Download Seed-borne Plant Virus Diseases

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
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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
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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
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128
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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
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143
144
145
145
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177
178
179
189
189
190
190
191
191
191
192
192
193
194
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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 . . . . . . .
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201
201
203
205
206
207
209
210
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214
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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 . . . . . . . . . . . .
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233
233
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241
243
243
244
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249
249
249
250
250
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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
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258
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266
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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
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Abdel-Salam AM, Amin AH (1990) An Egyptian isolate
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Abdullahi I, Ikotin T, Winter S, Thottappilly G, Atiri GI
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Abou-Elnasr HA, Jones AT, Mayo MA (1985) Detection
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Accotto GP, Boccardo G (1986) The coat protein and
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Adams DB, Kuhn CW (1977) Seed transmission of peanut
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Aftab M, Mughal SM, Ghafoor A (1989) Occurrence and
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Agarwal VK, Nene YL, Beniwal SPS (1977) Detection
of bean common mosaic virus in urdbean (Phaseolus
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Agarwal VK, Nene YL, Beniwal SPS, Verma HS (1979)
Transmission of bean common mosaic virus through
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Agrios GN (2005) Plant pathology. Academic, New York
Ainsworth GC, Ogilvie L (1939) Lettuce mosaic. Ann
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Al-Mabrouk O, Mansour AN (1998) Viruses affecting
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Albrechtsen SE (2006) Testing methods for seed transmitted viruses: principles and protocols. CABI publishing, Oxfordshire/Wallingford, pp 1–268
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Allen WR (1969) Occurrence and seed transmission of
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49:797–799
1
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Allen WR, Davidson TR (1967) Tomato bushy stunt
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Allen WR, Davidson TR, Briscoe MR (1970) Properties
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Allen DJ, Thottappilly G, Rossel HW (1982) Cowpea mottle virus: field resistance and seed transmission in virus tolerant cowpea. Ann Appl Biol
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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
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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.
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of seed borne Black eye cowpea mosaic potyvirus
to disease dynamics and loss of yield. Trop Sci
42:147–152
Puttaraju HR, Prakash HS, Shetty HS (2004a) Seed infection by Black eye cowpea mosaic potyvirus and yield
loss in different cowpea varieties. J Mycol Plant Pathol
34:41–46
Puttaraju HR, Shylaja H, Dharmesh M, Prakash HS,
Shetty HS (2004b) Black eye cowpea mosaic
potyvirus – polyclonal antibody production and its
application in seed health testing. J Mycol Plant Pathol
14:810–815
Rao GP, Gupta AK, Shukla K, Joshi RD (1990) Nature
of yield loss in French Bean (Phaseolus vulgaris L.)
infected with Bean Common Mosaic Virus. Int Natl J
Trop Plant Dis 8:129–135
Ravinder Reddy C, Tonapi VA, Varanarasiappan S, Navi
SS, Jayarajan R (2005a) Studies on seed transmission
of urd bean leaf crinkle virus on Vigna mungo. Indian
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Ravinder Reddy C, Tonapi VA, Varanarasiappan S, Navi
SS, Jayarajan R (2005b) Influence of plant age on
infection and symptomological studies on urd bean
leaf crinkle virus. Int J Agric Sci 1:1–6
Ross JP (1968) Effect of a single and double infections
of soybean mosaic and bean pod mottle viruses on
soybean yield and seed characters. Plant Dis Rep
52:344–348
Ross JP (1969a) Effect of time and sequence of inoculation of soybeans with soybean mosaic and bean
pod mottle viruses on yields and seed characters.
Phytopathology 59:1404–1408
Ross JP (1969b) Pathogenic variation among isolates of
soybean mosaic virus. Phytopathology 59:329–832
Ross JP (1977) Effect of aphid transmitted soybean mosaic virus on yields of closely related resistant and
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Ross JP (1986) Response of early and late-planted soybeans to natural infection by bean pod mottle virus.
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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
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5
Mechanism of Seed Transmission
Abstract
The specific mechanism by which some plant viruses are transmitted
through seed, while others are excluded, is known very little. Genetic tests
designed to identify the viral genes which control seed transmission may
aid in the discovery of this mechanism.
Cytoplasmic connections between the infected mother plant, flower
and the developing seeds influence the seed infection. 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
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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.
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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,
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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). Quarantines must
be strengthened by molecular marker methods to
detect the seed-transmitted viruses and to prevent
new introductions of viruses into a country.
145
6.7
Conclusions
Molecular diagnostic assays should be viewed
as tools to manage plant virus diseases. They
are not designed to be used in a vacuum, but
rather in conjunction with a knowledge of symptomatology, varietal responses to viruses and environmental effects on disease development. As
such, they will play a key role in crop management systems, permitting accurate and early
detection of virus and viroid diseases and help
in more efficient implementation of effective control measures.
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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
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Peanut clump virus and studies on its natural transmission. Ann Appl Biol 97:99–107
Thouvenel JC, Fauquet C (1981) Peanut clump virus. No
235 in Descriptions of plant viruses common Mycol.
Lust. Assoc. Appl. Biol, Kew surry, England 4 pp
Thresh JM (1974) Vector relationships and the development of epidemics: the epidemiology of plant viruses.
Phytopathology 64:1050–1056
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Weed plants as sources of cucumber mosaic virus. Ann
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transmissibility through seeds. Ann Phytopathol Soc
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Udayashankar AC, Nayaka CS, Kumar BH, Mortensen
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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
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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;
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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
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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
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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.
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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.
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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
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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.
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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
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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