiological build-up of infected pollen and to disastrous consequences in a couple of years. This is the reason why only the horizontal transmission through pollen is epidemiologically important for viruses of categories D1 and D2 (Mandahar 1981, 1985; Mandahar and Gill 1984). 7.5 Viruses There are nearly 231 viruses which are seed transmitted, and the frequency of seed transmission is more in potyvirus, nepovirus, cryptovirus, ilarvirus, tobamovirus, potexvirus, comovirus, carlavirus, carmovirus, cucumovirus, sobemovirus, furovirus, bromovirus and tymovirus groups (Table 2.1). The viruses are spherical, bacilliform, rigid or flexuous rods (Table 2.2). Some of the viruses appear highly specific affecting only one or two species or only species within one family like SMV, whereas others like TRSV or CMV may infect a large number of species in many families, including herbaceous and woody plants. The epidemiology of virus diseases also depends on different pathogen strains which vary in virulence, host range and transmissibility. Viruses continue to change by mutation and selective adaptation by passage through their hosts as in case of CMV and BCMV strains. Variation may 178 7 also result from pseudo-recombination as since, some of them posses divided genomes or by heterologous encapsidation. These phenomena are both examples of direct interaction between virus strains or viruses in mixed infections. Variation in viruses is often apparent from differences in such biological features such as symptom expression or host range and transmission by vectors or through seed. The epidemiological importance of strain variation has been recognised as in the studies on Sugarcane mosaic virus in western United States (Pelham et al. 1970). Seasonal and other changes in prevalence of strains have also been recorded in studying the incidence of CMV in vegetable crops and weeds in South-East France (Quiot et al. 1979). The strains that predominated in spring-sown crops of tomato, celery and pepper were much more sensitive to heat than those isolated from equivalent summer plantings (Thresh 1987). Other complex interactions between the virulence of strains and host response have been observed with BSMV in barley. Sensitive barley varieties infected with virulent strains produce a large proportion of infected seed, although few seeds develop and tend to be small with poor viability. By contrast, tolerant varieties produce abundant viable seed, but few were infected. Thus, the virus strains that persist readily for successive generations tend to be of intermediate virulence that infect substantial proportions of seed without drastic effects on fertility or on contact between plants (Timian 1974). The above examples illustrate complex factors influencing the overall epidemiological competence of viruses and their strains. However, much of the evidence on variation has come from ‘taxonomic’ or aetiological studies intended to provide definitive descriptions of viruses and to determine their interrelationships. There have been few comprehensive surveys of prevalence and distribution of various strains of even the most important viruses. This is a serious limitation in attempts to estimate crop loss or breed for disease resistance, as there can be greater differences between strains in their ability to infect or in their effects, with important interactions between varieties and strains. Ecology and Epidemiology of Seed-Transmitted Viruses Another important aspect in epidemics is the introduction of resistant genotypes which led to the emergence and spread of resistance-breaking strains. Experience with Sugarcane mosaic in Louisiana and Soybean mosaic in Korea illustrates the rapidity with which resistancebreaking strains have become prevalent following the introduction of resistant varieties (Pelham et al. 1970) and also with the introduction of TMV mild strain protection (Fletcher and Butler 1975). These changes were monitored using isogenic tomato lines and suggested that the resistance-breaking strain did not compete successfully with the former one in susceptible varieties and declined rapidly when the resistant varieties were replaced. Similarly, limited distribution of resistance-breaking strain of Raspberry ring spot virus in Scotland may be due to its limited epidemiological competence, as it is less invasive and less readily seed transmitted than the common strain (Hanada and Harrison 1977). 7.6 Conclusion In a nutshell, the ecology and epidemiology of seed transmission are important because it helps the virus to perpetuate during unfavourable environmental conditions. It provides early and randomised infection foci within a crop from which the virus spreads to other plants in a field either mechanically through pollen or primarily through vectors depending on the virus. Because it is seed transmitted, the introduction and establishment of a virus into new and distant locations take place in nature. Generally, forecasting of seed-transmitted diseases whose spread depends on interaction between virus, host and vector is difficult to frame as compared to other fungal or bacterial diseases. Attempts at forecasting are justified because the success indicates an understanding of the main factors influencing epidemiology. If the forecasting system is developed against a particular virus disease, it will help to operate an early warning system or to select growing seasons or areas for special crops where infection is unlikely. The quantitative epidemiological information has in a References few cases allowed forecasting the development of few seed-transmitted virus disease and helped in preventing or reducing the crop losses. References Alphey TJW, Boag B (1976) Distribution of Trichodorid nematodes in Great Britain. Ann Appl Biol 84: 371–381 Alvarez M, Campbell RN (1978) Transmission and distribution of squash mosaic virus in seeds of cantaloupe. Phytopathology 68:257–263 Amari K, Burgos L, Pallas V, Amelia M (2007) Prunus necrotic ring spot virus early invasion and its effect on apricot pollen grain performance. 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Phytopathology 46:662–664 8 Methods of Combating Seed-Transmitted Virus Diseases Abstract The basic principles in the management of seed-transmitted viruses are generally more or less similar regardless of type of virus involved. There are five approaches, namely, (1) avoidance of the virus from the seeds, (2) prevention/minimising the rate of spread through vector management, (3) legislation, (4) production of healthy seeds through certification programmes and (5) modern diagnostic molecular and biochemical approaches. Avoidance of virus inoculum from infected seeds can be done by removal of infected seeds, chemical seed disinfection, seed disinfection by heat and by irradiation. Implementing the cultural practices like field sanitation, roguing, crop rotation, planting dates, barrier and cover cropping will also reduce the virus disease incidence. The management of virus spread under field conditions is also possible by the application of insecticides, pyrethroids and mineral oils, which reduces the vector population. The role of quarantines, ISTA, biosafety measures, resistant cultivars, healthy seed production and certification schemes for healthy seed production was discussed in detail. 8.1 Introduction Seed-transmitted viruses cause enormous qualitative and quantitative yield losses, which are quite evident from the examples given in Chap. 3. Because of the serious losses to agricultural crops, the virus diseases have acquired great importance in plant pathology and entrusted for effective control measures. They are not amenable to seed treatment like fungi and bacteria, and no commercial viricides have yet been developed. They cause systemic infections after seed germination and are very effectively transmitted through vectors which are air or soilborne. Therefore, preventing their spread is a serious, complex problem, and hence, epidemiology has a profound influence in combating virus diseases. However, detection and elimination of infected seeds is essential since it will eliminate the primary source of infection and reduce the chances of establishment of the virus in commercial plantings. The basic principles in the management of seed-transmitted viruses are generally more or less similar regardless of type of virus involved. There are five approaches: K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 8, © Springer India 2013 185 186 8 Methods of Combating Seed-Transmitted Virus Diseases (1) avoidance of the virus from the seeds, (2) prevention/minimising the rate of spread through vector management, (3) legislation, (4) production of healthy seeds through certification programmes and (5) modern diagnostic molecular and biotechnological approaches. The control measures that have been applied against different virus–host combinations are discussed in this chapter. could be reduced by sieving out the smaller seeds (Inouye 1962). Mechanical seed cleaning helps in bringing down the initial virus inoculum under field conditions. In some instances, viruses do not exhibit any changes in the morphology of seed. Even in certain virus–host combinations, the seed coat mottling alone should not be considered as a reliable indication of seed-transmitted virus infection. The notable example is of soybean seed infected with Soybean mosaic virus (SMV), wherein the seed coat mottling has been viewed as generally inconsistent and an unreliable indicator of the presence of virus (Hill et al. 1980; Bryant et al. 1982; La et al. 1983). In such cases, the seed should also be tested through serological, molecular and biological techniques. 8.2 Avoidance of Virus Inoculum from Infected Seeds 8.2.1 Removal of Infected Seeds A careful and close look at the seed in certain cases gives an indication of the presence of virus(es). Seed morphological abnormalities like shrunkenness, shrivelled seed coats, discolourations, reduced size and weight, cracking, necrotic spots or bands are sometimes associated with the presence of virus in the seed (Table 6.1). For example, pea seeds infected by Pea early browning virus (PEBV) are wrinkled with the seed coat exhibiting a greenish grey discolouration (Bos and Van der Want 1962). Small and shrivelled seeds of barley, wheat and cowpea indicate the possible infection of BSMV, BMV and CPMV, respectively (Phatak 1974; Von Wechmar et al. 1984). SMV seed infection in soybean can be detected by characteristic seed discolouration and black bands or zones radiating from the long axis of the hilum (Phatak 1974). Even in other virus–host combinations like peanut with PMV (Paguio and Kuhn 1974) and peanut stunt virus (PSV) (Culp and Troutman 1968) and soybean with soybean stunt virus (Koshimizu and Iizuka 1963), seed infection is generally associated with reduction in seed size and mottled seed coat. Routine seed cleaning techniques minimise the extent of infection but can never completely eliminate the viruses. For example, Stevenson and Hagedorn (1970) reduced the transmission of Pea seed-borne mosaic virus in a given seed lot from 10 to 4% by removal of infected seeds with growth cracks. Even BSMV inoculum in barley seeds 8.2.2 Chemical Seed Disinfection Seed disinfection helps in removing external infection in fruit pulp remnants as in TMV on tomato, chilli and brinjal seeds and Cucumber green mottle mosaic virus (CGMMV) on cucumber seeds. The seed cleaning is usually done by fermentation of the seed containing fruit pulp with pectolytic enzymes, detergents, certain chemicals or by mechanical means. TMV infection in tomato seeds was greatly reduced by treatment of the pulp with one quarter of its volume of concentrated HCl for 30 min, followed by washing and drying of the seeds (Howles 1957; Crowley 1958; Broadbent 1965; McGuire et al. 1979). Even soaking the extracted seed in 10% solution of teepol for 2 h or soaking in a 10% solution of trisodium orthophosphate or 10% sodium carbonate solution helps in freeing the seed from TMV contamination (Alexander 1960; Nitzany 1960; Broadbent 1965; McGuire et al. 1979). Cordoba-Selles et al. (2007) have eradicated the Pepino mosaic virus infection from tomato seeds by immersing the infected seeds in 10% trisodium phosphate for 3 h which do not hinder the germination. AVRDC scientists (Berke et al. 2005) have also suggested soaking 2 g of chilli pepper seeds in 10 ml of 10% (w/v) trisodium phosphate (TSP) (Na3 PO4 12 H2 O) 8.2 Avoidance of Virus Inoculum from Infected Seeds for 30 min, transferring them to a fresh 10% TSP solution for 2 h, then rinsing in running water for 45 min, and this treatment can be done on freshly harvested or dry seeds. It is also suggested to soak seeds for 4–6 h in 5% (v/v) hydrochloric acid, then rinse in running water for 1 h and dry them for storage, or sow immediately for minimising Tobamoviruses including Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV) and Pepper mild mottle virus (PMMV) in chilli pepper. Seed cleaning with pectolytic enzyme (pectinase) and dilute HCL and drying at 80ı C for 24 h is found effective in bringing down TMV inoculum to a negligible level (Laterrot and Pecaut 1965). Tomato mosaic virus contaminated tomato seeds were treated with 1% HCL and obtained the healthy crop by eliminating the virus contamination (Pradhanang 2009). Gooding (1975) successfully eliminated the TMV by treatment of tomato seed with trisodium orthophosphate and sodium hypochlorite. The treatment comprised of soaking infected tomato seed in 1% aqueous solution of trisodium orthophosphate for 15 min and in 0.52% sodium hypochlorite for 30 min. The treated seed did not lose viability. Even treating the seed of the Capsicum spp. infected with TMV, with 9% calcium chloride or 10% trisodium phosphate (Na3 PO4 ) gave good virus elimination (Betti et al. 1983). In the Netherlands, it was observed that treating Capsicum seeds infected with Capsicum mosaic virus with 10% trisodium orthophosphate (100 g/l) solution for 2 h followed by dry heat at 70ı C for 7 days helps in eliminating the virus from seeds without affecting germination (Rast and Stijger 1987). In contrast, polishing dry cucumber seeds and treating them with detergents or chemicals did not completely eradicate cucumber virus 2 (Van Dorst 1967). Studies of Salamon and Kaszta (2000) and Svoboda et al. (2006) have indicated that capsicum seeds can be disinfected from Pepper mild mottle virus by using 2% NaOH. Even Madhusudhan et al. (2005) have proved salicylic acid (0.05 M) and neem oil (5%) seed/seedling treatment was found to be effective in reducing Tobamovirus concentrations in tomato and bell 187 pepper. Salicylic acid was proved to be an effective inducer. Several reports are available on induction of resistance against plant viruses by using chemicals, and one among them is salicylic acid which is used to control TMV (Murphy and Carr 2002; Singh et al. 2004). Elimination of some seed-transmitted viruses in certain crops was also achieved by soaking them in chemical solutions for varying periods. In India, malic hydrazide, NAA, 2-thiouracil, 24-D and teepol were tested and soaking in malic hydrazide at 40, 100 and 400 ppm for 90 min; 2-thiouracil at 500 and 700 ppm for 60 min; NAA at 40 ppm for 4 h and teepol (5 and 10%) for 4 h completely inactivated Cowpea banding mosaic virus (CpBMV) without affecting seed viability (Sharma and Varma 1975a, b). Cherry leaf roll virus was inactivated in infected seeds by giving a pre-germination imbibitions in an osmotic solution of polyethylene glycol (PEG) followed by high-temperature treatment during the early stages of germination (Cooper 1976). Subsequently, Walkey and Dance (1979) reported inactivation of Lucerne mosaic virus by imbibing diseased seeds in PEG and incubating for 6–10 days at 40ı C. Longer periods of treatments at 40ı C impaired seed viability, while seeds treated for 9 days at 22ı C remain infected. Even Spinach latent virus was eradicated from Nicotiana tabacum cv. Xanthi seed by imbibing in PEG solution at 40ºC for 20– 40 days (Walkey et al. 1983). Certain dimethyl sulfoxide solutions reduced the BSMV symptoms in barley when the infected seeds were treated (Miller et al. 1986). Disinfection of mechanically transmitted Potato spindle tuber viroid from knives and other equipments could be achieved by soaking in sodium hypochlorite solution at 2–3% concentration (Singh et al. 1989). 8.2.3 Seed Disinfection by Heat Most attempts to eliminate virus from seed by heat treatment have been done with high temperatures for relatively short periods or at low temperatures for longer periods by means 188 8 Methods of Combating Seed-Transmitted Virus Diseases of hot water or dry heat treatments. CPMV in cowpea seeds is inactivated when freshly infected seeds were exposed to 30ı C for 4 days or by hot air treatment at 55ı C for 15 min followed by keeping the seeds at 25ı C for 4 days (Verma 1971). Sharma and Varma (1983) studied the effect of dry heat on cowpea seeds infected with CpBMV for 15 min at 65ı C; this was followed by incubating the seeds at 30ı C for 4–8 days or by hot air treatment at 55ı C for 15 min followed by keeping the seeds at 25ı C for 4 days, which reduced the seed transmission from 13.1–24.7% to 0.9–3.3%. Heat therapy of seeds also reduced the field spread of this virus to 4.7–9.1% as compared to 23.7–29.7% in control. Seed yield from heat-treated seeds was 19.3–22.4% higher than untreated diseased seeds. TMV from infected tomato was successfully eliminated by subjecting the dried infected seeds to temperatures of 70ı C for 3 days or 80ı C for 1 day. This is one of the commonly used methods to eliminate the virus from seeds infected internally (Broadbent 1965; Rees 1970; Howles 1978). In Taiwan, dry heat treatment at 78ı C for 2 days against TMV was recommended (Green et al. 1987). Heat treatment delayed germination, but the seeds did not lose their viability when only dried seeds were used. Vovk (1961) reported disinfection of TMV by exposing infected tomato seeds at 50–52ıC for 2 days followed by 1 day at 78–80ıC. However, Howles (1961) could not eliminate TMV completely when infected seeds were subjected to 72ı C for 22 days, but further work has shown that most of the seeds can be freed from TMV during 1–3 days treatment at 70ı C and LMV in lettuce seed by treatment with hot air maintained at 80ı C for 3 days without loss of seed viability. LMV was also inactivated by dry air treatment for 80–120 days at 55ı C (Kristensen 1970). Earlier, Rohloff (1963) reported that treatment of mosaic-infected lettuce seeds at more than 100ı C reduced the percentage of seedlings carrying the virus to 0.2% but seed emergence was only 29%. Van Dorst (1967) reported complete inactivation of CGMMV in cucumber seeds by hot air treatment at 76ı C for 3 days. Similarly, Fletcher et al. (1969) found that treatment at 70ı C for more than 1 day was sufficient to eliminate the same virus (CGMMV) from infected cucumber seeds. These seeds could tolerate dry heat treatment with little adverse effect, although at 80ı C germination was delayed and the cotyledonary leaves were distorted. In a commercial trial, over 45,000 cucumber plants free from symptoms of the virus were raised from infected seeds treated at 70ı C for 3 days. Sharma and Chohan (1971) reported inactivation of Cucumis virus-1 in infected pumpkin (Cucurbita pepo) seed by hot air treatment at 70ı C for 2 days or 40ı C for 4 weeks. Hot water treatment at 55ı C for 60 min was found to be effective in eliminating infection without any adverse effects on seed germination. Similarly, Cherry necrotic ring spot and prune dwarf (PDV) viruses were eliminated from freshly extracted sour cherry seeds by exposing the soaked seed to 60ı C for 1 h or dry seed to 90ı C for 1 h without any adverse effects on seed viability. In another experiment, Necrotic ring spot virus in sour cherry seeds was inactivated by exposing at 55ı C for 6 weeks without affecting seed viability (Megahed and Moore 1969). Even Sharma and Varma (1975b) recorded complete reduction in seed transmission of CpBMV in cowpea by hot water treatment for 40 min at 45ı C and 20 min at 50ı C. Hot air treatment for 50 min at 50ı C, 30 min at 60ı C and 20 min at 65ı C also reduced infection, but viability of the seeds was considerably reduced at these temperatures. However, dry hot air treatment for 15 min at 65ı C followed by 2, 4 and 8 days of incubation at 30ı C proved comparatively better in eliminating seed transmission, while seed viability was affected to a lesser degree. Similarly, the seed-transmitted infection of ULCV was completely eliminated by treating the urd bean seeds in water bath for 30 min at 55ı C without affecting the seed germination (Beniwal et al. 1980). However, heat treatment did not always cure diseased seeds. For example, BSMV was not inactivated even when the seeds were subjected to 130ı C for 30 min (Timian 1965). Similarly, no inactivation of TRSV in the seeds of soybean without loss of viability was recorded (Owusu et al. 1968). 8.3 Reducing the Rate of Virus Spread Through Vector Management 8.2.4 Storage Effect Some viruses survive in seed only for a few months, and others persist for many years (Table 5.1). Long-term storage of infected seed also makes the seed virus-free, and the viruses which are externally seed transmitted are often inactivated than embryo-borne viruses which usually persist for longer periods. For example, BCMV was infective even after storage for 6 years (Polak and Chod 1967) and Bean (Western) mosaic virus for 3 years (Scotland and Burke 1961). Similarly, Bennett (1969) did not notice any decrease in Lychnis ring spot virus transmission in the seed of Lychnis divaricata even after 9 years of storage. In some virus–host combinations, where the virus was seed transmitted, the loss of seedtransmitted virus was achieved with seed storage, and some of the successful attempts are as follows. Substantial reduction in Brinjal mosaic virus in brinjal was noticed by storing seeds at room temperature (Mayee and Khatri 1975). They have also observed a 72.5% reduction in virus incidence content in the first 3 months after storage in the var S-16, and after 7 months, no viral incidence was reported without appreciable loss in viability. Rader et al. (1947) recorded a decrease in mosaic virus infection (3–5%) in freshly harvested seed of muskmelon. It was observed that CGMMV was minimised by storing the infected seed for 2–3 years before sowing. In tests conducted during 1961, infection of cucumber plants grown from seed obtained in 1960 was 44.0% and only 9.5% from a 1958 seed lot (Yakovleva 1965). Similarly, in 7 cantaloupe seed lots with 11–31%, Squash mosaic virus (SqMV) transmission in 1967 showed no transmission in 1969 (Powell and Schlezel 1970). Van Koot and Van Dorst (1959) recommended 1year storage for freeing cucumber seeds from the same virus. Similarly, Prunus necrotic ring spot virus (PNRSV) and Prune dwarf virus (PDV) in the seed of Prunus spp. were lost after a 5-year storage period, but seed viability decreased at a slower rate than that of virus infectivity (Fulton 1964; Megahed and Moore 1969). Retention of CpBMV in cowpea seeds of cv. Pusa Dophasli has been noticed when stored for up to 2 years 189 at room temperature (25ı C). After storage for 2 years, both virus and seed viability decreased (Sharma and Varma 1986). From a commercial point of view, the stored seed should have maximum germination, and so the method of eliminating the virus by storage will be effective only when the virus is active in seed only for a few months. For the viruses retained in the infected seed for longer periods, alternative methods like chemical and heat treatment should be worked out. 8.2.5 Irradiation Effect Gamma irradiation has been tried for the inactivation of seed-transmitted viruses. Megahed and Moore (1969) reported the inactivation of Necrotic ring spot and Prune dwarf viruses in Prunus spp. seeds after irradiation. Sharma and Varma (1975a) failed to obtain promising results in eliminating CpBMV from infected cowpea seeds by gamma ray treatment at 20 KR. There was very little reduction in seed transmission of the virus at higher doses (30 and 40 KR), but viability of seeds was severely affected. Although germination after treatment was good, a majority of seedlings died early due to a lethal mutation induced by gamma rays. Treating infected seed with effective chemical inhibitors like 2thiouracil or heat, followed by gamma irradiation may yield better results in curing infected seeds. 8.3 Reducing the Rate of Virus Spread Through Vector Management 8.3.1 Avoiding of Continuous Cropping Infection sources, generally from the same crop or from other crops which are susceptible and grown nearby, will lead to severe virus problems. Some of the crops like tomato, cucurbit, peanut and other legumes are grown throughout the year without any break, and inevitably new crops are 190 8 Methods of Combating Seed-Transmitted Virus Diseases grown near old ones. By breaking the disease cycle, it is possible to minimise the spread of virus diseases which have limited host range. Effective virus control would be significant only if a crop-free period is enforced or voluntarily agreed upon at a regional level. For example, a celery-free period has been enforced in California (USA) over four counties to control Celery mosaic virus, and this was also voluntarily imposed by growers in Florida (Zitter 1977). Similarly, in Salinas Valley (California, USA), a lettucefree period in December along with seed certification programme has reduced Lettuce mosaic incidence (Zink et al. 1956; Wisler and Duffus 2000). Many aphid species will be drastically reduced in hot dry climates when the daily maximum temperature exceeds 35ı C (Maelzer 1981). For this reason, lettuce seed was grown free from mosaic virus during summer in Australia (Stubbs and O’Loughlin 1962). In Southeast Asian countries where peanut is extensively cultivated, PMV and PStV which are seed transmitted are gaining importance since their aphid vector Aphis craccivora occurs in high proportions in almost all seasons on a number of legume crop and weed hosts. Selection of virus-free seed and skipping peanut in one season may help in reducing the virus spread. Certain nematode and fungal-transmitted viruses could also be reduced by avoiding planting of the susceptible crops in fields infected with soilborne vectors. in the soil for long periods. In certain cases, the presence of weeds in the field becomes more dangerous as they are symptomless virus carriers and consequently are difficult to assess. Controlling the weeds either manually or by weedicide application in and around the fields has given encouraging results in crops like cucurbits and celery against CMV. Volunteer infected peanut plants proved to be the source of PMV inoculum for soybean crop in Georgia (USA), and the disease spread was reduced by destroying volunteer plants (Demski 1975). In India, high incidence of PBNV is noticed in South India where ever Parthenium weed population occurs in high proportion (Reddy et al. 1983). Similar situation was also recorded in sunflower in relation to TSV incidence (Prasada Rao et al. 2003, 2009). All growers in a region should cooperate in a weed control programme involving removal of the weeds manually or by large-scale weedicide application. Even wild plants act as direct source of viruses and vectors. Their removal eliminates the sources of infection, reduces virus spread in seeds – if the virus is seed transmitted – and also prevents vectors from breeding on them. 8.3.2 Elimination of Weed, Volunteer and Wild Hosts Weed and volunteer plants being major components of the agro-ecosystem not only compete with crop plants for water and nutrients but also serve as source of inoculum, since they harbour virus diseases (Duffus 1971; Thresh 1981; Sastry 1984). It is well understood that weed hosts act as sources of virus inoculum for both the crop and the vector. In some of the weed hosts, the virus is seed transmitted, and the infected seeds survive 8.3.3 Roguing Even by roguing, the infected plants when the virus incidence is very low, especially in small plantings, help in minimising the spread of virus. Inouye (1962) reduced the incidence of BSMV in two varieties from 85 to 12% and 46% to nil through early roguing. Zink et al. (1957) found that roguing LMV-infected plants once soon after thinning had no effect on disease incidence at harvest (81.7%) presumably because much spread had already occurred, but roguing twice or thrice reduced the incidence, that is, from 73 to 43 and 36% and 17 to 9 and 6%, respectively, in two field trials. In most countries, the commercial growers rogue out the infected plants in seedbeds of crops like lettuce and brassicas, but it is difficult to implement roguing practice over large areas and also against the seed-transmitted virus diseases that spread rapidly and far from small initial foci. 8.3 Reducing the Rate of Virus Spread Through Vector Management 8.3.4 Crop Rotation Crop rotation has historically been a major means of disease control in production of annual and biennial crops. Generally fallowing is recommended against soilborne nematode diseases. A short period of fallow may or may not decrease the nematode vector population since they live for long periods. Encouraging results are available wherein the soilborne diseases are minimised by crop rotation with non-susceptible hosts of virus/vector. TMV remains infectious even after 2 years in the old infected root debris, and crop rotation is one of the ways of freeing the soil from TMV satisfactorily, as the root debris and viruses are eventually destroyed by fungi and bacteria in cultivated soil. Tomato mosaic virus on tomato crop was successfully eliminated by composting tomato residues, and it was due to biological elimination of the virus or due to heat inactivation (Avegelis and Manios 1989). This suggests the use of tomato compost in plastic houses since the virus is inactivated during composting. 8.3.5 Planting Dates Adjusting seeding/planting and harvesting time of the crops based on vector migration is a strategy of control for some seed-transmitted virus spread. From Germany, Kemper (1962) reported low incidence of LMV in lettuce plantings made during March when compared with the crop grown during June–August, when the aphid vector population reaches its maximum. In contrast, in Jordan, the incidence of LMV increased from mid-January to end of February (Al-musa and Mansour 1984). In Delhi (India), cowpea crop grown during March–May had considerably less CpBMV infection than that sown in the month of July (Sharma and Varma 1984). In the coastal areas of Israel, bell peppers are not planted before mid-April to avoid heavy infections of CMV. From the beginning of May, infection rates decreased in the vector population decreased because of drying of weeds and 191 removal of virus reservoirs (Raccah et al. 1980). At Davis (USA), Slack et al. (1975) reported reduced spread of BSMV in spring-seeded barley by producing seed sources in fall-seeded areas. At Pullman (Washington state, USA) wherein the USDA Phaseolus germplasm collections are maintained, Klein et al. (1989) could successfully reduced the BCMV incidence by over 66% in 26 Phaseolus vulgaris accessions by propagation in the greenhouse during the winter (January through June) because of the absence of aphid vectors. The decline in BCMV incidence was sufficient in most accessions as BCMV could be eliminated from the important germplasm collections by elimination of infected plants without affecting genetic diversity. In Nepal, PSbMV incidence was higher in late planted pea crops (January) than those sown in November– December (Dahal and Albrechtsen 1996). In India, even Puttaraju et al. (2002) have noticed higher incidence of BICMV in cowpea sown in March to May than in September to November. At Ranchi (India), Prasad et al. (2007) found that BCMV incidence in French bean would be lowest (5.4 and 7.3%) when sown on 5th and 20th September. Maximum BCMV incidence (25.4%) was recorded in late sown crop, that is, 19th November. It was because of aphid vector population which was least in September sown crop and maximum aphid population was recorded in November sown crop and BCMV incidence is directly related to aphid vector population. 8.3.6 Plant Density High density of plants generally reduces the number of infections per unit area, and the planting rate should be such that it should cover the soil without reducing the yield due to competition. Some of the outstanding examples wherein high plant density decreased the incidence of seedtransmitted viruses are PMV in soybean (Demski and Kuhn 1975) and CpBMV in cowpea (Sharma and Varma 1984). 192 8.3.7 8 Barrier and Cover Crops In developed countries, the protective cropping of some commercial crops like lettuce, tomato and cucurbits has been done by cultivating them in screenhouses/glasshouses. The scientists of the Asian Vegetable Research and Development Centre, Shanhua (Taiwan), found the tunnels of blue plastic screen of no. 200 mesh (13.5 lines/mm) placed over rows of Chinese cabbage was found to be effective in greatly reducing Turnip mosaic virus symptoms and in increasing yields (Anonymous 1977b). Because of the high cost involved in glasshouse or screenhouse construction using plastic sheet or nets, simple methods like barriers of either plants or muslin cloth screens were used in virus control. The barrier crops should be generally of fastgrowing taller species, which would be not susceptible hosts for virus and vector, and hence, monocot plants are planted as barriers for dicot crops and vice versa. The barrier crops are more effective against stylet-borne viruses, since the aphid vectors lose the virus while probing the barrier crop even for a brief period. The same principle also applies to cover crops. Devaux (1977) and Deol and Rataul (1978) reduced CMV infection in cucumber and chillies, respectively, by planting the crop behind barrier rows of corn, sorghum and pearl millet. In India, about 50% reduction of CpBMV incidence in cowpea was achieved by raising 2 rows of pearl millet at a distance of 3.75 and 5.25 m, but complete protection was achieved when the barrier rows were provided at 1.5 and 2.25 m apart; however, this adversely affected the growth of cowpea. Mixed cropping of cowpea with maize reduced the virus infection to 3.0%, as against 12.6% in pure stand of cowpea crop (Sharma and Varma 1984). The least incidence of mosaic diseases was recorded in muskmelon by growing wheat as a barrier crop (Toba et al. 1977). The wheat plants apparently provide additional probing sites, so that incoming viruliferous aphids lose their virus on the non-host plants before probing susceptible cantaloupes. Similarly, at Georgia (USA), a wide barrier of oat (Avena Methods of Combating Seed-Transmitted Virus Diseases sativa) significantly decreased the incidence of Watermelon mosaic virus (WMV) in watermelon (Chalfant et al. 1977). There is scope for reducing SMV incidence by having sunflower as a barrier crop in soybean (Irwin and Goodman 1981). Virus-free nurseries of brassica, chillies, brinjal, etc., could be raised by growing barrier crops like barley, wheat or sorghum around the seedbeds. Choice of the distance between the barriers for providing the maximum protection will depend upon many factors such as vigour, thickness and height of barrier crop and the environmental factors like the direction of the wind and its velocity. 8.4 Integrated Cultural Practices for Seed-Transmitted Virus Disease Management Cultural practices individually were proved to be effective in reducing the seed-transmitted virus incidence in different crops. However, some experimental results are available where in more effective virus management was achieved when integration of different cultural practices were followed. This approach was quite effectively proved by the experiments conducted by Bwye et al. (1999) in lupins against Cucumber mosaic virus. Seven experiments were conducted in Australia with lupin (Lupinus angustifolius) to examine the effects of cultural practices on incidence of Cucumber mosaic virus (CMV). The factors investigated were row spacing, banding fertiliser below seed, straw ground cover and tillage. The seed sown carried 5– 15% CMV infection. Seed-infected plants were the primary source for subsequent virus spread by aphids. Incidence of seed-infected plants and the extent of virus spread were gauged by counting numbers of lupin plants showing typical seed-transmitted and current-season CMV symptoms. Due to greater competition with other plants within wide than narrow rows, wide row spacing diminished the survival of seedinfected plants by 46%. Increased plant growth from banding superphosphate below seed did not significantly decrease numbers of seed-infected 8.5 Crops Hygiene plants surviving. Straw spread on the soil surface suppressed final CMV incidence by 25–40% and, when applied at different rates, diminished recorded CMV incidence more at 4 than 2 t/ha and least at 1 t/ha. Where there was no straw, CMV incidence increased faster with narrow spacing than wide spacing. Soil disturbance from sowing seed with double discs instead of types significantly increased incidences of both seed-transmitted and current-season infection and diminished grain yield. Neither straw nor row spacing treatments significantly affected grain yield, but the decrease in CMV spread due to straw ground cover significantly increased individual seed weight and overall yields were greater with straw. Myzus persicae was the main colonising aphid species, but Aphis craccivora and Acyrthosiphon kondoi also colonised the lupins. There were significantly fewer colonising M. persicae in plots with 4 t/ha of straw than in those with none. This work suggests that stubble retention, minimum tillage and wide row spacing should be included as components of an integrated disease management strategy for CMV in L. angustifolius crops. Effective management of nonpersistent aphid-transmitted viruses in Lucerne and CMV in lupins was achieved by developing integrated disease management strategies by Jones (2001, 2004b). Similar type of integrated approach with modifications depending on the locality and environmental conditions, trails should be tried with different seed-transmitted viruses in other crops. 8.5 Crops Hygiene Against highly stable viruses like TMV and Capsicum mosaic virus in tomato and capsicum crops and also against certain viroid diseases, hygiene is particularly important. Some of the recommended measures to combat the spread of these diseases during the cultural operations are as follows: For example, in tomato and pepper, TMV occurs as a contaminant on the seed coat, and as the radicle (root) grows, it comes into contact with the surface of the seed. TMV particles 193 present on the seed coat adhering to the seedlings or adhering to the root surface; at this point, the seedling is only contaminated but not infected. When the seedlings of tomato/pepper are pulled out from the ground for transplanting, root hairs are broken, giving way for entry of TMV particles, and thus, the actual infection of seedling takes place only at the time of transplanting. Seedlings are not infected when left undisturbed even though they are raised from diseased seed. At USSR, it was found in field experiments that TMV incidence in tomato crop was 10–15 times more when the crop was transplanted and that cultivation without transplanting increased yield by 19% and also improved fruit quality (Nikitina 1966). Taylor et al. (1961) recommended direct sowing instead of transplanting of tomato seedlings. Direct sowing has been adopted by some growers to eliminate pricking out effect. This method is easier now as it is possible to obtain pelleted seed. Goldin and Yurchenko (1958) observed infection of only 5 plants in a field in which tomato seed was directly sown in comparison to 71 infected plants when they were transplanted. However, raising the nurseries and transplantation is the popular way of raising crops. The risk of mechanical contamination could be greatly reduced, however, by spraying plant beds of tomato and pepper with milk just before plants are pulled out for transplanting. Dipping and washing hands with milk before handling the nursery is quite useful since TMV is rapidly inactivated by proteins present in milk (Crowley 1958; Hare and Lucas 1959). Besides sterilisation, the virus contamination on the farm implements can be reduced by washing with 10% trisodium phosphate or 2% formaldehyde plus 2% sodium hydroxide (Broadbent 1963; Patezas et al. 1989). The workers should not keep tools in their pockets which might contain tobacco debris. Even preferring non-smokers or non-snuffers for jobs which involve much handling of plant seedbeds, planting, etc., would minimise virus spread. If not, every effort must be made to prevent smoking or taking of snuff while work is in progress. 194 8 Methods of Combating Seed-Transmitted Virus Diseases It has been demonstrated that packing containers, if contaminated, also act as a probable source of infection. The spread of CGMMV was attributed to the use of contaminated packing containers used for harvest of cucumber fruits and also for the transport of young plants (Van Dorst 1988). During transport, the leaves might have touched the contaminated sides of the containers, resulting in early infection. Use of low-cost disposable packing containers will help in minimising contamination. It is also established that TMV spreads from one crop to the next on contaminated trellis, seedbed cloth, frame boards and wires. Nitzany (1960) reported retention of TMV for one and half to four months on trellis wires removed from infected fields when stored in a shaded store. He also reported virus inactivation by dipping infected trellis wires in 1% formalin for 5 min or 5% TSOP for 10 min or 0.1% caustic soda solution. In potato, PVX and Potato spindle tuber viroid diseases which are seed-transmitted spread in the field during tractor operations. Washing the machinery thoroughly with any one of the earlier mentioned virus-inactivating detergents before it is taken into the next healthy crop helps in reducing the spread of the disease. In the glass/mesh houses which are used for tomato cultivation in countries like the USA, Hungary, the UK, etc., and also for conducting research work under controlled conditions, contamination with TMV is a general problem as even the small fragments of the infected leaf debris either on the benches or the floor also serve as source of virus inoculum. Thorough washing of glass or wire mesh houses with a disinfectant when they are empty between crops will ensure that they do not become contaminated. (Fig. 8.1) which is followed as mentioned here. Germination varies depending on variety, seed quality and soil mixture. For optimum germination, sow seeds in a well-drained, sterile soilless mix at 25–28ıC, and water daily. Under these conditions, seeds will germinate in about 8 days. Seeds will germinate in 13 days at 20ı C and 25 days at 15ı C; they may not germinate at all if temperatures are below 15 or above 35ı C. For example, 1 g of chilli pepper contains approximately 220 seeds. Approximately 150 g may be needed to transplant 1 ha at a density of 30,000 plants/ha, assuming 90% germination and 90% of seedlings are of good quality. Fill the seedling tray with sowing medium, such as peat moss, commercial potting soil, or a potting mix prepared from soil, compost, rice hulls, vermiculite, peat moss and/or sand. The potting mix should have good water-holding capacity and good drainage and recommend a mixture of 67% peat moss and 33% coarse vermiculite. For use of non-sterile components, it is recommended to sterilise the potting mixture by autoclaving or baking at 150ı C for 2 h. If seedlings are to be grown on a raised soil bed, the soil should be sanitised by burning a 5-cm-thick layer of rice straw or other dry organic matter on the bed. This also adds small amounts of P and K to the soil for the seedlings. Sowing one seed per cell of potrays (or broadcast the seeds lightly in a seedbed) and covering or sow them inside a greenhouse or screenhouse. This provides shade and protects seedlings from heavy rain and pests, such as aphids, which transmit viruses. Use a fine sprinkler. Irrigate with a 0.25% (w/v) solution of water-soluble or liquid fertiliser (10-10-10) when two true leaves appear. If damping-off occurs, irrigate with a 0.25% (w/v) solution of Benlate or similar fungicide. If the seedlings have been grown in shade, harden them by gradually exposing them to direct sunlight over 4–5 days prior to transplanting. On the first day, expose them to 3–4 h of direct sunlight. Increase the duration until they receive full sun on the 4th day. The procedure described herein has been mentioned in one of the popular articles from AVRDC, Taiwan (Berke et al. 2005). 8.5.1 Raising Transplants A number of MNC seed companies and research organisations are rising the transplants of crops like tomato, capsicum, cucurbits, rice and other crops under protected environment to have good growth and protection from pests and diseases 8.6 Control of the Vectors 195 Fig. 8.1 Raising transplants in protected environment (Source: R.A. Naidu) 8.6 Control of the Vectors 8.6.1 Insecticides Chemical control of vectors to avoid or limit introduction of virus and subsequent spread has been in practice for long time. It proved to be successful in certain situations because of the ease of application, ready availability, low cost and general effectiveness against insects. More than 50% of seed-transmitted viruses are aphid-borne and nonpersistent type, wherein a few seconds of acquisition and inoculation periods are required. Adequate control methods are lacking since the insecticides applied do not inactivate or kill the vectors within a short period before acquisition and inoculation, and before they die, the vectors infect the plants. In recent years, photostable synthetic pyrethroids have been prepared, some being found to be fast-acting insecticides and have low mammalian toxicity (Naumann 1990). However, very limited information is available on the effect of insecticide application in reducing the spread of some seed-transmitted viruses. In India, the spread of seed-transmitted virus disease of cowpea was reduced by soil drenching with granular insecticides like aldicarb, phorate and dimethoate (Sharma and Varma 1972). Similarly, in the Primork region of the former USSR, the sprays of synthetic pyrethroid, Lcyhalothrin, at 5.0 and 7.5 g/ha induced aphid vector mortality and reduced the incidence of cowpea viruses (Atiri and Jimoh 1990). SMV was minimised by 12.3–24.3% by management of aphid vectors through the application of aphidon and disyston and increased yields by 5–12% (Smirnov Yu 1975). In general, insecticides do not seem to have any effect on vectors of nonpersistent viruses which infect the crop from sources outside and have long continuous flight periods. A more effective method is to use the insecticide against the vector either on the virus source outside the crop or on trap crops. However, controlling the soilborne vectors by application of chemicals to infested soil is difficult as some of the nematode vector species occur at considerable depths in the soil for long periods. Another factor is that the virus could be transmitted to a plant by a single nematode. The 196 8 Methods of Combating Seed-Transmitted Virus Diseases efficient management of nematode-transmitted virus disease requires application of a fumigant before planting to decrease nematode numbers. Application of granular systemic nematicide at the time of planting is necessary as this will decrease the efficiency of surviving nematodes and facilitate the establishment of the crop. This has to be followed by repeated application of a systemic foliar spray or a drench to maintain a toxic environment around surviving nematodes throughout the life of the crop. Such a control programme would be expensive and would require careful epidemiological and environmental evaluation. The future of systemic nematicides that control nematodes transmitting plant viruses will depend on the development of materials with greater direct nematicidal toxicity and/or greater persistence. Greater persistence could introduce the problem of toxic residues in the crop, so serial applications of short persistence materials could be preferable. Attempts were also made to control fungaltransmitted viruses by using biocides. The incidence of clump disease transmitted by P. graminis in peanut crop could be reduced by applying dibromochloropropane and furadon at nematicidal dosages. Nevertheless, their application on small forms is unlikely to be economical (Reddy et al. 1988). Germany. Some of the seed-transmitted virus diseases which are effectively controlled by oil spray are CMV in cucumber (Loebenstein et al. 1964, 1966), Pea mosaic virus in broad bean (Zschiegner et al. 1971), etc. At Georgia (USA), PMV and CMV were found to cause enormous yield losses in peanut and soybean, and encouraging results were achieved by spraying 0.75% oil at weekly intervals (Kuhn 1969). The cost of protecting a seed crop with oil could be about $35 per acre for material. Kuhn and Demski (1975) estimated that peanut losses from PMV in Georgia amount to $11,000,000 annually. The cost of oil to protect the 30,000 acres of crop needed for seed production and to plant the 512,000 acres of commercial peanut would be about $1,000,000 annually which provides the Georgia peanut growers an 11:1 return on investment in virus control. While working with CMV, Loebenstein et al. (1966) also observed an increase of 50% in the yield of marketable cucumbers with low-volume sprays of summer oil. The use of oil as sprays has several advantages. It is low cost, has good spread ability, is easy to mix and has low toxicity to man and animals. Another important consideration is the fact that insect vectors have not yet developed any resistance to them. The efficacy of oils could be enhanced by judicious application at suitable concentration with the appropriate spray nozzle for the proper stage of the crop. A great disadvantage, however, is that these oils have to be sprayed at frequent intervals for effective control of virus spread. Limitations to the use of oil sprays are plant toxicity, volatility or viscosity of the oils, adequate coverage of the leaves and removal of the oil cover by rain or irrigation water. 8.6.2 Mineral Oils Bradley (1963) showed that mineral oil sprays impeded virus infection. Control of a large number of stylet-borne seed-transmitted virus diseases with oil spray has been tried by researchers with varying success for the last 25 years (Peters1977; Vanderveken 1977; Zitter and Simons 1980; Simons and Zitter 1980; Simons 1981, 1982; Sharma and Varma 1982a; Sastry 1984). Of the different kinds of oils in use, namely, vegetable, mineral, synthetic and essential oils, effective control of virus diseases was achieved only with mineral oils in countries like the USA, the Netherlands, Israel, the UK, India and West 8.6.3 Repelling Surfaces Some of the seed-transmitted plant virus diseases of legumes, cucurbits and certain solanaceous crops have insect vectors like aphids, beetles, thrips and others which are responsible for virus spread. Like insecticide and mineral oil application for vector control, even some 8.7 Virus Avoidance repelling and attracting surfaces of aluminium, plastic and straw mulches were used in certain virus–host combinations. Aphids transmit a number of seed-transmitted viruses, and the aphids respond differently to various wavelengths of light; the use of attractive colours as traps or repellents to avoid landing of the vector on susceptible crops is advantageous in minimising the spread of virus diseases. The visible infrared and ultraviolet lights emitted from different surfaces are responsible for vector repellency. It was first demonstrated that white surfaces reflecting ultraviolet or short wavelength which was unattractive to alighting aphids were even avoided by them (Moericke 1954). In number of countries, aluminium mulches were used for controlling virus diseases in crops like bell pepper, tomato, cucumber, lettuce and in certain ornamental crops. These mulches are effective at least against 12 species of aphids (Smith et al. 1964). In French beans, aphid-transmitted BCMV is effectively reduced by using silver polythene film. Experiments conducted in Japan with reflective mulches in pea crop were effective in reducing Pea seed-borne mosaic virus. A number of attempts were also made to reduce the virus incidence by minimising the vector population by using plastic mulches. The plastic mulches are cheaper than the aluminium mulches, and partial success has been achieved in many countries by reducing the virus incidence by using different coloured plastic mulches. Jones and Chapman (1968) tested plastic sheets in nine different colours in addition to aluminium foil to determine their attractiveness to aphids. Yellow colour was most attractive, followed in order by pink, green, red and black, whereas white, orange, light blue and dark blue colours attracted the fewest aphids. In certain crops, straw of cereal crops was used as mulch and proved to be effective in reducing the insect vector population and virus incidence (Cradock et al. 2001; Summers et al. 2005). The straw as mulch will be effective for a short period when compared to aluminium or plastic mulches; however, due to biodegradation, the straw will mix with soil and enriches the soil. 197 8.7 Virus Avoidance 8.7.1 Exclusion In various countries and territories, legislation currently in force to prevent introduction of important seed-transmitted diseases and pests involves the following quarantine regulations: (1) embargo and import permit, (2) inspection (field inspection and laboratory testing) in the exporting country before shipment of the consignment, (3) seed treatment for diseases and pests that can be eliminated by disinfection or fumigation with reasonable certainty, (4) post-entry growth inspection by the importing country in closed quarantine and (5) certification. A detailed discussion of general quarantine principles and philosophy, except some information on seed-transmitted virus diseases, is beyond the scope of this book. More information on quarantine aspects can be obtained from the review articles of Hewitt and Chiarappa (1977), Kahn (1977, 1989), Neergard (1980), Lovisolo (1981), Chiarappa (1981), Reddy (1982), Raychaudhuri and Khurana (1984) Plucknett et al. (1987), Jalil et al. (1989), Frison et al. (1990), Kahn and Mathur (1999), Ebbels (2003), Khetarpal (2004), Khetarpal and Gupta (2006) and Munkvold (2009). Migrations, military conquests, voyages of discovery, religious expeditions and germplasm exchange have in many ways contributed to the spread of seed material. In addition, ship loads of grain for consumption or large quantities of seeds for direct sowing are imported by many countries. Most countries differentiate between the seed imported for scientific purposes and those imported for sowing or commercial purposes. Since more than 231 viruses are seed transmitted, there is a risk of introducing the virus diseases which are not known to occur in a country if proper testing is not carried out. Even minute quantities of soil and plant debris contaminating true seeds can introduce the virus/vector, or both. Now a days chances of virus introduction are greater with quick and efficient air transport enabling exchange 198 8 Methods of Combating Seed-Transmitted Virus Diseases of large quantities of seed material between plant breeders and crop scientists around the world. Thus, man is the direct or indirect cause of most epidemic imbalances that we know about, and the high virulence and extreme susceptibility of an epidemic situation is an unnatural imbalance usually brought about by human disturbance (Stace-Smith 1985; Jones 2000; Degirmenci and Acikgoz 2005). The basic objective of plant quarantine is to check the introduction and spread of potentially dangerous pests and diseases imported along with germplasm from region to region by official regulation. In various countries and territories, legislation currently is in force to prevent introduction of important seed-transmitted diseases and pests. There is need for systematic effort to reach the goal of virus-free germplasm collection and maintenance. Often the virus affected germplasm collections are received at quarantine stations as in cases like soybean with SMV (Goodman and Oard 1980) pea and lentil with PSbMV (Hampton 1982, 1983) and; Coutts et al. (2008), French beans with BCMV and CMV (Hampton and Braverman 1979; Davis et al. 1981). In postentry quarantine stations in peanut germplasm, PStV virus was intercepted (Demski et al. 1984b; Persley et al. 2001). Urd bean with ULCV (Beniwal et al. 1980) and the viruses involved were commonly detected in routine seed evaluation tests. Hampton et al. (1982) listed some of the viruses that could be expected to occur in germplasm accessions of major crops in the USA. Seed-transmitted infections by PSbMV in pea germplasm, sometimes reaches 90% or more, are the main sources of its distribution over long distances (Hampton and Mink 1975). The germplasm collection such as the USDA pea collection at the Northeast Regional Plant Introduction Station, Geneva, New York, is not only a source of valuable pea characteristics but also a source of seed-transmitted pathogens such as PSbMV. It has been estimated that 23% of the accessions in the collection were infected by the virus (Hampton and Braverman 1979). Similarly, Alconero and Hoch (1989) have recorded 189 isolates of PSbMV from seedlings of 435 pea germplasm introductions. Similar situation of the occurrence of high seed transmission of BCMV in bean germplasm and CpAMV in Vigna species is noticed in almost all countries. Klein et al. (1988) reported approximately 60% of the 207 French bean accessions contaminated with BCMV. Peanut stripe virus (PStV) was introduced into the USA in the 1980s through peanut seed in germplasm imported from China (Demski et al. 1984b). Even in India, the same virus was detected in peanut germplasm lines in Andhra Pradesh and Gujarat states (India) which was introduced through germplasm exchange (Prasada Rao et al. 1988, 2004). During 2001, PStV was intercepted in Australia from imported peanut seeds (Persley et al. 2001). At NBPGR, New Delhi (India), Chalam et al. (2005a, b) and Khetarpal et al. (2001) have reported viruses which were intercepted in germplasm of different crops like cowpea, mung bean and broad bean in the quarantine section, and the list of viruses is presented in Table 8.3. Chalam et al. (2007a) have screened the imported seed material by following the advanced serological techniques and could find five exotic viruses which were not recorded in India, namely, Barley stripe mosaic virus in barley and wheat; Broad bean stain virus in faba beans; Cherry leaf roll virus in soybean and French bean; Cowpea mottle virus in cowpea and Raspberry ring spot virus and Tomato ring spot virus in soybean. Hence, every care should be taken to minimise the seed-transmitted incidence of viruses in accession seeds since it will be a source of contamination of breeding stock as well as a cause of yield losses commercially. Its management in germplasm collection conditions should be taken into account for the possible loss of genetic diversity of some accessions (Alconero et al. 1985). Procedures used in the elimination of the virus should be consistent with the need to maintain genetic characters of the accessions. Therefore, FAO/United Nations Environment Programme/International Board of Plant Genetic Resources in a conference during 1981 have recommended that all germplasm exchanges should take place through National Quarantine Services. They have suggested that national and regional laboratories be established 8.8 Resistance to expedite the passage of germplasm through National Quarantine Services. The seed lots received at the quarantine stations should be initially evaluated by growing-on tests, indicatorinoculation tests, serological tests, radiography, etc. In recent years, serological tests like ELISA, RISA, SSEM, cDNA, PCR, RT-PCR, and western blot have been developed and are used for quick and judicious detection of the seedtransmitted infection to avoid the introduction of new viruses or severe strains of certain existing viruses. Hence, strict quarantine treatments are applied in almost all countries to prevent the entry of new viruses into an area where it is not known to occur – or is of limited distribution. Quarantine treatments can be divided into three groups: (1) chemotherapy, (2) thermotherapy and (3) cultural procedures. The impact of these treatments in different virus–host combinations has been discussed earlier in this chapter. In almost all countries, quarantine stations are located near airports or seaports, wherein vigorous testing of the seed lots is carried out despite the delay and inconvenience involved. From the facts cited above, it is clear that in general the quarantine policy towards seed is not adequate without sound seed health testing facilities for detection of seed-transmitted viruses. There is need for international cooperation, particularly in the alignment of seed health testing methods. Emphasis has to be laid on seed crop certification with a view to supply virus-free seed material to growers of all countries. In order to lower or minimise the virus risk associated with the import of seed, safeguards like the regulations themselves, phytosanitary certificates with or without added declarations, permits, inspection upon arrival, treatment, quarantine, etc., are to be undertaken. Embryo culture methodology is being used even though it has certain limitations as a safeguard in the international transfer of plant genetic stocks. Kahn (1977, 1989) has developed this technique by using tissue culture methodology. In this technique, instead of complete seed, only embryo axis is excised and used for culturing, and it can be mailed without any risk. The objective is to facilitate the exchange of 199 plant genes while lowering the chances of inadvertently exchanging some of the serious seedtransmitted viruses. Even though strict legislative measures are enforced, still a few new virus diseases are introduced into countries and are published as new records either to the host or virus. Some reasons attributed to this are as follows: The methods may not be sensitive enough to reveal latent infection and the lack of adequately trained personnel. Those responsible for quarantine measures should be properly trained and experienced. There is need for professional workers and the public to cooperate on an international scale for effective operation of quarantine regulations. The importance of quarantine has increased by many folds in the WTO regime and by adopting not only the appropriate techniques but also right strategies for virus detection would go a long way in ensuring virus-free seed trade and exchange of germplasm. 8.8 Resistance Plant resistance in crop plants having virus transmission through seed has the great advantage for a component that it usually enhances the effectiveness of other virus management measures. Plant resistance can reduce virus infection and disease development, and a large number of disease resistance genes were identified during 1986–1996. Although the scientific and technical advances have been rapid, additional studies must be carried out for the better understanding of the molecular basis of resistance of producing transgenic plants. It is well documented that host plant and vector resistance are the most effective control measures against certain seed-transmitted diseases. Usefulness and success of the resistance strategy depends on our knowledge of the mechanism(s) of resistance and its effects on the virus–vector– host interactions. More complex and probably more durable resistance is more difficult to establish and certainly more difficult to breed. This does not preclude their existence or possible future utilisation. Paradoxically, application of 200 8 Methods of Combating Seed-Transmitted Virus Diseases knowledge of the genetics of major gene resistance in breeding programmes may have militated against breeding for polygenic resistance by more empirical approaches. It is clear that before horizontal or polygenic resistances can be exploited in crop protection, there should be an attempt on cost–benefit assessment. The evidence for horizontal resistance against plant viruses is still sparse. It may be more effective to combine known major genes which are genetically well understood and therefore can be handled on a rational rather than empirical basis, to construct robust oligogenic systems. In some of the crops where seed transmission of virus is noticed, resistant/tolerant varieties were identified namely, soybean lines against Soybean mosaic virus (Almeida 1995; Provvidenti 1977; Pedersen et al. 2007), muskmelon lines against Cucumber green mottle mosaic virus (Rajamony et al. 1990), pea lines against Pea seed-borne mosaic virus (Khetarpal et al. 1990), cowpea against cowpea viruses (Sharma and Varma 1981) and soybean against Soybean mosaic virus (Cho and Goodman 1979; Lim 1985; Suteri 1986; Arif and Hassan 2002). Novel methods for revealing or creating variation and for transferring it between genotypes by nontraditional methods are already available and are increasingly being applied to resistance genes. The inability of even quite closely related species to hybridise creates a major barrier to transfer of potentially useful resistance genes. This may be especially relevant to wild relatives of crop species. Somatic hybridisation by protoplast fusion has already shown its ability to transfer virus resistance genes between species, where transfer by conventional breeding techniques has failed because of sexual incompatibility. At present, transfer of genes between genera by somatic hybridisation is more difficult than between species in one genus. Callus culture can induce mutation. Clones regenerated from callus cultures have shown an encouragingly high level of variability (somaclonal variation) in useful characteristics, with many clones outperforming the parent lines. Several of these instances involve improved disease resistance. Interestingly, it has been shown that variation induced by callus culture may be under monogenic or polygenic control. Culture techniques may provide a convenient, non-hybridisation method for selecting and handling polygenic resistance. The technique that leads to production of haploids, although spontaneous or chemically induced doubling of the chromosome number can give dihaploids. Production of a number of dihaploids can expose variation present in the parent, by eliminating dominance and complex hypostatic effects, and by offering a number of ‘cross sections’ of genetic variability which then can be usefully combined in accelerated breeding programmes. The process of haploidisation also may be mutagenic and leads to the association of virus resistance with other desirable characters. The basic techniques for transferring plant genes between taxonomically unrelated species, and for ensuring their expression in the recipient species, are now established. Many problems remain to be solved, but these are perhaps matters of degree rather than of fundamental principle. For resistance genes, the major difficulty remains in the isolation of useful genes which have been successfully transferred and coded. Known major proteins produced in large amounts, and these factors have undoubtedly eased gene identification. Knowledge of gene products and biochemistry of action is so scanty that isolation of resistant genes is going to be a major difficulty. Unless suitable ‘shotgun’ approaches to selection of resistance genes from gene libraries can be developed, there will remain a need for more fundamental research on mechanisms of gene activity. On the other hand, the demonstrated genetic simplicity of most of the presently understood mechanisms makes them primary targets for genetic manipulation. Furthermore, many viruses cause serious disease in several crop species, so a resistance gene made accessible to genetic manipulation might possibly be utilised several times. Care should be required, however, to ensure that this did not lead to placing the genetic control of virus diseases on too narrow base. 8.8 Resistance Manipulation of resistance genes from several sources against the same virus could be used to develop artificial polygenic systems. It seems unlikely that the current art of genetic manipulation could deal easily with natural polygenic systems or horizontal resistance, unless individual components could be recognised. It is established that many genetically understood resistance mechanisms involve gene dosage effects. It is possible that an increased effectiveness of resistance within a cultivar could be obtained by artificial amplification of gene number. Further reduction in virus multiplication might act to limit the frequency of evolution of virulence. Some authors have suggested that ability to transfer genes between non-hybridisable species might allow non-host immunity to be used for crop protection. This might be possible for nonhost immunity, although it would be difficult if a number of genes had to be transferred. Non-host immunity based on the ‘negative’ model would be impossible to transfer, being based on lack of a susceptibility gene. It does raise the idea of conferring resistance by deleting or nullifying a susceptibility gene in the normal host. In contrast to the complexities of the plant genome, the genomes of viruses are very simple to analyse and manipulate. The near future should see molecular explanations of the nature of virulence, which may provide valuable clues to mechanism of resistance gene function and host– virus interactions. Isolation and characterisation of the genes to be introduced are crucial steps in transformation, and various methods are currently available for the introduction of exotic genes into plant cells. Transformation using the Agrobacterium Ti-plasmid system is widely used directly in dicotyledonous plants. The utilisation of virus coat protein genes for the protection of virus infection is successful in crops like tomato, cucumber, capsicum, etc. The analysis of gene structures of economically important seed-transmitted viruses in commercial crops is to be carried out in order to use the information on the nucleotide sequence of the viruses and their satellite components for the cross protection. 201 8.8.1 Host Resistance Resistance to viruses manifests itself as absence of symptoms and/or restriction of virus multiplication and spread within the plant and is an effective measure to control seed-transmitted virus diseases. It may not often be practicable to adopt cultural and chemical control measures against seed-transmitted viruses in time because of cost and other considerations. However, use of resistant/tolerant varieties has been found to be the most efficient and economical method, provided stable sources of resistance are obtained. A precondition for the development of virusresistant varieties is that the plant breeder should be aware of the viruses which he is confronting and also with its host plant. There are instances of breaking down of resistance due to the evolution of new virus strains, and hence, the plant breeder aims to introduce disease resistance into cultivars that will provide useful disease control for a period long enough to ensure that the commercial life of a cultivar is not curtailed. The major advantage of breeding for resistance to viruses is that once a resistant cultivar is developed, no specific action is required by farmers to achieve control. More information on virus disease resistance can be had from review articles of Fraser and Van Loon (1986), Nene (1988), Boulcombe (1994), Khetarpal et al. (1998), Michael Deom (2004), Kang et al. (2005) and Gomez et al. (2009). 8.8.2 Sources of Resistance The examples of durable resistance include both monogenic and polygenic resistance, whereas theoretically polygenic resistance should be more durable. Some of the resistant sources identified against seed-transmitted diseases are discussed below. 8.8.2.1 Resistance in Cultivated Species Breeding for resistance to virus diseases which are seed transmitted has been successful in certain cases and is presented below. TMV in tomato (Honma et al. 1968; Besedina 1985) and Capsicum species (Provvidenti 1977; 202 8 Methods of Combating Seed-Transmitted Virus Diseases Rast 1982; Sowell 1982; Patezas et al. 1989); Tobacco streak virus (Kalyani et al. 2005, 2007); PMV in peanut (Kuhn et al. 1968) and soybean (Demski and Kuhn 1975); BCMV in peas (Provvidenti 1991) and beans (Temple and Morales 1986; Allavena 1989; Gupta and Chowfla 1990; Ogliari and Castarro 1992; Kelly 1997; Jones and Cowling 1995). BYMV and pea virus II in French beans (Provvidenti and Schrolder 1973); Bean pod mottel virus in Soybean (Hill et al. 2007); Soybean mosaic virus in soybean (Lim 1985; Arif and Hassan 2002; Cho and Goodman 1982; Hill et al. 2007; Pedersen et al. 2007; Domier et al. 2007) BYMV in soybean (Provvidenti 1975) and lupins (Mckirdy and Jones 1995a); subterranean clover (Mckirdy and Jones 1995b) and peas (Musil and Jurik 1990); PSbMV in lentils (Haddad et al. 1978; Latham and Jones 2001a; Anjum et al. 2005); faba beans (Fagbola et al. 1996) and peas (Hampton 1980; Hampton and Braverman 1979; Muehlbaur 1983; Baggett and Kean 1988; Provvidenti and Alconero 1988; Khetarpal et al. 1990; Maury et al. 1992; Thakur et al. 1995; Dhillon et al. 1995; Lebeda et al. 1999; Kraft and Coffman 2000; Latham and Jones 2001a, b); SMV in soybean (Koshimizu and Iizuka 1963; Ross 1977; Kwon and Oh 1980; Cho and Goodman 1979, 1982; Bowers and Goodman 1982, 1991; Hashimoto 1983; Buzzell and Tu 1984; Suteri 1986); Cowpea mottle virus in cowpea (Allen et al. 1982); CPMV in cowpea (Wells and Deba 1961; Robertson 1966; Beier et al. 1977); TRSV and CPMV in cowpea (Mali et al. 1987; Ponz et al. 1988); CpAMV in cowpea (Ladipo and Allen 1979; Mali et al. 1987); Black eye cowpea mosaic virus in cowpea (Ndiaye et al. 1993; Bashir et al. 2002; Lovely et al. 2006; Kamala et al. 2007); BCMV in French beans (Zaumeyer and Meiners 1975; Provvidenti 1977; Walkey and Innes 1979; Sastry et al. 1981; Naderpour et al. 2010); and Phasemy bean (Provvidenti and Braverman 1976); Melon mosaic virus in cucumber (Cohen et al. 1971); CGMMV in muskmelon (Rajamony et al. 1990) CMV in vegetable marrow, Cucurbita pepo (Pink and Walkey 1984; Walkey and Pink 1984); melons (Webb and Bohn 1962; Karchi et al. 1975; Lecoq and Pitrat 1983; Yang et al. 1986); mung bean (Sittiyos et al. 1979); and cowpea (Mali et al. 1987); ULCV in urd bean (Indu Sharma and Dubey 1984; Bashir et al. 2005; Ashfaq et al. 2007); Blackgram mottle virus in urd bean (Krishnareddy 1989); LMV in lettuce (Ryder 1970, 1976; Walkey et al. 1985; Pavan et al. 2008); BYMV in yellow lupin (Pospieszyn 1985). Alfalfa mosaic in Lupins (Jones et al. 2008) and CMV in lupins (Jones and Latham 1996). 8.8.2.2 Resistance in Graminaceous Cereal Crops Some of the examples for resistance for virus diseases of graminaceous crops are Sugarcane mosaic virus in sorghum (Teakle and Pritchard 1971; Henzell et al. 1982), BSMV in barley (Timian and Sisler 1955; Jackson and Lane 1981; Catherall 1984; Timian and Franckowiak 1987; Edwards and Steffenson 1996) and MDMV in maize (Miao-Hongqin et al. 1998). 8.8.2.3 Resistance in Fruit Crops Raspberry bushy dwarf virus (RBDV) is seed transmitted in Rubus idaeus to the extent of 22–60% (Cadman 1965; Converse 1973) and can be controlled through the use of gene Bu that confers resistance to D-type isolates of RBDV. In Rubus, new sources of virus resistance derived from pathogen themselves have been developed for RBDV in Scotland and North America. However, whether fruits produced from transgenic plants will be a susceptible socially remains to be determined (Martin 2002). Resistance gene factors for other virus diseases in certain important fruit crops are also well documented. 8.8.2.4 Resistance in Wild Species Certain wild species of crop plants have been screened when the resistant source is not identified among the cultivars. For example, Culver et al. (1987) identified resistant sources to Peanut stripe virus in Arachis diogio (PI46814 and PI-468142), A. helodes (PI-468144), Arachis sp. (PI-468345) and of the rhizomatosae section (PI-468174, PI-468363 and PI-468366). In India, peanut accessions of the Arachis 8.8 Resistance section A. cardenasii (PI-11558) were absolutely resistant to PStV even after sap, aphid and graft inoculation, while A. chacoense (PI4983), A. chiquitana (PI-11560), A. cardenasii (PI-11562 and PI-12168) and accessions of Erectoides section, A. stenophylla (PI-8215), and A. Paraguariensii (PI-8973) were resistant after aphid and sap inoculation only (Prasada Rao et al. 1989, 1991). Similarly, sources of resistance to Peanut mottle virus in eight peanut entries of Rhizomatosae and Arachis sections were also identified (Melouk et al. 1984). Jones et al. (2008) have reported that Lupinus angustifolius exhibited milder symptoms on sap inoculation with Alfalfa mosaic virus and seed transmission was only 0.8% in this host. From Canada, Singh (1985) reported clones 1,726 and 1,729 of PI473340 of Solanum berthaultii to be resistant to PStVd, which has high seed transmission. Provvidenti et al. (1978a) identified Cucurbita ecuadorensis and C. foetidissima to be resistant to CMV. Kalyani et al. (2007) identified 8 resistant accessions against Tobacco streak virus (TSV) in wild Arachis, which are cross compatible with A. hypogaea for utilisation in breeding programme. Regarding TSV, both positive and negative seed transmission reports are existing (Sharman et al. 2009; Prasada Rao et al. 2009). 8.8.3 Conventional Breeding of Natural Resistance Genes 8.8.3.1 Bean/BCMV Bean is originated in Latin America which is the largest producer. According to FAO (1990), the area of production in the world covers 24 millions ha, North America being the second largest producer followed by Africa. BCMV was found throughout the world wherever beans are grown. BCMV is also seed transmitted in other important legume crops like cowpea (previously BICMV – Blackeye cowpea mosaic virus) and peanut (previously PStV – Peanut stripe virus). Since the first bean cultivar resistant to BCMV (Spragg and Down 1921) improved cultivars of bean could be obtained by introduction of a dominant I gene conferring hypersensitivity, a 203 gene closely linked with resistance genes to other potyviruses BCMV (BICMV strain), CABMV, SMV and WMV-2 (Kyle and Provvidenti 1987). Resistance may also be conferred by recessive bc genes (Drijfhout 1978). Presently, resistance programmes have been achieved in developed countries giving satisfactory results. However, necrotic strains of BCMV induced severe epidemics in the USA (Provvidenti 1990). Further studies also indicated that the virus inducing such necrosis was classified as a potyvirus different from BCMV and named Bean common mosaic necrosis virus (BCMNV) (Vetten et al. 1992b). BCMNV is rare in bean crops in Europe and North America. The I gene-carrying cultivars resistant to BCMV are susceptible to BCMNV, but they would not transmit it through seed (Morales and Castano 1987). However, a virulent strain K causing mosaic on these I gene-carrying cultivars instead of necrosis has been identified (Buruchara and Gathuru 1979). Finally, I gene-carrying cultivars are resistant to both BCMV and BCMNV when they possess also the recessive bc resistance genes bc3 or bc22 (Davis et al. 1987). It is also interesting to analyse the situation in Africa, the third largest producer of bean: Africa often grows beans as traditional mixtures of 10–20 cultivars selected for taste, seed colour, speed of cooking and global resistance to the different pathogens. A survey in Rwanda indicated that 96% of farmers preferred planting mixtures because they resulted in higher and more predictable yields. In this country, as well as in Burundi, bean provides 45% of the total protein of human diet, which is the highest world percentage. Bean is also important in 22 other African countries. However, average bean production in Africa is 660 kg/ha and may be as low as 214 kg/ha, to be compared with 1,500 kg in North America and 2,000–3,000 in some parts of Europe (FAO 1990). BCMV/BCMNV is by far the most important virus disease of bean in Africa. BCMV was frequently isolated as seed transmitted from local farmers’ landraces of bean and was found prevalent in western Kenya. However, the widespread occurrence and predominance of 204 8 Methods of Combating Seed-Transmitted Virus Diseases BCMNV was clearly demonstrated in central eastern and southern Africa not only on beans but also, interestingly, on wild species of legumes of genera Cassia, Crotalaria, Macroptilium, Vigna and Rhynchosia, which also transmitted this virus through seed (Spence and Walkey 1993). BCMNV is not thought to occur naturally in South America, the centre of origin of P. vulgaris. When this virus has been recorded there or in North America, its occurrence has been traced to infected imported seed (Spence and Walkey 1993). In contrast, the widespread occurrence and predominance of BCMNV infecting both beans and wild species of legumes could qualify Africa as the geographical centre of origin of this virus. The observation that BCMNV is often latent on wild species of legumes would suggest host adaptation after long-term exposure and add support to such a hypothesis. To conclude about this system, the sources of resistance to BCMV/BCMNV are thus identified and satisfactorily used in industrialised countries; in Africa, a problem of virus diversification and a problem of ground to make up for controlling the same disease, the regional crop improvement programmes for providing African farmers with agronomically adapted resistant cultivars are not achieved. This situation thus appears as most critical in countries which have a nutritional dependence on this crop: It calls for other approaches for controlling this disease in the short term. programmes of incorporation of natural resistances have been achieved only in some countries. Such type of resistance is not completely satisfactory as the virus multiplies and virulence has been observed. Other approaches are necessary for controlling LMV. 8.8.3.2 Lettuce/LMV Two recessive genes g and mo govern a type of resistance named tolerance to LMV; that is, the virus multiplies but does not induce symptoms; seed transmission is also much reduced. The gene g was largely introduced in European varieties; in North America, a few varieties incorporate g or mo. These two genes, considered as identical, would rather be closely linked genes, the gene mo conferring a resistance to a larger range of strains. Virulence of LMV with respect to both genes has been observed, and seed transmission too. Other sources of resistance have been discovered in cv. Ithaca (single dominant gene) and in wild Lactuca species that would help improving the control (see Dinant and Lot 1992). For this crop, 8.8.3.3 Barley/BSMV Some sources of resistance to BSMV exist, and they are being mapped in relation to molecular markers, in view of being included in barley breeding programmes (Edwards and Steffenson 1996). 8.8.3.4 Soybean/SMV Soybean mosaic potyvirus is widespread in areas where this crop is grown. In the USA, six resistant soybean cultivars were used as a reference for classifying SMV strains into seven pathotypes. Several dominant resistance genes and also a recessive resistance gene have been identified. Particularly, a dominant gene Rsv2 conferred resistance by hypersensitivity to all the seven pathotypes of SMV identified in the USA (Buzzell and Tu 1984). However, in Japan, several strains of SMV had virulence properties different from strains identified in the USA. Another type of resistance, preventing the long-distance movement of SMV in the soybean plant and effective against all Japanese strains, has been detected in three cultivars (Lizuka and Yunoki 1983). 8.8.3.5 Tomato/TMV TMV and ToMV cause significant losses, in normal and soilless culture conditions because both are highly infectious and stable and easily transmitted by mechanical inoculation. Use of virusresistant varieties has provided effective control of these two tobamoviruses. Gene Tm-l from Lycopersicum hirsutum and gene Tm-2 or Tm22 from L. peruvianum are two well-known sources of resistance. Particularly, Tm-22 has proven very effective against TMV and ToMV wherever used (Watterson 1993). However, breeding for resistance has been laborious due to the tight linkage of Tm-22 with undesirable traits. 8.8 Resistance 8.8.3.6 Pepper/TMV, ToMV and PMMoV TMV, ToMV and PMMoV cause significant losses. Four different tobamoviral resistance genes are known in the Capsicum spp., namely, Ll from C. annuum, L2 from C. frutescens, L3 from C. chinensis and L4 from C. chacoense (Watterson 1993). These genes govern a hypersensitive response. Particularly, L3 confers a very effective resistance. Certain isolates of PMMoV are the only tobamoviruses able to overcome the L3 resistance. 8.8.3.7 Pea/PSbMV Three recessive genes sbml, sbm3 and sbm4 confer immunity against a large range of strains of Pea seed-borne mosaic virus (Provvidenti and Alconero 1988). These three sbm genes were grouped with resistance genes to other potyviruses of pea and conferred to these lines an excellent level of resistance to experimental check. Therefore, this resistance is being introduced in improved cultivars. However, lines having the 3 sbm genes could be experimentally infected at low frequency in the glasshouse by a strain of a virulent pathotype (Khetarpal et al. 1990, 1997). 8.8.3.8 Other Crops No resistance to PeMoV and PStV is known in cultivated peanut. Besides, sources of resistance are available for a number of seedtransmitted viruses of which important one are BCMV (BICMV)/cowpea, CABMV/cowpea, SBMV/French bean, SqMV/cucurbits, ULCV/ mung bean and urd bean. To summarise, conventional breeding of natural resistance genes is playing a significative role in the control of seed-transmitted viruses but far from a generalised implementation, either because in some cases, no source of useful resistance is available or because the natural available genes of resistance have not been incorporated in cultivars growing in important areas, or because virulent isolates overcome such natural resistances. The biotechnological approach, known for maintaining the important genetic traits of a cultivar to be transformed for resistance, is an exciting new field which has come to rescue the 205 cases where resistance sources are not available or to pyramid natural and engineered resistance in the search for durable resistance. 8.8.4 Cultivars with Low Seed Transmission Attempts have also been made to locate cultivars in which the virus transmission through seed is either low or nil, since the reduction of seed transmission has a major impact on virus spread as they form the primary source of virus inoculum from plant to plant spread in the field. Soybean lines PI-86736, improved Pelican and UFV-1 were found to possess low rate of seed transmission of SMV with superior agronomical qualities (Goodman and Oard 1980; Irwin and Goodman 1981). Against the same virus, Goodman and Nene (1976) also identified 12 lines of soybean in which seed transmission was not noticed. At Columbia (USA), the lowest incidence of BCMV (0–1%) was recorded in French bean lines, namely, Pinto-114, Imuna and Great Northern 123 and 31, while in susceptible lines, the extent of seed transmission was up to 54.4% (Morales and Castano 1987). PMV was not seed transmitted in the peanut lines EC-76446 (292) and NCAC 17133 (RF), and these are used in resistant breeding programme (Bharathan et al. 1984). In Montana (USA), Mobet barley germplasm (PI-467884) was developed for its resistance to seed transmission of three isolates of BSMV (Carroll et al. 1983). In Poland, yellow lupin variety Topaz had the least tendency to transmit BYMV through its seeds (Pospieszyn 1985). Combined resistance to seed transmission of four viruses, namely, BICMV, TMV, CpAMV and CMV, in Vigna unguiculata was identified by Mali et al. (1987) in genotypes like CoPusa-3, N-2-1 and V-16. While screening the germplasm, locating a resistant line against a number of virus strains with good agronomical characteristic is a major objective. Often the varieties found resistant to one virus strain turn out to be susceptible to another virulent virus. For example, several cultivars of pea were reported to be resistant to an 206 8 Methods of Combating Seed-Transmitted Virus Diseases isolate of Pea early browning virus at one site in Britain, whereas at another site, all of them were susceptible (Harrison 1966). Plant breeders are therefore advised not to rely on results from only one site in making selections for resistance. 201, 410 and 3273), although resistant to aphid (A. craccivora), could not establish resistance for CpAMV (Atiri et al. 1984). In India, Mali (1986) reported that cowpea genotype, P-1476, was resistant to A. craccivora. As resistance to a vector may result in an increased level of virus spread, it is incumbent upon those breeding for resistance to consider the probable effect of vector resistance on virus spread. In addition, since resistance to one arthropod species may be associated with altered levels of susceptibility to other species, the potential impact on virus spread of cross resistance to a vector species should not be ignored. Despite these dangers, the potential for controlling certain types of virus diseases through vector resistance is considerable. There exist a sufficient number of examples wherein vector resistance has contributed to disease reduction to justify continued efforts in this area. Research in vector behaviour (e.g. alighting and probing) as it is influenced by the host genotype may provide further insight into this aspect of resistance to vectors and its relationship to virus spread. In almost all the crops, resistance sources were identified and used in breeding programmes against majority of the seed-transmitted virus and vectors, and the approach is likely to be used increasingly in the future. However, resistance was broken in certain instances due to introduction of newer virus strains. Hence, resistance developed to strains in one location cannot be expected to operate against strains occurring elsewhere, and growing a cultivar in a new area may lead to serious epidemics. Furthermore, evidence is required on the durability of different types of resistance, on possible gene-for-gene relationships, on the relative merits of major and minor genes and on the possible advantages of using mixtures of different genotypes. At least, some of this information may soon be forthcoming because of the increased attention being given to breeding for some form of resistance against seed-transmitted viruses at many establishments and especially at the International Research Institutions. The inheritance pattern reported in different crop species against major virus diseases is presented in Table 8.1. 8.8.5 Vector-Resistant Cultivars Resistance to a virus vector is likely to exert a complex influence on virus spread. Since non-preference, antibiosis and tolerance are often combined into a single resistant cultivar, their relative contribution to the resistance as well as the overall magnitude of the resistance will influence the effect of resistance on virus spread. In addition, observed virus spread is the result of both primary and secondary spread and the relative importance of these and the effect of the resistance on them is important. The complexities involved are such that without a thorough understanding of the ecology of the virus and vector and the biology of vector resistance, it would be impossible to predict a priori the effect of vector resistance on virus spread. Each combination of virus, vector and host resistance must be considered separately. The effectiveness of a vector-resistant cultivar for the spread of seed-transmitted plant viruses will mostly depend on the type and effectiveness of resistance, its relative importance in primary (introduction of virus from outside the crop) and secondary (spread of virus within the crop) virus spread and virus–vector relationship (Kennedy 1976). The cumulative interaction of these factors may result in eventual differential effect on the spread of seed-transmitted viruses. For example, Wilcoxson and Peterson (1960) found less incidence of Pea seed-borne mosaic virus in fields of aphid-resistant red clover cv. Dollard than in adjacent fields of the relatively susceptible cv. Wegner. Lecoq et al. (1979) identified Cucumis melo Songwhan Charmi (PI-161375) to be resistant to CMV when tested with aphids (M. persicae and A. gossypii), and the mechanism was not specific to any virus strain. Studies conducted in Nigeria indicated that cowpea lines (TVU 408–2; 8.9 Immunisation 207 Table 8.1 Seed-transmitted viruses: inheritance pattern in different crop species Virus Alfalfa mosaic Barley stripe mosaic Bean yellow mosaic Blackeye cowpea mosaic Blackeye cowpea mosaic Cowpea mosaic Cowpea aphid-borne mosaic Cucumber mosaic Cucumber mosaic Pea seed-borne mosaic Crop Alfalfa Barley Cowpea Soybean Cowpea Cowpea Soybean Cowpea Mung bean Pea Type of resistance Monogenic Monogenic Monogenic Monogenic Monogenic Monogenic Monogenic Monogenic Monogenic Monogenic Pea seed-borne mosaic Soybean mosaic Lentil Soybean Monogenic Monogenic Tobacco ring spot Cowpea chlorotic mottle Peanut mottle Cowpea Soybean Soybean Monogenic Monogenic Monogenic Bean southern mosaic Bean common mosaic Bean Bean Monogenic Monogenic 8.9 Immunisation When field application is considered, the protecting strain is generally an isolate which induces mild symptoms and does not affect the marketable yield of the crops. However, when losses from the virus disease are great, some yield reduction induced by the protecting strain might be economically tolerable. Successful application of immunisation/cross protection in some seedtransmitted viruses in certain host plants is discussed herein. In Japan, the effects of Soybean mosaic virus (SMV) were reduced by immunisation technique under field conditions (Kosaka and Fukunishi 1994). In 1990 and 1991, c.20,000 and 78,000 seedlings, respectively, of black soybean (cv. Shin Tambaguro) were transplanted 1–3 days after protective inoculation with an attenuated isolate of SMV, Aa 15-M2. In the plants that received protective inoculation, virulent strains of SMV were effectively suppressed. Seeds virtually free from SMV were produced in 1990 (971 kg/ha) and free from SMV in 1991 (4,740 kg/ha). Black Reference Fraser (1986) Carroll et al. (1979b) Reeder et al. (1972) Provvidenti et al. (1983) Taiwo et al. (1981) Raj and Patel (1978) Provvidenti et al. (1983) Khalf-Allah et al. (1973) Sittiyos et al. (1979) Hagedorn and Gritton (1973) and Provvidenti and Alconero (1988) Haddad et al. (1978) Provvidenti et al. (1982), Buzzell and Tu (1984), and Provvidenti and Hampton (1992) Fraser (1986) Boerma et al. (1975) Boerma and Kuhn (1976) and Provvidenti and Hampton (1992) Zaumeyer and Harter (1943) Provvidenti and Hampton (1992) soybean seedlings grown from these seeds were widely transplanted in growers fields in 1991 (45 ha) and 1992 (50 ha) in Wachi, Kyoto (Japan). The incidence of virulent strains of SMV in late July was found to be 1.3% in 1991 and 0.8% in 1992, compared with the rates of >60% in 1989 and 1990. The use of seeds from A1 15-M2inoculated plants was also found to be effective for the control of SMV in other areas during 1992. In peanut, PStV is economically important, and Wongkaew and Dollet (1990) categorised 24 isolates from eight different countries into eight strains based on disease reactions on specific hosts and serology. Possibility exists for immunisation phenomenon at field level. Preliminary information is available on the use of this technique against PStVd, which is seed transmitted in true potato seed (Singh et al. 1990, 1993). Perring et al. (1995) and Walkey (1992) demonstrated that in vegetable marrow Zucchini yellow mosaic virus, incidence could be reduced by mild strain inoculation at seedling level prior to aphid transmission of severe strain. 208 8 Methods of Combating Seed-Transmitted Virus Diseases Although the use of a mild strain to protect the plants against more virulent strains has been suggested by Kunkel (1934), the technique has not been widely adopted by tomato growers until Rast (1972, 1975) from the Netherlands demonstrated the commercial benefits of inoculating crops with the avirulent TMV strain MII-16. Growers in the Isle of Wight (UK) were the first to adopt the immunisation technique in 1964, although a mild strain of TMV has not been available and the normal fairly severe strain of Tomato mosaic was used for inoculation in the seedling stage. The proportion of unsalable and poor quality fruit was reduced from about 30 to 3% in 1965. These results have led to the idea that the benefit gained from seedling infection would be greater if a mild strain of TMV that would protect the plants from severe strains could be used. In capsicum and tomatoes, TMV is both externally and internally seed transmitted. Extensive attempts have been made to combat TMV infection in tomatoes through immunisation, in which the plants are deliberately inoculated with avirulent strain which will give protection against the severe strain of the same virus. In the Netherlands, Rast (1972) isolated an almost symptomless mutant using the nitrous acid mutagenic technique which gave sufficient protection to five strains of TMV, when challenge inoculated. One of the effects observed on tomato plants following early infection with MII-16 was a temporary check of growth which delayed flowering and fruit set, indicating the necessity to advance the sowing date by about a week to compensate for this. He also found that plants inoculated at the seedling stage with MII-16 gave better yields than those inoculated at the same growth stage with the parent strain. Upstone (1974) reported that in 27 trials in the UK, plants inoculated with Rast’s MII-16 strain, on average, yielded 5% more than the uninoculated plants. The values of immunisation as a control measure against TMV infection in tomatoes grown in glasshouse conditions have been well established in different countries like Denmark (Paludan 1975), France (Migliori et al. 1972), the UK (Evans 1972; Fletcher and Rowe 1975; Channon et al. 1978), Israel (Zimmerman and Pilowsky 1975), Belgium (Vanderveken and Coutisse 1975), former USSR (Vlasov 1972), the Netherlands (Rast 1972, 1975), Japan (Oshima et al. 1965; Goto et al. 1966; Nagai 1977; Oshima 1981), New Zealand (Mossop and Procter 1975), German Democratic Republic (Schmelzer and Wolf 1975), the United States (Ali Ahoonmanesh and Shalla 1981) and China (Tien and Chang 1983). Most of these immunisation experiments were attempted with Rast’s MII-16 strain. In Denmark, Paludan (1975) worked with Danish attenuated TMV strain (K58-45-0) and found it not as encouraging as MII-16. The TMV strain K58-45-0 caused weak to moderate symptoms throughout the growing period. The TMV-MII-16 strain gave higher protection than the TMV strain K58-450. In Japan, Oshima et al. (1978) used LIIA, an attenuated tomato strain of TMV, and proved it to be ineffective for the existing TMV-resistant tomato cultivars having the Tm-1 gene. This was possibly because the strain multiplied very little in these cultivars and the plants were readily infected by virulent strains such as CH2 . In order to obtain another attenuated strain for protecting these cultivars, strain LllA 237 was isolated by successive passage of LIIA through TMV-resistant GCR 237 tomato bearing homozygous TMV-resistant gene Tm-1. The new strain also caused no symptoms in tomato and Samsun tobacco but differed from LllA in that it multiplied much faster in the existing TMV-resistant cultivars (Tm-1/T). It sometimes caused necrotic rings on Xanthi-nc tobacco in the environment in which LllA causes necrotic spots. In immunisation tests, a resistant cultivar, LllA 237, interfered more strongly with the multiplication of CH2 than did LllA. In glasshouse experiments, inoculation of resistant tomato Tanamo (Tm-1/T) with LllA 237 gave good control of the disease (Oshima et al. 1978; Oshima 1981). In China, a symptomless TMV mutant, N14 was produced by using nitrous acid mutagenic technique and this mild strain under field inoculations increased tomato yield by 5–60% (Tien and Chang 1983). As this immunisation technology proved beneficial to tomato growers in the Netherlands, the UK, Japan, New Zealand, etc., techniques 8.10 Approved Seed Certification Standards were developed for mass inoculation. Rast (1972) reported a spray gun method of inoculation to be better than manual inoculation because of the risk of contamination with the other strains. The MII-16 strain became commercially available in the UK in 1973 and is sold in 5 ml quantities of concentrated virus which has to be diluted 1,000 times before use. In addition to facilitating higher yields, this mild strain offers an advantage in seed production. Seedling inoculation with this strain results in negligible TMV infection of seeds. In contrast, infection is higher in plants inoculated with the virulent strains, where the virus is both exogenously and endogenously seed transmitted. So the deliberate inoculation with the mild strain could be used by seed growers and is an alternative to heat treatment. Steeply (1968) found a lower incidence of seed infection with a heat-attenuated strain. Prunus necrotic ring spot virus (PNRSV) and Apple mosaic virus (ApMV) are seed transmitted in some fruit crops; limited information is available on the use of cross protection technique to protect these crops. For example, Wood et al. (1975) found that the yields of Jonathan apple trees infected with ApMV almost doubled when these trees were top worked with scionwood containing a mild strain of ApMV and the symptoms of virus were alleviated. However, the yields obtained were still substantially lower than yields from mosaic-free trees, suggesting that replanting with healthy trees would be more economical in the long term. Mild strain protection of cherries against Cherry rugose mosaic strain of PNRSV may be possible. Mink (1983) reported a strain of PNRSV which produces no detectable effect on either tree vigour or fruit quality. Circumstantial evidence suggests that this symptomless strain might provide protection against later infection of trees by severe strains of the virus. The following points are to be seriously taken, while immunisation technique is followed under field condition in large-scale crop cultivation: 1. The mild strain of the virus should be fully systemic and invade all host tissues. Indeed, cross protection effectiveness depends on the presence of the mild strain in all tissues to be protected from severe strains. 209 2. The inoculated plants should induce milder symptoms than isolates commonly encountered in the fields and should not alter the yield and quality of the crop. It should also be mild in all its cultivated hosts including those which are not targets for the cross protection. 3. Mild strain multiplication should be conducted in highly protected under strict phytosanitary supervision in order to eliminate risk. 4. For mass multiplication, it is essential to provide to farmers and farm advisers an easy and efficient mild strain inoculation technique. However, some apprehensions have been expressed about immunisation. First, the mild or protecting strain could mutate to a highly virulent form, leading to crop losses rather than protection. Second, the protecting virus could act synergistically with an unrelated virus to create a disease combination that is more damaging than either virus on its own. Third, the mild strain could be virulent in other crops, making it unwise to spread it so extensively. Finally, the protecting strain generally causes a small but significant reduction in yield. Even with these limitations, plant immunisation has the potential for being highly effective in modern agriculture. For the success of this phenomenon in different crops, the selection of the mild strain is of paramount importance. An intensive approach is required to find a milder strain having desirable properties in terms of qualitative and quantitative crop yields. The strain should be tested regularly for both avirulence and protective power. The methods of application and timing of virus inoculation in relation to conditions of plant growth need careful study. 8.10 Approved Seed Certification Standards Seeds are the foundation to crop production, and seed health is related to food production in various ways. Seed being the foundation of successful agriculture, the demand for quality seeds of improved varieties/hybrids is growing fast and adoption of new technologies around the world 210 8 Methods of Combating Seed-Transmitted Virus Diseases by the farmers is happening at an amazing pace. Therefore, production and supply of high-quality seed of improved varieties and hybrids to the grower are a high priority in agricultural growth and development. Governments are strongly encouraged to implement a predictable, reliable, user-friendly and affordable regulatory environment to ensure that farmers have access to high-quality seed at a fair price. In particular, FAO member countries are participating in the internationally harmonised systems of the Organization for Economic Cooperation and Development (OECD), the International Union for the Protection of New Varieties of Plants (UPOV), the International Treaty on Plant and Genetic Resources for Food and Agriculture (ITPGRFA) and the International Seed Testing Association (ISTA). Participation in these systems will facilitate the availability of germplasm, new plant varieties and high-quality seed for the benefit of their farmers, without which their ability to respond to the challenges ahead will be substantially impaired. It is inevitable that governments need to develop and maintain an enabling environment to encourage plant breeding and the production and distribution of high-quality seed. multiplication. The agencies involved in the production of breeder seeds are agricultural universities and the ICAR institutes in India. Foundation seed is the progeny of breeder seed, and it is supervised and certified by the Seed Certification Agency. It should possess the minimum genetic purity of 99.00%. Expert handling is needed for producing foundation seed, and they are produced on seed farms where specialists and facilities are available. Agencies involved in the production of foundation seeds are the State Department of Agriculture, Horticulture, State Seed Corporation and the State Farms Corporations of their respective countries. Certified seed is the progeny of foundation seed and certified by the certification agency. The agencies involved in the production of certified seeds are the State Seed Corporation through their Registered Certified Seed Growers and the Private Seed Companies. Its genetic purity is 98.00% for varieties/composites/synthetics, and for cotton and castor hybrids, it is 85%. The seed should meet minimum standards of germination and purity prescribed and labelled as per Seed Act 1966. Use of quality seed and planting material which are free from seed-transmitted diseases including viruses contributes to the enhancement in crop production by better realisation of its own productivity potential (to the extent of 20–25% depending on the crop) and also by contributing to enhance the utilisation efficiency of other inputs to the extent of another 20–25%. Therefore, availability of good quality disease-free seeds of superior varieties/hybrids needs to be ensured for boosting the agricultural production. The central seed certification board, department of agricultural and cooperation, ministry of agricultural, and Government of India, New Delhi, have prescribed disease certification standards in foundation, and certified seed crops are provided in the table (Table 8.2). 8.11 Stages of Seed Multiplication Genetically pure, disease-free seeds are the prerequisite for a healthy, vigorous and highyielding crop. There are three important stages of seed multiplication. Nucleus seed, also known as original parental material, is the product of scientific breeding by crop breeders who evolved the varieties/hybrids. Breeder seed is the first and is the product of scientific breeding by crop breeders who evolved the particular variety. Breeder seed (sexually/asexually propagating material) is directly controlled by the original plant breeder. The production is also personally supervised by the breeder. It should possess the maximum genetic purity (99.5–100%). Breeder seed is made available in small quantities for further 8.12 Inoculum Threshold The level of the pathogen in seeds which gives rise to an unacceptable risk of disease is often referred to as ‘inoculum threshold’, although the 1.0 0.5 0.5 Severe mosaic Leaf roll Stage I seed Foundation seed 0.10 0.10 1.0 0.1 0.1 0.1 0.1 0.1 0.05 – Mild mosaic Virus Common mosaic Bean mosaic Jute chlorosis Cucumber mosaic Cucumber mosaic Watermelon mosaic Tomato mosaic Lettuce mosaic Mosaic Mild mosaic Severe mosaic Leaf roll Based on Tunwar and Singh (1988) and Narayan Rishi (2000) Potato tuber Tomato Lettuce Sweet potato Potato true seed Crop Cowpea French bean Jute Muskmelon Summer squash 0.75 0.75 2.0 Stage II seed 1.0 1.0 3.0 Certified seed 0.20 0.20 2.0 2.0 0.5 0.5 0.5 0.5 0.10 3.0 1.0 1.0 Certified seed Table 8.2 Maximum per cent virus disease certification standards in foundation and certified seed crops Stage II – 60 and 70 days in early and late varieties Stage I – 35 and 45 days in hills and plains, respectively Evaluation stage At flowering and fruiting stage At flowering and fruiting stage At maturity prior to harvest At maturity prior to harvest At fruit maturity Prior to harvesting Prior to harvesting Prior to harvesting At flowering stage At harvesting At harvesting At harvesting 8.12 Inoculum Threshold 211 212 8 Methods of Combating Seed-Transmitted Virus Diseases term ‘tolerance standard’ is perhaps less misleading and to be preferred. The risk of a ‘significant’ epidemic developing is dependent on the rate of transmission from seed to seedling and the rate of disease increase in the crop (both of which are highly dependent on environmental conditions). The use of infected seed for sowing is another major factor contributing to disease incidence and eventual crop loss. For instance, in England, lettuce crops are grown from 5 commercial seed lots with 2.2–5.3% infection of LMV, the plants became infected with mosaic were 25–96%, whereas 6 seed lots at <0.1% infection have produced crops with fewer than 0.5% of infected plants at the end of the growing season (Tomlinson 1962). However, where aphid population was high, as in the case of California (USA), lettuce seed at <0.1% infection has produced severe outbreaks (Grogan et al. 1952; Zink et al. 1956). Even in the case of peanut, the incidence of PMV depends partly on the initial level of seed infection (Paguio and Kuhn 1974). In irrigated plots, an average yield reduction among three barley cultivars of Betzes, Compana and Vantage ranged from 24 to 35% when plants were BSMV infected by mechanical inoculation (Carroll 1980). The reduction in barley grain yield, number of heads and seed weight due to BSMV was found to be linear in response to the level of seed infection (Nutter et al. 1984). Attempts on inoculum threshold were also made by Coutts et al. (2009) in pea infected with Pea seed-borne mosaic virus (PSbMV). If the primary virus inoculum through pea seed is 0.3– 6.5%, the final PSbMV incidence reached 98%, and the yield losses were 18–25% (Fig. 8.2). Neya et al. (2007) have studied epidemics development of Cowpea aphid-borne mosaic virus in cowpea by using 0–5% initial seed infection. Because of the high level of resistance in the cowpea varieties used, the progress of the disease was very low (<50%) in all treatments. Even Puttaraju et al. (2002) have also noticed that cowpea seeds with 5% Blackeye cowpea mosaic virus have resulted in yield loss of 34–53%, while 0.5% seed infection did not cause significant loss. Narrow-leafed lupin (Lupinus angustifolius) is one of the important cool season grain legumes in parts of Eastern Europe, Russia, southern Africa and Australia for green manure, forage and medicinal purposes, and CMV affects crop yields and also virus is seed transmitted to the extent of 12–18% (Jones 1988, 2000) and causes epidemics. An integrated virus disease management approach was used to minimise the yield losses, and sowing lupin seed stocks with minimal virus contents provide a key control measure within this strategy (Bwye et al. 1995, 1997, 1999; Jones 1988, 1991b, 2000, 2001; Jones and Proudlove 1991; Thackray et al. 2004). Since 1988, a commercial testing service for CMV in lupin seed samples has provided an estimate of per cent CMV infection based on a 1,000-seed test. This helps farmers to avoid sowing seed with infection levels likely to result in serious yield losses. Bwye et al. (1994) have reported that sowing 1% CMV infected lupin seed resulted in significantly decreased yields in 2 experiments, while 0.75 and 0.5% infected seed caused significant losses in 1 experiment (16–19% losses). For lupin-growing areas at lower risk from CMV, a threshold of <0.5% seed infection suffices to avoid serious yield losses in most years, but for high-risk areas, a threshold level of <0.1% is used for lupin grain crops, that is, a zero test result on a 1,000-seed sample (Bwye et al. 1995; Jones 2000, 2001). A model forecasting CMV epidemics in lupins was incorporated into a decision support system by the Department of Agriculture and Food, Western Australia, for use by farmers and agricultural consultants in planning CMV management (Thackray et al. 2004) and is accessed via the departments’ websites. From the four field experiments conducted at Mysore (India), Udayashankar et al. (2010) showed that sowing of cowpea seeds with 10, 5, 3, 2, 1, 0.75, 0.5 and 0.05% infection of Bean common mosaic virus (Blackeye cowpea mosaic strain) BCMV–BICM results in an average cumulative disease incidence of 90, 53, 37, 26, 12, 8, 6 and 1%, respectively. The seed yield reduction was directly correlated to levels of seed infection. Seed infection of 10, 5 and 1% resulted in 74, 54 and 18% seed yield loss per plant. Infection of 0.75 and 0.5% reduced the seed yield per 8.13 International Seed Testing Association (ISTA) 213 Fig. 8.2 Plots of field pea cv. Kaspa at Avondale in 2005 (experiment 1) originally sown with (a) 6.5% and (b), 0.3% Pea seed-borne mosaic virus (PSbMV)-infected seed. Note pale appearance and uneven canopy with obvious depressions caused by widespread PSbMV infection in (a) and vigorous growth with a uniform canopy in (b) (Source: Coutts et al. 2009; R.A.C Jones) plant by less than 1%. These field experiments demonstrated that sowing >1% BCMV–BICMinfected seed can lead to significant losses in grain yield, while the spread of BCMV–BICM infection resulting from sowing 1% infected seed did not significantly decrease seed yield. In some of the Prunus species and other stone fruit crops, which are propagated on root stocks also carry seed-transmitted virus diseases like Prunus necrotic ring spot virus and Prunus dwarf virus. Most of the stone fruit certification programmes specify that Prunus seedling lot must contain fewer than 5% virus-infected plants. Because the demand of certified seed is greater than the supply available in most years, nurseries often purchase large amounts of Prunus seed of unknown virus contents, which exceeds the 5% tolerance (Mink 1984). Actually even 5% tolerance level results in the production and distribution of contaminated bud wood. By the application of serological and molecular virus diagnostic tests, there is every reason to believe that a zero tolerance level for these two viruses in Prunus seedlings can be achieved and will be demanded in the future. There is requirement of maintaining the inoculum threshold levels in almost all certification schemes of fruit trees. More details on inoculum threshold level can be had from the review articles (Jones 2000; StaceSmith and Hamilton 1988; Roberts 1999). 8.13 International Seed Testing Association (ISTA) The International Seed Testing Association (ISTA) was founded in 1924 during the 4th International Seed Testing Congress held in Cambridge, United Kingdom. As on June 2012, its membership consists of about 201 member laboratories, 52 personal members and 42 associate members, from 79 countries around the world. Nearly 120 of the ISTA member laboratories that are accredited by ISTA are entitled to issue ISTA international seed lot analysis certificates. ISTA is independent and 214 8 Methods of Combating Seed-Transmitted Virus Diseases acts free from economic interest and political influence, and it is unbiased, objective and fair. Furthermore, the hitherto unsurpassed expertise of ISTA is based on the non-profit, cooperation of the international community of approximately 400 experienced, competent and energetic seed scientists and analysts. 8.13.2.1 Orange International Seed Lot Certificate An Orange International Seed Lot Certificate is issued when both sampling from the lot and testing of the sample are carried out under the responsibility of an accredited laboratory, or when sampling from the lot and testing of the sample are carried out under the authority of different accredited laboratories. Where the accredited laboratory carrying out the sampling is different to the one carrying out the testing, this must be stated. The procedure followed links the Orange International Seed Lot Certificate with the seed lot. The certificate is coloured orange. In the case of Orange International Seed Lot Certificates, the results reported refer strictly to the lot as a whole at the time of sampling. 8.13.1 Objectives of ISTA (a) The primary objective of the association is to develop, adopt and publish standard procedures for sampling and testing seeds and to promote uniform application of these procedures for evaluation of seeds moving in international trade. (b) The secondary objective of the association is to promote research in all areas of seed science and technology, including sampling, testing, storing, processing and distributing seeds; to encourage variety (cultivar) certification; to participate in conferences and training courses aimed at furthering these objectives and to establish and maintain liaison with other organisations having common or related interests in seed. Seed quality determination, as established by ISTA, on seed to be supplied to farmers is an important measure for achieving successful agricultural production. The establishment or maintenance of an appropriate infrastructure on the scientific as well as technical level in developed and developing countries is highly commendable. 8.13.2 ISTA Certificates Certificates like Orange International Seed Sample Certificate and Blue International Seed Sample Certificates are issued by accredited member and laboratories of ISTA and must only be issued in accordance with the ISTA rules currently in force after seed analysis. Well qualified and trained personnels are involved in ISTA labs and international standards are maintained in seed quality testing. 8.13.2.2 Blue International Seed Sample Certificate A Blue International Seed Sample Certificate is issued when sampling from the lot is not under the responsibility of an accredited laboratory. The accredited laboratory is responsible only for testing the sample as submitted. It is not responsible for the relationship between the sample and any seed lot from which it may have been derived. The certificate is coloured blue. In the case of Blue International Seed Sample Certificates, the results reported refer strictly to the sample at the time of receipt. The Blue International Seed Sample Certificate refers only to the sample submitted for testing. 8.13.2.3 Provisional Certificate A provisional certificate is an ISTA certificate issued before the completion of a test or tests. It is marked PROVISIONAL and must include a statement under ‘other determinations’ that a final certificate will be issued upon completion. 8.13.3 Conditions for Issuance of ISTA Certificates ISTA certificates must be issued only on forms obtained from the ISTA secretariat and approved by the ISTA executive committee. There are two 8.14 Seed Certification Against Plant Virus Diseases 215 kinds of certificates: Orange International Seed (h) For an Orange International Seed Lot Lot Certificates and Blue International Seed SamCertificate, each container in the lot must ple Certificates, as defined earlier. be marked, labelled and sealed in accordance An ISTA certificates may be issued only by with ISTA (ISTA 2011). the seed testing laboratory which either carried (i) An Orange International Seed Lot Certificate out all the tests to be reported or subcontracted shall be valid until it is superseded by sampling and/or some of the tests to be reported, another valid Orange International Seed Lot and under the conditions listed below: Certificate subsequently issued on the same (a) The issuing laboratory must be currently aulot. Not more than one Orange International thorised to do so by the executive committee. Seed Lot Certificate shall be valid for a lot at (b) The seed tested must be of species listed in one time for any particular test. the ISTA manual (ISTA 2011) according to (j) For an Orange International Seed Lot the ISTA rules and shall be considered to be Certificate, the submitted sample must be covered. tested by an accredited laboratory. The issuing Consequently, no certificates may be laboratory must ensure that sampling, sealing, issued for species not listed in the current identification, testing and issuance of the ISTA rules, nor for mixtures of species which certificate are in accordance with the ISTA are in the ISTA rules, as no procedures are rules, although subcontracting of sampling prescribed for mixtures. and/or testing to another accredited laboratory (c) The tests must be carried out in accordance is permissible. The laboratory which carries with the ISTA rules. However, additionally out sampling must provide all the information and on request, results of tests not covered that is necessary to complete the Orange by these rules may be reported on an ISTA International Seed Lot Certificate. certificate. Results of analyses not covered by the current ISTA rules may be included on a certifi- 8.13.4 Accredited Laboratory cate only if results of at least one test covered An accredited laboratory is an ISTA-accredited by the ISTA rules are also being reported. (d) For the result of determination of moisture member laboratory authorised by the ISTA excontent to be reported on an ISTA certificate, ecutive committee under Article VII of the ISTA the sample must be submitted in an intact, constitution to sample and test seeds and to ismoisture-proof container from which as much sue ISTA certificates. Blank ISTA certificates for seed analysis are produced by ISTA and only air as possible has been excluded. (e) To report results of tests which are in provided to accredited laboratories for reporting the ISTA rules, the laboratory must be the results of tests. These completed certificates accredited for these tests, either directly or are the property of ISTA and may only be issued through subcontracting to another laboratory under the authority of ISTA. The list of accredited laboratories of the member countries and abstract accredited for these tests. (f) The assessment of any attribute reported on of ISTA member laboratories are provided ISTA a certificate must be calculated from tests website. carried out on one submitted sample. (g) In the case of Orange International Seed Lot 8.14 Seed Certification Against Certificates: Plant Virus Diseases – The seed lot must comply with the requirements prescribed in ISTA manual Quality control of seed for viruses is not very (ISTA 2011). – The submitted sample must be drawn and common due to the relative ‘weight’ of previous available biological assays and to the high dealt with in accordance with ISTA rules. 216 8 Methods of Combating Seed-Transmitted Virus Diseases number of seeds to be tested for assessing low virus transmission rates. The enzyme-linked immunosorbent assay (ELISA) which offers greater sensitivity of detection and enables a large number of samples to be analysed per day has become a technique of choice for large-scale testing of seed-transmitted viruses (Maury and Khetarpal 1989). There is no doubt that the recent development of the polymerase chain reaction (PCR) technique has resulted in a thousand times gain in sensitivity of detection of viruses in plant tissues. Since 1992, using reverse transcription (RT-PCR), certain RNA viruses have been detected in seeds, namely, PSbMV in pea seeds (Kohnen et al. 1992), CMV in lupin seeds (Wylie et al. 1993), BCMV in bean seeds (Saiz et al. 1994) and AMV, Bean yellow mosaic virus (BYMV), Clover yellow vein potyvirus (CYVV) and Subterranean clover mottle virus (SCMoV) in germplasm of subterranean clover and medic seeds (Bariana et al. 1994). The use of PCR for seed testing of viruses in routine would require the step of extraction of RNA from large number of seeds, which in fact is laborious, time-consuming and inconvenient. Moreover, nucleic acid extraction of a sample cannot be exploited for simultaneous detection of seed-transmitted bacterial and fungal pathogens because the progress in the latter field comes from bio-PCR, which detects the pathogen after enrichment on a culture medium. In this case, a variant of PCR, that is, immunocapture-PCR (IC-RT-PCR) which has an inherent simplicity of amplifying sequences of viral RNA without prior RNA extraction, was presumed to facilitate use of PCR in routine. However, recently, a lower level of sensitivity and lack of reliability of IC-PCR has been observed for group testing of pea seed infected by PSbMV (Phan et al. 1997). Thus, this technique needs an adaptation to seed testing before becoming popular for routine. tested in number of hosts viz., BSMV/barley (Lister et al. 1981), LMV/lettuce (Ghabrial et al. 1982), PeMoV/peanut (Bharathan et al. 1984), PSbMV/pea (Hamilton and Nichols 1978; Maury et al. 1987b), SMV/soybean (Bossennec and Maury 1978; Lister 1978; Maury et al. 1983) and SqMV/cucurbits (Nolan and Campbell 1984) have shown a large variation in the concentration of virus among infected embryos. The virus has been found in the axis and not in the cotyledons of about 20% of the infected embryos of soybean and pea (Maury et al. 1983; Masmoudi et al. 1994b) and also in a proportion of peanut (Zettler et al. 1993) and bean embryos (Klein et al. 1992). However, such low concentration is positively detected by ELISA when extracting the whole soybean embryos at the 200 w/v dilution and pea embryos at 500 w/v for detecting SMV and PSbMV, respectively (Maury et al. 1983; Masmoudi et al. 1994a). Good correlations have been found between the percentage of ELISA-positive embryos in a given seed lot and the percentage of infected seedlings raised from the same seed lot for SMV/soybean seed (Maury et al. 1984), PeMoV/peanut seed (Bharathan et al. 1984) and PSbMV/pea seed (Maury et al. 1987b). Johansen et al. (1994) considered that infectivity assays often indicate an absence of intact virions in cotyledons. If the virus is inactivated in cotyledons, ELISA-positive embryos where the virus is present in the cotyledons and not in the axis would be false-positive embryos. Such a distribution of virus in embryos has been found rarely for SMV/soybean (Maury et al. 1985) and more frequently for BICMV/cowpea seed (Gillaspie et al. 1993). It was also established that the BICMV from cowpea cotyledons is not seed transmitted. (b) Avoidance of interference of non-embryonic tissues during extraction: Since the presence of virus in the seed coat does not relate to virus transmission from seeds to seedlings, whole-seed serological assays are not suitable for estimating rates of seed transmission (Johansen et al. 1994). While preparing samples for determining the transmission rate in routine conditions on large number of seeds, it is therefore necessary that the viral antigen from non-embryonic 8.14.1 The Quality Control by ELISA Studies of correlation between detection of virus in infected embryos and seed transmission. Were 8.14 Seed Certification Against Plant Virus Diseases tissues (i.e. the seed coat in general) be not simultaneously extracted in order to prevent false-positive reactions. That introduces a potential complication as manual processing of large number of seeds is poorly compatible with the routine. Surprisingly, a lack of viral antigen in testa has been reported for mature peanut seed transmitting BCMV (PStV) (Demski and Warwick 1986; Xu et al. 1991) and PeMoV (Adams and Kuhn 1977) as well as for LMV/lettuce seed (Falk and Purcifull 1983). This could also be due to a failure in extraction of the virus from testa as demonstrated in case of SMV/soybean (Maury et al. 1985), or a failure in detection as shown for PSbMV/pea (Masmoudi et al. 1994a). In the latter case, it was indeed observed that the PSbMV capsid is partially cleaved in testa of infected pea seeds, during the maturation process. Antisera to the cleaved polypeptide detected PSbMV specifically in the embryos but not in the testa, thus enabling use of whole seed for testing without prior decortications. In other virus/seed systems, testae have to be removed in order to avoid the false-positive reactions and to correctly determine the seedtransmission rate of a seed lot. 8.14.2 Certification Schemes Against Crops Among all the methods of obtaining healthy seed material, seed certification is the most dependable and prudent measure which helps to maintain reasonable health standards without affecting seed germination. Programmes against seedtransmitted viruses must start at the basic level of germplasm collection available to the plant breeder and continue through the subsequent development of varieties and the increase of seed through breeder seed (pre-basic seed), foundation seed (basic seed), registered seed (certified seed, first generation) and certified seed (second generation). Some of the details of certification schemes against crops like lettuce, barley, beans, pea and cowpea are outlined below. 217 8.14.2.1 Lettuce A field applicable seed certification programme of lettuce seed production has been developed against Lettuce mosaic virus was worked out by number of workers. Grogan et al. (1952) developed a system in which lettuce seed was produced from virus-free plants grown under glasshouse conditions. In isolated areas, only the healthy plants were grown to maturity after roguing out the infected plants. The tolerance levels of seed transmission varied from place to place depending on the ecology and epidemiology of the virus disease. Researchers in California (Zink et al. 1956; Wisler and Duffus 2000) and Florida (Purcifull and Zitter 1971) have established that if seed transmission exceeded 0.1%, control was not satisfactory because of large winter and spring aphid population. In England, when lettuce seed with 0.1% infection was sown, only 0.5% of the plants were infected (Tomlinson 1962). It was observed that the initial level of 1.6% Lettuce mosaic virus infection in lettuce seed reached to 29%, whereas zero starting infection reached only 3% (Zink et al. 1956). The number of infected lettuce seedlings emerging per acre at the 0.1% rate would be 200 per acre or 20,000 infected seedlings per 100 acres (Greathead 1966). This is based upon an estimated 400,000 seeds/lb, planting 1 lb/acre and with 50% emergence. Programmes in both the Salinas Valley and Imperial Valley of California have, however, stressed the use of seed which is double tested – both the seed companies and by a committee of lettuce growers – to show zero mosaic/30,000 seeds tested (Kimble et al. 1975; Grogan 1980, 1983). This has reduced losses by an estimated 95–100%, resulting in considerable increase in yield and quality. At Florida since 1974, the use of double-indexed seed has eliminated Lettuce mosaic virus, a major limiting factor in lettuce production (Zitter 1977). However, during 1995, in Florida, due to noncompliance with the lettuce mosaic certification rule 5B-38, an outbreak of LMV in commercial lettuce production was noticed (Raid and Nagata 1996). 218 8 Methods of Combating Seed-Transmitted Virus Diseases 8.14.2.2 Barley In barley, Barley stripe mosaic virus (BSMV) which has no biological vector, seed transmission is the major factor facilitating disease spread. The simplest way of ameliorating the losses caused by this virus is by planting uninfected seed. A seed certification programme to obtain barley seed free from BSMV was attempted at Kansas (Hampton et al. 1957), Montana and North Dakota in the USA (Carroll 1983). It began with the growing of samples of seed in glasshouses during late autumn and winter to select those which are virusfree, for further propagation. The control programme involved assaying seedlings for presence of BSMV by serology and roguing infected plants from certified seed plots which significantly reduced the incidence of the virus. In 1972, a zero tolerance for seed-transmitted BSMV was placed on all certified seeds sold in Montana, and as a result, losses due to BSMV declined dramatically. The certification scheme against BSMV in Montana involved inspection of each barley field being considered for certification according to the field standards published by the Montana Seed Growers Association and Montana State University. When a field intended for the production of registered or certified barley seed was inspected for quality and purity, it was also carefully checked for plants exhibiting symptoms of BSMV. One or two inspections of each field were made by the secretary-manager of the Montana Seed Growers Association or the Agronomist with the Montana Cooperative Extension Service, Bozeman-Montana or a member of his staff. If infected plants were found in such a field, that field was no longer eligible for certification. The barley harvested from an uncertifiable field was usually sold for feed purposes. The second procedure of the certification programme is the inspection of seed from each potentially certifiable barley field by employing serological tests. At Montana, because of the implementation of certification schemes, the percentage of infected seed lots denied appreciably from 1967 onwards. At North Dakota, the certification scheme against BSMV resembled the Montana scheme, which involved both a procedure for inspection of seed fields and seed lot examination. Only foundation seed determined by test to be BSMV-free was used to produce certified seed which was then made available to growers. It was reported that by 1971, for all practical purposes, BSMV was eliminated in North Dakota (Timian 1971). Similarly at Montana, the two major barley cultivars, namely, Compana and Uniton, suffered losses of approximately 4 bushels per acre from 1953 to 1967, but by 1967–1970, these declined to 2 bushels per acre. Since 1970, losses due to BSMV were reported to be about half a bushel per acre after using the certified seed (Carroll 1980; Jackson and Lane 1981). 8.14.2.3 Pea Among the viruses infecting peas, PSbMV has seed transmission up to 100%, and even 23% of pea accessions are infected in USDA collection (Hampton and Braverman 1979). Because of high seed transmission and potent aphid vector, schemes to produce virus-free seed are developed. In Canada, the mother plants in dry (field) pea breeding programmes are propagated under aphid-free conditions and screened by visual inspection and a serological assay based on SDS-gel immunodiffusion (Hamilton and Nichols 1978). Subsequent seed increase plots are grown in isolation in areas of Manitoba and Saskatchewan where populations of aphid vectors are minimal. Before release to the commercial seed trade, the standing crop of a new variety is inspected visually, and samples of harvested seed (200 seed/50 lb) are subjected to a growing-on test. Seedlings are examined, and five randomly selected seedlings are assayed for PSbMV by SDS-gel immunodiffusion. Virus-free lots are then released to the Canadian Seed Growers Association or to SeCan, a federal government agency for propagation in a seed certification programme. However, no further testing for PSbMV is done. In the USA, in the primary seedproducing areas (Washington and Idaho), there is a zero tolerance for PSbMV-induced symptoms observed during field inspections (10 days postemergence and 10 days prior to harvest). Visual inspection is being supplemented by direct assay of seed (200 seeds/seed lot irrespective of size of seed lot), and there is increasing use of ELISA for 8.14 Seed Certification Against Plant Virus Diseases 219 the testing of breeder seed, seed from breeding lines and seed for commercial growers. Official certification (phytosanitary certificate) of testing for PSbMV by ELISA is required for shipment of pea or lentil seed from Washington and Idaho to Australia, New Zealand or South Africa. monitoring remaining plants by ELISA. Seed from such lines has been exported to breeding programmes in other international institutes, but the continued maintenance of virus-free nursery becomes the responsibility of the recipient breeding programmes (Hamilton 1983). 8.14.2.4 Beans BCMV in French beans is seed transmitted to the tune of 83%, and economic yield losses are encountered, whenever virus-infected seed is used. Hence, in the USA and Canada, the certification programme of field and garden bean against BCMV is based only on field inspection of the crop. These crops are not allowed to BCMV diseased plants when grown for foundation seed, 0.5% mosaic diseased plants for registered seed and 1% for certified seed (Anonymous 1971). In Brazil, bean lines are screened visually for major virus diseases, primarily BCMV, and apparently virus-free lines are increased under insect-free conditions in greenhouses. That seed is multiplied under field conditions in arid regions of Brazil for two generations, with periodic visual inspection for virus symptoms before being sold to certified seed growers. Yield increases of about 30–100% have been reported following use of such certified seed (Hamilton 1983). 8.14.2.6 Cowpea In cowpea, out of the 12 seed-transmitted viruses, CpAMV, CMV and Cowpea mottle viruses are highly seed transmitted and are worldwide in distribution. In Nigeria, the International Institute of Tropical Agriculture (IITA) and the Institute of Agricultural Research and Training are the main centres for cowpea improvement, and it is their policy to test cowpea lines before being sent to other institutions for breeding purposes or for increase in other parts of Nigeria. Samples of 1,000 seeds are planted, and the resulting seedlings are examined visually for the presence of seed-transmitted viruses; differential hosts and serological tests are used to facilitate further identification. Seed lots showing 2% or more seed transmission are not distributed; those with less than 2% seed transmission are so indicated and the recipient is advised. Preliminary results indicate that the ELISA may be applicable in detecting Cowpea mottle virus in seedlings (Hamilton 1983). 8.14.2.5 Soybean In soybean, SMV, CMV, TRSV, Soybean stunt, and Tobacco streak viruses are seed transmitted at higher percentage (up to 100%) and are widespread in occurrence. Despite the fact that SMV is present and can cause serious damage wherever soybean is cultivated, there appears to be no bona fide seed certification programmes against this or other seed-transmitted viruses in this crop. Two major soybean improvement programmes (INTSOY at the University of Illinois, Urbana, and the Asian Vegetable Research and Development Center, Shanhua, Taiwan) are screening soybean lines for resistance to some strains, but neither of these agencies employs formal certification schemes (Hashimoto 1986). International Soybean Program (INTSOY) has eliminated SMV from a number of tropical soybean lines by roguing standing crops and 8.14.2.7 Peanut In peanut crop, PStV and PMV are very important, and seed transmission is recorded to the extent of 8.5–30% both for PMV and PStV. At Georgia (USA), a seed certification programme against PMV was successfully implemented (Kuhn and Demski 1975). Even against PStV, a well-worked out certification programme was framed in the USA. Inspection of the standing crop is the method used for detecting PStV-infected plants in peanut seed fields in Virginia, a major producer of peanut seed for commercial peanut growers in Georgia, which demands zero tolerance for PStV. Infected plants are rouged from seed fields. The effectiveness of the detection system has been monitored by serological assays of suspicious plants; correlation between diagnosis 220 8 Methods of Combating Seed-Transmitted Virus Diseases and confirmation has been essentially 100% (Demski and Lovell 1985). Even at Florida, Zettler et al. (1993) developed a scheme for production of peanut seed free of Peanut stripe and Peanut mottle viruses by using virus-indexed greenhouse-grown seed. interference of non-embryonic tissues (which do not play a role in transmission of virus through seeds) in routine assessment of seed transmission rate and its role in seed certification programmes. The germplasm samples are usually received as a few seeds/sample, and thus, it is often not possible to do sampling because of the few seeds and also because of the fact that a part of the seed is also to be kept as voucher sample in the gene bank of any country apart from the diseasefree part that has to be released. Hence, extreme precaution is needed to ensure that whatever is the result obtained in the tested part, it should as far as possible not to denote a false-positive or a false-negative sample. The importers need to be encouraged to get as much sample as possible to allow effective processing. More attention needs to be given to non-destructive techniques wherever possible, and as in case of groundnut, the whole seed is not used for ELISA testing for viruses, and only cotyledonary part of the seed is analysed. Manual processing of large number of seeds is not possible in a routine test (Maury and Khetarpal 1997). Therefore, efforts should be made to develop techniques which could detect virus (e.g. PSbMV) only in embryo, although the whole seed is used for extraction (Masmoudi, et al. 1994a, b). Moreover, if the number of imports is more, it would be difficult to subject all the samples for growing in post-entry quarantine (PEQ) greenhouses. The PEQ of bulk imports in India is being carried out under the supervision of the inspection authorities who are the senior level plant protection scientists of various universities and institutes. However, these inspection authorities, in general, lack sufficient funds to carry out their work effectively. This can be a major bottleneck in the functioning of this aspect of the quarantine operations (Khetarpal 2004) (Fig. 8.3). 8.15 Conclusion In seed certification schemes, precautions should be taken by restricting the regions for raising the seed lots along with the protective measures during the cropping period. If these precautions are not taken according to seed health certificate requirements, then (1) proper crop stand as expected by seed analysis will not be achieved, and (2) the disease will develop and lead to total devastation of the crop, resulting in deterioration in the quality of seeds and, consequently, their market value. If the diseased seed is distributed within the country or sent to other countries, these seed-transmitted virus diseases are further disseminated. The seed after harvest should be carefully analysed by some of the advanced virus diagnostic techniques described in Chap. 6 to decide whether the seed samples in question should be released for use or rejected. Review chapter of Maury et al. (1998) and Albrechtsen’s textbook (2006) are additional sources for seed certification. 8.16 Quality Control of Bulk Seed Lots The size of consignment received is very critical in quarantine from processing point of view. Bulk seed samples of seed lots need to be tested by drawing workable samples as per norms. The prescribed sampling procedures need to be followed strictly, and there is a need to develop and adapt protocols for batch testing, instead of individual seed analysis (Maury et al. 1985). Maury and Khetarpal (1989) discussed in depth the use of ELISA for detecting viruses in single embryo, determination of seed transmission by coupling it with group analysis, mode of eliminating the 8.16.1 Determination of Seed Transmission Rate If the per cent seed transmission of a seed lot is high, individually excised embryos can be tested 8.16 Quality Control of Bulk Seed Lots 221 Fig. 8.3 Growing-on test of legume germplasm in post-entry quarantine greenhouse at NBPGR, New Delhi (Source: NBPGR, R.K. Khetarpal; V.C. Chalam) by ELISA. However, if the per cent transmission is assumed to be low, a large number of embryos need to be tested that calls for use of a grouptesting method. Due to the variation in virus titre in different embryos, it is not possible to correlate, as previously suggested for BSMV (Lister et al. 1981), an ELISA absorbance value for a sample of embryos with the number of infected embryos in this sample. Therefore, the group-testing method adopted for determining seed transmission rates consists of dividing a representative sample of the seed lot to be analysed into a number of groups of n seeds, each made at random. As compared with single embryo tests, the number of tests is divided by n. The percentage of transmission can be estimated as a function of the number of ELISA-negative groups, with a confidence interval which determines the precision of the determination (Maury et al. 1985). For example, while testing 60 groups of 500 seeds of lettuce, if all the 60 groups are negative, the confidence interval is 0–0.012% at 95% level of probability. The number n of seeds per group has a limited value depending on the dilution limit of the em- bryos having the lowest titre of virus. Therefore, with a given antiserum, a workable group size limit is determined, coherent with a probability Psl D l of detecting one infected embryo in such a group of seeds (Maury et al. 1985; Maury et al. 1987b; Maury and Khetarpal 1989). The systematic use of such quality control has been efficiently protecting the lettuce crops in California and Florida from LMV outbreaks. The determination by this method of seed transmission rates is also used by the seed industry for classifying seed lots in classes of quality, but the following approach mentioned in (b) would make it simpler. According to the General Agreement on Tariffs and Trade (GATT), now known as World Trade Organization (WTO), any phytosanitary measure should be based on a pest risk analysis. The pest risk analysis enables each country to define its appropriate level of protection. The classification of seed lots according to seed transmission rates would much help in a pragmatic determination of tolerance thresholds if quality controls are now extended to other crops. 222 8 8.16.2 Infection Status of a Bulk Seed Lot with Respect to a Tolerance Limit Is the infection of a given seed lot under or above a non-tolerable level of infection (Int)? Answering this question does not require determining the seed transmission rate. After testing k groups of N seeds, a statistical approach which includes two probabilities of errors leads to the following decision rule: ‘the seed lot is above the threshold if at least one group of N seeds is found infected’ (Geng et al. 1983; Masmoudi et al. 1994b). Increasing each group size N reduces accordingly the number k of groups to be tested if it is also taken into account a probability PsI of detecting one infected embryo in a group of N seeds. This probability depends on the avidity of the antiserum used. As an example, to determine with a good accuracy the status of a pea seed lot with respect to a threshold of 0.1%, it was sufficient to analyse 31 groups of 200 seeds each, that is, one ELISA microplate (Masmoudi et al. 1994b). It is important to underline the significant simplification in the number of ELISA samples to be examined and the ease with which a seed lot can thus be classified by this approach in view of local or international trade. 8.16.3 Infection Status of a Bulk Seed Lot with Respect to a Virus Not Known in the Importing Country A zero tolerance should theoretically be applied to the international exchange of commercial seed lots when a destructive virus exists in the exporting country, that is, the country where the seed lot is produced and/or when the same is not known to occur in the importing country. Since it is not feasible to achieve zero tolerance while certifying bulk seed lots, it is in the larger interest to assess the risk associated with the possible introduction of a viral disease not known to occur in the importing country (Kahn 1979). (a) The Actual Difficulties Preventing a Generalised Use of Quality Control Methods of Combating Seed-Transmitted Virus Diseases The first concern is a complete lack of standardised protocols. To solve this gap, it is necessary to control all the causes of variability which would induce result discrepancy when several laboratories perform the same test on the same seed lot. An important cause is the variability of the antiserum, due to the specificity, the range of strains detected, the avidity and the epitopes detected on the virus particle. Another important difficulty is, as many laboratories from developing countries will be diffcult to perform these tests if they they do not have the technology and advanced facilities. Finally, the lack of accurate working sheet leaves each laboratory functioning in isolation with its rudimentary methodology. 8.17 World Trade Organization (WTO) Regime and Its Implications The World Trade Organization (WTO), established on 1 January 1995, is the legal and institutional foundation of the multilateral trading system. The main purpose of the WTO is to promote free trade, serves as a forum for trade negotiations and settles disputes based upon the principles of non-discrimination, equivalence and predictability. The WTO agreement contains more than 60 agreements covered under some 29 individual legal texts encompassing everything from services to government procurement, rules of origin and intellectual property (http://www.wto.org) of these, and the following four agreements have a direct bearing on agriculture and related activities: • Agreement on Agriculture is designed to ensure increased fairness in farm trade. • Agreement on Trade-Related Intellectual property rights is aimed at improving conditions of competition where ideas and inventions are involved. • Agreement on Technical Barriers to Trade is to ensure that technical regulations and certification procedures do not create obstacles to trade. 8.18 The Role of Plant Biosecurity in Preventing and Controlling. . . • Agreement on the Application of Sanitary and Phytosanitary (SPS) Measures is in fact the one which is going to have major implications on seed/plant health in trade (Khetarpal and Gupta 2002; Khetarpal et al. 2003, 2005a, b; Khetarpal 2004). 8.17.1 Examples of Introduced Plant Viruses Through Seed Exchange Among the various disease-inciting agents of plants, viruses assume special importance by virtue of being sub-light microscopic, causing systemic infection in the host and not being generally controlled by physical and chemical methods. Plant viral diseases are known to cause serious yield losses. Probably the greatest challenge is due to globalisation and the international trade in agricultural and horticultural produce, which is breaking down the traditional geographical barriers to the movement of viruses. There are several instances of inadvertent introduction of pests along with introduced seed or planting material into a country or region within a country. Trade and exchange of germplasm at international level play a key role in the long-distance dissemination of a destructive virus or its virulent strain. The worldwide distribution of many economically important viruses especially those attacking legumes, such as Bean common mosaic virus, Soybean mosaic virus (Goodman and Oard 1980), Pea seed-borne mosaic virus (Hampton and Braverman 1979) and Peanut mottle virus, is attributed to the unrestricted exchange of seed lots. Peanut stripe virus (later identified as a strain of BCMV) was introduced into the USA in the 1980s through groundnut seed germplasm imported from China (Demski et al. 1984b). The same pathogen was intercepted in Australia (Persley et al. 2001) from imported groundnut seed held in post-entry quarantine. Like in other countries, a number of exotic plant viruses have been introduced into India along with imported planting material causing serious crop losses. These included Banana bunchy 223 top virus (BBTV), Banana streak mosaic virus, Peanut stripe virus, etc., and BBTV was probably introduced into India from Sri Lanka in 1940 (Magee 1953) and has since spread widely in the country. The international spread of BBTV is primarily through infected planting material (Wardlaw 1961). In India, an annual loss of Rs.40 crores due to BBTV has been estimated in Kerala alone. These introductions highlighted the fact that increased pace of international travel and trade had exposed countries to the danger of infiltration of exotic viruses harmful to the agriculture. Since both seeds and vegetative propagules are efficient carriers of viruses, infection is progressively transmitted through generations. Further, if the in vitro cultures are raised from a virusinfected mother plant, the plantlets are bound to carry the virus infection. The successful detection and control of viruses in seed and other planting material depend upon the availability of rapid, reliable, robust, specific and sensitive methods for detection and identification of viruses. Over the years, a great variety of methods have been developed that permit the detection and identification of plant viruses. Virus indexing is done by deploying a combination of some of the wellknown virus detection techniques mentioned in Chap. 6 keeping in view the intended purpose. 8.18 The Role of Plant Biosecurity in Preventing and Controlling Emerging Plant Virus Disease Epidemics The emergence of a global community and an increase in plant virus discovery in the last 15 years have increased the requirement for countries and regions to protect their farming systems from these exotic pests. In a recent report, plant viruses were identified as the cause of 47% of the emerging infectious diseases of plants that were recorded on the PubMed database during the period of 1996–2002, and a further 4% were attributed to phytoplasmas, and it is likely that this trend will continue. 224 8 Methods of Combating Seed-Transmitted Virus Diseases Plant biosecurity can be defined as a set of measures designed to protect crops from emergency plant pests (EPPs) at national, regional and individual farm levels (Anonymous 2005). Countries are required to comply with international obligations as defined by the World Trade Organisation (WTO) Agreement on the Application of Sanitary and Phytosanitary Measures (WTO 1995). More specifically, international guidelines and standards are outlined under the International Plant Protection Convention to demonstrate pest-free areas (FAO 1995) that stipulate that a pest/pathogen is ‘known not to occur’ in a given geographical region (van Halteren 2000). This edict is significantly different from the previous wording of ‘not known to occur’ and has resulted in additional requirements for surveillance of EPPs in countries to demonstrate area freedom. Countries that import plant products can demand from exporting countries or regions evidence for the absence of pests which do not occur in that importing country. Importing countries also have an obligation to provide more justification for their quarantine measures and not conceal or fail to look for the presence of pests in their territory (van Halteren 2000). In this context, a number of research strategies have been initiated over the last decade to enhance our biosecurity capacity at the pre-border, border and post-border frontiers. In preparation for emerging plant virus disease epidemics, diagnostic manuals for economically important plant viruses that threaten local industries (e.g. Plum pox virus) have been developed and validated under local conditions. Contingency plans have also been documented that provide guidelines to stakeholders on diagnostics, surveillance, survey strategies, disease epidemiology and pest risk analysis. Reference collections containing validated positive controls have been expanded to support a wide range of biosecurity sciences. Research has been conducted to introduce highthroughput diagnostic capabilities and the design and development of advanced molecular techniques to detect virus families. These diagnostic tools can be used by post-entry quarantine agencies to detect known and unknown plant viral agents. Pre-emptive breeding strategies have also been initiated to protect plant industries if and when key exotic viruses become established in local communities. With the emergence of free trade agreements between trading partners, there is a requirement for quality assurance measures of pathogens, including viruses, which are present in both the exporting and importing countries. These measures are required to ensure market access for the exporting country and also minimise the risk of the establishment of a disease epidemic in the importing country (Khetarpal and Gupta 2007; Rodoni 2009). A number of research strategies have been initiated over the last decade to enhance plant biosecurity capacity at the pre-border, border and post-border frontiers. In preparation for emerging plant virus epidemics, diagnostic manuals for economically important plant viruses that threaten local industries have been developed and validated under local conditions. Contingency plans have also been prepared that provide guidelines to stakeholders on diagnostics, surveillance, survey strategies, epidemiology and pest risk analysis. Reference collections containing validated positive virus controls have been expanded to support a wide range of biosecurity sciences. Research has been conducted to introduce high-throughput diagnostic capabilities and the design and development of advanced molecular techniques to detect virus genera. These diagnostic tools can be used by post-entry quarantine agencies to detect known and unknown plant viral agents. Pre-emptive breeding strategies have also been initiated to protect plant industries if and when key exotic viruses become established in localised areas. With the emergence of free trade agreements between trading partners there is a requirement for quality assurance measures for pathogens, including viruses, which may occur in both the exporting and importing countries. These measures are required to ensure market access for the exporting country and also to minimise the risk of the establishment of a damaging virus epidemic in the importing country. 8.19 8.19 Pest Risk Analysis (PRA) Pest Risk Analysis (PRA) Pest categorisation is the key component of pest risk assessment. This is not just an identification of the pest species but an analysis of its potential danger as a pest. A pest of quarantine significance refers to a pest of potential economical importance to the area endangered, but not yet present there, or present but not yet widely distributed and being officially controlled. Pest categorisation includes the following major elements: • Identification of the pest • Definition of the PRA area • Distribution and official control programmes within the PRA area • Potential of the pest for establishment and spread in the PRA area • Potential economic impact potential in the PRA area • Endangered areas The other major components of PRA include Economical Impact Assessment (basically a thorough examination of the economic risk associated with the process) and the Probability of Introduction, which looks at the prospects of both entry and establishment of the pest. Essentially, pest risk management is the process of deciding how to react to a perceived risk, deciding whether action should be taken to minimise the risk, and, if so, what action should be chosen. These steps should provide a clear scientific basis for pest risk assessment, risk management and quarantine decisions, and prevent these from being used in an inconsistent manner and as barriers to international trade. 8.19.1 Pest and Pathogen Risk Analysis Availability of accurate and reliable information on the occurrence of the pests and diseases and the damage they cause is one of the essential requirements for the effective operation of the quarantine services. Unless such information is available, the plant quarantine or plant protection officer has no basis on which to act in regulating the importation of plants. The entry status for the 225 major pests and diseases is to be studied. The entry status refers to the entire range of decisions or policies or regulations that serve as guidelines for rules and regulations of that government whether or not a potential carriers like plants, plant products, cargo, baggage, mail, common carriers, etc., are enterable and if enterable, under what safeguards, into one geographical region to another. Pest and pathogen risk is based on an evaluation of biological variables. Examples of such variables are the (1) ecological range of a hazardous organism compared to the ecological range of its hosts in the importing country, (2) hitchhiking activity of the pest and (3) ease of colourisation of the pest. Attitudes towards entry status may range from ‘conservative’ to ‘liberal’, but ‘liberal’ in this context does not imply ‘lax’. The most conservative attitude is that the plant material is excluded without any exceptions, whereas the most liberal attitude is that the plant material may enter freely without agricultural regulatory restrictions. When the points of valid matchings are plotted and connected, a biological curve may be drawn. By illustrating the pathogen and pest risk analysis diagrammatically as a curve on a graph, one can effectively communicate generation philosophy, principles, policies and decisions not only to quarantine officers but to the scientific, commercial or growers. It is also required to explain a quarantine decision or activity that is against the interest of the importer. This curve is also useful in domestic quarantine matter in making understand the interaction of biological, economic and political factors to the trainees. It also helps the quarantine officers in diagnosing priorities or discussion budgetary matters. 8.19.2 Pest Risk Analysis for Viral Diseases of Tropical Fruits Virus and virus-like diseases have been causing serious damage to fruit production in tropical areas. They are not usually transmitted through seeds, but are often carried across national boundaries by infected bud wood, vegetatively propagated seedlings and transmission by insect vectors. 226 8 Methods of Combating Seed-Transmitted Virus Diseases With regard to disease risk analysis, one has to have sound knowledge about the major virus diseases of economically important cereals, vegetables, and fruit crops, in terms of the aetiology and epidemiology of the diseases, current diagnosis methods, their geographical distribution and their economic impact. The various strains of important viruses infecting fruit crops are to be critically identified. For framing suitable management measures in recent years tissue culture techniques have gained importance in producing virus-free planting material and advantages and disadvantages of this technique are to be studied depending on the virus and the crop. Plantlets propagated by tissue culture have been found to be more susceptible to Banana mosaic virus than seedlings of sucker origin. Since tissue culture is widely used to produce disease-free banana seedlings, this suggests that intensive vector control is essential in banana plantations where plantlets are raised by tissue culture are being grown. Greening is one of the most serious citrus diseases which is now identified to be Liberibacter asiaticus C gram-negative bacterium. It affects most citrus species and is vectored by psyllids in a persistent matter. Most natural spread probably occurs when young shoots are sprouting, at a time when succulent growth is present on donor and receptor plants, and psyllid populations are high. Recent work by an FFTC survey team, using new methods of diagnosis and indexing, had found greening to have a much wider distribution in the Pacific than previously recognised. Some strains are virulent to pomelo, which had been thought resistant to the disease. This illustrates the importance of using advanced scientific procedures when certifying planting materials, particularly imported ones, as virus-free. The pest and pathogen risk is based on two general precepts – (1) the benefits must exceed the risk, and (2) the benefits must exceed the costs. In international transfer of genetic stocks, the benefits usually consist of the opportunity to introduce new crops or new varieties of old crops or to introduce new genes to improve the old varieties. Such improvement may consist of increased yields through increased pest or pathogen resistance coupled with increased nutritive values also. When costs are entered into the pest risk analysis, plant quarantine officers consider the costs of adequate safeguards like virus indexing, heat therapy, meristem tissue culture, etc. The pest and the pathogen risk analysis curve can be exemplified with the little cherry disease, present in Washington. This little cherry disease is a phytoplasma disease spreads due to the supply of infected plant material by nursery men and secondary spread in the field by the vectors and is found in the dooryard cherries in a 40square-mile area in the Washington state. Studies on the risk analysis curve to illustrate specific problems in plant quarantine due to little cherry to US agriculture and various quarantine actions taken are (1) destruction of all species of Prunus in a given geographical area; (2) destruction of P. avium, P. cerasus and all following cherry species like P. serrulata, P. subhirtella and C. yedoensis in a given geographical area; (3) destruction of all sweet (P. avium) and sour cherries (C. cerasus) in the area; (4) destruction of only sweet and sour cherries showing diagnostic symptoms (either with or without confirmatory laboratory staining results); (5) survey for symptoms to determine the distribution of the agent in P. avium and P. cerasus in the Western States or the entire United States and (6) no regulatory action (Kahn 1989; Kahn and Mathur 1999). The problem presented by the little cherry agent in the dooryard cherry in Washington is to determine which one or more of the quarantine actions shown on the ‘Y’ axis should be implemented so that the quarantine action and pest risk interact somewhere on the pest risk analysis curve. Another good example of pathogen risk analysis is the Cadang-cadang disease, of the coconut palm, whose aetiology is proved to be of viroid in nature and the incubation period is long and uncertain. Similarly, there are risks involved in sending carnation and chrysanthemum cuttings to warmer climates to avoid the growing conditions of the northern European winter. At present, cuttings are sent from the United Kingdom to overwinter in places such as Malta, Sardinia, the Canary isles, Kenya and South Africa. The cuttings are grown as mother plants in the warmer climate until large enough to provide cuttings, which are then returned to Britain or 8.20 Biosafety other European countries. In these warmer countries, viroid diseases like chrysanthemum stunt, chrysanthemum chlorotic mottle, cucumber pale fruit, citrus exocortis and potato spindle tuber are highly hazardous and multiply much more rapidly and reach greater concentrations in plants at temperatures in the range of 30–35ıC. Under these conditions, each of them is able to infect the principal host plants of the other viroids. This factor should not be overlooked, for example, where chrysanthemums send to the Canary isles may be growing near to potato or cucurbit crops, so that infections might be transferred in either direction (Hollings and Stone 1973). Pest risk analysis is generally accepted as the principal strategy to make sure that plant quarantine standards are transparent and justified on a sound scientific basis. However, its application to individual pests will require great deal of further discussion and further detailed work. Once this is done, PRA can be expected to remove unnecessary barriers to trade. There is an urgent need for all countries to reach an equal level in plant quarantine, in terms of technology and equipment. An international network on quarantine pest monitoring is also needed, to meet the growing danger of exotic pest invasion as a result of growing international tourism and trade, and the long-distance migration of insect pests. Disease risk analysis has to be followed by control of the diseases. In general, these vectorborne systemic plant virus diseases can be effectively controlled by integrated control measures, including production and cultivation of virus-free seedlings, elimination of inoculum sources and prevention of reinfection through IPM of vector insects and cross protection with mild strains. The establishment of pathogen-free nursery systems is the most important way of preventing these diseases from spreading. 8.20 Biosafety Genetically modified plants can spread the transgene to other plants or – theoretically – even to bacteria. Depending on the transgene, this may pose a threat to the environment by changing 227 the composition of the local ecosystem. Therefore, in most countries, environmental studies are required prior to the approval of a GM plant for commercial purposes, and a monitoring plan must be presented to identify potential effects which have not been anticipated prior to the approval. 8.20.1 Biosafety Regulations Application of GM technology for commercial crop production is faced with a number of issues and challenges the most important being safety of environment, and human and animal health (Grumet and Gifford 1998; Khetarpal 2002; Philippe 2007). These concerns are based on the argument that recombinant DNA-based GM technology differs from traditional breeding in that totally new genes using potentially risky technology are transferred between widely unrelated organisms, and the location of these genes on the recipient genome is random, unlike when gene transfer takes place through conventional breeding. These differences demand that adequate laboratory safeguards are used and plants developed by GM technology are rigorously assessed for their performance as also for the likely risks they pose to the environment and human health and a few other concerns related to the application of biotechnology in agriculture. 8.20.2 History of Biosafety Protocol and Regulations Because of the risk associated with genetically modified organisms, there were concerns worldwide to control it. It is also important to protect the biological diversity of the nature while releasing or accidental escape of GMO to the environment. This leads to the formation of biosafety protocols dealing with genetically modified organisms. The origins of the biosafety protocol were found in the UN Convention on Biological Diversity, which was signed by over 150 governments at the Rio ‘Earth Summit’ in 1992, and which came into force in December 1993. In 228 8 Methods of Combating Seed-Transmitted Virus Diseases the Convention on Biological Diversity (CBD), it was acknowledged that release of GMOs (referred to in the CBD as ‘living modified organisms’ or LMOs) may have adverse effects on the conservation and sustainable use of biological diversity. All countries that signed up to the CBD were expected to: (a) ‘Establish or maintain means to regulate, manage or control the risks associated with the use and release of living modified organisms resulting from biotechnology which are likely to have adverse environmental impacts, taking also into account the risks to human health’. (b) ‘Consider the need for and modalities of a protocol setting out appropriate procedures in the field of the safe transfer, handling and use of any living modified organism resulting from biotechnology that may have adverse effect on the conservation and sustainable use of biological diversity’. In accordance with the precautionary approach contained in Principle 15 of the Rio Declaration on Environment and Development, the objective of the protocol is to contribute to ensuring an adequate level of protection in the field of the safe transfer, handling and use of living modified organisms resulting from modern biotechnology that may have adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health, and specifically focusing on trans boundary movements. The Cartagena Protocol on biosafety, the first international regulatory framework for safe transfer, handling and use of living modified organisms (LMOs), was negotiated under the aegis of the Convention on Biological Diversity. The protocol contains reference to a precautionary approach and reaffirms the precaution language in Principle 15 of the Rio Declaration on Environment and Development. The protocol also establishes a Biosafety Clearing-House to facilitate the exchange of information on LMOs and to assist countries in the implementation of the protocol. The protocol was adopted on 29 January 2000. The protocol has been signed by 103 countries (except the USA). The Cabinet (GOI) approved the proposal, and India signed the biosafety protocol on 23 January 2001. Subsequent to the Cabinet approval on 5 September 2002, India has acceded to the biosafety protocol on 17 January 2003. So far, 43 countries have ratified the protocol. The protocol will come into force on the 90th day after the date of deposit of the fiftieth instrument for ratification by countries that are parties to the convention. 8.20.3 Biosafety Regulations of Asia-Pacific Countries Beginning the late 1980s, Asia-Pacific countries initiated legislative measures to manage the potential risks associated with GM technology. In 1986, India enacted ‘Environment Protection Act’ and published ‘The Environment (Protection) Rules’ to regulate environmental pollution by managing hazardous substances, including hazardous microorganisms and GMOs. In 1990, ‘Philippines Presidential Order’ established a national biosafety committee, and during the early 1990s, India and Thailand published the first guidelines on research and environmental aspects of GMOs. The biosafety regulatory systems vary across countries; some developing an entirely new biosafety specific system while others making modifications in the existing regulatory systems to address biosafety issues. The legal instruments used for the purpose have been new or modified laws, acts, decrees, guidelines, rules, etc. Alongside, administrative systems have been developed to operationalise the legal instruments. Following one or the other approach, a number of countries have currently in place regulations on development, contained use, environmental release, commercialisation and import of GM crops and products. Several other countries have their biosafety regulations either in drafting or implementation phase. 8.22 8.21 Quarantines Risks Associated with Genetically Modified Crops Genetically modified crops deal with the growth of plants. Transgenic plants exposed to the open environment, and the interaction takes place with other organisms in the field. Many of them contain toxic gene and antibiotic resistance marker with them. Therefore, genetic modification in agriculture has become a more sensitive issue than those experiences with recombinant biotherapeutics. Transgenic plants need special attention because they are exposed to environment. Until today, there is no major risk concern associated with the marketed transgenic crops such as cotton, tomato, corn and soybean. Unfortunately, the public debate over the hazards of transgenic plant or transgenic food suffers from misinformation and misunderstanding of the basis of genetic manipulation in plant system. These GM foods carry a label and have been to extensive field trials for safety and environmental impact before they are approved for commercialisation. With the continuing accumulation of evidence of safety and efficiency and no harm to public or the environment, more and more transgenic plants and food are getting appreciated and used by the people. Nevertheless, thorough assessment of the risk and safety associated with GM crops and food needs complete evaluation before they are released to the environment. The major safety concerns associated with GM crops and GM food are as follows: 1. The effect of GM crops on environment and biodiversity 2. Gene pollution (escape of gene through pollen) 3. Toxicity of the GM plant due to altered metabolism 4. Safety, toxicity and allergenicity of the GM food 5. Insect- and herbicide-resistant varieties of plant 6. Undesirable effect of transgenic plant on nontargeted or beneficial insect or plant in the environment 229 8.22 Quarantines The term quarantine is derived from Latin word, quarantum, meaning forty. The objective of plant quarantines is to protect the agriculture, by preventing the introduction and spread of important pests and diseases by legislative restrictions on the movement of plants and plant products. In other words, quarantines are important disease control measures based on avoidance and exclusion technique. Migrations, military conquests and occupations, voyages of discovery, religious expeditions and germplasm exchange have in many ways contributed to the spread of plant material. A number of diseases that were not present earlier in certain countries have been introduced into new areas either on or in the plant materials which in turn had spread and caused disastrous crop losses. For increase of the food production by breeding techniques, there is a constant exchange of germplasm throughout the globe, and hence, a great impact is noticed in agricultural outputs. Most of our major crop plants now grown in areas where they did not evolve, but have been disseminated by man, are introductions. The phenomenal increase in advanced transport techniques during the recent years has increased the movement of the plant material by man from place to place irrespective of geographical barriers. While air travelling, a small bud wood stick or cutting can be carried in a viable condition without any protective measures. In the case of ship travel involving prolonged duration of time, much propagative material will be lost during the passage, and the inspection can be done leisurely at the port of entry. It is also established from Table 1.1 that nearly 231 viruses are seed transmitted, while on the contrary, the propagules derived from the virusinfected vegetative material are mostly infected. The dissemination of seed-transmitted viruses is also occurring due to the massive movement of seed, particularly under ‘green revolution’. In the developing countries, import of large quantities of food grains may result in certain inadvertent disease introduction, and in certain times, farmers use the same in smaller amounts as planting material. 230 8 All countries are involved in the export and import of agricultural products and are depending heavily on agriculture for their export earnings. The key concept in the new free trade situation is pest risk analysis, an objective assessment of the dangers of invasion by a particular known and unknown pests and diseases including plant virus and virus-like diseases which may damage agriculture production more drastically if they were accidentally introduced. Scientific community is also introducing the infected genetic stocks for research investigations unaware of national regulations or due to sheer ignorance. On several occasions, seeds are sent across the world by post enclosing them in envelopes or are brought by tourists. For scientific purposes, there is no embargo exchange of germplasm when safeguards are adequate and quarantine regulations of every country are playing major role in this regard. Plant quarantine is a government endeavour enforced through legislative measures to regulate the introduction of planting materials, plant products, soil, living organisms, etc., in order to prevent inadvertent introduction of pests (including fungi, bacteria, viruses, nematodes, insects and weeds) harmful to the agriculture of a country/state/region and, if introduced, prevent their establishment and further spread. It is evident from the third chapter that the virus and virus-like diseases are potentially dangerous in causing heavy toll of the crops. Sometimes, the catastrophic consequences suffered by agriculture through the introduction of exotic diseases compelled every nation or a group of government to impose legal restrictions on international trade of plant materials which sometimes unnoticingly carry pests and diseases. The entry and further spread of these dangerous pests and diseases in each country are controlled by quarantines. Khetarpal et al. (2004, 2005a, 2006a) and Khetarpal and Gupta (2007) have reviewed various methods for the safe movement of plant germplasm. Methods of Combating Seed-Transmitted Virus Diseases 8.22.1 Plant Quarantine Plant quarantine promulgated by a government or group of governments to restrict the entry of plants, plant products, soil, cultures of living organisms, packing materials and commodities, as well as their containers and means of conveyance to protect agriculture and the environment from avoidable damage by hazardous organisms. They exclude dangerous organisms while permitting plants and plant products to enter (Kahn 1988). The term exclusion conveys this objective more clearly than plant quarantine. Exclusion means the practice of keeping out any materials or objects that are contaminated with pathogens or disease plants and preventing them from entering the production system for which quarantine rules and regulations are to be implemented. 8.22.2 Plant Quarantine Measures A number of quarantine measures are available, and may be used, either separately or collectively, in an attempt to prevent the establishment of a new plant pest or disease in an area previously free of it. The governments enact regulations and stipulate safeguards in accordance with the risk presented by the importation of the plant materials from areas where hazardous pests and pathogens are known to occur. The quarantines operate between different countries or between adjacent states in one country. The regulations are formulated and implemented by the federal or state government or both. Persons desiring detailed information about the quarantine regulations of foreign countries should request their own country’s plant protection and quarantine service. In each country, the government regulations issued by ministries or Departments of Agriculture have been received and abstracted. In general, these regulations are as follows: (1) Specify requirements of import permits, (2) require phytosanitary certificates, (3) require certificates of origin, (4) stipulate inspection upon 8.22 Quarantines arrival, (5) prescribe treatment upon arrival to eliminate a risk, and (6) prescribe quarantine or post-entry quarantine depending on the nature of the risk. Some of the quarantine measures comprise the following: embargoes, inspection at the port of entry, inspection at the port of dispatch, field inspection during the growing season (preclearance), controlled entry (i.e. disinfection, treatments) and post-entry quarantine stations. Some details of these quarantine measures are mentioned in the subsequent pages. 8.22.3 Functions of Plant Quarantine The prime functions of plant quarantine are as follows: (1) enforcing the measures for the prevention of foreign species of plants with pests and diseases from overseas and it is defined as international quarantines; (2) quick identification, localisation and eradication of exotic pests and diseases which are invaded within a country itself and is called as domestic quarantine and (3) providing to the country with as many new varieties as possible at the same time without allowing entry of new diseases. International Quarantine: This will prohibit or regulate the entry of imports from other counties’ necessitation international cooperation on phytopathological and entomological problems. For long periods, plants and the organisms pathogenic to them in many instances coexisted in balanced equilibrium. Interference by man invariably disturbs this balance, and the pathogens of minor significance in one situation may cause epiphytotics when transferred to new environment/places. It may be because of plants developing in the absence of the pathogen, have no opportunity to select resistant factors specific against an introduced pathogen, as a consequence of which when they are grown in a place where they have been imported become extremely vulnerable to disease attack. In certain cases, the introduced diseases may also mutate to more destructive forms and cause epidemics. There are a number of examples, where the introduced plant viruses into new areas have resulted into heavy crop losses. For example, the 231 Asian greening and its vectors, once established in an area, have the potential of totally destroying citrus as an economic crop (Fraser et al. 1966; Salibe and Cortez 1966). There are new strains of Citrus tristeza virus which can spread very rapidly (Bar-Joseph and Loebenstein 1973) and can affect many citrus root stocks and scions once considered tolerant to the virus. Even the introduction of exocortis viroid into a country such as Japan or Uruguay, where the predominant root stock is P. trifoliata, is a distinct hazard because exocortis can be spread by unindexed bud wood or very effectively by cutting tools and is very damaging to Poncirus root stocks. Some of the important virus and virus-like diseases introduced in different countries are discussed under separate heading. For the international exchange of germplasm, the involved scientists or commercial nurseries whether importers or exporters should be cognizant of the quarantine regulations of the importing country. The quarantine regulations of the importing country will determine the enterability of such germplasm. Even the quarantine service of the exporting country is involved as a source of information about the requirements of the importing country and also in the issuance of phytosanitary certificates, if required by the importing country. Domestic Quarantines: In spite of the best precautions taken and stringent measures of quarantine applied, even the most advanced countries have not been able, on several occasions, to prevent the entry of the diseases. In such cases, man has taken recourse to domestic quarantine, which will help in the confinement of the diseases and pests to particular area only, due to the restriction in the movement of the plant material between the states. They will primarily deal with the new diseases and pests which are already entered in a country and will be made to eliminate them before they become established. The most effective quarantine action is that which is applied at the source of spread. The problems of domestic quarantine are basically similar to those of international quarantine. In practice, federal and state governments jointly enforce regulations to prevent intrastate as well as interstate spread. 232 8 Methods of Combating Seed-Transmitted Virus Diseases In each country, some legislations are also passed against certain devastating virus diseases; for example, in California, an ordinance No. 1053 of the county of Monterey was implemented to prevent losses of lettuce crop due to mosaic disease and reads in part as follows: In Sudan, legislation is passed for pulling of the volunteer cotton plants and the regrowth to be prevented for the control of Cotton leaf curl virus. Growing of okra before 15th September is strictly prohibited. Any programme of regulation and free registration can succeed only if it is supported by the growers and nursery men. In California, they have provided outstanding support and cooperation of various regulatory and quarantine programmes. Similarly, in South Africa, a cooperative programme between growers and the government regulated in legislation passed in 1927 providing for the eradication of all psorosis-infected citrus trees showing bark lesions (Doidge 1939). This programme carried out between 1930 and 1950, effectively reduced psorosis in South Africa from one of the most destructive diseases to one that is now rare. In the Philippines during 1962–1974, the area of mandarin, sweet orange and pomelo cultivation was greatly reduced in certain parts up to 60% due to leaf mottling (greening). In 1969, a quarantine measure was issued through an administrative order by the Secretary of the Department of Agriculture to prohibit entry of citrus planting materials into noninfected areas. All citrus for planting will be required to certification papers from the point of origin for verification in inspection centres and quarantine stations throughout the citrusgrowing areas of the Philippines (Altamirano et al. 1976). Similarly, in Florida, legislations were passed for certifying the peach nursery stocks. Prior to June 30th, of every year, Phony peach trees should be eradicated which are found are mile within the environs, of the nursery. Ultimately, the success of quarantine is dependent on a substantial degree of public support, but if regulations can be ignored with impunity by a few, they soon lose the support of many. ‘Mosaic is a virus disease which infects lettuce crops and is a direct cause of crop failures. It is a disease which is seed transmitted and carried by aphids and, if not prevented, spreads from field to field affecting large areas of production and causing great losses to growers : : : The disease threatens to seriously affect the general economy if prompt and effective measures are not taken at once to eradicate this menace. It shall be unlawful for any firm or corporation to plant any lettuce seed in the unincorporated area of the country of Monterey which has not been mosaic –indexed. Any violation of this ordinance constitutes a misdemeanour punishable by a fine of not more than $500.00 or by punishment in the county jail for a period not exceeding six (6) months’. This ordinance mentioned above has been effective in reducing losses in lettuce production for nearly 20 years. Similarly, Western celery mosaic virus is effectively controlled by the establishment of a legally defined period in which no celery can be grown in a given district was described by Milbrath (1948). Due to the implementation of this legislation in the Venice–Sawtelle region of California, celery yields were increased by 50% in the first 2 years following passage of the law and increased by another 50% in the subsequent years. In most of the countries, the legislations were passed for the nursery men for the supply of disease-free plants. The nurseries were inspected while growing, by the quarantine staff so that only certified planting stocks were sold which meets certain prescribed standards. In most of the developed countries, seed potatoes are certified. In India, the Directorate of Plant Protection, Quarantine and Storage takes necessary steps to regulate the interstate movements of plants and plant materials to prevent the further spread of destructive pests and diseases. Some of the successful test control aspects by legislations in different countries are as follows: 8.22.4 Pathways of Spread of Pests and Pathogens Pests and pathogens may move along natural or man-made pathways. Whether a pest or pathogen 8.22 Quarantines becomes established at the end of the pathway depends on the synchronisation of three factors: sufficient viable inoculum (or population threshold), the presence of susceptible hosts and a favourable environment. At the end of the pathway, if any one of the factors is not operative, an organism, although it may have gained entry, does not become established. In theory, countries are bombarded by exotic organisms entering on natural pathways: Yet, there is usually no establishment if the three factors are not operating when the pest or pathogen arrives (Kahn 1988). 8.22.5 Quarantine Status of Plant Importations A number of precautions are to be undertaken to minimise pest/disease risk. They include the regulations themselves, phytosanitary certificates, with or without added declarations, permits, inspection upon arrival, treatments, etc. Based on their quarantine status, when even a plant or seed material is received at the inspection station or port of entry, they are placed in one of the four categories mentioned here: (1) absolute prohibition, (2) quarantine, (3) post-entry quarantine and (4) restricted entry. Whenever the risk of introducing pests and pathogens is very high, then the importation of that particular plant material is completely prohibited, even for government services and such material will be included under absolute prohibition category. This is invoked by the importing country when there are no adequate safeguards and when isolation from commercial crop production is not possible. For example, an absolute prohibition against coconut from eight specified geographical areas was recommended by the South West Pacific Commission, because of the diseases like cadang-cadang (Hanold and Randles 1991). The second category, quarantine, covers the plant materials on which the pests and diseases have already been reported from the country. Admission of plants is usually reserved for government services and not for private agencies. In the third category, postentry quarantine, the genera are placed on which pests and pathogens have been reported 233 in some, but not all, foreign countries, but the importation originates from a country where such risks have not been reported. Post-entry quarantine is usually reserved for government services, in situations and qualified individuals whose facilities meet post-entry quarantine requirements. Safeguards consist of inspection upon arrival and during a special post-entry period, usually for at least two growing seasons, at the premises of the importer. The fourth and last category, restricted entry, is usually assigned to only restricted plants that are received by the general public, and they were subjected to inspection and treatment upon arrival at a port of entry or inspection station. 8.22.6 Types of Materials Received The genetic stocks may be in the form of ‘vegetative propagule’ or ‘seed’ type. Vegetative propagative material may be bud wood, scion material or unrooted cuttings, all of which, although dangerous, carry less risk than does rooted vegetative propagating material and can be transported quickly by air. The vegetative propagative material also includes plant materials like rooted cuttings or bulbs, tubers, corms or rhizomes. The plant material can also be exchanged in the form of ‘seed’. In the earlier chapter, the seed-transmitted viruses and the methods to make them virus-free were discussed. The introduced plant material should be tested at the quarantine stations and which involve some time and labour. The imported plant material should be tested by germination, indexing, serology, electron microscopy, etc., which were earlier described in this chapter. The seeds are less likely to carry virus diseases than is the vegetative propagative material. The propagules derived from virus-infected mother plants will be mostly infected. The import of seed is not considered to involve a risk of the same magnitude as that presented by living plant material, except that in some cases, viruses are carried through seed. Since no case of seedtransmitted disease transmitted by whiteflies has been definitely established, the required germplasm can be introduced through seeds. 234 8 Methods of Combating Seed-Transmitted Virus Diseases With the discovery of Potato spindle tuber viroid which is transmitted through true seed of tuber bearing species of Solanum can no longer be regarded as freely interchangeable among breeders and collectors. FAO–IBPGR have recommended the following steps for majority of the ‘seed’-type germplasm movement for majority of the crops, and with little modifications, one can plan depending on the size of the consignment. At International Institute of Tropical Agriculture (IITA), Nigeria, the in vitro techniques have been applied to the conservation and exchange of cassava germplasm to collaborators outside Nigeria, and plantlets regenerated from meristem culture were transplanted and indexed for African cassava mosaic virus (ACMV). Plantlets regenerated from nodal cuttings obtained from their virus-tested plant were used for distribution upon request. NARS has distributed these certified virus-tested plantlets for evaluation in more than 40 countries in Africa (Ng and Ng 1997). The transfer of germplasm should be carefully planned in consultation with quarantine authorities and should be in amounts that allow adequate handling and examination. The material should be accompanied with the necessary documentation. existing knowledge in all disciplines relating to the phytosanitary safety of germplasm transfer. This has prompted FAO and IBPGR to launch a collaborative programme for the safe and expeditious movement of germplasm reflecting the complementarity of their mandates with regard to the safe movement of germplasm. Frison et al. (1990) (Frison EA, Bos L, Hamilton RI, Mathur SB, Taylor JW) under the instructions of FAO/IBPGR have brought out technical guidelines for the safe movement of legume germplasm in which they have furnished the information on virus and virus-like diseases of legume crops and provided the guidelines for international crop improvement programmes, collecting, conservation and utilisation of plant genetic resources and their global distribution. In this preparation, the experienced crop experts, namely, Albrechtsen SE, Johnstone GR, Makkouk KM, Feliu E, McDonald D, Mink GI, Morales FJ, Reddy DVR, Vermeulen H, Rossel HW and Zhang Zheng, have provided the details of virus and virus-like diseases of leguminous crops and participated in the preparation of the guidelines. The movement of germplasm involves a risk of accidentally introducing plant quarantine pests along with the host plant material; in particular, pathogens that are often symptomless, such as viruses, pose a special risk. To minimise this risk, effective testing (indexing) procedures are required to ensure that distributed material is free of pests that are of quarantine concern. General recommendations on how best to move germplasm of the crop concerned and mention available intermediate quarantine facilities when relevant are provided. 8.23 Role of FAO/IBPGR in Germplasm Exchange FAO has a long-standing mandate to assist its member governments to strengthen their Plant Quarantine Services, while IBPGR’s mandate – inter alia – is to further the collecting, conservation and use of the genetic diversity of useful plants for the benefit of people throughout the world. The aim of the joint FAO/IBPGR programme is to generate a series of crop-specific technical guidelines that provide relevant information on disease indexing and other procedures that will help to ensure phytosanitary safety when germplasm is moved internationally. The ever increasing volume of germplasm exchanged internationally, coupled with recent, rapid advances in biotechnology, has created a pressing need for crop-specific overviews of the 8.23.1 Conceptual Guidelines for Exchange of Legume Germplasm, Breeding Lines and Commercial Seed Lots as Follows (a) Germplasm • All legume germplasm collections should be maintained free of known seedassociated pests (seed borne or seed 8.23 Role of FAO/IBPGR in Germplasm Exchange transmitted in the case of fungi and bacteria; seed transmitted in the case of viruses). Descriptor data should be obtained from pest-free germplasm. • Only seed lots certified to be free of such pests should be distributed. • In recipient countries, seed lots should be established and maintained for one generation under conditions of isolation (temporal and/or spatial) or containment, with periodic inspection, testing and roguing. (b) Breeding Lines • Legume seed lots to be exchanged among breeding programmes should be produced under conditions of isolation (with appropriate chemical protection) or containment, with periodic inspection and roguing to eliminate seed-associated pests. • Seed lots should be tested for seedassociated pests and certified by the appropriate regulatory agency before distribution. (c) Commercial Seed Lots • Commercial seed lots should continue to be subject to current regulatory procedures. 8.23.2 The Technical Guidelines for Exchange of Germplasm and Breeding Lines (a) General Recommendations • Vegetative material of legume species should go through intermediate or postentry quarantine and should be tested for absence of viruses. • Legume seed should not be moved internationally in pods. • Seed should be harvested at optimal time for the crop and care taken to ensure effective drying. • Seed samples should be cleaned to eliminate all soil, plant debris, seeds of noxious weeds and phanerogamic parasites. 235 • Unless specified otherwise, seeds should be surface disinfected (with sodium hypochlorite or a similar product) before being given appropriate fungicide and insecticide treatments. • Seed lots suspected to contain insects should be fumigated with an appropriate pesticide. • Parcels containing seeds should be unpacked in a closed packing material and should be incinerated or autoclaved. 8.23.3 Movement of Germplasm (a) Introduction of Germplasm • Introduction of new germplasm entries should satisfy local regulatory requirements. • Each new introduction should be grown under containment or isolation. • Plants should be observed periodically. Plants suspected to be affected with seedassociated pests should be destroyed. • All symptomless plants should be tested for latent infections by viruses known to occur in the place of origin of the material and in the country of maintenance. Ideally this testing should be carried out at this stage or, if not possible, it should be carried out before the germplasm is distributed (see ‘International Distribution of Germplasm’). Infected plants should be destroyed. • Seed should be collected from healthy plants only. (b) Further Multiplication of New Introductions or Rejuvenation of Germplasm Accessions • Seed should be sown under containment or isolation with appropriate chemical protection. • Plants should be observed periodically. Plants affected by seed-associated pests should be removed and destroyed. • Seed should be collected from healthy plants only. 236 8 (c) International Distribution of Germplasm • Germplasm accessions that have been introduced and multiplied according to the procedures described above can be certified and distributed internationally. • Germplasm accessions which are not yet in a pest-free state should be handled according to the same procedures as described for new introductions. • Movement of germplasm should comply with regulatory requirements of the importing country. • In addition to the phytosanitary certificate, a ‘germplasm health statement’, indicating which tests have been performed to assess the health status of the material, should accompany the germplasm accession. (d) Movement of Breeding Material • Seeds used for the multiplication of breeding material should be pest-free. • Breeding material under multiplication should be grown under containment or isolation with appropriate chemical protection. • Plants should be inspected soon after emergence and periodically thereafter. Plants infected with seed-associated pests should be destroyed. For field-grown plants, suitable precautions should be taken to prevent soil spread from infected plants and introduction of possible seedassociated pests from local sources of infection. • Seeds should be harvested only from symptomless plants. • Seed samples of appropriate size should be tested for seed-associated pests. • When non-destructive seed health tests are available, all seeds should be tested accordingly. • Movement of germplasm should comply with regulatory requirements of the importing country. • In addition to the phytosanitary certificate, a germplasm health statement, indicating which tests have been performed to assess the health status of the material, should accompany the breeding material. Methods of Combating Seed-Transmitted Virus Diseases 8.24 Steps in Technical Recommendations for Seed Germplasm Exchange in the Country of Origin or Destination • Seed production should be carried out in areas which are free from diseases of quarantine significance whenever possible. • Fruits should be harvested from healthy looking plants. • Seeds of normal size should be selected from healthy looking fruits. • Seeds should be treated according to the following recommendations, either in the country of origin or in the country of destination: – Immerse the seeds in water and discard any floating seeds. – Treat the seeds immersed in water in a microwave oven at full power until the water temperature reaches 73ı C and pour off the water immediately after the treatment. – If a microwave oven is not available, treat the seeds with dry heat for 2 weeks at 60ı C. – Dry the seeds and treat them with thiram dust. – Pack the seeds in a paper bag. – After arrival in the country of destination, the seeds should be inspected for the presence of insect pests. If found to be infected, they should be fumigated or destroyed (if fumigation is not possible). – Seeds should be sown under containment or in isolation and kept under observation until the plants are well established and normal healthy leaves are produced. If bud wood or scion material is to be exported, prior information is required to the stations which enable the officers involved to raise the test plants. Healthy stock seedlings should also be grown in advance, so that they will be suitable for grafting when the importation arrives. Any material received with roots, there are chances of carrying nematode or fungal vectors. Therefore, it should be first grown in sterile soil, if possible under glass. Cuttings then can be taken from the shoots of plants whose tops have been 8.25 Part of the Planting Material to Be Tested and Post-Entry Quarantine found to be healthy, and these can be rooted or grafted to healthy root stocks. If it is sap transmitted, it should be tested on susceptible hosts. The original roots must be destroyed. In the UK, the Carnation necrotic fleck virus (CNFV) had been detected in the imported carnation cuttings from the Netherlands (Stone and Hollings 1976). During March and April 1977, a series of consignments of Portuguese glasshouse carnation cuttings severely affected by CNFV were intercepted and destroyed at the UK. Similarly, daphne mosaic plants were found in a lot of 250 Daphne mezereum plants to the USA from Holland and were promptly destroyed under the supervision of state nursery inspector. When the unrooted cuttings were received, they should be treated with some insecticide and rooted in an appropriate medium, and transplanted to richer compost when rooted and indexed by all possible methods. In the USA between 1957 and 1967, a total number of 1,277 vegetatively propagated plant materials of citrus, ipomoea, prunus, solanum and vitis were received from various countries, and 62% of the plant material was infected with different viruses (Kahn et al. 1967). In the next decade, that is, from 1968 to 1978, the percentage of imported infected plant material was only 50%, out of the 1,913 vegetative propagules of Saccharam, Theobroma, Vitis, Solanum, Ipomoea, fruit trees, grasses and other plants (Kahn 1989). Some of the virus diseases detected in ornamental plant importations from different countries to the USA during 1968–1978 have also been reported by Kahn (1989) and Kahn (1988). For breeding programmes, the wild species are exchanged from one country to another, and viruses are carried even in these hosts. Kahn and Monroe (1970) recorded 39% virus incidence in the wild Solanum introductions collected as tubers. Similarly, Kahn and Sowell (1970) and Kahn (1988, 1989) reported 11% of the 46 wild Arachis species collected from Uruguay, Argentina and Brazil were virus infected. The above examples clearly indicate the need for effective measures to be taken while introducing any type of plant material. 8.25 237 Part of the Planting Material to Be Tested and Post-Entry Quarantine Methods available for detecting viruses include testing of seedlings using a combination of techniques after subjecting the seeds for post-entry quarantine (PEQ) growing as per the technical guidelines developed by FAO/Bioversity International (formerly International Plant Genetic Resources Institute, IPGRI) for safe movement of germplasm (http://www.bioversityinternational. org/scientific information/themes/germplasm health/). However, this includes growing for a season which requires good environmentcontrolled PEQ greenhouses involving funds for constructing greenhouses or a field in isolation, which is not easily available. Detection of viruses in embryo indicates that the viruses are seed transmitted. Since the presence of virus in the seed coat does not relate to virus transmission from seeds to seedlings, whole-seed serological assays sometimes are not suitable for determining seed transmission (Maury and Khetarpal 1989; Johansen et al. 1994). While preparing samples (i.e. extracting embryos) for determining the transmission rate on large number of seeds, it is therefore necessary that viral antigen from non-embryonic tissues (i.e. seed coat) is not simultaneously extracted, because only the virus in the embryo leads to seed transmission and extraction of virus from non-embryonic tissue may lead to false-positive reactions. Moreover, manual processing of large number of seeds is not possible in a routine test (Maury and Khetarpal 1997). Therefore, efforts should be made to develop techniques which could detect virus (e.g. PSbMV) only in embryo, although the whole seed is used for extraction (Masmoudi et al. 1994a, b). Moreover, if the number of imports is more, it would be difficult to subject all the samples for growing in PEQ greenhouses. The PEQ of bulk imports in India is being carried out under the supervision of the inspection authorities who are the senior level plant protection scientists of various universities and 238 8 Methods of Combating Seed-Transmitted Virus Diseases institutes. However, these inspection authorities, in general, lack sufficient funds to carry out their work effectively. This can be a major bottleneck in the functioning of this aspect of the quarantine operations (Khetarpal 2004). SPS measures are defined as any measure applied within the territory of the member state: (a) To protect animal or plant life or health from risks arising from the entry, establishment or spread of pests, diseases and diseasecarrying/disease-causing organisms (b) To protect human or animal life or health from risks arising from additives, contaminants, toxins or disease-causing organisms in food, beverages or foodstuffs (c) To protect human life or health from risks arising from diseases carried by animals, plants or their products, or from the entry, establishment/spread of pests (d) To prevent or limit other damage from the entry, establishment or spread of pests The SPS agreement explicitly refers to three standard-setting international organisations commonly called as the ‘three sisters’ whose activities are considered to be particularly relevant to its objectives: International Plant Protection Convention (IPPC) of Food and Agriculture Organization (FAO) of the United Nations, World Organization for Animal Health (OIE) and Codex Alimentarius Commission of Joint FAO/WHO. The IPPC develops the International Standards for Phytosanitary Measures (ISPMs) which provide guidelines on pest prevention, detection and eradication. To date, 34 standards (https://www.ippc.int/index. php?id=13399&type=publication&subtype=& category id=&tx publication pi1[pointer]=0& showAll=1#publication) as given below have been developed, and several others are at different stages of development: 1. ISPM 1: Principles of plant quarantine as related to international trade 2. ISPM 2: Guidelines for pest risk analysis 3. ISPM 3: Code of conduct for the import and release of exotic biological control agents 4. ISPM 4: Requirements for the establishment of pest-free areas 5. ISPM 5: Glossary of phytosanitary terms 6. ISPM 6: Guidelines for surveillance 7. ISPM 7: Export certification system 8. ISPM 8: Determination of pest status in an area 8.26 Exclusion of Exotic Plant Viruses Through Quarantine 8.26.1 International Scenario The recent trade-related developments in international activities and the thrust of the WTO agreements imply that countries need to update their quarantine or plant health services to facilitate pest-free import/export. The establishment of the WTO in 1995 has provided unlimited opportunities for international trade of agricultural products. History has witnessed the devastating effects resulting from diseases and pests introduced along with the international movement of planting material, agricultural produce and products. It is only recently, however, that legal standards have come up in the form of Sanitary and Phytosanitary (SPS) Measures for regulating the international trade. The WTO agreement on the application of SPS measures concerns the application of food safety and animal and plant health regulations. It recognises government’s rights to take SPS measures but stipulates that they must be based on science, should be applied to the extent necessary to protect human, animal or plant life or health and should not unjustifiably discriminate between members where identical or similar conditions prevail (http://www.wto. org). The SPS agreement aims to overcome healthrelated impediments of plants and animals to market access by encouraging the ‘establishment, recognition and application of common SPS measures by different members’. The primary incentive for the use of common international norms is that these provide the necessary health protection based on scientific evidence and improve trade flow at the same time. 8.26 Exclusion of Exotic Plant Viruses Through Quarantine 9. ISPM 9: Guidelines for pest eradication programmes 10. ISPM 10: Requirements for the establishment of pest-free places of production and pest-free production site 11. ISPM 11: Pest risk analysis for quarantine pests including analysis of environmental risks and living modified organisms 12. ISPM 12: Guidelines for phytosanitary certificates 13. ISPM 13: Guidelines for the notification of non-compliance and emergency action 14. ISPM 14: The use of integrated measure in a systems approach for pest risk management 15. ISPM 15: Guidelines for regulating wood packaging material in international trade 16. ISPM 16: Regulated non-quarantine pests: concept and application 17. ISPM 17: Pest reporting 18. ISPM 18: Guidelines for the use of irradiation as a phytosanitary measure 19. ISPM 19: Guidelines on list of regulated pests 20. ISPM 20: Guidelines for phytosanitary import regulatory system 21. ISPM 21: Pest risk analysis for regulated non-quarantine pests 22. ISPM 22: Requirements for the establishment of areas of low pest prevalence 23. ISPM 23: Guidelines for inspection 24. ISPM 24: Guidelines for the determination and recognition of equivalence of phytosanitary measures 25. ISPM 25: Consignments in transit 26. ISPM 26: Establishment of pest-free areas for fruit flies (Tephritidae) 27. ISPM 27: Diagnostic protocols for regulated pests 28. ISPM 28: Phytosanitary treatments for regulated pests 29. ISPM 29: Recognition of pest-free areas and areas of low pest prevalence 30. ISPM 30: Establishment of areas of low pest prevalence for fruit flies (Tephritidae) 31. ISPM 31: Methodologies for sampling of consignments 32. ISPM 32: Categorisation of commodities according to their pest risk 239 33. ISPM 33: Pest-free potato (Solanum spp.) micropropagative material and minitubers for international trade 34. ISPM 34: Design and operation of post-entry quarantine stations for plants Prior to the establishment of WTO, governments on a voluntary basis could adopt international standards, guidelines, recommendations and other advisory texts. Although these norms shall remain voluntary, a new status has been conferred upon them by the SPS agreement. A WTO member adopting such norms is presumed to be in full compliance with the SPS agreement. 8.26.2 National Scenario Plant quarantine is a government endeavour enforced through legislative measures to regulate the introduction of planting material, plant products, soil, living organisms, etc., in order to prevent inadvertent introduction of pests and pathogens harmful to the agriculture of a region and, if introduced, prevent their establishment and further spread. As early as in 1914, the Government of India passed a comprehensive Act, known as Destructive Insects and Pests (DIP) Act, to regulate or prohibit the import of any article into India likely to carry any pest that may be destructive to any crop, or from one state to another. The DIP Act has since undergone several amendments. In October 1988, new policy on seed development was announced, liberalising the import of seeds and other planting material. In view of this, plants, fruits and seeds (regulation of import into India) order (PFS order) first promulgated in 1984 was revised in 1989. The PFS order was further revised in light of the WTO agreements, and the plant quarantine (regulation of import into India) Order 2003 [hereafter referred to as PQ Order] came into force on 1 January 2004 to comply with the sanitary and phytosanitary agreement (Khetarpal et al. 2006b). Until 12 June 2010, 14 amendments of the PQ Order were notified, and ten draft amendments were prepared, revising definitions, clarifying specific queries raised by 240 8 Methods of Combating Seed-Transmitted Virus Diseases quarantine authorities of various countries, with revised lists of crops under the Schedules VI, VII and quarantine weed species under Schedule VIII. The revised list under Schedules VI and VII now includes 729 and 286 crops/commodities, respectively, and Schedule VIII now includes 31 quarantine weed species. The PQ Order ensures the incorporation of ‘Additional/Special Declarations’ for import commodities free from quarantine pests, on the basis of pest risk analysis (PRA) following international norms, particularly for seed/planting material (http://www.agricoop.nic. in/gazette.htm). The Directorate of Plant Protection, Quarantine and Storage (DPPQS) under the Ministry of Agriculture is responsible for enforcing quarantine regulations and for quarantine inspection and disinfestation of agricultural commodities. The quarantine processing of bulk consignments of grain/pulses, etc., for consumption and seed/planting material for sowing are undertaken by the 35 plant quarantine stations located in different parts of the country, and many pests were intercepted in imported consignments (http://www.plantquarantineindia.org/docfiles/ appendix-8.htm). Import of bulk material for sowing/planting purposes is authorised only through five plant quarantine stations. There are 41 inspection authorities who inspect the consignment being grown in isolation in different parts of the country. Besides, DPPQS has developed 21 standards on various phytosanitary issues such as on PRA, pest-free areas for fruit flies and stone weevils, certification of facilities for treatment of wood packaging material and methyl bromide fumigation. Also, two standard operating procedures have been notified on export inspection and phytosanitary certification of plants/plant products and other regulated articles and post-entry quarantine inspection (www.Plantquarantineindia.org/standards.htm). The National Bureau of Plant Genetic Resources (NBPGR), the nodal institution for exchange of plant genetic resources (PGR), has been empowered under the PQ Order to handle quarantine processing of germplasm including transgenic planting material imported for research purposes into the country by both public and private sectors. NBPGR has developed well-equipped laboratories and postentry quarantine greenhouse complex. Keeping in view the biosafety requirements, National Containment Facility of level-4 (CL-4) has been established at NBPGR to ensure that no viable biological material/pollen/pathogen enters or leaves the facility during quarantine processing of transgenics. At NBPGR, adopting a workable strategy, a number of viruses of great economic and quarantine importance have been intercepted on exotic material, many of which are yet not reported from India, namely, Barley stripe mosaic virus in barley, Broad bean stain virus in broad bean, Cherry leaf roll virus on French bean and soybean, Cowpea mottle virus in cowpea and Bambara groundnut, Raspberry ring spot virus and Tomato ring spot virus on soybean, etc. (Khetarpal et al. 2001, 2006a, b; Chalam et al. 2005a, 2008, 2009a, b, c; Chalam and Khetarpal 2008). In addition, many interceptions have also been made of viruses not reported on the host on which it is intercepted. In India, the seeds of cowpea, mung bean, soybean and broad bean received from Nigeria, Taiwan, Columbia, Brazil, Myanmar, Australia, Hungary, Spain and Bulgaria are screened at NBPGR, and about eight viruses which are seed transmitted are intercepted (Khetarpal et al. 2001; Parakh et al. 2008; Chalam et al. 2008, 2009a, b, c). Until to date, 8,700 samples of transgenic crops comprising Arabidopsis thaliana, Brassica spp., chickpea, corn, cotton, potato, rice, soybean, tobacco, tomato and wheat with different traits imported into India for research purposes were processed for quarantine clearance, wherein they are tested for associated exotic pests, if any, and also for ensuring the absence of terminator gene technology (embryogenesis deactivator gene) which are mandatory legislative requirements. Barley stripe mosaic virus and Wheat streak mosaic virus which are not reported from India were intercepted in wheat. Also, Maize dwarf mosaic virus not reported on wheat in India was intercepted (Chalam et al. 2009a). All the plants infected by the viruses were uprooted and incinerated. The harvest from virusfree plants was released to the indenters. If not 8.28 Quarantine for Germplasm and Breeding Material 241 intercepted, some of the above quarantine viruses could have been introduced into our agricultural fields and caused havoc to our productions. Thus, apart from eliminating the introduction of exotic viruses from our crop improvement programmes, the harvest obtained from virus-free plants ensured conservation of virus-free exotic germplasm in the National Genebank. The process requires detailed information on pest scenario in both countries importing and exporting the commodity. Efforts to develop a database for endemic pests have been made by the plant quarantine station in Chennai, and NBPGR has compiled pests of quarantine significance for cereals (Dev et al. 2005) and a checklist for viruses in grain legumes (Kumar et al. 1994). Compilation of pests of quarantine significance for grain legumes by Chalam and Khetarpal (2008) and the Crop Protection Compendium of CAB International, UK, are some of the useful assets to scan for global pest data (CAB International 2007). As one faces challenges to crops from intentional or non-intentional introductions of viruses, speed and accuracy of detection becomes paramount. The size of consignment received is very critical in quarantine from processing point of view. Bulk seed samples of seed lots need to be tested by drawing workable samples as per norms. The prescribed sampling procedures need to be followed strictly, and there is a need to develop/adapt protocols for batch testing, instead of individual seed analysis (Maury et al. 1985; Maury and Khetarpal 1989). On the other hand, germplasm samples are usually received as a few seeds/sample, and thus, it is often not possible to do sampling because of few seeds and also because of the fact that a part of the seed is also to be kept as voucher sample in the National Genebank in India apart from the pestfree part that has to be released. Hence, extreme precaution is needed to ensure that the result obtained in the test was not denoted a falsepositive or a false-negative sample (Khetarpal 2004; Chalam and Khetarpal 2008). 8.27 Challenges in Diagnosis of Pests in Quarantine The issues related to quarantine methodology and challenges in exclusion of plant viruses during transboundary movement of seeds were analysed/reviewed recently (Khetarpal 2004; Chalam and Khetarpal 2008). The challenge prior to import is preparedness for pest risk analysis (PRA). PRA is now mandatory for import of new commodities into India. The import permit will not be issued for the commodities not covered under the Schedules V, VI and VII under the PQ Order. Hence, for import of new commodities in bulk for sowing/planting, the importer should apply to the Plant Protection Adviser to the Government of India for conducting PRA. In case of germplasm, import permit shall be issued by the Director, NBPGR, after conducting PRA based on international standards (http://agricoop.nic.in/ Gazette/Psss2007.pdf). The ISPM-2, ISPM-11 and ISPM-21 deal with the guidelines for conducting PRA for quarantine pests and regulated non-quarantine pests (http:// www.ippc.int/ipp/en/standards.htm). There are two kinds of PRA, that is, pathway based and pest based. It consists of three stages: initiating the process for analysing risk, assessing pest risk and managing pest risk. Initiating the process involves identification of pests or pathways for which the PRA is needed. Pest risk assessment determines whether each pest identified as such, or associated with a pathway, is a quarantine pest, characterised in terms of likelihood of entry, establishment, spread and economic importance. Pest risk management involves developing, evaluating, comparing and selecting options for reducing the risk. 8.28 Quarantine for Germplasm and Breeding Material Scientists, hobbyists and growers worldwide have contributed to horticultural and silvicultural wellbeing. There have been a vast number of exotic plant introductions in agriculture. This plant movement was somewhat haphazard and uncontrolled until the end of the nineteenth century. 242 8 In 1898, the US Federal Government began conducting plant explorations to introduce new and unusual plants and began maintaining systematic and detailed records on plant introductions. Most germplasm enters the United States as a result of government-sponsored explorations or as a result of requests by nurserymen, botanical gardens, hobbyists and various scientists. Germplasm from original or secondary gene centres is now in great demand. With the high priority awarded to resistance, introduction often comes from developing countries with very limited quarantine resources and limited information on endemic diseases. In case of germplasm accessions and breeding lines, a zero tolerance is advocated. Plant genetic resources are actually the key elements in any crop improvement program. Germplasm which represents a rich variability in plant genotype is collected from diverse agro-climatic zones. Theoretically in those geographical regions where sources of resistance in wild genotypes are found, there is a strong possibility of the emergence of virulent isolates of the virus because of selective inoculum pressure. In the case of seedtransmitted viruses, there is also the possibility of perpetuating such pathotypes through infected seeds of susceptible germplasm lines. Therefore, it is essential to certify that seeds of genetic resources and breeding lines at different stages of utilisation and exchange are free from viruses (Hampton et al. 1993). (a) Germplasm Evaluation and Trials: Germplasm materials are generally multiplied and evaluated in the field. During the course of multiplication, seed-transmitted virus(es) serve as primary source(s) of inocula and, in the presence of insect vectors, are transmitted to neighbouring susceptible lines. Hence, the virus is finally spread to different lines of the collection in which it becomes seed transmitted. A very large percentage of the germplasm collections from different regions are thus found to be infected. Since germplasm is collected from diverse regions and is often exchanged internationally, considerable viral strain variation can be expected in these collections. Methods of Combating Seed-Transmitted Virus Diseases (b) Germplasm Exchange and Quarantine: The increase in exchange of seeds of germplasm accessions that are likely to be contaminated by viruses has necessitated the need to certify them to be free from viruses through stringent quarantine measures, keeping in mind the possibility of introducing unknown viruses or virulent strains into a country. SMV, PSbMV and BCMV (PStV) are the classical examples of economically significant, seed-transmitted viruses of soybean, pea and peanut, respectively, which are known to have spread to different countries through seeds of infected germplasm (Demski et al. 1984a, b; Goodman and Oard 1980; Hampton and Braverman 1979). Seeds of germplasm material are usually exchanged in small quantities. As quarantine processing of only a portion of the seed lot does not indicate the status of the untested portion, it is necessary to plant seed of an imported accession in isolation. Plant expressing suspicious symptoms should be rogued, and only seeds from plants shown to be virus-free by appropriate tests should be harvested and released to breeders for use in crop improvement programmes (Jones 1987; Khetarpal et al. 1991). By adopting such a procedure, a number of important seed-transmitted viruses, namely, CABMV, SBMV, SMV and many others, were intercepted in accessions of legumes imported into Australia (Jones 1987). Similarly, SMV, CPMoV, CABMV, BYMV, BBSV, BCMV and PSbMV were intercepted in seeds of legume germplasm and breeding lines imported from different countries into India (Khetarpal et al. 1993, 1994; Chalam et al. 2004, 2008, 2009a). The importance of interception can be highlighted by the fact that the economically damaging virus of cowpea, CPMoV, is still not known to occur in India where cowpea is an important source of protein for a predominantly vegetarian population. Moreover, other intercepted viruses are known to possess different virulent strains. Production of virus-free seeds for conservation can be achieved simply by identifying virusfree plants from which harvested seeds are to be 8.29 Role of IPGRI and NBPGR in Germplasm Maintenance and Exchange used for conservation. The more rapid alternative which consists in testing a representative sample of the seed lots for presence of viruses before being conserved would not be foolproof. Seeds of germplasm stored in a gene bank must also be periodically multiplied in the field for either replenishing the depleting stock or to circumvent the problems of seed viability. Such multiplication needs to be protected from vectors for ensuring freedom from seed-transmitted viruses. Technical guidelines jointly issued by FAO and IPGRI (Frison et al. 1990) and the checklist of seed-transmitted viruses (Kumar et al. 1994) are useful references for ensuring safe movement of germplasm material. 8.29 Role of IPGRI and NBPGR in Germplasm Maintenance and Exchange IPGRI: The International Plant Genetic Resources Institute (IPGRI) is an independent international scientific organisation that promotes the conservation and use of plant genetic diversity for the well-being of present and future generations. It is one of 15 centres supported by the Consultative Group on International Agricultural Research (CGIAR), an association of public and private members who support efforts to mobilise cutting-edge science to reduce hunger and poverty, improve human nutrition and health and protect the environment. More than 90% of respondents felt that agricultural biodiversity could help to meet these challenges and that it could make a ‘major’ contribution to food security and environmental conservation. IPGRI has its headquarters in Macanese, near Rome, Italy, with offices in more than 20 other countries worldwide. It has five regional groups, with collective global coverage. IPGRI’s main focus is on the developing countries, but the regional groups also establish contacts with institutions in the more developed countries. The institute operates through three programmes: (1) the Plant Genetic Resources Programme, (2) the CGIAR Genetic Resources Support 243 Programme and (3) the International Network for the Improvement of Banana and Plantain (INIBAP). The international status of IPGRI is conferred under an Establishment Agreement which, by January 2003, had been signed by the Governments of Algeria, Australia, Belgium, Benin, Bolivia, Brazil, Burkina Faso, Cameroon, Chile, China, Congo, Costa Rica, Côte d’Ivoire, Cyprus, Czech Republic, Denmark, Ecuador, Egypt, Greece, Guinea, Hungary, India, Indonesia, Iran, Israel, Italy, Jordan, Kenya, Malaysia, Mauritania, Morocco, Norway, Pakistan, Panama, Peru, Poland, Portugal, Romania, Russia, Senegal, Slovakia, Sudan, Switzerland, Syria, Tunisia, Turkey, Uganda and Ukraine. For IPGRI’s research, financial support is provided by more than 150 donors, including governments, private foundations and international organisations. For details of donors and research activities, please see IPGRI’s Annual Reports, which are available in printed form on request from [email protected] or from IPGRI’s website (www.ipgri.cgiar.org). 8.29.1 Objectives of NBPGR NBPGR (National Beauro of Plant Genetic Resources) is one of the ICR institutions established by GOI at New Delhi (India). The objective of this institution are: • To plan, organise, conduct and coordinate exploration and collection of indigenous and exotic plant genetic resources • To undertake introduction, exchange and quarantine of plant genetic resources • To characterise, evaluate, document and conserve crop genetic resources and promote their use, in collaboration with other national organisations • To develop information network on plant genetic resources • To conduct research, undertake teaching and training, develop guidelines and create public awareness on plant genetic resources NBPGR has a separate Division of Plant Quarantine to meet the quarantine requirements 244 8 Methods of Combating Seed-Transmitted Virus Diseases in respect of the germplasm materials being exchanged through it. The division has trained scientific and technical staff representing the disciplines of entomology, nematology and plant pathology, well-equipped laboratories, greenhouses and post-entry isolation growing field facilities to discharge its quarantine responsibilities efficiently. In case of certain crops, after laboratory examination at NBPGR, the exotic material is passed on to the specific crop-based institutes for post-entry isolation growing, before it is released to the indenters. These institutes have established adequate post-entry isolation growing facilities, and required expertise is also available with them. These are Central Potato Research Institute, Shimla; Central Tuber Crops Research Institute, Trivandrum; Central Tobacco Research Institute, Rajahmundry; Sugarcane Breeding Institute, Coimbatore; and Central Plantation Crops Research Institute, Kasaragod. NBPGR has established a regional plant quarantine station at Hyderabad to fulfil the quarantine requirements of the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Directorate of Rice Research and other research organisations in the region. It is also proposed to establish quarantine facilities for temperate fruit crops at NBPGR’s Regional Station, Bhowali, and an offshore Quarantine Station at Port Blair in the Andaman Group of Islands for vegetatively propagated tropical crops. During the last 12 years or so, a large number of exotic insects and mites, plant parasitic nematodes, plant pathogens and weeds have been intercepted from the imported germplasm materials, many of which are of major quarantine significance and are not yet known to occur in the country. While processing the germplasm for quarantine clearance, all out efforts are made to salvage the infested/infected materials so that valuable exotic germplasm could be made available in a healthy state for exploitation in crop improvement programmes in the country. In this direction, Chakrabarty et al. (2004) have listed the different pests intercepted in oil seed germplasm imported during 1986–2003. The Table 8.3 shows the list of intercepted plant viruses in India. The data presented in the table includes the seed-transmitted viruses intercepted in crops like cowpea, mung bean, beans, soybean and broad bean (Khetarpal et al. 2001; Chalam et al. 2004, 2008, Chalam et al. 2009a, b, c; Parakh et al. 2008). National Bureau of Plant Genetic Resources (NBPGR) is the nodal agency for quarantine processing of exotic germplasm and hence undertaken the quarantine processing of all germplasm including transgenic planting material under exchange for research purposes. Kumar et al. (1994) and Khetarpal et al. (2001) have summarised the viruses intercepted in leguminous crops during 1991–2000. Similar type of information is available on the list of intercepted plant viruses in almost all tropical countries. NBPGR also deals with testing for absence of terminator technology which is mandatory as per national legislation. This authorisation was vested upon NBPGR for germplasm vide Article 6 of PQ Order 2003 and for transgenic planting material vide Government of India Notification No. GSR 1067(E) dated 05.12.1989. 8.29.2 Methods of Testing at Quarantine Stations The testing of the plant material received is done by indexing on indicator hosts either by mechanical/insect transmission and also by grafting. The symptoms are expressed within a few days or weeks when herbaceous test plants are used, but may require months or years when woody plants are used in which case the entry of the genetic stocks will be delayed. The various methods involved in detecting the viruses in seeds and seed stocks are described in the Chap. 6. Indexing the viruses infecting fruit crops is difficult than the other crops. For example, for testing cocoa planting material, Amelonado plant (West African Cocoa Research Institute Selection C14) is the best known indicator plant. The surface-sterilised bud wood sent from the donor country is budded onto decapitated Amelonado plants under glasshouse conditions. The health of scion and 8.29 Role of IPGRI and NBPGR in Germplasm Maintenance and Exchange 245 Table 8.3 Virus and virus-like diseases intercepted in exotic germplasm at NBPGR and quarantine stations in India Virus intercepted TBRV AMV Sl. no 1 2 Crop Beans Broad bean 3 4 Broad bean Broad bean 5 Cowpea 6 7 8 9 10 11 12 Cowpea Cowpea Mung bean Mung bean Mung bean Peanut Soybean CABMV PStV AMV 13 Soybean BCMV 14 Soybean CABMV 15 Soybean CMV 16 Soybean SBMV 17 Soybean TRSV BYMV, PSbMV BYMV, BBSV, PSbMV AMV, CMV, TBRV CABMV CABMV CMV Source of country CIAT, Columbia ICARDA-Syria, Eritrea, Iraq, Spain Spain, Syria Bulgaria Reference Chalam et al. (2004) Chalam et al. (2009c) IITA, Nigeria Chalam et al. (2008) Eritrea USA China USA AVRDC, Taiwan Myanmar AVRDC-Taiwan, IITA-Nigeria, Brazil, Myanmar, USA AVRDC-Taiwan, IITA-Nigeria, USA AVRDC-Taiwan, IITA-Nigeria, Myanmar, USA AVRDC-Taiwan, IITA-Nigeria, Brazil, Myanmar, USA AVRDC-Taiwan, IITA-Nigeria, Australia, Brazil, Hungary, Thailand, USA IITA-Nigeria, Myanmar Chalam et al. (2008) Khetarpal et al. (2001) Chalam et al. (2008) Chalam et al. (2008) Chalam et al. (2008) Prasada Rao et al. (1990) Parakh et al. (2008) the indicator shoots from the root stock are checked weekly for virus symptoms. For safety, the scion should be top worked later with an Amelonado bud. If free from symptoms after three or four leaf flushes, bud wood is sent to the recipient country. In certain plant materials, some of the latent viruses cannot be detected during the routine inspection, as they do not exhibit well-marked symptoms. Some virus– host combinations have long incubation periods which vary from few weeks to several years. For example, certain of the citrus viruses have 3–8year incubation period. Inspection and treatment at ports of entry may not be adequate safeguards to prevent the entry of these latently infected plant materials. Plant experts and plant breeders also introduce the wild/cultivated plants which were not showing the symptoms, considering it as resistant/tolerant source (Kahn 1976). Some times more than one virus may become latent Chalam et al. (2009c) Khetarpal et al. (2001) Parakh et al. (2008) Parakh et al. (2008) Parakh et al. (2008) Baleshwar Singh et al. (2003) and Parakh et al. (1994, 2008) Parakh et al. (2008) or symptomless, and this has been demonstrated in certain fruit trees. For example, Kunkel et al. (1951) and Manns et al. (1951) have reported for peach yellows and little peach Myrobalan plum (Prunus cerasifera) as a symptomless carrier, and several varieties of the Japanese plum (Prunus salicina) are symptomless carriers of little peach. The Grape vine corky bark virus is latent in Vitis vinifera cv. ‘Emerald Riesling’, whereas in ‘Pinot Noir’ and ‘Almeria’, it causes severe disease (Hewitt 1975). In many grape varieties, viruses-like fleck (marbrure), vein mosaic and vein necrosis are latent. They can be detected by indexing on Vitis ruperstris St. George, Riparia gloire and Berlandieri x Rupestris (Hewitt et al. 1972; Legin and Vuittenez 1973). Even certain diseases are latent in some ornamentals like chrysanthemum, dahlias, gladiolus, carnations, etc. Sometimes, the latent virus may not damage the host plant, and it proves to be 246 8 Methods of Combating Seed-Transmitted Virus Diseases very economically important to other crops. For example, virus YN strain which is symptomless or causes only very mild symptoms in potatoes will spread rapidly by several aphid species and can completely destroy the tobacco crop in which it causes severe necrosis. Tomato spotted wilt virus in arums, begonias, chrysanthemums and dahlias often cause no visible symptoms, but if it is introduced, it causes a great loss to the crops like tomato, tobacco, lettuce, peas, beans, pineapple and other economic crops. Dodder (Cusanta californica) is reported by Bennett (1944) as a symptomless carrier of a virus causing damage to sugar beets, cantaloupes, tomatoes and several other crops. Even swollen shoot of cocoa and sunblotch of avocado are symptomless in some varieties. In such cases, plant materials are sent to special quarantine, where they are grown for one or two seasons under special greenhouses. Besides the indicator hosts, the other tests comprising histopathological, serological and electron microscopic techniques are also enabling for the quick and authentic identification of the virus and virus-like diseases of the imported plant materials of certain fruit crops. Based on these laboratory tests, the quarantine staff will decide the further measures to be taken against the materials under inspection (James et al. 2001). Only healthy plant material those guaranteed free from infection will be released. The details of the inspection techniques can be obtained from USDA Manual 1971 and also from the handbook of phytosanitary inspectors in Africa (Caresche et al. 1969). Since testing through indicator hosts is timeconsuming in almost all quarantine stations and research organisations are following molecularbased diagnostic tests for virus detection. Proper virus identification is always the key in developing appropriate practical solutions to manage plant virus diseases. Recent advances in biotechnology and molecular biology have played a significant role in the development of rapid, specific and sensitive diagnostic tests. The use of ELISA, by employing either polyclonal or monoclonal antibodies, was a significant step in adding sensitivity and precision to virus detection. The development of the tissue-blot immunoassay (TBIA), as a variant of ELISA, greatly simplified testing, reduced the cost and permitted virus testing at locations where facilities are limited or even absent. Immunochromatographic assay (ICA) is another ELISA variant which added speed to virus identification, where results can be obtained within 10–15 min, as compared to 2–3 h for TBIA. However, ICA is more expensive than TBIA. The development of nucleic acid-based tools was another new dimension of virus detection. The most common among these techniques are cDNA hybridisation and polymerase chain reaction (PCR). In addition, PCR can be used as a confirmatory test for TBIA, where processed blots can be cut individually and tested by PCR. This proved to work well with both DNA and RNA plant viruses. Furthermore, unprocessed plant tissue blots on nitrocellulose membrane represent a good sample for PCR amplification. PCR products can also be used for cloning and subsequent sequencing which is extremely useful for identification of new viruses or virus strains. 8.30 Important Cases of Introduction Lack of proper quarantine measures for the introduced plant materials has resulted in the introduction of some very highly devastative plant pathogens from one country to another and has proved to be catastrophic. This has been resulted into increased cost of food material. Evidence about the entry of disease into new areas is, however, often circumstantial, and it is rarely possible to say with certainly about how a disease has been introduced. A good example is tristeza virus of citrus, originated probably in China, and introduced into South Africa by 1900 and probably at times into the USA, but its presence was marked due to the use of tolerant varieties and the absence of vector (Broadbent 1964). Prior to 1920s, the citrus industry of Argentina and Brazil flourished despite the use of tristeza susceptible root stock, but 20 years after tristezainfected nursery stock was imported from South Africa and Australia, 20 million trees had perished due to the spread of the virus by an abundant local vector Aphis citricida (Costa 1956; 8.30 Important Cases of Introduction Ducharme et al. 1951). In North America and in the Mediterranean region, no disease state existed for decades. A few varieties infected with tristeza were introduced into North America at the end of the nineteenth century (Olson 1955b). In the Mediterranean region, tristeza was introduced in the 1930s (Reichert and Bental 1960), but natural spread was encountered in Spain only in 1960s and in Israel in 1970 (Bar-Joseph et al. 1974). Another example is of Star Ruby isolate of citrus Ring spot virus(CRSV-SR) discovered in a ‘Star Ruby’ grape fruit tree (Citrus paradisi) and was brought into Florida by a private grower from commercial sources in Texas, without authorisation from the State Department of Agriculture. CRSV-SV has not been found in ‘Star Ruby’ trees imported officially for testing and release (Garnsey et al. 1976). The other serious diseases of citrus like psorosis, exocortis, xylopsorosis, greening and stubborn are also proved to be quite dangerous as they were introduced through infected bud wood and nursery stock (Klotz et al. 1982; Knorr 1965). Plum pox virus (Sharka disease) was introduced into England during 1965, through the infected root stocks from Germany (Cropley 1968), and adequate precautions are to be taken in other parts of the world against introducing this disease in plum, peach, apricot and a variety of susceptible ornamental Prunus species. The imported cherry material from European and Asian sources to Canada exhibited virus symptoms, when indexed on commercial cherry, apricot, prunus and peach varieties (Welsh and James 1965). Stevens (1931) states an available evidence which indicates that cranberry false blossom first appeared in Wisconsin, probably as early as 1885, and spread from there to Massachusetts, New Jersey and Oregon in shipments of infected vines. Early spread of the disease was largely by diseased plants, but as the disease became established, its spread from plant to plant by the natural vector, Euscelis striatulus, increased in the eastern states. The disease has apparently been of little importance in the Pacific Northwest, possibly largely due to scarcity or absence of vectors. There has been little or no extension of infected area in the United States in the past 25– 247 30 years. Experts convened by EPPO in 1963, made surveys to estimate the situations of viruses of deciduous tree fruits and small fruits, which were already widespread or locally established in Europe and those not known to be present within the area (Granhall 1964). There is circumstantial evidence, too, that important virus diseases of raspberry and the strawberry might have been introduced into the USA when tolerant varieties (perhaps infected with the disease) were brought in around 1920. Banana bunchy top, which is threatening the banana industry in India, has been introduced from Ceylon through the infected suckers during 1940 (Vasudeva 1959). The Fiji disease of sugarcane is quite serious in Fiji islands, and from there, this disease is introduced to Madagascar and to other countries (Kahn 1989; Kahn and Mathur 1999). Potato spindle tuber viroid has its home in North America and was introduced into Russia and Poland from N. America and barred on to Rhodesia. The infected plants of Abutilon striatum Dickson var. thompsonii with Abutilon infectious variegation virus show attractive leaf variegation and hence were brought into Europe from Brazil more than 100 years ago (Hollings and Stone 1979). Circumstantial evidence suggests that Tomato aspermy virus in chrysanthemum may have been introduced into the USA with chrysanthemum imports from Japan (Brierley 1958) or from Europe in the early 1950s. Certain new cultivars of pelargonium have been imported into the United Kingdom from the USA in recent years, and a number of them have been infected with Tomato ring spot virus (Hollings and Stone 1979). According to Leppik (1964), Squash mosaic virus was introduced into the USA by the seed from Iran and disseminated in Iowa by cucumber beetles. After several years of intensive work, this disease has been eradicated. From the USA, the same virus has been introduced into New Zealand through the seed of Honey dew rock melon plants (Thomas 1973). In Great Britain, Barley stripe mosaic virus has been isolated only twice in barley seed imported from France (Kassanis and Slykhuis 1959; Watson 1959) and from Hordeum zeocriton seed imported from Denmark (Catherall 1972). Many more examples can be cited where interna- 248 8 Methods of Combating Seed-Transmitted Virus Diseases tional movement of planting material has helped in distributing pathogens from one part of the world to the other. Besides the infected plant material, in some cases, the insects which are acting as vectors were also reported with the early free movement of plant material and also which in due course were established and became potent vectors. For example, Aphis citricola (Dspiricola) has recently been introduced to the Mediterranean region and may also have come from Spanish and Portuguese overseas territories, although now widespread (Reichert 1959). Macrosiphum euphorbiae was apparently not present in Britain before 1917 and may have been introduced to Southern Europe from America some time before that could have been a direct introduction from North America on new varieties of potatoes. It is now thought that the disease and the vector, the leaf hopper Circulifer tenellus may have been introduced together to California on live plant material used as a animal fodder on ships sailing round Cape Horn during the ‘Gold Rush’ era (Bennett and Tanrisever 1957). The interception of known insect vectors of plant viruses that depend solely on specific insects for spread may prevent the establishment of the viruses, provided no indigenous species are capable of serving in a vector capacity. The best way of restricting the vector entry is by defoliating the plants to be imported and treating with some systemic insecticide, before its dispatch. seeds (Konate et al. 2001). In citrus some of the virus diseases which are emerging are at present confined to one or two countries, and some of the diseases mentioned below are infectious and might spread, if introduced into new areas. They are bud-union crease in Argentina, abnormal budunion problems in trees on rough lemon in South Africa, brittle twig yellows in Iran, decline of rangpur lime in Brazil, Dweet mottle and yellow vein in California, gum pocket disease of trees on Poncirus trifoliata in Argentina and South Africa, gummy bark in Egypt, gummy pitting of P. trifoliate in New South Wales, infectious mottling on navel orange in Japan, leaf curl in Brazil, leaf variegation in Spain and Greece, marchitamiento repentino or sudden wilt in Uruguay, the missiones disease in Argentina, multiple sprouting in South Africa and Rhodesia, narrow leaf in Sardinia, small fruit and stunting in Argentina and young tree decline in Florida. There should be thorough indexing against these diseases, whenever the plant material is dispatched from these countries. The need for strictly enforced quarantines in every citrus-producing country is self evident. Plum pox (Sharka disease) is at the moment confined to Europe, and adequate precautions are taken in other parts of the world against introducing this disease in plum, peach, apricot and a variety of susceptible ornamental Prunus species (Matheys 1975; Capote et al. 2006). Cocao swollen shoot virus and Cocao necrosis virus are restricted to West Africa Ghana and Nigeria. The plants belonging to the families Sterculiaceae, Bombacaceae, and Tiliaceae are potential carriers of swollen shoot, and their export should be strictly controlled. Abaca mosaic virus of banana is restricted to the Philippines. Yellow dwarf of potato is known only from North America. Cadang-cadang and coconut root wilt diseases are restricted to the Philippines and India, respectively. Another serious disease of coconut, lethal yellowing (Kaincope), is limited to Jamaica, Cuba, Cayman Islands, Bahamas, Haiti, West Africa and Florida. The movement of cassava vegetative material from Africa to American continent should be banned to avoid the introduction of Cassava 8.31 Important Diseases Restricted to Some Countries Many diseases have limited distribution and have been reported from only one or two countries. US plant quarantine regulations (1977) prohibit importation of plants and plant parts from specified countries because of the occurrence in these countries of pests and pathogens of quarantine significance to the United States. Rice yellow mottle virus is confined to some of the African countries only and the virus was found in the fresh harvested seeds, but the seed transmission of the virus was not noticed in the dried rice 8.32 Effective Methods of Plant Importations mosaic in the western hemisphere. An additional risk would be the introduction of whitefly vector (B. tabaci) races better adopted to breed on cassava than those existing in America. 8.32 Effective Methods of Plant Importations 8.32.1 Phytosanitary Certificates The risk of introducing the pathogen can be checked by phytosanitary certificates for exporting or importing the plant materials, and permits in the form of phytosanitary certificates issued by the quarantines in relation to its health are highly useful in restricting the entry of the diseased material into new area. It is understood that when the certificate is issued with the importing country policies and regulations, the plant materials are to be healthy. These phytosanitary certificates, the imported material may be liable to be destroyed at the port of entry of the importing country, as per the international agreement. ‘Certified’ refers that the plant material is free from virus and virus-like diseases and also from other pests and diseases. In some of the advanced countries, there are approved certified nurseries. For example, Canada and United States accept certified Prunus, Cydonia and Malus nursery stock from the certified nurseries in the UK, France, Belgium, the Netherlands and West Germany. Similarly, the East African Plant Quarantine Station at Kenya accepts the certification of chrysanthemums by the Nuclear Stock Association in the UK and the certification of grapevine by the Foundation Plant Materials Service of the University of California. These certificates are issued by the central and state governments after careful examination of the material. Although the variety of phytosanitary certificates varies from country to country, depending on their competence of their regulatory agency, their use has reduced the movement of plant diseases between countries. For evaluating the seed health, there are some international certificates like orange certificate, blue certificate, Rome certificate, etc. The Orange 249 certificate has international standard in testifying the quality of the seed for trade transactions which is issued after examining the seed lots according to the prescriptions laid down in the international rules of seed testing (ISTA 1966), whereas the Blue certificate is issued for samples which have not been drawn officially. The Rome certificate has the international standard, set up in accordance with FAO model phytosanitary certificate. These phytosanitary certificates are issued to the plant material which does not show any external symptoms and also tested by mechanical/graft transmission to the indicator hosts. It is also confirmed by using the other techniques like histopathology, serology and electron microscopy. In some of the virus–host combinations, visual observations and indexing do not help in complete restriction of diseased material. Generally, quarantine inspectors depend on symptoms to detect the presence of viruses. However, symptoms alone are totally unreliable if they are not diagnostic and if the plant is infected only recently and incubation period is incomplete. In some cases, the diseases will be latent. Since three decades the molecular diagnostic tests are followed for screening the plant materials at the quarantine stations. 8.32.2 Closed Quarantines Because of latent or symptomless behaviour of the virus in a particular consignment, certain serious diseases have escaped inspection in the country of origin. All these imported plant materials should be grown in secured glasshouses in a controlled atmosphere to avoid external contamination and tested for virus. This is known as closed quarantines. From the small consignment of plant material accepted for closed quarantine, a few should be grown, and all plants should be inspected daily. If necessary, propagations derived from the indexed mother plants or seeds from the second crop should be released. If suspicious symptoms are observed and the casual virus is determined to be one not yet established within the country, the safest procedure is the 250 8 Methods of Combating Seed-Transmitted Virus Diseases destruction of the entire consignments in the station incinerator. If the crop is very important, by means of thermotherapy and tissue culture, the plants can be raised, and the healthy plant material can be handed over to consignee, and the details are discussed in this chapter. While working in glasshouses, the precautions like the treatment of the floor of the entrance cubicle with a disinfectant, using gumboots and laboratory coats within the glasshouse unit, using sterilised soil for raising plants, washing hands and instruments with detergents, maintaining distance between plants and use of partition screens to avoid contact transmission, etc. are to be followed. The principles of the closed quarantine procedures have been described by Sheffield (1968). This type of plant quarantine stations are at, Maguga, Kenya, the Post-entry Plant Quarantine Station Ibadan, Nigeria and US Plant Introduction Station, Glenn Dale, Maryland. 8.32.4 Open Quarantine 8.32.3 Quantity of Plant Materials Examination of the plants before harvest under field conditions will help in detecting and assessing the incidence of some of the virus diseases but, once again, depends upon the virus, virus combinations and environment. For example, with annual imports of flower bulbs from Holland, US quarantine inspectors, following previous agreement between the parties involved, visit and inspect the flower fields in Holland. If they find the fields to be diseasefree, they issue inspection certificate allowing the import of such bulbs into the USA without further tests. The USA annually imports large quantities of vegetables from Mexico, and arrangements were made for the USA and Mexican personnel to conduct field surveys in the vegetable production areas of Mexico. Such cooperative field inspections during growth season at points of origin provide greater protection than checking at ports of entry. The growing season inspection is generally more sensitive than pre-export inspection of planting material or produce. This method will be very effective wherever the vegetatively propagated materials are exported. Stover (1997) described an effective method of exporting bananas for The risk of introducing the pathogen and pest can be minimised by reduced quantity of a given variety or clone. For vegetative propagative materials, small quantities are to be considered like 5–10 roots, tubers or corms; 50 buds, 20 unrooted cuttings or 10 rooted cuttings. In case of seeds, quantity required for sowing a 10-m row will do the purpose. Initially, these small quantities of planting materials should be maintained, and it should be free from all destructive pests and diseases. After confirmation as healthy material, it can be tested in the field. The vegetative propagative materials should be defoliated, which will avoid the entry of vectors, which are colonised on leaves. The plant material should be examined for the eggs and different stages of vector and should be dusted or sprayed with the insecticide before packing for exporting. The majority of vectors can be controlled by methyl bromide fumigation. It should be sent in unused cloth or paper bags or envelopes. The importers or the exporters of germplasm should ascertain well in advance the requirements of the importing country for avoiding unnecessary delay at quarantine stations. This is the quarantine of plants without using such physical confinement structures as glasshouses or screenhouses. This can be used to reduce or eliminate the risk of spread of pests by adhering to a quarantine protocol. The technique has been used successfully in East and Central Africa to exchange germplasm resistant to EACMV-UG. Open quarantine has facilitated safe introduction of large quantities of cassava germplasm, which would not have been possible through other means. The method was cheaper than micropropagation, and plant mortality was also low. This method can be used in germplasm exchange programmes where the climate and pest species are more or less similar. 8.32.5 Examination of Exportable Crops During Active Growth 8.32 Effective Methods of Plant Importations commercial or research purposes. The rhizomes should be taken from a disease-free area, at least for 1 year. They should be pealed free of all root stubs, until only white tissue remains. Later, they should be submerged in hot water at 54ı C for 10 min. The cartons having the suckers should be shipped air freight with the required phytosanitary documents at temperature above 14ı C. After receiving the rhizomes at quarantine stations, the plants should be raised for nearly 1 year, and later, they can be released. 8.32.6 The Intermediate Quarantine The genes for resistance to virus and virus-like diseases are likely to be found in regions where the plant in question originated (primary gene centres) or has subsequently been grown (secondary and tertiary gene centres). It is in such places that long association between pathogens and plants has occurred with consequent elimination of susceptible plant genotypes through natural selection. Hence, resistance material is generally searched for in what are thought to be as the gene centres of cultivated plants. While collecting the sources of resistance from the gene centres, one should not inadvertently introduce genes for virulence by collecting the new forms of the horizontal and vertical pathotypes. As discussed earlier, many virus diseases occur latent, without expressing any visual external symptoms. The risk of introducing the new pathogens, either from the gene centres or from exporting country, can be reduced by transferring a genetic stock to a third country instead of sending directly, where that crop is not grown and the pathogen would not establish there. Thirdcountry or intermediate quarantine is an international cooperative effort to lower the risk to country B associated with transferring genetic stocks from country A by passing these stocks through isolation or quarantine in country C. The plants are maintained in country C to test for obscure pathogens, or they are detained to allow incipient infections to surface or to permit treatment at a weak point in the life cycle of hazardous organisms. The salient feature of 251 this type of safeguard is that it advocates the passage through country C only of genera that pose no threat to country C because (1) the crop is not grown in country C, (2) the harmful organisms of that crop have narrow host ranges so they will not attack other crops of country C, (3) the harmful organisms that may gain entry to country C would not become established because susceptible hosts are absent or the climate is unfavourable. The third-country quarantine locations include the plant quarantine facility at Glenn Dale, Md; the US Sub-tropical Horticultural Research Unit, Miami; Kew Gardens, UK; and Royal Imperial Institute Wageningen and IRAT at Nogent-Sur-Marne, France. For example, the export of cacao propagating material primarily from West Africa to the other cacao-growing countries is first quarantined at an intermediate quarantine station of third-country quarantine. Facilities may exist within the tropics reasonably far from growing cacao as at Mayaguez in Puerto Rico or Salvador in Bahia, but safety usually required intermediate quarantine in a temperate country, for instance, at Kew, Glenn Dale; Miami or Wageningen. Similarly, sugarcane sets entering East Africa are first put into quarantine at the East African Agriculture and Forestry Research Organisation (EAAFRO) at Maguga to see if they carry viruses. 8.32.7 Aseptic Plantlet Culture Another safeguard in the international exchange of germplasm is to import only plantlets established as aseptic cultures. The required plant materials which are proved virus-free after indexing can be developed by tissue culture technique and can be sent to the other country without any hazards. As the size of the consignment will be small and in aseptic conditions, there will not be any chance of reinfection with other pathogens like fungi, bacteria and also with other pests like insects, mites and nematodes. In this technique, an imported variety or clone may be represented by meristem tips or exercised buds or embryos instead of several cuttings, scions, tubers, seeds, etc. The tissue culture technique in combination 252 8 Methods of Combating Seed-Transmitted Virus Diseases with thermotherapy or chemotherapy or both has been used in the production of virus or viruslike disease-free planting material of number of horticultural crops. During 2009, Wang et al. have developed cryotherapy technique and produced virus-free plants of citrus, grapes, Prunus spp. and certain tuber crops like potato and sweet potato. Prior to the establishment of tissue cultures, the mother plants were indexed and tested serologically for viruses. The use of this technique was first reported by Kahn (1976) for Asparagus officinalis L. In 1972, asparagus clones from France were indexed for viruses at Glenn Dale. From the plants that indexed negatively for viruses, aseptic plantlets were developed on agar medium in screw cap bottles and were shipped to Kenya. Roca et al. (1978) shipped 340 aseptic cultures of Solanum germplasm from the International Potato Center (CIP) in Lima, Peru, to the countries like Australia, Bolivia, Brazil, Canada, Colombia, Costa Rica, India, Indonesia, Kenya, Mexico, the Philippines, Turkey, the United Kingdom and the United States. The clones were successfully established in 12 of the 14 countries. At CIP, multiple shoots in tissue culture were produced in shake cultures. Plantlets of potato regenerated from nodal cuttings of multimeristem shoots were then shipped in culture tubes from CIP to corresponding countries (Roca et al. 1979). While developing the aseptic cultures, heat treatment is also employed for plant materials in which viruses were not easily eliminated. Waterworth and Kahn (1978) developed sugarcane plantlets of three varieties that indexed negatively for Sugarcane mosaic virus (SCMV) by hot water treatment and aseptic bud culture. The hot water treatment was three sequential exposures of cuttings at 24-h intervals for 20 min at 52, 57 and 57ı C. The SCMVinfected canes were sent from the East African Plant Quarantine Station, Kenya, to Glenn Dale where they were subjected to heat therapy followed by bud culture. The aseptic plantlets were returned to Kenya in screw cap bottles for transplanting and indexing (Kahn 1976, 1977). Aseptic cultures were also developed against crops like ginger, chrysanthemum, sweet potato and banana and used for exchanging virus-free genetic stocks between countries. This technique is also useful to the germplasm explorers, where they can collect the required perishable plant materials right in the field. They have to carry tubes having tissue culture media, and after thorough disinfection of the plant material, they can be transferred to the medium, with a higher risk contamination. 8.32.8 Embryo Culture As some of the viruses are internally seed transmitted, countries which have a zero tolerance against the seed-transmitted pathogens have prohibited the entry of the seed from other countries where these specified organisms are known to exist. For commercial purposes, the imports are strictly prohibited; however, a very small quantities are permitted for scientific purposes. Kahn (1979) developed during 1970–1972 an embryo culture technique by using tissue culture methodology. In this technique, instead of complete seed, only embryo axes were exercised and used for culturing, and it can be mailed without any risk. This technique is also useful for germplasm explorers. Guzman and Manuel (1975) used this technique for coconut plant material. Embryos collected in large numbers in diseased area can be maintained in laboratory culture for 16 weeks and grown in screened glasshouses for some months before they need to be planted. If no virus, phytoplasma, viroid and other pathogens are found, then adequate samples were screened by standard methods, it could be considered safe to plant embryo cultured seedlings of coconut. Braverman (1975) modified this technique so as to include one microbiological test and virus indexing. As a safeguard against the breaking of agar in the glass bottles or tubes during transit, it is desirable to add warm sterile agar before shipping, until the container is almost full. The plantlets are submerged in agar, but they ship well and usually arrive in excellent condition with the agar remaining undisturbed. 8.33 General Principles for the Overall Effectiveness of Quarantines 8.32.9 Use of Shoot Tip Grafting or Micrografting Micrografting is another technique which provides a means whereby plants can be transported from one country to another. The culturing of lateral buds in vitro to induce multiple shoots may have potentially important applications. This method is proposed by Navarro et al. (1975). Bud wood could be fumigated and shipped in test tubes or in a sealed container, and the lateral buds excised in the receiving country and cultured in vitro for production of multiple flushes, whose shoot tips then be used for grafting. The method involves holding the previously trimmed shoot with tweezers under the microscope, and the very tip end of the growing bud (0.88 mm) is removed with a razor knife blade and quickly transferred to the cut edge of the inverted ‘T’ on the seedlings. The resulting graft plants after 3–5 weeks could then be indexed for a broad spectrum of pathogens in a special quarantine facility and destroyed if pathogens were found. This should pose no serious problems with quarantine regulations to the country of origin or the country processing the bud wood, since all the bud wood, bud or explants would be under continuous cover. This method also helps in exchanging of superior cultivars. 8.33 General Principles for the Overall Effectiveness of Quarantines Here details of quarantine regulations are not included as they vary from one country to another, but a general set of principles contains the basic guidelines: 1. Seed rather than vegetative material should be introduced unless clonal propagation is necessary. 2. For clonal propagations, non-rooted propagative material such as scions or cuttings should take precedence over rooted plants. 3. Woody plant introductions should not be more than 2 years old. 253 4. Consignments of vegetatively propagated material should be small; that is, each variety or species should be presented by a few tubers, scions or cuttings. 5. A stock plant should not be reused for propagation if a foreign bud or scion failed to survive on the stock. Bud or graft union failures may be caused by pathogens such as viruses transmitted from the introduction to the stock. 6. It should never be assumed that all vegetative propagations of a given species or variety were derived from the same mother plant. 7. Each scion, cutting or tuber of a clonal introduction should be considered as a sub-clone. 8. When pest or pathogen detection tests indicate that a particular sub-clone is eligible for release from quarantine, propagations for release should come only from the sub-clone that was tested and not from other sub-clones that were not tested, even though these subclones constitute part of the original accession. 9. If introductions are received as roots, such as sweet potato, cuttings derived from the roots should be released rather than the original root itself, which should be destroyed. 10. Visual observation is not satisfactory for diagnosing virus diseases because neither the presence nor absence of virus-like symptoms is necessarily indicative of the presence or absence of virus. Besides the above ten principles, the following four types of quarantine actions will mostly restrict the entry of the diseases: 1. The consignment should be followed by embargo and airport permit. 2. Inspection (field inspection, laboratory tests, etc.) in the exporting country before shipment of the consignment and to be on the safe side the material must be given chemo- and thermotherapy. 3. Post-entry growth inspection of the importing country in closed quarantine. 4. Certification – phytosanitary certificate attested for freedom from disease and pests. 254 8.34 8 Quarantine Facilities The type of quarantine facilities depends on the climate at the introduction station, the crops and its temperature requirements, pest and pathogen risks and duration of the quarantine period as it affects plant size. Features that can be incorporated into a greenhouse to improve phytosanitation and thus facilitate quarantine include a series of small glasshouses, air conditioning, filtered air, humidity control, concrete floor with drains, partition screens, soil sterilisation, fumigation chambers, etc. Other features are pathogen-free water supply, heat therapy unit, tissue culture rooms, hot water treatment facilities, black-light insect traps, shoe disinfectants and fungicide and insecticide spray programmes. Big and well-equipped quarantine stations are started whenever a country is both agriculturally and scientifically well advanced, whereas less advanced countries will have small stations of nonexistent. A sufficient number of adequately trained and experienced inspectors are required for the effective examination of incoming plant material at airports, railway stations, seaports and frontier parts. 8.35 Need for Networking for the Developing Countries Networking within and between the subregions, and with institutes in developed countries and international agricultural research centres (IARCs), is critical to strengthen plant virus research in the world. There are numerous advantages of such international networking (Plucknett and Smith 1984). Networking is a rapidly growing mechanism, as funding becomes increasingly scarce, to optimise resource utilisation and to facilitate the efficient transfer of technologies to developing countries. It is in developing countries where the impact of these technologies can be felt almost immediately if the resources are available to utilise them. A good example of such an effort was the collaborative project on identification of cowpea viruses coordinated by the International Institute of Tropical Agriculture (IITA) Methods of Combating Seed-Transmitted Virus Diseases with monoclonal antibodies provided by, and financial support from, the International Development Research Centre (IDRC), Canada (Thottappilly et al. 1993). Many existing networks are crop based and are often divisive from the point of view of the virology community because the networks rarely link together. The networks such as the Cassava Biotechnology Network (CBN), ProMusa, East Africa Root Crops Research Network (EARRNET) and the Southern Africa Root Crops Research Network (SARRNET) take a big step in the right direction. However, until capacity and confidence are increased, many of the virologists from developing countries are unable to participate fully and gain maximum benefit from the interactions. The organisation of working group meetings at regular intervals, either at the regional or subregional level, is an ideal way to promote the networking concept. These meetings would involve scientists from all over the world who are working on the plant virology problems of their country. It would enable priority constraints to be identified and research needs of national programmes to be determined. A framework for collaborative research programmes in partnership with IARCs and research institutes in advanced countries must be developed and sustainable funding obtained. Sustainability of funding must be considered a key issue that must be addressed. Working groups on crop-specific virus disease problems have already been developed, for example, the International Working Group on Groundnut Viruses in Africa and the Virology Working Group of ProMusa. However, a formal network must be developed for the upcoming countries which these collaborative initiatives and other virus-specific subgroups can interact. This will ensure better coordination of efforts and synergy between different partners and stakeholders in addressing a broad range of plant virology needs of the national programmes. However, it is vital that any initiative that is taken regarding networks and collaboration is assured of funding for long enough for the activities to be self-sustaining. Any plant virology network must be endowed with sufficient funds to have a dynamic coordinator and secretariat with au- 8.36 Perspectives thority (perhaps under the auspices of the steering committee of the network) to initiate appropriate research and donor contacts. Regional and subregional organisations should take an active role in encouraging and supporting such initiatives. More information can be also obtained from the chapters in edited books and also review articles on plant quarantines (Chiarappa 1981; Kahn 1989; Ebbels 2003; Khetarpal et al. 2004). 8.36 Perspectives Detection and diagnosis of viruses are crucial for trade and for exchange of germplasm. But the level of confidence, knowledge and accuracy on the part of the workers needs to be improved for precise detection and diagnosis. Training is required for quarantine officials, germplasm curators and scientists who are involved in assessing the conformity to the international standards especially in developing and least developed countries. There is also a need to seek technical assistance of international agencies in the area of human resource development. Compared to the other pest detection techniques, virus indexing requires technically more advanced equipment, reagents, etc. Accreditation of few wellestablished laboratories would offer a reasonable solution for reduction of costs. India has limited laboratories, which are well equipped and have trained experts to deal with diagnosis of plant viruses. Such laboratories need to be accredited on priority to enhance their credibility at international level. At NBPGR, the legume germplasm was found to be infected by 29 viruses when tested by ELISA and, in some cases, by electron microscopy and RT-PCR. Jones (1987) emphasises the need for disease-free seed stocks in germplasm collections to enable countries with less sophisticated quarantine systems to import disease-free seeds, intended for improving crop production and not for introducing new pathological problems. However, this is not a problem in case of bulk consignments with the trade being under the gamut of Agreement on Application of Sanitary and 255 Phytosanitary Measures of WTO, wherein the consignments can be justifiably rejected, if the exporting country does not follow the importing country’s requirements. Strict regulatory measures together with growing new introductions under containment or isolation and collection of seeds from only virusfree plants must be followed to eliminate the risk of introducing seed-transmitted viruses/their strains if any. Conservation of virus-free seeds of a crop in gene banks will minimise the spread of ‘germplasm-borne’ viruses as these materials are multiplied and exchanged worldwide. The countries should take note of a series of technical guidelines for the safe movement of germplasm prepared by FAO/Bioversity International (formerly IPGRI), Rome (Frison and Diekmann 1998). Database on all seed-transmitted viruses, including information on host range, geographical distribution and strains, should be made available for its use as a ready reckoner by the quarantine personnel. There is a need to have antisera for all the seed-transmitted viruses in the quarantine laboratories to facilitate the interception of exotic viruses and their strains. Information access and exchange on diagnostics of plant viruses and quarantine are crucial for effective working. Establishment of a National Diagnostic Network for Plant Viruses including antisera bank, database of primers, seeds of indicator hosts, national DNA bank of plant viruses, lateral flow strips/dip sticks which can detect multiple viruses, microarray technology, DNA barcoding and, ultimately, a national biosecurity chip for diagnosis of all current threats to crop plants would be the backbone for strengthening the programme on plant quarantine. The National Diagnostic Network for Plant Viruses, if established, can be a storehouse of information on biology of plant viruses, diagnostic procedures and policies, international standards and related issues. Also Regional Working Groups of Experts for Detection and Identification of Plant Viruses thus need to be formed to explore future cooperation in terms of sharing of expertise and facilities, for example, in South Asia where the borders are contiguous. This would help in avoiding the 256 8 Methods of Combating Seed-Transmitted Virus Diseases introduction of plant viruses not known in the region and also the movement of plant viruses within the region. The importance of quarantine has increased manifold in the WTO regime and adopting not only the appropriate technique but also the right strategy for plant virus detection and diagnosis would go a long way in ensuring virus-free trade and exchange of germplasm, and is considered the best strategy for preventing transboundary movement of plant viruses. the danger of bringing in infected or infested plant materials from abroad through advertising leaflets, posters and broadcasting on radio. Unless quarantine regulations are scientifically sound and administratively feasible, they cannot be successful and may cause serious political and economic problems. On the other hand, there must be adequate legal authority and appropriate enforcement in prosecuting wilful violators. The enforcement of quarantines requires considerable expenditure of money, much interference in trade travel and other normal activities of man. The international cooperation and strengthening regional organisations are of paramount importance in attaining the objectives of plant quarantine. 8.37 Conclusion The foregoing discussion clearly stresses the need for effective quarantines. The expenses incurred in establishing and maintaining the quarantines are but a fraction of the economic losses that would be suffered, if plant diseases gain freely entry into the country. The success of the plant quarantine measures mainly depends on the proper composition of the state service of plant quarantine, the scientific background and the qualifications of the inspectors and the specialists and the availability of necessary equipments at the quarantine stations and laboratories. The post-entry quarantine service for all imported seed must be established, so that it can produce and distribute pathogen-free seed derived from the imported infected material. It will also be useful if world distribution of seedtransmitted pathogens is mapped. The safest source of the healthy materials should be from a country with efficient quarantine services which has talented staff who can diagnose the indigenous pests and diseases. There is a vital need for public awareness of the importance of quarantine regulations. It is well recognised by quarantine officials that success of a quarantine programme is dependent on cooperation between government agencies and the public. Generally, the public will co-understood. Quarantine action is better received and followed if it is based on cooperation between concerned groups rather than on a unilateral compulsion. In this age of rapid and greatly expanded movement of people and cargoes by air, it is extremely important to alert the public by all means to 8.38 Biotechnology and Virus-Derived Resistance Engineered resistance to viruses includes virusderived resistance, reviewed by Beachy (1997), and several other approaches which are being explored (Robaglia and Tepfer 1996; Varma et al. 2002). In some of the crops, partial success has been achieved in dealing the aspects of virus-derived resistance which have already been applied or are going to be applied for controlling seedtransmitted viruses. The concept postulates that the expression of a viral gene in a host can interfere with host–virus interactions, thus rendering the host resistant. Virus-derived resistance can include a variety of different viral gene sequences, most of which interfere at different points in the replication cycle. Two dates are important in the history of this approach: • The first capsid protein-mediated resistance was described in 1986: tobacco TMV. • The first commercial sale of virus-resistant transgenic crop in the USA in 1995: squash WMV2 C ZYMV sold by Asgrow Seed Company. Strategies for virus-derived resistance are divided into those that require the production of proteins and those that require only the 8.39 Molecular Approaches accumulation of viral nucleic acid sequences. In general, the former confers resistance to a broader range of virus strains and viruses, and the latter provides very high levels of resistance to a specific virus strain. The future challenge for scientists is to develop strategies that broaden the effect and increase the degree of resistance. 8.38.1 Capsid Protein-Mediated Resistance Capsid protein-mediated resistance can provide either broad or narrow protection. The CP of TMV provided effective levels of resistance to closely related strains of TMV and decreasing levels to tobamoviruses that share less CP sequence similarity. Apparently CP interferes with the disassembly of TMV, thereby preventing infection. Certain mutants of TMV CP can confer much greater levels of resistance than wild-type CP. Other viral proteins can similarly confer resistance, for example, replicase interrupted by an insertion sequence element produced high levels of resistance in tobacco plants to a wide range of tobamoviruses (Donson et al. 1993). For potyviruses, wild-type CP was not efficient, while CP deleted of its N-terminal part conferred capsid protein-mediated resistance. However, heterologous protection to another potyvirus has also been observed (Dinant et al. 1993). Nucleic Acid-Mediated Resistance: The most widely studied example of nucleic acid-mediated resistance is referred to as RNA suppression, a phenomenon that describes the targeted posttranscriptional destruction of RNA sequences (Baulcombe 1996). A high correlation exists between RNAmediated resistance and multiple copies of gene inserts. The cell detects abnormally elevated levels of RNA sequences and activates a destruction mechanism that involves nuclease destruction of the gene transcript and of viral RNA. Levels of RNA-mediated resistance may be high; it is effective only against genomes that contain sequences that are similar or identical to the transgene. 257 8.39 Molecular Approaches Molecular approaches have high level of sensitivity and discrimination over classical techniques. The nucleic acid-based methods do not depend on the metabolic state of the virus or the state of gene expression as both coding and noncoding regions of the genome (Torrance 1992; Matthews 1993; Hull 2004; Webster et al. 2004). The applications of molecular tools to detect the seed-transmitted virus diseases are relatively recent, and this method is useful for detection and strain determination of plant viruses and viruslike diseases. High sensitivity and specificity are the major goals of virus detection, and it is used in quarantine programmes. Rapid advances in the techniques of molecular biology have resulted in the cloning and sequence analysis of the genomic components of a number of plant viruses. Basic molecular approaches are used for detection and diagnosis of plant virus type and molecular sizes of the virion-associated nucleic acids, cleavage pattern of viral DNA or cDNA, hybridisation between nucleic acids and polymerase chain reaction (Hull 2002). Recent progress in biotechnology and the availability of novel techniques in molecular biology and genetic engineering now makes it possible to consider new approaches to plant virus disease management. It will involve genetic engineering techniques to introduce specific genes into plants or genetic codes, not only from plants but also from the viral genome itself. 8.39.1 Molecular Interactions of Seed-Transmitted Viruses Molecular mechanism of few viruses has illustrated full length, and infectious cDNA clones are available. Viral genes or products influence transmission mainly by regulating the regulation capacity and their movement in the reproductive tissues of infected hosts, though participation of host factors cannot be ruled out. Several host determinants participate in seed transmission. Pseudorecombinants of few bi- or 258 8 Methods of Combating Seed-Transmitted Virus Diseases multipartite RNA viruses have been studied. RNA 1 component of both CMV with a tripartite genome and some of the nepoviruses have been shown the determinant of seed transmissibility. Though pseudo-recombinants have lower seed transmissibility, other minor determinants are also suspected in reducing the rate of virus transmission. Helper component protease (HcPro), a non-structural gene in potyviruses is involved in aphid-mediated transmission, replication and long-distance translocation of virus. Hc-Pro regulates replication and movement of PSbMV in pea, thus influencing invasion of the embryo. Pea early browning virus, a tobravirus (PEBV), has 12 k gene resembles to the Hc-Pro of a potyvirus and RNA ” gene of a hordeivirus. 12 k gene influences the virus transmission to the gametes of the pea cultivars. BSMV contains RNA’, RNA“ and RNA”, and major seed determinants are located in 50 UTR and a 369-nt repeat present in the ”a and ”b genes. ”b genes of hordeiviruses share cystein-rich domains that regulate transmission, replication and movement of the virus in the embryo (Khan and Dijkstra 2007). (2009). Some of the approaches that are in use include antisense RNA, satellite RNA, coat protein genes and specific resistance genes to antiviral activities in plants, etc. The genomic sequences of all the important seed-transmitted viruses mentioned here are now established. The use of viral 2 sequences as transgenes depends now on advance in the technology of transformation of each species. The academic studies on plant transformation have concentrated first on model systems such as members of Solanaceae, tobacco and tomato. Other plants have been more recalcitrant to in vitro manipulation and particularly the legume species: Only soybean and peanut which are rich in proteins and in oil have received much attention in terms of improvement through biotechnology. 8.39.2 Transgenic Approach Transgenic technique is one of the approaches focused on the resistance of transgenic plant against viral infection and suggested this as ‘pathogen-derived resistance (PDR)’. The advantages and setbacks of genetic engineering for crop improvement are clearly exemplified by virus resistances in transgenic plants. Introducing more than one or two transgenes is usually problematic because of ‘transgenic silencing’. Hence, at present, state-of-the-art genetic engineering is short of causing a remedy to all the pathogens of a given crop. It is not surprising that genetic transformation for virus resistance became one of the first approaches of its kind to protect plants against pathogens. Protection of crops against viral pathogens by genetic engineering was reviewed by Lomonossoff (1995), Beachy (1995), Galun and Breiman (1997), Dudhare and Deshmukh (2007), Varma and Praveen Shelly (2007) and Reddy et al. 8.39.2.1 Tomato/TMV and ToMV Engineered protection to TMV and ToMV has already been achieved, improving elite tomato varieties without altering their desirable characteristics. In the first field trial ever done of plants engineered for virus resistance, Nelson et al. (1988) evaluated resistance conferred by transformation with the CP gene of TMV. Transgenic plants displayed nearly complete resistance to mechanical infections by TMV, and only 5% had symptoms at the end of the trial, as compared with 99% of the control. Fruit yield was identical for inoculated transgenic and uninoculated control plants. Sanders et al. (1992) extended the field characterisation of these TMV-resistant tomato plants and found they had a limited ability to protect against ToMV since 56–89% of them were infected with ToMV causing 11–25% yield loss. Tomato plants expressing the CP of ToMV were developed to introduce ToMV resistance. Transgenic line 4174 showed substantial resistance to both tobamoviruses. The evaluation in Japan of the impact of release of transgenic tomatoes on the environment concluded that released transgenic plants could be cultivated in the fields and that resistance was maintained throughout generations (Asakawa et al. 1993). 8.39 Molecular Approaches Tomato was also successfully transformed with the well-known resistance gene N from Nicotiana glutinosa which confers in Nicotiana durable resistance by hypersensitivity to both TMV and ToMV (Whitham et al. 1996). This is a single example of an isolated resistance gene introduced by bioengineering and the demonstration that it is effective in a new biological context. 8.39.2.2 Lettuce/LMV Lettuce transformation has been achieved by Agrobacterium-mediated transfer or by protoplast electroporation in France, Japan and the USA. Transgenic lettuce expressing the CP gene of LMV has been obtained (Dinant et al. 1997). Constitutive expression of the CP gene was demonstrated by ELISA. In 60 plants of the R2 progeny, different phenotypes of protection were observed: 8 plants were completely resistant, whereas 51 plants recovered after a first phase of active multiplication of LMV. A breakdown of recovery was observed in greenhouse growth conditions, leading to a late progression of viral infection in a significant number of plants. Interestingly, both types of resistance were effective towards all strains including the virulent ones. This lack of strain specificity could be due to the high degree of CP similarity between strains. 8.39.2.3 Squash/SqMV Development of the transgenic squash is a significant breakthrough for squash improvement considering the economic importance of ZYMV and WMV-2 and the difficulties in developing resistant cvs by traditional breeding (Fuchs and Gonsalves 1995). Moreover, multiple CP genes enable controlling several aphidtransmitted viruses: Transgenic squash lines expressing the CP genes of ZYMV, WMV-2 and CMV have been developed (Tricoli et al. 1995), and field tests have demonstrated the potential of such transgenic squash in controlling mixed infections by these three viruses. This strategy could thus be extended to the control of SqMV. 259 8.39.2.4 Pepper/PMMoV, TMV and ToMV Transformation of pepper appears as more difficult than transformation of tomato. In view of its transformation for resistance to PMMoV, a preliminary study of the engineered resistance was studied in Nicotiana benthamiana (Tenllado et al. 1995). The gene N would also be a candidate to be introduced, although the only known strain Ob virulent on N genotype was selected from pepper. 8.39.2.5 Soybean/SMC Soybean genetic engineering is the most advanced among all grain legumes, and this reflects the economic importance of the crop in the industrialised world. Since SMV is economically important seed-transmitted disease, coat protein – mediated resistance has been developed by using a non-aphid transmissible isolate of SMV to reduce the risk of spreading new pathogenic strains by recombination (Reverse et al. 1996). Furutani et al. (2007) have reported the virus resistance in transgenic soybean was caused by post transcriptional gene silencing. Intensive researches on transgenics are carried out in majority of the soybean growing countries. 8.39.2.6 Peanut/BCMV (PStV), PeMoV and PCV Two methods have been used successfully for the Agrobacterium-mediated transformation of peanut (McKently et al. 1995) and particle bombardment (Ozias-Akins et al. 1993). Particularly, by bombardment of shoot meristems of mature embryonic axes, the nucleocapsid protein of Tomato spotted wilt virus was introduced into peanut (Brar et al. 1994). Resistance to seed-transmitted viruses is now a realistic target. 8.39.2.7 Bean Biotechnology programmes relating to bean improvement and utilisation have been ongoing in Mexico, Colombia and other countries. For bean too, the particle bombardment appears as the most promising method of gene delivery (Russell 260 8 Methods of Combating Seed-Transmitted Virus Diseases et al. 1993; Kim and Minamikawa 1996; Christou 1997). Transgenic beans expressing the coat protein of a non-seed-transmitted virus, Bean golden mosaic begomovirus, have been obtained (Russell et al. 1993). This first success makes the control of BCMV/BCMNV a realistic target. should thus progressively help in controlling many viruses. Particularly, for seed-transmitted viruses, even an incomplete or a delayed resistance could prevent the transmission of virus through seed that would solve the agricultural problem of the primary inoculum. It is important to mention that, after release of transgenic soybean, a patent issued by the US patent office and by the European patent office to a US Plant Biotechnology Company provided broad patent protection for all transgenic soybean (Lehrman 1994). Such type of right of intellectual property should be revised because it is going to prevent the achievement of programmes, the objectives of which are to satisfy the basic needs of a major part of world population. Genetic modification of plants with viral CP genes is an example of pathogen-derived resistance that has been used successfully to produce viral-resistant plants (Lomonossoff 1995; Beachy 1997). CP-mediated resistance in peas against Pea enation mosaic virus (Chowrira et al. 1998), Pea seed-borne mosaic virus (Jones et al. 1998) and Alfalfa mosaic virus (Timmerman-Vaughan et al. 2001). Transgenic squash resistant to Cucumber mosaic virus, Zucchini yellow mosaic virus and Watermelon mosaic virus was released during 1996 (Tricoli et al. 1995). Resistance to viral infections in plants has been exploited in two ways – first one is resistance gene-dependent responses and second one is pathogen-derived resistance. Out of these two approaches, the pathogen-derived resistance is more appropriate; thus, the creation of crop plants with broad resistance to more number of viruses is a potential area of future research. 8.39.2.8 Pea/PSbMV Production of transgenic pea plants was obtained with difficulty, by Agrobacterium-mediated transfer (Puonti-Kaerlas et al. 1990; Bean et al. 1997). Significant progress has been reported in the last 4 years in pea transformation. However, efficiencies are still low, and methods are variety dependent, an inherent difficulty with Agrobacterium-mediated transfer. Transformation with PSbMV-derived constructs is under study in Europe to complement the multiresistance conferred by three recessive sbm genes. Effective genetic engineering has started, and certain transgenic varieties of crops have been launched on the market. For