K.

Subramanya Sastry

Plant Virus and

Viroid Diseases
in the Tropics
Volume 1: Introduction of Plant Viruses
and Sub-Viral Agents, Classification,
Assessment of Loss, Transmission
and Diagnosis
Plant Virus and Viroid Diseases in the Tropics
K. Subramanya Sastry

Plant Virus and Viroid

Diseases in the Tropics
Volume 1: Introduction of Plant Viruses
and Sub-Viral Agents, Classification,
Assessment of Loss, Transmission
and Diagnosis

123
K. Subramanya Sastry
Department of Virology
SV University
Tirupathi, Andhra Pradesh
India

ISBN 978-94-007-6523-8 ISBN 978-94-007-6524-5 (eBook)

DOI 10.1007/978-94-007-6524-5
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Foreword

The detection of a contagium vivum fluidum associated with a mosaic disease of

tobacco in Europe at the close of the nineteenth century, was the beginning of a
century of major achievements in the advancement of biological sciences. The
demonstration in 1937 that Tobacco mosaic virus (TMV), was a nucleoprotein,
and that its nucleic acid (RNA), contained the genetic information necessary to
induce disease in tobacco, set the stage for the advent of genetics, molecular
biology, transgenic technology, and the use of viruses as molecular tools. The
physicochemical characterization of TMV also lead to the diffusion of modern
technologies, such as virus purification (centrifugation), immunology, electro-
phoresis, electron microscopy, protein and nucleic acid sequencing, and atomic
structure of nucleoproteins (X-ray analysis). These developments would eventu-
ally make a major contribution to the understanding of the structure of DNA by
Watson and Crick in 1953. Finally, these breakthroughs then paved the way to the
advent of Molecular Biology, bringing about the greatest revolution in the multiple
fields of biological sciences.
However, TMV had a humble origin in the lowlands of tropical South America,
where tobacco had been cultivated by the native societies, until the Spanish
conquistadores turned it into a commercial export commodity during colonial
times. In the nineteenth century, tobacco was being widely grown in Europe as a
medicinal plant and, consequently, the stage was set for the emergence of one of
the first global epiphytotics of a highly contagious plant virus. In 1887, Dmitri
Ivanovsky was sent from the University of Saint Petersburg, the imperial capital of
Russia, to investigate a disease affecting tobacco plantations in Ukraine. In 1892,
Ivanovsky demonstrated that the causal agent was not excluded by a porcelain
filter capable of retaining bacteria, the only known microbial pathogen at that time.
In 1898, Martinus Beijerinck confirmed Ivanovsky0 s observations in The Nether-
lands and, thus, the science of Plant Virology was born.
Despite the significant progress made in plant virology in the twentieth century,
the detection and characterization of many plant viruses of economic importance
remained elusive until the 1980s, particularly in the Tropics, where plant virology
facilities were non-existent or very poorly equipped due to the difficult nature of

v
vi Foreword

plant viruses (non-culturable) and lack of the expensive equipments needed to

characterize these pathogens up to that decade. Consequently, the early plant
virologist had to be thoroughly trained in the various fields of the agricultural
sciences: agronomy, genetics, plant breeding, plant physiology, epidemiology,
entomology, and plant pathology, in order to manage the viral diseases of crops,
often without knowing the causal agent. The advent of molecular biology and the
application of molecular techniques, such as the Polymerase Chain Reaction
(PCR), to the detection and characterization of plant viruses possessing RNA or
DNA genomes, completely changed the field of Plant Virology in the 1980s. All of
the sudden, plant virologists only needed partial nucleic acid sequences and a
relatively inexpensive PCR machine to detect and identify plant viruses, without
the need to visualize, purify, conduct serological assays, or undertake lengthy and
complex physicochemical assays to characterize plant viruses. All that was needed
to identify viruses was a suitable pair of primers (a strand of nucleic acid that
serves as a starting point for DNA synthesis) to obtain partial or total viral genome
sequence data to compare to reported viral sequences freely available in databases
such as GenBank.
The adoption of molecular techniques not only facilitated research on plant
viruses, but it also changed agricultural education and research in areas of critical
importance to the science of Plant Virology. Advances in tissue culture techniques,
molecular markers, and the genetic manipulation of plant genomes rapidly shifted
the attention from traditional plant breeding and traditional virus screening tech-
niques to the promise of selection of virus resistant plant genotypes in molecular
biology laboratories using molecular markers. More important, acquiring a basic
knowledge in agricultural sciences was no longer required. Instead, a new gen-
eration of molecular biologists was formed to deal with any phytopathological
problem regardless of the causal organism, be it a fungus, bacterium, or virus.
Thus, the new virologist is usually a molecular biologist who chose to work with
plant viruses, without former training in agricultural sciences.
Whereas the science of Plant Virology has immensely benefited from the
adoption of the new molecular techniques; and conducting plant virus research
without a basic working knowledge of molecular biology is no longer possible or
desirable in this new millennium, the new generation of molecular virologists need
to know the foundations of Plant Virology. Basically, the science of plant
pathology, the agronomy of the plant species affected, and the genetic interaction
of plant viruses with their plant hosts and vectors. Finally, any virologist must
understand how plant viruses are disseminated in nature, and the various control
measures available to manage the viral diseases of economically important food
and industrial crops. Hence, the importance of a comprehensive book like this one
written by Dr. K. Subramanya Sastry, presented in different volumes which
describe the nature of plant viruses and viroids, their classification and identifi-
cation, and the main viral and viroids pathogens that affect food production in
the most challenging and dynamic agricultural system in the world: the Tropics.
Foreword vii

The virus detection techniques described are completely up-to-date, including the
latest molecular techniques developed in the world for the detection and charac-
terization of viruses and viroids in general. The interested readers, professors, and
students of agricultural sciences, and specially plant pathologists, will find this
publication a complete source of information on the science of Plant Virology in
the Tropics.

Francisco J. Morales
Former Head Plant Virology Laboratory
Emeritus Scientist
International Centre for Tropical Agriculture
Palmira, Valle, Columbia
Preface

Virus and viroid diseases have become increasingly important constraints to

sustainable crop production in the tropical countries. The climatic changes that are
occurring throughout the world have impact on plants, vectors, and viruses causing
increasing instability within virus–host ecosystems. Some of the threatening and
economically important virus diseases in tropical zone which affect the food
production are tungro, yellow mottle, and hoja blanca in rice; mosaic in sugarcane,
mosaic in cassava; tristeza in citrus; swollen shoot in cacao; sterility mosaic in
pigeonpea; rosette, clump, and bud necrosis in peanut; necrosis in sunflower and
legumes, vegetables, and ornamental crops; yellow mosaic in legumes; leaf curl in
cotton and tomato; and ring spot in papaya. Key factors for emergence of new
plant virus and virus-like diseases include the intensification of agricultural trade
(globalization), changes in cropping systems (crop diversification), and climate
change.
Largest group of plant viruses exist in the family Potyviridae followed by
Geminiviridae and Bunyaviridae. In tropical countries, whitefly transmitted
begomoviruses are responsible for heavy crop losses in cassava, cotton, tobacco,
tomato, potato, pepper, squash, okra. etc. The tospo- and ilarviruses are wide
spread in tropics and affect several important field, horticultural and ornamental
crops resulting in serious economic damage in crops like groundnut, sunflower,
onion, watermelon, and vegetables like tomato, chillies, and potatoes. Divergence
exists in the type of vectors and their population from country to country, for
example Hemipterans (aphids, whiteflys, leafhoppers, mealybugs, and others) are
the major vectors of plant virus and virus like diseases, comprising more than
80 % of insect-transmitted viruses which represents close to 400 virus species
within 39 different genera.
The primary aim of this book is to provide to readers with latest information on
different virus and viroid diseases of crops in tropical countries. This volume
comprises of five chapters that give an overview of the progress made on virus and
viroid diseases of crops of tropics. The first chapter deals with general information
on tropics and climate, tropical countries and tropical agriculture; second chapter
provides information on viruses, viroids, phytoplasma, and other subviral agents;
third chapter on impact of virus and viroid disease on tropical crops; the fourth
chapter on various modes of transmission of virus and virus-like agents. Various

ix
x Preface

methods for detection and diagnosis of viruses and viroid disease of tropical crops
are extensively reviewed in the fifth chapter.
Since the inception of plant virology, phytoplasma is dealt along with plant
viruses, hence a few pages were devoted in this book for providing background
information about phytoplasma for traditional scientists/researchers. Even though
the attempt is only to include the examples from tropical zone but it was not
possible to confine to tropical examples as successful research outcomes are there
from temperate zone; hence, some examples from temperate zone were also
referred. If any omissions have occurred inadvertently in seeking permissions for
figures and tables, it may please be condoned.
It is hoped that the information provided in this volume on various aspects of
virus and viroid diseases of tropical crops would be useful to research scientists,
seed companies, quarantine personnel, and institutions of both research and
teaching.

K. Subramanya Sastry
Acknowledgments

To bringout the two volumes on ‘‘Plant Virus and Viroid Diseases in the Tropics’’, a
large number of plant virologists of both National and International have immensely
helped. I have been benefited by the critical suggestions and comments made by
Dr. G. P. Rao, Dr. M. Hema, Dr. C. I. Chacko, Prof. Jawaid Khan, Prof. P. Anan-
dakumar, Dr. P. Lavakumar, D. D. R. Reddy, Dr. B. Viswanath, Dr. K. Vemana,
Dr. SK Raj, Dr. K Bikas Mandal, Prof. H. R. Pappu, Dr. R. A. Naidu, Dr. A. M.
Anthony Johnson, Dr. D. C. Sastri, Prof. S. M. H. Khurana, Dr. S. E. Albrechtsen,
Dr. R. Selvarajan, Dr. G. Nagaraja and others. The single person who has taken all the
brunt and hardship in finalizing the manuscript is Prof. P. Sreenivasulu, Former Head
of the Department of Plant Virology, SV University, Tirupathi (India) and I record
appreciation for his sincere hard work and devotion to the plant virology subject. I am
highly indepted to late Prof. M. V. Nayudu, my mentor, who has introduced me to
plant virology subject. I profusely thank my friend Dr. D. V. R. Saigopal, Head of the
Department of Plant Virology, SV University, Tirupathi, for providing me space in
the department, timely guidance, and critically going through all the chapters. I am
highly grateful to Prof. T. A. Zitter and Prof. F. J. Morales for providing suitable
suggestions and modifications for the improvement of the text. I thank Elsevier,
CABI, Springer, and other publishers for providing permission for using the illus-
trations and photographs from their earlier publications. Throughout preparation of
the volume 1, Mr. C. Nagaraja has devoted maximum time in computerizing the book
for which the author is highly indebted. My wife B. N. K. Kumari and my family
members K. Sreedhar, M. Padmavathi, and M. Muralidhar for their ever-lasting love,
for their ceaseless support, and were my constant companions during the course of
preparation of the books. The meticulous care taken by staff of Springer publishers in
bringing out this publication at an early date with nice getup of the book is gratefully
appreciated and acknowledged.
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.

K. Subramanya Sastry

xi
Acronyms

A1MV Alstroemeria mosaic virus

ABMV Azuki bean mosaic virus
AbMV Abaca mosaic potyvirus
AbMV Abutilon mosaic virus
ACLSV Apple chlorotic leaf spot
ACMV African cassava mosaic virus
AGVd Australian grapevine viroid
AMV Alfalfa mosaic virus
APLV Andean potato latent virus
ApMV Apple mosaic virus
ArMV Arabis mosaic virus
ARSV Apple ring spot virus
ASBVd Avocado Sunblotch viroid
ASGV Apple stem grooving virus
ASPV Apple stem pitting virus
ASSVd Apple scar skin viroid
AYRSV Artichoke yellow ring spot virus
BaMMV Barley mild mosaic virus
BaMV Bamboo mosaic virus
BaYMV Barley yellow mosaic virus
BBMV Broad bean mottle virus
BBrMV Banana bract mosaic virus
BBSV Broad bean stain virus
BBTMV Broad bean true mosaic virus
BBTV Banana bunchy top virus
BBWV Broad bean wilt virus
BCaMV Bean calico mosaic virus
BCMNV Bean common mosaic necrotic virus
BCMV Bean common mosaic virus
BCTV Beet curly top virus
BDBV Banana dieback virus
BDMV Bean dwarf mosaic virus
BGMV Bean golden mosaic virus

xiii
xiv Acronyms

BGYMV Bean golden yellow mosaic virus

BICMV Black eye cowpea mosaic virus
BLMV Blue berry leaf mottle virus
BLRV Bean leaf roll virus
BlShV Blueberry Shock Ilarvirus
BMCTV Beet mild curly top virus
BMoV Blackgram mottle virus
BMV Brome mosaic virus
BMYV Beet mild yellowing virus
BNYV Broccoli necrotic yellows virus
BNYVV Beet necrotic yellow vein virus
BPMV Bean pod mottle virus
BRSV Beet ringspot virus
BSGFV Banana streak GF virus
BSMV Barley stripe mosaic virus
BSMV Beet stripe mosaic virus
BSMyV Banana streak Mysore virus
BSOLV Banana streak OL virus
BSUgIV Banana streak Uganda I virus
BSUgLV Banana streak Uganda L virus
BSUgMV Banana streak Uganda M virus
BSV Banana streak virus
BtMV Beet mosaic virus
BWYV Beet western yellows virus
BYDV Barley yellow dwarf virus
BYMV Bean yellow mosaic virus
BYSV Bean yellow stipple virus
BYSV Beet yellows stunt virus
BYV Beet yellows virus
BYVMV Bhendi yellow vein mosaic virus
CABMV Cowpea aphid borne mosaic virus
CaCV Capsicum chlorosis virus
CaMV Cauliflower mosaic virus
CarMV Carnation mottle virus
CBDV Colocasia bobone disease virus
CBMV Common bean mosaic virus
CbMV Calibrachoa mottle virus
CBRV Cabbage black ring virus
CBSV Cassava brown streak virus
CBSUV Cassava brown streak Uganda virus
CbVd-1 Coleus blumei viroid 1
CbVd-2 Coleus blumei viroid 2
Acronyms xv

CCCVd Coconut cadang–cadang viroid

CChMVd Chrysanthemum chlorotic mottle viroid
CCMV Cowpea chlorotic mottle virus
CCSV Cucumber chlorotic spot virus
CCSV Calla lily chlorotic spot virus
CCSV Cassava Colombian symptomless virus
CdMV Cardamom mosaic virus
CeMV Celery mosaic virus
CEVd Citrus exocortis viroid
CFDV Coconut foliar decay virus
CFMMV Cucumber fruit mottle mosaic virus
CFSV Cassava frogskin virus
CGMMV Cucumber green mottle mosaic virus
CGMV Cassava green mottle virus
ChiLCV Chilli leaf curl virus
CIBV Cassava ivorian bacilliform virus
CiLV Citrus leprosis virus
CiMV Citrus mosaic virus
CiTLV Citrus tatter leaf virus
CIVV Citrus infectious variegation virus
CLCrV Cotton leaf crumple virus
CLCuAV Cotton leaf curl Allahabad virus
CLCuBV Cotton leaf curl Bangalore virus
CLCuBuV Cotton leaf curl Burewala virus
CLCuKV Cotton leaf curl Kokhran virus
CLCuMV Cotton leaf curl Multan virus
CLCuRV Cotton leaf curl Rajasthan virus
CLCuV Cotton leaf curl virus
CLRV Cherry leaf roll virus
CLVd Columnea latent viroid
ClYMV Clover yellow mosaic virus
ClYVV Clover yellow vein virus
CMBV Citrus mosaic badnavirus
CMDV Carrot mottley dwarf virus
CMV Cucumber mosaic virus
CNV Cocao necrosis virus
CoYMV Commelina yellow mottle virus
CpBMV Cowpea banding mosaic virus
CpCDV Chickpea chlorotic dwarf virus
CpCSV Chickpea chlorotic stunt virus
CPFVd Cucumber pale fruit viroid
CpGMV Cowpea golden mosaic virus
CpMMV Cowpea mild mottle virus
CPMoV Cowpea mottle virus
CpMV Cowpea mosaic virus
xvi Acronyms

CPSMV Cowpea severe mosaic virus

CPsV Citrus psorosis virus
CRSV Citrus ring spot virus
CsALV Cassava American latent virus
CsCMV Cassava common mosaic virus
CSNV Chrysanthemum stem necrosis virus
CSSV Cocoa swollen shoot virus
CSVd Chrysanthemum stunt viroid
CsVX Cassava virus X
CTLV Carrot thin leaf virus
CTV Citrus tristeza virus
CuNV Cucumber necrosis virus
CVMV Cassava vein mosaic virus
CVMV Chilli veinal mottle virus (Syn. Pepper vein banding mosaic
virus)
CVV Citrus variegation virus
CVYV Cucumber vein yellowing virus
CymMV Cymbidium mosaic virus
CymRSV Cymbidium ringspot virus
CYMV Chicory yellow mottle virus
CYMV Citrus yellow mosaic virus
CYSDV Cucurbit yellow stunt disorder virus
DAV Dapple apple virus
DBV Dioscorea bacilliform virus
DoYMV Dolichos yellow mosaic virus
DsMV Dasheen mosaic virus
EACMCV East African cassava mosaic Cameroon virus
EACMV East African cassava mosaic virus
ELCV Enation leaf curl virus
EMDV Eggplant mottled dwarf virus
EMV Eggplant mosaic virus
FBNYV Faba bean necrotic yellows virus
FLNV Freesia leaf necrosis virus
GBLV Grapevine Bulgarian latent virus
GBNV/PBNV Groundnut bud necrosis virus
GFkV Grapevine fleck virus
GFLV Grapevine fan leaf virus
GLRaV-1 Grapevine leafroll-associated virus-1
GLRaV-2 Grapevine leafroll-associated virus-2
GLRaV-3 Grapevine leafroll-associated virus-3
GLRV Grapevine leafroll virus
GMMV Gayfeather mild mottle virus
GRSPaV Grapevine rupestris stem pitting-associated virus
GRSV Groundnut ringspot virus
GRV Groundnut rosette virus
Acronyms xvii

GSLV Guar symptomless virus

GVA Grapevine Virus-A
GVB Grapevine virus B
GYSV Grapevine Yellow Speckle Viroid
HgYMV Horsegram yellow mosaic virus
HPV High plains virus
HSVd Hop stunt viroid
ICMV Indian cassava mosaic virus
INSV Impatiens necrotic spot virus
IPCV Indian peanut clump virus
IYSV Iris yellow spot virus
JMV Jatropha mosaic virus
JYMV Japanese yam mosaic virus
KGMMV Kyuri green mottle mosaic virus
KMV Konjac mosaic virus
LALV Lucerne Australian latent virus
LBGMV Lima bean golden mosaic virus
LBVV Lettuce big vein virus
LCV Lettuce chlorosis virus
LiYV Lettuce infectious yellows virus
LMV Lettuce mosaic virus
LNYV Lettuce necrotic yellows virus
LTSV Lucerne transient streak virus
LYSV Leek yellow stripe virus
MCDV Maize chlorotic dwarf virus
MCLCuV Melon chlorotic leaf curl virus
MCMV Maize chlorotic mottle virus
MDMV Maize dwarf mosaic virus
MeCMV Melon chlorotic mosaic virus
MLRV Myrobalan latent ringspot virus
MMV Maize mosaic virus
MNSV Melon necrotic spot virus
MPVd Mexican papita viroid
MRDV Maize rough dwarf virus
MRFV Maize rayado fino virus
MRMV Melon rugose mosaic virus
MRSV Mulberry ring spot virus
MSMV Melon severe mosaic virus
MSpV Maize stripe virus
MSV Maize streak virus
MYMV Mungbean yellow mosaic virus
MYSV Melon yellow spot virus
NVMV Nicotiana velutina mosaic virus
OGSV Oat golden stripe virus
OkMV Okra mosaic virus
xviii Acronyms

OLCV Okra leaf curl virus

OLV-1 Olive latent virus-1
OLV-2 Olive latent virus-2
ORSV Odontoglossum ringspot virus
OYDV Onion yellow dwarf virus
OYVMV Okra yellow vein mosaic virus
PaLCuV Papaya leaf curl virus
PAMV Potato aucuba mosaic virus
PapMV Papaya mosaic virus
PBCVd Pear blister canker viroid
PBNV Peanut bud necrosis virus
PCFV Peanut chlorotic fanspot virus
PCFVd Pepper chat fruit viroid
PCV Peanut clump virus
PDV Prune dwarf virus
PEBV Pea early browning virus
PEMV Pea enation mosaic virus
PepGMV Pepper golden mosaic virus
PepLCBV Pepper leaf curl Bangladesh virus
PepLCV Pepper leaf curl virus
PepMoV Pepper mottle virus
PepMV Pepino mosaic virus
PeSV Pea streak virus
PLMVd Peach latent mosaic viroid
PLRV Potato leafroll virus
PLRV Pea leaf roll virus
PMiMV Pea mild mosaic virus
PMMoV Pepper mild mottle virus
PMTV Pepper mild tigre virus
PMTV Potato mop top virus
PMV Panicum mosaic virus
PMV Pea mosaic virus
PMV Peanut mottle virus
PMWaV-1 Pineapple mealybug wilt associated virus-1
PMWaV-3 Pineapple mealybug wilt associated virus-3
PNRSV Prunus necrotic ringspot virus
PopMV Poplar mosaic virus
PoRSV Polygonum rings pot virus
PPSMV Pigeon pea sterility mosaic virus
PPV Plum pox potyvirus
PRMV Peach rosette mosaic virus
PRSV Papaya ring spot virus
PSbMV Pea seed-borne mosaic virus
PSMV Physalis silver mottle virus
PStV Peanut stripe virus
Acronyms xix

PSTVd Potato spindle tuber viroid

PSV Peanut stunt virus
PVA Potato virus A
PVC Potato virus C
PVS Potato virus S
PVT Potato virus T
PVX Potato virus X
PVY Potato virus Y
PYDV Potato yellow dwarf virus
PYMoV Piper yellow mottle virus
PYMV Pepper yellow mottle virus
PYMV Potato yellow mosaic virus
PYSV Peanut yellow spot virus
PYVHV Pepper yellow vein huasteco virus
PYVV Potato yellow vein virus
PZSV Pelargonium zonate spot virus
RBDV Raspberry bushy dwarf virus
RDV Rice dwarf virus
RGMV Rye grass mosaic virus
RGSV Rice grassy stunt virus
RHBV Rice hoja blanca virus
RMV Rice mosaic virus
RpRSV Raspberry ring spot virus
RRSV Rice ragged stunt virus
RSV Rice stripe virus
RTBV Rice tungro bacilliform virus
RTSV Rice tungro spherical virus
RTV Rice tungro virus
RTYV Rice transitory yellowing virus
RWSV Rice wilted stunt virus
RYEV Radish yellow edge virus
RYMV Rice yellow mottle virus
SACMV South African cassava mosaic virus
SALCV Solanum apical leaf curling virus
SbBMV Soybean blistering mosaic virus
SBMV Southern bean mosaic virus
SBWMV Soil-borne wheat mosaic virus
SBYV Sugarbeet yellows virus
SCBV Sugarcane bacilliform virus
SCLV Soybean crinkle leaf virus
SCMoV Subterranean clover mottle virus
SCMV Sugarcane mosaic virus
SCRLV Subterranean clover red leaf virus
SCSV Subterranean clover stunt virus
SCYLV Sugarcane yellow leaf virus
xx Acronyms

SCFDV Sugarcane Fiji disease virus

SgCSV Sorghum chlorotic spot virus
SLCMV Sri Lankan cassava mosaic virus
SLCV Squash leaf curl virus
SLRSV Strawberry latent ring spot virus
SLV Shallot latent virus
SMMV Soybean mild mosaic virus
SMV Soybean mosaic virus
SMoV Strawberry mottle virus
SMYEPV Strawberry mild yellow edge potexvirus
SMYEV Strawberry mild yellow edges virus
SNMoV Solanum nodiflorum mottle virus
SoMV Sowbane mosaic virus
SPCFV Sweet potato chlorotic fleck virus
SPCSV Sweet potato chlorotic stunt crinivirus
SPFMV Sweet potato feathery mottle potyvirus
SPLCV Sweet potato leafcurl virus
SPLL Sweet potato little leaf
SpLV Spinach latent virus
SPLV Sweet potato latent virus
SPMMV Sweet potato mild mottle virus
SPMSV Sweet potato mild speckling virus
SPSVV Sweet potato sunken vein virus
SPVD Sweet potato virus disease
SPVMV Sweet potato vein mosaic virus
SPYDV Sweet potato yellow dwarf virus
SqMV Squash mosaic virus
SRMV Sunflower rugose mosaic virus
SrMV Sorghum mosaic virus
SCSMV Sugarcane streak mosaic virus
SuCMoV Sunflower chlorotic mottle virus
SVBV Strawberry vein banding virus
SYMMoV Squash yellow mild mottle virus
SYNV Sonchus yellow net virus
SYVV Sowthistle yellow vein virus
TaBV Taro bacilliform virus
TASVd Tomato apical stunt viroid
TAV Tomato aspermy virus
TBRV Tomato black ring virus
TBSV Tomato bushy stunt virus
TBV Tulip breaking virus
TCSV Tomato chlorotic spot virus
TCV Turnip crinkle virus
TDLCV Tomato dwarf leafcurl virus
TEV Tobacco etch virus
Acronyms xxi

TFMV Taro feathery mosaic virus

TICV Tomato infectious chlorosis virus
TLCPuV Tomato leaf curl Pune virus
TLCrV Tomato leaf crumple virus
TLCV Tobacco leaf curl virus
TLCV Tomato leaf curl virus
TMV Tobacco mosaic virus
TNV Tobacco necrosis virus
ToCMoV Tomato chlorotic mottle virus
ToCV Tomato chlorosis virus
ToLCD Tomato leaf curl disease
ToLCGV Tomato leaf curl Gujarat virus
ToLCKV Tomato leaf curl Karnataka virus
ToLCNDV Tomato leaf curl New Delhi virus
ToMoV Tomato mottle virus
ToMV Tomato mosaic virus
ToRSV Tomato ringspot virus
ToSLCV Tomato severe leaf curl virus
ToTV Tomato torrado virus
ToYMV Tomato yellow mosaic virus
TPCTV Tomato pseudo-curly top virus
TPMVd Tomato planta macho viroid
TriMV Triticum mosaic virus
TRSV Tobacco ring spot virus
TRV Tobacco rattle virus
TStV Tobacco stunt virus
TSV Tobacco streak virus
TSWV Tomato spotted wilt virus
TuMV Turnip mosaic virus
TVMV Tobacco vein mottling virus
TYFRV Tomato yellow fruit ring virus
TYLCV Tomato yellow leaf curl virus
TYMV Turnip yellow mosaic virus
TYRV Tomato yellow ring virus
TYVSV Tomato yellow vein streak virus
TZSV Tomato zonate spot virus
ULCV Urd bean leaf crinkle virus
VTMoV Velvet tobacco mottle virus
WBNV Watermelon bud necrosis virus
WCMV White clover mosaic virus
WCSV Watermelon chlorotic stunt virus
WMV Watermelon mosaic virus
WMV-1 Watermelon mosaic virus-1
WMV-2 Watermelon mosaic virus-2
WSBMV Wheat soil borne mosaic virus
xxii Acronyms

WSMoV Watermelon silver mottle virus

WSMV Wheat streak mosaic virus
WSSMV Wheat spindle streak mosaic virus
YMMV Yam mild mosaic virus
YMV Yam mosaic virus
YVMV Yellow vein mosaic virus
ZGMMV Zucchini green mottle mosaic virus
ZLCV Zucchini lethal chlorosis virus
ZYMV Zucchini yellow mosaic virus
Contents

1 Introduction to Plant Virus and Viroid Diseases in the Tropics . . . 1

1.1 Introduction. . . . . . . . . . . ........................ . . 1
1.2 Tropics and Climate . . . . . ........................ . . 1
1.3 Tropical Countries . . . . . . ........................ . . 4
1.4 Tropical Crops. . . . . . . . . ........................ . . 5
1.5 Plant Virus Diseases in the Tropics . . . . . . . . . . . . . . . . . . . . 6
1.6 Conclusions. . . . . . . . . . . ........................ . . 8
References . . . . . . . . . . . . . . . . ........................ . . 9

2 Viruses and Sub-Viral Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2 Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.1 Virus Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.3 Sub-Viral Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
2.3.1 Viroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
2.4 Phytoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
2.5 Spiroplasma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
2.6 Other Sub-Viral Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
2.6.1 Satellite Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
2.6.2 Defective Interfering Particles (DI Particles) . . . . . . . . . 90
2.7 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

3 Impact of Virus and Viroid Diseases on Crop Yields . . . . . . . . . . . 99

3.1 Crop Losses Due to Virus and Viroid Diseases . . . . . . . . . . . . 99
3.2 Yield Losses in Different Crops. . . . . . . . . . . . . . . . . . . . . . . 104
3.2.1 Cereals and Millets . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.2.2 Food Legumes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
3.2.3 Vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
3.2.4 Tuber Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
3.2.5 Fruit Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
3.2.6 Industrial Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
3.2.7 Edible Oil Seed Crops . . . . . . . . . . . . . . . . . . . . . . . . 136

xxiii
xxiv Contents

3.2.8 Spice Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

3.2.9 Bio Fuel Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.3 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

4 Transmission of Plant Viruses and Viroids . . . . . . . . . . . . . . . . . . 161

4.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
4.1.1 Transmission Through Vegetative Propagules . . . . . . . . 161
4.1.2 Transmission Through Seed . . . . . . . . . . . . . . . . . . . . 162
4.1.3 Transmission Through Pollen . . . . . . . . . . . . . . . . . . . 190
4.1.4 Transmission Through Contact and Mechanical. . . . . . . 191
4.1.5 Transmission Through Water . . . . . . . . . . . . . . . . . . . 193
4.1.6 Transmission Through Vectors . . . . . . . . . . . . . . . . . . 193
4.2 Arthropod vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
4.2.1 Aphids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
4.2.2 Leaf, Plant and Tree Hoppers . . . . . . . . . . . . . . . . . . . 198
4.2.3 Whiteflies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
4.2.4 Thrips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
4.2.5 Beetles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
4.2.6 Mealybugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
4.2.7 Mirids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
4.2.8 Mites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
4.3 Non-Insect Vectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
4.3.1 Nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
4.3.2 Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
4.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

5 Diagnosis and Detection of Plant Virus and Viroid Diseases . . . . . 233

5.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
5.1.1 Need and Progress in Plant Virus Diagnostics . . . . . . . . 234
5.1.2 Detection Specificity and Sensitivity . . . . . . . . . . . . . . 234
5.2 Approaches Used for Identification of Plant Virus
and Viroid Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
5.2.1 Biological Approaches . . . . . . . . . . . . . . . . . . . . . . . . 236
5.2.2 Physical Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
5.3 Antibody-Based Tests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
5.3.1 Precipitation Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
5.3.2 Agglutination Tests . . . . . . . . . . . . . . . . . . . . . . . . . . 240
5.3.3 Immuno Diffusion Tests . . . . . . . . . . . . . . . . . . . . . . . 241
5.3.4 Immunochromatography . . . . . . . . . . . . . . . . . . . . . . . 243
5.3.5 Immuno Electron Microscopy (IEM) . . . . . . . . . . . . . . 244
5.3.6 Labeled Antibody Based Assays . . . . . . . . . . . . . . . . . 245
Contents xxv

5.4 Viral
Nucleic Acid-Based Tests . . . . . . . . . . . . . . . . . . . . . . . 263
5.4.1Molecular Hybridization . . . . . . . . . . . . . . . . . . . . . . . 263
5.4.2Polymerase Chain Reaction (PCR). . . . . . . . . . . . . . . . 268
5.4.3PCR Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
5.4.4Real Time Quantitative PCR. . . . . . . . . . . . . . . . . . . . 281
5.4.5PCR-RFLP for Detection of Plant Virus Diseases . . . . . 284
5.4.6PCR Application for the Detection of Viruses
in the Vectors . . . . . . . . . . . . . . . . . . . . . . . . . ..... 285
5.5 Recombinant DNA Technology . . . . . . . . . . . . . . . . . . ..... 294
5.5.1 Production of Recombinant Antibodies
by Phage Display Technology . . . . . . . . . . . . . . ..... 295
5.5.2 Single Chain Variable Fragment Antibody (scFv) ..... 296
5.5.3 Plantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 297
5.5.4 Induction of Polyclonal Antibodies (PAbs)
by rDNA Based Immunization . . . . . . . . . . . . . . . . . . 299
5.6 Array Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
5.6.1 Macroarray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
5.6.2 Microarray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
5.7 Rolling Circle Amplification (RCA) in Plant
Virus Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 304
5.8 Metagenomics in Plant Viral Diagnosis . . . . . . . . . . . . ..... 305
5.9 Biosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 306
5.10 DNA Barcodes Use as Genetic Markers for Identifying
Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 309
5.11 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 310
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 312

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Chapter 1
Introduction to Plant Virus and Viroid
Diseases in the Tropics

1.1 Introduction

There are more than 840 million under-nourished people worldwide who would
benefit from substantial food production increases in the Tropics. Protecting the
crops from pests and diseases would significantly reduce food deficits (FAO 2003).
There are numerous ways by which agricultural productivity may be increased in a
sustainable way, but farmers usually lack technical assistance other than that
provided by agro-chemical companies. Unfortunately, fungal and bacterial dis-
eases, and most arthropod pests can be chemically controlled, but plant viruses
cannot, although some of their vectors can be chemically controlled. In this book
the major virus and virus-like diseases of tropical plants are described in relation to
their socio-economic importance, and the disease management practices shown to
control crop yield losses caused by these pathogens.
Table 1.1 lists 169 countries that have part of their land mass between the
Tropics of Cancer and Capricorn. Countries which are in brackets have less than
half of their land in the tropics, while the rest, i.e., (Algeria), (Australia), (Baha-
mas), (Bangladesh), (Chile), (China), (Egypt), (Lybia), (Paraguay), (Saudi Arabia),
(Taiwan), (United Arab Emirates), and (Western Sahara) have \ 50% land area in
the tropics (Table 1.1).

1.2 Tropics and Climate

Despite the position of the tropics with respect to the sun, some tropical regions
can experience marked differences in temperature, particularly diurnal and noc-
turnal, during certain months of the year, both in the lowlands and highlands.
These climate variations are usually related to the occurrence of ’dry’ and ’rainy’
seasons. These dry and wet seasons may present a uni-modal or bi-modal distri-
bution during the year. Dry seasons are often associated with low diurnal or
nocturnal temperatures, depending on their proximity to the equator and altitude.

K. S. Sastry, Plant Virus and Viroid Diseases in the Tropics, 1

DOI: 10.1007/978-94-007-6524-5_1, Ó Springer Science+Business Media B.V. 2013
 2

Table 1.1 List of Tropical Countries

North Central South Caribbean Central Africa East Africa West Africa South
America America America East Asia
Mexico Belize Bolivia Anguilla Angola Burundi Benin Brunei
Costa Rica Brazil Antigua and Barbuda Cameroon Comoros Burkina Faso Burma
El Salvador Colombia Aruba Central African Djibouti Côte d’Ivoire (Myanmar)
Guatemala Ecuador Bahamas Republic Eritrea (Ivory Coast) Cambodia
Honduras French Barbados Chad Ethiopia The Gambia East Timor
Nicaragua Guiana British Virgin Islands Congo Kenya Ghana Indonesia
Panama Guyana Cayman Islands Democratic Madagascar Guinea India
Paraguay Cuba Republic of Congo Malawi Guinea-Bissau Laos
Peru Dominica (Zaire) Mauritius Liberia Malaysia
Suriname Dominican Republic Equatorial Guinea Mayotte Mali Maldives
Venezuela Grenada Gabon Mozambique Mauritania Philippines
Guadeloupe Sudan Reunion Niger Singapore
Haiti Zambia Rwanda Nigeria Sri Lanka
Jamaica Seychelles Saint Helena Thailand
Martinique Somalia Sao tomé and Vietnam
Montserrat Tanzania Principe
Netherlands Antilles Uganda Senegal
Puerto Rico Sierra Leone
Saint Barthelme Togo
Saint Kits and Nevis
Saint Lucia
Saint Martin (France)
Saint Vincent and the
Grenadines
Trinidad and Tobago
Turks and Cacaos Islands
U.S. Virgin Islands
1 Introduction to Plant Virus and Viroid Diseases in Tropics
1.2 Tropics and Climate 3

These phenomena create a large number of different eco-systems and a diverse

biodiversity of plant and animal life in the tropics, which has allowed some plant
pathogens and pests from Temperate countries to adapt to tropical and sub-tropical
environments. The sub-tropics include regions adjacent to the Tropics of Cancer
and Capricorn, which may suffer a ’spill-over’ invasion of tropical pathogens and
pests, or which may act as an entry point for temperate pathogens and their vectors
into the tropics.
An additional problem encountered in the tropics is the extreme and highly
variable environmental conditions found, particularly the high temperature and
high humidity conditions, which cause accelerated degradation of tropical soils,
making them highly acidic (pH \ 5), toxic (high aluminum content), and deficient
in critical nutrients, such as phosphorus. In the humid tropics, the relative
importance of acid soils is greatest in Latin America (81 %), but also significant in
Africa (56 %) and Asia (38 %). In rainforests and mountain slopes, the rapid
degradation of tropical soils is noticed when there is total crop failure or when
mountain soils lose their protective vegetation. In some tropical regions, the dry
season may last for six months on average, impeding the cultivation of plants,
unless irrigation is available in some wet-and dry tropical regions. This season is
also associated with a significant increase in the increase of arthropod pests, many
of which can act as virus vectors. However, irrigation tends to aggravate salinity
problems and also favours the population increase of insect vectors of plant
viruses. On the contrary, the wet season may be so intense that flooding occurs and
crops are totally lost.
Based on the quantity of rainfall, tropical zones are defined as (1) arid: less than
400mm rainfall/year; (2) semi-arid: 400mm to 599mm rainfall/year; (3) sub-
humid: 600mm to 1200mm rainfall/year; (4) humid: over 1200mm rainfall/year.
Besides the abiotic stresses, the dry weather, adequate humidity and tempera-
ture are quite favourable for insects like aphids, leafhoppers, whiteflies and thrips
which are active vectors of some plant viruses and can cause severe direct damage
to crops.
In West Africa, Atiri et al., (2000) have extensively studied some climatic
factors in relation to the epidemiology of economically important virus diseases.
Case studies of the Maize streak mastre virus, Okra mosaic tymovirus and African
cassava mosaic begomovirus demonstrate that the most important factor that
influences the incidence and spread of virus diseases is climate. In these cases,
climate influences: (1) virus disease outbreaks; (2) the rate of development and
activity of virus vectors and their migration; and (3) the phenology of crops, weeds
and wild hosts that harbour plant viruses. Rainfall, temperature and wind are
identified as key weather components in virus patho-systems involving maize
(cereal), okra (vegetable) and cassava (root crop), and are therefore important
factors determining the most suitable period in which to undertake crop protection
measures. Loebenstein and Thottappilly (2003); Anderson and Morales (2005);
Thresh (2006) and Sastry and Zitter (2013, II Volume) have also provided more
details about virus and viroid disease situation, epidemiology and management
aspects in tropical and sub-tropical countries.
4 1 Introduction to Plant Virus and Viroid Diseases in Tropics

1.3 Tropical Countries

The names of the tropical countries in Mesoamerica, Central America, South

America, the Caribbean, Central Africa, East Africa, West Africa and South East
Asia are listed Table 1.1 and in Fig. 1.1.
In the Western Hemisphere, tropical countries include part of Mexico; all of
Central America; the Caribbean islands; and in South America; Colombia,
Ecuador, Peru, Bolivia, Colombia, Venezuela, Guyana, Suriname, French Guiana,
Brazil, northern Paraguay and the northern-most portions of Chile and Argentina.
In Africa, the only nations that cannot be called tropical countries are Morocco
and Tunisia in the north and Lesotho and Swaziland in the south. All the rest lie
either entirely, or at least partly, in the tropics. The Middle East has four tropical
countries: Yemen, which is entirely in the tropics, and parts of Saudi Arabia,
Oman, and United Arab Emirates. India, in southern Asia, lies mostly in the
tropics, and all countries of Southeast Asia are tropical countries. Parts of Aus-
tralia, Micronesia, the Marshall Islands, Kiribati, and most of the other island
nations of Oceania in the South Pacific are tropical countries, as well.
The strongest link in explaining the wealth and poverty of nations is the rela-
tionship between ecological zones and per capita income, according to National
Bureau of Economic Research (NBER). Yet, most recent cross-country analyses of
economic growth have neglected the importance of physical geography.
Despite their varied economic, political, and social histories, almost all of the
tropical countries remain underdeveloped at the start of the twenty-first century.
Only two tropical-zone countries, Hong Kong and Singapore, rank among the 30
countries classified as high-income by the World Bank. All of the high-income
regions (North America, Western Europe, Northeast Asia, the Southern Cone of

Fig. 1.1 World map with the tropical zone

1.3 Tropical Countries 5

Latin America, and Oceania) are outside of the tropics. Sea navigable regions are
generally richer than land-locked nations. Those that are both tropical and land-
locked-including Bolivia, Chad, Niger, Mali, Burkina Faso, Uganda, Rwanda,
Burundi, Central African Republic, Zimbabwe, Zambia, Lesotho and Laos are
among the very poorest in the world (Sachs 1999).
At the core of this long-term growth was the continued development of tech-
nology, a process that has benefitted the temperate-zone countries much more than
the tropics. Production technology in the tropics has lagged behind temperate-zone
technology in the two critical areas of agriculture and health. The difficulty of
mobilizing energy resources in tropical economies has also contributed to wid-
ening the income gap between climate zones. The problems of applying temperate-
zone technological advances to the tropical setting have amplified these factors.
Agricultural, health, and some manufacturing-related technologies that could
diffuse within ecological zones could not diffuse across them.
In the Temperate-zone the productivity of the major crops like rice, maize and
wheat is considerably higher than in the tropics. Sachs (1995) estimated that the
productivity per hectare of grain produced was approximately 50 % higher in
temperate-zone countries. The explanation lies in soil formation and erosion, pests,
water availability, environmental, technological and economic factors. Poor
nutrition, resulting from low agricultural productivity, then leads to poor health.
Sachs argues that economic development in tropical eco-zones requires a con-
certed international effort: agricultural technologies must be specific to the needs
of tropical agriculture (Sachs 1999). For instance, between 1961 and 1991 the so-
called ‘Green Revolution’, exponentially increased the yield of maize, wheat and
rice in developing countries, demonstrating that it is possible to increase food
production in the tropics with technological know-how. Unfortunately, the
intensive agricultural practices implemented in the past century, were not always
environmentally friendly. Nevertheless, it is possible to increase food production
in the tropics in a sustainable manner. Food production also varies in the tropics.
For instance, in Africa, the annual increase (1.3%) in yield per hectare of maize,
wheat and rice is less than a one third of that achieved (4.5%) in Asia (Persley
2002).

1.4 Tropical Crops

The tropics are either the center of origin or of domestication of many of the most
important food crops currently cultivated in the world: maize, rice, potato, sweet
potato, cassava, cocoa, sorghum, millet, tomato, peppers, many cucurbits, peanut,
rubber, tobacco, cotton, lima bean, common beans, oil palm, coconut, sugarcane,
coffee, cocoa, and many fruit crops, such as banana, pineapple, mango, sweet
pepino, passion fruit, guava, avocado and papaya. However, a myriad of other food
crops were also domesticated and consumed by the early civilizations that
developed in the tropics, particularly in Latin America.
6 1 Introduction to Plant Virus and Viroid Diseases in Tropics

1.5 Plant Virus Diseases in the Tropics

Plant viruses greatly affect food production in the tropics. Virus genera such as the
Begomovirus, Potyvirus, Tospovirus, and Cucumovirus affect crops that feed the
greatest number of people in tropical countries, often causing 100% yield losses and
widespread famine, as is the case with several Begomoviruses transmitted by
whiteflies in Africa, Asia and Latin America and the Caribbean. The third chapter of
this book will cover the extent of yield losses in different crops grown in the tropics.

Table 1.2 Virus diseases of Tropical Countries

Central/East Africa
African cassava mosaic virus
African cereal streak virus
Alfalfa mosaic virus
Banana bunchy top virus
Banana dieback virus
Banana streak virus
Barley stripe mosaic virus
Barley yellow dwarf virus
Bean calico mosaic virus
Bean common mosaic virus
Bean yellow dwarf virus
Brome mosaic virus
Cassava brown streak virus
Cassava common mosaic virus
Cassava Ivorian bacilliform virus
Cassava kumi virus
Cassava ‘Q’ virus
Cereal chlorotic mottle virus
Chick pea chlorotic dwarf virus
Citrus tristeza virus
Cocoa swollen shoot virus
Cotton leafcurl virus
Cotton leaf mottle virus
Cowpea aphid borne mosaic virus
Cowpea golden mosaic virus
Cowpea mild mottle virus
Cucumber mosaic virus
East African cassava mosaic virus
Groundnut ringspot virus
Groundnut rosette virus
Impatiens necrotic spot virus
Iris yellow spot virus
Limabean golden mosaic virus
Macroptilium yellow mosaic virus
Maize dwarf mosaic virus
Maize line virus
Maize mottle/chlorotic stunt virus
Maize mottle virus
Maize pellucid ringspot virus
Maize rayado virus
Maize rough dwarf virus
North/Central/South America
Abutilon infectious variegation
virus
Andean potato latent virus
Arracacha virus A
Banana streak virus
Barley yellow dwarf virus
Bean calico mosaic virus
Bean common mosaic necrosis
virus
Bean dwarf mosaic virus
Bean golden mosaic virus
Bean golden yellow mosaic virus
Bean leaf crumple virus
Bean rugose mosaic virus
Bean yellow stipple virus
Bidens mosaic virus
Cacao swollen shoot virus
Cassava American latent virus
Cassava Caribbean mosaic virus
Cassava Colombian symptomless
virus
Cassava common mosaic virus
Cassava frogskin virus
Cassava Ivorian bacilliform virus
Cassava latent rhabdo virus
Cassava vein mosaic virus
Cassava virus X
Chinodel tomato virus
Chrysanthemum stem necrosis
virus
Citrus tristeza virus
Clitoria falcate mosaic virus
Corn lethal necrosis virus
Cotton antho cyanosis virus
Cotton leafcrumple virus
Cowpea aphid borne mosaic virus
Cowpea mild mottle virus
Cowpea mosaic virus
Cowpea severe mosaic virus
Cucumber mosaic virus
Dasheen mosaic virus
South East Asia
Alfalfa mosaic virus
Banana bract mosaic virus
Banana bunchy top virus
Banana streak Mysore virus
Banana streak OL virus
Barley stripe mosaic virus
Barley yellow dwarf virus
Bean common mosaic virus
Bhendi yellow vein mosaic virus
Bittergourd yellow mosaic virus
Cacao swollen shoot virus
Capsicum chlorosis virus
Cassava Colombian symptomless
virus
Cassava common mosaic virus
Cassava green mottle virus
Chick pea chlorotic dwarf virus
Chilli leafcurl virus
Chrysanthemum stem necrosis
virus
Citrus infectious variegation virus
Citrus mosaic virus
Citrus psorosis virus
Citrus tristeza virus
Cotton leaf crumple virus
Cotton leafcurl virus
Cowpea golden mosaic virus
Cowpea mild mottle virus
Cucumber chlorotic spot virus
Cucumber green mottle mosaic
virus
Cucumber mosaic virus
Dolichos yellow mosaic virus
Eastern wheat striate virus
Groundnut eye spot virus
Groundnut ringspot virus
Hibiscus chlorotic ring spot virus
Horsegram yellow mosaic virus
Impatiens necrotic spot virus
Indian cassava mosaic virus
Indonesian soybean dwarf virus

(continued)
1.5 Plant Virus Diseases in Tropics
Table 1.2 (continued)
Central/East Africa
Maize streak virus
Maize stripe virus
Moroccan Watermelon mosaic
virus
Okra leafcurl virus
Okra mosaic virus
Papaya leafcurl virus
Papaya mosaic virus
Papaya ringspot virus
Pea leaf roll virus
Peanut clump virus
Peanut mottle virus
Peanut stunt virus
Peanut yellow mottle virus
Pepper leafcurl virus
Pepper mildmottle virus
Pepper veinal mottle virus
Potato leafroll virus
Potato virus S
Potato virus X
Potato virus Y
Rice stripe necrosis virus
Rice yellow mottle virus
Rhynchosia golden mosaic virus
Soil-borne wheat mosaic virus
Sorghum mosaic virus
South African cassava mosaic virus
Soybean golden yellow mosaic
virus
Sugarcane bacilliform virus
Sugarcane chlorotic streak virus
Sugarcane mosaic virus
Sugarcane yellow leaf virus
Sunflower yellow blotch virus
Sunflower yellow ringspot virus
Sweet potato chlorotic fleck virus
Sweet potato chlorotic stunt virus
Sweet potato leaf curl virus
Sweetpotato feathery mottle virus
Tobacco bushy top virus
Tobacco leaf curl virus
Tobacco mosaic virus
Tobacco ringspot virus’
Tobacco vein mottle virus
Tomato dwarf leafcurl virus
Tomato mosaic virus
Tomato spotted wilt virus
Tomato vein-yellowing virus
Tomato yellow leafcurl virus
Turnip mosaic virus
Watermelon chlorotic stunt virus
Wheat dwarf virus
Wheat spindle streak mosaic virus
North/Central/South America
Eggplant mosaic virus
Elephant grass mosaic virus
Groundnut ringspot virus
Impatiens necrotic spot virus
Iris yellow spot virus
Lettuce mosaic virus
Lima bean golden mosaic virus
Macroptilium yellow mosaic virus
Maize chlorotic mottle virus
Maize dwarf mosaic virus
Maize rayado finovirus
Maize streak virus
Maize stripe virus
Mal de Rio cuarto virus
Melon chlorotic leafcurl virus
Melon chlorotic mosaic virus
Melon severe mosaic virus
Merremia mosaic virus
Mirafiori varicosavirus
Pangola stunt virus
Papaya mosaic virus
Papaya ringspot virus
Peanut chlorotic fanspot virus
Peanut mottle virus
Pepper golden mosaic virus
Pepper Hausteco yellow vein virus
Pepper mild tigre virus
Pepper yellow vein huasteco virus
Plum pox virus
Potato black ringspot virus
Potato leafroll virus
Potato virus T
Potato virus Y
Potato yellow mosaic virus
Potato yellow vein virus
Rice hoja blanca virus
Rice stripe necrosis virus
Rhynchosia golden mosaic virus
Solanum apical leafcurling virus
Sorghum mosaic virus
Sowbane mosaic virus
Soybean golden mosaic virus
Soybean mosaic virus
Soybean yellow shoot virus
Squash leafcurl virus
Squash yellow mild mottle virus
Sugarcane bacilliform virus
Sugarcane mosaic virus
Sugarcane yellow leaf virus
Sunflower chlorotic mottle virus
Sweet potato chlorotic stunt virus
Sweet potato feathery mottle virus
Tobacco leaf curl virus
7
South East Asia
Iris yellow spot virus
Kokke kondu carla virus
Limabean golden mosaic virus
Maize dwarf mosaic virus
Maize streak virus
Melon yellow spot virus
Mungbean yellow mosaic virus
Mungbean yellow mosaic
India virus
Okra leafcurl virus
Pangola stunt virus
Papaya leafcurl virus
Papaya ringspot virus
Peanut bud necrosis virus
Peanut chlorotic streak virus
Peanut green mosaic virus
Peanut mottle virus
Peanut stripe virus
Peanut yellow spot virus
Physalis silver mottle virus
Pigeonpea sterility mosaic virus
Plum pox virus
Potato apical leafcurl virus
Potato leafroll virus
Potato virus S
Potato virus X
Potato virus Y
Rice black streaked dwarf virus
Rice chlorotic streak virus
Rice grassy stunt virus
Rice mosaic virus
Rice ragged stunt virus
Rice stripe virus
Rice transitory yellowing virus
Rice tungro virus
Sorghum mosaic virus
Soybean crinkle leaf virus
Soybean mosaic virus
Squash leafcurl china virus
Srilankan cassava mosaic virus
Sugarcane bacilliform virus
Sugarcane fiji disease virus
Sugarcane mosaic virus
Sugarcane streak mosaic virus
Sugarcane yellow leaf virus
Sweet potato feathery mottle virus
Sweet potato leafcurl virus
Tea phloem necrosis virus
Tobacco leafcurl virus
Tobacco mosaic virus
Tobacco streak virus
Tobacco vein banding mosaic virus
Tomato leafcurl New Delhi virus
(continued)
8 1 Introduction to Plant Virus and Viroid Diseases in Tropics

Table 1.2 (continued)

Central/East Africa
Wheat streak mosaic virus
Zucchini yellow mosaic virus
Note: The plant virus names in italics are ICTV recognized while not in italics are unrecognized viruses
North/Central/South America South East Asia
Tobacco leaf rugose virus Tomato leafcurl virus
Tobacco mosaic virus Tomato yellow leafcurl virus
Tobacco rattle virus Tomato yellow ring virus
Tobacco streak virus Tomato zonate spot virus
Tomato chino virus Watermelon bud necrosis virus
Tomato chlorotic mottle virus Watermelon mosaic virus
Tomato chlorotic spot virus Watermelon silver mottle virus
Tomato dwarf leafcurl virus Wheat spindle streak mosaic virus
Tomato golden mosaic virus Zucchini yellow mosaic virus
Tomato golden yellow mosaic
virus
Tomato leafcrumple virus
Tomato leafcurl Sinaloa virus
Tomato leaf deformation virus
Tomato mosaic Havana virus
Tomato mottle virus
Tomato mottle taino virus
Tomato severe rugose virus
Tomato severe leafcurl virus
Tomato spotted wilt virus
Tomato yellow leafcurl virus
Tomato yellow mosaic virus
Tomato yellow mottle virus
Tomato yellow veinstreak virus
Ullucus mild mosaic virus
Ullucus virus C
Wheat soil borne mosaic virus
Zucchini lethal chlorosis virus
Zucchini yellow mosaic virus

1.6 Conclusions

In the context of combating a global food crisis and problems of chronic hunger,
child malnutrition and infant mortality in the tropics, the detection and identifi-
cation of plant viruses and their vectors becomes of critical importance for the
implementation of suitable disease management strategies against these pests.
The development of plant genotypes possessing genetic resistance to viruses in all
food crops is the most sustainable viral disease management practice. Unfortunately,
this important disease control practice requires the concerted collaboration of plant
breeders, virologists and agronomists, among other agricultural specialists.
Regarding R&D needs in tropical developing countries, there is urgent need to
increase the agricultural productivity. As of 1995, whereas 57 % of the labour
force in low-income countries (classified by the World Bank as those countries
with income per capita below $745 in 2001) was engaged in agriculture (World
Bank 2002), and 797 million people in the developing world remained under-
nourished in 1999 (FAO 2002). Kremer and Zwane (2005) have given reasons and
1.6 Conclusions 9

recommendations for encouraging private sector research for increasing food

production in tropical countries.
The R&D needs of tropical agriculture is distinct from that of temperate
countries for several reasons. Some staple crops grown in tropical countries, such
as cassava and millet, are neither grown nor imported by rich countries on a
significant scale (Binenbaum et al. 2003). Tropical countries have distinct agro-
ecological systems, including higher average temperatures, relatively fragile soils,
a lack of a seasonal frost to control pests, and an abundant incidence of pests,
fungal, bacterial and viral diseases (William and Wiebe 2000). Zone-specific
productivity constraints mean that increased productivity in maize in temperate
countries cannot be immediately transferred to tropical regions. Thus, it cannot be
expected that the adoption of technology developed in industrialized nations of
Europe and North America can be rapidly put into practice as ‘‘spill-over’’ tech-
nology (Diamond 1997; Johnson and Evenson 2000).
Fortunately, efforts to encourage agricultural research and development (R&D)
in developing, tropical countries has significantly increased in recent years.
Innovation and technological change in agriculture, especially in biotechnology,
hold out the promise of major productivity advances as long as it is not undertaken
in an isolated manner (Huang et al. 2002).
In Latin America, Asia and Sub-Saharan Africa, agricultural research capa-
bilities are being improved; but the most recent figures still show public expen-
ditures to be about 0.85% of agricultural GDP as compared to 2.64% in developed
countries (Pardey and Beintema 2001).
In the past four decades, intensive research on increased food production in the
Tropics was undertaken by several International Centers belonging to the Con-
sultative Group for International Agricultural Research (CGIAR), sponsored by
many industrialized nations, World Bank and other foundations in the United
States and Europe. The International Agricultural Research Centers are: IITA,
Ibadan (Nigeria); CIAT, Cali (Colombia); CIMMYT, El Batan (Mexico); IRRI,
Los Banos (Philippines); ICRISAT, Hyderabad (India); ICRAF, Nairobi (Kenya);
ICARDA, Beirut (Lebanon); CIP, Lima (Peru); CIFOR, Bogor (Indonesia);
WARDA, Cote d’Ivoire (Cotonou. Benin) and IWMI, Battaramulla (Sri Lanka).
These centers are carrying out research in different disciplines and are maintaining
the databases of information on agriculture within their specific mandate crops.
However, further efforts are needed to strengthen national agricultural research
programmes in most tropical countries, in order to bring these efforts into fruition
and increased food production.

References

Anderson PK, Morales FJ (eds) (2005) Whitefly and whitefly-borne viruses in the tropics:
building a knowledge base for global action. Publication 341. CIAT, Colombia, 351 pp
Atiri GI, Njukeng AP, Ekpo EJA (2000) Climate in relation to plant virus epidemiology and
sustainable disease management in West Africa. J Sustain Agr 16:17–30
10 1 Introduction to Plant Virus and Viroid Diseases in Tropics

Binenbaum E, Pardey PG, Wright BD (2003) The CIAT-Papalotla agreement: Intellectual

property in a partnership that may help transform tropical cattle farming. Unpublished
manuscript
Diamond J (1997) Guns, germs, and steel. W.W. Norton & Co., New York
FAO (2002) State of food insecurity in the world. FAO, Rome
FAO (2003) The international year of rice, 2004. Concept paper
Huang J, Pary C, Rozelle S (2002) Enhancing the crops to feed the poor. Nature 418(8):678–684
Johnson DKN, Evenson RE (2000) How far away is Africa? Technology spillovers to agriculture
and productivity. Am J Agric Econ 82:743–749
Kremer M, Zwane AP (2005) Encouraging private sector research for tropical agriculture
(abstract). World Dev 33:87
Loebenstein G, Thottappily G. (eds.). (2003) Viruses and Virus-Like Diseases of Major Crops in
Developing Countries. Dortrecht, The Netherlands: Kluwer Academic Publishers. 840 pp
Pardey PG, Beintema NM (2001) Slow magic: agricultural R&D a century after mendel. IFPRI
food policy report. IFPRI, Washington
Persley GJ (2002) Agricultural biotechnology: global challenges and emerging science. In:
Persley GJ, Mac Intyre LR (eds) Agricultural biotechnology: country case studies. CAB
International, pp 1–37
Sachs DJ (1995) ‘Do we need an International Lender of Last Resort?’ Frank Graham Lecture,
Princeton University
Sachs DJ (1999) Helping the world’s poorest. Economist 352(8132):17–20
Sastry KS, Zitter TA (2013) Plant virus and viroid diseases in the tropics Volume-II.
Epidemiology and Management of plant viruses, Springer, (In Press).
Thresh JM (2006) Crop viruses and virus diseases: a global perspective. In: Virus Diseases and
Crop Biosecurity. (JI Cooper, T Kuehne, VP Polischuk, ed.), IOS Press, Amsterdam, The
Netherlands, 9–32
William MA, Wiebe KD (2000) Climate and agricultural productivity. Manuscript processed, 15
Oct 2000
World Bank (2002) World development indicators [CDROM]. World Bank, Washington
Chapter 2
Viruses and Sub-Viral Agents

2.1 Introduction

Throughout the tropics the emerging, re-emerging and endemic plant pathogens are
challenging our ability to safeguard plant health. Further globalization, climate
change, increased human mobility and pathogen and vector evolution have con-
tributed to increase the spread of invasive plant pathogens. Plant diseases including
viruses and viroids are responsible for enormous losses worldwide ($30–50 billion
annually) in cultivated and stored crops, and thus are a major impediment to
effective food production and distribution. Virus and viroid diseases of short-lived
vegetables and herbaceous annual crops (eg. tomato, capsicum, cucurbits, etc.)
which are grown using true seed also show maximum infections. If viruses spread
rapidly and infect a large population of the crop within few weeks or months, there
will be maximum loss in yields. Even certain vegetable and fruit crops that are
propagated vegetatively (potato, sweet potato, yam, citrus, apple, etc.) are partic-
ularly prone to damage by viruses, as infection tends to buildup in successive cycles
of propagation. The emergence of a global community and the increasing numbers
of plant viruses identified in the last two decades have increased the requirement for
countries and regions to protect their farming systems from exotic viruses. The
ProMED database (http://www.promedmail.org) is one of the global electronic
reporting systems for detecting outbreaks of emerging infectious diseases and
toxins, and these outbreak reports that has been generated since 1994. It is one of
the most comprehensive plant emerging infectious disease database available
(Anderson et al. 2004). Plant viruses including sub-viral agents and phytoplasmas
were identified as the cause of 51 % of the emerging infectious diseases of plants
that were recorded in the ProMED database during the period of 1996–2002 and it
is likely that this trend will continue. The factors cited as responsible for emerging
diseases caused by viruses included structure, genome organization and effective
mode of spread. Tropical crops are affected by numerous viruses, the list includes
those that are propagated vegetatively and also through true seed to various extents.
In this chapter basic information of viruses and sub-viral agents is briefly
presented to realize their significance as an important category of plant pathogens.

K. S. Sastry, Plant Virus and Viroid Diseases in the Tropics, 11

DOI: 10.1007/978-94-007-6524-5_2,  Springer Science+Business Media B.V. 2013
12 2 Viruses and Sub-Viral Agents

2.2 Viruses

Plant viruses are infectious, intracellular and obligate pathogens that are too small
to be seen with a light microscope, but despite their small size, they can cause lethal
diseases in plants. The simplest viruses are composed of a small piece of nucleic
acid, either RNA or DNA but not both, surrounded by a protein coat. The structure
(morphology) of virus is given by its coat of proteins which surrounds the viral
genome. As is the case with other organisms, viruses carry genetic information in
their nucleic acid which typically specifies four or more proteins. All plant viruses
are obligate parasites that depend on the cellular machinery of their hosts to
reproduce. Viruses are not active outside of their hosts, and this has led some people
to suggest that they are not alive. All types of living organisms including animals,
plants, fungi, and bacteria are hosts for viruses, but most viruses infect only one
type of host. Viruses cause many important plant diseases and are responsible for
losses in crop yield and quality in all parts of the world (refer Chap. 3).
The beginnings of plant virology date back to the late 19th century, when
Dutch microbiologist Martinus Beijerinck and Russian researcher Dmitrii Iwa-
nowski were investigated the cause of a mysterious disease of tobacco. Subse-
quently, numerous scientists from around the world are responsible for the growth
of the discipline of plant virology it occurs today. Some of the major developments
in the history of plant virology are enumerated below.
1. Virus disease of plants was known long before the discovery of bacteria.

i) Eupatorium yellow vein disease is the first record of viral disease by

Japanese empress Koken in her poem in 752 AD.
ii) Breaking of flower colour of tulips (as early as 1576).
iii) Transmission of leaf variegations from the scion to the stock of woody
plants (as early as 1700).
2. Tobacco mosaic was identified by Swietch in Holland during 1857.
3. In 1882, Adolph Mayer (1843–1942) described a condition of tobacco plants,
which he called ‘‘mosaic disease’’ (‘‘mozaïkziekte’’). The diseased plants had
variegated leaves that were mottled. Adolph Mayer (1886) was the first to
point out that tobacco mosaic (TMV) is readily transmissible and infectious.
4. Iwanowski (1892) confirmed some of the results of Adolph Mayer. He
demonstrated that the power to infect was lost if the sap was previously
heated. He reported that infectiousness was retained even when sap was
passed through bacteria proof filters indicating that the causal agent is different
from bacteria.
5. In 1898, the Dutch microbiologist Martinus Beijerinck (1851–1931) repeated
the experiments and became convinced that the filtered solution contained a
new form of infectious agent. He observed that the agent multiplied only in
cells that were dividing and he called it a contagium vivum fluidum (soluble
living germ) and coined the word virus, which in Latin means toxin or poison.
2.2 Viruses 13

6. Stanley (1935) crystallized tobacco mosaic virus with ammonium sulphate

and concluded that the virus was an autocatalytic protein that could multiply
within the living cells. For his discovery he was awarded the Nobel Prize.
7. Bawden and Pirie (1938) from TMV infected plants isolated a liquid crys-
talline nucleoprotein containing nucleic acid of the pentose type.
8. Kausche et al. (1939) saw virus particles for the first time with the electron
microscope. They confirmed that TMV was rod shaped.
9. Bernal and Fankuchen (1941) were the first to study X-ray diffraction pictures
of the crytallized virus. On the basis of pictures, Rosalind Franklin in 1955
discovered the full structure of the virus. In the same year, Heinz Fraenkel-
Conrat and Robley Williams showed that purified tobacco mosaic virus RNA
and its coat protein can assemble by themselves to form functional viruses,
suggesting that this simple mechanism was probably the means through which
viruses were created within their host cells.
10. Muller (1942): Williams and Wycoff (1944) developed as shadow casting
technique with heavy metals which was useful for determining the overall size
and shape of the virus particles.
11. Markham and Smith (1949) isolated Turnip yellow mosaic virus (TYMV) and
showed that purified preparations contained two classes of particle, one an
infectious nucleoprotein with about 35% of RNA, and the other an apparently
identical protein particle that contained no RNA and that was not infectious.
12. Gierer and Schramm (1956) showed that the protein could be removed from
the virus and that the nucleic acid carried the genetic information so that
inoculating with the nucleic acid alone cause infection and could reproduce
the complete virus.
13. Kassanis (1962) was the first to describe the satellite virus (Sv) which was
found only in association with tobacco necrosis virus.
14. Doi et al. (1967) recognized MLO disease (Yellow Witches’ broom). Ishiie
et al. (1967) showed that the MLO bodies and the symptoms disappeared
temporarily when the plants were treated with tetracycline antibodies.
15. In 1971, Diener identified that the potato spindle tuber disease was caused by a
small (250–400 nucleotide long), single stranded circular molecule of infec-
tious RNA which he called a viroid.
16. Davis and Worley (1973) observed motile, helical microorganisms associated
with corn stunt disease and named it spiroplasma.
More details about history of plant virology can be found in reviews by
Scholthof (2001); Harrison (2009); Van der Want and Dijkstra (2006).
Disease and economic importance
Depending on the particular combination of virus and host, and on environmental
conditions, a plant’s response to infection may range from a symptomless con-
dition (latent infection) to severe disease and subsequent plant death. In some
cases, small necrotic or chlorotic spots called local lesions develop at the site of
infection. In most cases, viruses cause systemic infection and spread throughout
the whole plant. Typical leaf symptoms of viral diseases include mosaic patterns,
14 2 Viruses and Sub-Viral Agents

chlorotic or necrotic lesions, yellowing, stripes or streaks, vein clearing, vein

banding, and leaf rolling and curling. Symptoms on flower include deformation
and changes in the color of the flowers including dramatic color mosaics called
color breaking (e.g., tulip flower breaking). In fruit and vegetable crops, the
general symptoms produced are mosaic patterns, stunting, discoloration or mal-
formation, and chlorotic ringspots. Infected stems of plants, develop stem pitting
and grooving or tumors in response to virus infection.
The symptoms induced by plant viruses lead to reduced crop quality and yield.
The extent of these losses is demonstrated by the following three examples. Cacao
swollen shoot virus (CSSV) (Bowers et al. 2001) is estimated to cause an annual
loss of 50,000 tons of cocoa beans in Africa with an estimated value of
$28 million dollars. In South east Asia, infection of rice with rice tungroviruses
leads to an estimated annual loss of $1.5 billion dollars (Hull 2002). Tomato
spotted wilt virus (TSWV) infects a wide variety of plants including tomato,
peanuts, and tobacco (Sherwood et al. 2003), and the estimated annual losses due
to infection by this virus worldwide are estimated at $1 billion dollars (Hull 2002;
Thresh 2003).
The end result of virus infection is a reduction in plant growth, lower yield,
inferior product quality, and economic loss to individuals who work in the plant
industry. Most of the symptoms induced by viruses can also occur due to adverse
environmental conditions or diseases caused by other plant pathogens (viroids,
phytoplasmas). Because of this, correct diagnosis of viral diseases normally
requires laboratory tests (Hull 2002).
Harrison (2009) narrated the development of plant virology in the twentieth
century. These researchers independently described an unusual agent that caused
mosaic disease in tobacco (Zaitlin 1998). What distinguished this agent from other
disease-causing agents was its much smaller size compared to that of other
microbes. This agent, later named Tobacco mosaic virus (TMV), was the first virus
described and since then, a large number of diverse viruses have been found in
plants, animals, humans, fungi, and bacteria. The current estimate of recognized
viruses is 2284 of which about 1020 are plant viruses (King et al. 2012). The
majority of the virus diseases which are affecting crop plants are responsible for
heavy yield losses. Historically, viruses are perceived almost exclusively as a
health threat to humans, livestock, and crop plants. However, recent progress in
understanding virus-host interactions has transformed viruses into important
molecular tools (gene vectors, regulatory elements of transcription and translation)
of biomedicine and biotechnology. According to 9th ICTV report and subsequent
online updates, there are 349 genera and 2284 virus species (King et al. 2012).
Recently, Scholthof et al. (2011) have made a comprehensive review on the fol-
lowing top 10 ranking of plant viruses in the world. (1) Tobacco mosaic virus
(TMV), (2) Tomato spotted wilt virus (TSWV), (3) Tomato yellow leaf curl virus
(TYLCV), (4) Cucumber mosaic virus (CMV), (5) Potato virus Y (PVY), (6)
Cauliflower mosaic virus (CaMV), (7) African cassava mosaic virus (ACMV), (8)
Plum pox virus (PPV), (9) Brome mosaic virus (BMV) and (10) Potato virus X
(PVX). In addition to these top ten viruses, there are other plant viruses of global
2.2 Viruses 15

economic importance e.g., Citrus tristeza virus (CTV), Banana bunchy top virus
(BBTV), Barley yellow dwarf virus (BYDV), Potato leaf roll virus (PLRV),
Tomato bushy stunt virus (TBSV) and Rice tungro virus complex.
Rybicki (2012) based on his rich experience in plant viruses, he has listed the
following ten plant viruses in alphabetical order.
(1) African cassava mosaic begomovirus (ACMV) (Begomovirus complex)
(2) Banana bunchy top nano virus (BBTV)
(3) Banana streak badna virus (BSV)
(4) Barley yellow dwarf disease (BYDV) (Luteo virus complex)
(5) Cucumber mosaic cucumovirus (CMV)
(6) Maize streak masterovirus (MSV)
(7) Maize dwarf mosaic/Sugarcane mosaic potyviruses (MDMV/SCMV)
(8) Rice tungro disease complex (RTBV/RTSV)
(9) Rice yellow mottle sobemovirus (RYMV)
(10) Sweet potato feathery mottle potyvirus (SPFMV)
In addition to the above 10 viruses Rybicki (2012) has mentioned that tomato
begomoviruses are worldwide especially in Asia. Even tospoviruses are also
equally wide spread worldwide. In Brazil and some South American countries,
begomoviruses are economically important in vegetable crops. In Asia, various
potyviruses infecting vegetable crops are very important. In South east Asian
countries Rice tungro virus complex is the major limiting factor for successful rice
cultivation. In recent years Tomato yellow leaf curl virus and Tomato torrado virus
transmitted by Bemisia tabaci and Trialeurodes vaporariorium respectively are
causing heavy losses in tomato in majority of the countries.
Many of the above viruses occur in tropics and subtropics and inflict significant
losses in crops like cassava, tomato and potato. For instance, plant viruses are
being used to produce large quantities of proteins of interest in plants (Pogue et al.
2002) and to develop safe and inexpensive vaccines against human and animal
viruses (Walmsley and Arntzen 2000; Canizares et al. 2005; Grasso and Luca
2010). Some plant viruses like TMV, PVX and Brome mosaic virus (BMV) have
been exploited as model systems for varied purposes in plant biotechnology
(Scholthof 2004; Ding et al. 2006).
More details about plant virus and viroid diseases can be found from the following
sources: Hull (2002); Khan and Dijkstra (2002); Nayudu (2008); Mahy and Van
Regenmortel (2008); Ahlawat (2010). The following websites will also provide more
details of various aspects of plant virus and viroids viz., http://www.virology.net/
garryfavwebplant.html, http://www.dpvweb.net, http://www.vegetablemdonline.
ppath.cornell.edu/, http://www.actahort.org/, http://www.ncbi.nlm.nih.gov/ICTV
db/Ictv/fr-fst-g.htm, http://www.pk.uni-bonn.de/ppigb/, http://www.apsnet.org/
edcenter/intropp/PathogenGroups/Pages/PlantViruses.aspx, http://www.pvo.bio-
mirror.cn/refs.htm, http://www.isaaa.org/, http://www.q-bank.eu/Virus/. An over-
view of plant viruses with the basic concepts of virology like the structure of virus
particles, genome, pathogenicity, replication, and other aspects are briefly presented
here.
16 2 Viruses and Sub-Viral Agents

(a) Viral genome

Viruses are small enough to avoid our watchful eyes, hiding themselves in infected
hosts for most of the time and only occasionally reveal their presence by causing
symptoms. Plant viruses are a diverse group infecting hosts from unicellular plants
to trees. Viruses are ultramicroscopic and have genomes, either ribonucleic acid
(RNA) or deoxyribonucleic acid (DNA). The nucleic acid may be single stranded
(ss) or double stranded (ds) and it may be linear or circular. Each plant virus
consists of at least a nucleic acid and a protein. Some viruses consist of more than
one size of nucleic acid and proteins, and some of them contain enzymes or
membrane lipids. The majority of plant viruses possess single-stranded (ss),
positive-sense RNA genomes, and these viruses are called positive-strand RNA
viruses. Examples of the most economically important families of the positive-
strand RNA viruses are Bromoviridae, Secoviridae, Tymoviridae, Tombusviridae,
Virgaviridae, Alphaflexiviridae, Betaflexiviridae, Closteroviridae, Luteoviridae,
and Potyviridae. Relatively few plant viruses, exemplified by the families Bun-
yaviridae, Ophioviridae and Rhabdoviridae, possess negative-sense RNA gen-
omes. Reoviridae, Partitiviridae, Endornaviridae are the families of the double-
stranded (ds) RNA viruses. There is only one family of the plant viruses with
dsDNA genomes, the Caulimoviridae family or so-called pararetroviruses, and
replication of these viruses involves an RNA intermediate. The ssDNA viruses are
represented by the large and economically important family Geminiviridae and
also Nanoviridae. These viruses possess ssDNA genomes, and they have a dsDNA
intermediate in their life cycle (Hull 2002). The evolutionary relationships among
the positive-strand, negative-strand, and dsRNA viruses, as well as the pararet-
roviruses and ssDNA viruses appear to be extremely distant if not absent. Most
plant viruses (about 540) contain ssRNA, 40 contain dsRNA, 50 contain ssDNA
and about 30 contain dsDNA (Agrios 2005). Based on 9th ICTV classification
(King et al. 2012) genome nature and viruses (ssRNA, dsRNA, ssDNA, dsDNA)
along with systematic position are presented in Table 2.1. Additions have been
made as appears within the 2012 on line version (http://ictvonline.org/
virusTaxonomy.asp).
The total genome size of plant viruses ranges from just over 1 kb for satellite
viruses, which require helper virus for replication, and Nanoviruses (e.g., Banana
bunchy top virus) to 28.9 kb for members of Reoviridae (Sugarcane Fiji disease
virus). Nearly half of them are elongate (rigid rods or flexuous threads), and almost
as many are spherical (isometric or polyhedral), with the remaining being cylin-
drical bacillus like rods.
Many plant viruses have segmented genomes, consisting of two or more distinct
nucleic acid strands encapsidated in different sized particles made of the same
protein subunits. Viruses with segmented genome include Alfamoviruses, Brom-
oviruses, Bymoviruses, Comoviruses, Cryptoviruses, Dianthoviruses, Benyviruses,
Hordeiviruses, Ilarviruses, Nepoviruses, Tenuiviruses, Tobraviruses, Reoviruses,
Enamoviruses and Tospoviruses. Mandahar (1989) have reviewed the multi
component viruses. For example Tobacco rattle virus consists of 2 rods: a long one
(195 by 25 nm) and a shorter one (43 by 25 nm). They are also called as multi-
2.2 Viruses 17

Table 2.1 Nature of Genome, Families, Genera, and Species of Plant Viruses
Nature of genome Family or Genus Type species
unassigned
genus
(+) sense ssRNA Potyviridae Potyvirus Potato virus Y
Viruses Rymovirus Ryegrass mosaic virus
Macluravirus Maclura mosaic virus
Tritimovirus Wheat streak mosaic virus
Ipomovirus Sweet potato mild mottle virus
Bymovirus Barley yellow mosaic virus
Poacevirus Triticum mosaic virus
Brambyvirus Blackberry virus Y
Unassigned genus Spartina mottle virus
Unassigned genus Tomato mild mottle virus
Secoviridae Sequivirus Parsnip yellow fleck virus
Waikavirus Rice tungro spherical virus
Comovirus Cowpea mosaic virus
Fabavirus Broad bean wilt virus 1
Nepovirus Tobacco ringspot virus
Sadwavirus Satsuma dwarf virus
Cheravirus Cherry rasp leaf virus
Torradovirus Tomato torrado virus
Luteoviridae Luteovirus Barley yellow dwarf virus-PAV
Polerovirus Potato leafroll virus
Enamovirus Pea enation mosaic virus-1
Tymoviridae Tymovirus Turnip yellow mosaic virus
Marafivirus Maize rayado fino virus
Maculavirus Grapevine fleck virus
Tombusviridae Tombusvirus Tomato bushy stunt virus
Carmovirus Carnation mottle virus
Alphanecrovirus Tobacco necrosis virus A
Machlomovirus Maize chlorotic mottle virus
Dianthovirus Carnation ringspot virus
Avenavirus Oat chlorotic stunt virus
Aureusvirus Pothos latent virus
Panicovirus Panicum mosaic virus
Bromoviridae Bromovirus Brome mosaic virus
Alfamovirus Alfalfa mosaic virus
Cucumovirus Cucumber mosaic virus
Ilarvirus Tobacco streak virus
Oleavirus Olive latent virus 2
Closteroviridae Closterovirus Beet yellows virus
Crinivirus Lettuce infectious yellows virus
Ampelovirus Grapevine leafroll-associated
virus 3
Alphaflexiviridae Potexvirus Potato virus X
Allexivirus Shallot virus X
Mandarivirus Indian citrus ringspot virus
(continued)
18 2 Viruses and Sub-Viral Agents

Table 2.1 (continued)

Nature of genome Family or Genus Type species
unassigned
genus
Betaflexiviridae Carlavirus Carnation latent virus
Capillovirus Apple stem grooving virus
Trichovirus Apple chlorotic leaf spot virus
Foveavirus Apple stem pitting virus
Vitivirus Grapevine virus A
Virgaviridae Tobamovirus Tobacco mosaic virus
Tobravirus Tobacco rattle virus
Hordeivirus Barley stripe mosaic virus
Furovirus Soil-borne wheat mosaic virus
Pomovirus Potato mop-top virus
Pecluvirus Peanut clump virus
Unassigned Benyvirus Beet necrotic yellow vein virus
genera Sobemovirus Southern bean mosaic virus
Idaeovirus Raspberry bushy dwarf virus
Umbravirus Carrot mottle virus
(-) sense ssRNA Rhabdoviridae Cytorhabdovirus Lettuce necrotic yellows virus
Viruses Nucleorhabdovirus Potato yellow dwarf virus
Bunyaviridae Tospovirus Tomato spotted wilt virus
Ophioviridae Ophiovirus Citrus psorosis virus
Unassigned Tenuivirus Rice stripe virus
genera Varicosavirus Lettuce big-vein associated
virus
dsRNA viruses Reoviridae Phytoreovirus Wound tumor virus
Fijivirus Fiji disease virus
Oryzavirus Rice ragged stunt virus
Partitiviridae Alphacryptovirus White clover cryptic virus 1
Betacryptovirus White clover cryptic virus 2
Endornaviridae Endornavirus Vicia faba endornavirus
ssDNA Viruses Geminiviridae Mastrevirus Maize streak virus
Curtovirus Beet curly top virus
Topocuvirus Tomato pseudo-curly top virus
Begomovirus Bean golden mosaic virus
Nanoviridae Nanovirus Subterranean clover stunt virus
Babuvirus Banana bunchy top virus
dsDNA (Reverse Caulimoviridae Caulimovirus Cauliflower mosaic virus
transcribing Soymovirus Soybean chlorotic mottle virus
viruses) Cavemovirus Cassava vein mosaic virus
Petuvirus Petunia vein clearing virus
Badnavirus Commelina yellow mottle virus
Tungrovirus Rice tungro bacilliform virus
2.2 Viruses 19

partide viruses. However, there are many variations in the structure of the viral
genomes. Viruses have one or more protein coats or capsids surrounding their
perimeter. These capsid layers are composed of protein subunits and may be
composed of the same type or different types of protein.

(b) Basic biology

Viruses are fundamentally different from other pathogens. Unlike all other living
organisms, viruses are non-cellular. In contrast to cells, which multiply by dividing
into daughter cells, viruses assemble from pools of their structural components.
Mature virus particles are dormant; they come alive and reproduce only inside
infected cells. In other words, viruses are obligate parasites that cannot be culti-
vated using any growth media suitable for bacterial, fungal, plant or animal cell
types. All viruses lack protein-synthesizing and energy-producing apparatuses. As
a rule, virus particles are immobile outside the infected host; they rely on the aid of
other organisms (arthropods, nematodes, fungi) or the environment (water) for
their dissemination. The plant virus stability outside of its host cell is variable. For
example, TMV is stable for months to years whereas TSWV survives only for few
hours.
Regarding the biological function of viral components, the infectivity of viruses
is strictly the property of their genomic nucleic acid. The protein coat of a virus not
only provides a protective sheathing for the nucleic acid of the virus, but also plays
a vital role in determining vector transmissibility of a virus and the kinds of
symptoms it causes. Protein itself has no infectivity, but its presence generally
increases the infectivity of the nucleic acid. Among the various viral genes, one
distinguishes those encoding for a structural protein, the coat protein, and those
encoding for non-structural proteins such as the polymerase, the helicase, the
movement protein, the transmission protein (helper component), or the protease,
e.g., TMV genome.
Another important biological aspect in plant virology is of vector transmission.
The mode of transmission is a useful characteristic of some groups of plant
viruses. For example in the family Potyviridae, members of the largest genus
(Potyvirus) are transmitted by aphids, while viruses in the genera Rymovirus and
Tritimovirus are transmitted by mites of the genus Abacarus or Aceria respec-
tively, and those in the genus Ipomovirus are transmitted by whiteflies and those in
the genus Bymovirus by plasmodiphorids.

(c) Virus architecture

Viruses are the smallest among all known organisms. The shape and size of virions
distinguish rod-shaped, filamentous, icosahedral, or large enveloped particles. On
the other hand, viruses sharing the same shape and size are difficult to distinguish
by their appearance. There is a simple structural principle that applies to virtually
all viruses in their mature form. Virus particles (virions) are composed of two
principal parts, the genome that is made of nucleic acid (4–5 % in viruses with rod
or filamentous morphology, 15–45 % in case of icosahedral viruses), and a pro-
tective coat that is made of protein. A definite number of protein subunits present
20 2 Viruses and Sub-Viral Agents

Fig. 2.1 Families and Genera of viruses infecting plants. Courtesy MHV van Regenmortel
et al. eds., Virus Taxonomy: 7th Report of ICTV (Elsevier)

on surface of viruses are arranged spirally in the elongated viruses and packed on
the sides of the polyhedral particles of the spherical viruses. In cross section, the
elongated viruses appear as hollow tubes with the protein subunits forming the
outer coat and the nucleic acid, also arranged spirally, embedded between the inner
ends of two successive spirals of the protein subunits. In spherical viruses the
2.2 Viruses 21

visible shell consists of protein subunits, while the nucleic acid is inside the shell.
The viral proteins consist of amino acids. The amino acid content and sequence for
identical protein subunits of a given virus are constant, but vary for different
viruses. In addition, some virus particles (tospoviruses, plant rhabdoviruses) are
enveloped by an outer membrane containing lipids and proteins (lipoprotein
membrane). The enveloped spherical tospoviruses are slightly pleomorphic and
their diameter ranges 80–110nm. The protein coat of plant viruses (capsids) are
assembled in accord with one of the two fundamental types of symmetry (Fig. 2.1).
The first type of virion is helical (roughly elongated). The elongated viruses come
in two major variants, rigid rods (e.g., tobamoviruses) and flexuous filaments (e.g.,
potyviruses). Over 50% of known plant viruses are rod shaped and the length of
the particle is normally dependent on the genome, but it is usually between
300–500nm with a diameter of 15–20nm. Some of the filamentous viruses reach the
length of *2000nm (Closteroviruses). In both of these variants, the nucleic acid is
highly ordered: it assumes the same helical conformation as the proteinaceous
capsid. The second type of virus particle is icosahedral/roughly spherical (e.g.,
Cucumoviruses) and the general diameter will be 30nm. In cases where there is only
a single coat protein, the basic structure consists of 60 T subunits, where T is an
integer. Some viruses may have two coat proteins or associate to form an icosa-
hedral shape particle. In icosahedral virions, the genomic nucleic acid forms a
partially ordered ball inside the proteinaceous capsid. The smallest spherical virus is
Tobacco necrosis virus (15 nm in diameter); Nanoviruses (18–20 nm in diameter).
The diameter of plant Reoviruses and Caulimoviruses is 65–70 and 45–50 nm,
respectively. For instance, small spherical viruses may be difficult to distinguish
from each other and from plant ribosomes. The variations of this basic shape include
bacilliform virions (e.g., Badnaviruses, 300–400 9 95 nm). In geminate particles,
twin virions composed of two joined incomplete icosahedra (e.g., 18 9 30 nm) as
seen in geminiviruses. The icosahedral and elongated virions alike can self-
assemble in a test tube if the nucleic acid and protein subunits are incubated under
proper conditions (Rao 2006; Atabekov et al. 2007). The particle morphology of
some of the plant viruses is presented in Fig. 2.2.
Few viruses have their genome distributed in different particles (split genome) and
accordingly they are divided into monopartite (e.g., tobamoviruses, potexviruses),
bipartite (e.g., tobraviruses, pecluviruses), tripartite (e.g., hordeiviruses) or multi-
partite (e.g., Alfamoviruses, Phytoreoviruses, Nanoviruses). All the morphological
components are essential for the infectivity of these viruses (Hull 2008).
The properties used to distinguish the viruses are the type of nucleic acid in the
virus genome (single or double stranded DNA or RNA), the shape, size and
number of their particles and the presence or absence of an envelope around the
virus particles.
Viruses like geminiviruses, badnaviruses and phyto rhabdoviruses can be easily
identified based on their characteristic virion morphology (Zechmann and Zelling
2009). The rigid, rod-shaped TMV particle is 300 9 18 nm and consists of an
RNA genome of about 6,400 nucleotides encapsidated by 2,130 copies of the
TMV coat protein.
22 2 Viruses and Sub-Viral Agents

Fig. 2.2 Electron micrographs—particle morphology of different plant viruses. Source http://
www.virology.net/big picture book of viruses

(d) Pathogenicity
Generally different viruses elicit similar symptoms, but the disease phenotype can
provide only limited information for disease diagnosis. More specific and reliable
methods of virus identification are based on various properties of the viruses like
biological, physical, antigenic and molecular (Matthews 1993; Webster et al. 2004;
2.2 Viruses 23

Fig. 2.3 Symptoms of diseases induced by certain begomoviruses. Source http://

www.virology.net/big picture book of viruses

Nayudu 2008). Researchers during last four decades have precisely identifying the
unknown viruses mainly based on their host range, physical properties and bioas-
says using indicator plants. Some plant genera, such as Nicotiana tabacum
(tobacco), Vigna sinensis (cowpea), Phaseolus vulgaris cultivars (French bean) and
Chenopodium species are hosts for a number of viruses. Since the responses of these
plants to viral infections under greenhouse conditions are consistent and distinctive,
they are commonly used as indicator plants (Nayudu 2008). In virus infected plants,
two major types of responses are noticed. Local lesions, which are confined to
inoculated leaves (local lesion hosts), and systemic infections which produce
symptoms on leaves distant from the inoculation site (systemic hosts). Many plant
viruses are transmissible to indicator plants by means of mechanical transmission or
grafting. But for the past three decades plant viruses are identified and classified
based on host and symptoms, particle morphology, physico-chemical properties,
virus protein composition, virus NA sequence analysis, molecular tests and
other factors (Murphy et al. 1995; van Regenmortel et al. 2000; Fauquet et al. 2005;
King et al. 2012).
Plant viruses induce variety of systemic symptoms, sometimes they may be
diagnostic useful to identify the causal virus. Symptoms induced by most prevalent
begomoviruses and TSWV in tropics are given in Figs. 2.3 and 2.4.
In the tropics, the major fruit crops are banana, citrus, papaya, pineapple, grape,
passion fruit and avocado which are affected by virus and viroid diseases. The
symptoms of some of these diseases are shown in the Fig. 2.5. In some of the
virus-host combinations, the flowers, fruits/parts will show viral symptoms viz.,
24 2 Viruses and Sub-Viral Agents

Fig. 2.4 Symptoms induced by TSWV on different crops

leaves with varied degrees of mosaic symptoms, ring spots, yellowing, reduced
size, flower break/irregular colour streaks, fruits with reduced size, distortion,
blistering and some times ring spot symptoms.
2.2 Viruses 25

Fig. 2.5 Symptoms of virus diseases of fruit crops. Source www.virology.net/big picture book of
viruses

After mechanical sap inoculation in host range studies, some plants may not
show symptoms or virus multiplication for a period and the time interval is called
‘‘latency period’’ or ‘‘incubation period’’. After some time period, during which
the virus will replicate to a critical concentration, but will not induce symptoms, as
seen in Carnation latent virus. Whereas in some hosts at particular temperature,
the symptoms will disappear temporarily, but after some period, the symptoms will
be expressed as seen in Pea leafroll virus and Prune dwarf virus.

(e) Plant virus replication

The plant virus genome has the information for replication, assembly, movement,
various interactions with its host etc. However, the genome of nearly 80% of plant
viruses are made up of RNA. Most of the plant viruses are composed with ssRNA that
is the same (positive-sense) polarity as the messenger RNAs of the cell (e.g., viruses
belonging to families: Potyviridae, Bromoviridae, Virgaviridae, Tombusviridae,
Secoviridae, Luteoviridae, Tymoviridae, Closteroviridae, Alphaflexiviridae,
Betaflexiviridae) which follows the Baltimore strategy IV of virus genome
26 2 Viruses and Sub-Viral Agents

replication and expression. There are some RNA viruses which are ssRNA of neg-
ative polarity (e.g., viruses belonging to families: Rhabdoviridae, Bunyaviridae,
Ophioviridae). The basic mechanism of replication of positive sense RNA genome is
that the virus encoded replicase synthesizes a complementary negative strand by
using the positive strand as template and then new positive strands are synthesized
from the negative strand template through semi conservative mode of replication.
Synthesis of new RNA is from the 3’ and 5’ ends of the templates. Replication occurs
in a replication complex that comprises the templates, newly synthesized RNA, the
replicase and host factors. The positive and negative forms of the viral genome
contain the signals that control both the specificity and timing of their replication.
Sometimes the divided genome may be encapsidated either in single particle or
morphologically distinct particles.
Viruses do not produce any kind of reproductive structures and they multiply by
using host cell machinery. The life cycles start by penetration of the virion into the
cell and to replication sites in the cell. Plant viruses are unable to penetrate the
plant cuticle and cell wall, it is believed that the virion enters the cytoplasm of the
cell passively through wounds caused by mechanical damage to the cuticle and
cell wall or vector transmission. The next phase of virus infection is the partial or
complete removal of the coat protein of the virion in the cytoplasm. In the next
step, the cell mediates expression of the viral genome by providing a transcrip-
tional apparatus and a translation apparatus.
Replication of single stranded RNA viruses starts with the entry of the virions
in to the cell cytoplasm; the viral nucleic-acid is released from the coat protein and
induces the viral RNA polymerase. This enzyme is being utilized for the synthesis
of viral RNA as template and forms complimentary RNA. The initially synthe-
sized RNA is not viral RNA but a mirror image of that RNA (complimentary
copy). This complimentary RNA is temporarily connected to the viral strand and
synthesizes double stranded RNA, later it synthesizes the progeny viral RNA
which is a mirror image of negative strand. This cycle repeats for number of times
and synthesizes more positive sense RNA strands. As soon as the progeny viral
RNA is produced, some of these are translated to induce the protein molecules of
viruses. When progeny virus RNA and virus protein sub-units have been produced,
the RNA organizes the protein sub-units around it and the two are assembled
together to form the provirion in the cytoplasm.
All viruses must direct the formation of at least three types of proteins: repli-
cation proteins that are essential for nucleic acid production, structural proteins
that form the protein shell and other components (e.g., Vpg) contained in the
virions, and movement proteins that mediate virus transport between plant cells
(Hull 2002). The viral replication proteins combine with cellular proteins to
produce a complex of proteins that manufactures multiple copies of the virus
genome. These newly made genomes interact with the structural proteins to form
new virions. The DNA genomes are replicated in the nucleus and RNA genomes in
the cytoplasm of plant cells.
The next step in the virus replication cycle is movement of the virus into
neighboring cells. There are two basic routes by which a virus moves through the
2.2 Viruses 27

plant to give full systemic infection i.e., cell to cell movement and long distance
movement. Depending on the virus, the viral genome or the virions are transported
into neighboring cells through small channels called plasmodesmata. Many viruses
produce Movement Proteins (MP) that modify the plasmodesmata channels and
facilitate viral movement into neighboring cells. The process of cell-to-cell
movement is relatively slow: it takes from one to few hours for a virus to move to
the next cell. The successful translocation of virus to entire plant, needs to enter in
to the vascular system of the plant. The process of systemic or long-distance
transport normally proceeds through the phloem sieve elements where viruses
move passively with the flow of photosynthates. After quite rapid systemic spread
of the virus (centimeters per hour) in the phloem, the virus moves from the phloem
into surrounding cells where it reproduces and spreads by cell-to-cell movement.
The time between initial infection of one or a few cells and systemic infection of
the plant varies from few days to a few weeks depending on the type of virus, host
plant and environmental conditions. Transmission of the virus from one plant to
another completes the virus life cycle.
Some viruses have genome made up of dsRNA (e.g., viruses belonging to
families: Reoviridae, Partitiviridae, Endornaviridae). Some of the plant viruses
have genome that are composed of ssDNA (e.g., viruses belonging to families:
Geminiviridae, Nanoviridae). There are very few plant viruses belonging to
family: Caulimoviridae which have dsDNA genome. The genome may be either
linear (e.g., tobamoviruses; cucumoviruses) or circular (e.g., caulimoviruses,
geminiviruses, nanoviruses).
Since the virus replication steps vary for dsRNA, ssDNA and dsDNA viruses,
more details can be obtained from reviews and references (Hull 2002, 2008;
Mandahar 2006; Nayudu 2008; Laliberte and Sanfacon 2010).

(f) Plant-virus interactions

Plant viruses are capable of infecting virtually all species of cultivated and wild
plants. Either mechanically or through vectors or both. However, host ranges of
individual viruses vary from very narrow to very broad. For instance Citrus tris-
teza virus (CTV) infects only a few species in the Citrus genus, Similarly Banana
bunchytop virus (BBTV) and Cocoa swollen shoot virus (CSSV) have limited host
range, whereas CMV infects over 1000 species in 85 plant families. Even TSWV
infects over 925 plant species, including both monocots and dicots, belonging to
70 botanical families. Because of the wide host range, the viruses like CMV, PVY
and TSWV are worldwide in distribution and also have potential insect vectors.
Susceptibility or resistance of plant species and cultivars to viruses is determined
primarily by the plant genotype. Plants possess active and passive means of pre-
venting virus infection. Passive defenses are due to the failure of the plant to
produce one or more host factors required for virus reproduction and spread within
the host. Active defenses include detection and destruction of the virus-infected
cells due to the function of specific resistance genes in the plant. Normally,
resistance genes are active only against a particular virus. In addition, plants
possess a general defense system that is somewhat analogous to the animal
28 2 Viruses and Sub-Viral Agents

immune system. The major difference between the two is that the immune system
in animals targets a pathogen’s proteins, whereas the plant defense system, which
is called RNA silencing, detects and degrades viral RNAs (Wassenegger and
Pelissier 1998).
On the other hand, in some of the plant viruses due to their inherent genetic
differences and influence of the host and the environment, there will be synergistic
or antagonistic effects due to multiplication of two or more related and unrelated
viruses in the same host. The synergistic reaction will result into severe symptoms
and heavy yield losses. To quote few examples viz., in Sub-Saharan Africa (SSA)
the emerge of a new variant, EACMV-Ug is the result of recombination between
two distinct viruses, namely African cassava mosaic virus (ACMV) and East
African cassava mosaic virus (EACMV), and is responsible for epidemics in
Uganda (Zhou et al. 1997; Otim-Nape et al. 1997). Another example is Corn lethal
necrosis disease resulted from the combination of Maize chlorotic mottle virus
with Maize dwarf mosaic virus (MDMV) (Uyemoto 1983). In Africa, severe Sweet
potato virus disease (SPVd) which is due to infection of Sweet potato chlorotic
stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV) (Nome et al.
2007). In general when a susceptible crop is infected by a single virus, the impact
on yield losses will not be as great as when two viruses interact in the same host
(Goldberg and Brakke, 1987). Due to a synergistic reaction increased seed
transmission was noticed in certain virus-host combinations. For example seed
transmission of 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).
Almost all plant viruses can exhibit strainal variation and the strains may be
mild, moderate or severe. The antagonism results due to mild strains which
produces mild symptoms due to mild strains of plant viruses as seen in crops like
tomato, potato, sweet potato, cucumber, soybean, citrus, passion fruit, cocoa and
papaya and in these crops the cross protection aspect has been studied by different
workers (Balaraman 1981; Fraser 1998; Tripathi et al. 2008; Zhou and Zhou 2012).

(g) Plant viruses as agents of agroterrorism

The majority of people are aware of bioterrorism with regard to introduction of
severe strains of virus or insect vectors which transmit human and live stock
viruses resulting in high mortality/devastation. There are also very few examples
of viruses which are of crop devastating nature when a severe strain enters into a
new country in which this particular virus was not previously known.
In recent years attention has been given to the threat posed by the deliberate
introduction of plant pathogens that are not already present in a country, or of
novel and particularly aggressive strains of pathogens already present. However,
the risks are likely to be less than those posed by highly infectious viruses, or other
pathogens of humans or livestock (Gewin 2003). Moreover, wind-borne plant
pathogenic fungi seem to present a greater threat than plant viruses, which usually
spread less rapidly and require an insect or fungus vector.
2.2 Viruses 29

In certain countries of Africa and Asia in the earlier years, due to early infection
of plant viruses, there was total financial shortage and partial food shortage for
human and livelihood. On the other hand, the developed countries can compensate
for any losses incurred by imports and purchase of food grains from elsewhere.
There are very few opportunities for terrorists to exploit the ignorance of the
general public on plant pathological issues. This makes it easy to provide mis-
leading information and initiate unease and even panic or hysteria, especially by
targeting fresh fruit or vegetable crops intended for immediate consumption.
Out of the two possibilities of bioterrorism, one possibility is to introduce
particularly damaging strains of a plant virus or viruses that is/are already present,
but having relatively benign effects. The scope for adopting this approach is
apparent from the devastation caused by the recombinant strain of a Cassava
mosaic virus that appeared naturally and is causing food shortages in large regions
of East and Central Africa (Otim-Nape et al. 2000). Problems have also been
caused by particularly virulent strains of Citrus tristeza virus (Bar-Joseph et al.
1981) and by novel strains of Sugarcane mosaic virus that seriously damage
cultivars selected for their resistance to the strain(s) occurring previously (Thresh
1989). The chances for agro-terrorism is more in case of whitefly and thrips
transmitted gemini and tospoviruses respectively, when introduced into a new
areas. The last two viruses have very large number of new strains and variants as
seen in case of TLCV in tomato, YVMV in okra, CoLCV in cotton and tospovi-
ruses in vegetables. These two virus groups have very wide host-ranges. Even the
vectors of this two viruses viz., whitefly and thrips have number of biotypes.
Because of these factors wherever and whenever these viruses are introduced into
new areas, there are maximum chances of causing epidemics if susceptible host is
available. Clearly, considerable expertise will be required to select the most
appropriate viruses for this approach and to develop suitable strains by selection
from those occurring naturally, or following some sort of genetic manipulation.
The second possibility of agro-terrorism is the introducing an entirely new
vector or a novel biotype of an existing vector, which will spread virus diseases
and causes heavy losses. The consequence could be very damaging, as evident
from the apparent ease with which the western flower thrips, the brown citrus
aphid, and other aphids and the ‘‘B-biotype’’ of B. tabaci have become established
recently in new areas. However, there is again a requirement for expertise, rearing
facilities, an effective means of introduction and sufficient time for the vectors to
become established and build up damaging populations.
The bio-safety measures and strict quarantine rules and regulations in each
country, would certainly solved the problems that arise because of bioterrorism
due to the entry of new virulent virus/viroid and insect vectors. Generally plant
pathogens seem to pose a lesser threat than pathogens of humans and livestock.
These agents would undoubtedly have a greater and more immediate impact on
public sentiment, attitudes and actions, especially if reinforced by an effective
propaganda campaign designed to initiate panic and an irrational behavior and
response. The scope for this form of bioterrorism and the risks posed are discussed
by Madden and Wheelis (2003).
30 2 Viruses and Sub-Viral Agents

2.2.1 Virus Classification

Classification or taxonomy of plant viruses is a method of categorization of viruses

reported from different parts of the world and have two faces, systematic and
nomenclature.
Classification is the arrangement of biological entities into taxonomic catego-
ries (taxa) on the basis of similarities and/or relationships, whereas nomenclature
is the assignment of names to taxa according to international rules. To apply these
(apparently) simple concepts to viruses, the Virology Division of the International
Union of Microbiological Societies (IUMS) established, some 30 years ago, the
International Committee for the Nomenclature of Viruses (ICNV), whose name
was subsequently changed to the present International Committee on Taxonomy of
Viruses (ICTV).

(a) The objectives of the ICTV are

(i) to develop an internationally agreed taxonomy for viruses,
(ii) to develop internationally agreed names for virus taxa, including species and
subviral agents,
(iii) to communicate taxonomic decisions to the international community of
virologists and
(iv) to maintain an index of virus names.

More details of the history of virus taxonomy can be obtained from the review
articles of Gibbs (1969), Francki (1981), Matthews (1983), Martelli (1992), Mayo
and Brunt (2007).

(b) Systematics and background information

When we consider plant virus classification, we should remember that it was the
mid 1930s before the first plant virus was purified and characterized. Prior to this
time, most plant virologists named a virus based on the host plant in which it was
found and the type of major symptom that the virus caused in the plant. For
example, TMV was first described in tobacco in which it induces a mosaic pattern
in the leaves Virus names are usually shortened to an acronym, for example,
tobacco mosaic virus is shortened to TMV, and tomato spotted wilt virus is
shortened to TSWV. The names of many virus genera and families are derived
from an important virus within the family. For example, the family name
Bromoviridae is derived from Brome mosaic virus which is in this family.
As early as 1927 Johnson, drew attention to the need for a system of classifi-
cation and nomenclature of plant viruses. In the following four decades, many
schemes were introduced but none proved acceptable. Since little was known
about the intrinsic properties of the viruses, great weight was placed on characters
such as symptoms, host range, physical and chemical parameters.
After World War II, viruses were recognized as constraints for production
throughout the world. Legume virus researchers were among the first to
2.2 Viruses 31

standardize the techniques for identifying the viruses (Bos et al. 1960). An
International Working Group on Legume Viruses (IWGLV) was established in
1961 for the exchange of seeds, antisera and of information. The tentative list of
viruses reported from naturally infected leguminous plants animated further
worldwide assemblage of information in computerized form by the Australian
Virus Identification Data Exchange project (VIDE). Its microfiche publication on
VIDE viruses of legumes was soon followed by a printed version in 1983 (Boswell
and Gibbs 1983). Similar books on viruses of plants in Australia (1988) and of
tropical plants (1990) were succeeded by viruses of plants in 1996 which was also
distributed on the internet and later contributed to the database of ICTV.
Within the structure of the ICTV, plant virus taxonomic matters are first han-
dled by the Plant Virus Subcommittee (PVS) and there are similar subcommittees
concerned with viruses of vertebrates, bacteria, invertebrates, and fungi. Plant
virology is represented by the chairman of a subcommittee that itself consists of 19
study group chairs and eight other members. The study groups are concerned with
particular taxa or groups of taxa (e.g., the Potyviridae Study Group). The sub-
committee chair appoints the chair of the study groups, who then appoint study
group members as is appropriate.
Ideas for taxonomic change, either creation or modification of plant virus taxa,
or decisions about names of these taxa, usually originate in study group deliber-
ations. These ideas are scrutinized by the Plant Virus Subcommittee, largely for
their acceptability within the overall taxonomic scheme for plant viruses. If
approved, the proposals are then submitted to the Executive Committee members
who examine their acceptability in the context of all virus taxonomy. Approved
proposals are then put to the membership of ICTV for a final vote as to their
acceptability. After a favorable vote by ICTV, the proposals become part of the
taxonomic scheme for viruses. These decisions are then published in Virology
Division News in Archives of Virology and/or in the regular ICTV reports (e.g.,
van Regenmortel et al. 2000).
The president of the ICTV publishes a report every 3 years that has become the
standard handbook on virus taxonomy. The latest 9th ICTV report is published by
(King et al. 2012).
ICTV has been very active in the last 30 years, incrementally increasing the
number of taxa and virus names from 369 in 1985 to 7,881 in 2004 (Martelli
1997). Not only have the numbers increased exponentially (more than 20-fold), but
the complexity of virus nomenclature and taxonomy has become tremendously
complicated and controversial. However, the overall stability of this virus classi-
fication, established in 1962 (Lwoff et al. 1962), is quite remarkable in that, for
example, names of all genera and families established in the 1980s are still in use
till 2011. The advancement with the most impact was the definition of a virus
species (van Regenmortel et al. 1991; Mayo and Fauquet 2000), which still is not
fully understood by most virologists.
Taxonomic decisions are taken by ICTV, which is authorized by statutes
approved by the Virology Division of the International Union of Microbiological
Societies (Mayo and Pringle 1998; Mayo and Horzinek 1998 and references therein).
32 2 Viruses and Sub-Viral Agents

Thus, decisions are subjected to representative international scrutiny. The ICTV is

organized and advised by an Executive Committee that consists of 18 members: four
officers, chairs of six subcommittees representative of each major branch of virology,
and eight elected members.

(c) Nomenclature
Nomenclature is the assignment of names to taxa according to international rules.
To apply these (apparently) simple concepts to viruses, the Virology Division of
the IUMS established, some 30 years ago, the ICNV, whose name was subse-
quently changed to the present ICTV.

(d) Principles of nomenclature

The essential principles of virus nomenclature are to:
(i) aim for stability,
(ii) avoid or reject the use of names which might cause error or confusion and
(iii) avoid the unnecessary creation of names.

Nomenclature of viruses and sub-viral agents is independent of other biological

nomenclature. Virus and virus taxon nomenclature are recognized to have the status
of exceptions in the proposed International Code of Bio nomenclature (Bio Code).
The primary purpose of naming a taxon is to supply a means of referring to the
taxon, rather than to indicate the characters or history of the taxon. The application of
names of taxa is determined, explicitly or implicitly, by means of nomenclatural
types. The name of a taxon has no official status until it has been approved by ICTV.

(e) Generating the ratified list of taxa

Using Taxonomic Proposals Management System (TPMS), it will be possible to
generate a complete list of approved taxa at any time, and this will be possible either
alphabetically or taxonomically. Accepted taxa also will be presented according to
the order of presentation of virus taxonomy: dsDNA, ssDNA, rtDNA, rtRNA,
dsRNA, ssNRNA, ssPRNA, SAT (Satellites), VIR (Viroids), UN (unassigned).
Within each category, the families will be classified according to the order of
presentation of the virus. Taxonomy species will be assigned to a genus and clas-
sified alphabetically within the genus. The species taxa should comprise at least one
isolate but include as many as described.

(f) Rules about species

The definition of a virus species as a polythetic class means that all members of the
species do not have a single defining property in common that is necessary and
sufficient for class membership. It is also defined as a polythetic class of viruses
that constitutes a replicating lineage and occupies a particular ecological niche. It
is not appropriate, therefore, to search for an elusive, single property that would
define a virus species.
2.2 Viruses 33

Therefore, a virus species is defined as follows: A virus species is a polythetic

class of viruses that constitutes a replicating lineage and occupies a particular
ecological niche (van Regenmortel 1989, 1990).

(g) Construction of a species name

While dealing the virus taxonomy aspect, the names of orders, families, subfam-
ilies, and genera are always printed in italics and the first letters of the names are
capitalized. At its meeting in San Diego in March 1998, the Executive Committee
of the ICTV decided to extend this practice to the names of species taxa to clearly
indicate that the species name had been approved as the official, internationally
recognized name (Pringle 1998).
The order name shall be a single word ending in… virales.
The family name shall be a single word ending in… viridae.
The subfamily name shall be a single word ending in… virinae.
The genus name shall be a single word ending in… virus.

(h) Latin and binomial nomenclature in virus taxonomy

When one talks about the virus species, the new ICTV rules demand that it be
written ‘‘Tobacco mosaic virus’’, i.e. in italics and with a capital initial, whereas
Dr. Bos, in his textbook, chose instead to use the name ‘‘Tobacco mosaic tob-
amovirus’’, i.e. a binomial name written in lower case Roman characters and
without a capital initial.
The value of using italics is that it visibly reinforces the status of the corre-
sponding species as a taxonomic entity, i.e., a formal, abstract class, distinct from
the concrete viral objects that replicates and cause disease and that are written in
Roman characters. Only if it is necessary to draw attention to the taxonomic
position of the virus under study will it be necessary to refer to the official species
name written in italics. Even then, the official name need be given only once,
probably in the introduction or materials and methods sections (e.g., Cucumber
mosaic virus, genus Cucumovirus, family Bromoviridae). In publications written
in languages other than English, the use of italics for the English official species
name would also indicate the alien nature of the term. In such publications, the
common names of viruses will be those used in that language and not the English
names. The use of italicized English instead of italicized Latin for the names of
virus species reflects the emergence of English as the modern language of inter-
national scientific communication, and it also does away with the invidious task of
having to coin new Latin names for all virus species (van Regenmortel 2000).
By introducing italicized virus species names, the ICTV in no way intended to
replace the existing vernacular or common names of viruses written in Roman
characters (van Regenmortel 2000; van Regenmortel and Fauquet 2002a). The
viruses studied by virologists are concrete, disease-causing entities and not abstract
classes, and they should continue to be referred to by their common, non-italicized
names. As reiterated by Drebot et al. (2002), only the names of viral taxonomic
classes are written in italics, not the names of viruses. In scientific articles, authors
34 2 Viruses and Sub-Viral Agents

need to refer most of the time to the virus as a physical entity rather than as a
member of a taxonomic class. Therefore, the common name written in Roman
characters will most often be used; the species name, in italics, will appear only
once for the purpose of taxonomic placement of the virus being discussed.

(i) A proposed binomial nomenclature for virus species

For many years, some plant virologists have been using an unofficial binomial
system for referring to virus species (as well as to viruses). In this system, the
italicized word virus appearing at the end of the current official species name is
replaced by the genus name, which also ends in ‘‘-virus’’ (Gibbs 2000; Drebot
et al. 2002). Thus Tobacco mosaic virus becomes Tobacco mosaic tobamovirus
and Cucumber mosaic virus becomes Cucumber mosaic cucumovirus. The
advantage of such a system is that inclusion of the genus name in the species name
indicates relationships with other viruses and therefore provides additional infor-
mation about the properties of the members of the species.
Such a binomial system for species names would also have the advantage of
clearly distinguishing between the species name written in italics (Tobacco mosaic
tobamovirus) and the common, non-italicized virus name, measles virus. At
present, the distinction between the species name and the virus name in most cases
relies only on typography (i.e., Tobacco mosaic virus versus tobacco mosaic
virus), which can lead to confusion (van Regenmortel and Fauquet 2002a).
Whether nonlatinized binomials should become the official species names of
viruses has been debated within the ICTV for many years (Bos 1999; van Re-
genmortel 2000; van Regenmortel et al. 2000; van Regenmortel 2001; Drebot et al.
2002; Bos 2002). Although most plant virologists have favored the use of bino-
mials for many years (Albouy and Devergne 1998), to what extent human and
animal virologists would find the system acceptable has not been known. As the
ICTV strives to develop a universal system of nomenclature approved by all
virologists (Mayo and Horzinek 1998; Mayo and Brunt 2007), it is bound to move
cautiously before changing all the current, official names of virus species. Since
very few virologists express their views on matters of taxonomy (van Regenmortel
2000), successive ICTV Executive Committees have always found it difficult to
poll the representative opinion of virologists worldwide (Matthews 1983), and it is
not clear what sort of democratic process would satisfy those who criticize ICTV
decisions. During 2002, efforts were made to canvass virologists regarding their
acceptance of a binomial system of species names; the results of two ballots
showed that a sizeable majority (80–85 %) of the 250 virologists who expressed an
opinion were in favor of a binomial system (van Regenmortel and Fauquet 2002b;
Mayo 2002). The new ICTV Executive Committee established at the 12th Inter-
national Congress of Virology, held in Paris in July 2002, decided the introduction
of binomial names of virus species. A list of current virus species names, together
with their binomial equivalents, can be found on ICTV net (available from: URL:
http://www.danforthcenter.org/ILTAB/ICTVnet/). The details of the 1st to 9th
ICTV reports furnishing the number of approved orders, families, subfamilies,
genera, and virus species are furnished in the Table 2.2.
2.2 Viruses 35

Table 2.2 Virus taxa dealt in different ICTV reports

ICTV International Congress of Number of approved ‘families, sub References
report Virology held at families, groups/genera and species
First Helsinki, 1968 43 families and groups Wildy (1971)
Second Budapest, 1971 and 47 families and groups Fenner (1976)
Madrid, 1975
Third The Hague, 1978 50 families and groups Matthews
(1979)
Fourth Strasbourg, 1981 54 families and groups Matthews
(1982)
Fifth Sendai, 1984, Edmonton, 2420 viruses Francki et al.
1987, and Berlin, 1990 belonging to 73 (1991)
families or groups
Sixth Glasgow, 1993 1 order, Murphy et al.
50 families, 9 (1995)
subfamilies,
164 genera and
more than 3,600
virus species
Seventh Jerusalem, 1996 3 orders, van
63 families, Regenmortel
9 subfamilies, et al. (2000)
240 genera,
1550 species
Eighth Sydney, 1999 and Paris, 3 orders, Fauquet et al.
2002 73 families, (2005)
11 subfamilies,
289 genera and 1898 species
Nineth 2011 6 orders, King et al.
87 families, (2012)
19 subfamilies,
349 genera; and
2284 species

ICTV IXth report

The current ICTV IXth report lists 2284 species distributed amongst 349 genera,
19 sub-families, 87 families and six orders. More details are provided in Part II
about the unassigned viruses that provides information on a number of viruses that
have not yet been classified but which are probably representatives of new genera
and/or families (King et al. 2012). The 9th report is being published both as a book
and also online. ICTV expects to make regular updates to keep the online version
in step with the latest taxonomic decisions (see http://ictvonline.org/virus
Taxonomy.asp). Only the list of plant viruses included in the ICTV IXth report
(King et al. 2012) is presented in the following Table 2.3.
Each genus contains a type species (the representative used in defining the
genus) and often a number of other species. For each species, authors have been
asked to provide details of a single isolate, a characterized virus that is
Table 2.3 9TH ICTV Classification of plant viruses
36

Order Family Subfamily Genus Type species Species

Mononegavirales Rhabdoviridae Cytorhabdovirus 0 Barley yellow striate mosaic virus
Mononegavirales Rhabdoviridae Cytorhabdovirus 0 Broccoli necrotic yellows virus
Mononegavirales Rhabdoviridae Cytorhabdovirus 0 Festuca leaf streak virus
Mononegavirales Rhabdoviridae Cytorhabdovirus 1 Lettuce necrotic yellows virus
Mononegavirales Rhabdoviridae Cytorhabdovirus 0 Lettuce yellow mottle virus
Mononegavirales Rhabdoviridae Cytorhabdovirus 0 Northern cereal mosaic virus
Mononegavirales Rhabdoviridae Cytorhabdovirus 0 Sonchus virus
Mononegavirales Rhabdoviridae Cytorhabdovirus 0 Strawberry crinkle virus
Mononegavirales Rhabdoviridae Cytorhabdovirus 0 Wheat American striate mosaic virus
Mononegavirales Rhabdoviridae Nucleorhabdovirus 0 Datura yellow vein virus
Mononegavirales Rhabdoviridae Nucleorhabdovirus 0 Eggplant mottled dwarf virus
Mononegavirales Rhabdoviridae Nucleorhabdovirus 0 Maize fine streak virus
Mononegavirales Rhabdoviridae Nucleorhabdovirus 0 Maize Iranian mosaic virus
Mononegavirales Rhabdoviridae Nucleorhabdovirus 0 Maize mosaic virus
Mononegavirales Rhabdoviridae Nucleorhabdovirus 1 Potato yellow dwarf virus
Mononegavirales Rhabdoviridae Nucleorhabdovirus 0 Rice yellow stunt virus
Mononegavirales Rhabdoviridae Nucleorhabdovirus 0 Sonchus yellow net virus
Mononegavirales Rhabdoviridae Nucleorhabdovirus 0 Sowthistle yellow vein virus
Mononegavirales Rhabdoviridae Nucleorhabdovirus 0 Taro vein chlorosis virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Andean potato mottle virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Bean pod mottle virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Bean rugose mosaic virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Broad bean stain virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Broad bean true mosaic virus
Picornavirales Secoviridae Comovirinae Comovirus 1 Cowpea mosaic virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Cowpea severe mosaic virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Picornavirales Secoviridae Comovirinae Comovirus 0 Glycine mosaic virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Pea green mottle virus
2.2 Viruses

Picornavirales Secoviridae Comovirinae Comovirus 0 Pea mild mosaic virus

Picornavirales Secoviridae Comovirinae Comovirus 0 Quail pea mosaic virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Radish mosaic virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Red clover mottle virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Squash mosaic virus
Picornavirales Secoviridae Comovirinae Comovirus 0 Ullucus virus C
Picornavirales Secoviridae Comovirinae Fabavirus 1 Broad bean wilt virus 1
Picornavirales Secoviridae Comovirinae Fabavirus 0 Broad bean wilt virus 2
Picornavirales Secoviridae Comovirinae Fabavirus 0 Gentian mosaic virus
Picornavirales Secoviridae Comovirinae Fabavirus 0 Lamium mild mosaic virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Apricot latent ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Arabis mosaic virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Arracacha virus A
Picornavirales Secoviridae Comovirinae Nepovirus 0 Artichoke Aegean ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Artichoke Italian latent virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Artichoke yellow ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Beet ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Blackcurrant reversion virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Blueberry leaf mottle virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Cassava American latent virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Cassava green mottle virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Cherry leaf roll virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Chicory yellow mottle virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Cocoa necrosis virus
(continued)
37
Table 2.3 (continued)
38

Order Family Subfamily Genus Type species Species

Picornavirales Secoviridae Comovirinae Nepovirus 0 Crimson clover latent virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Cycas necrotic stunt virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Grapevine Anatolian ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Grapevine Bulgarian latent virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Grapevine chrome mosaic virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Grapevine deformation virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Grapevine fanleaf virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Grapevine Tunisian ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Hibiscus latent ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Lucerne Australian latent virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Melon mild mottle virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Mulberry ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Myrobalan latent ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Olive latent ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Peach rosette mosaic virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Potato black ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Potato virus U
Picornavirales Secoviridae Comovirinae Nepovirus 0 Raspberry ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 1 Tobacco ringspot virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Tomato black ring virus
Picornavirales Secoviridae Comovirinae Nepovirus 0 Tomato ringspot virus
Picornavirales Secoviridae Cheravirus 0 Apple latent spherical virus
Picornavirales Secoviridae Cheravirus 1 Cherry rasp leaf virus
Picornavirales Secoviridae Cheravirus 0 Stocky prune virus
Picornavirales Secoviridae Sadwavirus 1 Satsuma dwarf virus
Picornavirales Secoviridae Sequivirus 0 Carrot necrotic dieback virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Picornavirales Secoviridae Sequivirus 0 Dandelion yellow mosaic virus
Picornavirales Secoviridae Sequivirus 1 Parsnip yellow fleck virus
2.2 Viruses

Picornavirales Secoviridae Torradovirus 0 Tomato marchitez virus

Picornavirales Secoviridae Torradovirus 1 Tomato torrado virus
Picornavirales Secoviridae Unassigned 0 Black raspberry necrosis virus
Picornavirales Secoviridae Unassigned 0 Strawberry latent ringspot virus
Picornavirales Secoviridae Unassigned 0 Strawberry mottle virus
Picornavirales Secoviridae Waikavirus 0 Anthriscus yellows virus
Picornavirales Secoviridae Waikavirus 0 Maize chlorotic dwarf virus
Picornavirales Secoviridae Waikavirus 1 Rice tungro spherical virus
Tymovirales Alphaflexiviridae Allexivirus 0 Garlic mite-borne filamentous virus
Tymovirales Alphaflexiviridae Allexivirus 0 Garlic virus A
Tymovirales Alphaflexiviridae Allexivirus 0 Garlic virus B
Tymovirales Alphaflexiviridae Allexivirus 0 Garlic virus C
Tymovirales Alphaflexiviridae Allexivirus 0 Garlic virus D
Tymovirales Alphaflexiviridae Allexivirus 0 Garlic virus E
Tymovirales Alphaflexiviridae Allexivirus 0 Garlic virus X
Tymovirales Alphaflexiviridae Allexivirus 1 Shallot virus X
Tymovirales Alphaflexiviridae Lolavirus 1 Lolium latent virus
Tymovirales Alphaflexiviridae Mandarivirus 1 Indian citrus ringspot virus
Tymovirales Alphaflexiviridae Potexvirus 0 Alstroemeria virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Alternanthera mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 0 Asparagus virus 3
Tymovirales Alphaflexiviridae Potexvirus 0 Bamboo mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 0 Cactus virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Cassava common mosaic virus
(continued)
39
Table 2.3 (continued)
40

Order Family Subfamily Genus Type species Species

Tymovirales Alphaflexiviridae Potexvirus 0 Cassava virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Clover yellow mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 0 Commelina virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Cymbidium mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 0 Daphne virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Foxtail mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 0 Hosta virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Hydrangea ringspot virus
Tymovirales Alphaflexiviridae Potexvirus 0 Lettuce virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Lily virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Malva mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 0 Mint virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Narcissus mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 0 Nerine virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Opuntia virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Papaya mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 0 Pepino mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 0 Phaius virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Plantago asiatica mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 0 Plantago severe mottle virus
Tymovirales Alphaflexiviridae Potexvirus 0 Plantain virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Potato aucuba mosaic virus
Tymovirales Alphaflexiviridae Potexvirus 1 Potato virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Schlumbergera virus X
Tymovirales Alphaflexiviridae Potexvirus 0 Strawberry mild yellow edge virus
Tymovirales Alphaflexiviridae Potexvirus 0 Tamus red mosaic virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Tymovirales Alphaflexiviridae Potexvirus 0 Tulip virus X
Tymovirales Alphaflexiviridae Potexvirus 0 White clover mosaic virus
2.2 Viruses

Tymovirales Alphaflexiviridae Potexvirus 0 Zygocactus virus X

Tymovirales Betaflexiviridae Capillovirus 1 Apple stem grooving virus
Tymovirales Betaflexiviridae Capillovirus 0 Cherry virus A
Tymovirales Betaflexiviridae Carlavirus 0 Aconitum latent virus
Tymovirales Betaflexiviridae Carlavirus 0 American hop latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Blueberry scorch virus
Tymovirales Betaflexiviridae Carlavirus 0 Cactus virus 2
Tymovirales Betaflexiviridae Carlavirus 0 Caper latent virus
Tymovirales Betaflexiviridae Carlavirus 1 Carnation latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Chrysanthemum virus B
Tymovirales Betaflexiviridae Carlavirus 0 Cole latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Coleus vein necrosis virus
Tymovirales Betaflexiviridae Carlavirus 0 Cowpea mild mottle virus
Tymovirales Betaflexiviridae Carlavirus 0 Dandelion latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Daphne virus S
Tymovirales Betaflexiviridae Carlavirus 0 Elderberry symptomless virus
Tymovirales Betaflexiviridae Carlavirus 0 Garlic common latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Helenium virus S
Tymovirales Betaflexiviridae Carlavirus 0 Helleborus net necrosis virus
Tymovirales Betaflexiviridae Carlavirus 0 Honeysuckle latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Hop latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Hop mosaic virus
Tymovirales Betaflexiviridae Carlavirus 0 Hydrangea latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Kalanchoë latent virus
(continued)
41
Table 2.3 (continued)
42

Order Family Subfamily Genus Type species Species

Tymovirales Betaflexiviridae Carlavirus 0 Ligustrum necrotic ringspot virus
Tymovirales Betaflexiviridae Carlavirus 0 Lilac mottle virus
Tymovirales Betaflexiviridae Carlavirus 0 Lily symptomless virus
Tymovirales Betaflexiviridae Carlavirus 0 Melon yellowing-associated virus
Tymovirales Betaflexiviridae Carlavirus 0 Mulberry latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Muskmelon vein necrosis virus
Tymovirales Betaflexiviridae Carlavirus 0 Narcissus common latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Narcissus symptomless virus
Tymovirales Betaflexiviridae Carlavirus 0 Nerine latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Passiflora latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Pea streak virus
Tymovirales Betaflexiviridae Carlavirus 0 Poplar mosaic virus
Tymovirales Betaflexiviridae Carlavirus 0 Potato latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Potato virus M
Tymovirales Betaflexiviridae Carlavirus 0 Potato virus P
Tymovirales Betaflexiviridae Carlavirus 0 Potato virus S
Tymovirales Betaflexiviridae Carlavirus 0 Red clover vein mosaic virus
Tymovirales Betaflexiviridae Carlavirus 0 Shallot latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Sint-Jan’s onion latent virus
Tymovirales Betaflexiviridae Carlavirus 0 Strawberry pseudo mild yellow edge virus
Tymovirales Betaflexiviridae Carlavirus 0 Sweet potato chlorotic fleck virus
Tymovirales Betaflexiviridae Carlavirus 0 Verbena latent virus
Tymovirales Betaflexiviridae Citrivirus 1 Citrus leaf blotch virus
Tymovirales Betaflexiviridae Foveavirus 1 Apple stem pitting virus
Tymovirales Betaflexiviridae Foveavirus 0 Apricot latent virus
Tymovirales Betaflexiviridae Foveavirus 0 Grapevine rupestris stem pitting-associated virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Tymovirales Betaflexiviridae Foveavirus 0 Peach chlorotic mottle virus
Tymovirales Betaflexiviridae Tepovirus 1 Potato virus T
2.2 Viruses

Tymovirales Betaflexiviridae Trichovirus 1 Apple chlorotic leaf spot virus

Tymovirales Betaflexiviridae Trichovirus 0 Apricot pseudo-chlorotic leaf spot virus
Tymovirales Betaflexiviridae Trichovirus 0 Cherry mottle leaf virus
Tymovirales Betaflexiviridae Trichovirus 0 Grapevine berry inner necrosis virus
Tymovirales Betaflexiviridae Trichovirus 0 Peach mosaic virus
Tymovirales Betaflexiviridae Unassigned 0 African oil palm ringspot virus
Tymovirales Betaflexiviridae Unassigned 0 Banana mild mosaic virus
Tymovirales Betaflexiviridae Unassigned 0 Cherry green ring mottle virus
Tymovirales Betaflexiviridae Unassigned 0 Cherry necrotic rusty mottle virus
Tymovirales Betaflexiviridae Unassigned 0 Sugarcane striate mosaic-associated virus
Tymovirales Betaflexiviridae Vitivirus 1 Grapevine virus A
Tymovirales Betaflexiviridae Vitivirus 0 Grapevine virus B
Tymovirales Betaflexiviridae Vitivirus 0 Grapevine virus D
Tymovirales Betaflexiviridae Vitivirus 0 Grapevine virus E
Tymovirales Betaflexiviridae Vitivirus 0 Heracleum latent virus
Tymovirales Betaflexiviridae Vitivirus 0 Mint virus 2
Tymovirales Tymoviridae Unassigned 0 Poinsettia mosaic virus
Tymovirales Tymoviridae Maculavirus 1 Grapevine fleck virus
Tymovirales Tymoviridae Marafivirus 0 Bermuda grass etched-line virus
Tymovirales Tymoviridae Marafivirus 0 Blackberry virus S
Tymovirales Tymoviridae Marafivirus 0 Citrus sudden death-associated virus
Tymovirales Tymoviridae Marafivirus 0 Grapevine Syrah virus 1
Tymovirales Tymoviridae Marafivirus 1 Maize rayado fino virus
Tymovirales Tymoviridae Marafivirus 0 Oat blue dwarf virus
(continued)
43
Table 2.3 (continued)
44

Order Family Subfamily Genus Type species Species

Tymovirales Tymoviridae Marafivirus 0 Olive latent virus 3
Tymovirales Tymoviridae Tymovirus 0 Anagyris vein yellowing virus
Tymovirales Tymoviridae Tymovirus 0 Andean potato latent virus
Tymovirales Tymoviridae Tymovirus 0 Belladonna mottle virus
Tymovirales Tymoviridae Tymovirus 0 Cacao yellow mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Calopogonium yellow vein virus
Tymovirales Tymoviridae Tymovirus 0 Chayote mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Chiltepin yellow mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Clitoria yellow vein virus
Tymovirales Tymoviridae Tymovirus 0 Desmodium yellow mottle virus
Tymovirales Tymoviridae Tymovirus 0 Dulcamara mottle virus
Tymovirales Tymoviridae Tymovirus 0 Eggplant mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Erysimum latent virus
Tymovirales Tymoviridae Tymovirus 0 Kennedya yellow mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Melon rugose mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Nemesia ring necrosis virus
Tymovirales Tymoviridae Tymovirus 0 Okra mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Ononis yellow mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Passion fruit yellow mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Peanut yellow mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Petunia vein banding virus
Tymovirales Tymoviridae Tymovirus 0 Physalis mottle virus
Tymovirales Tymoviridae Tymovirus 0 Plantago mottle virus
Tymovirales Tymoviridae Tymovirus 0 Scrophularia mottle virus
Tymovirales Tymoviridae Tymovirus 1 Turnip yellow mosaic virus
Tymovirales Tymoviridae Tymovirus 0 Voandzeia necrotic mosaic virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Tymovirales Tymoviridae Tymovirus 0 Wild cucumber mosaic virus
Unassigned Avsunviroidae Avsunviroid 1 Avocado sunblotch viroid
2.2 Viruses

Unassigned Avsunviroidae Elaviroid 1 Eggplant latent viroid

Unassigned Avsunviroidae Pelamoviroid 0 Chrysanthemum chlorotic mottle viroid
Unassigned Avsunviroidae Pelamoviroid 1 Peach latent mosaic viroid
Unassigned Bromoviridae Alfamovirus 1 Alfalfa mosaic virus
Unassigned Bromoviridae Anulavirus 1 Pelargonium zonate spot virus
Unassigned Bromoviridae Bromovirus 0 Broad bean mottle virus
Unassigned Bromoviridae Bromovirus 1 Brome mosaic virus
Unassigned Bromoviridae Bromovirus 0 Cassia yellow blotch virus
Unassigned Bromoviridae Bromovirus 0 Cowpea chlorotic mottle virus
Unassigned Bromoviridae Bromovirus 0 Melandrium yellow fleck virus
Unassigned Bromoviridae Bromovirus 0 Spring beauty latent virus
Unassigned Bromoviridae Cucumovirus 1 Cucumber mosaic virus
Unassigned Bromoviridae Cucumovirus 0 Gayfeather mild mottle virus
Unassigned Bromoviridae Cucumovirus 0 Peanut stunt virus
Unassigned Bromoviridae Cucumovirus 0 Tomato aspermy virus
Unassigned Bromoviridae Ilarvirus 0 American plum line pattern virus
Unassigned Bromoviridae Ilarvirus 0 Apple mosaic virus
Unassigned Bromoviridae Ilarvirus 0 Asparagus virus 2
Unassigned Bromoviridae Ilarvirus 0 Blackberry chlorotic ringspot virus
Unassigned Bromoviridae Ilarvirus 0 Blueberry shock virus
Unassigned Bromoviridae Ilarvirus 0 Citrus leaf rugose virus
Unassigned Bromoviridae Ilarvirus 0 Citrus variegation virus
Unassigned Bromoviridae Ilarvirus 0 Elm mottle virus
Unassigned Bromoviridae Ilarvirus 0 Fragaria chiloensis latent virus
(continued)
45
Table 2.3 (continued)
46

Order Family Subfamily Genus Type species Species

Unassigned Bromoviridae Ilarvirus 0 Humulus japonicus latent virus
Unassigned Bromoviridae Ilarvirus 0 Lilac leaf chlorosis virus
Unassigned Bromoviridae Ilarvirus 0 Lilac ring mottle virus
Unassigned Bromoviridae Ilarvirus 0 Parietaria mottle virus
Unassigned Bromoviridae Ilarvirus 0 Prune dwarf virus
Unassigned Bromoviridae Ilarvirus 0 Prunus necrotic ringspot virus
Unassigned Bromoviridae Ilarvirus 0 Spinach latent virus
Unassigned Bromoviridae Ilarvirus 0 Strawberry necrotic shock virus
Unassigned Bromoviridae Ilarvirus 1 Tobacco streak virus
Unassigned Bromoviridae Ilarvirus 0 Tulare apple mosaic virus
Unassigned Bromoviridae Oleavirus 1 Olive latent virus 2
Unassigned Bunyaviridae Tospovirus 0 Groundnut bud necrosis virus
Unassigned Bunyaviridae Tospovirus 0 Groundnut ringspot virus
Unassigned Bunyaviridae Tospovirus 0 Groundnut yellow spot virus
Unassigned Bunyaviridae Tospovirus 0 Impatiens necrotic spot virus
Unassigned Bunyaviridae Tospovirus 0 Tomato chlorotic spot virus
Unassigned Bunyaviridae Tospovirus 1 Tomato spotted wilt virus
Unassigned Bunyaviridae Tospovirus 0 Watermelon silver mottle virus
Unassigned Bunyaviridae Tospovirus 0 Zucchini lethal chlorosis virus
Unassigned Caulimoviridae Badnavirus 0 Aglaonema bacilliform virus
Unassigned Caulimoviridae Badnavirus 0 Banana streak GF virus
Unassigned Caulimoviridae Badnavirus 0 Banana streak MY virus
Unassigned Caulimoviridae Badnavirus 0 Banana streak OL virus
Unassigned Caulimoviridae Badnavirus 0 Banana streak VN virus
Unassigned Caulimoviridae Badnavirus 0 Bougainvillea chlorotic vein banding virus
Unassigned Caulimoviridae Badnavirus 0 Cacao swollen shoot virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Caulimoviridae Badnavirus 0 Canna yellow mottle virus
Unassigned Caulimoviridae Badnavirus 0 Citrus yellow mosaic virus
2.2 Viruses

Unassigned Caulimoviridae Badnavirus 1 Commelina yellow mottle virus

Unassigned Caulimoviridae Badnavirus 0 Dioscorea bacilliform AL virus
Unassigned Caulimoviridae Badnavirus 0 Dioscorea bacilliform SN virus
Unassigned Caulimoviridae Badnavirus 0 Gooseberry vein banding associated virus
Unassigned Caulimoviridae Badnavirus 0 Grapevine vein clearing virus
Unassigned Caulimoviridae Badnavirus 0 Kalanchoë top-spotting virus
Unassigned Caulimoviridae Badnavirus 0 Pineapple bacilliform CO virus
Unassigned Caulimoviridae Badnavirus 0 Pineapple bacilliform ER virus
Unassigned Caulimoviridae Badnavirus 0 Piper yellow mottle virus
Unassigned Caulimoviridae Badnavirus 0 Rubus yellow net virus
Unassigned Caulimoviridae Badnavirus 0 Schefflera ringspot virus
Unassigned Caulimoviridae Badnavirus 0 Spiraea yellow leaf spot virus
Unassigned Caulimoviridae Badnavirus 0 Sugarcane bacilliform IM virus
Unassigned Caulimoviridae Badnavirus 0 Sugarcane bacilliform MO virus
Unassigned Caulimoviridae Badnavirus 0 Sweet potato pakakuy virus
Unassigned Caulimoviridae Badnavirus 0 Taro bacilliform virus
Unassigned Caulimoviridae Caulimovirus 0 Carnation etched ring virus
Unassigned Caulimoviridae Caulimovirus 1 Cauliflower mosaic virus
Unassigned Caulimoviridae Caulimovirus 0 Dahlia mosaic virus
Unassigned Caulimoviridae Caulimovirus 0 Figwort mosaic virus
Unassigned Caulimoviridae Caulimovirus 0 Horseradish latent virus
Unassigned Caulimoviridae Caulimovirus 0 Lamium leaf distortion virus
Unassigned Caulimoviridae Caulimovirus 0 Mirabilis mosaic virus
Unassigned Caulimoviridae Caulimovirus 0 Strawberry vein banding virus
(continued)
47
Table 2.3 (continued)
48

Order Family Subfamily Genus Type species Species

Unassigned Caulimoviridae Caulimovirus 0 Thistle mottle virus
Unassigned Caulimoviridae Cavemovirus 1 Cassava vein mosaic virus
Unassigned Caulimoviridae Cavemovirus 0 Sweet potato collusive virus
Unassigned Caulimoviridae Petuvirus 1 Petunia vein clearing virus
Unassigned Caulimoviridae Solendovirus 0 Sweet potato vein clearing virus
Unassigned Caulimoviridae Solendovirus 1 Tobacco vein clearing virus
Unassigned Caulimoviridae Soymovirus 0 Blueberry red ringspot virus
Unassigned Caulimoviridae Soymovirus 0 Cestrum yellow leaf curling virus
Unassigned Caulimoviridae Soymovirus 0 Peanut chlorotic streak virus
Unassigned Caulimoviridae Soymovirus 1 Soybean chlorotic mottle virus
Unassigned Caulimoviridae Tungrovirus 1 Rice tungro bacilliform virus
Unassigned Closteroviridae Ampelovirus 0 Grapevine leafroll-associated virus 1
Unassigned Closteroviridae Ampelovirus 1 Grapevine leafroll-associated virus 3
Unassigned Closteroviridae Ampelovirus 0 Grapevine leafroll-associated virus 5
Unassigned Closteroviridae Ampelovirus 0 Little cherry virus 2
Unassigned Closteroviridae Ampelovirus 0 Pineapple mealybug wilt-associated virus 1
Unassigned Closteroviridae Ampelovirus 0 Pineapple mealybug wilt-associated virus 2
Unassigned Closteroviridae Ampelovirus 0 Pineapple mealybug wilt-associated virus 3
Unassigned Closteroviridae Ampelovirus 0 Plum bark necrosis stem pitting-associated virus
Unassigned Closteroviridae Closterovirus 0 Beet yellow stunt virus
Unassigned Closteroviridae Closterovirus 1 Beet yellows virus
Unassigned Closteroviridae Closterovirus 0 Burdock yellows virus
Unassigned Closteroviridae Closterovirus 0 Carnation necrotic fleck virus
Unassigned Closteroviridae Closterovirus 0 Carrot yellow leaf virus
Unassigned Closteroviridae Closterovirus 0 Citrus tristeza virus
Unassigned Closteroviridae Closterovirus 0 Grapevine leafroll-associated virus 2
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Closteroviridae Closterovirus 0 Mint virus 1
Unassigned Closteroviridae Closterovirus 0 Raspberry leaf mottle virus
2.2 Viruses

Unassigned Closteroviridae Closterovirus 0 Strawberry chlorotic fleck-associated virus

Unassigned Closteroviridae Closterovirus 0 Wheat yellow leaf virus
Unassigned Closteroviridae Crinivirus 0 Abutilon yellows virus
Unassigned Closteroviridae Crinivirus 0 Bean yellow disorder virus
Unassigned Closteroviridae Crinivirus 0 Beet pseudoyellows virus
Unassigned Closteroviridae Crinivirus 0 Blackberry yellow vein-associated virus
Unassigned Closteroviridae Crinivirus 0 Cucurbit yellow stunting disorder virus
Unassigned Closteroviridae Crinivirus 0 Lettuce chlorosis virus
Unassigned Closteroviridae Crinivirus 1 Lettuce infectious yellows virus
Unassigned Closteroviridae Crinivirus 0 Potato yellow vein virus
Unassigned Closteroviridae Crinivirus 0 Strawberry pallidosis-associated virus
Unassigned Closteroviridae Crinivirus 0 Sweet potato chlorotic stunt virus
Unassigned Closteroviridae Crinivirus 0 Tomato chlorosis virus
Unassigned Closteroviridae Crinivirus 0 Tomato infectious chlorosis virus
Unassigned Closteroviridae Unassigned 0 Alligatorweed stunting virus
Unassigned Closteroviridae Unassigned 0 Grapevine leafroll-associated virus 7
Unassigned Closteroviridae Unassigned 0 Little cherry virus 1
Unassigned Closteroviridae Unassigned 0 Megakepasma mosaic virus
Unassigned Closteroviridae Unassigned 0 Mint vein banding-associated virus
Unassigned Closteroviridae Unassigned 0 Olive leaf yellowing-associated virus
Unassigned Endornaviridae Endornavirus 0 Oryza rufipogon endornavirus
Unassigned Endornaviridae Endornavirus 0 Oryza sativa endornavirus
Unassigned Endornaviridae Endornavirus 0 Phaseolus vulgaris endornavirus
Unassigned Endornaviridae Endornavirus 1 Vicia faba endornavirus
(continued)
49
Table 2.3 (continued)
50

Order Family Subfamily Genus Type species Species

Unassigned Geminiviridae Begomovirus 0 Abutilon mosaic virus
Unassigned Geminiviridae Begomovirus 0 African cassava mosaic virus
Unassigned Geminiviridae Begomovirus 0 Ageratum enation virus
Unassigned Geminiviridae Begomovirus 0 Ageratum leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Ageratum yellow vein Hualian virus
Unassigned Geminiviridae Begomovirus 0 Ageratum yellow vein Sri Lanka virus
Unassigned Geminiviridae Begomovirus 0 Ageratum yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Alternanthera yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Bean calico mosaic virus
Unassigned Geminiviridae Begomovirus 0 Bean dwarf mosaic virus
Unassigned Geminiviridae Begomovirus 0 Bean golden mosaic virus
Unassigned Geminiviridae Begomovirus 1 Bean golden yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Bhendi yellow vein mosaic virus
Unassigned Geminiviridae Begomovirus 0 Bitter gourd yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Boerhavia yellow spot virus
Unassigned Geminiviridae Begomovirus 0 Cabbage leaf curl Jamaica virus
Unassigned Geminiviridae Begomovirus 0 Cabbage leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Chayote yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Chilli leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Chino del tomate virus
Unassigned Geminiviridae Begomovirus 0 Clerodendron golden mosaic virus
Unassigned Geminiviridae Begomovirus 0 Corchorus golden mosaic virus
Unassigned Geminiviridae Begomovirus 0 Corchorus yellow spot virus
Unassigned Geminiviridae Begomovirus 0 Corchorus yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Cotton leaf crumple virus
Unassigned Geminiviridae Begomovirus 0 Cotton leaf curl Alabad virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Geminiviridae Begomovirus 0 Cotton leaf curl Bangalore virus
Unassigned Geminiviridae Begomovirus 0 Cotton leaf curl Gezira virus
2.2 Viruses

Unassigned Geminiviridae Begomovirus 0 Cotton leaf curl Kokhran virus

Unassigned Geminiviridae Begomovirus 0 Cotton leaf curl Multan virus
Unassigned Geminiviridae Begomovirus 0 Cowpea golden mosaic virus
Unassigned Geminiviridae Begomovirus 0 Croton yellow vein mosaic virus
Unassigned Geminiviridae Begomovirus 0 Cucurbit leaf crumple virus
Unassigned Geminiviridae Begomovirus 0 Desmodium leaf distortion virus
Unassigned Geminiviridae Begomovirus 0 Dicliptera yellow mottle Cuba virus
Unassigned Geminiviridae Begomovirus 0 Dicliptera yellow mottle virus
Unassigned Geminiviridae Begomovirus 0 Dolichos yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 East African cassava mosaic Cameroon virus
Unassigned Geminiviridae Begomovirus 0 East African cassava mosaic Kenya virus
Unassigned Geminiviridae Begomovirus 0 East African cassava mosaic Malawi virus
Unassigned Geminiviridae Begomovirus 0 East African cassava mosaic virus
Unassigned Geminiviridae Begomovirus 0 East African cassava mosaic Zanzibar virus
Unassigned Geminiviridae Begomovirus 0 Erectites yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Eupatorium yellow vein mosaic virus
Unassigned Geminiviridae Begomovirus 0 Eupatorium yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Euphorbia leaf curl Guangxi virus
Unassigned Geminiviridae Begomovirus 0 Euphorbia leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Euphorbia mosaic virus
Unassigned Geminiviridae Begomovirus 0 Hollyhock leaf crumple virus
Unassigned Geminiviridae Begomovirus 0 Honeysuckle yellow vein Kagoshima virus
Unassigned Geminiviridae Begomovirus 0 Honeysuckle yellow vein mosaic virus
Unassigned Geminiviridae Begomovirus 0 Honeysuckle yellow vein virus
(continued)
51
Table 2.3 (continued)
52

Order Family Subfamily Genus Type species Species

Unassigned Geminiviridae Begomovirus 0 Horsegram yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Indian cassava mosaic virus
Unassigned Geminiviridae Begomovirus 0 Ipomoea yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Kudzu mosaic virus
Unassigned Geminiviridae Begomovirus 0 Lindernia anagallis yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Ludwigia yellow vein Vietnam virus
Unassigned Geminiviridae Begomovirus 0 Ludwigia yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Luffa yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Macroptilium mosaic Puerto Rico virus
Unassigned Geminiviridae Begomovirus 0 Macroptilium yellow mosaic Florida virus
Unassigned Geminiviridae Begomovirus 0 Macroptilium yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Malvastrum leaf curl Guangdong virus
Unassigned Geminiviridae Begomovirus 0 Malvastrum leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Malvastrum yellow leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Malvastrum yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Malvastrum yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Malvastrum yellow vein Yunnan virus
Unassigned Geminiviridae Begomovirus 0 Melon chlorotic leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Merremia mosaic virus
Unassigned Geminiviridae Begomovirus 0 Mesta yellow vein mosaic virus
Unassigned Geminiviridae Begomovirus 0 Mimosa yellow leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Mungbean yellow mosaic India virus
Unassigned Geminiviridae Begomovirus 0 Mungbean yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Okra yellow crinkle virus
Unassigned Geminiviridae Begomovirus 0 Okra yellow mosaic Mexico virus
Unassigned Geminiviridae Begomovirus 0 Okra yellow mottle Iguala virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Geminiviridae Begomovirus 0 Okra yellow vein mosaic virus
Unassigned Geminiviridae Begomovirus 0 Papaya leaf curl China virus
2.2 Viruses

Unassigned Geminiviridae Begomovirus 0 Papaya leaf curl Guandong virus

Unassigned Geminiviridae Begomovirus 0 Papaya leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Pedilenthus leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Pepper golden mosaic virus
Unassigned Geminiviridae Begomovirus 0 Pepper huasteco yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Pepper leaf curl Bangladesh virus
Unassigned Geminiviridae Begomovirus 0 Pepper leaf curl Lahore virus
Unassigned Geminiviridae Begomovirus 0 Pepper leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Pepper yellow leaf curl Indonesia virus
Unassigned Geminiviridae Begomovirus 0 Pepper yellow vein Mali virus
Unassigned Geminiviridae Begomovirus 0 Potato yellow mosaic Panama virus
Unassigned Geminiviridae Begomovirus 0 Potato yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Pumpkin yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Radish leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Rhynchosia golden mosaic Sinaloa virus
Unassigned Geminiviridae Begomovirus 0 Rhynchosia golden mosaic virus
Unassigned Geminiviridae Begomovirus 0 Senecio yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Sida golden mosaic Costa Rica virus
Unassigned Geminiviridae Begomovirus 0 Sida golden mosaic Florida virus
Unassigned Geminiviridae Begomovirus 0 Sida golden mosaic Honduras virus
Unassigned Geminiviridae Begomovirus 0 Sida golden mosaic virus
Unassigned Geminiviridae Begomovirus 0 Sida golden yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Sida leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Sida micrantha mosaic virus
(continued)
53
Table 2.3 (continued)
54

Order Family Subfamily Genus Type species Species

Unassigned Geminiviridae Begomovirus 0 Sida mottle virus
Unassigned Geminiviridae Begomovirus 0 Sida yellow mosaic China virus
Unassigned Geminiviridae Begomovirus 0 Sida yellow mosaic virus
Unassigned Geminiviridae Begomovirus 0 Sida yellow mosaic Yucatan virus
Unassigned Geminiviridae Begomovirus 0 Sida yellow vein Madurai virus
Unassigned Geminiviridae Begomovirus 0 Sida yellow vein Vietnam virus
Unassigned Geminiviridae Begomovirus 0 Sida yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Siegesbeckia yellow vein Guangxi virus
Unassigned Geminiviridae Begomovirus 0 Siegesbeckia yellow vein virus
Unassigned Geminiviridae Begomovirus 0 South African cassava mosaic virus
Unassigned Geminiviridae Begomovirus 0 Soybean blistering mosaic virus
Unassigned Geminiviridae Begomovirus 0 Soybean crinkle leaf virus
Unassigned Geminiviridae Begomovirus 0 Spilanthes yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Squash leaf curl China virus
Unassigned Geminiviridae Begomovirus 0 Squash leaf curl Philippines virus
Unassigned Geminiviridae Begomovirus 0 Squash leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Squash leaf curl Yunnan virus
Unassigned Geminiviridae Begomovirus 0 Squash mild leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Sri Lankan cassava mosaic virus
Unassigned Geminiviridae Begomovirus 0 Stachytarpheta leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Sweet potato leaf curl Canary virus
Unassigned Geminiviridae Begomovirus 0 Sweet potato leaf curl China virus
Unassigned Geminiviridae Begomovirus 0 Sweet potato leaf curl Georgia virus
Unassigned Geminiviridae Begomovirus 0 Sweet potato leaf curl Lanzarote virus
Unassigned Geminiviridae Begomovirus 0 Sweet potato leaf curl Spain virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Geminiviridae Begomovirus 0 Sweet potato leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Tobacco curly shoot virus
2.2 Viruses

Unassigned Geminiviridae Begomovirus 0 Tobacco leaf curl Cuba virus

Unassigned Geminiviridae Begomovirus 0 Tobacco leaf curl Japan virus
Unassigned Geminiviridae Begomovirus 0 Tobacco leaf curl Yunnan virus
Unassigned Geminiviridae Begomovirus 0 Tobacco leaf curl Zimbabwe virus
Unassigned Geminiviridae Begomovirus 0 Tomato chino La Paz virus
Unassigned Geminiviridae Begomovirus 0 Tomato chlorotic mottle virus
Unassigned Geminiviridae Begomovirus 0 Tomato curly stunt virus
Unassigned Geminiviridae Begomovirus 0 Tomato golden mosaic virus
Unassigned Geminiviridae Begomovirus 0 Tomato golden mottle virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Arusha virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Bangalore virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Bangladesh virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl China virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Comoros virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Guangdong virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Guangxi virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Gujarat virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Hsinchu virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Java virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Joydebpur virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Karnataka virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Kerala virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Laos virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Madagascar virus
(continued)
55
Table 2.3 (continued)
56

Order Family Subfamily Genus Type species Species

Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Malaysia virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Mali virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Mayotte virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl New Delhi virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Philippines virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Pune virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Seychelles virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Sinaloa virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Sri Lanka virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Sudan virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Taiwan virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Uganda virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl Vietnam virus
Unassigned Geminiviridae Begomovirus 0 Tomato leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Tomato mild yellow leaf curl Aragua virus
Unassigned Geminiviridae Begomovirus 0 Tomato mosaic Havana virus
Unassigned Geminiviridae Begomovirus 0 Tomato mottle Taino virus
Unassigned Geminiviridae Begomovirus 0 Tomato mottle virus
Unassigned Geminiviridae Begomovirus 0 Tomato rugose mosaic virus
Unassigned Geminiviridae Begomovirus 0 Tomato severe leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Tomato severe rugose virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl Axarquia virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl China virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl Guangdong virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl Indonesia virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl Kanchanaburi virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl Malaga virus
2.2 Viruses

Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl Mali virus

Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl Sardinia virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl Thailand virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl Vietnam virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow margin leaf curl virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow spot virus
Unassigned Geminiviridae Begomovirus 0 Tomato yellow vein streak virus
Unassigned Geminiviridae Begomovirus 0 Vernonia yellow vein virus
Unassigned Geminiviridae Begomovirus 0 Watermelon chlorotic stunt virus
Unassigned Geminiviridae Curtovirus 0 Beet curly top Iran virus
Unassigned Geminiviridae Curtovirus 1 Beet curly top virus
Unassigned Geminiviridae Curtovirus 0 Beet mild curly top virus
Unassigned Geminiviridae Curtovirus 0 Beet severe curly top virus
Unassigned Geminiviridae Curtovirus 0 Horseradish curly top virus
Unassigned Geminiviridae Curtovirus 0 Pepper curly top virus
Unassigned Geminiviridae Curtovirus 0 Spinach curly top virus
Unassigned Geminiviridae Mastrevirus 0 Bean yellow dwarf virus
Unassigned Geminiviridae Mastrevirus 0 Chloris striate mosaic virus
Unassigned Geminiviridae Mastrevirus 0 Digitaria streak virus
Unassigned Geminiviridae Mastrevirus 0 Eragrostis streak virus
Unassigned Geminiviridae Mastrevirus 1 Maize streak virus
Unassigned Geminiviridae Mastrevirus 0 Miscanthus streak virus
Unassigned Geminiviridae Mastrevirus 0 Panicum streak virus
(continued)
57
Table 2.3 (continued)
58

Order Family Subfamily Genus Type species Species

Unassigned Geminiviridae Mastrevirus 0 Setaria streak virus
Unassigned Geminiviridae Mastrevirus 0 Sugarcane streak Egypt virus
Unassigned Geminiviridae Mastrevirus 0 Sugarcane streak Reunion virus
Unassigned Geminiviridae Mastrevirus 0 Sugarcane streak virus
Unassigned Geminiviridae Mastrevirus 0 Tobacco yellow dwarf virus
Unassigned Geminiviridae Mastrevirus 0 Urochloa streak virus
Unassigned Geminiviridae Mastrevirus 0 Wheat dwarf virus
Unassigned Geminiviridae Topocuvirus 1 Tomato pseudo-curly top virus
Unassigned Luteoviridae Enamovirus 1 Pea enation mosaic virus-1
Unassigned Luteoviridae Luteovirus 0 Barley yellow dwarf virus-MAV
Unassigned Luteoviridae Luteovirus 0 Barley yellow dwarf virus-PAS
Unassigned Luteoviridae Luteovirus 1 Barley yellow dwarf virus-PAV
Unassigned Luteoviridae Luteovirus 0 Bean leafroll virus
Unassigned Luteoviridae Luteovirus 0 Rose spring dwarf-associated virus
Unassigned Luteoviridae Luteovirus 0 Soybean dwarf virus
Unassigned Luteoviridae Polerovirus 0 Beet chlorosis virus
Unassigned Luteoviridae Polerovirus 0 Beet mild yellowing virus
Unassigned Luteoviridae Polerovirus 0 Beet western yellows virus
Unassigned Luteoviridae Polerovirus 0 Carrot red leaf virus
Unassigned Luteoviridae Polerovirus 0 Cereal yellow dwarf virus-RPS
Unassigned Luteoviridae Polerovirus 0 Cereal yellow dwarf virus-RPV
Unassigned Luteoviridae Polerovirus 0 Chickpea chlorotic stunt virus
Unassigned Luteoviridae Polerovirus 0 Cucurbit aphid-borne yellows virus
Unassigned Luteoviridae Polerovirus 0 Melon aphid-borne yellows virus
Unassigned Luteoviridae Polerovirus 1 Potato leafroll virus
Unassigned Luteoviridae Polerovirus 0 Sugarcane yellow leaf virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Luteoviridae Polerovirus 0 Tobacco vein distorting virus
Unassigned Luteoviridae Polerovirus 0 Turnip yellows virus
2.2 Viruses

Unassigned Luteoviridae Unassigned 0 Barley yellow dwarf virus-GPV

Unassigned Luteoviridae Unassigned 0 Barley yellow dwarf virus-RMV
Unassigned Luteoviridae Unassigned 0 Barley yellow dwarf virus-SGV
Unassigned Luteoviridae Unassigned 0 Chickpea stunt disease associated virus
Unassigned Luteoviridae Unassigned 0 Groundnut rosette assistor virus
Unassigned Luteoviridae Unassigned 0 Indonesian soybean dwarf virus
Unassigned Luteoviridae Unassigned 0 Sweet potato leaf speckling virus
Unassigned Luteoviridae Unassigned 0 Tobacco necrotic dwarf virus
Unassigned Nanoviridae Babuvirus 0 Abaca bunchy top virus
Unassigned Nanoviridae Babuvirus 1 Banana bunchy top virus
Unassigned Nanoviridae Babuvirus 0 Cardamom bushy dwarf virus
Unassigned Nanoviridae Nanovirus 0 Faba bean necrotic stunt virus
Unassigned Nanoviridae Nanovirus 0 Faba bean necrotic yellows virus
Unassigned Nanoviridae Nanovirus 0 Milk vetch dwarf virus
Unassigned Nanoviridae Nanovirus 0 Pea necrotic yellow dwarf virus
Unassigned Nanoviridae Nanovirus 1 Subterranean clover stunt virus
Unassigned Nanoviridae Unassigned 0 Coconut foliar decay virus
Unassigned Ophioviridae Ophiovirus 1 Citrus psorosis virus
Unassigned Ophioviridae Ophiovirus 0 Freesia sneak virus
Unassigned Ophioviridae Ophiovirus 0 Lettuce ring necrosis virus
Unassigned Ophioviridae Ophiovirus 0 Mirafiori lettuce big-vein virus
Unassigned Ophioviridae Ophiovirus 0 Ranunculus white mottle virus
Unassigned Ophioviridae Ophiovirus 0 Tulip mild mottle mosaic virus
Unassigned Partitiviridae Alphacryptovirus 0 Alfalfa cryptic virus 1
(continued)
59
Table 2.3 (continued)
60

Order Family Subfamily Genus Type species Species

Unassigned Partitiviridae Alphacryptovirus 0 Beet cryptic virus 1
Unassigned Partitiviridae Alphacryptovirus 0 Beet cryptic virus 2
Unassigned Partitiviridae Alphacryptovirus 0 Beet cryptic virus 3
Unassigned Partitiviridae Alphacryptovirus 0 Carnation cryptic virus 1
Unassigned Partitiviridae Alphacryptovirus 0 Carrot temperate virus 1
Unassigned Partitiviridae Alphacryptovirus 0 Carrot temperate virus 3
Unassigned Partitiviridae Alphacryptovirus 0 Carrot temperate virus 4
Unassigned Partitiviridae Alphacryptovirus 0 Hop trefoil cryptic virus 1
Unassigned Partitiviridae Alphacryptovirus 0 Hop trefoil cryptic virus 3
Unassigned Partitiviridae Alphacryptovirus 0 Radish yellow edge virus
Unassigned Partitiviridae Alphacryptovirus 0 Ryegrass cryptic virus
Unassigned Partitiviridae Alphacryptovirus 0 Spinach temperate virus
Unassigned Partitiviridae Alphacryptovirus 0 Vicia cryptic virus
Unassigned Partitiviridae Alphacryptovirus 1 White clover cryptic virus 1
Unassigned Partitiviridae Alphacryptovirus 0 White clover cryptic virus 3
Unassigned Partitiviridae Betacryptovirus 0 Carrot temperate virus 2
Unassigned Partitiviridae Betacryptovirus 0 Hop trefoil cryptic virus 2
Unassigned Partitiviridae Betacryptovirus 0 Red clover cryptic virus 2
Unassigned Partitiviridae Betacryptovirus 1 White clover cryptic virus 2
Unassigned Pospiviroidae Apscaviroid 0 Apple dimple fruit viroid
Unassigned Pospiviroidae Apscaviroid 1 Apple scar skin viroid
Unassigned Pospiviroidae Apscaviroid 0 Australian grapevine viroid
Unassigned Pospiviroidae Apscaviroid 0 Citrus bent leaf viroid
Unassigned Pospiviroidae Apscaviroid 0 Citrus dwarfing viroid
Unassigned Pospiviroidae Apscaviroid 0 Citrus viroid V
Unassigned Pospiviroidae Apscaviroid 0 Citrus viroid VI
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Pospiviroidae Apscaviroid 0 Grapevine yellow speckle viroid 1
Unassigned Pospiviroidae Apscaviroid 0 Grapevine yellow speckle viroid 2
2.2 Viruses

Unassigned Pospiviroidae Apscaviroid 0 Pear blister canker viroid

Unassigned Pospiviroidae Cocadviroid 0 Citrus bark cracking viroid
Unassigned Pospiviroidae Cocadviroid 1 Coconut cadang–cadang viroid
Unassigned Pospiviroidae Cocadviroid 0 Coconut tinangaja viroid
Unassigned Pospiviroidae Cocadviroid 0 Hop latent viroid
Unassigned Pospiviroidae Coleviroid 1 Coleus blumei viroid 1
Unassigned Pospiviroidae Coleviroid 0 Coleus blumei viroid 2
Unassigned Pospiviroidae Coleviroid 0 Coleus blumei viroid 3
Unassigned Pospiviroidae Hostuviroid 1 Hop stunt viroid
Unassigned Pospiviroidae Pospiviroid 0 Chrysanthemum stunt viroid
Unassigned Pospiviroidae Pospiviroid 0 Citrus exocortis viroid
Unassigned Pospiviroidae Pospiviroid 0 Columnea latent viroid
Unassigned Pospiviroidae Pospiviroid 0 Iresine viroid 1
Unassigned Pospiviroidae Pospiviroid 0 Mexican papita viroid
Unassigned Pospiviroidae Pospiviroid 0 Pepper chat fruit viroid
Unassigned Pospiviroidae Pospiviroid 1 Potato spindle tuber viroid
Unassigned Pospiviroidae Pospiviroid 0 Tomato apical stunt viroid
Unassigned Pospiviroidae Pospiviroid 0 Tomato chlorotic dwarf viroid
Unassigned Pospiviroidae Pospiviroid 0 Tomato planta macho viroid
Unassigned Potyviridae Brambyvirus 1 Blackberry virus Y
Unassigned Potyviridae Bymovirus 0 Barley mild mosaic virus
Unassigned Potyviridae Bymovirus 1 Barley yellow mosaic virus
Unassigned Potyviridae Bymovirus 0 Oat mosaic virus
(continued)
61
Table 2.3 (continued)
62

Order Family Subfamily Genus Type species Species

Unassigned Potyviridae Bymovirus 0 Rice necrosis mosaic virus
Unassigned Potyviridae Bymovirus 0 Wheat spindle streak mosaic virus
Unassigned Potyviridae Bymovirus 0 Wheat yellow mosaic virus
Unassigned Potyviridae Ipomovirus 0 Cassava brown streak virus
Unassigned Potyviridae Ipomovirus 0 Cucumber vein yellowing virus
Unassigned Potyviridae Ipomovirus 0 Squash vein yellowing virus
Unassigned Potyviridae Ipomovirus 1 Sweet potato mild mottle virus
Unassigned Potyviridae Ipomovirus 0 Ugandan cassava brown streak virus
Unassigned Potyviridae Macluravirus 0 Alpinia mosaic virus
Unassigned Potyviridae Macluravirus 0 Cardamom mosaic virus
Unassigned Potyviridae Macluravirus 0 Chinese yam necrotic mosaic virus
Unassigned Potyviridae Macluravirus 1 Maclura mosaic virus
Unassigned Potyviridae Macluravirus 0 Narcissus latent virus
Unassigned Potyviridae Macluravirus 0 Ranunculus latent virus
Unassigned Potyviridae Poacevirus 0 Sugarcane streak mosaic virus
Unassigned Potyviridae Poacevirus 1 Triticum mosaic virus
Unassigned Potyviridae Potyvirus 0 Algerian watermelon mosaic virus
Unassigned Potyviridae Potyvirus 0 Alstroemeria mosaic virus
Unassigned Potyviridae Potyvirus 0 Alternanthera mild mosaic virus
Unassigned Potyviridae Potyvirus 0 Amaranthus leaf mottle virus
Unassigned Potyviridae Potyvirus 0 Amazon lily mosaic virus
Unassigned Potyviridae Potyvirus 0 Angelica virus Y
Unassigned Potyviridae Potyvirus 0 Apium virus Y
Unassigned Potyviridae Potyvirus 0 Araujia mosaic virus
Unassigned Potyviridae Potyvirus 0 Arracacha mottle virus
Unassigned Potyviridae Potyvirus 0 Artichoke latent virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Potyviridae Potyvirus 0 Asparagus virus 1
Unassigned Potyviridae Potyvirus 0 Banana bract mosaic virus
2.2 Viruses

Unassigned Potyviridae Potyvirus 0 Basella rugose mosaic virus

Unassigned Potyviridae Potyvirus 0 Bean common mosaic necrosis virus
Unassigned Potyviridae Potyvirus 0 Bean common mosaic virus
Unassigned Potyviridae Potyvirus 0 Bean yellow mosaic virus
Unassigned Potyviridae Potyvirus 0 Beet mosaic virus
Unassigned Potyviridae Potyvirus 0 Bidens mottle virus
Unassigned Potyviridae Potyvirus 0 Brugmansia suaveolens mottle virus
Unassigned Potyviridae Potyvirus 0 Butterfly flower mosaic virus
Unassigned Potyviridae Potyvirus 0 Calanthe mild mosaic virus
Unassigned Potyviridae Potyvirus 0 Canna yellow streak virus
Unassigned Potyviridae Potyvirus 0 Carnation vein mottle virus
Unassigned Potyviridae Potyvirus 0 Carrot thin leaf virus
Unassigned Potyviridae Potyvirus 0 Carrot virus Y
Unassigned Potyviridae Potyvirus 0 Celery mosaic virus
Unassigned Potyviridae Potyvirus 0 Ceratobium mosaic virus
Unassigned Potyviridae Potyvirus 0 Chilli ringspot virus
Unassigned Potyviridae Potyvirus 0 Chilli veinal mottle virus
Unassigned Potyviridae Potyvirus 0 Chinese artichoke mosaic virus
Unassigned Potyviridae Potyvirus 0 Clitoria virus Y
Unassigned Potyviridae Potyvirus 0 Clover yellow vein virus
Unassigned Potyviridae Potyvirus 0 Cocksfoot streak virus
Unassigned Potyviridae Potyvirus 0 Colombian datura virus
Unassigned Potyviridae Potyvirus 0 Commelina mosaic virus
Unassigned Potyviridae Potyvirus 0 Cowpea aphid-borne mosaic virus
(continued)
63
Table 2.3 (continued)
64

Order Family Subfamily Genus Type species Species

Unassigned Potyviridae Potyvirus 0 Cowpea green vein banding virus
Unassigned Potyviridae Potyvirus 0 Cypripedium virus Y
Unassigned Potyviridae Potyvirus 0 Daphne mosaic virus
Unassigned Potyviridae Potyvirus 0 Dasheen mosaic virus
Unassigned Potyviridae Potyvirus 0 Datura shoestring virus
Unassigned Potyviridae Potyvirus 0 Diuris virus Y
Unassigned Potyviridae Potyvirus 0 East Asian Passiflora virus
Unassigned Potyviridae Potyvirus 0 Endive necrotic mosaic virus
Unassigned Potyviridae Potyvirus 0 Euphorbia ringspot virus
Unassigned Potyviridae Potyvirus 0 Freesia mosaic virus
Unassigned Potyviridae Potyvirus 0 Fritillary virus Y
Unassigned Potyviridae Potyvirus 0 Gloriosa stripe mosaic virus
Unassigned Potyviridae Potyvirus 0 Groundnut eyespot virus
Unassigned Potyviridae Potyvirus 0 Guinea grass mosaic virus
Unassigned Potyviridae Potyvirus 0 Hardenbergia mosaic virus
Unassigned Potyviridae Potyvirus 0 Helenium virus Y
Unassigned Potyviridae Potyvirus 0 Henbane mosaic virus
Unassigned Potyviridae Potyvirus 0 Hibbertia virus Y
Unassigned Potyviridae Potyvirus 0 Hippeastrum mosaic virus
Unassigned Potyviridae Potyvirus 0 Hyacinth mosaic virus
Unassigned Potyviridae Potyvirus 0 Iris fulva mosaic virus
Unassigned Potyviridae Potyvirus 0 Iris mild mosaic virus
Unassigned Potyviridae Potyvirus 0 Iris severe mosaic virus
Unassigned Potyviridae Potyvirus 0 Japanese yam mosaic virus
Unassigned Potyviridae Potyvirus 0 Johnsongrass mosaic virus
Unassigned Potyviridae Potyvirus 0 Kalanchoë mosaic virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Potyviridae Potyvirus 0 Konjac mosaic virus
Unassigned Potyviridae Potyvirus 0 Leek yellow stripe virus
2.2 Viruses

Unassigned Potyviridae Potyvirus 0 Lettuce mosaic virus

Unassigned Potyviridae Potyvirus 0 Lily mottle virus
Unassigned Potyviridae Potyvirus 0 Lycoris mild mottle virus
Unassigned Potyviridae Potyvirus 0 Maize dwarf mosaic virus
Unassigned Potyviridae Potyvirus 0 Malva vein clearing virus
Unassigned Potyviridae Potyvirus 0 Meadow saffron breaking virus
Unassigned Potyviridae Potyvirus 0 Moroccan watermelon mosaic virus
Unassigned Potyviridae Potyvirus 0 Narcissus degeneration virus
Unassigned Potyviridae Potyvirus 0 Narcissus late season yellows virus
Unassigned Potyviridae Potyvirus 0 Narcissus yellow stripe virus
Unassigned Potyviridae Potyvirus 0 Nerine yellow stripe virus
Unassigned Potyviridae Potyvirus 0 Nothoscordum mosaic virus
Unassigned Potyviridae Potyvirus 0 Onion yellow dwarf virus
Unassigned Potyviridae Potyvirus 0 Ornithogalum mosaic virus
Unassigned Potyviridae Potyvirus 0 Ornithogalum virus 2
Unassigned Potyviridae Potyvirus 0 Ornithogalum virus 3
Unassigned Potyviridae Potyvirus 0 Papaya leaf distortion mosaic virus
Unassigned Potyviridae Potyvirus 0 Papaya ringspot virus
Unassigned Potyviridae Potyvirus 0 Parsnip mosaic virus
Unassigned Potyviridae Potyvirus 0 Passiflora chlorosis virus
Unassigned Potyviridae Potyvirus 0 Passion fruit woodiness virus
Unassigned Potyviridae Potyvirus 0 Pea seed-borne mosaic virus
Unassigned Potyviridae Potyvirus 0 Peanut mottle virus
Unassigned Potyviridae Potyvirus 0 Pennisetum mosaic virus
(continued)
65
Table 2.3 (continued)
66

Order Family Subfamily Genus Type species Species

Unassigned Potyviridae Potyvirus 0 Pepper mottle virus
Unassigned Potyviridae Potyvirus 0 Pepper severe mosaic virus
Unassigned Potyviridae Potyvirus 0 Pepper veinal mottle virus
Unassigned Potyviridae Potyvirus 0 Pepper yellow mosaic virus
Unassigned Potyviridae Potyvirus 0 Peru tomato mosaic virus
Unassigned Potyviridae Potyvirus 0 Pfaffia mosaic virus
Unassigned Potyviridae Potyvirus 0 Pleione virus Y
Unassigned Potyviridae Potyvirus 0 Plum pox virus
Unassigned Potyviridae Potyvirus 0 Pokeweed mosaic virus
Unassigned Potyviridae Potyvirus 0 Potato virus A
Unassigned Potyviridae Potyvirus 0 Potato virus V
Unassigned Potyviridae Potyvirus 1 Potato virus Y
Unassigned Potyviridae Potyvirus 0 Ranunculus leaf distortion virus
Unassigned Potyviridae Potyvirus 0 Ranunculus mild mosaic virus
Unassigned Potyviridae Potyvirus 0 Ranunculus mosaic virus
Unassigned Potyviridae Potyvirus 0 Rhopalanthe virus Y
Unassigned Potyviridae Potyvirus 0 Sarcochilus virus Y
Unassigned Potyviridae Potyvirus 0 Scallion mosaic virus
Unassigned Potyviridae Potyvirus 0 Shallot yellow stripe virus
Unassigned Potyviridae Potyvirus 0 Sorghum mosaic virus
Unassigned Potyviridae Potyvirus 0 Soybean mosaic virus
Unassigned Potyviridae Potyvirus 0 Spiranthes mosaic virus 3
Unassigned Potyviridae Potyvirus 0 Sugarcane mosaic virus
Unassigned Potyviridae Potyvirus 0 Sunflower chlorotic mottle virus
Unassigned Potyviridae Potyvirus 0 Sunflower mosaic virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Potyviridae Potyvirus 0 Sweet potato feathery mottle virus
Unassigned Potyviridae Potyvirus 0 Sweet potato latent virus
2.2 Viruses

Unassigned Potyviridae Potyvirus 0 Sweet potato mild speckling virus

Unassigned Potyviridae Potyvirus 0 Sweet potato virus 2
Unassigned Potyviridae Potyvirus 0 Sweet potato virus C
Unassigned Potyviridae Potyvirus 0 Sweet potato virus G
Unassigned Potyviridae Potyvirus 0 Telfairia mosaic virus
Unassigned Potyviridae Potyvirus 0 Telosma mosaic virus
Unassigned Potyviridae Potyvirus 0 Thunberg fritillary mosaic virus
Unassigned Potyviridae Potyvirus 0 Tobacco etch virus
Unassigned Potyviridae Potyvirus 0 Tobacco vein banding mosaic virus
Unassigned Potyviridae Potyvirus 0 Tobacco vein mottling virus
Unassigned Potyviridae Potyvirus 0 Tradescantia mild mosaic virus
Unassigned Potyviridae Potyvirus 0 Tropaeolum mosaic virus
Unassigned Potyviridae Potyvirus 0 Tuberose mild mosaic virus
Unassigned Potyviridae Potyvirus 0 Tuberose mild mottle virus
Unassigned Potyviridae Potyvirus 0 Tulip breaking virus
Unassigned Potyviridae Potyvirus 0 Tulip mosaic virus
Unassigned Potyviridae Potyvirus 0 Turnip mosaic virus
Unassigned Potyviridae Potyvirus 0 Twisted-stalk chlorotic streak virus
Unassigned Potyviridae Potyvirus 0 Vallota mosaic virus
Unassigned Potyviridae Potyvirus 0 Watermelon leaf mottle virus
Unassigned Potyviridae Potyvirus 0 Watermelon mosaic virus
Unassigned Potyviridae Potyvirus 0 Wild potato mosaic virus
Unassigned Potyviridae Potyvirus 0 Wild tomato mosaic virus
Unassigned Potyviridae Potyvirus 0 Wisteria vein mosaic virus
(continued)
67
Table 2.3 (continued)
68

Order Family Subfamily Genus Type species Species

Unassigned Potyviridae Potyvirus 0 Yam mild mosaic virus
Unassigned Potyviridae Potyvirus 0 Yam mosaic virus
Unassigned Potyviridae Potyvirus 0 Yambean mosaic virus
Unassigned Potyviridae Potyvirus 0 Zantedeschia mild mosaic virus
Unassigned Potyviridae Potyvirus 0 Zea mosaic virus
Unassigned Potyviridae Potyvirus 0 Zucchini yellow fleck virus
Unassigned Potyviridae Potyvirus 0 Zucchini yellow mosaic virus
Unassigned Potyviridae Rymovirus 0 Agropyron mosaic virus
Unassigned Potyviridae Rymovirus 0 Hordeum mosaic virus
Unassigned Potyviridae Rymovirus 1 Ryegrass mosaic virus
Unassigned Potyviridae Tritimovirus 0 Brome streak mosaic virus
Unassigned Potyviridae Tritimovirus 0 Oat necrotic mottle virus
Unassigned Potyviridae Tritimovirus 0 Wheat eqlid mosaic virus
Unassigned Potyviridae Tritimovirus 1 Wheat streak mosaic virus
Unassigned Potyviridae Unassigned 0 Spartina mottle virus
Unassigned Potyviridae Unassigned 0 Tomato mild mottle virus
Unassigned Pseudoviridae Pseudovirus 0 Arabidopsis thaliana Art1 virus
Unassigned Pseudoviridae Pseudovirus 0 Arabidopsis thaliana AtRE1 virus
Unassigned Pseudoviridae Pseudovirus 0 Arabidopsis thaliana Evelknievel virus
Unassigned Pseudoviridae Pseudovirus 0 Arabidopsis thaliana Ta1 virus
Unassigned Pseudoviridae Pseudovirus 0 Brassica oleracea Melmoth virus
Unassigned Pseudoviridae Pseudovirus 0 Cajanus cajan Panzee virus
Unassigned Pseudoviridae Pseudovirus 0 Glycine max Tgmr virus
Unassigned Pseudoviridae Pseudovirus 0 Hordeum vulgare BARE-1 virus
Unassigned Pseudoviridae Pseudovirus 0 Nicotiana tabacum Tnt1 virus
Unassigned Pseudoviridae Pseudovirus 0 Nicotiana tabacum Tto1 virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Pseudoviridae Pseudovirus 0 Oryza australiensis RIRE1 virus
Unassigned Pseudoviridae Pseudovirus 0 Oryza longistaminata Retrofit virus
2.2 Viruses

Unassigned Pseudoviridae Pseudovirus 0 Solanum tuberosum Tst1 virus

Unassigned Pseudoviridae Pseudovirus 0 Triticum aestivum WIS-2 virus
Unassigned Pseudoviridae Pseudovirus 0 Zea mays Hopscotch virus
Unassigned Pseudoviridae Pseudovirus 0 Zea mays Sto-4 virus
Unassigned Pseudoviridae Sirevirus 0 Arabidopsis thaliana Endovir virus
Unassigned Pseudoviridae Sirevirus 1 Glycine max SIRE1 virus
Unassigned Pseudoviridae Sirevirus 0 Lycopersicon esculentum ToRTL1 virus
Unassigned Pseudoviridae Sirevirus 0 Zea mays Opie-2 virus
Unassigned Pseudoviridae Sirevirus 0 Zea mays Prem-2 virus
Unassigned Pseudoviridae Unassigned 0 Phaseolus vulgaris Tpv2-6 virus
Unassigned Reoviridae Sedoreovirinae Phytoreovirus 0 Rice dwarf virus
Unassigned Reoviridae Sedoreovirinae Phytoreovirus 0 Rice gall dwarf virus
Unassigned Reoviridae Sedoreovirinae Phytoreovirus 1 Wound tumor virus
Unassigned Reoviridae Spinareovirinae Fijivirus 1 Fiji disease virus
Unassigned Reoviridae Spinareovirinae Fijivirus 0 Garlic dwarf virus
Unassigned Reoviridae Spinareovirinae Fijivirus 0 Maize rough dwarf virus
Unassigned Reoviridae Spinareovirinae Fijivirus 0 Mal de Rio Cuarto virus
Unassigned Reoviridae Spinareovirinae Fijivirus 0 Nilaparvata lugens reovirus
Unassigned Reoviridae Spinareovirinae Fijivirus 0 Oat sterile dwarf virus
Unassigned Reoviridae Spinareovirinae Fijivirus 0 Pangola stunt virus
Unassigned Reoviridae Spinareovirinae Fijivirus 0 Rice black streaked dwarf virus
Unassigned Reoviridae Spinareovirinae Oryzavirus 0 Echinochloa ragged stunt virus
Unassigned Reoviridae Spinareovirinae Oryzavirus 1 Rice ragged stunt virus
Unassigned Tombusviridae Aureusvirus 0 Cucumber leaf spot virus
(continued)
69
Table 2.3 (continued)
70

Order Family Subfamily Genus Type species Species

Unassigned Tombusviridae Aureusvirus 0 Johnsongrass chlorotic stripe mosaic virus
Unassigned Tombusviridae Aureusvirus 0 Maize white line mosaic virus
Unassigned Tombusviridae Aureusvirus 1 Pothos latent virus
Unassigned Tombusviridae Avenavirus 1 Oat chlorotic stunt virus
Unassigned Tombusviridae Carmovirus 0 Ahlum waterborne virus
Unassigned Tombusviridae Carmovirus 0 Angelonia flower break virus
Unassigned Tombusviridae Carmovirus 0 Bean mild mosaic virus
Unassigned Tombusviridae Carmovirus 0 Calibrachoa mottle virus
Unassigned Tombusviridae Carmovirus 0 Cardamine chlorotic fleck virus
Unassigned Tombusviridae Carmovirus 1 Carnation mottle virus
Unassigned Tombusviridae Carmovirus 0 Cowpea mottle virus
Unassigned Tombusviridae Carmovirus 0 Cucumber soil-borne virus
Unassigned Tombusviridae Carmovirus 0 Galinsoga mosaic virus
Unassigned Tombusviridae Carmovirus 0 Hibiscus chlorotic ringspot virus
Unassigned Tombusviridae Carmovirus 0 Honeysuckle ringspot virus
Unassigned Tombusviridae Carmovirus 0 Japanese iris necrotic ring virus
Unassigned Tombusviridae Carmovirus 0 Melon necrotic spot virus
Unassigned Tombusviridae Carmovirus 0 Nootka lupine vein clearing virus
Unassigned Tombusviridae Carmovirus 0 Pea stem necrosis virus
Unassigned Tombusviridae Carmovirus 0 Pelargonium flower break virus
Unassigned Tombusviridae Carmovirus 0 Saguaro cactus virus
Unassigned Tombusviridae Carmovirus 0 Soybean yellow mottle mosaic virus
Unassigned Tombusviridae Carmovirus 0 Turnip crinkle virus
Unassigned Tombusviridae Carmovirus 0 Weddel waterborne virus
Unassigned Tombusviridae Dianthovirus 1 Carnation ringspot virus
Unassigned Tombusviridae Dianthovirus 0 Red clover necrotic mosaic virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Tombusviridae Dianthovirus 0 Sweet clover necrotic mosaic virus
Unassigned Tombusviridae Machlomovirus 1 Maize chlorotic mottle virus
2.2 Viruses

Unassigned Tombusviridae Necrovirus 0 Beet black scorch virus

Unassigned Tombusviridae Necrovirus 0 Chenopodium necrosis virus
Unassigned Tombusviridae Necrovirus 0 Leek white stripe virus
Unassigned Tombusviridae Necrovirus 0 Olive latent virus 1
Unassigned Tombusviridae Necrovirus 0 Olive mild mosaic virus
Unassigned Tombusviridae Necrovirus 1 Tobacco necrosis virus A
Unassigned Tombusviridae Necrovirus 0 Tobacco necrosis virus D
Unassigned Tombusviridae Panicovirus 0 Cocksfoot mild mosaic virus
Unassigned Tombusviridae Panicovirus 1 Panicum mosaic virus
Unassigned Tombusviridae Tombusvirus 0 Artichoke mottled crinkle virus
Unassigned Tombusviridae Tombusvirus 0 Carnation Italian ringspot virus
Unassigned Tombusviridae Tombusvirus 0 Cucumber Bulgarian virus
Unassigned Tombusviridae Tombusvirus 0 Cucumber necrosis virus
Unassigned Tombusviridae Tombusvirus 0 Cymbidium ringspot virus
Unassigned Tombusviridae Tombusvirus 0 Eggplant mottled crinkle virus
Unassigned Tombusviridae Tombusvirus 0 Grapevine Algerian latent virus
Unassigned Tombusviridae Tombusvirus 0 Havel River virus
Unassigned Tombusviridae Tombusvirus 0 Lato River virus
Unassigned Tombusviridae Tombusvirus 0 Limonium flower distortion virus
Unassigned Tombusviridae Tombusvirus 0 Moroccan pepper virus
Unassigned Tombusviridae Tombusvirus 0 Neckar River virus
Unassigned Tombusviridae Tombusvirus 0 Pelargonium leaf curl virus
Unassigned Tombusviridae Tombusvirus 0 Pelargonium necrotic spot virus
Unassigned Tombusviridae Tombusvirus 0 Petunia asteroid mosaic virus
(continued)
71
Table 2.3 (continued)
72

Order Family Subfamily Genus Type species Species

Unassigned Tombusviridae Tombusvirus 0 Sitke waterborne virus
Unassigned Tombusviridae Tombusvirus 1 Tomato bushy stunt virus
Unassigned Tombusviridae Unassigned 0 Maize necrotic streak virus
Unassigned Tombusviridae Unassigned 0 Pelargonium line pattern virus
Unassigned Unassigned Benyvirus 1 Beet necrotic yellow vein virus
Unassigned Unassigned Benyvirus 0 Beet soil-borne mosaic virus
Unassigned Unassigned Cilevirus 1 Citrus leprosis virus C
Unassigned Unassigned Emaravirus 1 European mountain ash ringspot-associated virus
Unassigned Unassigned Emaravirus 0 Fig mosaic virus
Unassigned Unassigned Idaeovirus 1 Raspberry bushy dwarf virus
Unassigned Unassigned Ourmiavirus 0 Cassava virus C
Unassigned Unassigned Ourmiavirus 0 Epirus cherry virus
Unassigned Unassigned Ourmiavirus 1 Ourmia melon virus
Unassigned Unassigned Polemovirus 1 Poinsettia latent virus
Unassigned Unassigned Sobemovirus 0 Blueberry shoestring virus
Unassigned Unassigned Sobemovirus 0 Cocksfoot mottle virus
Unassigned Unassigned Sobemovirus 0 Imperata yellow mottle virus
Unassigned Unassigned Sobemovirus 0 Lucerne transient streak virus
Unassigned Unassigned Sobemovirus 0 Rice yellow mottle virus
Unassigned Unassigned Sobemovirus 0 Ryegrass mottle virus
Unassigned Unassigned Sobemovirus 0 Sesbania mosaic virus
Unassigned Unassigned Sobemovirus 0 Solanum nodiflorum mottle virus
Unassigned Unassigned Sobemovirus 1 Southern bean mosaic virus
Unassigned Unassigned Sobemovirus 0 Southern cowpea mosaic virus
Unassigned Unassigned Sobemovirus 0 Sowbane mosaic virus
Unassigned Unassigned Sobemovirus 0 Subterranean clover mottle virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Unassigned Sobemovirus 0 Turnip rosette virus
Unassigned Unassigned Sobemovirus 0 Velvet tobacco mottle virus
2.2 Viruses

Unassigned Unassigned Tenuivirus 0 Echinochloa hoja blanca virus

Unassigned Unassigned Tenuivirus 0 Maize stripe virus
Unassigned Unassigned Tenuivirus 0 Rice grassy stunt virus
Unassigned Unassigned Tenuivirus 0 Rice hoja blanca virus
Unassigned Unassigned Tenuivirus 1 Rice stripe virus
Unassigned Unassigned Tenuivirus 0 Urochloa hoja blanca virus
Unassigned Unassigned Umbravirus 0 Carrot mottle mimic virus
Unassigned Unassigned Umbravirus 1 Carrot mottle virus
Unassigned Unassigned Umbravirus 0 Groundnut rosette virus
Unassigned Unassigned Umbravirus 0 Lettuce speckles mottle virus
Unassigned Unassigned Umbravirus 0 Pea enation mosaic virus-2
Unassigned Unassigned Umbravirus 0 Tobacco bushy top virus
Unassigned Unassigned Umbravirus 0 Tobacco mottle virus
Unassigned Unassigned Varicosavirus 1 Lettuce big-vein associated virus
Unassigned Virgaviridae Furovirus 0 Chinese wheat mosaic virus
Unassigned Virgaviridae Furovirus 0 Japanese soil-borne wheat mosaic virus
Unassigned Virgaviridae Furovirus 0 Oat golden stripe virus
Unassigned Virgaviridae Furovirus 0 Soil-borne cereal mosaic virus
Unassigned Virgaviridae Furovirus 1 Soil-borne wheat mosaic virus
Unassigned Virgaviridae Furovirus 0 Sorghum chlorotic spot virus
Unassigned Virgaviridae Hordeivirus 0 Anthoxanthum latent blanching virus
Unassigned Virgaviridae Hordeivirus 1 Barley stripe mosaic virus
Unassigned Virgaviridae Hordeivirus 0 Lychnis ringspot virus
(continued)
73
Table 2.3 (continued)
74

Order Family Subfamily Genus Type species Species

Unassigned Virgaviridae Hordeivirus 0 Poa semilatent virus
Unassigned Virgaviridae Pecluvirus 0 Indian peanut clump virus
Unassigned Virgaviridae Pecluvirus 1 Peanut clump virus
Unassigned Virgaviridae Pomovirus 0 Beet soil-borne virus
Unassigned Virgaviridae Pomovirus 0 Beet virus Q
Unassigned Virgaviridae Pomovirus 0 Broad bean necrosis virus
Unassigned Virgaviridae Pomovirus 1 Potato mop-top virus
Unassigned Virgaviridae Tobamovirus 0 Brugmansia mild mottle virus
Unassigned Virgaviridae Tobamovirus 0 Cucumber fruit mottle mosaic virus
Unassigned Virgaviridae Tobamovirus 0 Cucumber green mottle mosaic virus
Unassigned Virgaviridae Tobamovirus 0 Frangipani mosaic virus
Unassigned Virgaviridae Tobamovirus 0 Hibiscus latent Fort Pierce virus
Unassigned Virgaviridae Tobamovirus 0 Hibiscus latent Singapore virus
Unassigned Virgaviridae Tobamovirus 0 Kyuri green mottle mosaic virus
Unassigned Virgaviridae Tobamovirus 0 Obuda pepper virus
Unassigned Virgaviridae Tobamovirus 0 Odontoglossum ringspot virus
Unassigned Virgaviridae Tobamovirus 0 Paprika mild mottle virus
Unassigned Virgaviridae Tobamovirus 0 Pepper mild mottle virus
Unassigned Virgaviridae Tobamovirus 0 Rehmannia mosaic virus
Unassigned Virgaviridae Tobamovirus 0 Ribgrass mosaic virus
Unassigned Virgaviridae Tobamovirus 0 Sammons’s Opuntia virus
Unassigned Virgaviridae Tobamovirus 0 Streptocarpus flower break virus
Unassigned Virgaviridae Tobamovirus 0 Sunn-hemp mosaic virus
Unassigned Virgaviridae Tobamovirus 0 Tobacco latent virus
Unassigned Virgaviridae Tobamovirus 0 Tobacco mild green mosaic virus
Unassigned Virgaviridae Tobamovirus 1 Tobacco mosaic virus
(continued)
2 Viruses and Sub-Viral Agents
Table 2.3 (continued)
Order Family Subfamily Genus Type species Species
Unassigned Virgaviridae Tobamovirus 0 Tomato mosaic virus
Unassigned Virgaviridae Tobamovirus 0 Turnip vein-clearing virus
2.2 Viruses

Unassigned Virgaviridae Tobamovirus 0 Ullucus mild mottle virus

Unassigned Virgaviridae Tobamovirus 0 Wasabi mottle virus
Unassigned Virgaviridae Tobamovirus 0 Youcai mosaic virus
Unassigned Virgaviridae Tobamovirus 0 Zucchini green mottle mosaic virus
Unassigned Virgaviridae Tobravirus 0 Pea early-browning virus
Unassigned Virgaviridae Tobravirus 0 Pepper ringspot virus
Unassigned Virgaviridae Tobravirus 1 Tobacco rattle virus
Source King et al. (2012)
75
76 2 Viruses and Sub-Viral Agents

representative of the species as a whole. Some sub-groups (SGs) are working to

define official ‘‘type isolates’’ for each species; probably this is a desirable goal
although it has not been adopted as ICTV policy.

(j) ICTV on the internet

A plan to develop a universal virus database was first discussed around 1990 and
led to the development of ICTV data base. The known properties of virus isolates
and species were encoded and ‘‘translated’’ for the user in natural language text.
Enormous efforts were made to maintain and develop this database and to link with
other important databases on biological taxonomy, publications, sequences etc.
This was managed by the Virus Data Subcommittee of ICTV and relied heavily on
the energy and commitment of Cornelia Buchen-Osmond. The database contains a
wealth of important information but it has proved difficult to sustain funding and
personnel at a time when taxonomic information is expanding rapidly.
In recent years, ICTV has also had a web presence providing lists of the
currently recognized taxa and information on the Executive Committee, Sub-
committees and Study Groups. Templates and other information to assist in
writing and submitting taxonomic proposals have also been provided. For some
years, this was hosted by Fauquet at the Danforth Center, St Louis, but since 2008
the Virus Data subcommittee has overseen the development and maintenance of an
official ICTV website (http://www.ictvonline.org) that now provides a central
point of reference for all ICTV matters. A separate website (http://www.talk.
ictvonline.org) is used to host taxonomic proposals and allows for comment and
discussion to which all virologists are invited to contribute.

2.3 Sub-Viral Agents

2.3.1 Viroids

A group of diseases resembling those caused by viruses are now known to be due
to viroids. Viroids are circular, single stranded, non-coding RNAs that are able to
infect certain plants. The infectious RNAs cause a number of economically
important plant viroid diseases (Hadidi et al. 2003; Flores et al. 2005; Flores and
Owens 2008). In 1971 Diener, for the first time used the term ‘viroid’ after
studying the molecular nature of potato spindle tuber pathogen.
Viroids have not been found in man or animal although several diseases have
been considered to be caused by viroid-like agents. Plant viroids are similar to
some plant viruses in that they contain an RNA genome, but they differ from RNA
plant viruses in two key ways. First, viroids are composed of ‘‘naked’’ RNAs, that
is, they lack a protein coat. Second, they cannot specify any proteins in spite of the
fact that they are made of RNA. The RNA genome of viroids is a small, circular
molecule that contains between 246 and 375 nucleotides. Even though viroids do
2.3 Sub-Viral Agents 77

Fig. 2.6 Electro micrographs in dark-field illumination of viroids as relaxed circles. Magnifi-
cation is 185,000. Courtesy (Riesner et al. 1983)

not produce their own proteins, they are capable of using the host cell machinery to
reproduce their RNA and move into other cells to infect the whole plant (Fig. 2.6).
For many years prior to the discovery of viroids, the diseases caused by them
were classified as plant virus diseases. One reason for this confusion is that the
types of symptoms induced by viroids in plants are similar to those induced by
plant viruses. These symptoms can be visualized by listing the imaginative names
given to some viroids (note that viroids are named in a manner similar to plant
viruses, but that the name ends in ‘‘d’’): Potato spindle tuber viroid (PSTVd),
Apple scar skin viroid (ASSVd), Avocado Sunblotch viroid (ASBVd), and Pear
blister canker viroid (PBCVd). Despite their small size, the economic effects of
viroids can be devastating.
PSTVd is the first viroid disease to be studied by number of plant pathologists.
In 1923, its infectious nature and ability to spread in the field led Schultz and
Folsom (1923) to group potato spindle tuber disease with several other ‘degen-
eration diseases’ of potatoes. Only in 1971 Diener, observed that the molecular
properties of its causal agent, PSTVd, were fundamentally different than those of
conventional plant viruses and for the first time Diener coined the term ’viroid’
which means ’virus-like’.

(a) Geographic distribution

Among economically important viroid diseases, Citrus exocortis viroid (CEVd) is
present in most of the citrus-growing areas where susceptible rootstock is used and
is widespread in S. America (especially Brazil and Argentina), Australia and the
78 2 Viruses and Sub-Viral Agents

Mediterranean region (especially Spain) but is of limited occurrence in the USA,

N. Africa and certain Asian countries. Coconut cadang-cadang viroid (CCCVd) is
limited to the central eastern Philippines (Quezon, Bicol provinces, Samar prov-
inces and Biliran). In many avocado-growing countries including Australia, Israel,
Peru, South Africa, USA and Venezuela, ASBVd is recorded. PSTVd is common
in the potato-growing regions of northern and north eastern USA and to an
unknown extent in the USSR and South Africa. In some of the Asian countries like
India, PSTVd is reported in very low incidence. Even viroid diseases affecting
perennial woody fruit crops like grapes, pome and stone fruits and also in vege-
tatively propagated ornamental plants like chrysanthemum, viroid diseases are
reported to a limited extent in certain tropical countries. The primary factor for
increase and rapid spread of viroid diseases is the international exchange of plant
germplasm and also the latent infections (asymptomatic).

(b) Host range and transmission

Depending on the viroid, the host range is either wide or narrow; for example the
host range of CEVd is largely confined to Rutaceae and some species in the
Solanaceae (potato, tomato, petunia) and some Compositae members. PSTVd has
wide host range and can replicate in about 160 primarily solanaceous hosts, while
only two members of the family Lauraceae are known to support ASBVd repli-
cation. Hop stunt viroid (HSVd) has a particularly wide host range that includes
herbaceous species as well as woody perennials. On the other hand, CCCVd is
confined to members of Palmae (Arecaceae). Viroids spread to other plants
through vegetative propagation, mechanical contamination, pollen and seed.
All viroids are mechanically transmissible, and most of them are naturally
transmitted from plant to plant by farm implements, pruning tools clothing and
human hands. Individual viroids vary greatly in their ability to infect different
plant species. Many natural hosts are either vegetatively propagated or crops that
are subjected to repeated grafting or pruning operations. PSTVd, ASBVd, and
Coleus blumei-1 viroid (CbVd-1) are vertically transmitted through pollen and/or
true seed, but the significance of this mode of transmission in the natural spread of
disease is unclear.
Seed transmission has been demonstrated for many but not all viroids and
pollen-borne transmission is also known to occur in tomato. Reports of seed
transmission of Chrysanthemum stunt viroid (CSVd) are contradictory; Monison
et al. (1973) presented evidence for seed transmission, where as Hollings and
Stone (1973) reported that CSVd is not seed transmitted. Vertical transmission has
been demonstrated for ASBVd for Avocado. Vertical transmission has been
demonstrated for ASBVd in avocado and PSTVd in tomato and pepino. Doubtful
reports of aphid transmission of Tomato planta macho viroid (TPMVd) and
PSTVd is reported (De Bokx and Pirone 1981; Salazar et al. 1995).
Commonly used techniques for the experimental transmission of viroids include
the standard leaf abrasion methods developed for conventional viruses, ‘razor
slashing’ methods in which phloem tissue in the stern or petiole is inoculated via
cuts made with a razor blade previously dipped into the inoculum, and, in the case
2.3 Sub-Viral Agents 79

Fig. 2.7 Symptoms of viroid diseases. Source http://www.virology.net/big picture book of

viruses

of CCCVd, high-pressure injection into folded apical leaves. Viroids can also be
transmitted by either plant transformation or ‘agroinoculation’ during which a
modified Agrobacterium tumefaciens. Ti plasmid is used to introduce full-length
viroid-complementary DNA into the potential host cell. Under field conditions in
general, contaminated cutting knives and tractor wheels also aid in the spread of
viroid diseases. Identification of the molecular mechanism(s) that determine viroid
host range remains an important research goal.

(c) Symptomatology
Stunting and leaf epinasty (a downward curling of the leaf lamina) are considered
the classic symptoms of viroid infection. Other commonly observed symptoms are
vein clearing, veinal discoloration or necrosis, and the appearance of localized
chlorotic/necrotic spots or mottling in the foliage. Symptoms may also be
expressed in flowers, on bark, on fruits and on tubers. The viroid-infected plants
may be abnormally shaped, discolored (Fig. 2.7). For more photographs of
symptoms associated with specific viroid diseases, see Hadidi et al. (2003) and the
disease compendia series of the American Phytopathological Society for specific
crops (APS Press).
Viroid infection of certain citrus rootstock/scion combinations may result in
tree dwarfing. Viroid infections are often latent and rarely kill the host. It is
80 2 Viruses and Sub-Viral Agents

Fig. 2.8 The general structure and organization of viroids (Based on Keese and Symons 1985).
Courtesy Randles and Ogle

estimated that more than 30 million coconut palms in the Philippines have died
due to CCCVd.
Viroid infections are also accompanied by a number of cytopathic effects like
chloroplast and cell wall abnormalities, the formation of membranous structures in
the cytoplasm, and the accumulation of electron-dense deposits in both chloro-
plasts and cytoplasm. Metabolic changes include dramatic alterations in growth
regulator levels.

(d) Molecular biology

Viroids are small, circular, noncoding RNAs and are the smallest self-replicating
genetic units known. Without encoding proteins and requirement for helper
viruses, these small RNAs contain all the information necessary to mediate
intracellular trafficking and localization, replication, systemic trafficking, and
pathogenicity. All or most of these functions likely result from direct interactions
between distinct viroid RNA structural motifs and their cognate cellular factors.
Viroid RNAs are rich in structural motifs that likely are involved in the diverse
biological functions necessary to establish infection. Therefore, viroids will allow
investigation of the many aspects of RNA–protein interactions to achieve specific
biological functions. The multi-functionality of some viroid motifs is of excep-
tional value for investigating the structure–function relationships of RNAs in
greater detail.

(e) Genome structure

The majority of the plant viroids reported today, have been sequenced. Despite
their differences in sequences, certain domains have been recognized as common
to all of them. These are also indicated in Fig. 2.8. The central conserved domain
has been used as the basis for grouping viroids.
Intensive studies are made to understand viroid replication process. We know
that viroid RNA does not code for any protein. The replication mechanism
involves RNA polymerase II, an enzyme normally associated with synthesis of
messenger RNA from DNA, which instead catalyzes ‘‘rolling circle’’ synthesis of
2.3 Sub-Viral Agents 81

new RNA using the viroid’s RNA as template. Some viroids are ribozymes, having
catalytic properties which allow self-cleavage and ligation of unit-size genomes
from larger replication intermediates.
The complete sequences of nearly 29 distinct viroid species plus a large number
of sequence variants have been determined (Table 2.4). All are single-stranded
circular RNAs containing 246–401 unmodified nucleotides. Theoretical calcula-
tions and physicochemical studies indicate that PSTVd and related viroids assume
a highly base-paired, rod-like conformation in vitro (Fig. 2.8). Pair wise sequence
comparisons suggest that the series of short double helices and small internal loops
that comprise this so-called ‘native’ structure are organized into five domains
whose boundaries are defined by sharp differences in sequence similarity. The five
structural domains termed are: central(C), variable(V), pathogenic(P), and termi-
nal left and right (TL and TR) respectively (Keese and Symons 1985). The ‘central
domain’ is the most highly conserved viroid domain and contains the site where
multimeric PSTVd RNAs are cleaved and legated to form circular progeny. The
‘pathogenicity domain’ contains one or more structural elements which modulate
symptom expression, and the relatively small ‘variable domain’ exhibits the
greatest sequence variability between otherwise closely related viroids. The two
‘terminal domains’ appear to play an important role in viroid replication and
evolution. Although these five domains were first identified in PSTVd, ASSVd and
related viroids also contain a similar domain arrangement.

(f) Replication
The lack of protein-coding capacity of viroids entails that their replication
mechanism is much more host-reliant than that of RNA viruses, which at least
encode a subunit of the RNA-dependent RNA polymerase catalyzing initiation and
elongation of viral strands.
Viroid RNA does not code for any protein. The replication mechanism involves
RNA polymerase II, an enzyme normally associated with synthesis of messenger
RNA from DNA, which instead catalyzes ‘‘rolling circle’’ synthesis of new RNA
using the viroid’s RNA as template. Some viroids are ribozymes, having catalytic
properties which allow self-cleavage and ligation of unit-size genomes from larger
replication intermediates.
Based on the site of viroid replication in the cell, the viroids are classified into
two families, the Pospiviroidae and the Avsunviroidae. Intriguingly, viroids have
evolved the ability to replicate in two cellular organella, the nucleus (family
Pospiviroidae) and the chloroplast (family Avsunviroidae). Viroid replication
proceeds through an RNA-based rolling-circle mechanism with three steps cata-
lysed by: (i) host deoxyribonucleic acid (DNA)-dependent RNA polymerases
redirected to accept RNA templates, (ii) processing enzymes or, in the family
Avsunviroidae, hammerhead ribozymes and (iii) RNA ligases. When infecting a
cell, the viroid RNA must travel to its replication organelle, with the resulting
progeny moving cell-to-cell through plasmodesmata and reaching distal parts
through the phloem.
Table 2.4 Officially recognized viroid species (VIII Report, ICTV)
82

Genusa Species Variantsb Length(nt) Natural host(s) Emerging hosts

Family Pospiviroidae
Pospiviroid Potato spindle tuber (PSTVd) 109 341–364 Potato tomato, avocado
Chrysanthemum stunt (CSVd) 19 348–356 chrysanthemum
Citrus exocortis (CEVd) 86 366–475 citrus, tomato tomato
Columnea latent (CLVd) 17 359–456 Columnea, Brunfelsia, tomato
Nemathanthus
Iresine (IrVd) 3 370 Iresine
Mexican papita (MPVd) 6 359–360 Solanum cardiophyllum
Tomato apical stunt (TASVd) 5 360–363 tomato tomato
Tomato chlorotic dwarf (TCDVd) 2 360 Uncertain tomato
(tomato?)
Tomato planta macho (TPMVd) 2 360 tomato
Hostuviroid Hop stunt (HSVd) 144 294–303 citrus, grapevine, Prunus spp. hop, cucumber
Cocadviroid Coconut cadang–cadang (CCCVd) 8 246–301 coconut palm African oil palm, other
monocots
Coconut tinangaja (CTiVd) 2 254 coconut palm
Citrus bark cracking (CBCVd) 6 284–286 citrus
Hop latent (HLVd) 10 255–256 hop
Apscaviroid Apple scar skin (ASSVd) 8 329–333 apple, pear
Apple dimple fruit (ADFVd) 2 306 apple
Apple fruit crinkle (AFCVd)c 29 368–372 apple
Australian grapevine (AGVd) 1 369 grapevine
Citrus bent leaf (CBLVd) 24 315–329 citrus
Citrus dwarfing (CDVd) 53 291–297 citrus
Grapevine yellow speckle 1 (GYSVd-1) 49 365–368 grapevine
Grapevine yellow speckle 2 (GYSVd-2) 1 363 grapevine
Pear blister canker (PBCVd) 18 314–316 pear, quince
(continued)
2 Viruses and Sub-Viral Agents
Table 2.4 (continued)
Genusa Species Variantsb Length(nt) Natural host(s) Emerging hosts
Coleviroid Coleus blumei-1 (CbVd-1) 9 248–251 Coleus,
Mentha spp.
Coleus blumei-2 (CbVd-2) ? 295–301
Coleus blumei-3(CbVd-3) 3 361–364 Ocimum basilicum, Melissa
officinalis
2.3 Sub-Viral Agents

Family Avsunviroidae
Avsunviroid Avocado sun blotch (ASBVd) 83 239–251 avocado
Pelamoviroid Chrysanthemum chlorotic mottle 21 397–401 chrysanthemum
(CChMVd)
Peach latent mosaic (PLMVd) 168 335–351 peach, nectarine
Elaviroid Eggplant latent (ELVd) 9 332–335 eggplant
a
Names of viroid genera are derived from those of the respective type species (listed first)
b
Sequences available online from the Subviral RNA Database. [http://subviral.med.uottawa.ca]
c
Provisional species (not officially recognized)
Source Subviral RNA Database [http://subviral.med.uottawa.ca]
83
84 2 Viruses and Sub-Viral Agents

(g) Movement
Limited information is available about the pathways and mechanism of viroid
movement inside the host plant. The viroids after entering in a potential host cell, it
has to move to either the nucleus (Pospiviroidae) or chloroplast (Avsunviroidae)
before beginning replication. Available data suggest that PSTVd enters the nucleus
as a ribonucleo protein complex formed by the interaction of cellular proteins with
specific viroid sequence or structural motifs. Ding et al. (1997) have observed that
PSTVd moves from cell to cell via plasmodesmata and this movement is mediated
by a specific sequence of structural motif. VirPl, a bromodomain-containing
protein isolated from tomato, has a nuclear localization signal and hinds to the
terminal right domain of PSTVd. Proteins such as TFIIIA and ribosomal protein
L5 that bind to the loop E motif may also be involved in viroid transport into the
nucleus (Flores and Owens 2008).
To establish a systemic infection, viroids leave the initially infected cell-
moving first from cell to cell and then long distances through the host vasculature.
Long-distance movement of viroids occurs in the phloem where it follows the
typical source-to-sink pattern of photoassimilate transport. Viroid movement in the
phloem almost certainly requires formation of a ribonucleoprotein complex,
possibly involving a dimeric lectin known as phloem protein 2 (Pp-2), the most
abundant protein in phloem exudate.

(h) Diagnosis of viroid diseases

In the earlier years, biological methods have been used in detecting the viroids
based on the reaction on diagnostic host plants. Since this test takes long time the
scientists have switched over to molecular tests. Nucleic acid based techniques are
the one that can help in reliable and rapid detection of viroids. Early molecular
detection methods involved a combination of native and denaturing polyacryl-
amide gel electrophoresis that relies on the circular properties of the viroid RNA
molecule to resolve it from other plant RNAs (Schumacher et al. 1986). This
method is not as useful for the identification of specific viroids, as several viroids
may have the same electrophoretic mobility.
Molecular method (dot-blot hybridization) is quite useful for viroid detection
and is reliable for viroids of known sequence and very sensitive. However this
method cannot be relied upon to detect new viroids where sequence information is
unavailable. A number of reverse transcription-polymerase chain reaction (RT-
PCR) protocols have been developed for detection of different viroids, including
real-time RT-PCR (Shamloul et al. 1995; Hadidi et al. 1997; Boonham et al. 2004;
Bagherian et al. 2009). Through microarray and macroarray technology a number
of viroid diseases in different crop plants were identified (Agindotan and Perry
2008). Even tissue print immuno assay or tissue blot immuno binding were also
used for viroid diagnosis (Hadidi et al. 1991; Hurtt and Podleckis 1995). The dot-
blot hybridization techniques are widely used for the detection of viroid diseases in
different crops (Podleckis et al. 1993).
2.3 Sub-Viral Agents 85

2.3.1.1 Viroid Classification

The structural and functional properties, as well as their evolutionary origin of

viroids differ fundamentally from those of viruses, posing specific problems for the
classification of these sub viral pathogens. Rules concerned with the classification
of viruses shall also apply to the classification of viroids.
Viroids can be classified into two major families, the Pospiviroidae and the
Avsunviroidae, based on where the viroid replicates in the cell (Tabler and Tsagris
2004). Viroids in the Pospiviroidae family replicate in the nucleus while viroids in
the Avsunviroidae family replicate in the chloroplast.
Pospiviroidae is further subdivided into three subfamilies: Pospiviroinae (from
PSTVd), Apscaviroinae (from Apple scar skin viroid, ASSVd), and Coleviroinae
(from Coleus blumei viroid 1, CbVd1). The subfamily Pospiviroinae contains
three genera: Pospiviroids (from PSTVd), Hostuviroids (from Hop stunt viroid,
HSVd), and Cocadviroids (from Coconut cadang-cadang viroid, CCCVd). The
Apscaviroinae and Coleviroinae subfamilies each contain one genus, Apscaviroids
and Coleviroids, respectively. The Avsunviroidae family contains three genera:
Avsunviroids (from ASBVd) and Pelamoviroids (from Peach latent mosaic viroid,
PLMVd) (Flores et al. 1998), and Elaviroid (from Eggplant latent viroid ELVd).
The formal endings for taxa of viroids are the word ‘‘viroid’’ for species, the
suffix ‘‘-viroid’’ for genera, the suffix ‘‘-viroinae’’ for sub-families (should this
taxon be needed) and ‘‘-viroidae’’ for families. For example, the species Potato
spindle tuber viroid is classified in genus Pospiviroid, and the family Pospivi-
roidae. The list of recognized viroid species and their properties are provided in
the Table 2.4.
More details about viroids can be obtained from the articles of Diener (1979,
1999); Flores (2001); Hadidi et al. (2003); Flores et al. (2005); Hammond and
Owens (2006); Flores and Owens (2008).

2.4 Phytoplasma

During the past 35 years it has become apparent that yellows and witche’s broom
type of diseases are caused by agents similar to mycoplasmatales (Pleuro pneu-
monia-like organisms) and not due to viruses. The first report is by Doi et al.
(1967) who discovered mycoplasmas-like bodies (MLO) in the phloem sieve
elements of yellows-infected plants. They also observed that tetracyclines induced
temporary remission of symptoms. Since then about 200 different plant species in
59 families are demonstrated to be affected by phytoplasma (Bertaccini and Duduk
2009; Rao et al. 2011). In recent years MLO diseases are renamed as phytoplasma
diseases. The phytoplasma infected plants exhibit growth of adventitious shoots,
chlorosis without spotting; clearing of the veins, growth stimulation of normally
dormant axillary buds; malformation, stunting and the transformation of floral
structures into green leaf like structures known as phyllody (Fig. 2.9).
86 2 Viruses and Sub-Viral Agents

Fig. 2.9 Some important phytoplasma and spiroplasma infected plants. Source http://
www.virology.net/big picture book of viruses

The diseases associated with phytoplasmas have been divided into the fol-
lowing four types: Aster yellows (elongation of internodes, leaf yellowing);
Stolbour (apical dwarfing, stunting, leaf roll, epinasty, wilting, virescence); Wit-
ches’ broom (proliferation of axillary shoots) and decline (degeneration). The
morphology and structure of phytoplasma are similar to true mycoplasmas of
animals and are usually spheroidal to ellipsoid, ranging from 70 to 1100 nm
diameter with some elementary bodies of 50–10 nm. They are bounded by single
unit triple-layered membrane, devoid of rigid cell wall and are highly pleomorphic
(Cousin et al. 1970). In some cases irregularly tubular to filamentous structures are
also noticed. They have cytoplasm and central nuclear areas comprised of a loose
net work of double stranded DNA strands or more rarely a distinct nucleotide
(Nasu et al. 1970). Ribosomes which are 10–15 nm in their size are either scattered
throughout or clustered about the periphery of the cell and are smaller than host
ribosomes (Hirumi and Maramorosch 1969). Vacuoles, which are only occasion-
ally found in filamentous bodies, are frequently encountered in the large globular
bodies (Fig. 2.10).
2.4 Phytoplasma 87

Table 2.5 Some of the major taxonomic groups and the candidatus species that belong to
phytoplasma
16Sr group Group name Species
16SrI Aster yellows Ca. Phytoplasma asteris
Japanese hydrangea phyllody Ca. Phytoplasma japonicum
16SrII Peanut witch’s broom Ca. Phytoplasma aurantifolia
16SrIII X-disease Ca. Phytoplasma pruni
16SrIV Coconut lethal yellowing Ca. Phytoplasma palmae
Ca. Phytoplasma castaneae
Ca. Phytoplasma cocosnigeriae
16SrV Elm yellows Ca. Phytoplasma ulmi
Rubus stunt Ca. Phytoplasma rubi
Jujube witche’s broom Ca. Phytoplasma ziziphi
16SrVI Clover proliferation Ca. Phytoplasma trifolii
16SrVII Ash yellows Ca. Phytoplasma fraxini
16SrVIII Luffa witch’s-broom Ca. Phytoplasma luffae
16SrIX Pigeon pea witch’s broom Ca. Phytoplasma phoenicium
16SrX Apple proliferation Ca. Phytoplasma mali
Pear decline Ca. Phytoplasma pyri
European stone fruit yellows Ca. Phytoplasma prunorum
Spartium witche’s broom Ca. Phytoplasma spartii
16SrXI Rice Yellow Dwarf Ca. Phytoplasma oryzae
16SrXII Stolbur Ca. Phytoplasma solani
Australian grapevine yellows Ca. Phytoplasma australiense
16SrXIII Mexican periwinkle virescence Undefined
16SrXIV Bermuda grass white leaf Ca. Phytoplasma cynodontis
16SrXV Hibiscus witch’s-broom Ca. Phytoplasma brasiliense

The phytoplasma bodies have been reported in the sieve elements of the phloem
and less often phloem parenchyma and parenchyma cells near the problem (Doi
et al. 1967; Worley 1970), in phloem companion cells or cortical parenchyma
(Cousin et al. 1970). However electron micrographs by several workers have
shown that the bodies are capable of the deformation required to press through
sieve pores. In the infected plants blockage of movement of the energy storage
compounds like sugars from leaves to roots could account for the progressive
decline and often death. Phytoplasma diseases are not transmissible to plants by
mechanical inoculation, but they are transmitted to healthy plants by grafting
diseased material or by using dodder. Natural spread is by insect vectors usually
leaf-hoppers, although in few cases psyllids and planthoppers are also responsible.
The leafhopper vectors are have a very long incubation period which ranges from
10 to 45 days and they are viruliferous throughout their life after incubation
period. In some cases transovarial transmission was also noticed. Phytoplasma
diseases were detected by nucleic acid based techniques like dot-blot hybridization
assay and PCR. Even some success is achieved by Dienes stain for the detection of
phytoplasmal infection. In tropical countries the phytoplasma diseases are
88 2 Viruses and Sub-Viral Agents

Fig. 2.10 Phytoplasmas (arrows) in the phloem cells of Catharanthus roseus L. (bar = 0.5 l).
Courtesy Rita Musetti, and Maria Augusta Favali

economically important and some of them are: Rice yellow dwarf, Sugarcane
white leaf, Coconut lethal yellowing, Coconut root-wilt, Sandal spike, Cotton
virescence, Tomato big bud, Pear decline, and Bois noir phytoplasma diseases of
grapes (Table 2.5).
In recent years the phytoplasma is grouped under bacillus and is considered
along with bacterium. Hence more details about diagnosis, epidemiology and
management measures of phytoplasma are not dealt in this text book. However
more information on phytoplasma and the diseases they cause can be obtained
from review and text book chapters (Varma and Ahlawat 1994; Randles and Ogle
1997; Lee et al. 2000; Cousin and Boudon-Padieu 2002; Seemuller et al. 2002;
Bertaccini and Duduk 2009).

2.5 Spiroplasma

Some of the yellows type of diseases which were earlier grouped under phytopl-
asma were identified to be due to spiroplasma organisms. The genus Spiroplasma
has been placed in the family Spiroplasmataceae, under the order Mycoplasma-
tales (Skripal 1974). They are pleomorphic cells that vary in shape from spherical
or slightly ovoid, 100–250 nm or larger in diameter and 3–25 lm in length. They
often seem attached to spherical structures called blebs. They do not have true cell
2.5 Spiroplasma 89

wall and are bounded by a single triple layered unit membrane. Although spi-
roplasmas are morphologically distinguish able from mycoplasmas, they are very
similar in most respects. They can be easily cultured on nutrient media and they
produce mostly helical forms in liquid media. For the first time, the name ‘Spi-
roplasma’ was proposed for the corn stunt organism by Davis and Worley (1973).
They etiology of the term is as follows: Spiro (Greek noun ‘Speira’) meaning coil
and Greek noun ‘plasma’ meaning something formed or molded to denote shape or
form. The movement exhibited by the helical filaments is yet another characteristic
divergent from members of the class mollicutes. The helical filaments are motile,
moving by a slow undulation of the filament and probably by a rapid rotary or
‘screw’ motion of the helix (Brownian movement).
The common diseases caused by spiroplasmas are citrus stubborn, corn stunt,
Bermuda grass white leaf, Opuntia tunamonstrosa witche’s broom and aster yel-
lows. Most of these spiroplasmas are cultured and they require sterol for their
growth. The spiroplasmas have ribosomes consisting of RNA and a coil of DNA as
their genome. Most probably they multiply by binary fission. They are resistant to
penicillin; however, tetracyclines, erythromycin, amphotericin and neomycin
inhibit these organisms. Serology and polyacrylamide gel electrophoresis are
commonly used to find out the inter relationships of cultured spiroplasmas.

2.6 Other Sub-Viral Agents

2.6.1 Satellite Viruses

Satellite viruses are defined as sub-viral agents lacking genes that could encode the
enzymes needed for their replication and they cannot cause infection by them-
selves. Instead, they must always be associated with certain typical viruses (helper
viruses) because they depend on the latter for multiplication and plant infection.
Satellite viruses often reduce the ability of the helper viruses to multiply and cause
disease i.e., satellite viruses act like parasites of the associated helper viruses.
Therefore, their multiplication depends on the co-infection of a host cell with a
helper virus. A satellite virus is genetically distinct from its helper virus by virtue
of having a nucleotide sequence substantially different from it, although some
satellites share short sequences often at the termini of their RNA, with their helper
viruses. Satellite viruses are not classified by species or genera because they are
not a homogeneous group of agents and information on their properties (e.g.,
nucleotide sequence) is in sufficient to deduce their evolutionary origins.
For the convenience, satellite viruses are divided into two major categories:
(1) ‘‘Satellite viruses’’ (resembling Tobacco necrosis satellite virus) and the
examples are Single-stranded RNA satellite viruses, Subgroup 1: Chronic bee-
paralysis satellite virus, Subgroup 2: Tobacco necrosis satellite virus.
90 2 Viruses and Sub-Viral Agents

(2) ‘‘Satellite nucleic acid’’ is divided into (1) Single-stranded satellite DNAs,
e.g., Alphasatellites, Tomato leaf curl virus satellite DNA, Betasatellites. (2)
Double-stranded satellite RNAs, e.g., Saccharomyces cerevisiae M virus
satellite, Trichomonas vaginalis T1 virus satellite. (3) Single-stranded satellite
RNAs, e.g., Subgroup 1: Large satellite RNAs: Arabis mosaic virus large
satellite RNA, Bamboo mosaic virus satellite RNA, Chicory yellow mottle virus
large satellite RNA, Grapevine Bulgarian latent virus satellite RNA, Grape-
vine fanleaf virus satellite RNA, Myrobalan latent ringspot virus satellite RNA,
Tomato black ring virus satellite RNA, Beet ringspot virus satellite RNA,
Subgroup 2: Small linear satellite RNAs: Cucumber mosaic virus satellite
RNA, Cymbidium ringspot virus satellite RNA, Pea enation mosaic virus
satellite RNA, Groundnut rosette virus satellite RNA, Panicum mosaic virus
small satellite RNA, Peanut stunt virus satellite RNA, Turnip crinkle virus
satellite RNA, Tomato bushy stunt virus satellite RNA B10, Tomato bushy stunt
virus satellite RNA B1, Subgroup 3: Circular satellite RNAs or ‘‘virusoids’’:
Arabis mosaic virus small satellite RNA, Cereal yellow dwarf virus-RPV
satellite RNA, Chicory yellow mottle virus satellite RNA, Lucerne transient
streak virus satellite RNA, Solanum nodiflorum mottle virus satellite RNA,
Subterranean clover mottle virus satellite RNA, Tobacco ringspot virus
satellite RNA, Velvet tobacco mottle virus satellite RNA.

The genomes of satellites range upward from 359 nucleotides in length for
Satellite Tobacco Ringspot Virus RNA (STobRV). Satellite viral particles should
not be confused with satellite DNA. The aspect of plant virus satellites has been
reviewed by Francki (1985) and Roossinck et al. (1992).

2.6.2 Defective Interfering Particles (DI Particles)

DI Particles are virus particles which contains genomes that are grossly altered
genetically, usually by significant deletion of essential functions, but which nev-
ertheless retain critical replication origins and packaging signals, allowing for
amplification and packaging in co-infections with complimenting wild-type helper
virus. These particles usually display a replication advantage relative to wild-type
virus, resulting from increases in the copy number or efficiency of replications
origins. DI particles actively inhibit replication of wild-type virus, presumably by
competing for limiting essential replication factors. Study of DI particles has
provided significant insight into the viral replication in particular structure and
function of replication origins (Condit 2007). The DI genome is replicated only in
a cell that is infected with infectious virus of the type from which the DI genome
was generated as this is needed to supply replicative enzymes and structural
proteins.
2.6 Other Sub-Viral Agents 91

The production of subgenomic DNA, so-called defective interfering DNA, (DI

DNA) has been observed in all geminivirus genera (Frischmuth and Stanley 1993).
DI DNA generally have one or more parts of a genome deleted. It is assumed that
the production of DI DNA is due to intramolecular recombination, and most DI
DNAs have short sequence duplication at the deletion border. Characteristically,
DI DNAs contain only viral derived sequences. They are dependent for their
replication and movement on the parental virus and interfere with viral prolifer-
ation in transgenic plants (Frischmuth and Stanley 1993). Besides these virus
derived DI DNAs are generally small DNA molecules described as satellite. Even
nano virus like DNAs have been found in geminivirus infected plants. One of the
examples of geminivirus group is Cotton leafcurl virus (CLCuV) infected with a
small DNA molecule (designed as DNAI) related to nano viruses has been iden-
tified (Mansoor et al. 1999). More information on DI Particles can be obtained
from Huang (1973).

2.7 Conclusions

The tropical zone has nearly 169 countries out of the total 270 countries of the
world covering nearly 62.5 % of our planet. Besides the fungal, bacterial and
insect pests, even the virus and virus-like diseases also cause extensive yield
losses. Plant virus is basically a tiny bundle of genetic material-either DNA or
RNA carried in a shell called viral coat or capsid which is made up of protein
called capsomeres. Apart from the virus diseases, the viroid diseases also cause
catastrophic yield losses of the crops. The viroids are low molecular weight,
covalently closed circular RNA molecules and are distinguished from viruses by
the absence of protein coat, lack of mRNA activity and by the homogenous
structure, structural transitions and hydrodynamic behavior of their RNA mole-
cules. Even the satellite viruses and DI particles cause diseases in plants. The
identification of the etiological agent is most important and we have sufficient
information on the particle morphology genomic composition, epidemiology and
transmission mode for the majority of the virus diseases. Well established sero-
diagnosis and molecular techniques are available for accurate identification of
virus and virus-like pathogens. Since 1968, attempts have been made to classify
plant viruses by a number of research workers. At present the ICTV in the 9th
report which was updated in 2012, has a total of 87 families, 349 genera, and 2284
species. Similarly the viroids are also identified and classified into two families
viz., Pospiviroidae and Avsunviroidae. Since the etiological agents and their
epidemiological and pest risk analysis data is available for some of the diseases,
progress on the management measures against the major virus diseases have been
developed and these details are provided in Volume 2 of this series.
92 2 Viruses and Sub-Viral Agents

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Chapter 3
Impact of Virus and Viroid Diseases
on Crop Yields

3.1 Crop Losses Due to Virus and Viroid Diseases

The assessment of disease incidence and crop loss is a key factor in problems
involving the economic aspects of disease management. The prevention of crop
loss is the economic justification for plant pathology in general, and epidemiology
in particular. Estimates of yield reduction for a particular crop and pathogen have
no general validity, may be catastrophic or mild or insignificant. Certain plant
viruses and virus-like diseases in some crops damage all the plant parts like leaves,
stems, roots, seeds or flowers, and the extent of loss will greatly depend upon the
value of the crop, which may be qualitative or quantitative or both. In the case of
quantitative loss, the crop loss will be in weight or in number, where as in qual-
itative loss this usually involves reduced size of the product, besides its variation in
chemical constitution and taste. Lower market values for fruits and vegetables
results from reduced size and distortion of the commodity, for flowers with color
breaking and reduced sizes, and for root crops with reduced size.
Crop losses are generally estimated in units of yields like kilograms, bushels
etc. and also expressed as per cent reduction of the ‘potential’ yield, i.e. of the
yields assumed to be that of a healthy crop. It can be also expressed in monetary
units, which depends on the fluctuating market values. In an open market, the
relationship between weight loss and financial loss for individual growers is fur-
ther complicated by the question as to whether other growers supplying to the
markets have had similar losses. If losses due to diseases are fairly evenly dis-
tributed, price rises will tend to compensate. The financial losses to certain growers
may be much more when only a few growers are affected. The virus or virus-like
diseases of fruit crops like citrus, grapes, banana, avocado or any other econom-
ically important perennial crop not only leads to the loss of the crop for one year,
but the loss of time and cost in bringing the trees to bearing, the losses of other
crops that could have been grown on the land during that time, and the differences
in the value of the land with and without a productive orchard. Disease losses
affect not only the farmer but also the consumer. When diseases limit production
due to heavy infection and result in a shortage of crop yields, then prices usually

K. S. Sastry, Plant Virus and Viroid Diseases in the Tropics, 99

DOI: 10.1007/978-94-007-6524-5_3, Ó Springer Science+Business Media B.V. 2013
100 3 Impact of Virus and Viroid Diseases on Crop Yields

increase. In the case of the fruit crops, to estimate the economic effects one must
consider four factors of prime importance. First, each fruit tree consists of two
different varieties or species, the root stock and scion. Secondly, varieties differ
markedly in their reaction to infection by a single virus. Thirdly, viruses occur in a
range of strains often varying from virulent to those causing no symptoms.
Fourthly, viruses may interact i.e., one virus may greatly influence the effect of
another in the same plant, and the influence may be synergistic and increase in the
severity of symptoms or antagonistic and protective. These four factors influence
the result of virus infection in a particular tree to the extent that no prediction can
be made with any certainty unless the identity of the scion variety and root stock,
the strain of the virus with which one is concerned, and the extent of latent
infection with other viruses are known. Usually only one of these factors is known.
Few viruses that affect only the fruit, such as strong pit of pear, can be assessed
merely by taking into account the number of infected trees and the proportion of
fruit rendered unsalable. Most virus diseases affect the growth and productivity of
the tree, however, as seen with Apple proliferation virus which has reduced the
fruit size and color, it has paramount importance in some markets. In stone fruit
trees, virus infection has been found to influence the survival of scions, particularly
of bud grafts, in the nursery. This effect has not been reported for pome fruits, but
several viruses (e.g., pear vein-yellows, apple mosaic and rubbery wood) reduce
scion growth and thereby the proportion of first grade trees. Nursery men may
offset this effect by retaining the smaller trees for a second year’s growth in the
nursery, but this obviously increases cost of production.
Availability of information on crop losses varies considerably in precision and
is difficult task as the severity of the disease varies greatly with the factors like
locality, the crop variety, the severity of the virus strain, the activity of the vectors,
the nutritional status of the crop and crop season. Young plants are particularly
vulnerable to virus infection. The role of variety of the host on the yield loss can be
well exemplified by the work of Dedic (1975). In a three year trial, the yield of
potato infected with virus was reduced by 22.4–31.3 % in the var. Rajka,
21.7–39.0 % in the var. Krsava, 9.8–34.8 in the var. Radka and 9.8–36.4 in the var.
Rea, compared with healthy plants. Similarly, Chiko and Zimmer (1978) also
recorded that Pea seed borne mosaic virus which caused losses of only 11.0 % in
the cv. Trapper, but had 36.0 % in the cv. Century. A number of examples can be
cited with reference to extent of yield losses in relation to crop age at the time of
infection. Tomato infected with Tobacco mosaic virus (Heuberger and Norton
1933; Mena 1973), sugarcane inoculated with Sugarcane mosaic virus (Abbott
1961), okra infected with Bhendi yellow vein mosaic virus (Sastry and Singh
1974), potato infected with Potato leaf roll virus (Knutson and Bishop 1964) and
cowpea infected with Cowpea aphid-borne mosaic virus, Southern bean mosaic
virus and Cowpea mottle viruses (Kareem and Taiwo 2007). It was also established
that the crop loss figures vary with inoculum load, which was exemplified in
sugarcane variety CB 46/47, when the initial infection was 100 % resulted in 71 %
3.1 Crop Losses Due to Virus and Viroid Diseases 101

loss, whereas with an initial infection of 25 % resulted in a yield reduction of only

19 % (Matsuoka and Costa 1974).
The resultant losses in economic terms in the same crop will also vary with
different viruses and virus-like pathogens, and it is also influenced by the viru-
lence of the virus strain involved. For example in India, in the potato variety
‘Kufri red’, the losses caused by Potato virus Y, Potato leaf roll virus, Potato
virus S, and witch’s broom were 71, 19, 22, 72 and 17 %, respectively (Nagaich
1971). Duffus (1961) reported that Beet yellows virus and Beet western yellows
virus caused 16.6 and 14.6 %, respectively, while the combined infection caused
30.6 % reduction.
With respect to losses due to virus strains, milder ones will be less damaging
than more virulent strains. Singh et al. (1971) reported that three isolates of mild
strains of Potato spindle tuber viroid reduced the yield in var. ‘Saco’ by 17, 24 and
24 % respectively and the severe strain reduced yields by 64 %. Shepherd and Till
(1965) also noticed that the losses in beetroot yield with very mild strain of Beet
mosaic virus was 7.6 % as against 20.4 % with a necrotic strain. From India,
Balaraman and Ramakrishnan (1979) noticed that in Kagzi lime, two mild strains
of Citrus tristeza virus MI and MII yielded 1,430 and 1,310 fruits, respectively,
while the severe strain produced only 80 fruits/plant in a 4- year of observation
period. A similar type of observation were also recorded by Kuhn et al. (1978)
with Peanut mottle virus in groundnut cultivars and also by Tu (1989) with
Soybean mosaic virus in 8 soybean cultivars.
Experimental results have also indicated that double infection with two or more
viruses also causes more severe losses than with a single virus infection. For
example, Strawberry mottle virus (SMoV) reduced yields by 20 %, but with the
yellows virus complex a 36 % reduction (Aerts 1973). Bolton (1974) also
observed that SMoV decreased the yields by 11.5 % in the third year, whereas the
combination of Strawberry vein banding virus and Strawberry latent C virus
reduced strawberry yields by 88.2 %. Similarly, double infection with Soybean
mosaic virus (SMV) and Bean pod mottle virus caused reductions up to 80 %,
where as SMV alone caused losses of only 8–25 %. Similar results were noticed
by several workers in tomato (Brack 1979), in black eye cowpea (Pio-Ribeiro et al.
1978); and in soybean (Quiniones et al. 1971) with different virus combinations.
The extent of losses will vary from place to place and country to country. In
some areas a particular grower or a group of growers may lose an entire crop in
terms of weight and financial loss with devastating virus diseases. While at the
same time another grower or group of growers in a different location will receive
an increased harvest because of the law of supply and demand and also based on
the virus strain and stage of infection. If losses due to a disease are fairly evenly
distributed, price rises will tend to compensate. Whereas only a few growers are
affected, the financial loss to certain growers may be much more severe for the
same loss of crop. Thus all crop losses are only estimates on an individual basis
and exploration of such losses to a world market is meaningless.
102 3 Impact of Virus and Viroid Diseases on Crop Yields

The variation in the quality of the produce is an important factor in the eco-
nomics of virus disease losses. In a crop picked over a season and also where the
product is graded, the time at which the losses occur and the effect of infection on
quality is important. Viruses of fruit trees will cause the fruit to be small, some-
times disfigured and, of poor flavor which are unacceptable. Wallace et al. (1944)
reported that in the apple variety Lord Lambourne, the Chat-fruit virus causes the
failure in the development of the red colour, typical of the variety and remained
small (chat) and delayed ripening is noticed. Posnette and Cropley (1965) recorded
25 % in the reduction of the fruit size with Chat-fruit virus infection and they will
be green even after maturity.
Some of the virus and virus-like diseases which caused epidemics and heavy
losses in different countries are as follows:
(a) Citrus quick decline (Citrus tristeza virus) in Africa, America, Brazil, India,
Australia etc., which causes continuous heavy losses.
(b) Swollen shoot of cacao (Cacao swollen shoot virus) is serious constraint to
cocoa production in West Africa, particularly in Ghana. Severe strains of this
virus can kill susceptible cocoa trees within 2–3 years. The virus is trans-
mitted from tree to tree by mealybug vectors.
(c) Bunchy top of banana (Banana bunchy top virus) is destructive in Asia,
Australia, Egypt, and Pacific Islands. In recent years Banana streak virus,
Banana bract mosaic virus are also gradually spreading through infected
suckers in different parts of tropical countries.
(d) Papaya ringspot virus which is a member of Potyviridae causes heavy yield
losses wherever papaya is grown in the tropical zone. When infected at the
seedling stage or within 2 months after planting, trees do not normally
produce mature fruits. A severe PRSV isolate from Taiwan is also known to
induce systemic necrosis and wilting along with mosaic and chlorosis.
(e) Sugar beet yellows virus is distributed worldwide and causes great losses
every year.
(f) Sugarcane mosaic virus is distributed worldwide, and results in great losses in
sugarcane and corn.
(g) Cassava mosaic virus complex (African cassava mosaic virus, East African
cassava mosaic virus, and South African cassava mosaic virus) are distinct
species of single-stranded DNA viruses that are a major destructive factor
affecting the cassava crop in Africa. Related species of these begomoviruses
do occur elsewhere. Currently this disease is pandemic in Africa, affecting
nine countries in East/Central Africa causing estimated losses of 47% of
production.
(h) Sweet potato virus diseases (SPVD) are widespread in Asia and African
highlands (Uganda, Rwanda, Burundi and Kenya). SPVD is a devastating
disease due to the dual infection and synergistic interaction of Sweet potato
feathery mottle potyvirus (SPFMV), spread by aphids and Sweet potato
chlorotic stunt crinivirus (SPCSV) (recognized by ICTV) or Sweet potato
sunken vein crinivirus (SPSVV), both vectored by whiteflies.
3.1 Crop Losses Due to Virus and Viroid Diseases 103

(i) Potato viruses including Potato virus S, Potato virus X, Potato virus Y, Potato
leaf roll virus and some phytoplasma diseases are destructive wherever
potatoes are grown.
(j) Groundnut rosette virus is major disease in eastern, western and southern
Africa where it is spread by aphids in a pesistent manner. In India and in some
south Asian countries Groundnut (peanut) bud necrosis virus is economically
important and is thrips-transmitted, and belongs to the tospovirus group.
(k) Cadang-Cadang viroid disease of coconuts (CCCVd), has killed more than 15
million trees in the Philippines to-date. Another major disease of this crop is
Lethal yellowing, a phytoplasma disease that is destructive in Central
America and the US.
(l) Rice tungro disease is prevalent in almost all rice growing areas of South east
Asia. It is an associated disease with two viruses, an RNA virus, Rice tungro
spherical virus (RTSV), a member of the family Sequiviridae and a DNA
virus, Rice tungro bacilliform virus (RTBV), a member of the family Caul-
imoviridae. Rice yellow mottle virus is present only on the African continent.
Hoja blanca (white tip) of rice, Rice hoja blanca virus, is presently confined to
Central America.
(m) Streak disease of maize Maize streak virus, spreads throughout sub-Saharan
Africa on sugarcane, corn, wheat, etc. is responsible for reduced yields. It is
a leafhopper-transmitted mastrevirus in the family Geminiviridae.
(n) In vegetables like tomato and capsicum, Tomato spotted wilt virus and
Capsicum chlorosis virus, are tospoviruses, transmitted by thrips, and are
highly destructive in different parts of the world.
(o) Begomoviruses like Tomato leafcurl virus, Bean golden yellow mosaic virus,
Tomato yellow leafcurl virus, Okra yellow mosaic virus etc., are whitefly-
transmitted and highly devastating in members belonging to Solanaceae,
Malvaceae, Cucurbitaceae and Leguminosae, and are wide spread in the
tropics.
The above cited examples are some of the outstanding threatening plant dis-
eases, which emphasize the need to prevent future catastrophes. The losses caused
by different virus and virus-like diseases in annual and perennial crop plants are
more frequently affected. Crops such as cassava, potatoes, citrus, bananas, taro,
yam, sugarcane, sweet potatoes, etc. which are vegetatively propagated will collect
various viruses and eventually suffer severe losses. In the case of perennial crops,
not only the immediate losses are very high, but also the cost of replacement is
equally great if not greater than seen in annual crops. The intensive monoculture of
crop plants today invites the epidemic spread of many virus diseases. Rapid
vegetative propagation and a flourishing international trade have made matters
worse. Some of the financial losses in different virus-host combinations are pre-
sented in (Table 3.1).
104 3 Impact of Virus and Viroid Diseases on Crop Yields

Table 3.1 Crop losses due to certain viruses

Virus Crop Countries Loss
African cassava mosaic virus Cassava Africa $ 2000 millions
Barley yellow dwarf virus Barley UK £ 6 millions
Barley yellow dwarf virus Wheat UK £ 5 millions
Barley stripe mosaic virus Barley Montana $ 30 million
Beet yellows virus Sugar beet UK £ 5–50 millions
Cacao swollen shoot virus Cacao Ghana £ 3,650000 millions
Cassava mosaic virus Cassava SSA 2 9 109 US $
Cassava mosaic virus Cassava Uganda US $ 60 million
Cassava brown streak virus Cassava Malvi US $ 5-7 millions
Citrus tristeza virus Citrus World-wide £ 9–24 millions
Cocao swollen shoot virus Cocoa SSA 1.9 9 108
(No. of trees eradiated)
Cotton leaf curl virus Cotton Pakistan $ 5 billion
Groundnut rosette virus Groundnut SSA 1.56 9 108US $
Groundnut bud necrosis virus Groundnut Asia US $ 89 millions
Potato leaf roll virus Potato UK £ 30–50 millions
Rice hoja blanca virus Rice S. America $ 9 millions
Rice ragged stunt virus Rice SE Asia $ 140 millions
Rice tungro viruses Rice SE Asia $ 1500 millions
Rice yellow mottle virus Rice West Africa 3.3 9 106 tons
Tomato yellow leaf curl virus Tomato Domican Republic $ 10 million
Yellow mosaic virus Legumes India $ 30 million
Source Modified from Wilson and Davies (1992), Arif and Hassan (2000) and Hughes et al. (2001)

3.2 Yield Losses in Different Crops

Some more information one can obtain from the reviews and text book chapters
viz., Large 1966; Bos 1982; Agrios 1990; Gaunt 1995; Waterworth and Hadidi
1998, where the yield loss data have been furnished through tabular forms. In the
present book, the available crop loss estimates from the published data in different
crops like cereals, fruits, vegetables, legumes, oil seeds, fiber crops, spices,
ornamentals and tuber crops are as follows.

3.2.1 Cereals and Millets

In the tropics, cereals and millets are the main staple food for more than half of the
world’s population and the major cereal crops are rice, maize, oats, sorghum,
barley, wheat, pearl millet, triticale, finger millet and rye. The four most important
cereals grown for human food in the tropics are rice, maize, sorghum and wheat.
There are other minor cereals of warm temperate or tropical origin which are of
local importance in the tropics. These include finger millet (Eleusine coracana), a
staple food in parts of East and Central Africa; barnyard millet (Echinochloa
3.2 Yield Losses in Different Crops 105

Fig. 3.1 Symptoms of some economically important virus diseases of cereals

frumentacea), cultivated in India and southeast Asia; foxtail millet (Setaria italica),
grown in parts of India; and teff (Eragrostis tef), which is confined to the Ethiopian
highlands. The temperate cereals wheat and barley are also grown to a limited
extent in the tropics, largely at high altitudes: for example, wheat in Kenya and
barley in Ethiopia. Rice and wheat are important crops in the Indian subcontinent,
and depending on the water availability, one to two crops of rice is largely grown in
a majority of the states and wheat is grown in the cool season at low altitudes. Even
these cereal crops are also affected with number of virus diseases and some of the
disease symptoms due to viruses affecting cereals are presented in Fig. 3.1. These
viruses induce epidemics in some areas and are responsible for heavy yield losses;
for each crop the extent of yield losses due to plant viruses are presented herein.

(a) Rice (Oryza sativa)

Rice is the main staple food for more than half of the world’s population, including
seventeen countries in Asia and the Pacific. Half of the world’s population depends
on rice, particularly in Asia, and Asia produces almost 90 % of the rice cultivated
in the world. Among the important rice viruses and Phytoplasma diseases are,
tungro disease (Rice tungro spherical virus and Rice tungro bacilliform virus),
Rice hoja blanca virus (RHBV), Rice yellow mottle virus, Rice grassy stunt virus,
Rice stripe virus (RSV), and yellow dwarf phytoplasma cause serious losses in
almost all rice growing countries. RSV is another serious disease of rice in certain
106 3 Impact of Virus and Viroid Diseases on Crop Yields

regions of East Asia. It can cause high yield losses when severe epidemics occur.
It has affected several thousand hectares of rice-growing areas and severe infection
at the seedling to early tillering stage was reported to cause yield losses of
50–100 % (Lin et al. 1990). In eastern China, RSV caused yield losses of 30–40 %
in 2003–2004 (Zhang et al. 2007).
RHBV also causes losses in yield and during 1956, the rough estimates of 25 %
loss in the Cuban rice crop and more than 50 % in Venezuela were recorded
(USDA 1960). From Philippines, Jennings (1963) also estimated the yield loss due
to this virus. In Japan, RDV also annually damages about 100,000 ha and the
losses in yields were by 15,000 tons of grain annually (Iida 1969).
Among the virus diseases infecting rice, tungro is the most common in South
east Asia. Infection of rice with tungro viruses leads to an estimated annual eco-
nomic loss of $ 1.5 billion dollars annually (Hull 2002). This disease in Indonesia
affected 30,000–50,000 ha of rice in the 1930s and in Thailand over 300,000 ha
were severely damaged in 1966. In 1971, thousands of hectares were affected in
Philippines (Ou 1973). Studies conducted at IRRI, Philippines, showed that IR-8
plants inoculated at 15, 30, 45, 60 and 75 days after sowing, the yield reductions
were 68, 57, 30, 16 and 7 %, respectively (IRRI Ann Rept. 1966). From India,
Ghosh and John (1979) and John and Ghosh (1981) reported the yield losses at
similar stages of inoculation in Taichung (Native) 1 where 54 and 48 % loss for
early inoculations, but negligible for late inoculations. The field tolerant variety
IET-5061 did not show any significant difference in yield or number of tillers,
following infection, although height was affected. Even Srinivasan (1979) recorded
yield losses of up to 98.5 % in a highly susceptible variety Cv. ADT-31 during 1978
in Tanjavur (dist.), Tamil Nadu, India when a severe epidemics of this disease
occurred.
In India during the 1975–2001 period, rice tungro disease occurrence caused
considerable damage to rice production only in 48 districts mainly of Andhra
Pradesh, Bihar, Punjab and Tamil Nadu which were under irrigated and rain fed
low land ecosystems. An epidemics outbreak of tungro during 2001 in three dis-
tricts of west Bengal caused rice production losses of 0.5 mt valued at Rs. 2,911
millions. Using 2003 prices, a study demonstrated that tungro epidemics could
cause a maximum production loss of 53 % in a district, 23 % in a state and 2 % in
the country as a whole (Muralidharan et al. 2003).
RYMV is quite prevalent in 17 countries in Africa. This virus has caused yield
losses of 56–68 % in Niger (Reckhaus and Amadou 1986), 84–97 % in Sierra
Leone (Taylor 1989), 19–44 % in Burkina Faso (Sere 1991) and 64–100 % in Mali
(Sy et al. 1993). Yield losses due to RYMV fluctuated between 10 and 100 %
(Kouassi et al. 2005). In several rice cultivars almost total yield losses have been
reported when infected at an early stage of growth (Abo et al. 1998). Some farmers
have suffered complete crop failure in Cote d’Ivoire (Yoboue 1989).

(b) Wheat (Bread, Triticum aestivum; Durum, Triticum durum)

The losses in U.S. wheat were about 2 % annually during 1951–1960 due to the
combination Barley yellow dwarf virus (BYDV), Wheat streak mosaic virus
3.2 Yield Losses in Different Crops 107

(WSMV) and Soil-borne wheat mosaic virus (SBWMV). BYDV caused annually
an average loss of $ 500,000. McKirdy et al. (2002) from Western Australia have
reported yield gaps which ranged from 0 to 2,700 kg/ha and the relationship
between yield gap and incidence of BYDV was always linear in wheat and oats.
More information is also available from this centre on BYDV yield losses which is
provided by Thackray et al. (2005) who have recorded the yield decrease due to
BYDV infection was 55–72 kg/ha. The variation in seed weight was 88 % and
protein content by 69 %. Hoffman and Kolb (1998) reported the yield reduction in
eight soft red winter wheat cultivars in response to BYDV infection and stated that
yield is the product of three components: number of spikes per unit area, number
of kernels per spike, and kernel weight. They have observed that both kernels per
spike and kernel weight were reduced by infection. Reductions in number of
kernels per spike ranged from 11 to 30 %, while kernel weight reductions ranged
from 3 to 19 %.
Epidemics of WSMV, a mite (Aceria tosichella) transmitted virus occurred in
1953, 1954 and 1959 and estimated reduced yields by 20 % in Kansas during
1959. Fitzgerald and Timian (1960) reported yield losses due to Barley stripe
mosaic virus (BSMV) in winter wheat were 50.4 bushels/acre, while disease-free
plots yielded 62.2 bushels and an average reduction was almost 19 %. Individual
reductions ranged from 13.4 % for cv. Wasatch to 25.9 % for cv. Cache. In
southern Alberta (Canada) due to epiphytotics of WSMV, loss in winter wheat
exceeded 700,000 bushels or 18 % of the potential yield. Diseased plants yielded
72 % less than the healthy ones, but since disease intensities range from 27 to
55 %, yield reductions varied from 20 to 40 %. Severely diseased plants die
without heading. Quality analysis showed a loss of 1.9 % in milling yield
(Atkinson and Grant 1967). WSMV causes yield losses of 10–99 % in wheat
(Murugan et al. 2011).

(c) Barley (Hordeum vulgare)

In the U.S. the estimated average annual losses for the period 1951–1960 due to
Barley stripe mosaic virus (BSMV) and Barley yellow dwarf virus (BYDV) dis-
eases were 4.8 %. This presents an average loss of £1.90/ha to the farmer. In U.S.
BYDV infects annually and causes an average loss of $ 300,000 (USDA 1965).
During 1951, BYDV infection has caused a 10 % loss in the barley crop in
California due to new outbreak of the disease (Oswald and Houston 1951, 1953).
From Montana, Gill (1970) reported an epidemic of BYDV during 1969 which
caused an estimated loss of 1,380.860 bushels of two rowed Herta barley in an area
of approximately 2,700 square miles. Yield losses of more than 70 % have
occurred in susceptible barley varieties as a result of BYDV infection (Watson and
Mulligan 1960; Doodson and Saunders 1970). From Carlow, grain yield reductions
in spring barley due to BYDV were also reported by Kennedy and Connery (2005).
BSMV is another economically important virus disease of this crop and from
Montana, Eslick (1953) demonstrated that this virus yielded 35–40 % less than
disease-free plantings and the average yield reduction was 31 %. Since 1970,
losses due to BSMV were about half a bushel per acre after using the certified seed
108 3 Impact of Virus and Viroid Diseases on Crop Yields

(Carroll 1980). Carroll, further reported that 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). Where as Hagborg (1954) found a reduction of 64 % yield loss
with this virus. From North Dakota, Timian and Sisler (1955) reported the losses
with this virus ranged from 17 to 24 %.

(d) Maize (Zea mays)

Maize dwarf mosaic virus (MDMV) infected corn in Ohio, during 1962 and by 1974
it had spread all over the state and caused an annual loss of 500,000 bushels
(Johnson et al. 1965; Williams and Alexander 1965). In Idaho, the same virus caused
yield reductions of 75 % in severely infected fields (Forster et al. 1980). Rosenkranz
and Scott (1978) also noticed 23 % yield reduction with this virus (strain A), when
inoculated at the five-leaf stage. Kingsland (1980) studied the effect of this virus
infection on yield of corn cvs. 72–44 A and 76–29, and the average yield reduction
of 62 % occurred under conditions of 100 % disease incidence.
In many parts of Africa, Maize streak virus (MSV) caused severe losses in
irrigated maize crops. From Rhodesia, Rose (1974) observed that plants infected
less than a week after germination, produced no yield, at three weeks about half
yield, and at 8 weeks nearly full yield of maize. During the epidemics nearly every
maize plant has been infected within eight weeks after germination and it was not
worth harvesting. Guthrie (1978) also recorded yield losses ranging 25–60 % due
to this virus under field conditions.
From England, Panayotou (1977) reported that Barley yellow dwarf virus
(BYDV) has reduced the fresh weight of maize, both the stalks and ears of the
varieties Anjou 210, Pioneer 131 and Austra 290 by 24, 56 and 34 %, respectively.
Beuve et al. (1999) have noticed that the grain yield of maize infected with the
PAV serotype of BYDV was 15–20 % less than controls because infected plants
had fewer kernels per ear, where as in barley, infected with the same serotype
virus, yield reductions of up to 38 % were recorded (Edwards et al. 2001). In
Colombia, Pineda and Martinez (1977) estimated the yield losses in green matter
and grain yield of maize cv. ICA-V-504, infected with maize Colombian stripe
virus at different stages of infection. Losses in grain yield reached 89 % at stage 1
compared with 81 % reduction in green matter. At stages 2, 3 and 4 grain yield
losses were[50 %; at stage 5 and 6 they were 38 and 8 %: Losses in green matter
were [45 % for stages 2 and 3; but insignificant or nil at stages 5 and 6. Pre-
liminary studies of yield losses of Central American varieties with Maize rayado
fino virus transmitted by corn leaf hopper, Dalbulus maidis in American Tropics,
indicated reductions of 40–50 % of the weight of the mature ear, and yield losses
reached 100 % in introduced foreign or newly developed varieties (Gomez 1983).
In Columbia, total losses due to wilting and quick death of infected plants, due to
new virus disease have been reported for some cultivars and losses as high as
89–100 % for the weight of the green forage or grain was recorded by others
(Martinez and Lopez 1977). Sugarcane mosaic virus also reduced maize yields in
East Africa by 25 % (Kulkarni 1973). Louie and Darrah (1980) also reported the
3.2 Yield Losses in Different Crops 109

mean yield reduction which varied from 18 to 46 % in ten Kenyan hybrids of

maize due to the same virus infection. Maize chlorotic mottle virus caused crop
losses of 10–15 % in natural field infections and losses of up to 59 % in experi-
mental maize plots (Loayza 1977).

(e) Sorghum (Sorghum bicolor)

In Texas, Bockhalt and Toler (1968) found that sorghum breeding lines and
hybrids inoculated with Maize dwarf mosaic virus (MDMV) suffered yield
reductions of zero to 47.6 % in seven lines and zero to 18.9 % in six hybrids
involving these lines. Toler (1985) has reported that the losses due to MDMV
infection in sorghum was up to 100 % in highly susceptible cultivars, while it was
less than 5 % in resistant lines. From Queens land, Australia Henzell et al. (1979)
studied the yield losses in 11 grain sorghum with Sugarcane mosaic virus infec-
tion. The effect on cultivars producing mosaic symptoms ranged from tolerance to
yield losses of ca 25 %. In East Africa, the same virus reduced the yield by 73 %
(Kulkarni 1973).

(f) Pearl Millet (Pennisetum glaucum)

From India, Choudhary and Singh (1978) reported that pearl millet plants com-
pletely infected with pennisetum strain of Maize streak virus caused
98.68–98.84 % reduction in grain yield, while it was 74.86–77.00 % reduction
when partially diseased, when compared with healthy plant yield.

(g) Oats (Avena sativa)

The estimated average annual loss due to Barley yellow dwarf virus (BYDV) in the
U.S. was 3.8 % during 1951–1960. In 1959, an epiphytotic of this disease caused
severe damage in the oat producing North Central States and the estimated yield
losses as follows: Missouri 37 %, Indiana 27.5 %, Kansas 25 %, Iowa 12 %, and
Wisconsin 5 %. The estimated losses from BYDV in Oregon were 9.1 %, in 1957,
9.5 % in 1958, and 14.8 % in 1959. Severe damage also occurred in the Northeast in
1960 (USDA 1965). From Wisconsin, Shands and Cruger (1959) recorded 35.1 %
yield reduction with the same virus infection. Endo and Brown (1957, 1963) also
estimated the yield reductions at 3-leaf and boot leaf stages in 3 cultivars. The yield
reductions were: Fayette, 92.5 and 10.1 %, Clint land 94.4 and 21.8 % and Rodney
75.8 and 15.0 % respectively. McKirdy et al. (2002) have also reported the quan-
tification of yield losses due to BYDV from Western Australia. In winter oats, soil
borne oat mosaic is a major threatening problem and losses were up to 100 % and
susceptible varieties failed to head (Toler and Hebert 1963).

3.2.2 Food Legumes

(a) Common Bean (Phaseolus vulgaris)

Among the virus diseases affecting French bean Bean common mosaic virus
(BCMV), Beet curly top virus, Bean yellow stipple virus, Bean yellow mosaic virus
110 3 Impact of Virus and Viroid Diseases on Crop Yields

(BYMV), Tobacco necrosis virus, etc. are widely occurring throughout the bean
growing countries and are economically important. Hampton (1975) reported that
moderate and severe BCMW isolates caused 50 and 64 % reductions in the number
of pods/plant, respectively and 53 and 68 % reductions in the seed yields. From
India, Sastry (unpubl. data) reported out that French bean plants inoculated at 10 and
25 days after germination produced on average 11.5 and 27.0 pods/plant, weighing
40.54 and 191.45 g/plant, respectively; on the other hand the healthy control plants
produced 33 pods and weight was 252.99 g/plant. The calculated percentage loss in
yield was 83.98 and 24.32 in plants infected at 10 and 25 days. The pods from early
infected plants were small, disfigured and mostly unmarketable.
BYMV caused a 33 % reduction in the number of pods/plant and a 41 %
reduction in seed yield. The total reduction in yield was 40–45 % less than the
yields of adjacent control plants (Hampton 1960, 1975). In Cuba, Bean yellow
stipple virus caused 44–75 % yield loss, in five commercial bean cvs. (Blanco
Sanchez and Bencomo 1979). Kaiser (1972) observed 100 and 96 % seed loss in
bean cv. Bountiful infected with Pea leaf roll virus at pre-bloom stage (3–5 weeks
after planting), and full bloom stage (8–10 weeks after planting), respectively. In
El. Salvador, Rodas (1975) reported 37.7 % yield reductions due to Bean golden
mosaic virus (BGMV). The same virus in Jamaica, caused 57 and 25 % yield
losses when the plants were infected at 17 and 32 days after sowing (Pierre 1975).
From Brazil, Costa and Cupertino (1976) observed 75 and 100 % yield loss when
the plants were infected at 15 and 30 days after sowing. The average weight of 100
seeds was 12.2 and 10.6 g respectively, compared with 21.1 g in control. In
common bean, Aragao and Faria (2009) have reported 40–100 % yield losses due
to BGMV. In Latin America BGMV in beans has caused 80–100 % yield losses
during summer months of the year and in Argentina Bean dwarf mosaic virus
(BDMV) has destroyed more than 20,000 ha every year from 1979–1982
(Francisco J. Morales, personal observation).

(b) Pea (Pisum sativum)

Pea leaf roll virus and Pea mosaic virus attack peas in Germany, Belgium,
Holland, U.K., Europe, U.S., Australia, Japan, India, New Zealand and other
countries and caused yield losses. The estimated annual loss caused by virus
diseases was 6 % in the U.S. for the period 1951–1960 (USDA 1965). In the UK at
least 5 % loss in green peas was caused by different viruses (Ramaswamy 1972).
Chiko and Zimmer (1978) reported the average high yield losses due to Pea seed
borne mosaic virus (PSbMV) infection to be 11 and 36 % in cv. Trapper and
Century, respectively. Kraft and Hampton (1980) also recorded the yield losses
due to PSbMV virus infection at different weeks after emergence of seeds. Tomato
spotted wilt virus has also reduced yield losses to the tune of 57.1–84.4 per plant in
pea (Fajardo et al. 1998).
Kaiser (1972) reported that the percentage of seed yield loss due to Pea leaf roll
virus was 81 and 19 in the cv. ‘Rondo’ infected at pre-and full bloom stages,
respectively. Blaszczak and Weber (1978) recorded the various degrees of yield
losses due to BYMV.
3.2 Yield Losses in Different Crops 111

(c) Soybean (Glycine max)

In recent times the cultivation of soybean has increased dramatically for oil pro-
duction, and yields continue to be hampered by different virus diseases. From
Indiana, Kendrick and Gardner (1924) demonstrated yield reductions of 30–75 %
due to Soybean mosaic virus (SMV) during 1920 and 1922. Ross (1968, 1969a, b)
recorded yield losses of 8–25 % with the same virus strains in certain soybean
varieties. Synergistic yield reductions of up to 80 % were observed when soybean
plants were inoculated with SMV and Bean pod mottle virus, which produced
severe symptoms. Quiniones et al. (1971) also recorded the reduced seed yields in
three varieties by 18 % due to infection with SMV, which caused losses of
approximately 60 % when 85 % of the plants were infected.
In Taiwan Peanut stripe virus (PStV) has caused significant yield reduction of
up to 90 % and seed weight loss of 100 seed by 50 % in soybean (Green and Lee
1989). By early inoculation of soybean plants with Tobacco ring spot virus
(TRSV) yield was reduced by 79 % (Crittenden et al. 1966). In India, Soybean
yellow mosaic virus reduced the number of pods by 39–47 % and weight by
39.5–41.2 % (Suteri and Srivastava 1975). From Brazil, Costa (1975) noticed the
yield losses of 13–87 % in 13 soybean varieties affected with Soybean crinkle
mosaic disease, transmitted by B. tabaci. In Georgia, Cowpea chlorotic mottle
virus (CCMV, soybean strain) reduced the seed yield of soybeans cv. Davis by 23
and 31 % in 1968 and 1969 respectively. Reduced seed number accounted for
78 % of the yield loss and reduced seed size accounted for the remaining 22 %
loss (Harris and Kuhn 1971). Peanut mottle virus (PMV) also reduced the soybean
yields by 5–28 % during 1972–1975 (Demski and Kuhn 1977). Demski and Jellum
(1975) studied the yield losses due to synergistic reaction with four viruses. The
average yield loss was 18, 31, 66 and 76 % for single infection by PMV, CCMV,
SMV and TRSV, respectively. From doubly infected plants the yield loss was 46,
78, 80, 82, 87 and 98 % for PMV-CCMV, PMV-SMV, PMV-TRSV, CCMV-
TRSV, CCMV-SMV and SMV-TRSV, respectively. They also observed decreased
oil content due to infection. Generally PMV and CCMV had the least effect and
SMV and TRSV had greater effect on seed protein and oil. Tobacco streak virus in
soybean at U.S. has caused yield loss up to 25 % (Fagbenle and Ford 1970) and the
same virus in Brazil, caused yield losses up to 100 % (Costa and Carvalho 1961).
In Taiwan, Peanut stripe virus caused up to 90 % reduction in yield of soybean
(Green and Lee 1989).
Bean pod mottle virus (BPMV) transmitted by beetles has reduced soybean
yields ranging from 3 to 52 % (Gergerich 1999). Over a broad geographic range,
yield reductions between 10 and 40 % have been reported (Horn et al. 1973; Ross
1968, 1986). Impact of BPMV on yield depended upon the time of virus infection
relative to plant development, with early infection, the highest yield reduction was
recorded (Gergerich and Scott 1996). Ross (1968) showed that mixed infection
with BPMV and SMV reduced yield up to 85 %. In Louisiana, it was determined
that the BPMV infection level needed to be between 20 and 40 % of the plant
population in order to cause economic loss (Horn, et al. 1973). Yield effects on
soybean with Peanut stripe virus was studied by Gillaspie and Hopkins (1991) in
112 3 Impact of Virus and Viroid Diseases on Crop Yields

six soybean cvs., no significant decrease in plant height seed weight for any
cultivar was recorded.

(d) Chickpea (Cicer arietinum)

From ICRISAT (India), Horn et al. (1995) have reported the yield losses due to
Chickpea chlorotic dwarf virus at two locations in India. When infection was
before flowering, yield losses of individual plants amounted to nearly 100 % in the
three cultivars studied. They have also recorded that the plants that became
infected during flowering had yield loss of 75–90 %.

(e) Cowpea (Vigna unguiculata)

In no country where cowpeas are grown are there any reports that indicate freedom
from virus infection. In Nigeria, yield losses in cowpea of up to 87 % was
attributed to virus infection (Shoyinka et al. 1997). Complete loss of the cowpea
crop in northern Nigeria was reportedly due to Cowpea aphid-borne mosaic virus
(Raheja and Leleji 1974). In western Nigeria, Cowpea mosaic virus early infec-
tions (7 days after emergence) reduced yield by 40–60 %, where as late infections
(after flowering) caused only 5–10 % reductions (Gilmer et al. 1973). Kaiser and
Mossahebi (1975) in Iran studied the assessment of loss with Cowpea mosaic virus
in 17 cultivars and it varied from 13–87 %. The loss in seed yield of cowpea due to
Cowpea banding mosaic virus and Cowpea chlorotic spot virus varied from 11.6–
43.8 % and 24.2–66.7 %, respectively (Sharma and Varma 1981).
Yield reductions of up to 60–100 % due to Cowpea yellow mosaic virus
infection were reported (Chant 1960; Shoyinka 1974; Gilmer et al. 1974). The
earlier infection has resulted in greater yield reduction, although even with
infections as late as six weeks after planting, significant reductions were noticed
(Chant 1960).
From Georgia, U.S., Pio-Ribeiro et al. (1978) recorded 14.2 and 2.5 % reduction
in the yield of California black eye seed following with infections of Cucumber
mosaic virus and Black eye cowpea mosaic virus, respectively. Whereas yield on
doubly infected plants (cowpea stunt) was reduced by 86.4 %. Losses due to mixed
infections of Bean yellow mosaic virus, Cowpea chlorotic mottle virus and
Cucumber mosaic virus varied significantly (Harrison and Gudauskas 1968).
Experiments conducted at I.I.H.R. Bangalore (India) revealed that inoculation with
BICMV at different stages of cowpea cvs Arka Garima and C-152 resulted in yield
losses of 65.7–89.7 % when infected 10–20 days after sowing (Anon 2011).

(f) Greengram/Mungbean (Vigna radiata)

Nene (1972) reported the yield losses of mungbean due to Mungbean yellow
mosaic virus, which varied from 10 to 100 % depending on the stage at which the
plants were infected. There is also a report that this virus decreases the grain yield
to the extent of 22.3–61.7 % (Yadav and Brar 2010).
Premchand and Varma (1983) reported the changes in growth parameters and
yield reduction due to yellow mosaic virus infection ranged from 9.6 to like
38.2 % in height, 7–28.5 % in fresh weight of shoot and 4.3–22.1 % in dry weight,
3.2 Yield Losses in Different Crops 113

25.7 % in 1,000 seed weight of susceptible cultivar. However, the germinability of

seeds was apparently unaffected by yellow mosaic.
Bisht et al. (1988) studied the yield loss estimates due to Mung bean yellow
mosaic virus (MYMV) in four mungbean promising cultures viz., PIMS-1, PIMS-
2, PIMS-3 and PIMS-4, and recorded the average reduction in plant height and
fresh plant weight was statistically non significant. Average reduction in number
of pods was 5.90, 5.83; 25.65 and 24.76; in 100 grain weight was 4.95, 4.85, 27.42
and 27.02 and in yield per plant was 6.47, 6.35, 26.07 and 25.58 % over healthy
plants, respectively, in varieties PIMS-1, PIMS-2, PIMS-3 and PIMS-4. Even
Quaiser Ahmed (1991) reported a yield loss of 83.9 % and a maximum growth
reduction of 62.94 % on Vigna radiata cv. Pusa baisakhi due to Mungbean yellow
mosaic gemini virus infection. Losses in yield and yield components of greengram
infected with the same virus was reported by Khattack et al. (2000).

(g) Blackgram/Urdbean (Vigna mungo)

From India Nair and Nene (1974a, b) estimated the yield losses in blackgram
infected with Mungbean yellow mosaic virus (MYMV) at different plant stages
and observed that the plants infected at the first, second and third week did not
produce any seed, whereas at four week infection, plants yielded 0.69 g seeds/
plant. The total seed yield gradually increased with increase in age of the plant.
The yields were 1.90, 2.93, 3.78 and 4.15 g/plant for 5, 6, 7 and 8 weeks at which
the inoculations were made. While the healthy plant produced 4.69 g seed/plant.
The total number of pods/plant at these stages (4–8 weeks and healthy plants) were
3.63, 10.00, 15.13, 19.00, 20.13 and 22.87, respectively. Similar trend was noticed
even in seed number/pod. Vohra and Beniwal (1979); Premchand and Varma
(1983) and Jain et al. (1995) also observed similar type of yield losses with the
same virus MYMV. Significant decrease in the number of pods and grain yield was
noticed in blackgram up to 50 days after planting, insignificant losses were noticed
in infections at or after 60 days of planting.
Urdbean leaf crinkle virus (ULCV) is another economically important virus
disease of blackgram and on inoculation, the average number of pods produced by
the diseased plant was 41.77 as compared to 84.96 in healthy plant. The yield was
6.31 and 16.64 g in diseased and healthy plants, respectively. Estimated (esti-
mation based on 80,000 plants/hec grain yield in case of diseased and healthy
plants was about 5.04 and 13.31 quin/ha respectively and the percentage of loss in
yield was 62 % (Nene 1972). In Pakistan, Bashir et al. (1991) have also reported
that ULCV in Pakistan decreases the blackgram grain yield between 35 and 81 %
depending upon the genotype and time of infection.
Blackgram sterility mosaic virus also greatly reduced the number of blackgram
pods when infection occurred at 15 days of age and the percentage of reduction in
the number of pods decreased with increases in the age (30 and 45 days) of the
plants at the time of inoculation. Similar trend was also noticed with Black gram
mosaic virus infection (Narayanaswamy and Jaganathan 1974). Nene (1972)
114 3 Impact of Virus and Viroid Diseases on Crop Yields

reported that mosaic mottle disease reduced the yield by about 71.5 %. Bean
common mosaic virus also caused the losses of blackgram yields up to 83.6 %
(Agarwal et al. 1976).

(h) Broad Bean/Faba bean (Vicia faba)

Plants infected with Broad bean true mosaic, Pea mosaic and Pea enation mosaic
viruses reduced the seed yield by c. 60, 81 and 83 %, respectively, (Blaszczak and
Jamrog-Janicka 1972). In Sudan, Pea mosaic virus and Broad bean mottle virus
infected broad bean plants yielded 9.0 and 17.5 pods/plant respectively, while it
was 28.5 pods/healthy plant (Hussein and Freigoun 1978). In Manitoba (USA),
Bean yellow mosaic virus infection at pre-bloom (35 days from seeding), full
bloom (48 days from seeding) and post bloom (61 days from seeding) reduced
yields by 59, 48 and 17 % with mild isolate and 96, 70 and 17 % with severe
isolate, respectively (Frowd and Bernier 1977). Earlier, from Iran, Kaiser (1973)
also demonstrated 44, 42 and 23 % yield reductions, when infection of broad bean
occurred at pre-bloom, full bloom and post-bloom stages. Under Tasmanian
conditions, Subterranean clover red leaf virus, reduced the yield by 83 and 98 %
in faba bean cvs. Coles dwarf and Triple white respectively, when the infection
took place before pod set (Johnstone 1978).

(i) Redgram/Pigeonpea (Cajanus cajan)

In redgram, Pigeonpea sterility mosaic disease, transmitted by mite, Aceria cajani
causes an estimated annual loss of 205,000 tons of grains in India alone
(Kannaiyan et al. 1984). A susceptible genotype infected in the early stages (first
45 days) of crop growth shows near complete sterility and yield losses up to
100 %. As the plants grow older ([45 days), their susceptibility to sterility mosaic
disease decreases, such that plants show partial sterility (Reddy and Nene 1981). In
the case of early infection, yield reduction is related to the percentage of infected
plants, but in later infections, yield reduction is not correlated to the percentage of
infected plants, since they show only partial sterility. Genotypes such as ICP 2376
that have ring spot symptoms do not show any sterility, and thus suffer no obvious
yield loss. Genotypes such as NP (WR) 15, that develop mild mosaic symptoms
are partially sterile, and their yield loss is less (19–64 %). Disease incidence is
usually higher in ratoons and perennial pigeonpea crops.

(j) Lentil (Lens culinaris)

Lentils play a major role in the food and nutritional security of millions, particularly
among low income Asian families, because of the high protein content of their seed.
Latham et al. (2004) reported loss estimates of seed yield and dry weight due to
Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV) in lentil. In plants
of lentil cv. Matilda, infected with AMV decreased shoot dry weight by 74–76 %,
seed yield by 81–87 % and individual seed weight by 10–21 %, while CMV
diminished shoot dry weight by 72–81 %, seed yield by 80–90 % and individual
seed yield by 17–25 %.
3.2 Yield Losses in Different Crops 115

3.2.3 Vegetables

(a) Tomato (Solanum lycopersicum, Family Solanaceae)

Virus and virus-like diseases causing losses to tomato include Tomato leaf curl
virus (TLCV), Tomato yellow leaf curl virus (TYLCV), Tobacco mosaic virus
(TMV), Beet curly top virus (BCTV), Tomato bushy stunt virus (TBSV) and
Tomato spotted wilt virus (TSWV). Due to virus and other diseases, the mean yield
per hectare in Southern African countries ranged from 1.5 to 14 tons/ha, as
compared to the world average of 25 tons/ha (AVRDC 1998). In South Florida, a
virus complex, including mosaic and curly top was a limiting factor for tomato
production. Tomatoes grown in Florida are worth about 2,300/ha and estimated
average losses range between 10 and 30 % or 200 and 700/ha (USDA 1965).
Twardowicz-Jakuszowa (1961) recorded a decrease of 25–79 and 11–56 % in
tomato yield with TMV and Tomato streak virus, respectively. In UK, Broadbent
(1964) reported that with TMV infection, the fruits have shown necrotic pitting,
bronzing or severe mottling and sometimes irregular shape. The percentage of
diseased fruits ranged from 19 to 33 % depending on the stage of infection. The
infection at the stage when most of the fruits were set, will greatly affected the fruit
quality and leads to maximum financial loss. Similar fruit symptoms were also
earlier reported by Murakishi (1960). This loss in yield was due to ‘dry-set’ i.e.
failure of the flowers to set fruits. Weber (1960) reported yield from the healthy
controls were 16.4 tons/acre, where as yields from early inoculated and later
inoculated plants ranged between 13.9 to 15.0 tons/acre. In 1990–1991, crop losses
due to Tomato mottle virus (ToMoV) (whitefly-transmitted) were estimated at $
140 million (Schuster 1992). Tom GV1 virus and Tom GV2 virus another whitefly
transmitted virus, with incidences ranging from 20 to 100 % has caused crop
losses up to 100 % (Polston and Anderson 1997). Losses were still high with
mixed infections (Brack 1979). Losses with TMV in tomato were also recorded
from different countries (Heuberger and Norton 1933; Alexander 1950; Klin-
kowski 1958; Twardowicz-Jakuszowa 1961; Crill et al. 1973; Mena 1973). Pepino
mosaic of tomato which is highly mechanically transmitted in glass houses and
fields has caused losses to the extent of 20–40 % (Soler et al. 2000).
Sastry and Singh (1973) recorded reduced tomato yield losses which varied from
28.9 to 92.3 % depending on the stage of Tomato leaf curl virus (TLCV) infection.
Saikia and Muniyappa (1989) have also reported yield losses due to TLCV in
Karnataka, south India. Tomato fruit yields of 1.2, 2.3 and 5.1 t/ha (95, 90 and 78 %
loss in yield) were obtained when tomato plants were infected at 2, 4 and 6 weeks
after planting, respectively. However, when the plants were infected 10 weeks after
planting, the yield was 20.6 t/ha (10 % loss in yield) compared with healthy plants
(22.9 t/ha). Another economically important virus disease of tomato which is
worldwide in distribution is Tomato yellow leafcurl virus (TYLCV) is causing
heavy yield losses. Even though TYLCV is introduced into the Dominican
Republic during 2007, it has caused tomato crop losses up to 100% which were
estimated to cost more than $10 million (Gilbertson et al. 2007). From Lebanon,
116 3 Impact of Virus and Viroid Diseases on Crop Yields

Makkouk et al. (1979) reported that TYLCV, another whitefly transmitted virus has
reduced the yield by 63 %, if plants were inoculated three weeks after transplan-
tation. Yield losses reached 80 % according to Mazyad et al. (1979). In the surveys
at CIAT, the average perceived yield losses due to TYLCV in tomato were up to
40 % in Malawi, 50 % in Kenya, 75 % in Tanzania and 100 % in Sudan (CIAT
1998). In Lebanon, the yellow leaf curl diseases decreased yields by more than
80 % in many tomato crops (Makkouk et al. 1979). The losses due to TYLCV in
Dominican Republic during 1989–1995 were estimated at $ 50 million (Polston and
Anderson 1997). Petershmitt et al. (1999) have reported the estimated yield losses
due to TYLCV in Morocco, Tunisia and Libya were 65–100, 30–100 and 50–80 %
and value lost (million $) was 245–404; 179–450 and 79–150 million dollars,
respectively. The same virus in South Africa was responsible for yield losses to the
extent of 49–100 % (Pietersen and Smith 2002). They have also recorded tomato
yield losses due to Tomato curly stunt virus which is distantly related to TYLCV.
Depending on the time of infection yield losses due TYLCV ranged 50–82 %
(Ioannau and Iordanou 1985). While reviewing the economic losses due to TYLCV,
Pico et al. (1996) have reported economic losses up to 100 % in tomato crop in
tropical and subtropical regions.
In Nigeria, yield losses due to bunchy top disease in tomato was up to 34 %
(Ladipo 1973). Fernandex and Goborjanyi (1978) noticed 55 % yield loss in cv.
Placero Lobulado due to Tobacco etch virus infection. Pico et al. (1996) have also
reported the yield losses due to TYLCV

(b) Chilli/Pepper (Capsicum annuum and C. frutescens, Family Solanaceae)

In all the pepper and chillies growing areas of the world, the viral diseases like
CMV, PVY, TMV, TEV, pepper veinal necrosis, Tobacco leaf curl virus etc. have
caused losses to various extent. In the U.S. the average annual loss caused by
TMV, CMV, TEV and PVY was 1.0, 1.0, 0.5 and 0.5 %, respectively for the
period 1951–1960 (USDA 1965). Feldman et al. (1969) from Argentina, reported
40 and 80 % yield loss in susceptible pepper cvs. like California Wonder and
Perfection, where as the yields of the cv. Yolo Wonder was statistically unaffected
as it is resistant to TMV. During 1967–1968, the early inoculation (10 days after
planting) and late (40 days after planting) yielded 2.8 and 13.0 kg whereas the un-
inoculated control plants yielded 20.4 kg. In the cv. Perfection, similar yield losses
were 7.6, 16.8 and 34.0 kg, respectively. Chilli plants inoculated with PVY at 15,
45 and 90 days after sowing produced 9.0, 16.7 and 29.4 fruits. The number and
weight of the fruits at late infection (90 days after transplanting) was on par with
healthy control plants. Saha et al. (2005) from India have reported fruit yield losses
in chillies due to Chilli leaf curl virus during summer season to the extent of 81.3,
63.1 and 22.7 % from plants infected at 30, 45 and 60 days after transplanting,
respectively. Whereas in the case of winter season, the loss in yield was found to
be 74.6, 51.2 and 21.3 % at the same stages of infection. Sastry (unpubl. data)
observed that the yield loss of green chillies in the cv. NP 46A due to leaf curl
disease (a strain of Tobacco leaf curl virus), ranged from 57.0 to 70.4 %. Joshi and
Dubey (1973) recorded the total loss in fruit weight when the chilli plants had
3.2 Yield Losses in Different Crops 117

100 % CMV infection. From Bulgaria, Kovachevsky (1940) reported that in some
places a 100 % infection of ‘Resigkrankheit’ (CMV strain) was not uncommon
and in such cases the yield was reduced to 10–30 % of the normal. Danco (1964)
also noticed 20 % reduction in yield with the same virus. More marked reduction
in yield was observed with TMV when infection occurred before flowering. The
yield of capsicums cv. Yolo Wonder due to TEV was decreased by 47 % when
infected 3–6 weeks after transplanting (Fernandex and Goborjanyi 1978).
From Everglade’s area of Florida, Simons (1956) noticed yield loss of up to
50 % in cv. California Wonder peppers infected with Pepper vein banding mosaic
virus. Ajroldi (1939) estimated loss in pepper yield of 50–70 % due to Italian
pepper mosaic virus. Lamptey and Bonsi (1977) recorded 46–90 % loss in fruit
yield due to Pepper veinal mosaic virus and it depended on the time of inoculation.
On the other hand, Atiri and Dele (1985) could observe the symptom severity
which was negatively correlated with fruit number weight and length in all the cvs.
although this was not always statistically significant.

(c) Eggplant/Brinjal (Solanum melongena)

This crop suffers with mosaic and little leaf diseases. Singh and Singh (1975)
reported the yield loss due to mosaic disease which ranged from 9.51 to 27.03 %
during 1969 and 1970. Tobacco ring spot virus infected plants caused yield
reductions ranging from 65.2 to 70.3 and the fruits from the early infected plants
were pale in color and had concentric rings (Sastry and Nayudu 1978). Chakrabarti
and Chowdhury (1979) estimated the yield losses due to little leaf disease and they
reported that when compared with the healthy control, the diseased plants reduced
the number of fruits per plant inoculated at 4, 6, 8 and 10 weeks after sowing by
100.0, 100.0, 76.5 and 67.2 %, respectively.

(d) Ladies Finger (Okra/Bhendi) (Abelmoschus esculentus, Family Malvaceae)

During summer months okra crop will show more than 70 % Yellow vein mosaic
virus (YVMV) infection. Sastry and Singh (1974) estimated the fruit yield losses at
different stages of infection. Plants infected with YVMV at 35, 50 and 65 days
after germination, could produce only 2, 4 and 11 fruits/plant, which are small and
pale yellow in color, whereas control healthy plants had 20 fruits/plant. The
percentage of loss in yield when compared to healthy plants was 93.80, 83.63 and
49.36 when they were infected at 35, 50 and 65 days after germination. The
corresponding figures of calculated loss were Rs. 3,526.75, Rs. 3,257.70 and Rs.
2,355.95/ha, respectively, when the cost of okra was Rs. 0.50/kg. A similar type of
yield losses were also noticed by Chellaiah and Sellammal Murugesan (1976) with
the same virus-host combinations. Sinha and Chakrabarthi (1978) reported that the
loss in weight of okra seed was 86.1 % in plants produced YVMV symptoms on
33rd day after sowing and was lowest (32.9 %) in the plants which showed
symptoms on 75th day after sowing.
Bhagat (1999) carried out the quantitative assessment of growth and yield
parameters of bhendi against Bhendi yellow vein mosaic virus (BYVMV) (Syn.
YVMV) incidence in tolerant, susceptible and highly susceptible cvs. viz.,
118 3 Impact of Virus and Viroid Diseases on Crop Yields

Parbhani kranti, Vaishali vadhu and Pusa sawani, respectively. Yield and other
attributes such as number of fruits per plant, number of leaves, plant height, length,
girth and fruit weight were found less affected in the resistant cv., Parbhani kranti
compared to Vaishali vadhu (susceptible) and Pusa sawani (highly susceptible)
cvs. Even Ahmed (2001) has recorded reduction in plant height (44.43, 61.55 and
71.40 cm), fruits per plant (5.0, 8.3 and 14.0) and fruit size (6.39 9 0.87 cm,
8.46 9 1.28 cm and 11.34 9 1.5 cm) when bhendi plants were infected at
different stages (35, 45 and 55 days after planting) by yellow vein mosaic virus,
as compared to healthy.
Okra leaf curl virus (OLCV) was recorded for the first time in Nigeria by Lana
(1976) later it was reported from India, Burkina Faso, Ivory Coast and Pakistan.
The disease has caused 30–70 % economic losses and the magnitude of yield loss
was, however, depended on the age of infection of the crop. Losses were higher
when there was combined infection of YVMV and OLCV. Singh (1990) found that
Enation leaf curl virus (ELCV) caused as much as 93.10 % losses in yield of okra
fruits in case of early infections. Another important virus of okra in India is
Tobacco stunt virus which has reduced yield losses to the extent of 63 %
(Krishnareddy et al. 2003).

(e) Cucurbits( family Cucurbitaceae)

Viruses like Cucumber mosaic virus (CMV) , Watermelon mosaic virus (WMV-2),
Cucumber green mottle mosaic virus (CGMMV) and Tobacco ring spot virus
(TRSV) are known to cause yield losses wherever cucurbits like cucumbers,
melons, squash, pumpkin, water melon etc. are grown. In the U.S. during the
period from 1951–1960, the average yield losses in cantaloupes due to virus
diseases were 3.5 % annually. The cucumbers grown for fresh market, viruses like
CMV, TRSV and WMV caused 2.0, 1.0 and 1.0 % losses, respectively. The
average annual yield losses in cucumbers grown for pickling was 2.5 and 2 %,
respectively with CMV and TRSV infection (USDA 1965).
Doolittle (1924) reported that losses in cucumbers due to CMV in one locality
cost $ 75,000. Losses were so continuous that in some areas cucumbers were being
replaced by other crops. From Lea Valley (England), Fletcher et al. (1969)
reported that CGMMV reduced the yield of cucumbers by 15 %, when infected
before or at planting, but if the infection was delayed for six weeks, the loss in
yield was far less. Hills et al. (1961) observed a 32 % reduction over the un-
inoculated checks in the yield of marketable melons from plants inoculated with
Beet curly top virus alone, 40 %, reduction from CMV alone and 68 % reduction
from plants inoculated with both viruses.
From New Zealand, Wayne (1971) reported that Watermelon mosaic virus
WMV-2 reduced the yield in butter cup squash, golden hubbard squash and pumpkin
by 63, 53 and 49 %, respectively. Demski and Chalkley (1972) estimated the losses
in summer squash (Cucurbita pepo) with WMV-2 which averaged 43, 28 and 9 %,
respectively from early, mid and late inoculation. The former two treatments caused
nearly 100% loss in marketability due to distortion and discolorization of the fruits
and the latter inoculation resulted in 70 % loss.
3.2 Yield Losses in Different Crops 119

In 1974, Demski and Chalkley noticed a 73 and 18 % loss in yield of water

melon with Watermelon mosaic virus in early and late infected plants. The
symptoms on fruits were rings and green spots and the fruits were also misshapen.
In South Florida, Watermelon mosaic viruses 1 and 2 (Papaya ringspot virus and
WMV-2) caused losses every year and some years as much as 60 % of the crop
was lost (Adlerz 1969). In Texas, Tobacco ring spot virus caused up to 50 % loss
in water melons (Rosberg 1953).
In India, Watermelon bud necrosis virus (WBNV) is the most devastating in
cucurbitaceous crops. The studies carried out at Indian Institute of Horticultural
Research, Hessaraghatta, Bangalore (India) revealed that assessment of losses due
to watermelon bud necrosis disease was 37.4–100 % depending on the stage of
infection (Anon 2011). Yield reduction in pumpkins which varied from 9.1 to
96.4 % depending on the time of WMV-2 infection. The early inoculated plants of
bottle gourd, pumpkin and watermelon exhibited mosaic or ugly blisters on the
fruits, resulting in un-marketability and reported the extensive yield losses in
pumpkin infected with strain of WMV (Singh 1981). An epidemic of WMV was
also observed on pumpkin and other cucurbits in Central New York, and early
infection had tremendous adverse effect on plant growth and fruit yield (Provv-
identi and Schroeder 1970). Similar type of loss estimates were also recorded by
Demski and Chalkley (1972, 1974), Fischer and Lockhart (1974), Nome et al.
(1974), and Bhargava (1977) in various virus-cucurbit host combinations.

(f) Carrot (Daucus carota, Family Apiaceae)

In the U.S., aster yellows phytoplasma caused an average estimated loss of 2 %
annually during 1951–1960 (USDA 1965). In the UK, Watson and Serjeant (1964)
estimated loss in carrot roots due to motley dwarf virus at Woburn in 1959 and
1961 was about 60 %. They also reported that at Rothamsted and Woburn, virus
infection at late May or early June yielded 6 tons of roots per acre. While the
yields were 24–25 tons/acre during 1962 at which almost all the crop was unin-
fected. Howell and Mink (1979) reported yield reduction in root weight due to
Carrot motley dwarf virus and Carrot thin leaf virus were 78 % and 14–22 %
respectively.

(g) Crucifers (family Brassicaceae)

From various parts of the world, different virus and phytoplasma diseases were
reported on crops like radish, swedes, canola, rape, cabbage, turnip, and cauli-
flower. Ling and Yang (1940) reported that a mosaic of rape destroyed more than
30 % of the crop in China and that the reduction in seed yield ranged from 37 to
86 % of the samples examined. In the Coastal areas of the Central California,
commercial plantings of Cauliflower were severely affected by a virus disease that
caused 20–30 % loss in some fields (Tompkins 1934). A similar virus disease was
reported to be wide spread in Devon and Cornwall and have affected as many as
75 % of the plants in a field and entire crops were rendered unmarketable
(Caldwell and Prentice 1942). Strains of Turnip mosaic virus have reported to
damage commercial plantings of horse radish in Wisconsin, Illinois, Missouri and
120 3 Impact of Virus and Viroid Diseases on Crop Yields

Washington. In some areas 100 % of the crop was infected (Pound 1948). Sch-
melzer (1976) reported that Cauliflower mosaic virus (CaMV) reduced the number
of pods by 51 % and seed weight by 67 % in the garden radish (Brassica sativus).
In canola, due to Beet western yellows virus (BWYV) the estimated loss of 34 and
46 % have been reported in Europe and Western Australia, respectively (Kathi
et al. 2004). Subsequently Jones et al. (2007) have also estimated the losses in seed
yield and quality of Brassica napus (canola, oilseed rape) caused by infection
with Beet western yellows virus (BWYV) alone or in combination with direct
feeding damage by Myzus persicae (green peach aphid). When BWYV infection
at sites A and B reached 96 and 100 % of plants, it decreased seed yield by up to
46 and 37 %, respectively. Also, variation in BWYV incidence explained 95 %
(site A) and 96 % (site B) of the variation in yield gaps, where for each 1 %
increase in virus incidence there was a yield decrease of 12 (site A) and 6 (site B)
kg/ha. At both sites, this yield decline was entirely due to fewer seeds formed on
infected plants. At site B, BWYV infection significantly diminished oil content of
seeds (up to 3 %), but significantly increased individual seed weight (up to 11 %)
and erucic acid content (up to 44 %); significant increases in seed protein content
(up to 6–11 %) were recorded at both sites. In cabbage, early infection with Turnip
mosaic virus are Cauliflower mosaic virus and CaMV can reduce yields by 75 %
although late infection has been reported to have little effect on yield (Sherf and
MacNab 1986).

(h) Lettuce (Lactuca sativa, Family Asteraceae)

In the Imperial Valley of California where more than 40,000 acres were planted by
iceberg type lettuce, it was estimated that the loss due to the Lettuce big vein virus
(LBVV) every year was at least 700,000 cartons of the crop, the market value of
which exceeds $ 2.8 millions. It was also estimated that not more than 10 of the
diseased plants would be marketable size and of good quality (Kontaxis 1978).
Lettuce mosaic virus (LMV) is another world distributed disease, which is seed
transmitted, and in U.S. estimated average annual loss, particularly Cabbage lettuce
(Lactuca sativa var. capitata) is about 4 %. The other diseases like big vein and
aster yellows caused loss of 1.0 % each annually during 1951–1960 (USDA 1965).
Lettuce seed yields were decreased by 44 % with Beet western yellows virus in
glasshouse tests (Ryder and Duffus 1966). In California, Beet yellow stunt virus
occurred in lettuce fields in epidemic proportions in small areas, and the estimated
crop loss was between 50 and 85 % (Duffus 1972). Yield loss studies were con-
ducted by Davis et al. (1997) with LBVV which is transmitted by fungal vector
Olpidium brassicae. This virus impaired the formation of hearts and the proportion
of symptomatic plants that lacked hearts was 24–36 % when leaf symptoms first
appeared 5–7 weeks after transplanting, but 14–16 % after 8–9 weeks. When leaf
symptoms first appeared after 5–7 weeks, there was a fresh weight loss of
14–15 % of heads (all plants) and 39 % for hearts (excluding plants without
hearts). When leaf symptoms first appeared 7 weeks after transplanting, there was
no significant yield loss for heads and only a 14 % loss of hearts. At 8–9 weeks
there was no significant yield loss for heads or hearts.
3.2 Yield Losses in Different Crops 121

3.2.4 Tuber Crops

Among the tuber crops sugar beet, potato, cassava, sweet potato etc., suffer a great
deal of losses with diseases due to beet yellows, leaf roll, viruses X, Y, M and S,
African mosaic and sweet potato mosaic viruses and Witches’ broom or little leaf
diseases. The late infection of these crops is very important not because of
immediate losses but because the virus is carried over in the late infected plants to
the crops of the following year in which the losses may be heavy.

(a) Potato (Solanum tuberosum, Family Solanaceae)

Virus diseases of potato are prevalent in almost all the potato growing countries
and responsible for heavy yield losses. From India, Nagaich (1975) has estimated
the percentage of losses on account of mild viruses vary from 10 to 25 % and by
severe viruses and phytoplasma diseases caused about 20–90 % yield losses.
Potato virus Y (PVY) decreased yields up to 95 % in up-to-date cvs. and
77.5–87.0 % in Craig’s Defiance (Anonymous 1949–1956). Nagaich and Agrawal
(1969) estimated the average losses up to 40–85 % due to PVY depending on the
cvs. From over all annual losses estimated on the basis of 40–45 % as an infection
value and yield reduction value of about 40 %, it can be seen that an additional
produce of about 1.5–2 million metric tons could be obtained with an existing
resources by controlling viral and phytoplasma diseases by the use of disease-free
stock. The additional produce at Rs. 100/- per quintal can fetch about 1,500–2,000
million rupees every year.
In southern part of England potatoes also suffer from several viral and phy-
toplasma diseases and cause loss of one million tons per annum. The extensive
trade in ‘‘Seed potatoes’’ totaling over half a million tons which takes place
between England and Scotland every year is entirely due to the prevalence of virus
disease in England.
Potato leaf roll virus (PLRV) reduces the yield from 20.0 to 98.0 % depending
on the variety (Loughnane 1941; Nagaich and Agrawal 1969; Allam et al. 1974
and Nelson and Tarfason 1974; Killick 1979). Hoyman (1963) estimated that the
loss due to PLRV during 1959 at Washington was 3–4 million dollars. In Ban-
gladesh, with 100 % PLRV infected plants in the field caused 78 % yield loss
(Hossain et al. 1989). In the Cardial potato cv., Verhocks (1965) recorded 58.43 %
reduction in plant height over control due to 100 % inoculum level of PLRV. It
seems that yield loss as well as extent of degeneration due to PLRV infection in
plants was correlated with time of infection. i.e., early infection caused severe
damage (Robertson 1978; Khurana and Singh 1986). Rahman and Akanda (2010)
recorded significant reduction in plant height, tuber number and tuber yield when
20 % of the infected tubers were used for planting.
In Brazil, Cupertino et al. (1973) observed that Potato virus - A reduced yield
in cv. Eva by 25 % on an average. The same virus in the cv. Rajka reduced the
losses by 22.4–31.3 % (Dedic 1975). In Bruinswick (Canada), a severe strain of
Potato spindle tuber viroid (PSTVd) reduced tuber yield by 64 % and on the other
122 3 Impact of Virus and Viroid Diseases on Crop Yields

hand in cv. Saco three mild strains of PSTVd have lowered the tuber yields only by
17, 24 and 24%, respectively (Singh et al. 1971). Bald and Norris (1941) assumed
that in Australia, more than 90 % of the potatoes grown were infected with Potato
virus X (PVX). The estimated losses from this virus were as heavy as from all
other viruses combined and probably amounted to a loss of $ 1,750,000 a year.
Depending on the strain of PVX and the potato cv. the yield losses were 5–75 %
(Norris 1953). Bald (1943) reported yield reductions in the variety up-to-date, of
12 and 45 % due to mild strain of PVX, and the most severe naturally occurring
strain mixture of PVX, respectively. PVX caused 57.2 and 17.6 % losses in cv.
Kaster and Ora. In India, an average loss of 20.89 % has been reported in different
potato cvs. due to PVX (Khurana and Singh 1988). Bonde and Merriam (1951)
have also earlier found PVX to reduce yield in Chippewa by 13.7 %, in Sebago by
16.2 %, in Katahdin by 14.9 %, in Kennebec by 11.2 %, in Teton by 18.3 % and
in Mohawk by 7.3 %. Hoyman (1964) found PVX to reduce tuber yield by
21.85 %. Lim et al. (1966) observed 17.5 % yield reduction in potato var. Sebago.
Beemster and Rozendaal (1972) observed that certain necrosis evoking strains of
PVX, caused more than 50 % loss in some cvs. where as losses caused by Potato
virus S (PVS) were 10–15 %. Much literature is available stating low yield losses
due to PVS. The loss in yield due to PVS in cv. Craig’s Defiance was 17.6 and
28.9 % in primary and chronic infection respectively (Anonymous 1964; Khurana
2000). Nagaich and Agrawal (1969) also observed similar amount of losses:
Manzer et al. (1978) reported that yield weights from PVS infected seed averaged
about 3 % lower than those of virus-free seed. When PVS was combined with mild
PVX or moderate PVX, additional reduction of only 2 and 5 %, respectively were
obtained.
The reduction in yield due to Alfalfa mosaic virus has ranged from 22 to 46 %
in different potato varieties (Anonymous 1965). Geminiviruses are also wide
spread in potato and early infection of plants with Tomato leaf curl New Delhi
virus (TLCV-New Delhi), has resulted in higher tuber transmission ([90 %) and
reduced size. Percent tuber transmission of the TLCV-New Delhi was positively
correlated with disease incidence in plants. High level of transmission of virus in
plants (98 %) and the tubers (97 %) was observed when plants got infection prior
to tuberization. The potato plants infected after 80 days, did not produce any
infected tubers (Lakra 2009). In India Potato stem necrosis disease caused by
Groundnut bud necrosis virus (GBNV) reduced potato tuber yield losses up to
29% (Singh et al. 1997). PSTVd has also reduced potato yields as high as 64 %
(Singh et al. 1971). The size of potato tuber was very much reduced due to hairy
sprout which is a phytoplasmal disease and the yield was depressed up to
41.7–94.0 % (Kaley and Nagaich 1971). Another phytoplasma disease, purple top
roll, also caused reductions up to 40–70 %. The yield of Kufri Jyoti and up-to-date
was reduced by 83.69 and 86.02 % due to marginal flavescence, a phytoplasmal
disease (Nagaich et al. 1973).
3.2 Yield Losses in Different Crops 123

(b) Sweet Potato (Ipomoea batatas, Family, Convolvulaceae)

In sweet potato, viral and phytoplasmal diseases are responsible for the greatest
yield losses and have been described from all over the world. Presently available
stocks of some cultivars appear to be 100% infected with one or other of these
virus and phytoplasma diseases which include virus A, vein mosaic, russet crack,
feathery mottle and witche’s broom (little leaf). Nome and Docampo (1976)
reported that due to Sweet potato vein mosaic virus, there was a 7 % reduction in
number/plant and the loss in yield by weight was 84 %. In Uganda, Mukiibi (1977)
observed a 57 % reduction in yield in the cv. Kyebandula due to Sweet potato
mosaic virus. The average weight of sweet potato plants infected with the same
virus was 73 g as compared with 342 g per plant in control. In Nigeria, Hahn
(1979) observed 78 % reduction in fresh tuber yield due to sweet potato virus
diseases. Even Mukasa et al. (2003) and Ndunguru and Kapinga (2007) reported
yield reductions of 56–98 % with SPVD. In Uganda, Sweet potato virus disease
(SPVD) is caused by combination of aphid-transmitted Sweet potato feathery
mottle virus (SPFMV) and a whitefly transmitted Sweet potato chlorotic stunt virus
(SPCSV) and yield losses of over 90 % have been observed (Aritua et al. 2003).
Yield loss studies with SPVD were also conducted by Ngeve 1990; Karyeija et al.
1998; and Gutierrez et al. 2003. In U.S. during the period of 1951–1960, the
average annual loss caused by yellow dwarf and internal cork virus was 2.0 and
1.5 %, respectively (USDA 1965).

(c) Cassava (Manihot esculenta, Family Euphorbiaceae)

Cassava is the third largest source of carbohydrate in the world and is a major food
crop in 39 African countries and its adjacent islands and the African Cassava
mosaic virus (ACMV) affects the food supply of millions of Africans. Cassava
mosaic virus disease (CMD) is an important disease in all cassava-growing regions
of Africa and also in other parts of the world wherever this crop is grown and yield
losses are variable from the unsignificant to significant reduction. Fauquet and
Fargette (1990) have reported that 55–75 % yield losses would be of CMV
infected cuttings were used as planting material and 35–60 % losses from the later
infection by whiteflies. Recently a pandemic of an unusually severe form of CMD
spread within and beyond the borders of Uganda into Kenya, Tanzania, Demo-
cratic Republic of Congo, Congo Brazzaville and Sudan and caused by a new
strain of the virus known as Ugandan variant of East African cassava mosaic virus
(EACMV-Ug), which was first reported in Uganda in late 1980s (Deng et al. 1997;
Zhou et al. 1997; Legg 1999). EACMV-Ug is a recombinant hybrid derived from
the two principal cassava mosaic geminivirus species known to occur in Africa,
African cassava mosaic virus (ACMV) and East African cassava mosaic virus
(EACMV) (Zhou et al. 1997). In Tanzania, severe and rapidly spreading CMD was
first recorded in September 1998 in Missenye and Kiziba divisions of Bukoba
district, in the Lake Zone (Jeremiah and Mukandala 1998) causing significant
losses. Subsequent diagnostic tests confirmed the association of severe CMD with
the presence of EACMV-Ug (Legg and Okao-Okuja 1999). Since then the disease
has spread rapidly, covering Bukoba, Karagwe, Muleba and Biharamulo districts
124 3 Impact of Virus and Viroid Diseases on Crop Yields

in Kagera Region and Geita and Sangerema districts in Mwanza Region; Kibondo
District in Kigoma Region and Bukombe District in Shinyanga (Jeremiah and
Ndyetabula 2002). Padwick (1956) brought together the available information on
yield losses and estimated that the yearly loss in yield due to CMD was equivalent
to about 11 % of the crop in Africa. Yield losses on individual susceptible cassava
varieties range from 20 to 95 % (Beck and Chant 1958). Annual economic losses
in East and Central Africa were estimated to be 1.9–2.7 billion USD (Patil and
Fauquet 2009).
In Africa, ACMV has caused yield losses of 20–90 % (Terry and Hahn 1980;
Fargette et al. 1988; Thresh et al. 1994, 1997). From India, Alagianagalingam and
Ramakrishnan (1967) and Malathi et al. (1985) have reported the losses of 10–20
and 88 %, respectively. Narasimhan and Arjunan (1976) have also found that the
sets planted from infected plant (over two years continuously) showed severe
reduction in yield (84.2 %) compared to the crop planted from one year infected
sett (53.3 %) and the tubers showed severe splitting. Jennings (1970) worked out
separately the yield losses, which were found to range from 14.5 to 83.0 %. Bock
and Guthrie (1978) reported that in Kenya, the mean loss in a moderately resistant
hybrid (No. 46106/27) was 70 % and in a susceptible cv. (F 279) it was 86 %.
Terry and Hahn (1980) reported that within each variety plants established from
Cassava mosaic virus infected cultivars had considerably reduced root weights at
two months (77 and 93 %) compared with weights from plants established from
Cassava mosaic virus-free cuttings. By seven months this reduction in root weight
was 32 % from the more resistant TMS 30395, but was still as high as 69 % for the
more susceptible Isunikakiyan, cultivar. Thresh et al. (1997) have assumed
15–24 % loss estimates due to cassava mosaic disease which is equivalent to
15–28 million tons compared with the FAO production estimates of 84 million
tons. The annual economic losses were in the range of US $ 1,300–2,300 million in
Africa alone (Thresh et al. 1998). The epidemic of cassava mosaic disease in
Uganda in the 1990s led to starvation in some districts in the country and is
estimated to have resulted in a loss of US $ 60 million per annum during the height
of epidemics (Thresh and Cooter 2005). In Kenya, cassava mosaic disease has
caused significant yield losses ranging from 24 to 75 % (Seif 1982; Lwanga 2000).
Cassava brown streak virus is another economically important disease of
cassava is Malawi. Gondwe et al. (2002) have reported that infection with this
virus has caused 18–25 % yield loss. This loss translates to about MK 400 million
to MK 500 million or US $ 5 million to US $ 7 million annually based on farm-
gate prices. Even in Tanzania, the same virus has caused crop losses of up to 64 %
as reported by Mtunda et al. (2003).

(d) Aroids (Family Araceae)

Among aroids taro (Colocasia esculenta), elephant-foot-yam (Amorphallus com-
panulatus), tannia (Xanthosoma species) are grown in some of the tropical
countries and are affected with number of virus diseases. In Solomon Islands and
Papua New Guinea in taro Taro bacilliform virus and Colocasia bobone disease
3.2 Yield Losses in Different Crops 125

virus were thought to cause the lethal alomae disease which was considered as the
most destructive virus disease of taro (Rodoni et al. 1994).
In taro (var. ‘Kalpao’), the effect of Taro feathery mosaic virus (TFMV)
infection on the growth and yield was determined by using mechanically-inoculated
and naturally-infected taro seed pieces. Mechanical inoculation of TFMV
employed in both pot and field experiments showed a reduction in yield of taro
ranging from 2.80 to 58 % and 20.2 to 30.1 %, respectively. Inoculation of plants at
the earlier stages of growth generally showed higher disease incidence and yield
loss than inoculated at the later stage. Plants infected with TFMV produced more
suckers than uninfected ones (Gapasin 1986).
At Pune (India), Chatterjee et al. (1971) studied the effect of aphid transmitted
mosaic disease on elephant-foot-yam and reported the increased their bud prolif-
eration on the mother corms of diseased plants as also their separation in soil was
more in number (22) as compared to the healthy corms. They have also noticed
reduced corm size and growth of roots.
Among the viruses infecting cocoyam Dasheen mosaic virus (DsMV) is very
common and although not lethal it does retard the plant growth and reduces the
yield (Zettler et al. 1989). The yield per plant in virus-free plants was significantly
higher than yield obtained in virus infected plants. The estimated yield ha-1 for
virus-free plants was 18.2 tons and 13.4 tons for infected plants.
The estimated yield obtained from virus-free plants (18.2 ton ha-1) was 2.5 times
higher than the current national yield average (7.2 ton ha-1), and very close to the
former national yield average (19–22 ton ha-1) reported by Instituto Nicaraguense
de Tecnologia Agropecuaria (INTA) in the year 2000 (Reyes Castro 2006).

(e) Yam (Dioscorea spp., Family Dioscoreaceae)

Yam is a valuable source of carbohydrate to the people of the tropical and sub-
tropical Africa, Central and South America, and parts of Asia. Potyviruses infect
yam foliage throughout the Caribbean and West Africa, often accounting for yield
losses in the region of 25 % (Mohamed and Mantell 1976; Thouvenel and
Dumont, 1990). More information can be obtained from the book chapters by
Loebenstein and Thottappilly (2004). In the Ivory Coast Thouvenel and Dumont
(1990) have conducted studies on crop losses caused by Yam mosaic virus (YMV)
of the potyvirus group. Healthy yam plants (cv. Florido) gave 25 % higher yields.
The virus did not affect the number of tubers produced. Small sized tubers showed
a higher disease incidence. The selection of these small tubers as planting material
resulted in an increased number of infected plants.

3.2.5 Fruit Crops

(a) Citrus (Citrus spp., Family Rutaceae)

Citrus tristeza virus (CTV) is the most devastating disease of citrus and thousands
of citrus trees in several countries like U.S., Brazil, Argentina, Israel, India,
126 3 Impact of Virus and Viroid Diseases on Crop Yields

Uruguay, Australia, Spain, Nigeria and many other citrus growing countries were
affected by CTV and also with greening-fungal complex. Bennett and Costa (1949)
reported that in about 12 years, 60,00,000 or about 75 % of the orange trees were
destroyed due to CTV. In Argentina the losses were as high as 20 million bearing
trees, worth approximately 500 million dollars (Nolla and Fernandez 1976). In the
two decades, following introduction of tristeza in South America, 20 million citrus
trees were destroyed in Argentina alone (Klotz 1961). The real magnitude of this
dreadful disease is vividly indicated by Wallace (1959) who estimated that tristeza
has threatened to destroy more than half of the world’s citrus. The virus diseases in
Florida, along the Gulf Coast and in California have killed up to one million trees
each year. Bar-Joseph et al. (1989) while reviewing the control aspects of citrus
tristeza, have also mentioned that the losses caused by CTV - induced decline was
more than 10 million trees in Argentina, more than 6 million trees in Brazil and
more than 3 million, trees in the U.S.
Spain is the region of the world that has most suffered from the tristeza disease,
with more than 40 million trees killed between the first reported outbreak, in 1956
until 2000, what represent more than 35 % of sweet orange and mandarin trees
grafted on sour orange (C. aurantium) root stock (Cambra et al. 2000; Piquer et al.
2005). About half of these losses occurred between 1957 and 1989, and the other
half between 1990 and 2000, because of a faster field dispersal of the disease when
the efficient vector Aphis gossypii becomes more prevalent and also even the other
aphid species like A. spiraecola, Toxoptera citricidus and T. aurantii are also
responsible for spread of tristeza in number of countries (Cambra et al. 2000). CTV
isolates in Spain only cause decline in sweet orange and mandarins when grafted on
sour orange, and are efficiently controlled by grafting on tolerant rootstocks.
The average annual loss of lemons for the period 1951–1960 was 9.9 % due to
sieve tube necrosis, 6 % due to shell bark and 0.5 % due to psorosis. In oranges
and grape fruit the average annual loss was 1.9 and 0.1 % due to tristeza, due to
Citrus psorosis virus 2.0 and 0.4 %, due to Citrus exocortis viroid 1.9 and 0.2 %,
due to stubborn disease 0.1 and 0.1 %, and due to Xylopsorosis was 0.1 and 0.1 %
(USDA 1965).
Calavan and Carpenter (1965) estimated that the number of stubborn trees in
the California State probably exceeded 2,000,000. Further observations in Cali-
fornia citrus growing areas indicated that there were about more than 10,000,000
trees affected by stubborn. Tidd (1944) estimated that stubborn disease reduced the
production of navel oranges by 30–50 %. Calavan and Christiansen (1966)
reported that a severe form of stubborn reduced yield of young Frost ‘Lisbon’
lemon trees by 59 %, Frost Washington Navel by 92 %, tangelos by 41–95 % and
tangerines by 81–98 %. Calavan (1969) recorded the yield losses of 44–74 % in 6
year-old Frost Valentia orange trees on five root stocks, due to stubborn disease.
In Brazil, Rodriguez et al. (1974) recorded the yield losses of 5 year old
Hamilton orange on Rangpur lime due to severe strains of exocortis. The average
production of the trees affected with severe strain reached 59.1 kg per tree during
four year period, which is 13 % less than the healthy control plants, which yielded
68.3 %.
3.2 Yield Losses in Different Crops 127

In India, much of the decline threatening the lime industry is by CTV (Reddy
1965, 1968). Balaraman and Ramakrishnan (1977) have reported that CTV when
infected Kagzilime plants produced an average of 69 fruits/plant when compared
the yield of 550 fruits/control plant. In Spain, during 1972, CTV affected 82,000 ha
of citrus. Only one out of 13,000 Navelina sweet orange trees was free of virus and
virus-like diseases (Guardiola 1974; Guardiola et al. 1974). A similar situation was
found in Florida, U.S. where less than 1% of the old lime trees were found to be
free from the virus diseases (Childs and Knorr 1965; Knorr and Childs 1968). Even
though CTV is more problematic on almost all citrus species, no definite infor-
mation is available on crop losses in different citrus species.

(b) Banana (Musa acuminate, Family Musaceae)

Bunchy top disease is one of the most devastating diseases in almost all banana
growing countries. This disease was introduced into Australia though infected
banana suckers in 1913 and caused heavy infections of young banana industry,
with over 5,000 acres abandoned in New South Wales (Magee 1927). In Fiji
islands, during 1892, the banana bunches of about 788,000 were exported and
within 3 years due to floods and bunchy top disease, only 1,44,000 bunches were
exported (Magee 1953; Wardlaw 1972). This disease has also caused serious
losses in Egypt, Ceylon (Sri Lanka), India, Pakistan, Philippines, various parts of
Pacific islands, Africa, Vietnam etc., Total loss will be encountered if once the
banana plants were infected in the very early stage, no bunch will be produced.
Whereas plants infected at 8–9 months old (late infection), produced a bunch of
poor quality which were ten times lesser in weight than the bunch from a healthy
plant. Fruit size from such bunches was reduced and were unmarketable (Sastry,
unpubl.). A loss of 400 million (US $ 8.5 million) annually has been estimated due
to this disease in Kerala alone (Selvarajan and Balasubramanian 2008).
Banana streak virus (BSV), which was reported in India in 1996, though infects
many banana cultivars, in poovan, an important banana cultivar has caused yield
loss of 48 % in India (Thangavelu et al. 2000). Another economically important
virus of banana in India is Banana bract mosaic virus (BBrMV) that has caused
maximum yield reduction in cvs. Robusta (70 %) followed by Nendran (52 %) in
Kerala, India (Anitha Cherian et al. 2002). An extrapolation made out of survey
and yield loss assessment studies in Kerala (India) revealed a loss of Rs. 38.7
crores per annum in Nendran cultivar alone (Selvarajan et al. 1997; Selvarajan and
Jeyabaskaran 2006). The yield loss assessment studies due to BBrMV done during
1995–1996, showed 68.34, 50.00 and 46.34 % reduction in bunch weight in cvs.
Nendran, Poovan and Ney Poovan over uninfected healthy plants (Thangavelu
et al. 2000). Earlier Selvarajan et al. (1997) have recorded that the infected banana
plants showed significant reduction in height, girth, leaf area and finger weight
over healthy plants. Even in the Philippines in banana cvs. Cardaba and Lakatan
have caused yield loss of 40 % due to BBrMV (Magnaye 1994).
128 3 Impact of Virus and Viroid Diseases on Crop Yields

(c) Grapes (Vitis vinifera, Family Vitaceae)

Among several virus diseases recorded on grapevines from different countries,
Grapevine fan leaf virus (GFLV) and Grapevine leaf roll virus (GLRaV) were
wide spread in all viticulture areas of the world and substantially caused great crop
losses and/or lower fruit quality. A few viruses cause great losses locally, but
because their geographic distribution is limited, they have limited economic
importance. Other viruses like Tobacco ring spot virus, Tobacco necrosis virus,
Tobacco mosaic virus, Tomato bushy stunt virus and Sowbane mosaic virus have
been isolated occasionally from grapevines in which they were latent and appeared
to have little economic importance.
GLRaV, another important disease caused losses of 8.4 % or 15,057,000
annually in U.S. (USDA 1965). Goheen and Cook (1959) found that leaf roll virus
delayed spring growth of all varieties under their observation and demonstrated
that it reduced yield and sugar content, which varied from season to season. In six
cultivars yield reduction ranged from 46 to 85 % and the sugar content was
lowered by 0 to 16 %. In California, it was estimated that this disease caused an
annual loss of about 5 % of the total grape crop.
GFLV in Europe has caused yield losses of up to 50 % and is responsible for
grapevine degeneration (Vuittenez 1966). During 2004 Andret-Link et al. have
reported severe yield losses to the tune of 80 % in grapes with the same virus
along with alteration in fruit quality. It also affected plant vigor and productive life
span of vineyards.
Losses due to fan-leaf degeneration vary according to the tolerance of the
cultivar to the virus. Tolerant cvs. were little affected in their production, while
losses in the most susceptible ones can amount to 80 % of the yield (Martelli and
Savino 1990). For example, yield reductions were of 78–98 % in cvs. Chasselas,
Merlot and Pinot Noir in France (Bovey 1970), of 44–94 % in cv. Traminer in
Germany (Rudel 1985), 55–65 % in cv. Savagnin and 30 % in cv. Nebbiolo in
Italy (Legin et al. 1993; Mannini et al. 1994a). Fruit quality is also affected, with a
reduction of titratable acidity of –1.33 (Mannini et al. 1994b). Vigour of the vine is
reduced by 30–50 % (Legin et al. 1993; Mannini et al. 1994b), leading to pro-
gressive decline and reduced productive life of the vineyard. In France, 65 % of
the acreage was affected by GFLV, and 30 % was severely affected (Demangeat
et al. 2005).

(d) Papaya (Carica papaya, Family Caricaceae)

South and South east Asia, Australia, Philippines, Cuba, Puerto Rico, India,
Sri Lanka, Hawaii, Texas, Florida, South and Central America, Trinidad and Tobago
and Jamaica. Papaya ring spot virus (PRSV) is of major concern and it was in the
period of 1970s and 1980s that PRSV caused disasters to papaya production in much
of the South and South-east Asia. This disease has earlier caused considerable losses
in Hawaii, Florida and Taiwan. Initially the disease appears as oil streaks on stems
and petioles, subsequently mottling of leaves becomes evident. Severely infected
plants do not flower and die at young stage. Losses of up to 70% in yield have been
reported in some infested areas of papaya at Brisbane and Gold Coast (Australia) due
3.2 Yield Losses in Different Crops 129

to PRSV infection (Shannon Dillon 2005). The disease has continued to spread
steadily, causing large yield losses. PRSV is wide spread wherever crop in grown in
Karnataka state, India. This disease incidence ranges from 50 to 100 % and the extent
of yield loss depends on the stage of the infection (Byadgi et al. 1995). From Pune
(India), Sharma et al. (2006) have reported that PRSV infection caused 41.12 %
yield losses in papaya when plants were infected between flowering and fruit set
and 34.43 % yield losses when plants were infected after fruit set when compared
with the yield of plants infected after fruit development stage.

(e) Apple (Malus domestica, Family Rosaceae)

The majority of the viruses, viroids and phytoplasma diseases infecting apples
cause fruit symptoms on most of the apple cvs. and these varied from severe
distortion, cracking and reduction in size to mild pitting or simple resetting.
Estimated annual losses due to virus diseases in the U.S. for the period 1951–1960
was 0.2 % (USDA 1965). Apple fruits from Apple mosaic virus (ApMV) infected
plants were poorer in color and acidic in taste. In field trails, Posnette and Cropley
(1958) recorded that two strains of ApMV reduced yield of mature Cox’s Orange
Pippin and Allingaton Pippin trees by about 30 % over four years, while yield of
Worcester Pear main and Newton Wonder was only slightly reduced. Similar
results were also noticed by Posnette and Cropley (1959) and Meijneke et al.
(1963). Campbell et al. (1976) reported the effect of latent viruses on the growth of
apples. Yield losses attributed to the combination of four latent viruses during the
first five years was 6.7 tons/ha in ‘Cox’, 23.4 tons/ha in ‘Laxton’s Superb’,
3.7 tons/ha in ‘Discovery’ and 0.35 tons/ha in ‘Golden Delicious’. In Germany,
Schmidt (1972) used the same virus combinations as Prof. Campbell tested on 20
root stocks and reported yields as 67 % from virus-free trees, 28 % from trees
infected with latent viruses and 16 % from virus-free trees, 28 % from trees
infected with latent viruses and 16 % from trees with latent viruses plus rubbery
wood. Reduced growth due to latent viruses was also reported by Schmidt (1972)
in Germany; by Wood (1974) in New Zealand and by Johnstone and Boucher
(1973) and Sampson and Johnson (1974) in Australia.
Chat fruit virus inhibits the typical color of fruit and delayed ripening, and the
fruit remain small by 25 % (Wallace et al. 1944; Posnette and Cropley 1965). The
other viruses like Apple leaf pucker, Dapple apple, Apple green crinkle, Apple
ring spot, Apple rough skin, Apple star crack and Apple scar skin viruses cause
various changes both quantitatively and qualitatively. Due to the reduced fruit size,
nonuniform ripening and also conspicuous crackings and wart-like swellings and
rings, some of these viruses have resulted in severe losses.
From U.K. Campbell et al. (1978) have compared the effects of multiple virus
infections in four cvs. of apple on MM 106 root stocks. The virus infections
usually reduced crop and tree size in the same proportion.

(f) Strawberry (Fragaria ananassa, Family Rosaceae)

The vigor and yield of strawberries was reduced due to different viruses, for which
aphids, leaf hoppers and nematodes are vectors. The estimated average annual loss
130 3 Impact of Virus and Viroid Diseases on Crop Yields

due to virus diseases for the period 1951–1960, in U.S. was 5.0 % (USDA 1965).
From New Hampshire, Becker and Rich (1956) compared the yield losses of virus-
free plants with the three locally grown virus infected strawberry cvs. The average
yield per clone for the virus-free Catskill, Premier and Sparkle was 10.6, 13.5 and
10.9 quarts respectively, as compared to 3.9, 8.0 and 7.2 quarts for the three
locally-grown cvs.
Mottle virus alone may reduce the marketable yield of strawberries by 25–30 %
(Freeman and Mellor 1962; Horn and Carver 1962). When it occurs in combi-
nation with crinkle, vein banding or mild yellow edge viruses, the yields still
further reduced. Aerts (1973) estimated the yield losses in eight straw berry cvs.
infected with mottle virus and yellows complex at only 20 % with the farmer
virus, where as the latter reduced the yield up to 36 %. The reduction in loss was
due to less number of fruits, rather than small fruits. Bolton (1974) also reported
that during third year that mottle virus reduced the yield by 11.5 % where as vein
banding C virus reduced the yield by 88.2 % (total fruit) and 100 % (salable fruit)
in the third year. From Washington state, Barritt and Loo (1973) studied the effect
of mottle, crinkle, and mild yellow-edge viruses on growth and yield of Hood and
North west strawberries. Strawberry crinkle virus (SCrV) alone caused a signifi-
cant yield reduction (30 %) where as the other viruses together reduced the yield
by 60 %. Even Martin and Converse (1977) with the same viruses noticed definite
differences in yield losses in Hood strawberries. Losses caused by Strawberry mild
yellow edge virus (SMYEV) alone have not been estimated, but the virus usually
occurs as part of the yellows complex which in insensitive cvs. may reduce yield
by 75 % (Stitt and Breakey 1952).
In Brazil, Betti et al. (1979) studied the effect of three strawberry viruses singly
and of two virus complexes on the vigor and productivity of strawberry cv.
Campinas. The plants were graft inoculated with Strawberry mottle virus (SMV),
Strawberry vein banding virus (SVBV) and SMYEV viruses singly and in com-
binations, SMV ? SMYEV and SMV ? SMYEV ? SVBV. Singly the virus did
not reduce the plant vigor and yield. SMV and SMYEV reduced yield by 26 % in
the first 10 weeks of picking and total yield in 31 weeks by 15 %, with the average
fruit weight remaining unchanged. SMV ? SMYEV ? SVBV reduced yield by
78 and 68 % and average fruit weight by 25 and 22 % for early and total leaves,
leaf size and number of crowns were reduced by 34, 27, 29, 40 and 17 %,
respectively by the same complex. Earlier Lawrence and Miller (1968) recorded
significant reduction in the number of runners on the strawberry cv. North West,
by SMYEV, SCrV, SMYEV plus SMV and SMYEV plus SCrV. The mean
number of runners per plant in healthy control was 19.6 % when compared to
12.5–13.2 % in the other virus combinations.
It was shown by Horn and Carver (1962) that Strawberry chlorotic fleck virus
reduced the yields of Headliner strawberry plants and fruit. Virulent strains of
crinkle virus severely reduced vigor and productivity and even symptomless
strains, such as latent A, reduced vigor, runner production, yield and fruit size of
some cvs. (Freeman and Mellor 1962; McGrew and Scott 1964). More important is
the synergistic effect of SCrV with SMV, SVBV or SMYEV. Even though this
3.2 Yield Losses in Different Crops 131

crop is susceptible to more than 26 viruses and phytoplasma diseases, except for a
few diseases, there is not much information is available on crop loss.

(g) Pineapple (Ananas comosus, Family Bromeliaceae)

In Hawaii, Pineapple mealybug wilt-associated virus- (PMWaV-1) infection has
been correlated with 5–15 % of ratoon crop yield reduction and losses of up to
30 % of production due to premature or asynchronous fruit ripeness. However, in
that region the most widespread virus species is PMWaV-2 which causes up to
100 % fruit loss (Sether and Hu 2002).

3.2.6 Industrial Crops

(a) Sugarcane (Saccharum officinarum, Family Poaceae)

In some countries the cultivation of sugarcane was abandoned due to enormous
losses with Sugarcane mosaic virus (SCMV) and grassy stunt diseases. Losses
depend on the cv. of the cane involved, the strain of virus, the age of infection and
the climate of the area. It has been stated that the SCMV is milder in its effect in
tropical than in subtropical areas (Abbott 1961). Summers (1943) found that the
loss in yield in Co. 281 varied from 2.5 to 33.4 % and in Co. 290 from 6.0 to
14.8 %. Gonzalez-Rios and Adsuar (1953) reported 29 % reduction in cane crop
and 32 % in first ratoon in the cv. B. 34–104 due to SCMV. Edgerton (1959) stated
that losses in Louisiana with varieties P.O.J. 281 and Co. 290 were of the order of
8–15 % with the green mosaic strain of SCMV and 30–40 % with the severe strain
of SCMV. Hansford and Murray (1926) found that the effect of SCMV was
cumulative, the yields from successive ratoons progressively declining. Similar
observations have been made by Brandes (1919) who also found that losses varied
from 0.5 to 45 % depending on the cultivar of cane grown. The SCMV epidemics
in the 1920s caused a near collapse of the sugar industry in Argentina, Brazil and
Louisiana. In the 1930s SCMV brought the sugar industry to its knees in south
Africa (Anon 1980). Koike and Gillaspie (1989) reported the estimated yield
losses of 30–40 % and sometimes up to 60–80 % due to SCMV. Rassaby et al.
(2003) conducted detailed studies on yield losses of Sugarcane yellow leaf virus
(SCYLV) on sugarcane growth and yield on Reunion. There was 46 % reduction
of stalk weight and 13 % reduction of stalk diameter and 37 % reduction in
tonnage. In India, the same virus has caused significant reduction in cane weight,
number of internodes and cane diameter in the yellow leaf virus diseased canes.
Average juice yields of 429.6, 347.0 and 279.5 ml/kg were recorded in disease-
free, asymptomatic and diseased canes respectively by the 12th month in the
popular cv. Co86032. In Brazil, SCYLV has caused 25 % losses in the yield in SP
71-6163 (Vega et al. 1997). Yield losses of 15 to 20 % also have been reported due
to the same virus in Louisiana (Grisham et al. 2002). In India, also due to the
disease, cane productivity in the popular cv. showed a steady decline and reached
to the lowest of 77.5 t/ha from 95 t/ha in 10 years (Viswanathan 2002, 2012).
132 3 Impact of Virus and Viroid Diseases on Crop Yields

From India, Mc Rae (1932) reported that mosaic affected sugarcane cv. Co-213
showed 14.8 % less yield of stripped cane, 8.9 % lower in yield of juice and
slightly less in brix and 4 % lower in sucrose than healthy canes. Even, Chona
(1944) recorded 10–12 % reduction in yield of sugar in the same sugarcane cv. and
did not notice any change in the quality of juice. In India, the incidence of 10 % of
SCMV involved a 1 % reduction in cane yield and resulted in a loss of 3.3 million
rupees per annum. Strain A of SCMV caused a 16.8 % reduction in weight of
entire plants and 31.2 % of millable cane, while strain F caused only 8.0 and
4.8 %, respectively. Strain A also showed a greater reduction in the juice quality
and purity coefficient of sucrose than strain F. (Rishi et al. 1975). In Brazil, in the
cv. CB 46/47, Matsuoka and Costa (1974) obtained 71 and 19 % losses with
100 % initial infection rates. In Louisiana, the production of the cane sugar
decreased from 400,000 to 50,000 tons because of SCMV. It destroyed the old
established cvs. worldwide, and only with replacement using highly tolerant, cvs.
was total ruin avoided.
Grassy shoot phytoplasma disease is also economically important and the
studies conducted at the Sugarcane Breeding Institute, Coimbatore (India) showed
that grassy shoot infection can cause 35 % reduction in stalk height and 15 %
reduction in stalk girth. In addition, 50–60 % reduction in length of internodes was
observed. Above all, the infection caused a significant reduction in millable cane,
especially in ratoon crops. About 50–75 % crop infection resulted in 100 % failure
in millable cane production in the ratoon crop of clones such as IS152. In mod-
erately infected cvs. up to 40 % reduction in sugar yield was noticed (Viswanathan
and Rao 2011).

(b) Sugarbeet (Beta vulgaris var. Vulgaris, Family Chenopodiaceae)

Beet yellows virus (BYV) is the major disease of sugarbeets in Great Britain, U.S.,
Germany, Poland, France, Spain, Italy, Denmark and causes tremendous economic
losses. In Great Britain severe epidemics occurred in 1949, 1952, 1957, 1959 and
1961. Watson (1952) estimated that early infection of late sown beets caused a loss
of 67 % of the root and 71 % of the sugar yield. The loss of yield of sugar was
proportional to the number of weeks the plants took to show symptoms (infected-
plant-weeks = IPW). The beet plants lost 4–5 % of their potential yield for every
week which was equivalent to a loss of £ 12/ha. Beet yellows under the same
conditions reduced the sugar yield by 50 %. In Germany, Ludecke and Neet
(1956) reported that Beet mosaic virus reduced the yields of beetroots, foliage and
sugar by 6, 10 and 9 %, respectively. In similar experiments beet yellows caused
losses of about 9- fold greater than mosaic alone. In plants infected with both
yellows and mosaic, the effects of the two were additive Wiesner (1959) obtained
similar results. Mosaic virus reduced sugar yields by 6–10 % where as yellows
alone caused losses of 35–55 %. Again in doubly infected plants the effects of the
viruses were additive. Hull (1953, 1963 and 1968) estimated loss in yield of roots
during 1957, a year of severe yellows, was 1,184,000 tons. In Great Britain alone
between 1942 and 1952, the average annual loss was 2.5 million pounds. The loss
of sugar yield of about 4.5 % per 1 P.W., when both Beet yellows virus and Beet
3.2 Yield Losses in Different Crops 133

mild yellowing virus (BMYV) were present. The average loss of 1.5 per IPW when
a single virus was present (BYV or BMYV). Heathcote (1974, 1978) found that the
yield of cvs. Sharpe’s Kleion E and Maris Vanguard was decreased by 2–3 % by
Beet yellows virus and 2 % by Beet mosaic yellow virus alone for every week the
plants showed symptoms. Estimated national loss of yield, together with financial
loss was 18 %. The average financial loss per annum in England over the six year
period (1970–1975) was £ 4,23,3000.

(c) Cotton (Gossypium hirsutum, Family Fabaceae)

In Pakistan, the average cotton yield was reduced by nearly 30 % resulting in losses
of US $ 5 billion between 1992 and 1997 due to Cotton leaf curl virus (CLCV)
infection in cotton (Briddon and Markham 2000). From India, quantification and
qualitative losses with this virus were estimated in two cotton varieties. An average
percent reduction of 56.44 in seed cotton yield, 53.13 in number of harvestable
bolls, 13.63 in boll weight and 16.13 in height per plant was estimated in cv. F-846.
These reductions were 49.86, 42.64, 5.71 and 10.08 %, respectively in case of
cotton variety RST-9. Among quality parameters, reduction in fiber length was
3.55 % and in fiber strength 6.15 % in cv. F-846. The parameters were reduced to
2.6 and 3.4 % respectively in cv. RST-9. However, micronaire value increased to
1.25 and 2.25 % in cvs. F-846 and RST-9, respectively (Ajmera 2000). Sharma
(2002) and Sharma and Rishi (2007) have also have reported the yield losses due to
Cotton leaf curl virus CLCV of up to 60 % in Haryana state, India. Further they
have observed 17.48 % reduction in boll weight, 32.57 % reduction in seed weight
and 33.77 % in seed number. Losses resulting from Cotton leaf crumple virus
(CLCrV) infection ranged from 21 to 86 % depending on the age of plants at the
time of infection (Brown et al. 1987; Van Schaik et al. 1962).

(d) Tobacco (Nicotiana tabacum, Family Solanaceae)

In almost all the tobacco growing countries, Tobacco mosaic virus (TMV), Potato
virus Y (PVY), Tobacco etch virus (TEV) or Tobacco leaf curl virus (TLCV) are
some of the major viruses affecting tobacco production. When these viruses cause
severe leaf malformations they can make the tobacco leaf useless for processing.
From Holland, Mayer (1886) estimated the losses due to TMV to be 25 %. Mc
Murtrey (1928, 1929) estimated that losses would range from 30 to 35 %, when
the inoculation was done at the time of transplanting. The USDA calculated annual
loss for the period from 1951–1960 due to TMV at 1.4 % (USDA 1965). From
North Carolina, Gooding (1969) reported that the yield losses due to TMV varied
from 5–16 %. During 1970, he further reported that TEV caused only 3 % losses
in the tolerant cv. NC 2512, while the yield of other 10 cvs. were reduced by
6–18 %. Pirone (1974) recorded that Tobacco vein mottling virus reduced the yield
in Kentucky-10 (tolerant) by 35 %, Burley-21 (intermediate) by 45 % and Burley-
37 (sensitive) by 63 %. PVY reduced the weight of cured leaf by 28.5 % and yield
by 35.5 %, in flue cured tobacco (Thomson and Wright 1966). Sievert (1971)
reported that in Burley-49 tobacco, early infection by PVY resulted in tobacco
production of only 1,590 lb/acre, later infection resulted in 1,993 lb/acre, while the
134 3 Impact of Virus and Viroid Diseases on Crop Yields

un-inoculated control yielded 2,155 lb/acre. There was significant reduction in

yield with TMV or PVY, but the reduction was greater with PVY (about 35 %)
than with TMV (about 28 %). In the case of the combination the two viruses, this
caused an additive reduction in yield of about 64 %. The resultant tobacco from
the plants infected with these two viruses had a very low grade index as did by the
individual viruses. Reduction by TMV, PVY and TMV ? PVY was about 47, 62
and 82 %, respectively. As a result of infection, the value per kg was reduced by $
0.035, $ 0.085 and $ 0.148 for TMV, PVY and TMV ? PVY, respectively.
Compared to healthy tobacco, PVY infected tobacco had less nicotine and TMV-
infected tobacco had less water soluble acids, nicotine, phenols and amino nitro-
gen. Flowering was delayed in plants infected with TMV or TMV ? PVY; but
PVY infection alone had little effect on flowering (Sievert 1978). From Costa Rica,
Paniagua (1978) also observed the yield losses due to PVY infection in two types
of tobaccos. Hidaka et al. (1956) from Japan recorded varied yield losses due to
Tobacco stunt virus.

(e) Cacao (Theobrama cacao,Family Sterculiaceae)

Cacao swollen shoot virus (CSSV) (preferred by ICTV) was first recognized in
1936 by Steven, but almost certainly occurred in West Africa in 1920. CSSV is
estimated to cause an annual loss of 50,000 tons of cacao (or cocoa) beans in
Africa with an estimated value of $ 28 million (Bowers et al. 2001). In Ghana,
CSSV infection reduced the yield of mature trees by 25 % after one year, by 50 %
after second year and 100 % after third year, by which time almost plants were
dead or drying. This disease is responsible for taking the livelihood of many
people in the world (Crowdy and Posnette 1947).This disease has killed half of the
mature trees in a 250,000 acre area of cocao (Wellman 1954). Over 140 million
trees have been rogued out in Ghana in an eradication programme (Brunt and
Kenten 1971). In the eastern province of the Gold Coast, 1000,000 plants were
destroyed by this disease and production was reduced from 116,000 tons in 1936
to 64,000 tons of cocoa in 1945. On one farm the total production decreased from
30 tons/annum during 1926–1929, to 20 tons in 1936–1939, and to only 6 tons in
1943–1944, due to this disease (Posnette 1945). Philips (1961) reported an annual
loss of over 20,000 tons in cacao production in eastern Ghana. Beckett (1972) also
reported that the total production fell down from 118,000 tons in 1936 to 39,000
tons in production, and has remained at about this level in spite of replanting
programmes. Padwick (1956) estimated that the proportion of the world’s cacao
crop lost through CSSV was about 10 % ([10 % in Ghana and\10 % in Nigeria).
Longworth (1963) and Longworth and Thresh (1963) reported that in Nigeria virus
infection alone reduced the yield of well-maintained Amelonado cocoa trees by
20 % or less.
Estimates of annual yield losses due to this virus vary from above 20,000 tons
to approximately 120,000 tons of cacao from the eastern region of Ghana alone.
The average annual loss between 1946 and 1974 in Ghana was estimated to be
worth over £ 3,650,000 (CABI/CPC 2002).
3.2 Yield Losses in Different Crops 135

(f) Coffee (Coffea arabica, Family Rubiaceae)

Coffee ring spot virus (CoRSV) or mancha annular (Spanish) naturally infects
coffee and was first described by Bitancourt (1938) from Brazil. It is apparently
limited to Brazil and Costa Rica (Rodrigues et al. 2002) and in the Philippines,
where the seed transmission of CoRSV has been reported (Reyes 1959). Recently
large scale infection of this virus was reported in Minas Gerais state of Brazil that
resulted in yield losses. It is the only virus known to naturally infecting coffee.
Conspicuous symptoms are chlorotic ringspot on leaves, berries and less fre-
quently on twigs.
The virus is member of the Rhabdovirus group and consists of bullet-shaped
particles measuring 40 nm 9 100 to 110 nm, containing single-stranded RNA
(Boari et al. 2004). It is spread by the mite, Brevipalpus phoenicis. Trans ovarial
transmission within the mite does not occur. The virus is mechanically sap
transmitted. The virus presumably spreads from a native host in South America. It
is of low incidence and causes no significant damage to coffee (Kitajima and
Chagas 2009).

(g) Tea (Camellia sinensis, Family Theaceae)

Phloem necrosis of tea, previously reported from Ceylon (Sri Lanka), India,
China, Tibet, and Australia in estates at high altitude (4,000 ft. and up), has been
shown to be of virus nature through transmission by inarch grafting. Some plan-
tations show a disease incidence up to 50 %. Systemic infection occurs, and can be
detected in roots, stems and leaves. The most prominent symptoms are backward
curling or arching of leaves, angular conditions of nodes resulting in a zig zag
stem, general loss of vigor, and phloem necrosis. The disease was shown to be
transmissible by grafting to certain types of tea, nine clones of various origin
having been infected so far (Bond 1944). Yield loss studies were conducted in
Ceylon by Bond (1944). The disease was reduced by rouging the infected bushes
and replanting with healthy tea plants.

(h) Rubber (Hevea brasiliensis, Family Euphorbiaceae)

In Cambodia some young rubber trees showed malformed leaves with yellow
discolorations along the veins. Such leaves contained elongated virus-like particles
(rigid or slightly flexible) of various lengths (60–880 nm) (Brcak and Pozdena
1976). No information is available on this disease in terms of host-range, vector
transmission, and other aspects. Another disease reported on rubber is trunk
phloem necrosis reported from the Ivory Coast, and no further details are available.
In recent years, Tapping panel dryness syndrome (TPD) of rubber is reported from
India and the causal agent is identified to be a viroid. The TPD-infected rubber
plants exhibited symptoms of bark scaling, cracking, drying, necrotic streaking,
and browning of internal bark leading to the decay of internal tissues. Often
prominent abnormal bulges on the lower part of tree trunks occur where the first
panel begins to dry. Based on the molecular tests conducted by Ramachandran
et al. (2000) the etiological agent was proved to be a viroid pathogen.
136 3 Impact of Virus and Viroid Diseases on Crop Yields

3.2.7 Edible Oil Seed Crops

(a) Groundnut/Peanut (Arachis hypogaea, Family Fabaceae)

Groundnuts (peanuts) suffers enormous losses every year from diseases like
Groundnut rosette virus (GRV), Tomato spotted wilt virus (TSWV), Groundnut bud
necrosis virus (GBNV) (per ICTV), Peanut mottle virus (PeMoV), Peanut clump
virus (PCV), Peanut stunt virus (PSV), and Peanut stripe virus (PStV) in many
groundnut growing countries. Vander Merwe and Subrahmanyan (1997) have
predicted that GRV can cause yield losses up to 100 % in association with drought.
GBNV in peanuts in India, China, Indonesia, Nepal, Pakistan, Philippines, Sri
Lanka and Thailand is caused by a virus serologically different from TSWV. TSWV
that causes similar symptoms in peanut in Argentina, Brazil, Kenya, Malawi,
Nigeria, South Africa, Uganda and the U.S. In India, GBNV causes crop losses up
to 80 %, but early infections can cause 100 % yield losses, while late infection may
cause up to 70 % (Prasada Rao et al. 1979, Narayanaswamy and Ramiah 1977). The
losses due to GBNV alone have been estimated to be more than US $ 89 million per
annum in Asia (Reddy et al. 1995). From Georgia, Kuhn (1969) reported losses of
20–32 % in pod and seed weights due to PeMoV. Yield losses caused during 1973
was 5.2 % or 10.3 million dollars. Severe losses of up to 48 % when the PeMoV
infection was observed in the first five weeks of planting and 26, 22 and 18 %
losses occurred when the infection was observed at 7, 9 and 12 weeks after
planting, respectively. The number and size of seed were progressively reduced by
longer infection periods (Paguio and Kuhn 1974).
PSV caused the reduction in total seed number by 77, 31 and 34 % and the total
weight of seeds reduced by 91, 29 and 36 % at 60, 80 and 100 days after ger-
mination Kuhn (1969). Culp and Troutman (1967) have also observed the yield
reduction of the stunted plants increased from 25 to 100 % with the same virus.
In India, another important virus disease is peanut stem necrosis caused by
Tobacco streak virus (TSV), which has caused epidemics during Kharif 2000, in
Andhra Pradesh and crop losses were estimated to exceed 300 crores (Reddy et al.
2002). In Australia, TSWV caused yield losses up to 90 % (Saint-Smith et al. 1972).
Another virus disease of peanut which occurs in light sandy soils of India is Indian
peanut clump virus (IPCV) which caused yield losses as high as 60 % even in late
infected groundnut crop (Reddy et al. 1988). Yield diminution in Burkina Faso due
to clump and chlorosis disease has been estimated to be between 40 and 70 %
(Germani and Dhery 1973a, b). Lynch et al. (1989) recorded yield reduction of
5–7 % in Florunner peanut with Peanut stripe virus.

(b) Coconut (Cocos nucifera, Family Arecaceae [Palmae])

Cadang–Cadang disease which is of viroid nature (Randles 1975) has destroyed
250,000 trees in San Miguel Island. Twenty millions of trees have been killed in the
limited areas of the Philippines since 1926 (Price and Nitzany 1969; Randles 1975),
and is a disease of enormous economic importance in the Philippines; indeed it is
considered to be the threat to coconut production (Randles 1975). Losses in yield of
3.2 Yield Losses in Different Crops 137

copra due to cadang–cadang had been estimated to be $ 16,646,000 annually

(Price 1971). Strip surveys showed that the disease had destroyed an estimated
5,527,000 coconut trees by 1953. By 1959, as the number of diseased trees was
estimated to be 7,900,000 (Bigornia et al. 1960, as quoted by Price 1971). Coconut
foliar decay pacific region In India, Kerala State is facing an economic threat due to
coconut root wilt disease and nearly one third of the coconut trees are affected.
A conservative assessment of the loss is approximately Rs. 300 million annually.
Disease palms generally yield fewer nuts. The extent of decline in yield is 43% in
diseased early palms and 74% in diseased advanced palms, over root (wilt) free
palms. The oil content in the root (wilt) free, diseased early and diseased advanced
palms is respectively 69.7%, 68.3% and 67.5%. The loss in husk per nut of diseased
palm is around 25.8% and that of copra is 9% while 92.7% of leaves in the root
(wilt) free palms are palatable only 27.4% and 0.4% are palatable in diseased early
and diseased advanced palms, respectively (Jayasankar et al. 1989).

(c) Oil palm (Elaeisguineensis, Family Arecaceae [Palmae])

Oil palm crop is native to Africa and also grown in countries of Pacific region,
Solomon Islands, Malaysia, Thailand, Papua New Guinea, Philippines, Indonesia,
Columbia, Brazil, Peru, India and other oil palm growing countries. Like other
crops, this crop suffers from plant pathogens due to virus, viroid and phytoplasma
diseases. A viroid disease showing orange spotting of oil palms which is similar to
Coconut cadang-cadang viroid (CCCVd) was reported by Imperial et al. (1985).
Viroids are the smallest plant pathogens known, with single stranded, circular,
covalently closed, autonomously replicating RNA as genomes. The infected oil
palms show bright orange spotting on leaflets and appear from a distance to be
bronze-colored to necrotic. The youngest fronds are free of spots, and the size and
number of spots increase with increasing age of fronds (Hanold and Randles 1991).
In young fronds, the irregular shaped and non-necrotic orange spots are about
2–3 mm long and occur between the veins of the leaflets. As the age of the frond
increases, the spots coalesce into large circular patches, and distal necrosis of
leaflets is seen in the oldest fronds (Forde and Leyritz 1968). The height of the
infected plants is reduced with smaller fruit bunches and are significantly less
productive. Yield from infected palms was 25–50 % lower than adjacent healthy
plants (Forde and Leyritz 1968; Hanold and Randles 1991). This disease is seed
transmitted in oil palms. Studies conducted by Selvaraja et al. (2012) showed that
the visually-observed incidence of Orange Spotting (OS) disease (CCCVd) inci-
dence was 74.3 % from a 15 year old oil palm stand.
Among the virus diseases infecting oil palm, chlorotic ring (Anillo clorotico)
disease was described in nurseries in western Ecuador (Chinchilla et al. 1995,
Genty 1996) and in advanced stages this disease poses a potential risk for the
industry. A similar condition (if not the same) was described in Karnataka and
Andhra Pradesh states in India in 1994 (Solomon and Kochu Babu 1998). The
disease incidence in India is very low, where as in some nurseries in Ecuador it
was up to 80 %. Based upon the mor-phology of cytoplasmic inclusions and
particle morphology, Solomon and Kochu Babu (1998) have tentatively placed the
138 3 Impact of Virus and Viroid Diseases on Crop Yields

virus within sub-division I of Potyviridae. No information is available on host-

range, vector transmission and other aspects.
The blast disease is very destructive in nurseries in Africa and Malaysia. A
disease with similar characteristics has been described in Colombia. However,
blast symptoms could be easily confused with dry bud rot (ring spot disease or
‘‘Mancha anular’’). The real causes of the disorder are not resolved, so control is
somewhat empirical. Losses of 5–80 % of nursery plants were common in the past
(Turner 1981). The causal agent (non-cultivable mollicute?) seems to be trans-
mitted by sucking insects (Recilia mica : Cicadellidae).
Dry bud rot (Mancha anular) is another suspected virus disease in several South
American countries (Peru, Ecuador, Colombia, Brazil) and in Africa (Ivory Coast).
The disease incidence is higher and the symptoms are more severe in palms
between 2 and 4 years in the field. The death of the palm can occur between 3
months and 1 year from the time the symptoms first appeared. Quillec and Renard
(1985) were able to reproduce the symptoms of the disease using two cicadae:
Sogatella kolophon and S. cubana (Homoptera) and the vector transmission aspect
needs to be confirmed.
‘Marchitez lethal’ is a major disease affecting oil palm, has been observed with
increasing frequency in Colombia. Incidences of up to 30 % have been recorded in
several commercial fields in production areas of Villanueva and Casanare. Its
symptoms are similar to those caused by infection with phytoplasma and include
leaf discoloration, lower leaves will turn brown and hang downwards like a col-
lapsed umbrella. Due to rapid progress of disease the plants will die within
3–6 months. On the basis of DNA sequences, serology and presence of phytopl-
asma bodies, the oil palm phytoplasma was classified as a member of the 16 SrI in
the aster yellows group.
Kerala wilt disease is another phytoplasma disease which occurs on oil palm in
India. The insect vectors Proutista moesta (Homoptera: Derbidae) and Stephanitis
typica (Heteroptera: Tingidae) are responsible for transmission. Initial symptoms
appear on youngest leaf as chlorosis followed by necrosis. Emerging leaf rot and
smaller leaf size than normal affect the emergence of flowers resulting in a dropped
in productivity. This disease has killed 15,000 palm trees in Kerala province alone.

(d) Sunflower (Helianthus annuus, Family Asteraceae)

Among the viruses infecting sunflower Tobacco streak virus (TSV) is the most
destructive in India and in some other sunflower growing countries. Since
1999–2000 this virus disease is wide spread in Andhra Pradesh, Karnataka,
Maharashtra and Tamilnadu states (Prasada Rao et al. 2000; Kumar et al. 2008; Jain
et al. 2008). Kumar et al. (2008) stated that the TSV epidemics in 2002 on sunflower
in Karnataka state had caused yield losses of Rs. 110 crores. TSV infection at
seedling stage results in premature death of plants and infection during midstage of
the plant growth will result in necrosis of the leaves and severe reduction in yield.
Infection at the late stage of the plant growth results in mild chlorotic symptoms
with little apparent effect on plant growth and yield. It is estimated that an
approximate minimum loss in Rs. 332 millions per annum would have occurred to
3.2 Yield Losses in Different Crops 139

sunflower in India had it not been intercepted by the regional station of NBPGR,
Hyderabad, India (Prasada Rao et al. 2012).
From Argentina, Lenardon et al. (2001) have reported that Sunflower chlorotic
mottle virus infection in sunflower at all stages significantly reduced plant height
(16–39 %), stem diameter (21–51 %), capitulum diameter (27–57 %), achene
yield (58–87 %), seed width (13–15 %), seed length (10–16 %) and weight of
1,000 seeds (26–28 %) compared with healthy controls. Oil content determined by
magnetic nuclear resonance showed no significant differences among treatments.

3.2.8 Spice Crops

(a) Onion and Garlic (Allium cepa; Allium sativum, Family Alliaceae)
Studies conducted in eastern France during 1951 at the plant pathology station
located in Colmar, revealed that the Onion yellow dwarf virus (OYDV) had
reduced onion seed yields by about 55 % and bulb weight in white onions by
40 %, in shallots by 70 % and in leeks from 17 to 45 %, according to symptom
severity (Anon 1951). From Argentina, Munoz and Docampo (1975) estimated the
yield losses in bulb production by OYDV when inoculated immediately or 1.5
months later, and they recorded more bulb weight in control plants as compared to
the treatments. Bulbs of the infected plants were malformed and difficult to store
and were more susceptible to facultative parasites.
In India, OYDV infection in onion cv. Hisar reduced plant height, bulb weight
and bulb size by 39.6 cm, 79.7 g and 25.5 cm2 compared with 40.6 cm, 88.4 g and
27.6 cm2 in healthy plants. Heavy lodging of scapes caused by this disease is
responsible for heavy losses in seed production and seed quality. Bulb weight and
other yield qualities were reduced due to mixed virus infections in garlic (Takaichi
et al. 2001).
From Brazil, the crop loss estimates in onion infected with Iris yellow spot
virus was recorded by Pozzer et al. (1999). Even in India, this virus in addition to
OYDV has caused heavy yield losses in onion bulbs and seed crop production
under field conditions (Ravi et al. 2006; Kumar and Dhawan 2010).

(b) Cardamom (Elettaria cardamomum, Family Zingiberaceae)

Among the diseases of small cardamom, Katte, marble or mosaic disease is most
devastating. Varma (1962) estimated the yield losses due to this disease during
1958–1960. The total yield from 300 healthy plants for a period of three years was
26.7 kgs of dry cardamom, when compared to 7.8 kgs from the Katte affected
plants. The production of cardamom in India, has fallen sharply, although the areas
57,173 ha in 1935–1964 had increased to 75,412 ha in 1968–1969; the yield
figures were 2,200 and 2,100 tons, respectively, in which Katte disease is the
major limiting factor. Crop losses of 10–60 %, 26–91 % and 82–92 % were
reported under cardamom-areaca mixed cropping in the first, second and third
years of production, respectively (Varma 1962). The yields of large cardamom
140 3 Impact of Virus and Viroid Diseases on Crop Yields

(Amomum subulatum) is also reduced by mosaic streak (Chirke) disease. The

diseased large cardamom plants developed lower number of flowers, fruits and
seeds as compared to healthy plants of the same age, with the percentage loss
being 85.2 and 80.1 in total number of fruits and seeds, respectively by the end of
the third year of the crop. The chirke-affected plants had 1–5 flowers in an
inflorescence as opposed to 16–20 flowers borne by the inflorescence of the
healthy plant (Raychaudhuri and Ganguly 1965, 1968).

3.2.9 Bio Fuel Crops

(a) Jatropha (Jatropha curcas, Family Euphorbiaceae)

Over the past 20 years, the Jatropha crop has seen expansion for seed oil use as a
safe alternative source of energy (diesel and liquefied petroleum gas) as well as
used in soaps, resins, polish, gas. This crop is grown in the Philippines, Brazil,
Puerto Rico and India. Primarily Jatropha species suffers from whitefly-transmitted
Jatropha mosaic virus (JMV). In India this disease is quite prevalent (Ashwatha
Narayana et al. 2006). Jayanna (2006) from Dharwad, India has conducted studies
on crop loss estimates of J. curcas with JMV inoculations. He has reported that the
number of fruits per plant in the infected plants was reduced from 189.9 compared
to 353.69 in healthy plants. The size of the fruits was also reduced from
5.3 9 5.1 cm in infected plants as compared to 8.2 9 7.9 cm in healthy plants.
The capsules in the infected plants had 1.23 good seeds and 1.06 shriveled seeds as
compared to 2.41 and 0.49 in healthy plants, respectively.

3.3 Conclusions

From the foregoing examples, it is clear that the loss estimates in different crops
with virus, viroid and phytoplasma diseases vary from zero to 100 %, depending
on many factors. In plant pathology one of the most difficult problems is that of
accurately assessing the incidence of the diseases in crops and accurately assessing
the loss in terms of crop yield and money. Neither the disease agent nor crop losses
are static and they will change from year to year in a given location. Experiments
should be conducted at least for 3–4 years at a number of locations. Such infor-
mation needs to be up-to-dated, perhaps for every five years, because of rapidly
changing cultural practices, the introduction of new plant varieties and agricultural
chemicals. Absolute values for losses due to single virus/viroid/phytoplasma/
spiroplasma are always difficult because most of the time the same crop will be
affected with other pathogens and pests like fungi, bacteria and insects. The latter
can be controlled to some extent through the use of chemical sprays. Certain
estimates of losses where the values are very low can convey the false impression
that a disease is of no economic importance, whereas over estimates may result in
3.3 Conclusions 141

limited resources being concentrated on a particular disease to the neglect of other

more destructive ones. However, useful results can be obtained from properly
designed and well conducted field experiments. It is important to the the grower
who has to decide whether the monetary losses due to these diseases, warrant the
trouble and expense of applying control measures. The monetary losses caused by
diseases or the expense of controlling them are likely to be passed on to the
consumer in the form of higher retail prices or the government subsidies to the
farmer, so that all users of agricultural produce will be adversely affected.
Commonly crop loss experiments have been based solely on reduction of yield.
Assessment of loss should also include the reduction in the market value of the
crop due to the lowering of the quality by these virus, viroid and phytoplasma
pathogens. Variation in the market standards from area to area prevent an uniform
approach to the quality problem. In order to apply general knowledge to local
conditions and to give guidance on the development and the use of crop loss
appraisal methods, workshops or consultations should be planned in different
regions by the organizations like FAO and EPPO. Intensive research is needed in
the development of new crop loss appraisal methods.

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Chapter 4
Transmission of Plant Viruses and Viroids

4.1 Introduction

Plant virus transmission takes place through vertical and horizontal modes.
Vertical transmission is the transmission of a virus from a parent plant or insect
vector to their progeny and seed transmission plays a major role. In horizontal
transmission, the virus spreads from one plant to another through mechanically or
contact or both. Under field conditions, virus spread takes place by different types
of vectors like insects, nematodes, fungi etc. Horizontal transmission also occurs
by certain artificial methods of vegetative reproduction typically employed by
horticulturists and farmers.

4.1.1 Transmission Through Vegetative Propagules

The widespread use of vegetative propagules for the multiplication of many hor-
ticultural crops results in the spread of plant viruses and viroids through propagules
such as stem cuttings, tubers, runners, suckers, corms, rhizomes, and bulbs. Since
infection by most of the viruses and viroids is completely systemic, any propagule
arising from infected plants, is likely to be infected. Thus, vegetative propagation
presents a very efficient method of virus and viroid spread, without the virus/viroid
having the difficulty of entering and establishing infection in a new healthy plant.
Although virus/viroid spread through vegetative propagules might be expected
to occur over short distances in nature by natural scattering of infected propagules
such as tubers, but man has been responsible for the worldwide movement of many
virus and viroid diseases by means of exporting and importing the true seed and
vegetative propagative materials.
The importance of virus/viroid infection of vegetatively propagated plants, and
methods that may be used to eradicate viruses and viroids from plant clones that
are totally infected are discussed in the Chapter-II of Volume II. Some of the
highly economically important viruses and viroids which are widely distributed in

K. S. Sastry, Plant Virus and Viroid Diseases in the Tropics, 161

DOI: 10.1007/978-94-007-6524-5_4,  Springer Science+Business Media B.V. 2013
162 4 Transmission of Plant Viruses and Viroids

tropics through vegetative plant material are the viruses and viroids infecting the
crops like banana, citrus, pineapple, cassava, sugarcane, grapevine, potato, sweet
potato and taro. Certain properties of virus and viroid diseases of crops in the
tropics are given in Table 4.1a and b.

4.1.2 Transmission Through Seed

(a) Seed transmission

Transmission of nearly 231 plant viruses and viroids through seed is a property
intrinsic to members of at least 24 of the known groups of plant viruses.
Approximately 18 % of the described plant viruses are seed-transmitted in one or
more hosts, and it is estimated that one-third of the plant viruses will eventually
prove to be seed-transmitted in at least one host (Johansen et al. 1994; Khan and
Dijkstra 2002; Albrechtsen 2006; Sastry 2013). Sometimes viruses alter the size,
shape and colour of the seeds (Fig. 4.1).
Transmission through seeds is the most serious problem when a vector is
present and it primarily depends on the initial virus inoculum load. For example,
Bean common mosaic virus shows a very high percentage of transmission through
seed and has a very efficient aphid vectors. When even a small amount of infected
seed is planted, aphids will spread the infection to healthy plants under field
conditions. For example, cowpea seeds with 5% of Blackeye cowpea mosaic virus
have resulted the yield losses of 34-53%, while 0.5% seed infection did not cause
significant loss (Puttaraju et al. 2002).

(b) Controversial seed transmission

In tomato Pepino mosaic virus (PepMV) is seed transmitted to the extent of 1.84 %
(Cordoba-selles et al. 2007). During 2008, Ling has proved that PepMV is not seed
transmitted in large scale field trails and confirmed by ELISA and RT-PCR.
However the PepMV was detected in the seed coat fraction in both immature and
mature tomato seeds, but not in the embryos. Hence efficient mechanical trans-
mission of this virus from virus contaminated seed to seedlings could initiate the
disease epidemics, recommending to use certified seed. Even in rice, beetle
transmitted Rice yellow mottle virus in Africa, the virus was detected in all rice
seed parts, but no seed-borne infection was found (Konate et al. 2001) and field
spread takes place through beetle vectors.

(c) Mode of seed transmission

• Externally, on the seed cover which happens through contamination of the pulp
of the infected fruit, which is in contact with the seed cover (e.g., Tobacco
mosaic virus in tomato and hot peppers). In this case, the seed can be disinfected
with chemicals such as HCl (37 %) diluted to 1/20 and incubated for 4–8 h.
• Internally, in the embryo and the endosperm; in this case, the virus cannot be
eliminated by disinfection with chemical products. The virus remains inside the
Table 4.1a Details of virus diseases transmitted through vegetatively propagated materials of tropical crop plants
Name of the Scientific name of the Virus Genus Morphology Genome
crop plant crop plant
Apple Malus domestica Apple Chlorotic leaf spot Trichovirus Filamentous, NE, ssRNA 7.55 Kb
720–740 9 12 nm
4.1 Introduction

Apple Mosaic Ilarvirus Isometric, NE, 25–29 nm in ssRNA

diameter
Malus sylvestris Apple stem grooving Capillovirus Filamentous, NE, ssRNA
600–700 9 12 nm
Apple stem pitting Foveavirus Filamentous, NE, ssRNA, linear
800 9 12–15 nm
Banana Musa sapientum Banana Bract mosaic Potyvirus Filamentous 750 nm ssRNA
Musa sp. Banana Bunchy top (or) Abaca Nanovirus Isometric, NE, 18–20 nm ssDNA, circular
bunchy top
Banana streak Badnavirus Bacilliform 119 9 30 nm dsDNA
Beetroot Beta vulgaris Beet temperate Alpha crypto virus NE, Isometric 30 nm dsRNA, linear
Beet cryptic Beet 2 alpha NE, Isometric 30 nm dsRNA
cryptovirus
Beet cryptic Beet 3 alpha NE, Isometric 30 nm dsRNA
cryptovirus
Beet curly top Hybrigeminivirus Geminate NE, 18–22 nm, rounded ss-DNA, circular,
uni partite
Beet mosaic Potyviruses Filamentous, NE, flexous, ss-RNA, linear,
695–770 nm unipartite
Cacao Theobroma cacao Cacao necrosis Nepovirus Isometric, 24–26 nm, NE ssRNA, linear, 2
parts
Cacao swollen shoot Badnavirus Bacilliform, NE, 130 9 28 nm dsDNA, circular
Cacao yellow mosaic Tymovirus Isometric, NE, 28 nm ssRNA, Linear
(continued)
163
Table 4.1a (continued)
164

Name of the Scientific name of the Virus Genus Morphology Genome

crop plant crop plant
Cassava Manihot esculenta Cassava African mosaic Bigeminivirus Geminate, NE, 20 nm ssDNA, circular, 2
parts
Cassava brown streak Carlavirus Filamentous, NE, 650–690 nm ssRNA, linear
associated
Cassava brown streak Potyvirus Filamentous, NE, 750 nm ssRNA, linear
Cassava caribbean mosaic Potexvirus Filamentous, NE, 460 nm ssRNA, linear
Cassava Colombian, Potexvirus Filamentous, NE, 460 9 13 nm ssRNA, linear
symptomless
Cassava common mosaic Potexvirus Filamentous, NE ssRNA, linear
495–525 9 15 nm
Cassava green mottle Nepovirus Isometric NE, 27 nm ssRNA, linear, 2
parts
Cassava Indian mosaic Bigeminivirus Geminate, 16–18, 30 nm len ssDNA, circular
Cassava ivorian bacilliform Ourmiavirus Bacilliform NE, 42–76 9 18 nm ssRNA, 3 parts
Cassava symptom less Rhabdo virus Bacilliform ssRNA
Cassava vein mosaic Caulimovirus Isometric, 45–50 nm dsDNA, circular
Cassava X Potexvirus Filamentous ssRNA, unipartite
Citrus Citrus sp. Citrus enation woody gall Luteovirus Isometric 24–26 nm ssRNA
Citrus leaf rugose Ilarvirus Unusually shaped 25–32 nm ssRNA, linear 3
parts
Citrus ring spot Filamentous NE, ssRNA, 2 parts
300–500 9 8–10 nm
Citrus variegation Ilarvirus Usually shaped 26–35 nm –
Citrus limon Citrus tatter leaf Capillovirus Filamentous 650 9 19 nm ssRNA
Citrus sinensis Citrus leprosis Rhabdovirus Bullet shaped 100–110 9 30 nm ssRNA
Citrus tristeza Closterovirus NE, filamentous 2000 9 12 nm ssRNA, unipartite
(continued)
4 Transmission of Plant Viruses and Viroids
Table 4.1a (continued)
Name of the Scientific name of the Virus Genus Morphology Genome
crop plant crop plant
Dasheen Colocasia esculenta Colocasia bobone disease Rhabdovirus Bullet shaped, ssRNA
300–335 9 50–55 nm
4.1 Introduction

Dasheen mosaic Potyvirus Filamentous NE flexous ssRNA linear, uni

partite
Garlic Allium sativum Garlic common latent Carlavirus Filamentous 650 nm ssRNA
Garlic dwarf Potyvirus Isometric NE 65–70 dsRNA 10 segment
Ginger Zinfiber officinale Ginger chlorotic fleck Sobemovirus Isometric NE 28–33 nm ssRNA unipartite
Grapevine Vitis vinifera Grapevine A Trichovirus Filamentous NE 800 9 12 nm ss RNA
Grapevine ajinashika disease Luteovirus Isometric NE 28 nm ssRNA
Grapevine algerian latent Tombusvirus Isometric NE, 30 nm ssRNA, unipartite
Grapevine B Trichovirus Filamentous NE, 1800 nm –
Grapevine Bulgarian latent Nepovirus Isometric, NE, 30 nm ssRNA linear, 2
parts
Grapevine chrome mosaic Nepovirus Isometric, NE, 30 nm ssRNA linear, 2
parts
Grapevine corky bark Closterovirus Filamentous, flexuous, –
associated 1400–2000 nm
Grapevine fan leaf Nepovirus Isometric NE 30 nm in dia ssRNA linear, 2
parts
Grapevine fleck Isometric NE 30 nm in dia ssRNA
Grapevine leaf roll associated Closterovirus Filamentous, flexuous ssRNA
1800–2200 nm
Grape line pattern Ilarvirus Isometric NE 24 nm ssRNA
Grapevine stem pitting Closterovirus Filamentous, flexuous, ssRNA
associated 800 9 11–12 nm
Grapevine stunt – Isometric, NE, 25 nm –
(continued)
165
Table 4.1a (continued)
166

Name of the Scientific name of the Virus Genus Morphology Genome

crop plant crop plant
Onion Allium cepa Onion mite borne latent Potexvirus Filamentous, flexuous, NE, ssRNA, linear
775 nm L
Onion yellow dwarf Poty virus Filamentous, flexuous,NE, ssRNA, linear
772 9 823 nm L
Onion yellow mosaic Tymovirus Virions, isometric NE, 25–30 nm ssRNA
Passiflora Passiflora caerulea Passiflora latent Carlavirus Filamentous, NE, straight, 648 nm, –
L
P. edulis Passiflora ring spot Potyvirus Filamentous, flexuous, NE, ssRNA, linear
810 9 15 nm
Pineapple Annanus comosus Pineapple chlorotic leaf streak Nucleorhabdovirus Bullet shaped, 200–250- ssRNA
9 60–70 nm
Pineapple wilt associated Closterovirus Filamentous, flexuous NE, ssRNA, linear,
1200–1500 9 12 nm unipartite
Potato Solanum tuberosum Potato A Potyvirus Filamentous, NE, flexous, ssRNA
730 9 11 nm
Potato andean latent Tymovirus Isometric, NE, 30 nm in diameter ssRNA, linear,
unipartite
Potato andean mottle Comovirus Isometric, NE, 28 nm ssRNA, linear
Potato aucuba mosaic Potexvirus Filamentous, NE, flexous ssRNA
Potato black ringspot Nepovirus Isometric, NE, 25 nm in diameter ssRNA
Sugarcane Saccharum officinarum Sugarcane bacilliform badna Badnavirus Bacilliform, NE, 131 9 31 nm –
Sugarcane mosaic Potyvirus Filamentous, NE, ssRNA, unipartite
730–755 nm 9 13 nm
(continued)
4 Transmission of Plant Viruses and Viroids
Table 4.1a (continued)
Name of the Scientific name of the Virus Genus Morphology Genome
crop plant crop plant
Sweet potato Ipomea batatus Sweet potato caulimo Caulimovirus Isometric, NE, 50 nm in diameter dsDNA
Sweet potato feathery mottle Potyvirus Filamentous, flexuous, NE, ssRNA, linear,
4.1 Introduction

830–850 nm unipartite
Sweet potato latent Potyvirus Filamentous, flexuous, NE, ssRNA, linear, uni
700–750 nm partite
Sweet potato leaf curl Badnavirus Bacilliform, NE ssDNA
Sweet potato mild mottle Ipomavirus Filamentous, 800–950 nm ssRNA, linear
Sweet potato ring spot Nepovirus Isometric, NE, 28 nm in diameter ssRNA, linear, 2
parts
Sweet potato sunken vein Closterovirus Filamentous, flexuous, ssRNA
850 9 12 nm
Sweet potato vein mosaic Potyvirus Filamentous, flexuous, NE761 nm ssRNA, linear
L
Sweet potato yellow dwarf Ipomavirus Filamentous, flexuous, NE, ssRNA
750 nm, L
Yam Dioscorea alata Internal brown spot Badnavirus Bacilliform NE, 130 9 29 nm dsDNA, circular
D. caynesis mosaic Potyvirus Filamentous, flexuous NE, ssRNA, linear
785 9 15 nm
167
 168

Table 4.1(b) Details of viroid diseases transmission through vegetatively propagated materials of tropical crop plants
Natural host Propagation Genus Species Variants Length (nucleotides)
Apple Bud sticks, Grafting, Root cuttings Apscaviroid Apple scar skin (ASSVd) 8 329–333
Apple Bud sticks, Grafting, Root cuttings Apscaviroid Apple dimple fruit (ADFVd) 2 306
Apple Bud sticks, Grafting, Root cuttings Apscaviroid Apple fruit crinkle (AFCVd) 29 368–372
Avocado Bud sticks, Grafting Avsunviroid Avocado sun blotch (ASBVd) 83 239–251
Chrysanthemum Sucker, Cuttings Pospiviroid Chrysanthemum stunt (CSVd) 19 348–356
Chrysanthemum Sucker, Cuttings Pelamoviroid Chrysanthemum chlorotic mottle (CChMVd) 21 397–401
Citrus Bud sticks, Budding, Grafting Pospiviroid Citrus exocortis (CEVd) 86 366–475
Citrus Bud sticks, Budding, Grafting Apscaviroid Citrus bent leaf (CBLVd) 24 315–329
Citrus Bud sticks, Budding, Grafting Apscaviroid Citrus dwarfing (CDVd) 53 291–297
Citrus Bud sticks, Budding, Grafting Cocadviroid Citrus bark cracking (CBCVd) 6 284–286
Coleus Stem cuttings Coleviroid Coleus blumei-1 (CbVd-1) 9 248–251
Coleus Stem cuttings Coleviroid Coleus blumei-2 (CbVd-2) ? 295–301
Coleus Stem cuttings Coleviroid Coleus blumei-3(CbVd-3) 3 361–364
grapevine Stem cuttings, Bud sticks, Grafting Apscaviroid Australian grapevine (AGVd) 1 369
Hop Runners, Rhizomes Hostuviroid Hop stunt (HSVd) 144 294–303
Hop Runners, Rhizomes Cocadviroid Hop latent (HLVd) 10 255–256
Pear Budsticks, Budding, Grafting Apscaviroid Pear blister canker (PBCVd) 18 314–316
Potato Tubers Pospiviroid Potato spindle tuber (PSTVd) 109 341–364
4 Transmission of Plant Viruses and Viroids
4.1 Introduction 169

Fig. 4.1 Virus induced foliar and seed symptoms on broad bean and soybean Source http://
www.virology.net/big picture book of viruses

seed for a long time, and therefore long-distance dissemination of the virus
occurs and Bean common mosaic and Tobacco ring spot viruses are most
common examples.

The AVRDC-the world’s vegetable center, Taiwan had developed a Trisodium

phosphate (TSP) treatment protocol for eliminating the TMV infection on/in pepper
seeds. In this procedure, the freshly harvested capsicum seeds were taken in a cloth
or mesh sack (1/2 full) and immersed (hanged) it in a 10 % (W/V) solution of TSP
for 30 min, making sure that seeds are always covered by TSP. Transfered the sack
to a fresh solution of TSP for 2 h and rinsed the seeds in running water for 45 min
stirring continuously. The seeds were placed in a drier at 20 C for 2–3 days and the
above procedure helps in eliminating TMV from the capsicum seeds.

(d) Factors responsible for seed transmission

• Species and variety of host plant: For example, Nepoviruses are transmitted in
many host plants. Similarly, seed transmission of Barley stripe mosaic virus
varies from 0 to 75 %, depending on the variety. Similarly, Pea seed-borne
mosaic virus in peas, the percentage of seed transmission was 65–90 %
depending on the variety (Mink et al. 1969).
170 4 Transmission of Plant Viruses and Viroids

• Stage of plant infection: Early infected plants usually show a higher probability
of virus transmission by sexual seed. In beans, transmission occurs only if the
infection takes place before flowering. Similarly seed transmission of SMV
recorded in soybean was 18–19 % when inoculated at 3–4 weeks and only
3–4 % when inoculated at 9–10 weeks after sowing (Bowers and Goodman
1979; Irwin et al. 2000).
• Age of seed: The infectivity of some viruses in the seed decreases very rapidly
with storage, and virus can be lost before the seed loses its capacity to germi-
nate. The Cherry necrotic ringspot virus disappears after six years of storage.
Some viruses can be detected during the seed formation stage, but will disappear
at maturity. This happens because mature or germinating seeds contain
in-activators that are absent in young seeds. In most of the seed-transmitted
viruses, the virus apparently comes from the ovule of the infected plant.
However, in several reported cases, the virus found in the seed seems to come
just as frequently from the infected pollen fertilizing the flowers.

(e) Percentage of seed transmission

For testing virus transmission through seed, seeds collected from infected plants
are to be sown to observe the percentage of infected plantlets. Seeds from healthy
plants of the same variety are simultaneously planted as a control. A few hundred
seeds may suffice to determine the percentage of seed transmission for viruses
showing a high degree of transmissibility. If transmissibility is low, however,
several thousand seeds are necessary to test. The percentage of seed transmission
which varies from 0 to 100 % of plant viruses and viroids in different plants is
furnished in the Table 4.2.
Till today there are nearly 231 plant virus and viroid diseases to be seed-borne
and distributed in 24 virus groups including alfamo, bromo, carla, carmo, faba,
furo, como, cucumo, hordei, ilar, poty and tymovirus groups have greater number
of seed-borne viruses (Sastry 2013). It was also observed that high degree of seed
transmission is noticed with viroid diseases like Australian grapevine viroid,
Avocado sunblotch viroid, Citrus exocortis, Potato spindle tuber viroid, and Apple
scar skin viroid diseases. Crypticviruses which induce little or no disease symp-
toms are transmitted through seed at the highest percentage. These cryptic viruses
are not transmitted in the ordinary way and the virus particles are present in very
low concentrations, and hence escape casual identification (Boccardo et al. 1983).
Throughout the world the majority of the germplasm sources of the cultivars of
some crops like peas, peanuts, beans, cowpea and soybean, are generally con-
taminated with seed-borne viruses at some of the germplasm banks of international
institutions (Hampton et al. 1982; Alconero and Hoch 1989) and are affecting the
crop yields. To combat this type of situation, IBPGR and other organizations have
developed suitable quick diagnostic techniques and measures to increase healthy
seed lots to maintain healthy germplasm.
Table 4.2 Percent transmission of viruses and viroids through the seeds of different plants
Virus/viroid Host Per-cent Reference
Alfalfa mosaic virus Capsicum annuum 1–5 Sutic (1959)
Lathyrus sativus 0.9–4 Latham and Jones (2001b)
Lupinus angustifolius 0.8 Jones et al. (2008)
4.1 Introduction

Medicago polymorpha 80–100 Jones and Nicholas (1992)

Medicago sativa 6 Belli (1962)
M. sativa 55 Zschau and Janke (1962)
M. sativa 1–4 Frosheiser (1964); Jones (2004)
M. sativa 0.2–6 Frosheiser (1970)
M. sativa 0.6–17 Beczner and Manninger (1975); Tosic and Pesic (1975); Hemmati and
McLean (1977)
M. sativa 4 Ekbote and Mali (1978)
M. sativa 10.6 Pesic and Hiruki (1986)
M. sativa 0.3–74 Jones and Pathipanawat (1989); Pathipanawat et al. (1995, 1997);
Jones (2004)
M. sativa 3.5–6 Avgelis and Katis (1989)
Phaseolus vulgaris 0.7–4.9 Kaiser and Hannan (1983)
Vicia sativa 0.04–0.7 Latham and Jones (2001b); Latham et al. (2004)
Arabis mosaic virus Beta vulgaris 13 Lister and Murant (1967)
Glycine max 6.3 Lister (1960)
Lactuca sativa 60–100 Walkey (1967)
Lycopersicon esculentum 1.8 Lister and Murant (1967)
Artichoke yellow ring spot virus Vicia faba 9–33 Avgelis et al. (1992)
Asparagus bean mosaic virus Vigna sesquipedalis 35 Snyder (1942)
Avocado sun-blotch viroid Persea americana 76 Wallace and Drake (1962)
P. americana 86–100 Thomas and Mohamed (1979)
Azuki bean mosaic virus Vigna angularis – Tsuchizaki et al. (1978); Hampton et al. (1978)
(Strain of BCMV)
(continued)
171
Table 4.2 (continued)
172

Virus/viroid Host Per-cent Reference

Barley mottle mosaic virus Hordeum vulgare 2–45 Dhanraj and Raychaudhuri (1969)
Barley stripe mosaic Avena fatua 22 Chiko (1975)
(Syn. Barley false stripe) A. sativa 0–9.5 Mckinney and Greely (1965)
Bromus inermis 8 Inouye (1962)
Commelina communis 4 Inouye (1962)
Hordeum depressum 3 Inouye (1962)
H. glaucum 2 Inouye (1962)
H. glaucum 58 McKinney (1951)
H. glaucum Up to 90 McKinney (1953)
H. glaucum 50–100 Gold et al. (1954)
H. glaucum 4–64 Eslick and Afanasiev (1955)
H. glaucum 38–86 Inouye (1962)
H. glaucum 3–53 Mckinney and Greely (1965)
H. vulgare 55–75 Phathak and Summanwar (1967)
H. vulgare 38 Catherall (1972)
H. vulgare 5-60 Phatak (1974)
H. vulgare 38–45 Slack et al. (1975)
H. vulgare 61–70 Carroll and Mayhew (1976a, b)
H. vulgare 52 Lange et al. (1983)
H. vulgare 45 Makkouk et al. (1992)
Lolium spp 3–8 Inouye (1962)
Triticum aestivum 71 Hagborg (1954)
T. aestivum 6.7–80 McNeal and Afanasiev (1955)
T. aestivum 70 Lange et al. (1983)
(continued)
4 Transmission of Plant Viruses and Viroids
Table 4.2 (continued)
Virus/viroid Host Per-cent Reference
Bean common mosaic (Syn. Bean western Arachis hypogaea 2–16 Choi et al. (2006b)
mosaic) Cyamopsis tetragonoloba 94 Gillaspie et al. (1998b)
Lupinus luteus 16 Frencel and Pospieszny (1979)
4.1 Introduction

Macroptilium lathyroides 5–33 Kaiser and Mossahebi (1974); Provvidenti and Braverman (1976)
Phaseolus vulgaris 1–18 Puttaraju et al. (1999)
Phaseolus acutifolius var. 7–34 Lockhart and Fischer (1974)
latifolius
P. acutifolius var. latifolius 7–20 Provvidenti and Cobb (1975)
P. angustifolius 1.0 Klein et al. (1988)
P. vulgaris 50 Reddick and Stewart (1919)
P. vulgaris 43 Archibald (1921)
P. vulgaris 10–25 Kendrick and Gardner (1924)
P. vulgaris 50 Burkholder and Muller (1926)
P. vulgaris 21–51 Merkel (1929)
P. vulgaris 39 Nalini et al. (2006)
P. vulgaris 10–30 Fazardo (1930); Nalini et al. (2004)
P. vulgaris 20–60 Harrison, (1935)
P. vulgaris 2–66 Smith and Hewitt (1938)
P. vulgaris 10–86 Medina and Grogan (1961)
P. vulgaris 2–3 Skotland and Burke (1961)
P. vulgaris 5–33 Ordosgoitty (1972)
P. vulgaris 7–20 Phatak (1974)
P. vulgaris 20–80 Drijfhout and Bos (1977); Muniyappa (1976)
P. vulgaris 33.5 Meiners et al. (1978)
P. vulgaris 12–66 Capoor et al. (1986)
P. vulgaris 93 Edwardson and Christie (1986)
(continued)
173
Table 4.2 (continued)
174

Virus/viroid Host Per-cent Reference

P. vulgaris 39.7–54.4 Morales and Castano (1987)
P. vulgaris 12.4 Njau and Lyimo (2000)
Vigna mungo 67 Dinesh Chand et al. (2007)
Vigna mungo 2–10 Agarwal et al. (1979)
Vigna radiata 78 Dinesh Chand et al. (2007)
Vigna radiata 25 Kaiser et al. (1968)
Vigna radiata 4.1–7.2 Tsuchizaki et al. (1986)
Vigna radiata 8–32 Kaiser and Mossahebi (1974)
Vigna radiata 1–4.9 Choi et al. (2006)
Vigna sesquipedalis 37 Snyder (1942)
V. sinensis 25–40 Sachchidananda et al. (1973)
V. mungo 20–48 Provvidenti (1986)
Bean common mosaic necrosis virus Phaseolus vulgaris 36.6 Njau and Lyimo (2000)
Bean pod mottle virus Glycine max 0.1 Lin and Hill (1983)
Bean red node virus Phaseolus vulgaris 27 Thomas and Graham (1951)
Bean southern mosaic (Syn. Southern bean Phaseolus vulgaris 1–30 Crowley (1959); Smith (1972); Jayasinghe (1982); Morales and
mosaic virus) Castano (1985)
Vigna sinensis 1–40 Shepherd and Fulton (1962); Givord (1981); O’Hair et al. (1981)
V. unguiculata 1–3 Shepherd and Fulton (1962); Lamptey and Hamilton (1974)
Bean western mosaic virus Phaseolus vulgaris 2–3 Scotland and Burke (1961)
Bean yellow mosaic virus L. luteus 5–6.2 Mastenbroek (1942); Corbett (1958); Zschau (1962)
L. luteus 7–21 Porembskaya (1964)
Melilotus alba 3–5 Phatak (1974)
Phaseolus vulgaris 7 Crowley (1957)
Pisum sativum 10–30 Inouye (1967)
Trifolium pratense 12–15 Hampton (1967)
Vicia faba Low Quantz (1954); Bos (1970)
4 Transmission of Plant Viruses and Viroids

(continued)
Table 4.2 (continued)
Virus/viroid Host Per-cent Reference
V. faba 0.1–2.4 Kaiser (1972, 1973); Evans (1973)
V. faba 0.1–0.2 Fiedorow (1980)
V. faba 1.8 Eppler and Kheder (1988)
4.1 Introduction

V. faba 2.6 Aftab et al. (1989)

V. faba 9.8 El-Dougdoug et al. (1999)
Vigna sinensis 1–37 Snyder (1942)
Beet curly top virus Beta vulgaris 11–25 Abdel salam and Amin (1990)
Beet temperate virus Beta vulgaris 100 Natsuaki et al. (1983b)
Blackgram mild mottle virus Vigna mungo 4.9–14.9 Krishnareddy (1989)
Blackgram mottle virus Vigna radiata 8 Phatak (1974, 1983); Scott and Phatak (1979)
Vigna radiata 1–1.5 Saleh et al. (1986)
Vigna mungo 5.3–16.70 Krishnareddy (1989)
Vigna mungo 1.3–15.9 Varma et al. (1992)
Vigna mungo 5–10 Dinesh Chand et al. (2004)
Blackeye cowpea mosaic virus Vigna sinensis 30.9 Anderson (1957); Zettler and Evans (1972)
Vigna sinensis 1.8 Lin et al. (1981); Tsuchizaki et al. (1984)
Vigna mungo 14 Provvidenti (1986)
Vigna sinensis 1.2–30.9 Edwardson and Christic (1986)
Vigna sinensis 2.3–38.4 Sumana and Keshava Murthy (1992)
Vigna unguiculata 6–41.6 Pio-Ribeiro et al. (1978); Mali and Kulthe (1980)
Mali et al. (1987, 1988, 1989)
Bashir and Hampton (1996); Puttaraju et al. (2004)
Broad bean true mosaic virus Vicia faba 0–28 Brunt (1970); Neergaard (1977), Mali et al. (2003)
Broad bean mottle virus Cicer arietinum – Erdiller and Akbas (1996)
Phaseolus vulgaris 7 Phatak (1974)
Vicia faba 1.37 Makkouk et al. (1988)
(continued)
175
Table 4.2 (continued)
176

Virus/viroid Host Per-cent Reference

Broad bean wilt viruses Vicia faba 0.6 Putz and Kuszala (1973)
Makkouk et al. (1990)
Brome mosaic virus Triticum aestivum [50 Von Wechmor et al. (1984)
Cocoa swollen shoot virus Theobroma cocao 34–54 Quainoo et al. (2008)
Clover yellow mosaic virus T. pratense 7.6 Hampton (1963)
Cowpea aphid-borne mosaic virus Arachis hypogaea 0.15 Gillaspie et al. (2001)
Vigna sinensis 23 Capoor and Varma, (1956)
Vigna sinensis 0.3–1.6 Lovisolo and Conti (1966)
V. sinensis 5–16 Chenulu et al. (1968)
V. sinensis 35 Phatak (1974)
V. unguiculata 27 Snyder (1942)
V. unguiculata 0–73 Mazyad et al. (1984)
V. unguiculata 0–30 Kaiser et al. (1968); Phatak (1974)
V. unguiculata 0–2 Bock (1973a)
V. unguiculata 3–19 Phatak (1974)
V. unguiculata 1.1–39.8 Kaiser and Mossahebi (1975); Ndiaye et al. (1993)
V. unguiculata Upto 20.9 Ladipo (1977)
V. unguiculata 6.3–18.3 Ata et al. (1982); Bashir and Hampton (1996)
V. unguiculata 4.7 Chang and Kno (1983)
V. unguiculata 5.0–20 Mali et al. (1987, 1988 and 1989)
V. unguiculata 5.9 Pio-Ribeiro et al. (2000)
V. unguiculata 0.67–13.49 Udayashankar et al. (2009)
(continued)
4 Transmission of Plant Viruses and Viroids
Table 4.2 (continued)
Virus/viroid Host Per-cent Reference
Cowpea mild mottle virus Glycine max 90 Brunt and Kenten (1973)
G. max 0.9 Iwaki et al. (1982)
G. max 0.5 Thouvenel et al. (1982)
4.1 Introduction

Phaseolus vulgaris 6 Brunt and Kenten (1973)

V. unguiculata 90 Brunt and Kenten (1973)
Cowpea mosaic virus Vigna catjang 17 Capoor and Varma (1956)
V. sinensis 17.5 Diwakar and Mali (1977)
V. sinensis 23 Capoor and Varma (1956)
V. sinensis 0–55 Anderson (1957)
V. sinensis 1–5 Gilmer et al. (1974)
Vigna unguiculata 75–84 Mahalakshmi et al. (2008)
Cowpea mottle virus V. unguiculata 3–10.3 Shoyinka et al. (1978)
V. unguiculata 0.4 Allen et al. (1982)
Voandzeia subterranea 2 Robertson (1966); Bird and Corbett (1988)
Cowpea ringspot virus V. unguiculata 15–20 Phatak et al. (1976)
V. unguiculata 10–30 Phatak (1974)
Cowpea severe mosaic virus Vigna sesquipedalis 8 Dale (1949)
Vigna sinensis 3.3–5.8 Haque and Persad (1975)
Vigna unguiculata 10 Shepherd (1964); Vide (1996)
Cowpea severe mottle virus Vigna sinensis 0.7 Dos (1987)
Cucumber green mottle mosaic virus Citrullus vulgaris 5 Komuro et al. (1971)
Cucumis sativus 44 Yakovleva (1965)
C. sativus 4.2 Kawai et al. (1985)
(continued)
177
Table 4.2 (continued)
178

Virus/viroid Host Per-cent Reference

Cucumber mosaic virus Arachis hypogaea 1.3 Xu and Barnett (1984)
Cucumis melo 2.1 Kendrick (1934)
C. melo 16 Mahoney (1935)
C. melo 11.37–23.07 Sandhu and Kang (2007)
C. sativus 1.4 Doolittle (1920)
Cucurbita moschata 0.7 Sharma and Chohan (1974)
C. pepo 0.07 Reddy and Nariani (1963); Sharma and Chohan (1974)
Echinocystis lobata 9.1 Doolittle and Gilbert (1919)
E. lobata 55 Doolittle and Walker (1925)
E. lobata 15 Lindberg et al. (1956)
Lamium purpureum 4 Tomlinson and Carter (1970)
Lupinus angustifolius 12–18 Alberts et al. (1985); Jones (1988)
L. luteus 14 Porembskaya (1964)
Medicago sativa 0.1–0.3 Jones (2004)
Phaseolus vulgaris 41 Bos and Maat (1974)
P. vulgaris 30 Marchoux et al. (1977)
P. vulgaris 33.5 Meiners et al. (1978)
P. vulgaris 0–49 Davis and Hampton (1986)
P. vulgaris 30–100 Bhattiprolu (1991)
Spergula arvensis 2 Tomlinson and Carter (1970)
Stellaria media 1–30 Hani (1971); Hani et al. (1970)
S. media 5–8 Tomlinson and Carter (1970)
S. media 3-40 Tomlinson and Carter (1970)
S. media 1–4 Tomlinson and Walker (1973)
Spinacea oleracea 15 Yang et al. (1997)
Trifolium subterraneum 8.8 Jones and McKirdy, (1990); Jones (1991)
Vigna radiata 8–32 Kaiser et al. (1968); Kaiser and Mossahebi (1974)
4 Transmission of Plant Viruses and Viroids

(continued)
Table 4.2 (continued)
Virus/viroid Host Per-cent Reference
V. radiata 5 Phatak (1974)
V. radiata 11 Iwaki (1978)
V. sesquipedalis 4–28 Anderson (1957)
4.1 Introduction

V. sinesis 30 Meiners et al. (1977)

V. sinesis 10 Iwaki (1978)
V. unguiculata 4–28 Anderson (1957)
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 (1996)
V. unguiculata 1.3–25.8 Mali et al. (1989)
V. unguiculata 1.5–37 Gillaspie et al. (1998a)
V. unguiculata 10–30 Abdullahi et al. (2001)
Guar symptomless virus Cyamompsis tetragonoloba 12–28 Hansen and Leseman (1978); Behncken (1983)
High plains virus Zea mays Low Forster et al. (2001)
Lettuce mosaic virus Lactuca sativa 3.1 Newhall (1923)
L. sativa 10 Ogilvie et al. (1935)
L. sativa 2–8 Ainsworth and Ogilvie (1939)
L. sativa 6–15 Kramer et al. (1945)
L. sativa 1–8 Grogan and Bardin (1950); Grogan et al. (1952)
L. sativa 3–10 Couch (1955)
L. sativa 11 Herold (1956)
L. sativa 13 Rohloff (1962)
L. sativa 5 Ryder (1964)
L. scariola 0.2–6.2 van Hoof (1959)
Senecio vulgaris 2.3 Phatak (1974)
(continued)
179
Table 4.2 (continued)
180

Virus/viroid Host Per-cent Reference

Lettuce yellow mosaic virus L. sativa 30 Mandahar (1978)
Lima bean mosaic virus Phaseolus limensis 25 Mandahar (1978)
P. limensis 2.2 Sawant and capoor (1983)
P. lunatus 0.3 Gay (1972)
Lucerne Australian latent virus Medicago sativa 8 Taylor and Smith (1971); Black Stock (1978); Jones et al. (1979)
Lucerne transient streak virus Melilotus albus 2.5 Paliwal (1983)
Maize dwarf mosaic virus Zea mays 0.02–1.65 Williams et al. (1968); Hill et al. (1974); Tosic and Sutic (1977)
Melon rugose mosaic virus Cucumis melo var. 3.8 Mahgoub et al. (1997)
flexaosus
Melon necrotic spot virus Cucumis melo 20–22.5 Kishi (1966); Gonzalez-Garza et al. (1979); Avgelis (1985); Campbell
et al. (1996)
Mulberry ring spot virus Glycine max 10 Tsuchizaki et al. (1971)
Muskmelon mosaic virus Cucumis melo 12–93 Rader et al. (1947)
C. melo 22.5 Avgelis (1985)
Nicotiana velutina mosaic virus Nicotiana glutinosa \72 Randles et al. (1976)
Olive latent virus—1 Olive 35–82 Saponari et al. (2002)
Onion yellow dwarf virus A. cepa 6–29 Hardtl (1964, 1972)
Papaya ring spot virus Crica papaya 0.15 Bayot et al. (1990)
Pea early browning virus Pisum sativum 37 Bos and van der Want (1962)
P. sativum 1–2 Harrison (1973)
P. sativum 61 Fiedorow (1983)
Vicia faba 5 Cockbain et al. (1983)
V. faba 0.3–8 Fiedorow (1983)
Pea enation mosaic virus P. sativum 1.5 Blattny (1956)
P. sativum 4–5 Kheder and Eppler (1988)
Pea false leaf roll virus P. sativum 40 Thottappilly and Schmutterer (1968)
(continued)
4 Transmission of Plant Viruses and Viroids
Table 4.2 (continued)
Virus/viroid Host Per-cent Reference
Pea mild mosaic virus P. sativum 15 Clark (1972)
Pea mosaic (Syn.bean yellow mosaic virus) Trifolium hybridum 0.7 Mandahar, (1978)
T. pratense 47 Mandahar (1978)
4.1 Introduction

Pea seed-borne mosaic (syn. pea fizzle top Lathyrus clymenum 5 Latham and Jones (2001a)
and Pea leaf rolling virus) Lens culinaris 0.8 Al-Mabrouk and Mansour (1998); Erdiller and Akbas (1996)
Lens culinaris 32–44 Hampton and Muehlbauer (1977); Eppler et al. (1988)
L. culinaris 0.5–5 Goodell and Hampton (1984); Hampton (1982); Bayaa et al. (1998)
L. culinaris 6 Coutts et al. (2008)
Pisum arvense 9 Zimmer and Ali-Khan (1976)
Pisum sativum 8–30 Inouye (1967)
P. sativum 0–88 Stevenson and Hagedorn (1969, 1973)
P. sativum 20–80 Hampton (1969)
P. sativum 65–90 Mink et al. (1969); Alconero and Hock (1989); Cockbain (1988)
P. sativum 2–55 Musil (1970)
P. sativum 4–32 Hampton (1972)
P. sativum 0.5–5.8 Chiko and Zimmer (1978)
P. sativum 30–60 Thakur et al. (1985); Rishi and Singh (19870
P. sativum 23–24 Kheder and Eppler (1988)
P. sativum 10 Kumar et al. (1991)
P. sativum 58 Mc Keown and Biddle (1991)
P. sativum 10 Zimmer and Lamb (1993); Sontakke and Chavan (2007)
P. sativum 1–18 Latham and Jones (2001b); Deepthi Anand et al. (2006)
P. sativum 1.9–32.7 Gallo and Jurik 1995
P. sativum 5–30 Coutts et al. (2008)
Vicia spp 0.11–3 Hampton and Mink (1975); Musil (1980); Boulton et al. (1996)
Vicia faba 0.2–2 Latham and Jones (2001b); Coutts et al. (2008)
(continued)
181
Table 4.2 (continued)
182

Virus/viroid Host Per-cent Reference

Pea streak virus Pisum sativum 1.7 Kheder and Eppler (1988)
Peanut clump virus (African) A. hypogaea 24–48 Thouvenel et al. (1978)
Eleusine coracana 5.2 Dieryck et al. (2009)
Pennisetum glaucum 0.9 Dieryck et al. (2009)
Peanut clump virus (Indian) A. hypogaea 3.5–17 Reddy et al. (1998)
A. hypogaea 9–11 Reddy et al. (1988 and 1989)
Eleusine coracana 5.2–6.5 Reddy et al. (1989, 1998)
Pennisetum glauca 0.93 Reddy et al. (1989, 1998)
Setaria italica 9.7–10.2 Reddy et al. (1989, 1998)
Triticum aestivium 0.5–1.3 Delfosse et al. (1999)
Peanut mottle virus A. hypogaea 0.02–2 Kuhn (1965); Demski et al. (1983)
A. hypogaea 20 Bock (1973b)
A. hypogaea 3.7 Paguio and Kuhn (1974)
A. hypogaea 0–8.5 Adams and Kuhn (1977)
A. hypogaea 1.3 Bharatan et al. (1984); Iizuka and Reddy (1986)
A. hypogaea 1–7 Puttaraju et al. (2001)
Glycine max 0.22 Iwaki et al. (1986)
Lupinus albus (white lupin) 0.37 Demski et al. (1983)
Phaseolus vulgaris 1.0 Demski et al. (1983)
Vigna unguiculata 0.8 Demski et al. (1983)
Voandzeia subterranea 10.5 Li et al. (1991)
Peanut stunt virus A. hypogaea 3–4 Iizuka and Yunoki (1974)
Glycine max 0.2 Troutman et al. (1967); Kuhn (1969)
(continued)
4 Transmission of Plant Viruses and Viroids
Table 4.2 (continued)
Virus/viroid Host Per-cent Reference
Peanut stripe virus (Syn. peanut mild A.hypogaea 5–20 Xu et al. (1991)
mottle virus) A. hypogaea 1.3–4.8 Xu et al. (1983)
A. hypogaea 19.3–37.6 Demski et al. (1984)
4.1 Introduction

A. hypogaea 28.8 Prasada Rao et al. (1988)

A. hypogaea 43 Ohki et al. (1989)
A. hypogaea 12.5 Chang et al. (1990)
A. hypogaea 60.0 Matsumoto et al. (1991)
Glycine max 28.0 Warwick and Demski (1988)
Glycine max 2–3 Green and Lee (1989); Vetten et al. (1992)
Pelargonium zonate spot virus Lycopersicon esculentum 19 Lapidot et al. (2010)
Pepino mosaic virus Lycopersicon esculentum 1.84 Cordoba-Selles et al. (2007)
Pepper chat fruit viroid Capsicum annuum 19 Verhoeven et al. (2009)
Potato spindle tuber viroid Lycopersicon esculentum 2–11 Singh (1970); Kryczynski et al. (1988)
Physalis peruviana 29 McClean (1948)
Scopolia sinensis 71 Singh and Finnie (1973)
Solanum incanum 53 McClean (1948)
S. tuberosum 6–66 Singh (1970); Singh et al. (1992)
S. tuberosum 87–100 Hunter et al. (1969); Fernow et al. (1970); Grasmick and Slack (1986)
Potato virus T Solanum demissum-A 39 Salazar and Harrison (1978)
Solanum tuberosum cv. 33–59 Jones (1982)
Cara
Potato virus X Solanum tuberosum 0.6–2.3 Darozhkin and Chykava (1974)
S. tuberosum 14–16 Mandahar (1978)
Radish yellow edge virus Raphanus sativus 80–100 Natsuaki et al. (1979; 1983a)
Safflower mosaic virus Carthamus tinctorius 2.2–5.0 Chauhan and Singh (1979)
Soybean mild mosaic virus Glycine max 22–70 Takahashi et al. (1974 and 1980)
(continued)
183
Table 4.2 (continued)
184

Virus/viroid Host Per-cent Reference

Soybean mosaic virus Glycine max 0–68 Kendrick and Gardner (1924)
G. max 40 Heinze and Kohler (1941)
G. max 1–18 Ross (1963)
G. max 1–24 Kennedy and Cooper (1967)
G. max 34 Iizuka (1973)
G. max 55.9 Phatak (1974)
G. max 10.6–29.1 Suteri (1981)
G. max 20.5–29.5 Kim and Lee (1986)
G. max 64 Edwardson and Christie (1986)
G. max 10 Nakano et al. (1988)
G. max 11.2–41.1 Tu (1989)
G. max 25.7–91.7 Pacumbaba (1995)
G. max 43 Domier et al. (2007)
G. max 32.9 Patil and Byadgi (2005)
G. max 5.3 Golnaraghi et al. (2004)
G. soja 10–25 Mandahar (1978)
Lupinus albus 1.2 Vroon et al. (1988)
Phaseolus vulgaris 1.6 Castano and Morales (1983)
Soybean streak virus Glycine max 95 Iizuka (1973)
Soybean stunt virus G. max 50 Koshimizu and Iizuka (1963)
G. max [70 Honda et al. (1988)
G. max 95 Iizuka (1973)
Vigna unguiculata 5 Iizuka (1973)
(continued)
4 Transmission of Plant Viruses and Viroids
Table 4.2 (continued)
Virus/viroid Host Per-cent Reference
Spinach latent virus Celosia cristata 53 Bos et al. (1980)
Chenopodium quinoa 90 Bos et al. (1980)
C. quinoa 60 Stefanac and Wrischer (1983)
4.1 Introduction

Nicotiana clevelandii 90 Stefanac and Wrischer (1983)

N. megalosiphon 95 Stefanac and Wrischer (1983)
Nicotiana rustica 30 Bos et al. (1980)
N. tabacum white Burley 90 Bos et al. (1980)
N. tabacum xanthi 94 Bos et al. (1980)
Spinacea oleracea 50 Bos et al. (1980)
S. olaracea 56 Stefanac and Wrischer (1983)
Squash mosaic virus Citrullus vulgaris 1.5 Nelson and Knuhtsen (1969)
Cucumis melo 1–27 Mahoney 1935
C. melo 12–93 Rader et al. (1947); Mukhayyish and Makkouk (1983)
C. melo 7–21 Grogan et al. (1959)
C. melo 9–10 Kemp et al. (1972)
C. melo 3 Nelson and Knuhtsen (1973)
C. melo 4 Phatak (1974)
C. melo 0–34.6 Alvarez and Campbell (1978)
C. melo 30 Lange et al. (1983)
C. maxima 0.2–1.5 Grogan et al. (1959)
Cucumis melo 6.6–20 Grogan et al. (1959)
C. mixta 0.3 Grogan et al. (1959)
C. pepo 2.2 Middleton (1944)
C. pepo 5 Grogan et al. (1959); Nelson and Knuhtsen (1973)
Chenopodium murale 23 Lockhart et al. (1985)
C. quinoa 20 Lockhart et al. (1985)
(continued)
185
Table 4.2 (continued)
186

Virus/viroid Host Per-cent Reference

Subterranean clover mottle virus Trifolium subterraneum 0.5–3 Francki et al. (1988); Njeru et al. (1997)
Sugarcane mosaic virus Zea mays 0.1–0.4 Shepherd and Holdeman (1965); Williams et al. (1968); Baudin
(1969); Von Wechmar et al. (1984); Mikel et al. (1984)
Zea mays 4.81 Li et al. (2007)
Sunflower rugose mosaic virus Helianthus annuus 5.6 Singh (1979)
Sunn-hemp mosaic virus Vigna unguiculata 2.5–17.5 Mali et al. (1989)
Sunn-hemp rosette virus Crotalaria juncea 10–20 Verma and Awasthi (1978)
Tobacco mosaic virus Arabidopsis thaliana High de Assis Filho and Sherwood (2000)
Capsicum annum 45 Glaeser (1976); Demski (1977, 1981)
Capsicum annuum 13.5–29.6 Tosic et al. (1980); Chitra et al. (2002) and (1999a, b)
Capsicum frutescens 22 McKinney (1952)
Capsicum frutescens 18.4 Cicek and Yorganci (1991)
Lycopersicon esculentum 2-6 Doolittle and Beecher (1937); Taylor et al. (1961); Chitra et al. (2002)
and (1999a, b)
Lycopersicon esculentum 98.1 Cicek and Yorganci (1991)
Malus platycarpa 38 Gilmer and Wilks (1967)
Malus pumila 21 Allen (1969)
M. sylvestris 3–37 Gilmer and Wilks (1967)
Pyrus communis 35 Gilmer and Wilks (1967)
Vigna unguiculata 1–4 Phatak (1974)
V. unguiculata 14.6–22.6 Mali et al. (1987)
Vitis vinifera 20 Gilmer and Kelts (1968)
(continued)
4 Transmission of Plant Viruses and Viroids
Table 4.2 (continued)
Virus/viroid Host Per-cent Reference
Tobacco ring spot virus Cucumis melo 3–7 McLean (1962)
Gladiolus sp. 4 Sushak (1976)
Glycine max 54–78 Desjardins et al., (1954)
4.1 Introduction

G. max 78–82 Kahn (1956)

G. max 100 Athow and Bancroft (1959)
G. max 40 Kahn et al. (1962)
G. max 100 Owusu et al. (1968)
G. max 100 Iizuka (1973)
G. max 94–97 Yang and Hamilton (1974); Hamilton (1985)
G. max 2.1 Golnaraghi et al. (2004)
Lactuca sativa 3 Grogan and Schnathorst (1955)
L. sativa 21 Iizuka (1973)
N. tabacum 4.9–17 Valleau (1941)
Phaseolus aureus 68–91 Shivanathan (1977)
Solanum melongena 3.2–9.8 Sastry and Nayudu (1976)
Solanum tuberosum 2–9 Jones (1982)
Vigna sinensis 82 Kahn, (1956)
Zinnia elegans 5 Iizuka (1973)
Tobacco streak virus Glycine max 2.6–30 Ghanekar and Schwenk (1974)
G. max 90 Kaiser et al. (1982)
G. max 30–80 Truol et al. (1987)
G. max 2.3 Golnaraghi et al. (2004)
Lycopersicon esculentum 40–76 Shoodee and Teakle (1988)
Parthenium hysterophorus 6.8–48 Sharman et al. (2009)
Phaseolus vulgaris 1–26 Thomas and Graham (1951); Kaiser et al. (1991)
(continued)
187
Table 4.2 (continued)
188

Virus/viroid Host Per-cent Reference

Tomato apical stunt viroid Lycopersicon esculentum 80 Antignus et al. (2007)
Tomato aspermy virus Phaseolus vulgaris 18.7 Wang (1982)
Tomato black ring virus Beta vulgaris 3–27 Gibbs and Harrison (1964)
B. vulgaris 56 Lister and Murant (1967)
Capsella bursapastoris 90 Lister and Murant (1967)
Cerastium vulgatum 33–100 Lister and Murant (1967)
Fragaria x ananassa 40 Lister (1960)
Fumaria officinalis 100 Lister and Murant (1967)
Glycine max 83 Lister (1960)
Lactuca sativa 3 Morand and Poutier (1978)
Lamium amplexicaule 10–48 Lister and Murant (1967)
Lingustrum vulgare 5.7–8.3 Lister and Murant (1967)
Lycopersicon esculentum 19 Lister and Murant (1967)
Myosotis arvensis 100 Lister and Murant (1967)
N. rustica 4.4–8.8 Lister and Murant (1967)
N. tabacum 6 Hanada and Harrison (1977)
Petunia violacea 29.1 Phatak (1974)
Poa annua 2.7 Lister and Murant (1967)
Vigna sinensis 23 Lister (1960)
Tomato bushy stunt virus Lycopersicon esculentum 50–65 Tomlinson and Faithfull (1984)
Prunus avium High Allen and Davidson (1967)
Tomato ringspot virus Glycine max 76–80 Kahn (1956); Lister and Murant (1967)
G. max 1.4 Golnaraghi et al. (2004)
Lycopersicon esculentum 3 Hollings et al. (1972)
Nicotiana tabacum 11 Hollings et al. (1972)
Trifolium pratense 3–7 Hampton (1967)
(continued)
4 Transmission of Plant Viruses and Viroids
Table 4.2 (continued)
Virus/viroid Host Per-cent Reference
Tomato streak virus Lycopersicon esculentum 66 Berkeley and Madden (1932)
Turnip mosaic virus Raphanus raphanistrum 4 Tomlinson and Walker (1973)
Turnip yellow mosaic virus Arabidopsis thaliana 72.6 de Assis Filho and Sherwood (2000)
4.1 Introduction

Brassica chinensis var 9.5 Benetti and Kaswalder, (1982/1983)

Parrachinensis
Urd bean leaf crinkle virus (syn. Black Vigna mungo 18.3 Kolte and Nene (1972); Beniwal and Chaubey (1984)
gram leaf crinkle virus Syn. bean urd V. mungo 20.3–41.8 Narayanaswamy and Jaganathan (1975); Patel et al. (1999); Mahajan
leaf crinkle virus) and Joi (1999); Prasad et al. (1998)
V. mungo 17.6 Dubey and Sharma (1985)
V. mungo 8.6 Ravinder Reddy and Jeyarajan (1989); Ravinder Reddy et al. (2005)
V. mungo 1–83 Pushpalatha et al. (1999)
V. mungo 2.2–28.7 Sharma et al. (2007)
Vigna unguiculata 6–15 Beniwal et al. (1980)
Watermelon mosaic virus Echinocystis lobata 2 Lindberg et al. (1956)
Wheat mosaic virus Triticum sp. 0.01 Panarin and Zabavina (1978)
Zea mays 0.03 Panarin and Zabavina (1978)
Wheat soil borne mosaic virus Secale cereale 3 Jezewska (1995)
Wheat streak mosaic virus Zea mays 0.01–0.1 Hill et al. (1974); Panarin and Zabavina (1978); Jones et al. (2005)
Z. mays 0.2–1.5 Jones et al. (2005)
Zucchini yellow mosaic virus Cucurbita pepo 1.4–1.6 Schrijnwerkers et al. (1991); Riedle–Bauer et al. (2002); Tobias et al.
(2008); Simmons et al. (2011)
Source Sastry (2013)
189
190 4 Transmission of Plant Viruses and Viroids

4.1.3 Transmission Through Pollen

Infected pollen plays an integral role in the spread of viruses of some woody and
herbaceous plants. This topic has been reviewed by number of workers viz.
(Shepherd 1972; Hardtl 1978; Mandahar 1981, 1985; Mink 1993 and Sastry 2013).
Plant virus transmission through pollen takes place both vertically and horizon-
tally. In case of vertical transmission of viruses, the pollination and fertilization of
healthy ovules by infected pollen results 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 economi-
cally important in cross pollinated woody perennial plants than with annual crops
wherein both vertical and horizontal transmission takes place. In sunflower,
Tobacco streak virus is not seed-borne, but primary spread takes place under field
conditions through the virus infected pollen entering through the wounds caused
by the thrips vector feeding (Prasada Rao et al. 2003; Shukla et al. 2005).
Transmission of certain seed-borne plant viruses through pollen is prevalent in
bromo, nepo, crypto, alfamo, cucumo and ilar virus groups. Virus particles are
externally or internally pollen borne and have been observed in electron micro-
graphs 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). Raspberry bushy dwarf idaeovirus and Blue
berry shock ilarvirus in raspberry and blue berry, respectively are pollen trans-
mitted and geographical distribution for both viruses is Pacific North West (Mink
1983a, b; Bristow and Martin 1999). AMV was also effectively transmitted
through pollen upto 26.5% (Frosheiser 1974). 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 fertilization 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 size, morphology, viability and pollen tube size have been recorded (Yang and
Hamilton 1974). High level of pollen sterility resulting in poor fertilization has
been observed in certain virus-host combinations (Ryder 1964). Even some of the
plant viroid diseases which are seed transmitted are also pollen transmitted as in
case of Potato spindle tuber viroid (PSTVd) in potato and tomato (Fernow et al.
1970; Kryczynski et al. 1988); Chrysanthemum stunt viroid, Cucumber pale fruit
viroid in tomato (Kryczynski et al. 1988) and Peach latent mosaic viroid in peach
(Barba et al. 2007).
Pollen carrying insects primarily honey bees play a major role in transfer of
virus infected pollen to the stigma of flowers. Mink (1983b) indicated the possible
role of honey bees for long distance spread if PNRSV from California to sweet
4.1 Introduction 191

cherry orchards in Washington, USA. Antignus et al. (2007) have observed the
secondary spread of Tomato apical stunt viroid in green house tomatoes takes
place through bumble bees (Bombus Terrastris) which cause wounding of flowers
during their visits, in addition the spread also takes place through workers infested
hands and tools.
In India, the intensive studies carried out by Shukla et al. (2005) and Prasada
rao et al. (2003) have indicated that Tobacco streak virus (TSV) in sunflower,
certain legumes and other crops spreads through virus infected pollen dispersed
through wind currents and also on the body parts of the insects. However, TSV
infection is specifically associated with the thrips (Thysanoptera: Thripidae)
feeding and damage on the leaves and presence of TSV infected pollen at the
feeding sites are responsible factors for virus spread.
Generally, a greater percentage of plant 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 were used to pollinate healthy plants. But
the studies of Vemana and Jain (2010) have revealed that TSV is not seed
transmitted in number of leguminous hosts. Vertesy (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 plants com-
bined 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 earlier by Medina and Grogan (1961)
while working with BCMV infection in French beans. More information on pollen
transmission is furnished in the first Chapter of Volume-II (Sastry and Zitter 2013).

4.1.4 Transmission through Contact and Mechanical

The term mechanical transmission has often been used for transmission by contact
between plant parts, but it seems more appropriate for transmissions in which the
infective virus is a passive contaminant of anything that may come in contact with
a healthy plant. The confusion in using these terms may have arisen because
mechanically transmitted viruses are also transmitted by contact between plant
parts. Generally mechanical transmission is referred when inoculated to the test
plants after extraction of the virus by using pestle or hand or pinprick method.
Field workers can spread viruses mechanically while handling plants without
taking hygienic precautions or by accidentally rubbing plants with contaminated
clothes and horticultural tools. Animals can also spread viruses in the same way
when rubbing plants as they walk in between plants. Under field conditions the
most important source of contamination, seems to be farm machinery and
implements. For instance, transmission of PSTVd was found to result from
192 4 Transmission of Plant Viruses and Viroids

contaminated tractor wheels and PVX from cultivating and hilling equipment
(Merriam and Bonde 1954; Manzer and Merriam 1961). Mechanical transmission
of viruses to tubers may result from contaminated cutting knives used to divide
seed tubers into smaller pieces for planting. PVX and PSTVd (Goss 1926) and
PVS have been transmitted in this way (Franc and Banttari 1984). Even viruses
like Andean potato latent virus and Apple mosaic virus are also spreading
mechanically under field conditions.
The viruses like TMV, ToMV, PVX and Pepino mosaic virus are highly
infectious in Solanaceous crops and spread mechanically under field conditions.
These viruses are highly contagious and are readily spread by contact, contami-
nated tools, hands or clothing, grafting and plant-to-plant contact. The virus can
remain viable and infectious on contaminated equipment and clothing for several
weeks, and can survive in crop debris for at least several months. Pepino mosaic
virus can spread by both hand pollination and by bumble bees used for pollination
in glasshouse-grown crops. This virus also spreads with seed coat contamination,
resulting in a low rate of seed transmission, which is sufficient to introduce the
virus into a new location or nursery with subsequent spread by contact. There is no
evidence that common virus vectors such as aphids and whiteflies can transmit
Pepino mosaic virus.
Some viruses and viroids are also transmitted by contaminated pruning tools.
Although Berg (1964) reported that infected pruning tools did not transmit Poplar
mosaic virus, the preponderance of the published literature indicates otherwise.
Citrus exocortis and other citrus viroids can be transmitted with contaminated tools
(Kyriakou 1992; Roistacher et al. 1969). Hadidi et al. (1997) were successful in
transferring Peach latent mosaic viroid to both lignified and green shoots of peach
plants using contaminated shears and the infection rate was up to the extent of
50–70%.
Viruses will also be spread by direct transfer of sap by contact of a wounded
plant with a healthy one. Such contact may occur during agricultural practices, as
the damage caused by tools or hands, or by an animal feeding on the plants.
Generally TMV, Potato virus X, Tomato mosaic virus and Pepino mosaic virus are
transmitted through touching of plants with contaminated hands, tools, even by
contaminated cloths brushing against plants, through wind currents in crops like
tomato, potato, tobacco, etc. In Africa, Rice yellow mottle virus transmission takes
place in rice in the nursery stage by leaf contact through air currents (Traore 2006).
From Australia, McKirdy et al. (1998, 2005) have reported that White clover
mosaic and Subterranean clover mottle viruses transmission takes place by grazing
animals in pastures and forage crops.
Some of the viruses are also transmitted through natural root grafts to adjacent
plants particularly trees. For several viruses infecting trees natural root grafts are
the only known means of tree-to-tree spread of virus within established orchards.
4.1 Introduction 193

4.1.5 Transmission Through Water

Some of the plant viruses of the carmo-, tombus-, tobamo-, potex-, and furo- virus
groups, have been found in large amounts in surface water. Researchers in dif-
ferent countries have shown that certain plant viruses like Carnation ringspot
virus, Carnationation mottle virus, Tobacco necrosis virus, Potato virus X,
Tobacco mosaic virus, Tomato mosaic virus, Tomato bushy stunt virus and
Grapevine Algerian latent virus have been isolated from rivers, lakes, brooks,
ditches, outlets of sewage plants etc. (Block 1983; Tosic and Tosic 1984; Koenig
and Leseman 1985; Koenig 1986; Plazolla et al. 1986; Natasa Mehle and Maja
Ravnikar 2012). Most of the viruses discussed here have no known aerial vectors
and have intrinsically stable virus particles which may derive additional protection
when adsorbed to particulate inorganic or organic matter or when present with
plant debris. They can apparently be transmitted to the roots of healthy plants
without the aid of a vector. There are reports of involvement of fungal vector,
Polymyxa sps. as in case of Indian peanut clump virus, Soil-borne wheat mosaic
virus etc.,
Some of the viruses like Beet necrotic yellow vein virus may become associated
with a fungal vector Polymyxa betae that provides more effective host-directed
spread and also helps in long distance virus spread. From New Delhi (India) Vani
and Varma (1993) have isolated Cucumber green mottle mosaic virus from the
water of river Jamuna and noticed high virus incidence in cucurbit crops irrigated
with Jamuna river water. Some of the viruses which are proved to be present in
surface water are Carnation mottle virus, Tomato bushy stunt virus, Tobacco
necrosis virus, Tobacco mosaic virus, and Beet necrotic yellow vein virus and their
mode of spread is established (Block 1983; Koenig 1986).

4.1.6 Transmission Through Vectors

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 car-
rying pathogenic agents. Majority of the plant viruses spread through arthropod
vectors like aphids, leafhoppers, planthoppers, whiteflies, beetles, thrips, and
mealybugs. Subsequent vector studies have revealed that certain mites, nematodes
and fungi have also proved to be vectors of some plant viruses. Most of the viruses
are actively transmitted to healthy plants within the matter of seconds, hours, or
days by vectors (Hull 2002). Hohn (2007) has reported the factors that regulates
binding vs release of the virus particles during the insect vector transmission.
Vector-virus transmission consists of several successive steps: acquisition of
virions from an infected source, stable retention of acquired virions at specific sites
through binding of virions to ligands, release of virions from the retention sites
upon salivation or regurgitation, and delivery of virions to a site of infection in a
194 4 Transmission of Plant Viruses and Viroids

Table 4.3 Vectors and the plant virus groups that they transmit
Vector taxa Vector Virus groups Total %
group
Icosahedral Rod-shaped Viruses Enveloped
particles with particles with with viruses with
RNA genome RNA genome DNA RNA
genome genome
Hemiptera Aphids 26 153a 13 5 197 28
Whiteflies – 13 115b – 128 18
Leafhoppers 8 – 15 3 26 4
Planthoppers 10 4c – 4 18 3
Other hemiptera – 8 5 – 13
2
Thysanoptera Thrips 2 – – 14 16 2
Coleoptera Beetles 50 1 – – 51 7
Acari Mites 10 9 – – 10 1
Nematoda Nematodes 45 3 – – 48 7
Mycota Fungi 8 16 – – 24 3
No of 84 60 19 3d 166 24
identified
vectors
Total 233 268 167 30 697
% 33 39 24
a
Includes 110 virus species of the genus Potyvirus, family Potyviridae
b
Virus species of the genus Begomovirus, family Geminiviridae
c
These are all Tenuiviruses that have multiple shapes
d
These viruses probably have insect vectors
Source Hogenhout Saskia et al. (2008)

viable plant cell. Each step of this sequence is needed for transmission to be
successful (Andret-Link and Fuchs 2005). Information on vector taxa, particle
morphology, genome structure and other details are furnished in the Table 4.3.

(a) Vector specificity

Most of the plant viruses depend on vectors for their survival for two principal
reasons. (1) As the cuticle of the epidermis of some of the plants are impermeable,
the entry of the virus particles in the plants is prevented. Most of the vectors are
insects and non-insect vectors include mites, nematodes and fungi. (2) Plants are
rooted and lack of independent mobility therefore many viruses depend on insects
for transport among hosts.
Transmission of viruses by a vector is a very specific process. Transmission
specificity can be broad or narrow, but it is a prominent future for numerous viruses
and vectors. For instance, a virus transmitted by aphids is not transmitted by
nematodes or, among arthropod vectors. Each particular virus can be transmitted by
only one vector type (e.g., aphid), and not by another vector taxa (e.g., whitefly).
On the other hand, some vector species (e.g., green peach aphid Myzus persicae),
can transmit more than 70 non-persistent viruses (e.g., Lettuce mosaic virus,
4.1 Introduction 195

Cucumber mosaic virus, Potato virus Y, Bean common mosaic virus etc.). Even
viruses like Cucumber mosaic virus, Citrus tristeza virus, Potato virus Y and Bean
common mosaic virus have large number of aphid vectors. Where as Banana
bunchy top virus is transmitted by the only aphid vector, Pentalonia nigronervosa.

(b) Mode of insect vector transmission

The interaction between a virus and its specific vector that results in virus trans-
mission, varies for different virus vectors. The relationships that have developed
between viruses and their vectors are complex and of considerable interest to plant
virologists because vectors provide the main method of spread for many viruses
that cause severe economic losses. Based on the transmission of plant viruses by
insect vectors which feed as piercing-sucking manner and also depending on the
time taken to transmit the virus by the insect vector and the duration of the virus
retention in the vector, there are four categories viz., (1) The non-persistently
transmitted stylet-borne viruses. (2) The semi-persistently transmitted fore-gut
borne viruses. (3) The persistently transmitted circulative viruses. (4) The per-
sistently transmitted propagative viruses (Watson and Roberts 1939; Sylvester
1956). In the first category, in some virus/vector combinations, the interaction
between a virus and its vector is very superficial and is the result of virus
attachment to the external surfaces of the vector mouthparts. For example, viruses
in the Potyvirus genus produce a special protein called helper component that
‘‘glues’’ the virions to aphid stylets. In this case, acquisition of the virus from
infected plants and inoculation of virus to healthy plants takes from seconds to
minutes (Pirone and Blanc 1996). This type of viruses are called non-persistent or
stylet-borne viruses which do not require a latent period in the vector and are
transmitted by number of aphid species. Cucumovirus, Macluravirus, Carlavirus,
Potyvirus, Alfamovirus, and Fabavirus are some of examples for this category. The
second category is semi-persistent viruses which have some characteristics of non-
persistent and some of persistent viruses. They need longer periods (hours) for
acquisition and transmission and they do not circulate within their vector like
persistent viruses. They are usually associated with the phloem and hence virus
transmission is usually more efficient if acquisition feeding time is several hours.
The viruses are remained mainly in the foregut. The mechanism of transmission is
thought to be similar to that of non-persistent viruses. Virus particles accumulate
in the anterior portion of the alimentary canal of the vector and do not multiply.
They have a narrow range of vector species. Some of the viruses belonging to
genus Caulimovirus, Closterovirus, Badnavirus, Trichovirus, Waikavirus and
Sequivirus are some of the examples for this category. In the third category of
circulative transmission, viruses move from the foregut further to the mid and
hindgut, from where they are transported to the hemolymph and further to the
salivary gland, from where they are released into the plant tissue during feeding.
The minimum acquisition and transmission times for this type of virus transmission
are 20 minutes. Viruses are transmitted in a persistent, circulative, non-propagative
manner. Some of the viruses belonging to the genus Luteovirus, Polerovirus,
Babuvirus, Begomovirus, Curtovirus, Masterovirus, Enamovirus, Umbravirus,
196 4 Transmission of Plant Viruses and Viroids

Carmovirus, Comovirus, Sobemovirus, Tymovirus and Nanovirus are transmitted in

this manner. In the fourth category of persistent viruses, once acquired from
infected plants, viruses are associated with the vector for the remainder of their
lifetime. They require long acquisition times (hours to days) and long latent periods
(one day to several weeks). In persistent viruses, the longer the acquisition and
inoculation times the higher is the rate of transmission. Successful transmission of
persistent viruses requires an internalization of the ingested viruses that are actively
transported across several cell membranes. Thus, they are found in the hemocoel of
vectors and retained by vectors after molting. In this category, the viruses actually
multiply in the cells of their insect vector. For example, Tomato spotted wilt virus
when once thrips vectors acquire the virus, they can transmit it for the rest of their
life (Sherwood et al. 2003). Some of the viruses belonging genus belonging to
Tospovirus, Marafivirus, Phytoreovirus, Fijivirus, Oryzavirus, Phytorhabdovirus,
Cytorhabdovirus, Nucleorhabdovirus and Tenuivirus. Principal characteristics of
the modes of virus transmission by different types of insect vectors are provided in
Table 4.4. The virus vector relationship of aphids in Table 4.5, leafhopper vector in
Table 4.6 and whitefly vectors in Table 4.7 is provided. For more information about
virus vector studies can be obtained from the books and reviews of Harris and
Maramorosch (1980); Campbell (1996); Nault (1997); Taylor and Brown (1997);
Gray and Banerjee (1999); Spence (2001); Ng and Perry (2004); Nayudu (2008).

(c) Transmission specificity

Significant progress has been made over the last two decades on the interaction
between viruses and their vectors through biological, biochemical, and molecular
studies. Virus transmission by a vector is often characterized by some degree of
specificity. Numerous studies suggest the involvement of a virus-ligand interaction

Table 4.4 Principal characteristics of the modes of virus transmission by insect vectors
Feature External (noncirculative) Internal-circulativea
Nonpersistent Semipersistent Persistent
Duration of retention Brief (few hours) Intermediate (few Long (days to
days) months)
Duration of acquisition and Brief (seconds) Intermediate (hours) Long (hours to days)
transmission
Latent period Not required Not required Required
Tissue where virus is Epidermis and Epidermis, Mostly parenchyma
acquired and inoculated parenchyma parenchyma and and phloem
phloem
Pre-acquisition fasting Increase No effects No effect
transmission
Passage through moult Negative Negative Positive
Insect species specificity Low Intermediate High
Sequential inoculation Poor Intermediate Good
a
Internal-circulative = virus cross gut and salivary gland barriers
Source Raccah and Fereres (2009)
4.1 Introduction 197

in transmission specificity. The coat protein (CP) and its derivatives (readthrough
CP and minor CP), and nonstructural proteins, such as a helper component (HC) or
a transmission factor, are major viral determinants of transmission specificity.
A number of virion-binding vector proteins have been identified as potential
receptors. For example non-persistent aphid transmitted viruses like CMV, the CP
is the only virus-encoded protein involved in transmission. Similarly, CP gene is a
major determinant of vector specificity for the whitefly transmitted viruses of
genus Begomovirus. For nematode transmitted Tobraviruses, one or two non
structural proteins in addition to the CP involved in transmission. Even in certain
beetle transmitted viruses, efficiency of virus transmission is mediated by the CP
properties. Andret-Link and Fuchs (2005) have provided more information on the
molecular aspects of virus transmission with a major emphasis on the specificity of
transmission.

4.2 Arthropod Vectors

4.2.1 Aphids

Among the virus vectors, aphids constitute the most important group which
transmit more viruses than any others. About 50 % of plant viruses are aphid
transmitted and belong to carla-, clostero-, cucumo-, luteo-, enamo-, cavemo-,
soymo-, babu-, nano-, polero-, alfamo-, caulimo-, faba-, poty- viruses and also
certain plant rhabdo viruses. Both the acquisition and inoculation probes of
15–60 s each are for optimal transmission in a non-persistent relationship. Both
winged and wingless single aphids can transmit the virus. High percent of trans-
mission 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 virus present in
the sap usually adhere to their mouth parts and is 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 non-persistent plant 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 by 60 aphid species (Kennedy
et al. 1962) and SMV by 31 aphid species (Irwin and Goodman 1981). Some of the
aphid transmitted viruses which cause considerable yield losses throughout the
world are Sugarcane mosaic virus transmitted by Rhopalosiphum maidis, Banana
198 4 Transmission of Plant Viruses and Viroids

Table 4.5 Plant virus species of different taxa transmitted by aphids

Family Genus Mode of vector transmission
Bromoviridae Alfamovirus Non circulative capsid strategy
Cucumovirus Non circulative capsid strategy
Caulimoviridae Caulimovirus Non or semipersistent
Circoviridae Babuvirus, Nanovirus Circulative non propagative
Closteroviridae Closterovirus Semi persistant
Comoviridae Fabavirus Non circulative
Flexiviridae Carlavirus Non circulative
Luteoviridae Luteovirus, Polerovirus Circulative non propagative
Potyviridae Potyvirus Non circulative helper strategy
Macluravirus Non circulative
Rhabdoviridae Cytorhabdovirusa Circulative, propagative
Nucleorhabdovirusa
Sequiviridae Sequivirus Semipersistent
a
Some virus species of these two genera are aphid transmitted

bract mosaic virus by Aphis gossypii and Pentalonia nigronervosa, Groundnut

rosette virus by Aphis craccivora, Citrus tristeza virus by Toxoptera citricida,
Potato virus Y in potato and other vegetables by number of aphid species. Some of
the details on aphid transmission, virus family, genus and virus-vector relationship
are provided in Table 4.5.

4.2.2 Leaf, Plant and Tree Hoppers

(a) Leafhoppers
Leafhoppers belongs to the vector family Cicadellidae and Delphacidae which
transmit number of virus and phytoplasma diseases of economically important
crops. Of the 60 subfamilies, Agallinae and Deltocephalinae have vectors of plant
viruses where as plant hopers occur in families Fulgoroidea and Delphacidae have
vectors of cereal viruses. Leafhoppers transmit the viruses in semi persistently or
persistently. The persistent leafhopper transmitted viruses are of circulative and
propagative type. There are 12 circulative type viruses in two genera Mastrevirus
and Curtovirus there are more than 70 viruses which are propagatively persistently
transmitted. Generally, nymphs are more efficient in transmitting the virus. Viruses
transmitted by leafhoppers, mainly cause yellowing and leaf rolling symptoms in the
infected host plants and only a few are mechanically sap transmitted. The vectors
feed only on the phloem tissues as the viruses are concentrated in phloem tissues.
Some of the leafhopper transmitted viruses are Rice tungrovirus by Nephotettix
nigropictus, Maize streak virus by Cicadulina mbila, Beet curly top virus by Agalina
albidula, Wheat streak virus by Javesella pellucida, Maize rayado fino virus
by Dalbulus maidis and Potato yellow dwarf virus by Aceratagallia curvata.
For more details, one can refer Maramorosch and Harris (1979); Nault (1997).
4.2 Arthropod Vectors 199

Table 4.6 Leafhopper transmission of plant virus species of different families and modes of
transmission
Family Vector Mode of vector transmission
Bunyaviridae Leafhopper Non circulative helper strategy
Geminiviridae Leafhopper Circulative non propagation
Reoviridae Leafhopper Circulative propagation
Rhabdoviridae Leafhopper Circulative propagation

The leaf hoper vectors are so active that even a few infected plants (1 %) in the field
are enough for secondary spread and cause cent percent disease incidence. Some of
the details of transmission by leafhopper are presented in Table 4.6.

(b) Planthoppers
Planthoppers have received far less attention from vector researchers than the
aphids and leafhoppers. Till now, 13 genera and 23 vector species are recorded,
and these are responsible for the transmission of 24 viruses. The transmissions are
of circulative, and most, if not all, of the viruses also appear to be propagative.
Planthoppers occur in only family Delphacidae has vectors that feed on poaceae
members like rice wheat and maize.
Some of the examples of planthopper transmitted viruses are viz., Maize mosaic
virus (Peregrinus maidis), Rice stripe virus and Rice black streaked dwarf virus
(Laodelphaxstriatellus), Sugarcane fiji disease fiji virus (Perkinsiella saccharicida),
Oat sterile dwarf fijivirus (Javasella pellucida), Ramu stunt disease (Eumetopina
flavipes); Rice hoja blanca virus (Sogatodes orizicola), Rice ragged stunt virus
(Nilaparvata lugens), Sorghum stripe virus (Peregrinus maidis). In general, nymphs
and female adults tranmit these viruses in more efficient manner (Narayana and
Muniyappa 1996; Falk and Tsai 1998; Ammar and Nault 2002; Anderson et al.
2007).

(c) Tree hoppers

The only known instance of virus transmission by a treehopper is Micrutalis
malleifera which transmits Tomato pseudo-curly top virus belonging to family
Geminiviridae, genus Topocuvirus, and infects dicotyledonous plants. Data relating
to the vector transmission characteristics of the virus indicate that it is circulative.
For more details refer review article of Ammar and Nault (2002).

4.2.3 Whiteflies

In tropical and subtropical countries whitefly-transmitted virus diseases on cotton,

cassava, legumes, tobacco, sweet potato, papaya and on number of vegetable crops
have been reported for which Bemisia tabaci is the principal vector. This vector
has been recorded from more than 1000 crops and weed host species in tropics and
sub-tropics, and occupies a great diversity of niches with variable ecological
200 4 Transmission of Plant Viruses and Viroids

conditions. Nearly 90 % of the plant viruses transmitted by B. tabaci belong to the

genus Begomovirus, family Geminiviridae. These are unique single-stranded DNA
viruses encapsidated in two quasi-isometric mono or twin particles (Fauquet et al.
2005). B. tabaci can also transmit filamentous, single-stranded RNA plant viruses
belonging to three different genera, Crinivirus, Carlavirus, and Ipomovirus
(Fauquet et al. 2005; Jones 2003). The Criniviruses transmitted by B. tabaci are:
Cucurbit yellow stunting disorder virus, Lettuce chlorosis virus, Lettuce infectious
yellows virus, Sweet potato chlorotic stunt virus (SPCSV) and Tomato chlorosis
virus (ToCV). Cowpea mild mottle virus, and Melon yellowing associated virus
belonging to genus carlavirus, are transmitted by B. tabaci and latter has been
detected in Brazil affecting melon (Nagata et al. 2005). The Ipomoviruses trans-
mitted by B. tabaci are: Cassava brown streak virus, Cucumber vein yellowing
virus, Squash yellow leaf curl virus, Sweet potato mild mottle virus (SPMMV), and
Sweet potato yellow dwarf virus (Jones 2003).
Some of the important whitefly transmitted Begomoviruses which are respon-
sible for heavy yield losses in economically important crops are Cassava mosaic
virus, Cotton leafcurl virus, Tomato leaf curl virus, Tomato yellow leaf curl virus
and Okra yellow vein mosaic virus. The whiteflies are small, piercing and sucking
insects belonging to the family Aleyrodidae in the order Homoptera. One can
recognize the adults, nymphs and pupae of the whitefly, Bemisia tabaci, as they
occur on the undersides of leaves. Adults of the whitefly are small (about 1 mm
long). The males are slightly smaller than the females. The wings are brilliant
white, as are those of the spiraling whitefly, Aleurodicus disperus. However, adults
of B. tabaci are much smaller than those of spiraling whiteflies, are not covered
with large amounts of white waxy material and do not lay their eggs in distinctive
spirals. Older nymphs of B. tabaci are sessile and appear as pale yellow oval
specks to the naked eye. Begomo- viruses are persistently and circulatively
transmitted by whiteflies with 4-48 h latent period. Females were found to transmit
the virus more frequently than males. A female B. tabaci can lay up to 160 eggs on
the undersides of leaves in its life time of up to two months. The eggs hatch into
nymphs (larvae) within a week. Newly hatched nymphs (or crawlers) are the only
mobile nymph stage of the insect. Crawlers move to, and settle at, suitable feeding
locations on lower leaf surfaces and become sessile throughout the remaining
nymphal stages. The B. tabaci whitefly can produce 11–15 generations in a year.
Only whiteflies in the Bemisia and Trialeurodes genera are virus vectors. In the
genus Bemisia, only B. tabaci has been shown to be a vector of over 200 viruses
(Jones 2003; Morales 2007); Bemisia afer was considered as vector of Cassava
brown streak virus and Sweet potato chlorotic stunt virus (Malathi et al. 2004;
Gamarra et al. 2010); whereas in the Trialeurodes genus, Trialeurodes vapora-
riorum, T. abutilonea and T. ricini transmit viruses like Potato yellow vein virus in
potato (Salazar et al. 2000) and Tomato infectious chlorosis and Tomato torrado
virus in tomato (Wisler et al. 1998; Duffus et al. 1996). There are biotypes of
whitefly (B. tabaci) like B, Q and A. Naming of the biotypes has been based on the
esterase profiles, dendrograms produced from genetic similarities based on ran-
domly amplified polymorphic DNA (RAPD)-PCR and Amplified fragment length
4.2 Arthropod Vectors 201

polymorphism (AFLP) analysis (Guirao et al. 1997). According to Stanley et al.

(2005) and also of Fauquet et al. (2008), in the VIIIth report ICTV on virus
taxonomy, more than 178 species of whitefly-transmitted viruses are grouped in
the genus Begomovirus and family Geminiviridae. Their genome is circular single
stranded DNA and may be bipartite (containing both genomic DNA-A and B) or
monopartite (containing DNA-A like genome only). Whitefly instar nymphs and
adults feed by inserting their proboscises into the leaf, penetrating into the phloem
and withdrawing sap. It is during this feeding process that plant viruses are
acquired. Adult whiteflies will disperse and transmit the virus to new plants while
feeding. In general whiteflies are poor fliers and their long distance movements
are likely assisted by human beings involvement (Byrne and Bellows 1991). The
whitefly transmitted viruses do not normally multiply within the vectors i.e., they
are not propagative and transovarian transmission through the eggs has not been
shown, however, Ghanim et al. (1998), observed transovarial transmission of
Tomato yellow leaf curl virus (TYLCV) through eggs of Bemisia tabaci. In the
subsequent studies, Ghanim and Czosnek (2000) have showed that TYLCV was
transmitted from viruliferous males to females and from viruliferous females to
males but not among insects of the same sex. Hunter et al. (1998) have studied the
location of gemini viruses in the whitefly, B. tabaci and they detected both ToMoV
and CabLCV in the anterior region of the midgut and filter chamber of adult
whiteflies.
Viruses like Tomato leaf curl virus, Cassava mosaic virus, Mungbean yellow
mosaic virus, Bhendi yellow vein mosaic virus and Cotton leaf curl virus causes
highly devastating crop losses and molecular biology of these viruses was
extensively studied (Varma and Malathi 2003; Jose and Usha 2003; Sinha et al.
2004; Girish and Usha 2005; Surendranath et al. 2005; Patil et al. 2005). In central
Pakistan, Cotton leaf curl Burewala virus is highly prevalent. According to
sequence analysis of the available sequences in the gene bank, there are viruses
belonging to seven different species involved in the etiology of Cotton leaf curl
disease (CLCuD) viz., Cotton leaf curl Multan virus (CLCuMV); Cotton leaf curl
Kokhran virus (CLCuKV); Cotton leaf curl Rajastan virus (CLCuRV); Cotton leaf
curl Allahabad virus (CLCuAV); Cotton leaf curl Bangalore virus (CLCuBV);
Cotton leaf curl Burewala virus (CLCuBuV) and Papaya leaf curl virus (PaLCuV)
(Fauquet and Nawaz-Ul-Rehman 2008).
Mc Grath and Harrison (1995) have reported three vector biotypes of B. tabaci.
According to Briddon (2003) Geminiviruses are not seed transmitted. However,
Muniyappa and Reddy (1983) reported Cowpea mild mottle virus in soybean to be
seed transmitted which requires confirmation.
Some of the details on virus family and virus-vector relationship are presented
in Table 4.7. The additional information on whitefly transmission can be obtained
from the articles of Costa 1976; Muniyappa 1980a, b; Muniyappa and Veeresh
1984; Alegbejo 2001; Brown and Czosnek 2002; Jones 2003; Fauquet et al. 2003,
2008; Varma and Malathi 2003; Boulton 2003; Malathi et al. 2004; Malathi and
Sumiya 2006; Castillo et al. 2011.
202 4 Transmission of Plant Viruses and Viroids

Table 4.7 Modes of transmission of plant virus species of different taxa by whiteflies
Family Genus Vector Mode of vector transmission
Closteroviridae Crinivirus Trialeurodes vaporariorum Non circulative
Geminiviridae Begomovirus Bemisia tabaci Circulative and non propagative
Potyviridae Ipomovirus B. tabaci Non circulative

4.2.4 Thrips

Thrips transmitted viruses are becoming limiting factor for successful cultivation
of majority of crop and ornamental plants. Thrips belongs to Thysanoptera and are
generally reproduce by parthenogenitically. The first instar larvae alone acquires
the virus and both larvae and adult transmit the virus. Viruses from four virus
families or groups viz., Tospovirus, Ilarvirus, Carmovirus and Sobemovirus are
transmitted by thrips vectors. Frankliniella, Thrips and Scirtothrips species are the
major vectors, which are polyphagus and have piercing-sucking mouth parts.
Thrips tabaci, the most common species and efficient virus vector feeds on 140
plant species in over 40 families. Similarly, Frankliniella occidentalis has 148
hosts (Ullman et al. 2002). In tropical and sub-tropical Asia, Thrips palmi is the
predominant vector which transmits several different Tospoviruses viz., Peanut
(groundnut) bud necrosis virus, Capsicum chlorosis virus, Melon yellow spot
virus, Watermelon bud necrosis virus, Watermelon silver mottle virus and Lily
chlorotic spot virus. The T. palmi is widely distributed in several countries
including India, Indonesia, Japan, the Philippines and Thailand (Jones 2005;
Cannon et al. 2007; Pappu et al. 2007), while Frankliniella occidentalis, another
important thrips vector is prevalent in Australia, Europe and middle East, North
and South America and transmits Tospoviruses like Tomato spotted wilt virus,
Tomato chlorotic spot virus, Groundnut ring spot virus, Impatiens necrotic spot
virus and Chrysanthemum stem necrosis virus (Jones 2005; Pappu et al. 2009).
Because of the wide host range of virus and the vector, the tospo- and Ilarviruses
are affecting number of economically important crops and resulting in heavy yield
losses.
Throughout the world, except in certain Asian countries Tomato spotted wilt
virus (TSWV) is economically very important virus and transmitted by eight thrips
species viz., Frankliniella occidentalis (western flower thrips); F. schultzei,
F. fusca (tobacco thrips); Thrips tabaci (onion thrips); T. setosus, T. moultoni;
F. tenuicornis, and Scirtothrips dorsalis. The first four are considered to be the
most important vectors because of their wide distribution and the overlapping host
ranges of these thrips species and TSWV (Whitefield et al. 2005). Food crops like
peanut, watermelon, capsicum, tomato, zucchini, celery, eggplant, cucumber,
lettuce, pineapple, grape, many legumes as well as ornamental species (gladiolus,
dahlia, lily, impatiens, chrysanthemums, iris) are affected by TSWV and economic
losses caused, demands effective management measures.
4.2 Arthropod Vectors 203

In India Peanut or Groundnut bud necrosis virus (GBNV/PBNV) is wide

spread in peanut, potato, tomato, taro, capsicum and number of legumes and
transmitted by different thirps vectors like T. palmi, F. schultzei and Scirtothrips
dorsalis (Mandal et al. 2012). Similarly in onion Iris yellow spot virus transmitted
by T. tabaci has worldwide distribution. In India, potato stem necrosis disease
caused by a strain of PBNV-Po is seen in potato growing states. Jain et al. (1998)
have reported Watermelon bud necrosis tospovirus on watermelon to be eco-
nomically important in South India.
Another thrips transmitted Peanut yellow spot tospovirus is reported from
India and its vector is Scirtothrips dorsalis. The infected peanut plants exhibit
small chlorotic local lesions, which coalesce and become necrotic (Satyanarayana
et al. 1996).
Sunflower necrosis disease caused by Tobacco streak virus (TSV) belonging to
Ilarvirus, is transmitted by Thrips palmi and is prevalent in India, Australia and
other sunflower growing countries. The virus spreads through infected pollen
while thrips feeding. Both virus and thrips have wide host range and responsible
for heavy crop losses in number of crops like mungbean, cotton, peanut, sunn-
hemp, etc., Regarding virus vector relationship, TSV is persistently and propa-
gatively transmitted by thrips. The larvae of thrips acquire the virus while feeding
on virus-infected tissue. The virus crosses through the midgut barrier and enters
the salivary glands. The virus must be acquired by larval stage of thrips as the adult
thrips cannot acquire the virus (Pappu et al. 2009; Riley et al. 2011). The virus
passes from larvae to adult thrips as it under goes pupation and changes associated
with maturity. This is known as transstadial passage (Whitefield et al., 2005).
Thrips retain infectivity for their life time and the virus titer has been shown to
increase as the virus replicates in the thrips. There is no evidence of passage of
virus through the egg. For more details of thrips vectors and TSV, one can refer
Francki et al. 1981; German et al. 1992; Jones 2005; Whitefield et al. 2005; Pappu
et al. 2009; Riley et al. 2011.

4.2.5 Beetles

Another active vector group of plant viruses are beetles, which belong to Coleoptera
whose members transmit viruses belonging to genera Bromovirus, Carmovirus,
Comovirus, Machlomovirus, Sobemovirus, and Tymovirus (Fulton et al. 1980 and
1987; Meier et al. 2008). There is a high degree of specificity between the beetle
vectors and the viruses they transmit. Cowpea mottle and Southern bean mosaic
viruses are transmitted by the Ootheca mutabilis and Medythia quarterna, both
belonging to Chrysomelidae (Allen et al, 1981). Several species of Chrysomelidae
beetles are vectors of Rice yellow mottle virus (RYMV), viz., Sessielia pusilla,
Chaetocnema pulla, Trichispa serica and Dicladispa viridicyanea (Bakker 1974;
Abo et al. 2000). Maize chlorotic mottle virus in maize is also transmitted by six
species of beetles of the family Chrysomelidae (Scheets 2008).
204 4 Transmission of Plant Viruses and Viroids

The beetle vectors are confined to the families of Chrysomelidae, Coccinellidae

and Meloidae. They have biting-chewing type mouth parts and transmit viruses
mechanically by carrying them on the mouth parts. The beetles become virulif-
erous and transmit the virus in a single bite but a longer acquisition access period
and inoculation access period make the probability of transmission higher. These
vectors acquire the virus during acquisition feeding periods ranging from 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 envi-
ronmental 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 one or two 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 hemolymph, but apparently do not multiply in their beetle vectors.
Virus-vector relationship appears to be similar to semi-persistent type.
Markham and Smith (1949) have also pointed out that beetles regurgitate
during feeding which contain viruses over several days. The viruses are also
detected in the fecal matter of beetles which have fed on infected plants. It has
been established that the regurgitant fluid is a key factor in determining whether a
virus will or will not have beetle vectors (Gergerich et al. 1983).

4.2.6 Mealybugs

When compared to aphids and leafhoppers, the mealybug vectors are less mobile
on the plant and hence relatively inefficient vectors. Viruses belonging to the genus
Ampelovirus, Badnavirus, Trichovirus and Vitivirus have plant viruses, which have
mealybugs as the vectors. Mealybugs feed on the phloem and spread through wind
currents and also carried by the ants. The virus is acquired within 20 min period
and takes 16 min to inoculate to the host plant and the virus persists in the vectors
for less than 3 h. The mealybug vector can retain the virus for a maximum period
of 3–4 days and nymphs are the more efficient vectors than adults. The virus -
vector relationship is of semi-persistent type. Only the nymphs of the first, second
and third larval stages and the adult females are able to transmit the virus. Among
the mealybug transmitted viruses, Cocoa swollen shoot virus (CSSV) is eco-
nomically important disease in African countries and is transmitted by fourteen
species of mealybugs and Planococcoides njalensis and Planococcus citri are the
primary vectors. CSSV is also transmitted by seed to the extent of 34–54 %
(Quainoo et al. 2008) and virus was located in testa, cotyledons and embryo and
virus is pollen-borne. Even Grapevine leafroll associated virus is transmitted by
mealybug vectors viz., Planococcus ficus and Pseudococcus longispinus (Tsai
et al. 2008, 2010). Another virus which is transmitted by mealybug in sugarcane
is Sugarcane bacilliform virus and the vector is Saccharicoccus sacchari.
4.2 Arthropod Vectors 205

Even Sugarcane mild mosaic virus is transmitted by Saccharicoccus sacchari

(Lockhart et al. 1992). The citrus mealybug, Planococcus citri is the vector of
Citrus mosaic badna virus (CMBV) (Garnsey et al. 1998). Planococcus citri and
Saccharicoccus sacchari transmits Banana streak virus (Lockhart and Jones
1999).
Pineapple mealybug wilt associated virus-3 infecting pineapple in Cuba is
transmitted by Dysmicoccus brevipes (Hernandez et al. 2010). Earlier Pineapple
mealybug associated virus-1 which was transmitted by the same mealybug vector
in semi persistent manner and was first reported in Hawaii. In India, Piper yellow
mottle virus belonging to genus Badnavirus of black pepper spreads through
mealybug vectors like Ferrsia virgata and Planococcus citri (Bhat et al. 2003).

4.2.7 Mirids

Mirids belong to the family Miridae, which is one of the largest families in the
suborder Heteroptera. Their mouthparts, which are modified for sucking plant sap,
consist of two pairs of flexible stylets enclosed by a labium. Adult mirids are
winged and highly mobile and they are important pests of a wide range of crops
(Woodward et al. 1979; Wheeler 2001). The only report of the natural transmission
of plant virus by mirids is that of Velvet tobacco mottle virus (VTMoV) by the
mirid, Cyrtopeltis nicotianae and the virus vector relationship semi-persistent type.
Virus does not multiply in the vector and does not require a helper virus for vector
transmission (Gibb and Randles 1988). As mirids generally are poor vectors of
plant viruses, the possibility of identifying significant new vector species in the
Miridae seems unlikely (Wheeler 2001).

4.2.8 Mites

Mite vectors belong to Phylum-Arthropoda and class Arachnida. Members of the

mite families Eriophyidae and Tetranychidae are the plant virus vectors. The mite
retains infectivity through moulds and may remain infective for 6–9 days. The
mites become infective as nymphs but not as adults when fed on infected plants.
As early as 1963, Thresh has demonstrated that Cecidophioposis ribis, the mite as
the vector of black currant reversion. Some of the mite transmitted viruses are
Wheat streak mosaic virus and Triticum mosaic virus transmitted by Aceria tuli-
pae, Triticum mosaic virus by Aceria tosichella (McMechan 2012). Peach mosaic
virus by Eriophyes insidiosus, Cherry mottle leaf virus by E. inaequalis, Garlic
virus C and Garlic Virus D (Alexiviruses) by Aceria tulipae and Onion mosaic
virus by Aceria tulipae (Brakke 1971a, b). From Brazil, Rodrigues et al. (2000)
have reported that Brevipalpus phoenicis to be the mite vector of Citrus leprosis
206 4 Transmission of Plant Viruses and Viroids

virus in mandarins. B. phoenicis is the vector for Coffee ringspot nucleorhabdo

virus (Chagas et al. 2003).
For the first time, Khetarpal (1989) reported the transmission of the Pea seed-
borne mosaic virus in pea by the mite Tetranychus urticae under glass house
conditions and requires confirmation. Wheat streak mosaic virus which is also a
seed-borne virus in wheat, is transmitted by mite vector, Aceria tosichella (Scifers
et al. 1997; Jones et al. 2005). This mite vector also transmits High plains virus in
maize. Pigeon pea sterility mosaic virus (PPSMV) in pigeon pea (Cajanus cajan)
is transmitted by eriophyid mite Aceria cajani. Kulkarni et al. (2002) have
reported the transmission efficiency of single A. cajani to be up to 53 %, but was
100 % when [5 mites per plant were used. A. cajani acquired PPSMV after a
minimum acquisition access period (AAP) of 15 min and inoculated virus after a
minimum inoculation access period (IAP) of 90 min. No latent period was
observed. In general, wind is the main means of mites dispersal in nature, because
of their small size and are carried like dust particles. More information on mite-
transmitted viruses can be obtained from the review articles of Slykhuis (1960);
Oldfield (1970); Agrios (2005); Nayudu (2008).

4.3 Non-Insect Vectors

4.3.1 Nematodes

The soil inhabiting nematodes are responsible vectors for transmission of tobra-
and nepo- virus groups which are also seed-borne (Table 4.2) and this aspect has
been reviewed exhaustively (Taylor 1980, 2002). Four nematode genera viz.,
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). Viruses can be transmitted both
non-persistently and persistently by nematode vectors. The virions attach to the
stylet (feeding organ) or to the gut when they feed on the infected plant and can
transmit viruses during feeding healthy susceptible plants. The virus retention as a
mono layer adhering to the cuticular lining of the oesophagus track was observed
in X. diversicaudatum carrying Arabis mosaic virus and also in X. index carrying
GFLV (Taylor and Robertson 1973). Both adult and larval stages can transmit the
viruses with equal efficiency. L. elongatus retains the virus for 8–9 weeks and the
Xiphinema sp. for many months and there is no evidence that viruses multiply
inside the nematode vector and is not transmitted to its progeny.
4.3 Non-Insect Vectors 207

Based on the particle morphology of the nematode transmitted viruses, Cadman

(1963) and Harrison (1964) categorized them into nepo and netu 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 an 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 vector distribution. Some of the important nematode
transmitted viruses with their vectors are Grape fan leaf virus- Xiphinema index,
Arabis mosaic virus- X. diversicaudatum, Tobacco rattle virus- Trichodorus
pachydermis, Raspberry ring spot virus- Longidorus elongatus and Pea early
browning virus- Paratrichodorus and Trichodorus spp (Hull 2002). Demangeat
et al. (2010) have reported that even a single female xiphinema index has capacity
to transmit Grapevine fanleaf virus (GFLV). More details on nematode vectors can
be had from Taylor and Brown (1997) and Nayudu (2008).

4.3.2 Fungi

Certain fungi like Polymyxa sp., Olpidium sp., and Synchytrium sp. are also
implicated as vectors of plant viruses belonging to necro-, furo-, peclu- virus
groups. Plasmodiophoroids (Polymyxa, Sporosphaera and Spongospora species)
are only known to transmit the viruses internally and these are ssRNA (+) viruses
with divided genomes. Some have rod-shaped virions (genera: Benyvirus,
Furovirus, Pecluvirus, Pomovirus) while others have filamentous particles (genus:
Bymovirus, Family: Potyviridae). A number of virus genera are transmitted both
persistently and non-persistently by these soil-borne zoosporic protozoa. These are
not phytopathogenic themselves but parasitic. Transmission of the virus takes
place when they become associated with the plant roots. In India, Indian peanut
clump virus (IPCV) belonging to Pecluvirus genus is present to a limited extent in
sandy soils of Punjab, Rajasthan and Andhra Pradesh states and is transmitted both
by Polymyxa graminis and also through seed (Reddy et al. 1983 and Delfosse et al.
1999). The life cycle of Polymyxa graminis, the vector of IPCV in its gramina-
ceous hosts has been studied (Ratna et al. 1991). Another species of Polymyxa,
P. betae transmits the Beet necrotic yellow vein virus in sugarbeet throughout the
world (Abe and Tamada 1986). Soil-borne wheat mosaic virus (SBWMV) is
transmitted by Polymyxa graminis and virus is seed transmitted in rye and wheat
crops (Delfosse et al. 1999). Viruses like Tobacco necrosis virus is transmitted by
Olpidium brassicae of the order Chytridiales and Melon necrotic spot carmovirus
transmitted by Olpidium bornovanus which is seed transmitted in melon both
externally and internally (Campbell et al. 1996). Mirafiori lettuce big vein virus
and Tobacco stunt virus are transmitted by Olpidium virulentus (Sasaya and
Koganezawa 2006). Olpidium species transmit some small monopartite ssRNA (+)
viruses that are carried on the outside of zoospores and resting spores and the
208 4 Transmission of Plant Viruses and Viroids

examples are members of the family Tombusviridae (some of the genera Aureus
virus, Carmovirus, Necrovirus and Tombusvirus). Olpidium species also transmit
some ssRNA viruses with divided negative sense genomes that are members of the
genera Ophiovirus and Varicosavirus. These genera are not currently assigned to
any family and the viruses are internally borne. Potato virus X, transmitted by the
fungus, Synchytrium endobioticum is seed-borne in potato to an extent of 0.6–2.3 %
and requires confirmation (Darozhkin and Chykava 1974). Another virus infecting
potato viz., Potato moptop virus, for which the vector is Spongospora subterranea
(de Bokx and van Der Want (1987). In Germany, for Grapevine viruses, the
Sporosphaera viticola is the most potential vector (Kirchmair et al. 2005). In South
Africa, Brome mosaic virus is transmitted by rust spores, Puccinia graminis tritici
(Von Wechmar 1980). Different aspects of fungus transmission of plant viruses
have been reviewed by Teakle (1980) and Nayudu (2008).

4.4 Conclusions

Due to the immobility of plants, the major virus spread between the crops over long
distances takes place primarily due to active and mobile forms of vector species.
Effective transmission by vectors ensure the perpetuation of a virus. Insect vector
plays a paramount importance in the epidemiology of plant viruses. We have both
air-borne and soil-borne vectors and there are different degrees of vector specificity
for all the plant virus vectors. There is lot of diversity both in vectors and viruses
and has been confirmed by advanced molecular tests. Depending on the size and
mobility of the vectors, the virus spread takes place in the crop and several
apparently distinct mechanisms of transmission is observed. Among the different
vectors discussed in this chapter, whiteflies transmit several viruses, mainly to the
tropical and subtropical crops. Virus spread by aphid vectors in vegetable crops and
leafhoppers in cereal crops plays a major role. Virus spread in field crops is very
high as insect vectors of plant viruses are found in 7 out of 32 orders of the class
insecta. The complex interactions between the virus, host and vector emphasize the
need for comprehensive biological studies of the virus epidemiology.

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Chapter 5
Diagnosis and Detection of Plant Virus
and Viroid Diseases

5.1 Introduction

Agricultural and horticultural crops are being threatened by a wide variety of biotic
stresses which lowers vegetable and fruit quality leading to wipe out of entire
harvests. About 42 % of the world’s total agricultural crop is destroyed yearly
by diseases and pests. Crop losses can be significantly minimized and specific
treatments can be tailored to combat specific pathogens if they are correctly
diagnosed and identified at an early stage. These need-based treatments also
translate to economic and environmental gains.
The early and accurate diagnosis of plant diseases including plant virus dis-
eases, is a crucial component of all crop-management systems. Symptoms are of
major importance because they are the main means by which a viral disease is
determined but precise identification of a virus or viroid is not feasible on
symptoms alone. As several unrelated viruses produce similar symptoms and
different strains of the same virus group can also produce very different symptoms.
Accurate diagnosis of virus diseases and diseases in general, is the first
important step for any crop management system. Virus diseases are managed most
effectively if control measures are applied before infection occurs. The use of
healthy (virus-free) plant propagation material is among the most effective
approaches to adopt by farmers. One of the elements essential for successful
certification programs to produce a disease-free propagation material is the
availability of sensitive diagnostic methods. Few decades ago, virus detection was
based mainly on biological techniques which are too slow and not amenable to
large-scale application. Advances in molecular biology and biotechnology over the
last three decades were utilized to develop rapid, specific and sensitive techniques
for the detection of plant viruses. This chapter, summarizes the development and
use of the main immunological and nucleic acid-based methods for virus detection.

K. S. Sastry, Plant Virus and Viroid Diseases in the Tropics, 233

DOI: 10.1007/978-94-007-6524-5_5,  Springer Science+Business Media B.V. 2013
234 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

5.1.1 Need and Progress in Plant Virus Diagnostics

For the past 30 years, identifications and characterization of plant viruses has
brought revolutionary change in virus diagnostics. Though symptoms are still the
major criterion for initial tentative virus identification, it should never be based on
symptoms alone. Proper identification is always the key in developing appropriate
practical solutions to manage the spread of 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. During 1960s, tests
like agarose immunodiffusion, electronmicroscopy, immuno-chromatography, and
polyacrylamide gel electrophoresis were used for virus diagnosis in different virus
infected plants. ELISA, originally a medical immunodiagnostic assay, has been
introduced in plant virology by Voller et al. (1976). During 1977, Clark and
Adams, have developed microplate method of Enzyme linked immunosorbent
assay (ELISA) for the detection of plant viruses. Because of its simplicity and
possibility of handling a large number of samples at one time, ELISA-based tests
are one of the most frequently used diagnostic tools. However, development of
molecular tests have changed the testing methodology for virus diagnostics.
Depending on the crops, the nature of the viruses, and the interests of grower and
consumer, one has to make a decision on the test to be used. It is safe to use more
than one detection method for important viral diseases. One of the primary
selection criterias for detection techniques are their cost of the reagents, chemicals,
required equipment, and labor. In addition, useful methods should be rapid, simple
to use, reliable, and specific enough to detect virus strains or mixed infections.
The assays described in this chapter can be potentially used to distinguish
closely related pathogens and in many cases to identify virus and viroids in
extracts made directly from infected plant material or the vectors. For detailed
information on the techniques of plant virus or viroid detection and identification,
one can also refer the following references books and review articles (Bar-Joseph
and Garnsey 1981; Torrance and Jones 1981; Jones and Torrance 1985; Lange
1986; Singh and Sharma 1989; Hampton et al. 1990; Khurana and Garg 1993;
Barbara et al. 1995; Bar-Joseph et al. 1995; Banttari and Khurana 1998; Torrance
1998; Finetti-Sialer et al. (2000); Lopez et al. 2003; Naidu and Hughes 2003;
Gallitelli 2004; Maxwell and Martin 2005; James et al. 2006; Makkouk and Ku-
mari 2006a, b; Punja and Boer 2007; Vincelli and Tisserat 2008; Baranwal and
Ahlawat 2008; Koenig et al. 2008; Rao and Maneesha Singh 2008; Kumar 2009;
Ahlawat 2010; Prakash and Singh 2010; Narayanasamy 2011; Jan et al. 2012).

5.1.2 Detection Specificity and Sensitivity

For sensitivity, effectiveness and specificity of virus detection system, numerous

measures are developed by different researchers (Peruski and Peruski 2003;
5.1 Introduction 235

Malorny et al. 2003). Diagnostic specificity is defined as a measure of the degree to

which the method is affected by non-target components present in a sample, which
may result in false positive responses. Diagnostic sensitivity is defined as a measure
of the degree to detect the target pathogen in the sample, which may result in false
negative responses (Malorny et al. 2003). Too low sensitivity often leads to false
negatives. Thus, a high degree of diagnostic accuracy is characterized by the ability
to detect, true and precisely the target pathogens from a sample without interference
from non target components. The high degree of sensitivity of molecular methods
made pre-symptomatic detection and quantification of pathogens possible. Poly-
merase chain reaction (PCR) is developed by Mullis and Faloona (1987) is a highly
sensitive technology. However, its sensitivity is greatly affected by the presence of
inhibitors present in plants which will prevent or reduce amplification (Yang and
Rothman 2004). Although the mode of action of inhibitor is not clear, they are
believed to interfere with the polymerase activity for amplification of the target
DNA. On the other hand, it is worth mentioning that the high sensitivity of PCR also
causes one of the limitations of PCR that is the false positive results from slight DNA
contamination (Yang and Rothman 2004). Hence, stringent conditions are necessary
in conducting the assay and proper negative and internal controls must be included in
the test. It is also recommended to have separate dedicated areas for pre- and post-
PCR handling. With time, number of new advanced tests like microarrays, DNA
barcode technologies etc., are developed and are being discussed in this chapter.

5.2 Approaches Used for Identification of Plant Virus

and Viroid Diseases

Management measures depend on proper identification of diseases and of the causal

agents. Without proper identification of the disease and the disease-causing agent,
disease management practices will be a waste of time and money and will lead to
further economic losses and hence proper disease diagnosis is therefore vital. There
are several approaches used for the identification of disease causing agents and some
of the first and foremost steps in identifying the disease causing agent as follows:
(a) Checking for the symptoms: Variation in symptoms on the inoculated
plants sometimes due to variation in environmental factors, the cultivar and the
virus strain used may lead to an improper diagnosis.
(b) Identifying the plant parts affected: The symptoms associated with spe-
cific plant parts, some times also will be one of the helpful factors for diagnosis are
most commonly seen on specific plant parts and this observation can be important
in diagnosis.
(c) Checking for the distribution of disease affected plants: One of the first
things that a diagnostician should note is how the diseased plants are distributed
over the affected area. Are they distributed uniformly across an area or are they
localized? Is there a definite pattern to the distribution?
236 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

(d) Checking for the host specificity: Is the problem occurring in only one
plant species or different plant species are affected? If different plant species are
affected, this suggests the possibility of a non-infectious problem which could be
related to cultural or environmental problems.

5.2.1 Biological Approaches

5.2.1.1 Bio-Assays

A number of methods have been used for the quantification of virus within infected
plants. The local lesion assay remains the simplest method to quantitatively
measure the virus concentration. Holmes (1939) was the first to utilize the
observation that mechanical inoculation of Tobacco mosaic virus (TMV) onto the
leaves of Nicotiana glutinosa led to the formation of local lesions, and that the
number of local lesions was inversely correlated to the dilution of the inoculum.
The use of indicator or diagnostic hosts against certain viruses and their strains
for virus diagnosis is furnished in Table 5.1.
For non-sap transmissible viruses, grafting of suspected plant part (e.g., bud-
wood) to indicator plants and vector transmission of suspected virus to indicator or
diagnostic hosts is sometimes useful for initial tentative diagnosis (Hull 2002;
Nayudu 2008; Sastry 2013).

5.2.2 Physical Tests

5.2.2.1 Electron Microscopy

The EM proved to be one of the valuable tools for the routine diagnosis of viral
diseases. Most viruses with distinct virion morphology (Fig. 2.1) could be iden-
tified at least up to genus level by electron microscopy. In many cases, however,
viruses within same genus or species can be morphologically very much related
and thus requires additional identification tests. Kitazima (2004) has reviewed the
importance of electron microscopy in plant virology.
Transmission electron microscopy (TEM) also useful for investigation of
ultrastructural alterations during virus infection of plants. With these methods,
virus types and diseases can be diagnosed reliably since size and ultrastructural
features are specific for each group of viruses. Negative staining of viruses and
following visualization by TEM can provide rapid and accurate results, and in
most cases they are sufficient for the identification of virus diseases (Zechmann
and Zelling 2009).
5.2 Approaches Used for Identification of Plant Virus and Viroid Diseases 237

Table 5.1 Indicator or diagnostic hosts for certain sap transmissible viruses of tropical crops
Genus Type species Local lesion host/comments
Alfamovirus Alfalfa mosaic Phaseolus vulgaris and Vigna unguiculata spp. Sinensis for
virus most strains; Chenopodium amaranticolor and C.
quinoa are also suitable
Badnavirus Commelina yellow Not mechanically transmissible
mottle virus
Bromovirus Brome mosaic C. hybridum and Datura stramonium
virus
Bymovirus Barley yellow None
mosaic virus
Capillovirus Applestem P. vulgaris cv. Pinto and C. quinoa
grooving virus
Caulimovirus Cauliflower Brassica campestris cv. Just Right
mosaic virus
Closterovirus Beet yellows virus Difficult to inoculate mechanically
Comovirus Cowpea mosaic P. vulgaris cvs. Pinto and Scotia, C. amaranticolor
virus
Cucumovirus Cucumber mosaic Vigna unguiculata spp. Sinensis, P. vulgaris, C.
virus amaranticolor, and C. quinoa
Fabavirus Broad bean wilt V. unguiculata spp. Sinensis for the broad bean strain
virus 1 (giving red/brown lesions); C. amaranticolor and C.
quinoa, for the nasturtium, parsley, and petunia strains
Fijivirus Fiji disease virus Not mechanically transmissible
Furovirus Soil-borne wheat C. quinoa and C. amaranticolor
mosaic virus
Mastrevirus Maize streak virus Not mechanically transmissible
Curtovirus Beet curly top None
virus
Begomovirus Bean golden P. vulgaris cv. Top crop, gives chlorotic lesions on primary
mosaic virus leaves
Hordeivirus Barley stripe C. quinoa and C. amaranticolor
mosaic virus
Idaeovirus Raspberry bushy P. vulgaris cv. The Prince and C. murale are reliable, but
dwarf virus the lesions are more difficult to count.
Ilar virus Tobacco streak Cyamopsis tetragonoloba, Vigna sinensis, V. unguiculata
virus spp. Cylindrical, Beta patellaris, Dolichos biflorus,
Gomphrena globosa, and P. vulgaris, cv. Monteiga
Luteovirus Barley yellow Not mechanically transmissible
dwarf virus
Machlomovirus Maize chlorotic None
mottle virus
Marafivirus Maize rayado fino Not mechanically transmissible
virus
Necrovirus Tobacco necrosis P. vulgaris and C. amaranticolor
virus
Nepovirus Tobacco ringspot V. unguiculata, Vigna sinensis, Nicotiana tabacum,
virus Nicotiana clevelandii, C. amaranticolor,
and Cassia occidentalis
(continued)
238 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

Table 5.1 (continued)

Genus Type species Local lesion host/comments
Potexvirus Potato virus X G. globosa (the middle leaves of plant with 8-10 leaves are
most suitable).
Potyvirus Potato virus Y C. amaranticolor, C. quinoa, Nicotiana repunda, N.
rustica, Physalis floridana, Solanum tuberosum cvs.
Duke of York or Saco, and Solanum demissum ‘‘Y’’;
the latter produces local lesions with most strains
Sobemovirus Southern bean P. aureus
mosaic virus
Tobamovirus Tobacco mosaic N. glutinosa, C. amaranticolor, P. vulgaris cv. Pinto, and
virus Nicotiana tabacum cvs. Xanthi nc. and Samsun NN
Tospovirus Tomato spotted N. glutinosa, Petunia hybrida cvs. Pink beauty and
wilt virus Ministrel, Vigna sinensis
Tombusvirus Tomato bushy C. amaranticolor, D. stramonium, and Ocimum bacillium
stunt virus
Trichovirus Apple chlorotic C. quinoa and P. vulgaris
leaf spot
Source Boonham and Wood 1998

5.2.2.2 Gel Electrophoretic Techniques

The major useful systems for separation, purification and identification of viruses
and viroids are agarose gel electrophoresis and polyacrylamide gel electrophoresis
(PAGE).

Agarose gel Electrophoresis

This is one of the standard methods used to separate, identify and purify nucleic
acid (RNA or DNA) fragments by electrophoresis through agarose gels. It is simple
and rapid to perform, and capable of resolving mixtures of nucleic acid RNA and
DNA fragments that cannot be separated adequately 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.

Polyacrylamide gel Electrophoresis (PAGE)

Polyacrylamide gels are used to analyze the proteins and fragments of DNA/RNA.
They may be casted in a variety of polyacrylamide concentrations ranging from
3.5 to 20 %, depending on the size (Sambrook and Russel 2001).
The other major usefulness of PAGE system is determination of the molecular
weights of the virus polypeptides or nucleic acids and analysis dsRNAs in virus or
viroid infected plant samples. Based on this technique Walia et al. (2008) have
5.2 Approaches Used for Identification of Plant Virus and Viroid Diseases 239

detected Apple scar skin viroid in India. OEPP/EPPO (1984); Owens et al. (2012)
have also stated that Polyacrylamide gel electrophoresis (PAGE) is successfully
used for the rapid identification of viroid-infected plants.

Reverse Polyacrylamide Gel Electrophoresis (R-PAGE)

A modification of PAGE specially designed for the rapid and sensitive detection of
circular RNAs and termed return PAGE (R-PAGE) and has facilitated viroid
detection in various crop plants (Schumacher et al. 1986; Schroeder and Weide-
mann 1989, Singh et al. 1988, 1992, 1993; Singh and Boucher 1987).
In R-PAGE assay, nucleic acids are subjected to two electrophoretic runs, one
under non-denaturing 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 rendering
them easily distinguishable from non-infected plant extracts. Viroid concentrations
as low as 15–20 pg can be detected reliably by R-PAGE (Singh 1989).
R-PAGE has been successfully applied 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). 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 and Boucher 1988; Singh 1989). R-PAGE
can be used to assess the PSTV 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).

5.3 Antibody-Based Tests

The application of serology is the most quick and reliable method for the diagnosis
of plant viruses. Various diagnostic tests often provide valuable clues to etiology,
but every test in vitro has its limitations. Antibody-based tests are not applicable
for detection of viroids as they are naked RNA molecules and does not encode
with proteins like viruses.
The detection of plant viruses depends upon the surface and sequence prop-
erties of viral proteins. Based on the availability of the facilities, earlier workers
have produced polyclonal or monoclonal antibodies (Sander and Dietzgen 1984).
In the case of polyclonal, which contains antibodies to all available epitopes on the
antigen, where as in monoclonal which contains antibodies to one epitope.
The results of the researches carried out with different virus-host combinations,
have indicated that monoclonal antisera are more specific than polyclonal antisera
and used to differentiate strains of many viruses. Several books/reviews are
240 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

available on monoclonal antibodies and their application in plant virus diagnosis

(Sander and Dietzgen 1984; Van Regenmortel 1984; Franssen and van der Hulst
1985; Thomas et al. 1986; Aiton and Harrison 1989; Swanson and Harrison 1993;
Cambra et al. 1994; Asensio et al. 1995; Cancino et al. 1995; Canto et al. 1995;
Dolores and Bajet 1995; Konate et al. 1995; Schots et al. 1995; Harrison et al.
1997; Candresse et al. 1998a; Karande et al. 1998; Wu et al. 2009).
In the following sections, the developments in diagnostic techniques and their
advantages as well as disadvantages of various tests have been presented.

5.3.1 Precipitation Tests

Antigen and antibody precipitation occurs when the reaction between these sub-
stances form a grid-like structure, preventing the passage of water molecules
(hydrophobic reaction). The formation of the grid structure requires a proportional
concentration of reacting substances. For every antigen molecule, a given number
of antibodies are needed. Antibodies usually act as bivalent molecules and the
antigens as multivalent molecules. The presence of too many antibodies will make
the antibodies act as monovalent molecules only to one particle of the antigen, thus
preventing the formation of the grid structure. Too many antigens also prevent the
formation of the grid, because the antigens will then act only as monovalent
molecules. The antigen–antibody reaction cannot be observed in either case. These
precipitation based tests performed in liquid media are interface ring, tube pre-
cipitation and microprecipitation tests, and those performed in semi-solid gel media
are passive single or double diffusion tests and immunoelectrophoresis in different
formats that facilitate quick antigen–antibody reactions (Van Regenmortel 1982).
These tests are currently not applied in plant virus diagnosis because of their
inherent limitations. However, agar gel double diffusion test is a valuable test for
identification of plant viruses and their strains, especially in laboratories with
minimal facilities.

5.3.2 Agglutination Tests

The main difference between precipitation and agglutination tests is that in the latter
the antigen is very often larger than the antibody. For this reason, few antibody
molecules are necessary to form a visible particle grouping. The principles gov-
erning reactions are similar to those of the precipitation tests (Van Regenmortel
1982). Passive agglutination is based on the use of inert substances that carry
antigens or antibodies. These substances (latex spheres, bentonite) are several times
larger than the reacting substances, thus making it possible to use soluble antigens
(viral particles) in agglutination tests. The latex test is based on the use of
polystyrene spheres (800 nm diameter) covered by immunoglobulin molecules.
5.3 Antibody-Based Tests 241

This technique is 10–100 times more sensitive than the traditional microprecipi-
tation tests for detecting plant viruses and reaction may be seen with the naked eye.
In this test the size of the reacting antigenic complexes is large and adsorbed 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 1000-fold 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 has
been used in the detection of seed-borne plant viruses and to detect one infected
seed per 100 seeds or 1 lg/ml of virus (Carroll 1979). This test has been routinely
employed for large scale testing of potatoes, both in certification schemes as well
as in disease resistance screening (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).

5.3.3 Immuno Diffusion 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 co-efficient and con-
centrations. Thus they are extremely useful for virus detection and identification
from extract of virus infected seeds and also from virus infected leaf samples.
There are two types of immunodiffusion tests: (1) Single or simple immuno-
diffusion, in which one of the reactants diffuse into the gel and (2) Double
immunodiffusion, where both the reactants diffuse into the 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 any 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 hydrochloride or by detergents such as sodium dodecyl
sulphate (SDS), sodium dibutylnapthalene sulphonate (Leonil SA) and sodium-
N-methyl-N-oleoyl taurate (Igepon T-73) or by physical treatments such as
freezing and thawing and ultrasonic vibrations.

(a) 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 plant viruses.
242 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

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. (1984).

(b) Radial immuno diffusion 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 lg/ml of
degraded virus. This test has been used for the detection of seed-transmitted 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 (Shepherd and Secor 1969; Shepherd
1972). The drawback of this test is that it requires large amount of reactants.

(c) Gel double immuno diffusion (Ouchterlony)

Even in the present day, wherever ELISA and PCR facilities are not available this
test is most widely used in plant virology research and in quarantine inspections.
Thin agar or agarose gel layers are prepared on glass slides, or in petridishes and
suitably arranged wells are cut to place the reactants. The antibody and antigen
(infected plant/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. The bands from serologically identical or very closely related
viruses fuse, where as those from more distantly related viruses can form spurs.
The gel double immuno diffusion method is generally quite specific and rea-
sonably sensitive. It can detect virus concentrations of 10–25 lg/ml and can assay
a single seed or parts of a seed or virus infected plant tissues. This method has been
extensively used for the detection of several seed-transmitted viruses viz., BSMV
in barley embryos (Hamilton 1964; Slack and Shepherd 1975; Carroll et al. 1979);
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. 1984) and CMV in french bean
(Padma and Chenulu 1985). This technique was used for the identification of
viruses in infected leaf materials of Rice yellow mottle virus (Fauquet and
Thouvenel 1977) and Tobacco ringspot virus in brinjal (Sastry and Nayudu 1976);
Cowpea mosaic, Cucumber mosaic, Southern bean mosaic and Squash mosaic
viruses (Purcifull et al. 1981); Citrus tristeza virus (Garnsey et al. 1979); Potato
viruses X, S and M (Shepard 1972).
Occasionally in gel diffusion method, non-specific 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 under low speed centrifugation.
5.3 Antibody-Based Tests 243

Depending on the type of seed material and the virus, double immuno diffusion
test was modified by many researchers. Carroll et al. (1979) 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 seed testing laboratory at Montana State university, Boezman (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 West Africa this test is extensively used for the identification and charac-
terization of Rice yellow mottle virus isolates and it was able to reveal the exis-
tence of serological diversity among virus isolates within and among west African
countries (Sere et al. 2005).

5.3.4 Immunochromatography

(a) Rapid immuno filter paper assay (RIPA)

RIPA is another simple, rapid, sensitive and virus specific detection technique which
has been adopted for detection of virus in plants, seeds and other vegetatively
propagated plant materials. In this test, at the bottom of the Whatman glass filter
paper, latex beads coated with virus antibodies were immobilized 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 colored band
appears on the line where the white latex has been immobilized 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 1 year in a dessicator and the coated strips can be used. To the dried
strips, the antigen is added and reactions are observed 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 and other virus infected plants.

(b) Lateral flow test

For onsite detection and identification of plant virus detections, kits based on
lateral-flow were developed. These systems were developed as a result of the need
for rapid pathogen detection and identification in field and/or greenhouse condi-
tions. They utilize specific monoclonal or polyclonal antibodies in an immuno-
chromatographic format, incorporating antibody-coated latex particles. The kits
are designed so that the end user doesn’t require any prior knowledge or expertise
in order to use it, and no facilities or equipment are required. Lateral-flow kits for
the detection of TYLCV have been developed and are available commercially
(Danks and Barker 2000). In this test, tissue taken from a suspected plant is
macerated in a small plastic bottle containing buffer and small plastic beads (or in
244 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

a plastic bag containing buffer) in the field. Two to three drops of the extract are
applied to a small window in a test cassette. After a few minutes, the appearance of
two lines in a viewing window indicates viral infection. One line serves as the
cassette internal control, the other serves as the test line. The appearance of the
control line alone indicates that the cassette worked properly and that the test plant
is negative for the virus in question. No appearance of any line indicates that the
test has failed. These devices can be used at ports, quarantine sites and nurseries.
Danks and Barker (2000) have developed One-step lateral-flow tests for the on-
site detection and identification of several plant viruses. They have utilized specific
monoclonal and polyclonal antibodies in an immunochromatographic format,
incorporating antibody-coated latex particles. These tests have been combined with
a novel extraction procedure to allow disease diagnosis in the field within 3 min.
Lateral-flow devices for two potato viruses (Potato Y potyvirus and Potato X
potexvirus) have been demonstrated as being 100 % accurate in preliminary trials,
when compared with traditional microplate Enzyme-linked immunosorbent assays.

5.3.5 Immuno Electron Microscopy (IEM)

IEM has proved to be very useful in the diagnosis of many viral pathogens of
number of tropical vegetables, fruits, and ornamental crops. Examination of
antigen–antibody complex through pelleting of virus-antibody clumps and resus-
pension of the pellet in neutral phosphotungstic acid before being applied to a
support film (filmed EM grid) and dried, and has been used in the diagnosis of viral
pathogens (Lafferty and Oertelis 1961). Clumping of the virus particles covered
with antibodies was clearly seen in electron microscope. Leaf dip serology,
another quicker method, was described by Ball and Brakke (1968). Leaf-dip
serology involves placement of a drop of diluted antiserum on a filmed grid and
then dipping the freshly cut edge of a virus-infected leaf in the drop for a few
seconds. The drop is then allowed to air-dry and then the grid stained negatively.
However, drying of the virus antibody mixture on the grid disturbs the final image
hence was a major drawback of this technique. Derrick (1973) laid down foun-
dation of the present day IEM. Coating of the filmed grid with antibodies for
trapping the virus and washing of the grid at two points i.e. after antibody
adsorption and after virus adsorption to the grid are advantageous. Thorough
washing to remove salts and other components provides a clear background of the
image. The sensitivity of the test can be increased by the length of time during
which virus-containing fluids are in contact with the antibody-coated grids. When
the incubation time is short (15 min or less), mainly the homologous and very
closely related viruses are trapped. However, after incubation overnight, the effi-
cacy of virus trapping increases and more distantly related viruses are also
detected. Milne and Luisoni (1975) made further modifications to improve IEM. If
IEM is properly standardized, it may prove even more sensitive and reliable than
ELISA (Garg and Khurana 1992; Garg et al. 2000). However, heavy capital cost
5.3 Antibody-Based Tests 245

and requirement of skilled manpower are, the main drawbacks of IEM. Efficiency
of the technique is influenced by pH of extraction buffer, types of ions in buffer,
pH of antiserum diluting buffer, incubation or trapping time, pretreatment of grids
(Garg and Khurana 1994; Garg et al. 2000).
The visualization 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 is in suspension or visual-
ized in thin sections of the infected tissue. In recent years, viruses in suspension
are visualized directly on the electron microscopic grid. This technique is unique
as it combines direct visualization of virus particles with the specificity of a
serological reaction.
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.
The application of ISEM in identification of viruses in true seed and vegeta-
tively propagated plant materials is furnished in Table 5.2.

5.3.6 Labeled Antibody Based Assays

5.3.6.1 Radio Immunoassays

The immuno globulins are marked with radioactive substances (I125, P32, I128).
Their presence is determined by a reaction against photographic material. Anti-
bodies labeled with radioisotopes have been employed in the detection of plant
viruses. These techniques are very sensitive and well suited to detect and quantify
the virus infection in plant and seed materials. 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. However, this technique is rapid and sensitive and could detect
0.1 mg of Turnip yellow mosaic virus and less than 1 mg of Cauliflower mosaic
virus per gram of turnip leaves (Melcher et al. 1980).
Some of the techniques mostly applied for plant virus detection are as follows:
(a) Solid phase radio immuno assay (SPRIA): There are two types of SPRIA. In a
competitive type of assay, unlabelled antigen was first incubated in an antibody
coated polystyrene centrifuge tubes and 125I-labelled antigen is added afterwards.
The radio isotope labeled 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. This test is simple,
economical, sensitive and requires very small quantity of reactants (Ball 1973). This
test was successfully employed to detect SMV in soybean seeds (Hill 1981). Later, a
method based on SPRIA was developed which detects all strains of SMV with equal
246 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

Table 5.2 Application of immunosorbent electron microscopy (ISEM) for virus detection in
seed and vegetative plant propagules of certain crop plants
Crop Virus Reference
Vegetative propagules
Banana Banana streak virus Geering et al. (2000), Agindotan et al. (2006),
Manoranjitham et al. (2012)
Cassava Indian cassava Harrison et al. (1991)
mosaic virus
Garlic Shallot latent virus Meenakshi et al. (2009)
Grapes Grape leaf roll Hu et al. (1991)
clostero virus
Pepper Piper yellow mottle Lockhart et al. (1997)
virus
Potato Potato leaf roll virus Roberts and Harrison (1979)
Potato virus Y and X Garg and Khurana (1992), Khurana et al. (1993)
Potato mop top virus Roberts and Harrison (1979)
Sugarcane Sugarcane mosaic Shukla and Gough (1984), Rao and Maneesha Singh
virus (2008)
Sweet potato Sweet potato Aritua et al. (2009)
chlorotic fleck
virus
True seed transmitted viruses and viroids

Barley Barley stripe mosaic Brlansky and Derrick (1979), Lister et al. (1981),
virus Lundsgaard (1985), Lange and Heide (1986)
Black gram Bean common mosaic Chand et al. (2004)
virus
Black gram mottle Krishnareddy and Varma (1994)
virus
Cacao Cocoa Swollen shoot Sagemann et al. (1985)
virus
Common Bean common mosaic Jafarpour et al. (1979), Russo and Vovlas (1981),
bean virus Lundsgaard (1983), Hagita and Tamada (1984),
Lange and Heide (1986), Raizada et al. (1990),
Nalini et al. (2004)
Cowpea Black eye cowpea Jeyanandarajah (1992), Taiwo and Gonsalves (1982),
mosaic virus Taiwo et al. (1982)
Cowpea aphid-borne Kositratana et al. (1986), Raizada et al. (1991)
mosaic virus
Tobacco streak virus Karunakaran et al. (2008)
Cucumber Cucumber green Mukhayyish and Makkouk (1983)
mottle mosaic
virus
Faba bean Broad bean true Anon (1983)
mosaic virus
Greengram Bean common mosaic Jeyanandarajah (1992)
virus
Melon Melon severe mosaic Ciuffo et al. (2009)
virus
Squash mosaic virus Lange et al. (1983)
(continued)
5.3 Antibody-Based Tests 247

Table 5.2 (continued)

Crop Virus Reference
Lettuce Lettuce mosaic virus Brlansky and Derrick (1979), Van Vuurde and Maat
(1983), Falk and Purcifull (1983)
Pea Pea seed-borne Hamilton and Nichols (1978)
mosaic virus
Peanut Indian peanut clump Reddy et al. (1983, 1998)
virus
Tobacco streak virus Reddy et al. (2002)
Soybean Soybean mosaic virus Brlansky and Derrick (1979)
Tobacco ringspot Brlansky and Derrick (1979)
virus
Squash Zucchini yellow Hosseini et al. (2007)
mosaic virus
Sweet corn Maize dwarf mosaic Mikel et al. (1984)
virus
Wheat Brome mosaic virus Von Wechmar et al. (1984)

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 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): This 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 125I-labelled gamma globulin is substituted for the
globulin enzyme conjugate. The 125I-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). Khan et al. (2003)
have used this technique for detection of important plant viruses in vitro regen-
erated potato plants.
(c) Electro-blot radioimmuno assay (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 and also in seed which
are all virus infected. This technique consists of sodium dodecyl sulphate (SDS)
polyacrylamide gel electrophoresis (SDS-PAGE) of the virus infected seed extract,
248 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

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
(prepared against intact virus), and detection of immune complex with 125I-labelled
protein-A for autoradiographic and scintillation counter detection of virus-antibody
immune complexes (O’Donnel et al. 1982; Shukla et al. 1983; Singh et al. 1991).
The Electro blot radio immunoassay technique has successfully employed in the
detection of Tobacco mosaic virus at a sap dilution of 1: 10,000; four strains of
Sugarcane mosaic virus in their perennial hosts infected for about 4 years, and five
different isolated of Potato leaf roll virus (O’Donnell et al. 1982). Subsequent
studies have developed more sensitive molecular techniques for virus detection and
also the radio immunoassays are unsuitable for many laboratories as the tests
involve the use of radiolabels even though they are sensitive for the virus detection.

5.3.6.2 Fluorescent Immunoassay

This technique uses substances transforming light in the ultraviolet range

(200–400 nm) into longer wave length radiation. A modified microscope (a
fluorescence microscope) allows to see the light emitted by the fluorescing sub-
stance (fluoresceine isocyanate, FITC; rhodamine B).
Fluorescent antibody technique or immunofluorescent assay (IFA):
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 also
employed for detecting and locating viruses in thin sections of seed and plant
tissues. This technique is perhaps the most specific and versatile among all the
histochemical methods, since it provides a means of observing an antigen–anti-
body reaction by chemically linking a fluorescent dye such as FITC or Rhodamin
B to specific antibody molecules. Such labeled antibodies retain the ability to react
specifically with their respective antigens and when viewed under a fluorescent
microscope and the reaction site is relatively more common. There are two ways of
performing the immunofluorescence techniques i.e., direct and indirect methods.
(a) Direct method: In this method antigens are mixed with FITC-labelled specific
antibodies. The reaction gives a brilliant yellow green fluorescence when exam-
ined under a microscope fitted with an ultraviolet light source. For example, this
test was used in Blackgram mottle virus detection in embryo and germinated seeds
of blackgram (Krishnareddy 1989).
(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 in which the virus specific
antiserum was produced viz., rabbit.
Citrus tristeza virus which is wide spread in almost all citrus growing countries,
has been detected in infected plant tissues by fluorescent antibody technique
(Tsuchizaki et al. 1978). Phatak (1974) used the indirect method of immuno-
5.3 Antibody-Based Tests 249

fluorescence for the detection of SMV in germinated seeds of soybean. Free hand
sections of seed and squashes of plummule and small shoots were fixed for
5–10 min in acetone, dehydrated with neutral phosphate buffered saline, followed
by treatment with SMV antiserum and subsequently with the conjugate for 30 min
at 37 C. Sections of virus infected material under a fluorescent microscope,
appeared with more bluish green fluorescence than healthy ones. Similarly, SqMV
was detected in infected embryos, seedlings protoplasts from cotyledons and
microtome sections of dry embryos or seedlings by staining with FITC-labeled
antibodies distributed in clusters of cells in epidermal, palisade and spongy
mesophyll tissues. The virus was detected only in sections of cotyledons from 6-
day old seedlings (Alvarez and Campbell 1978).

5.3.6.3 Enzyme-Linked Immunosorbent Assay (ELISA)

Application of ELISA, a serological technique based on enzymatic amplification

of antigen–antibody interaction for sensitive and reliable detection of plant viruses
initially, was developed by Clark and Adams (1977). It is a solid phase procedure
and is carried out in microtitre plates. The technique utilizes the ability of anti-
bodies raised in animals to recognize proteins, usually the coat protein of the virus
of interest. In this technique antibodies are coated to the surface of the wall of the
micro titre plate and a sap extracted from the plant is added to the well. If the virus
of interest is present in the plant, it binds to the antibodies fixed on the surface.
Any unbound extract is washed-off before a secondary antibody that recognizes
the first antibody which was earlier added. The secondary antibody allows for
indirect detection of the virus because it has a reporter molecule attached to it,
usually an enzyme, alkaline phosphatase (ALP) that acts on a substance, paranitro
phenylphosphate (PNP), a colour less substrate by producing a yellow coloured
product, paranitro phenol. Colour which is detected visually and is measured in
spectrophotometer. With careful calibration, ELISA can be quantitative as well as
qualitative. The substrate P-nitrophenylphosphate is added if alkaline phosphatase
is used to label the antibody or penicillin if penicillinase is used to label the
antibody. Yellow colour developed is measured at 405 nm in a spectrophotometer.
If penicillinase is used, the blue colour changes to orange yellow and finally to
yellow and is measured at 620 nm (Sudarshana and Reddy 1989). For simplifi-
cation, improvement of sensitivity and enhanced reliability, several variants of the
ELISA technique have been developed. In Table 5.3, the list of host-virus com-
binations where ELISA and their variants are successfully used is provided.
Depending on research needs, the following assays of ELISA are frequently
used for plant virus detection and diagnosis.
• DAS-ELISA (Double Antibody Sandwich)
• PTA-ELISA (Plate Trapped Antigen)
• TAS-ELISA (Triple Antibody Sandwich)
• PAS-ELISA (Protein-A Sandwich).
Table 5.3 Application of Enzyme-linked immunosorbent assay (ELISA) for the detection of viruses and viroids in seed and vegetative plant propagules of
certain tropical crops
Plant Virus
Vegetatively propagated plants
Apple Apple chlorotic leaf spot virus
Apple mosaic virus
Apple stem grooving virus
Banana Banana bract mosaic virus
Banana bunchy top virus
Banana streak mosaic virus
Cucumber mosaic virus
Black pepper Piper yellow mottle virus
Cucumber mosaic virus
Pepper yellow mottle virus
Cardamom Cardamom vein clearing virus
Cardamom mosaic virus
Cassava African cassava mosaic virus
Cassava X virus
Indian cassava mosaic virus
Citrus Citrus ring spot virus
Citrus tatter leaf virus
Citrus tristeza virus
Indian citrus ring spot virus
Grape Grape leaf roll clostero virus
Onion Iris yellow spot virus
Tobacco streak virus
Potato Potato viruses
250
Reference
Thakur and Handa (2000)
Bhardwaj et al. (1994), Lakshmi et al. (2011)
Hassan et al. (2008a)
Dhanya et al. (2007), Ariyaratne and Liyanage (2002)
Geering and Thomas (1996)
Thottappily et al. (1998), Ariyaratne and Liyanage (2002), Delanoy et al. (2003), Prakash et al. (2010), James
et al. (2011b)
Kiranmai et al. (1996), Rajasulochana et al. (2008), Hu et al. (1995)
Bhadramurthy et al. (2005)
Bhat et al. (2004), Aglave et al. (2007), de Silva et al. (2002)
de Silva et al. (2002)
Saigopal et al. (1992)
Saigopal et al. (1992)
Thomas et al. (1986), Konate et al. (1995), Ogbe et al. (1997), Malathi et al. (1988)
Martinez and Pinto (2001)
Aiton and Harrison (1989), Harrison et al. (1991), Konate et al. (1995)
Hoa and Ahlawat (2004)
Su and Tsai (1990)
Bar-Joseph et al. (1979), Cambra et al. (1991), Ochasan et al. (1996) , Garnsey and Cambra (1991), Roy and
Ramachandran (2002), Ahlawat and Pant (2003), Baranwal and Ahlawat (2008), Fisher et al. (2011)
Rustici et al. (2000a, b)
Hu et al. (1991), Bertazzon and Angelini (2004)
Bulajic et al. (2009)
Sivaprasad et al. (2010)
de Bokx and Maat (1979), de Bokx et al. (1980), Singh and Somerville (1986), Jordon and Hammond (1991),
Spiegel and Martin (1993), Barker et al. (1993), Salim Khan et al. (2003), Boonham et al. (2009),
Latvala-Kilby et al. (2009)
(continued)
5 Diagnosis and Detection of Plant Virus and Viroid Diseases
Table 5.3 (continued)
Plant Virus
Sugarcane Sugarcane mosaic virus
Sugarcane streak mosaic virus
Sugarcane yellow leaf virus
Sweetpotato Sweet potato feathery mottle virus
Vanilla Cucumber mosaic virus
Yam Cucumber mosaic virus
Yam mosaic virus
True seed-transmitted viruses
Bambarra groundnut Peanut mottle virus
Barley Barley stripe mosaic virus
Cucumber mosaic virus
Blackgram Bean common mosaic virus
Blackgram mild mottle virus
Blackgram mottle virus
Blue berry Blue berry leaf mottle virus
Broadbean Bean yellow mosaic virus
Broad bean stain virus
Capsicum Tobacco mosaic virus
Chickpea Broad bean mottle virus
Common Bean Bean common mosaic necrosis virus
Tobacco streak virus
Cowpea Bean common mosaic virus
Black eye cowpea mosaic virus
Cowpea aphid-borne mosaic virus
Cowpea mild mottle virus
Cowpea severe mosaic virus
Cucumber mosaic virus
Southern bean mosaic virus
Reference
Chen et al. (1998), Rao et al. (2002a, b), Balamuralikrishnan et al. (2004), Gawande et al. (2011), Subba
Reddy et al. (2011)
Hema et al., 2003, Subba Reddy et al. (2011)
Viswanathan and Balamuralikrishnan (2004), Goncalves et al. (2012)
Kashif et al. (2012)
Bhat et al. (2003)
Eni et al. (2008b )
5.3 Antibody-Based Tests
Eni et al. (2008a)
Li et al. (1991)
Qiu et al. (1982), Lange et al. (1983),Mukhayyish and Makkouk (1983), Miller et al. (1986), Huth (1988)
Von Wechmar et al. (1983),
Chand et al. (2004)
Varma et al. (1992), Krishna Reddy and Varma (1994)
Varma et al. (1992), Krishna Reddy and Varma (1994)
Childress and Ramsdel (1986)
Eppler and Kheder (1988), Raizada et al. (1991), El-Dougdoug et al. (1999), Chalam et al. (2007a, b)
Anon (1984), Makkouk et al. (1987), El-Dougdoug et al. (1999); Khetarpal et al. (2001)
Chitra et al. (1999a, b, 2002)
Erdiller and Akbas (1996)
Njau and Lyimo (2000)
Walter et al. (1992)
Chalam et al. ( 2007b), Hao et al. (2001), Udayashankar et al. (2010)
Jeyanandarajah (1992), Puttaraju et al. (2002, 2003, 2004)
Yilmaz and Ozaslan (1989), Hampton et al. (1992), Bashir and Hampton (1993), Ndiaye et al. (1993), Konate
and Neya (1996), Khetarpal et al. (2001), Orawu et al. (2005), Chalam et al. (2007b), Akinjogunla et al.
(2008), Ojuederie et al. (2009), Amayo et al. (2012)
Orawu et al. (2005), Amayo et al. (2012)
Hampton et al. (1992), Bashir and Hampton (1993), Orawu et al. (2005), Amayo et al. (2012)
Hampton et al. (1992), Bashir and Hampton (1992, 1993), Gillaspie et al. (1998a), Abdullahi et al. (2001),
251
Akinjogunla et al. (2008)
Hampton et al. (1992), Bashir and Hampton (1993)
(continued)
Table 5.3 (continued)
Plant Virus
Cucumber Cucumber green mosaic mottle virus
Cucumber mosaic virus
Squash mosaic virus
French bean Bean common mosaic virus
Cherry leaf roll virus
Cucumber mosaic virus
Tomato black ring virus
Guar Guar green-sterile virus
Lentil Bean yellow mosaic virus
Broad bean stain virus
Pea seed-borne mosaic virus
Lettuce Lettuce mosaic virus
Pea early browning virus
Lolium sp. Ryegrass seed borne virus
Maize Maize chlorotic mottle virus
Maize dwarf mosaic virus
Maize mottle chlorotic stunt virus
Maize streak virus
Sugarcane mosaic virus
Melon Cucurbit aphid-borne yellows virus
Melon necrotic ring spot virus
Melon rugose mosaic virus
Melon severe mosaic virus
Squash mosaic virus
Mungbean Bean common mosaic virus
Blackgram mottle virus
Onion Iris yellow spot virus
252
Reference
Kawai et al. (1985)
Ertunc (1992
Nolan and Campbell (1984)
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, 2006a, b), Chalam et al. ( 2007b)
Chalam et al. (2005a)
Davis and Hampton (1986), Hampton and Francki (1992)
Chalam et al. (2005a, 2007a)
Gillaspie et al. (1998b)
Makkouk et al. (1992), Erdiller and Akbas (1996)
Makkouk and Azzam (1986), Erdiller and Akbas (1996)
Varma et al. (1991)
Jafarpour et al. (1979), Ghabrial et al. (1982), Falk and Purcifull (1983), Van Vuurde and Maat (1983, 1985),
Falk and Guzman (1984), Gusenleitner (1985), Dolores-Talens et al. (1989)
Van Vuurde and Maat (1985)
Chester et al. (1983).
Jensen et al. (1991)
Hill et al. (1974), Mikel et al. (1984), Khetarpal et al. (2006)
Taiwo et al. (2006)
Taiwo et al. (2006)
Li et al. (2007)
Mnari-Hattab et al. (2009)
Avegelis and Barba (1986)
Mahgoub et al. (1997)
Ciuffo et al. (2009)
Lange et al. (1983), Avegelis and Katis (1989), Franken et al. (1990)
Jeyanandarajah (1992), Choi et al. (2006)
Saleh et al. (1986), Chand et al. (2004)
Crowe and Pappu (2005)
5 Diagnosis and Detection of Plant Virus and Viroid Diseases
(continued)
Table 5.3 (continued)
Plant Virus
Pea Pea early browning virus
Pea seed-borne mosaic virus
Peanut Cucumber mosaic virus
Indian peanut clump virus
Peanut clump virus
Peanut mottle virus
Peanut mild mottle virus
Peanut stripe virus
Peanut stunt virus
Tomato spotted wilt virus
Pepper Pepper mild mottle virus
Soybean Bean pod mottle virus
Cherry leaf roll virus
Cowpea mild mottle virus
Soybean mosaic virus
Tobacco ringspot virus
Tomato ringspot virus
Sweet corn High plains virus
Tomato Capsicum chlorosis virus
Pepino mosaic virus
Tobacco mosaic virus
Tomato mosaic virus
Reference
Van Vuurde and Maat (1985)
Hamilton and Nichols (1978), Maury et al. (1987), 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),
Coutts et al. (2009)
Reddy et al. (1984), Cai et al. (1986), Demski and Warwick (1986)
Reddy et al. (1988), Reddy et al. (1998)
Dieryck et al. (2009)
5.3 Antibody-Based Tests
Bharatan et al. (1984), Hobbs et al. (1987), Gillaspie et al. (2000), Puttaraju et al. (2001), Prasada Rao et al.
(2004), Khetarpal et al. (2006), Chalam et al. (2007a)
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), Khetarpal et al. (2006)
Cai et al. (1986)
Sreenivasulu et al. (1991)
Svoboda et al. (2006)
Krell et al. (2003)
Chalam et al. (2007a)
Iwaki (1986), Horn et al. (1991), Hampton et al. (1992),
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. (2005a, b, 2008), Andayani et al. (2011)
Lister (1978), Golnaraghi et al. (2004)
Golnaraghi et al. (2004), Chalam et al. (2007a, b)
Forster et al. (2001)
Premachandra et al. (2005), Kunkalikar et al. (2007)
Cordoba-Selles et al. (2007)
Cicek and Yorganci (1991), Chitra et al. (1999a, b, 2002)
Chitra et al. (1999a, b, 2002)
(continued)
253
Table 5.3 (continued)
Plant Virus
Wheat Barley stripe mosaic virus
Brome mosaic virus
Indian peanut clump virus
Wheat streak mosaic virus
Non Seed-transmitted viruses
Capsicum Capsicum chlorosis virus
Chickpea Chickpea chlorotic dwarf virus
Cotton Cotton leaf curl virus
Okra Okra leaf curl virus
Okra yellow vein mosaic virus
Papaya Papaya mosaic virus
Peanut Tobacco streak virus
Soybean Tobacco streak virus
Sunflower Tobacco streak virus
Tobacco Tobacco leafcurl virus
Tomato Peanut bud necrosis virus
254
Reference
Lister et al. (1981), Qiu et al. (1982), Khetarpal et al. (2006)
Von Wechmar et al. (1984)
Reddy et al. (1998), Delfosse et al. (1999)
Jones et al. (2005)
Krishnareddy et al. (2008)
Horn et al. (1996), Kumari et al. (2004)
Parveen et al. (2010)
Swanson and Harrison (1993)
Prakasha (2009)
Akanda et al. (1991), Tennant et al. (1994)
Prasada Rao et al. (2003b)
Arun Kumar et al. (2008)
Jain et al. (2000), Prasada Rao et al. (2000, 2003a), Ramiah et al. (2001), Bhat et al. (2001)
Swanson et al. (1998)
Manjunatha (2008), Reddy et al. (2008)
5 Diagnosis and Detection of Plant Virus and Viroid Diseases
5.3 Antibody-Based Tests 255

Fig. 5.1 Diagrammatic drawings of the most frequently used methods for detection of plant
viruses: a Direct ELISA (DAS-ELISA), b Indirect ELISA (PTA-ELISA), c Triple antibody
sandwich (TAS-ELISA) and d Protein A-sandwich (PAS-ELISA). Courtesy Albersio J

For details of the above ELISA assays, refer Fig. 5.1.

The first reported method is the double antibody sandwich-ELISA (DAS-
ELISA) where the antibody is bound to the solid phase (e.g. polystyrene micro titer
plate), then the test samples containing the antigen of interest is added. Disease
sandwiched by adding another antibody which has been conjugated with an
enzyme such as Alkaline phosphatase. When substrate for the enzyme is added
such as p - nitro phenyl phosphate, a colour reaction product is obtained. In a
positive test, the substrate solution turns colored, whereas a negative test remains
colorless. The color intensity, which is proportional to virus antigen concentration,
can be measured by spectrophotometrically. Since the report of Clark and Adams
in 1977, many ELISA variants were reported by using different enzymes on
universal conjugates (Cooper and Edwards 1986; Hsu and Lawson 1991).
256 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

Plate Trapped Antigen-ELISA (PTA-ELISA) is also known as antigen coated

plate and is to allow the virus, in the absence of any specific virus trapping layer as
in DAS-ELISA, to adsorb on the plate surface by adding the test sample directly to
the wells. In the second step, virus antibody (usually called as primary antibody) is
added either as IgG or crude antiserum. The primary antibody is then detected with
antispecies antibodies (secondary or detecting antibody) conjugated to an enzyme,
followed by addition of colour development reagents. The detecting antibody
binds specifically to the primary antibody, since the former is produced against
IgGs from the animal in which virus antibodies are raised (e.g., if virus antibodies
are produced in rabbits, antirabbit IgGs are produced in a second species such as
goats). It has certain disadvantages such as competitions between plant sap and
virus particles for sites on the plate and high background reactions.
Another technique is triple antibody sandwich-ELISA (TAS-ELISA) which is
similar to DAS-ELISA, except that an additional step is involved before adding
detecting antibody-enzyme conjugate. In this step, a monoclonal antibody (MAb)
produced in another animal (usually mice) different from the trapping antibody is
used. This MAb then detected by adding an enzyme-conjugated species-specific
antibody (e.g., rabbit antimouse IgG), that does not react with the trapping anti-
body followed by colour development reagents. By following this technique,
Prune dwarf ilarvirus in sweet cherry trees (Rampitsch et al. 1995); Apple mosaic
virus in apple (Pasquini and Barba 1991); Sugarcane streak mosaic virus in
sugarcane (Hema et al. 2003); Banana streak virus in banana (Manoranjitham
et al. 2012); Yam mosaic virus in Yam (Eni et al. 2012) and Tospoviruses in
vegetables (Kunkalikar et al. 2011) were detected.
In Protein-A Sandwich-ELISA (PAS-ELISA), Protein-A is used in two appli-
cations to sandwich antibody–antigen–antibody layers. The first applied layer of
protein A prepares the plate for the coating antibody layer. The second layer of
protein A is conjugated to the enzyme and detects the second antibody layer. The
orientation of the IgG induced in the coating layer of antibody prevents later
unwanted reaction with the conjugated protein-A. By PAS-ELISA, Prune dwarf
virus was detected in 18–36 % of tested Prunus avium seeds (Edwards and Cooper
1985). This technique was also proved to be effective in identifying Yam mosaic
virus in yam (Eni et al. 2012). This permits the use of unfractionated antisera and
is more sensitive than DAS-ELISA.
In DAC-ELISA method, the first step of coating the solid phase with antibodies
is deleted and is that the antigen/virus particles are adsorbed directly on the solid
phase and hence the method is known as Direct Antigen Coating - ELISA. To the
antigen coated wells, either monoclonal or polyclonal antibodies of antigen spe-
cific were added after washing with PBS-T and incubated at 37 C. The goat anti-
rabbit antibodies labeled with ALP diluted with PBS-TPO was added to the wells.
After incubation at 37 C, the enzyme substrate PNP was added to the wells and
incubated for colour development in the dark at room temperature. The reaction
was terminated by adding 3N NaOH solution at 50 ll/well. The reactions were
noted according to colour intensity. The color developed was read at A405nm and
5.3 Antibody-Based Tests 257

OD values recorded. This test is used by Rajasulochana et al. (2008) and Dheepa
and Paranjothi (2010) for identifying CMV in banana, Tospoviruses in vegetables
(Kunkalikar et al. 2011); Sugarcane yellow leaf virus in sugarcane (Rao et al.
2000); Peanut viruses in peanut (Reddy et al. 1988a) and Soybean mosaic virus in
soybean (Ahangaran et al. 2009).

(a) Modifications of ELISA

These modifications of ELISA can be grouped under two categories viz., direct
and indirect ELISA. When primary antibody-enzyme conjugate binds directly to
the antigen, it is known as direct ELISA, while if primary antibody-enzyme
conjugate does not bind directly to the antigen, it is referred to as indirect ELISA.
Further DAS-ELISA is more specific and shows narrow spectrum i.e. may not
detect distinct strains of the same virus while triple antibody sandwich (TAS-
ELISA) is less specific, broad spectrum and more sensitive and hence useful in the
establishment of serological relationship among strains of the same virus or
between different viruses (Koenig and Paul 1982).
In some of the variants of ELISA, immunoassay sensitivity can be enhanced by
the use of different amplification systems, with avidin-biotin being the most
common. In addition to the polystyrene plates, a number of solid phase supports
were found adequate. Assays in which antibodies or virus particles are bound to
nitrocellulose membranes were used as immunoblots or dot-blots. Dot blot-ELISA
tends to be rapid, easy to perform and conservative of reagents and often more
sensitive than ELISA, carried out in a microtiter plate and the details are provided
in the subsequent pages.
Another modification viz., Microarray-based multiplex ELISA has modernized
and enhanced the efficacy of hitherto used serological assays and specifically to
detect numerous antigens simultaneously (Mendoza et al. 1999). Production of
monoclonal antibodies (MAbs) of high affinity and specificity has increased the
reliability of ELISA. Due to reasonably high sensitivity, reliability, specificity and
amenability to automation, ELISA has become the most popular and useful
techniques particularly for large-scale indexing of seed/propagative stocks like
potato and many other horticultural crops (de Bokx and Maat 1979; Singh and
Somerville 1986; Khurana and Garg 1993; Hassan et al. 2008a; Bulajic et al. 2009;
Fisher et al. 2011). The recombinant antibodies used to detect the plant viruses is
called as Recombinant antibody ELISA (R-ELISA):
The technique is based on recombinant antibodies (rAb) produced in bacteria.
These are the fragments of variable heavy and light chains of antibodies (scFv).
Selected specific scFv fused with different recombinant-proteins, including alka-
line phosphatase (AP) with a high enzyme activity, have been used in ELISA for
the detection of Potato leaf roll virus (PLRV) (Al-Mrabeh et al. 2009). Viruses
like black currant reversion associated virus for which specific polyvalent anti-
bodies could not be produced, scFv R-ELISA has proved very effective. scFv
encoding DNA have indefinite storability. E.coli takes 2–4 weeks to produce scFv.
MAbs are not only expensive to produce and some hybridoma cell lines either die
or lose antibody production during storage.
258 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

(b) Electroblot immuno assay (EBIA)

Electro blot immuno assay (EBIA) is performed after extraction of proteins from
virus infected plants by SDS PAGE. The protein bands are transferred on nitro-
cellulose membranes at 20 V for 2 h as described in O’Donnell et al. (1982). The
free sites on the membrane are blocked with serum albumin or fat-free dried milk
powder. Virus specific antiserum is added to probe the viral protein be it in CP or
any other. The antigen-antibody complex is then detected by 125I-labelled protein
or ELISA. This method identifies a virus by two properties viz., the molecular
weight and serological specificity of the viral protein. Homologous antiserum
against CMV was used at 1:1000 and antirabbit IgG labelled with alkaline
phosphatase was used at 1:20,000 (Sigma Chemical Co., St: Louis, USA). Pre-
stained marker protein (Bio-Rad, Richmond, CA, USA) was used as size standard.
Madhubala et al. (2005) have used this technique for diagnosis of Cucumber
mosaic virus in vanilla. Sreenivasulu et al. (1991) have identified TSWV isolates
by EBIA. At Zimbabwe, Sibiya et al. (1998) have identified the Potyvirus infecting
maize to be sugarcane mosaic virus strain MDB by using EBIA technique.
Burgermeister and Koenig (1984) have studied serological relationships of dif-
ferent viruses by EBIA extensively and reported both advantages and disadvantages
in this technique. They have reported that the coat proteins of tymo-, tombus-,
como-, nepo-, tobamo-, potex-, carla- and Potyviruses were subjected to Sodium
dodecylsulphate (SDS)-polyacrylamide gel electrophoresis, electro-blotted onto
nitrocellular membranes and reacted with homologous and heterologous antìsera to
intact plant viruses. Immune complexes were detected after reaction with alkaline
phosphatase-labelled goat anti-rabbit antibodies. SDS coat proteins of Comoviruses
reacted only with antisera which were homologous for their intact particles.

(c) Dot-immuno binding assay (DIBA)

This technique has been variously described as dot-blot immuno binding assay
(DIBA) by (Berger et al. 1984). This technique is also known as Immuno blot
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;
Bantarri and Goodwin 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.
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
plant material or seed extract is spotted on Nitrocellulose membrane (NCM)
instead of a microplate. In this technique, a few microliters of sap can be spotted
on the NCM which absorbs small amounts. After incubating and washing the
antigen, a blocking agent is added to saturate any unoccupied binding sites on the
NCM. Then the NCM is incubated in a solution IgG rabbit antibody specific to
the antigen to be detected and unbound antibody is removed by washing. Horse
radish peroxidase or ALP conjugated anti-rabbit second IgG is added to NCM
incubated and washed to remove unbound antibody. Finally, the enzymes specific
5.3 Antibody-Based Tests 259

Fig. 5.2 Detection of

Soybean mosaic virus by
DIBA with substrate solution
NBT/BCIP. F5 Positive
control, and F3 Negative
control. Courtesy Ahangaran
et al. 2009

substrate is added which is hydrolysed to form water-insoluble colored spots or

dots on the membrane. Horse radish peroxidase, the most frequently used enzyme
that produces purple spots after addition of substrate. Alkaline phosphatase reacts
with napthol AS-MX phosphate mixed with 5-chloro-2-toludinediazonium chlo-
ride hemizene chloride (fast red TR salt) to produce red dots, or with diazotized
4-benzolamino-2,5-dimethoxyaniline ZnCl2 (fast blue BBN salt) to produce blue
dots on the white background of the NCM (see Fig. 5.2).

Application of DIBA
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 for each test virus and also a single generally
applicable enzyme conjugate, (4) cost of nitrocellulose is less than that of plastic
microplates, (5) while testing the seed-borne viruses, part of the seed can be used
for testing and the remaining can be used for sowing, and (6) DIBA can be directly
adopted to field situations.
For the first time this technique was used by Von Wechmar et al. (1984) 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), PDV and PNRV in plum and cherry (Otto et al.
1991); BSMV in barley (Lange and Heide 1986; Lange et al. 1989); PSbMV in
peas (Lange et al. 1989; Ligat et al. 1991; Ali et al. 1998); TSWV in impatiens sps.
(Hsu and Lawson 1991) and Soybean mosaic virus in soybean (Ahangaran et al.
2009) (Fig. 5.2). The Papaya ring spot virus (PRSV) infection was detected in the
field by using this technique (Byadgi 2008). Caciagli and Bosco (1996, 1997) have
quantitatively determined the Tomato yellow leaf curl virus by DIBA in plant and
whitefly extracts. Zein et al. (2007) from Egypt have extensively used this tech-
nique for the identification of Cucumber mosaic virus.
By using dot-blot ELISA, Cucumber mosaic and Banana streak viruses
infecting banana have been detected (Hu et al. 1995; Rajasulochana et al. 2008). In
India Sugarcane streak mosaic virus (SCSMV) in sugarcane leaves and stem
260 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

pieces was detected by Hema et al. (2003). At southern Tanzania, Ndunguru et al.
(2009) have applied DIBA techniques for the detection of sweet potato viruses.
Tripathi et al. (2008) tested the transgenic plants of banana by DIBA for the
detection of BBTV. The 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 six months to test samples.
Since nitrocellulose membrane is comparatively costly, alternative use of plain
paper and blotting paper were tried in DIBA. Heide and Lange (1988) have
established that Potato leaf roll virus and also Potato viruses M, S, X and Y could
be detected even by using plain paper which was equally sensitive like the use of
nitrocellulose membrane in DIBA tests. Even in India, positive results were
obtained by using Blotting paper in Digoxigenin-labeled dot blot test, for identi-
fication of katte disease of cardamom, Papaya ring spot virus in papaya and
Peanut green mosaic virus in peanut (Saigopal DVR, unpublished data).

(d) Tissue blot immuno binding assay (TBIA)

Tissue blot immuno binding assay and Tissue print immunoassay techniques are
similar to dot-blot immuno assay and tissue print immunoassay, involves tissue
imprinting on Nitrocellulose Membrane (NCM). This technique does not involve
disruption of tissue or extraction of antigen from the targeted plant sample. The
fresh infected plant material or imbibed seed material can be used as a imprint
tissue on NCM (Lin et al. 1990; Hsu and Lawson 1991). Viruses contained in the
sap of virus infected plant materials like leaf/ petiole/ stem, become adsorbed to
this membrane and are detected by means of antibodies labeled with an enzyme
that is able to convert a color less soluble substrate in to a colored insoluble
product. For Alkaline Phosphatase, 5’ bromo- 4’ chloro- 3’ indolylphosphate
(BCIP) is commonly used as substrate. The tissue blotted antigen samples can be
processed as in dot-blot immunoassay. To remove the interference of plant tissues,
detergents like triton-X-100 or sodium hypochlorite can be used. The antigen
blotted membrane can be stored for several weeks at 4 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 detected ten viruses of fababean by
using this technique. Similar technique has also been used earlier for the detection
of Barley yellow dwarf virus in different cereal crops (Makkouk and Comeau
1994).
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 CMV infected lentil mother plants were
tested by TBIA, the CMV infection range was 0.9–9.5 % in 2000–2001 and
0.1–1.17 % in 2000–2002. Khatab Eman et al. (2012) have tested this technique to
detect BBTMV in faba beans. This technique does not involve disruption of tissue
or extraction of antigen from the plant or seed sample. In addition, tissue
5.3 Antibody-Based Tests 261

imprinting can provide data on virus localisation within plant organs (Makkouk
et al. 1993; Knapp et al. 1995). By following this technique Garnsey et al. (1993)
have identified Citrus tristeza virus in stem cuttings of infected citrus. Even
Ahangaran et al. (2006) have used this technique for identifying Soybean mosaic
virus in soybean. As early as 1995, Louro has identified Tospovirus from tomato,
capsicum, chrysanthemum, gladiolus, hydrangea and oleander plants from the field
samples by this technique.
This TBIA technique also helps in diagnosis of viroid diseases. Based on this
technique Apple scar skin viroid (ASSVd) which is seed transmitted in apple and
pear were identified (Hadidi et al. 1991; Hurtt and Podleckis 1995). By following
this technique even the stone fruit viroids viz., Peach latent mosaic viroid and Hop
stunt viroid were detected (Matic et al. 2005).
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 4C and can be used effectively for processing of the bulk
samples and also testing at field level. 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.
Western blotting: It is another technique, where polyacrylamide gel electro-
phoresis and Tissue print immunoassay are combined for plant virus diagnosis. By
means of Western blotting, the correct sizes and the time course of the expression
of the structural and non-structural proteins of a virus can be determined. In this
technique plant virus proteins either partially purified or purified are separated on
Sodium dodecel sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and are
then transferred on to nitrocellulose membrane (NCM) by electro blotting either by
using wet transfer or semi-dry blot. The protein-binding capacity of NCM is much
higher than polysterene surface. The transferred virus proteins irreversibly bound
to the membrane, where the viral proteins are detected by means of enyzme-
labeled antibodies as described earlier for Tissue print immuno assay. Some of the
examples of successful application of this technique are viz., Naghavi et al. (2008)
identified Soybean mosaic virus strains based on the molecular weight of viral
proteins. Sherwood and Melouk (1986) have detected Peanut mottle virus and
Peanut stripe virus by following this technique. From Taiwan, Lin et al., (1989)
have demonstrated the practical application of this technique in diagnosis of
Passion fruit woodiness virus in passion fruit. A combination of native electro-
phoresis and western blot analysis (NEWeB), was quite effective in distinguishing
two different strains of Plum pox virus in single and mixed infections (Manous-
sopoulos et al. 2000). The western blotting provides a useful method for qualitative
identification and molecular weight determination of viral proteins and for
determination of serological relationship of plant viruses.
262 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

(e) Disperse dye immunoassay (DDIA)

Gribnau et al. (1982) have developed DDIA method which is as sensitive as
ELISA, seems promising as an alternative for ELISA in routine application.
Advantages of DDIA over ELISA are the replacement of relatively expensive
enzymes by cheap dye sol particles and the replacement of the substrate incubation
step by addition of a simple organic solvent to dissolve the dye molecules from the
dye sol conjugate. For DDIA this results in an immediate overall staining of the
well in case of infected samples.
DDIA is a solid-phase immunoassay like ELISA wherein the enzyme conjugate is
replaced by immediate dissolving of dye molecules with an organic solvent, dime-
thyl sulphozide (DMSO). After the addition of DMSO, the plates are shaken for a
minute and color intensity is read at 540 nm. Van Vuurde and Maat (1985) have used
this technique to detect LMV and PEBV in the seeds of lettuce and pea, respectively.
The advantages of DDIA are: (a) preparation of conjugate is simple and
cheaper, (b) eliminates substrate incubation step, and (c) possibility of simulta-
neous 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.

(f) Application of ELISA for virus detection in seeds

ELISA has been useful in revealing and detecting virus situation in different seed
parts of the planting material, tolerance limits and detection of latent infections. It
is so sensitive that even a single infected embryo can be detected when diluted up
to 2000 w/v in case of SMV in soybean (Bossennec and Maury 1978; Maury et al.
1983), 3600 w/v for PMV in peanut (Bharatan et al. 1984) or when mixed with
1400 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; Falk and Purcifull 1983), PMV in peanut (Bharatan
et al. 1984), PSbMV in pea (Maury et al. 1987; Khetarpal and Maury 1990) and
SqMV in cucurbits (Nolan and Campbell 1984). Excepting BSMV, a large vari-
ation of virus concentration has been found in different infected embryos form the
same seed lot (Carroll 1980). Amount of PMV detected in axis and cotyledon
extracts from the same peanut seed were found to be the same (Bharatan et al.
1984), contrary to another isolate of PMV which was not detected in cotyledons
(Adams and Kuhn 1977). It was found by using ELISA, that the concentration of
SMV and Blackgram mottle virus (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).
The other applications of ELISA are primarily in epidemiological studies for
testing the weed hosts and vectors for the presence of the virus and sometimes it is
also used for detection of viruses in water. In recent years this technique is being
used for testing transgene products and detection of virus gene encoded proteins in
replication. Already exhaustive information is provided regarding the role of ELISA
in diagnosing the seed-borne viruses at quarantine stations and also in gene banks.
5.3 Antibody-Based Tests 263

The ELISA techniques 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. More information on ELISA techniques, can be
obtained from the articles of (Bar-Joseph and Garnsey 1981).

(g) Application of ELISA for virus detection in insect vectors

With increased advantages of ELISA, it has been successfully employed for
detecting several virus diseases which are circulative and propagative type were
detected even in the body of the insect vector. For example Rice ragged stunt virus
was detected even in the single plant hopper (Nilaparvata lugens) by using ELISA
(Hibino and Kimura 1982). Muniyappa et al. (2000) have detected Tomato leaf curl
virus in whitefly vector, Bemisia tabaci. Even Inoue et al. (2010) could detect the
Iris yellow spot virus in the thrips vector, Thrips tabaci, by using the DAS-ELISA.
Depending on the necessity and requirement, the ELISA technique has taken lot of
improvements for use in virus diagnosis and in epidemiological studies.

(h) Application of ELISA for virus detection in vegetative propagules

Majority of the fruit crops and certain vegetable crops are multiplied through
vegetative propagules like bud sticks, rhizomes, runners, corms, tubers, etc., both
virus and viroid diseases are carried to next generation through vegetative struc-
tures. ELISA is extensively used for virus disease diagnosis in the vegetative
propagules and also in certification schemes. Some of the examples where viruses
are detected in different crops are viz., banana (Prakash et al. 2010; James et al.
2011b); citrus (Konate et al. 1995; Baranwal and Ahlawat 2008; Fisher et al. 2011);
cassava (Ogbe et al. 1997; Martinez and Pinto 2001); potato (Salim Khan et al.
2003; Latvala Kilby et al. 2009); sugarcane (Chen et al. 1998; Rao et al. 2002a, b)
and other crops. More details about ELISA application is presented in Table 5.3.

5.4 Viral Nucleic Acid Based Tests

5.4.1 Molecular Hybridization

Sensitivity and reliability of the molecular hybridization methods depend on the

concentration and distribution of the viruses, the virus recovery during sample
preparation, and the quality of probes used to detect viral nucleic acids. Detection
of viral pathogens in infected samples is based on the production of nucleic acids
by specific hybridization between the single-stranded target nucleic acid sequences
and complementary single-stranded probes.
Molecular hybridisation-based assays were first utilized in plant virology to
detect Potato spindle tuber viroid (Owens and Diener 1981). Furthermore, mul-
tiple RNA riboprobes or polyprobes have been used to detect different viruses
(Ivars et al. 2004; Herranz et al. 2005) and they can be associated with tissue
printed or squashed material in addition to the spotted extracts. Molecular
264 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

hybridisation can also be applied to the specific detection of PCR amplicons

thereby increasing their sensitivity and specificity levels (Bertolini et al. 2001) and
reducing time when a flow-through system is used (Olmos et al. 2007a).
These are stretches of nucleotides complementary to a specific region or whole
genome of a virus or viroid. Thus, application of nucleic acid probe helps in
molecular hybridization i.e. binding of two strands of nucleic acids (DNA and
RNA). This binding can be affected in solution (solution hybridization) or on solid
support like filter paper or membrane (filter hybridization). Solid support
hybridization is also called Nucleic Acid Spot Hybridization (NASH).
For large scale indexing of virus and viroids, nucleic acid probes have been
used (Owens and Diener 1981; Singh et al. 1994; Singh and Singh 1995). Both
radio-labeled (e.g., P32, I125, I128) non-radio active labeled, (e.g., digoxigenin,
chemiluminescent) probes (cDNA or cRNA) have been developed for the purpose
(Singh and Singh 1995; Podleckis et al. 1993). Increase in size of the cDNA probe
from ca 0.56 kb to ca 3.25 kb significantly enhance the sensitivity of detection of
PVY (Dhar and Singh 1994). More details on nucleic-acid hybridization proce-
dures can be had from reference books chapter of Hull (1993) and Nayudu (2008).

(a) Nucleic Acid Spot Hybridization (NASH)

NASH is also a powerful technique with widespread application in plant virus
diagnosis. It is based on hybridization (binding) of complementary DNA sequences.
The affinity of one strand of DNA for its complementary sequence is one of the
strongest and most exquisitely specific interactions found in nature. This specificity
has been exploited in developing nucleic acid hybridization assays, which are based
on the homology between two strands of nucleic acid. In these assays, a single-
stranded complementary nucleic acid (either DNA or RNA), which has been
‘‘labelled’’ with a reporter molecule is used as a probe to form a hybrid with the
target nucleic acid. The double-stranded probe-target hybrid molecules are then
detected by several methods, depending on the reporter molecule used. This method
is less sensitive than PCR but also generally simpler and cheaper.
By using NASH technique Foster and Millis (1990) have detected the strains of
Potato virus S in potato. Based on this method, cucurbit geminiviruses in plant
tissue extracts of squash by Polston et al. (1989) and Citrus mosaic virus in citrus
were detected by Bhaskara Reddy (1997) (Fig. 5.3).
Nucleic acid hybridization of DNA or RNA probes has the advantage of being
able to detect the nucleic acid of the virus in both forms, single-stranded and
double-stranded. cRNA probes can be labeled with isotopes or non radioactive
probes. cRNA probes are preferable to cDNA probes when used to detect RNA
viruses, because RNA/RNA hybrids are more stable than DNA/RNA hybrids. An
RNA extraction from infected tissue is blotted onto a membrane and the probe
hybridized to it and detected. For example Orchid viruses like Cymbidium mosaic
virus and Odontoglossum ringspot virus were detected by Hu and Wong (1998) by
following this technique. Since NASH is a simple and cheaper technique, it is
being used for identification of viruses in different host plants including greening
pathogen in citrus (Gopal et al. 2009).
5.4 Viral Nucleic Acid Based Tests 265

Fig. 5.3 Nucleic Acid Spot Hybridization (NASH). Courtesy M. K. Nakhla (APHIS) and D.
P. Maxwell (UW-Madison)

(b) Dot-blot hybridization (DBH)

This hybridization format simply answers the question of whether a plant is
infected or not infected by a virus. Dot blotting does not distinguish between the
number and size of hybridized molecules, since the hybridization signal is the sum
of all sequences recognized by the probe. However, the technique is rapid and
versatile in identifying specific nucleic acid sequences in samples ranging from
crude plant sap to highly purified preparations. The DBH was conducted by using
non-isotopic digoxigenin labeled probes and isotopic labeled probes for identifi-
cation of virus and virod diseases of plants. The application of spot hybridization
for the detection of DNA and RNA viruses in plant tissues has been reviewed by
Maule et al. (1983) and Pallas et al. (1998a).

Application of DBH for the detection of plant virus and viroid diseases: Dot-
blot hybridization (DBH) is extensively used for the detection of plant viruses and
viroid diseases of vegetable and fruit crops. Even though this test generally does
not distinguish types and sizes of nucleic acids, it can be very useful for qualitative
detection since this method can discriminate closely related but different target
sequences. Isotope hybridization by using P32, I125 and I128, the virus and viroid
diseases were tested, wherever lab facilities were existing. For example Plum pox
virus (PPV-D) was detected in infected orchards by using various lengths of radio
266 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

actively labeled probes (Wetzel et al. 1990). The detection limit was of about 5 pg
of purified virus per assay. In Columbia, a strain of Soybean mosaic virus infecting
passiflora spp. was identified by this method (Benscher et al. 1996). From India,
Borah et al. (2008) have used this technique for the detection of Citrus yellow
mosaic badna virus from citrus species. The DBH using radio labeled RNA probes
were able to detect serotypes of prunus was shown to be as sensitive as DBH using
radioactively labeled probes for Cherry leaf roll virus (CLRV) (Mas et al. 1993).
Rice tungro virus in rice (Mangrauthia et al. 2010). By following this technique
Pesic and Hiruki (1986) have detected AMV in infected Alfalfa pollen by using
AMV specific 32p labelled cDNA probe.
Potato and pome fruit viroids were also detected by this technique viz.,
Podleckis et al. (1993) and Khan et al. (2009). Citrus exocortis viroid was detected
by using both radioactive and non-radioactive probes (Flores 1986; Fonseca et al.
1996). From Iran, Hop stunt viroid and Citrus exocortis viroid diseases from
Washington Novel orange (Citrus sinensis) were diagnosed by following this
technique (Bagherian et al. 2009). Imprint-hybridization (IH) assay was used for the
detection of viroids that are not possible to detect using serological methods and
showed that IH is fast and sensitive, and provides additional information on the sites
of viroid accumulation (Romero-Durban et al. 1995) and is the preferred detection
method for viroid indexing, especially when handling a large number of samples.
Based on the host and pathogen RNA dot blot hybridization method was mod-
ified and named as slide hybridization. For the first time Zhiyou et al. (2007) have
developed this technique of using dot-blot hybridization on glass slides with fluo-
rescently labeled probes for detecting plant RNA viruses and a viroid. An optimum
efficiency of RNA binding onto surfaces of activated glass slides was achieved
using aminosilane-coated glass slides as a solid matrix and 5x saline sodium citrate
(SSC) as a spotting solution. Combined with a CY5-labelled DNA probe prepared
through PCR amplification, the optimized glass slide hybridization could detect as
little as 1.71 pg of Tobacco mosaic virus (TMV) RNA. The sensitivity of the
modified method was four times better than that of dot-blot hybridization on nylon
membrane with a P32 labeled probe and this method is of high specificity. By this
technique even Potato spindle tuber viroid was also detected specifically, showing
the extensive application of this method to plant virus/viroid diagnosis.
In the above examples the radio actively labeled probes have been commonly
employed but the concern is about the environmental impact, safety and cost of
using radioactive labels have prompted the development of alternative hybrid-
ization methods that employ non-radioactive labels. The use of such hybridization
methods for detection of plant viruses that increased with digoxigenin (dig) labeled
probes and were used in the plant virus detection.
The non-isotopic digoxigenin labeled probes are widely used successfully.
Presently molecular hybridization is encouraged using non-isotopic digoxigenin-
labelled probes. For example this technique has been employed for the past 20 years
detection of viruses like Apple mosaic virus (ApMV), Prunus necrotic ringspot virus
(PNRSV), Prune dwarf virus (PDV), Plum pox virus (PPV), and Apple chlorotic leaf
spotvirus (ACLSV) (Pallas et al. 1998b), Citrus psorosis virus (CPsV),
5.4 Viral Nucleic Acid Based Tests 267

Citrus variegation virus (CVV) and Citrus tristeza virus in citrus by (Loconsole
et al. 2009; Barbarossa and Savino 2006). Cherry mottle leaf virus in cherry (James
et al. 1999). Lettuce infectious yellows virus, Zucchini yellow mosaic potyvirus and
Beet yellows clostero virus (Harper and Creamer 1995). Two Potyviruses infecting
peanut (Dietzgen et al. 1994) and TSV from infected sunflower, gherkin, pumpkin,
marigold, globe amaranth (Sarovar and Saigopal 2010). Rodriguez et al. (2011)
identified three Begomoviruses, viz., Bean golden mosaic virus (BGMV); Soybean
blistering mosaic virus (SbBMV) in bean and soybean respectively by using this
technique.
Based on DBH technique, number of viroid diseases were also diagnosed; for
example, the presence of apple scar skin group viroid in infected sap extracts could
be detected by DBH, even at a minimum of 2.0–2.5 pg of purified viroid. Tissue
blot of cross-sectioned Chrysanthemum stunt viroid infected chrysanthemum
stems or leaf petioles gave positive reactions when hybridized with the digoxi-
genin-labeled probe (Hooftman et al. 2001). Viroid diseases like Potato spindle
tuber viroid has been detected by this technique by Khan et al. (2009); Welnicki
and Hiruki (1992) and Pome fruit viroids (Podleckis et al. 1993).
Even with Digoxigenin (DIG)-labeled probes the Geminiviruses like Squash
leaf curl virus and Beet curly top virus (Harper and Creamer 1995). Cucurbit
Geminiviruses by Maule et al. (1983). and Bean golden mosaic virus Rodriguez
et al. (2011) have been identified.
Although preparation of the viral probes requires a well equipped laboratory, it
has been found that laboratories with access to an available probe can readily adapt
this detection method for use with minimal equipment. This method is suitable for
routine and large scale testing, for example, phytosanitary certification schemes
that require processing of many samples in a short time. To save time and reduce
cost and labor, the simultaneous use of the six riboprobes in a hybridization
reaction was proposed for the phytosanitary certification of tomato seedlings in the
nursery (Saldarelli et al. 1996). This technique is also widely used in breeding
programs to screen for resistance and to detect viruses in their vector.

(c) Southern and Northern blot hybridization

These hybridization formats give more qualitative results than dot blot hybrid-
ization since they precisely identify the molecule recognized by the probe (Sam-
brook and Russel 2001). This technique is useful mainly in the determination of
the pattern of the viral nucleic acid, detection of the non encapsidated nucleic acid
(e.g., subgenomic RNAs, defective-interfering nucleic acids (DI), satellite RNAs,
small-interfering RNAs (siRNAs in RNA silencing studies) and detection of virus-
related transgenic inserts for basic research and for regulatory issues. This tech-
nique was used in the recognition of Rice yellow mottle virus (Brugidou et al.
1995). For detection of viroid diseases also this technique was used. For example
Bagherian et al. (2009) have identified HSVd and CEVd viroid diseases in Citrus
sinensis by following southern blot hybridization technique. Khan et al. (2009)
have detected PSTVd in the leaf tissue of potato plants by following northern blot
hybridization technique.
268 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

(d) In situ hybridization

In situ hybridization (ISH) is used to detect either specific viral sequences; it
combines microscopy observation and hybridization. In situ hybridization gives
information on the distribution of the target nucleic acid within a cell or tissue and
is routinely applied to the localization of specific viral sequences involved in
replication and movement (Cillo et al. 2002) and to detect the integration of viral
sequences in the plant chromosome (e.g., Banana streak virus in Musa sp.)
(Harper et al. 1999a, b).
This technology has been applied for detecting plant viruses such as Apple stem
pitting virus (Klerks et al. 2001), PPV (Olmos et al. 2007a), Potato virus Y, and
Arabis mosaic virus (ArMV) The sensitivity of this method has been proved to be
similar to that obtained by real-time RT-PCR when applied to Plum pox virus
(PPV) detection (Olmos et al. 2007a).

5.4.2 Polymerase Chain Reaction (PCR)

Nucleic acid based methods are sensitive, specific and allow genetic relationships
determination. These tests have several advantages over serological assays. The
antigenic determinants of viral coat proteins used for most serological assays
represent only about 2–5 % of the viral genome. Many characteristics in virus
strains and isolates are governed by other major portions of viral genomes and thus
cannot be differentiated by serological assays. The cloned or cDNA probes with
appropriate common or specific sequences of nucleotides can be prepared and
labeled in different ways. The polyvalence of the molecular hybridization assay was
further improved by using RNA probes corresponding to structural and non
structural protein encoding genes, which has been shown to diagnose and differ-
entiate virus strains. The sensitivity can be increased by amplification of desired
sequences by using PCR. PCR was developed over 30 years ago, and its use in the
diagnosis of plant diseases has become very common in laboratory practice. Its
advantages (speed, sensitivity, specificity) are far more important than its draw
backs (risk of contamination, sensitivity to inhibitors, complexity, cost), and several
modifications to solve these problems have been performed with success. In gen-
eral, PCR, with all its variants, is currently a basic tool in diagnosis, alone or
preferentially in combination with other techniques. As for any target, PCR effi-
ciency for detection of viruses is based on the primer specificity (Lopez et al. 2003).

5.4.2.1 Steps in PCR

The PCR has been used as the new standard for detecting a wide variety of
templates across a range of scientific disciplines, including virology. The method
employs a pair of synthetic oligonucleotides or primers, each hybridizing to one
strand of a double stranded DNA target, with the pair spanning a region that will
5.4 Viral Nucleic Acid Based Tests 269

Fig. 5.4 Steps in polymerase chain reaction (PCR) exponential amplification. Courtesy Tolin
and Chang

exponentially reproduced. The hybridized primer acts as a recognition site for a

DNA polymerase, which creates a complementary strand via sequential addition of
deoxynucleotides (dNTPS). The process can be summarized in three steps:
(i) dsDNA separation at temperatures above 90 C, (ii) primers annealing for at
*40–60 C, and (iii) optimal extension at 72 C. The rate of temperature change,
the length of the incubation at each temperature and the number of times each
cycle is repeated are controlled by a programmable thermal cycler. The amplified
DNA fragments will then be separated by agarose gel electrophoresis and the
bands are visualized by staining with ethidium bromide and irradiation with
ultraviolet light. The specificity of PCR testing is dependent on the primer sets
used. There are virus species specific primers and genus specific primers. The
primers can detect all species of the genus Nanovirus and other primer sets that can
detect an individual virus species within that genus.
The diagrammatic representation of PCR is shown in Fig. 5.4 which is
universally used for the detection of plant viruses.

5.4.2.2 Polymerase Chain Reaction-based Tests

Polymerase chain reaction (PCR) is an In vitro method in which DNA sequences

are rapidly amplified with very high specificity and fidelity using oligonucleotide
primers and thermostable DNA polymerase. The amplified fragment (amplicon) is
detected by gel electrophoresis or characterized by nucleic acid hybridization,
restriction enzyme digestion sequencing. The major advantage of PCR is that it
270 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

can be used to increase the concentration of pathogen-related sequences which in

naturally infected hosts are below detection level either because they occur in too
low amounts or localized in certain tissues (i.e., phloem-limited viruses), or are
erratically distributed.
The availability of nucleotide sequences of many viruses and viroids has
enhanced the use of PCR-based assays as diagnostic tool. The PCR is a very
powerful method that has greatly facilitated detection of plant viruses that would
be difficult or time consuming to detect using conventional assays (Hadidi et al.
1995; Candresse et al. 1998b; Rao and Maneesha Singh 2008). The PCR products
can be used (a) as a target for hybridization, (b) for direct sequencing of DNA, and
(c) as a specific probe. The advantages of PCR-based assays include high sensi-
tivity, high specificity, and high sample throughput. It has been reported that using
PCR-based assay one can claims the detection of around 10 femtograms (fg) of
viral RNA (Romaine and Schlagnhaufer 1995). In comparison with serological
assays, PCR primers with any degree of selectivity can be synthesized at a much
lower cost than that associated with the development of monoclonal or polyclonal
antibodies. Because very small amounts of nucleic acid are needed for PCR
amplification, the development of rapid, small-scale procedure would allow testing
of many samples and increase the efficacy of PCR as a tool for routine diagnostics.
Ironically, high sensitivity also increases the risk of sample carry-over contami-
nation restricting PCR-based assays for routine usage.

5.4.2.3 Detection of ssRNA Viruses

Reverse transcription polymerase chain reaction (RT-PCR)

Nearly 70 % of known plant viruses have RNA genome which is single
stranded. For detecting ssRNA viruses reverse transcription (RT) PCR is a
stranded method, which involves an initial step of reverse transcription that con-
verts single strand RNA to cDNA. Accordingly, the PCR procedure followed for
the detection of ssRNA viruses is known as Reverse transcription PCR. This
procedure is a sensitive, fairly inexpensive and requires minimal skill to perform.
In the case of RNA viruses, oligonucleotide primers, flanking part of the genome
of the virus are extended by a thermostable DNA polymerase in a series of
denaturation and extension steps that exponentially increase the target DNA. The
sensitivity of the method is its major advantage.
Some of the ssRNA which were detected by reverse transcriptase RT-PCR are
Tobacco streak virus in sunflower (Kumar et al. 2008; Sharman et al. 2008;
Sarovar et al. 2010); Onion yellow dwarf and Leek yellow stripe viruses in garlic
(Takaichi et al. 1998, 2001); Peanut stripe virus (Gillaspie et al. 2001; Dietzgen
et al. 2001) and Tomato spotted wilt virus in peanut (Jain et al. 1998b); Cowpea
aphid borne mosaic virus in cowpea (Salem et al. 2010); Sweet potato viruses
(Kokkinos and Clark 2006); Sugarcane mosaic virus in sugarcane (Viswanathan
et al. 2010; Subba Reddy et al. 2011); Zucchini yellow mosaic virus in squash
5.4 Viral Nucleic Acid Based Tests 271

(Hosseini et al. 2007; Simmons et al. 2011; Manju Sharma et al. 2013); Cucumber
mosaic virus in banana (Singh et al. 1995; Hu et al. 1995); Potato virus Y and
Potato leaf roll virus in dormant potato tubers (Singh and Singh 1996; Awan et al.
2010; Crosslin and Hamlin 2011).
The reverse transcription (RT)-PCR assays have been used for the detection of
several viruses infecting woody plants, viz., Plum pox virus (PPV) was detected by
PCR in infected bark of trees so that the assay can be performed throughout the
year (Korschineck et al. 1991; Wetzel et al. 1991; Glasa et al. 2011). Citrus
tristeza virus in citrus spp. (Fisher et al. 2011); Grapevine leafroll associated
virus-3 in grapes (Tsai et al. 2008); and Prune dwarf Ilarvirus in stonefruits
(Parakh et al. 1995) have been identified by following this technique. Kundu et al.
(2003) have detected Apple stem grooving virus by this technique. Borja and Ponz
(1992) also detected Cherry leaf roll virus (CLRV) in infected walnut buds and
twigs using virus specific probes that amplified a specific fragment of 448 bp from
30 non translated region of viral RNAs. These assays have been employed for the
detection of several other fruit tree viruses (Candresse et al. 1995b; Kokko et al.
1996; Nolasco et al. 1993; Rosner et al. 1997; Spiegel et al. 1994; Vitushkina et al.
1994). This technique is rapid, highly specific and sensitive for detection of plant
viruses in importing and exporting plant materials at quarantine stations. In Korea,
Lee et al. (2011a, b) have used this technique for the detection of five viruses
belonging to Poty and Tospovirus genus. In fruit crops because of phenols, tannins
and other virus inhibitors, the reverse transcription (RT)-PCR cannot be used
universally. Hence the development of rapid methods for RNA extraction from
infected tissue samples helps for over coming these limitations in the diagnosis and
characterization of viruses using reverse transcription (RT)-PCR.
Single stranded RNA viruses are present in virus families like potyviridae,
bromoviridae, bunyaviridae, closteroviridae, rhabdoviridae, comoviridae, tom-
busviridae, luteoviridae, sequiviridae, and viruses belonging to these families were
identified by reverse transcription (RT)-PCR or variants of PCR. Possible draw-
backs of the method include need for a thermo cycler and sequence information for
designing primers. As initial knowledge of the nucleotide sequence is required in
order to design oligonucleotide primers, it cannot be used in identifying an
unknown virus.

5.4.2.4 Single-Cell-RT-PCR (SC-RT-PCR)

For SC-RT-PCR, total nucleic acid extracts prepared by reversible binding on

silica particles in the presence of guanidinium thiocyanate proved to be suitable for
RT-PCR detection of PPV, ACLSV, PDV, and Apple stem pitting virus belonging
to different virus groups (Malinowski 1997). SC-RT-PCR seems to be useful.
Immuno-PCR is another highly sensitive assay that uses streptavidin-labeled DNA
fragments linked to antigen–antibody (protein A linked) complex. This complex is
272 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

then bound to biotin-labeled DNA sequences followed by PCR amplification. This

assay is shown to be 1051 times more sensitive than ELISA (Sano et al. 1992) and
only requires antigen-specific antibody.

5.4.2.5 Detection of dsRNA Viruses

Double stranded RNA viruses of Reoviridae, Betaflexiviridae and Partiviridae are

detected with the help of PCR method. Fang et al. (2001) identified Rice black
streaked dwarf fijivirus in maize which was dsRNA virus. Even Southern rice
black streaked dwarf virus, a new proposed Fijivirus species in the family Reo-
viridae was also identified by PCR (Zhou et al. 2008). Cherry mottle leaf virus
(CMLV) of Prunus avium belonging to genus Trichovirus was identified by
RT-PCR, which was hundred times more sensitive than dot blot hybridization.
Even IC/RT-PCR and PC/RT-PCR were effective for the detection of CMLV in
herbaceous and woody tissues (James et al. 1999)

5.4.2.6 Detection of ssDNA Viruses

Like ssRNA viruses, ssDNA viruses also require a template with the production of
a replicative intermediate. The ssDNA can be amplified by using a complementary
DNA and later the second strand synthesis is required.
The PCR technique is extensively used against whitefly transmitted DNA
viruses (e.g. viruses of the genera Geminivirus, and Nanovirus). Among whitefly
transmitted viruses Bean golden mosaic virus (BGMV) (Gilbertson et al. 1991);
Tomato yellow leafcurl virus (Navot et al. 1992; Leam Khang et al. 2005; Mason
et al. 2007); East African cassava mosaic cameron virus (Alibi et al. 2008); Indian
cassava mosaic virus (Makesh Kumar et al. 2005); Banana bunchytop virus
(Nanovirus) (Selvarajan et al. 2007; Prakash et al. 2010) were identified by PCR.
Even Rojas et al. (1993) have identified leafhopper transmitted Beet curly top
geminivirus by this technique. This aspect has been reviewed by number of
workers (Moriones and Garcia-Andres 2008).

5.4.2.7 Detection of dsDNA plant viruses

The direct PCR is being used to detect the double stranded DNA (dsDNA) viruses.
In this technique dsDNA is directly is used as template and the primers can be used
directly for amplication of the genome. The PCR cycles are performed like other
methods of amplification. This direct PCR method is beneficial for detection of
dsDNA viruses of family Caulimoviridae and the method is simple and within
short period virus can be diagnosed. Citrus mosaic virus (Baranwal et al. 2003),
Citrus yellow mosaic virus (Baranwal et al. 2003; Borah et al. 2009); Cocoa
swollen shoot virus (Quainoo et al. 2008) are detected by PCR and its variants.
5.4 Viral Nucleic Acid Based Tests 273

5.4.3 PCR Variants

(a) Immunocapture-PCR (IC-PCR)

This technique is used with plant extract or with immobolised targets allowing
detection of minimal quantities of RNA targets from plant material or insect
vectors without going for extract preparation. This combines capture of virus
particles by antibodies with amplification by PCR. In this method, the virus is
absorbed by the antibody bound to a surface, then removed by heating with a
nonionic surfactant such as Triton X-100. The nucleic acids are then amplified by
using PCR (DNA viruses) or RT-PCR (RNA viruses). Although PCR can be very
sensitive and specific, its introduction for routine detection has been hampered by
its lack of robustness and by the complexity of the post-amplification analysis
required. PCR sometimes fails to correctly diagnose both infected and non-
infected plant material since carry-over contamination of amplicons may lead to
false-positive results, and inhibitor components in sample extracts may yield false
negatives. The sensitivity of PCR amplification can be enhanced in a number of
ways. The template chosen that were first trapped on a solid support by a specific
antiserum. This method is especially useful in concentrating virus particles from
plant species where virus titre is low, or where compounds that inhibit PCR are
present; for example, plum tree sap containing Plum pox virus (Wetzel et al.
1992); Banana bunchy top virus in Banana (Selvarajan et al. 2007); and sugarcane
sap containing Sugarcane streak mosaic virus (Hema et al. 2003). It has also been
used for detection of the episomal Banana streak virus, parts of whose genome are
naturally present within the banana genome, and therefore there is a high chance of
false positives from standard PCR tests (Harper et al. 1999a; Agindotan et al.
2006). This system, named immunocapture-PCR (IC-PCR) (Wetzel et al. 1992),
makes it possible to use sample volumes 200–250 times greater than those utilized
in standard PCR, and has been used with plant extracts or with immobilized targets
on paper (print/squash-capture[PC/SC] RT-PCR) (Olmos et al. 1996) allowing
viral detection from plant material or insect vectors without extract preparation.
Candresse et al. 1995a, b have detected several strains from Orchard trees of apple,
pear, plum, cherry, apricot, peach and quince. Mulholland (2009) has reviewed the
application of immuno capture PCR for plant virus detection.

(b) Immunocapture-Reverse Transcriptase-PCR (IC-RT-PCR)

Detection of viral pathogens becomes more sensitive when antibody binding and
PCR are combined. The sensitivity of detection is 250-times that of direct PCR
(Wetzel et al. 1992). Immunocapture-reverse transcriptase-polymerase chain
reaction (IC-RT-PCR) assay has been developed for the detection of several
economically important RNA viruses (Nemchinov et al. 1995a; Nolasco et al.
1993; Jain et al. 1997; Fuji et al. 1998; Jacobi et al. 1998; Sharman et al. 2000;
Hema et al. 2003; Ahangaran et al. 2009). In IC-RT-PCR the immunocapture of
virions from crude plant extracts is carried out directly in RT-PCR tubes in the
same manner of ELISA. This step concentrates and pre-purifies the virus particles.
274 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

RT-PCR is carried out using RNA extracted from trapped virions. For IC-RT-PCR,
plant extracts are pre-incubated with specific antiserum in PCR tubes in a fashion
reminiscent of ELISA assay. This step concentrates and pre-purifies the virus
particles. Immuno-captured samples were then used for RT-PCR omitting the need
for nucleic acid extractions. This method shows increased detection sensitivity
compared to ELISA by several orders of magnitudes (Candresse et al. 1995b;
Hadidi et al. 1995; Jacobi et al. 1998; Werner et al. 1997; Wetzel et al. 1992;
Hema et al. 2003; Ahangaran et al. 2009; Sreenivasulu and Saigopal 2010; Eni
et al. 2012). IC-RT-PCR is reliable over a large part of the growing season for the
detection of Apple chlorotic leafspot virus (ACLSV) strains taken from orchard
trees of apple, pear, plum, cherry, apricot, peach, and quince (Candresse et al.
1995a; Haddi 1995). Sarovar et al. (2010) have detected Tobacco streak virus by
this technique in sunflower, gherkin and pumpkin. In Uganda, Mukasa et al. (2003)
have identified five viruses infecting sweet potato viz., SPCSV, SPFMV, SPMMV
and SPCFV by RT-PCR and IC-RT-PCR. Even Nemchinov et al. (1995b) have
also reported that ACLSV in apple and peach could be detected even by IC-PCR,
IC-RT-PCR and multiplex IC-PCR. Werner et al. (1997) have applied this sen-
sitive method in the detection of CLRV. IC-RT-PCR assay was sensitive enough to
detect minute amount of CLRV in several woody plant samples. A sensitive
duplex-IC-RT-PCR technique was developed by Subba Reddy et al. (2011) for
detection and discrimination of Sugarcane streak mosaic virus and Sugarcane
mosaic virus which are naturally infecting sugarcane and proved to be very sen-
sitive technique.

(c) 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 particles and debris, disruption 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.

(d) Duplex PCR

Duplex RT-PCR technique helps in identification of mixed infection of Tobam-
oviruses in tomato and bellpepper (Vinayarani et al. 2011) and was positive in
identification of number of viruses in different host combinations. Even both
tristeza and greening diseases in citrus were also detected (Dilip Ghosh and Das
2012). Gupta et al. (2007) have simultaneously detected Citrus mosaic virus and
Indian citrus ringspot virus by this technique. In peanut two Potyviruses and two
Cucumoviruses were differentiated by using duplex RT-PCR assays. These assays
would be useful for testing peanut leaves or seeds for virus identification in epi-
demiological studies, seed testing or in post entry quarantine (Dietzgen et al.
2001). Simultaneous detection of viruses causing mosaic in sugarcane has been
studied by Viswanathan et al. (2008).
5.4 Viral Nucleic Acid Based Tests 275

(e) Multiplex-PCR
Multiplex-PCR is very useful in plant virus detection because different viruses
frequently infect a single crop or alternate hosts. This method is helpful in
detecting multiple species and strains of different viruses that frequently infect a
single host as a step towards propagation of pathogen free plant material. Multi-
plex PCR allows simultaneous and sensitive detection of different DNA and RNA
targets in a single reaction (Lopez et al. 2006). There are several examples of
simultaneous detection of viruses (Periasamy et al. 2006; Olmos et al. 2007b). To
identify the strain mixtures of Potato virus Y, this technique was quite helpful
(Lorenzen et al. 2006). Nevertheless, there are still very few examples in which
more than three plant viruses are amplified in a single PCR-based assay, however
there were some difficulties in virus identification probably due to the technical
difficulties of involving so many compatible primers.
Multiplex-PCR (M-PCR) is also found to be very useful for the detection of
several viruses in a single reaction (Mumford et al. 1996; Nie and Singh 2001;
Kierks et al. 2001; Szemes et al. 2002; Verma et al. 2004; Avijit et al. 2005). The
simultaneous detection of two or more DNA or/and RNA targets can be afforded
by duplex or multiplex-PCR in a single reaction with several specific primers
included in the PCR cocktail. This technique is being used for simultaneous
detection of African cassava mosaic virus and East African cassava mosaic viruses
in sub-Saharan Africa (Alabi et al. 2008).
The design of a multiplex RT-PCR is based on the use of compatible primers
specific to different targets. The method is particularly useful where primers are
specific for different viruses. It is important that the amplicons are of different
lengths and that there is no cross-reactivity among them. Lorenzen et al. (2006)
have identified different isolates and strain mixtures in potato. Yokomi et al.
(2010) and Avijit et al. (2010) have also used this technique in differentiating the
severe strains of Citrus tristeza virus. Two successful examples are the simulta-
neous detection of six major characterised viruses affecting olive trees: CMV,
CLRV, SLRSV, Arabis mosaic virus (ArMV), Olive latent virus-1 and Olive latent
virus-2 (Bertolini et al. 2001) and the simultaneous detection of nine grapevine
viruses (Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virusA, Grape-
vine virus B, Rupestris stem pitting-associated virus, Grapevine fleck virus,
Grapevine leaf roll-associated virus-1, -2 and -3) (Gambino and Gribaudo 2006)
and seven Tospoviruses infecting vegetables (Kunkalikar et al. 2011). From India,
Viswanathan et al. (2010) have identified three major RNA viruses infecting
sugarcane by following this technique. Even this technique was useful in detecting
viroid diseases, for example Singh and Nie (2003) and Khan et al. (2009) detected
PSTVd in potato and other solanaceous plants. Ito et al. (2002) have detected six
citrus viroids and one virus by this technique.
Sharman et al. (2000) have developed a multiplex immunocapture PCR with
colorimetric detection for viruses of banana.
276 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

(f) Nested-PCR
In this method, two PCRs are carried out with the first reaction increasing the
amount of template for the second. The method is particularly useful where the
virus has very low titre or inhibitors of DNA polymerase are present in the plant
extract. Low-specificity oligonucleotides, usually degenerate, are used in the first
round of amplification. Then, an aliquot of the reaction is placed into a fresh tube
for a second PCR with primers that anneal within the first amplicon. This increases
the target molecule and dilutes inhibitors. Since, nested-PCR requires two rounds
of amplification in different tubes, risk of contamination is increased. In order to
avoid this problem, several alternatives with single closed tubes have been
developed (Yourno 1992). This method has been used successfully to detect
members of Vitivirus and Foveavirus species in grapevines (Dovas and Katis
2003b; Dovas et al. 2003). Adkins et al. (2008) have detected Squash vein
yellowing virus from Momordica charantia by following this technique.

(g) Multiplex nested-PCR

Multiplex nested PCR method in a single tube, combines the advantages of the
multiplex PCR with the sensitivity and reliability of the nested PCR. In this
technique two reactions are sequentially performed using a single reaction cock-
tail. In addition, it enables simultaneous detection of RNA and DNA targets. The
accurate design of compatible primers is necessary to avoid hair pins and primer-
dimer formation. Although there are some examples in which multiplex nested
PCR has been used for detection of phytoplasmas, fungi and viruses (Clair et al.
2003; Stukenbrock and Rosendahl 2005; Dovas and Katis 2003a), only in one case
this technology was performed in a single tube for specific detection of CMV,
CLRV, SLRSV, ArMV (Bertolini et al. 2003). This method was used to detect six
citrus viroids and Apple stem grooving virus (Ito et al. 2002).

(h) Co-operational-PCR (Co-PCR)

A new PCR concept, based on the simultaneous action of three or four primers, has
also been developed (Olmos et al. 2002). This technique, named co-operational
amplification (Co-PCR), can be performed easily in a simple reaction increasing
the sensitivity level and using ten times less reagent than in conventional PCR. The
reaction process consists of the simultaneous reverse transcription of two different
fragments from the same RNA target, one internal to the other, the production of
four amplicons by the combination of the two pairs of primers, one pair external to
the other, and the co-operational action of amplicons for the production of the
largest fragment (Lopez et al. 2003). Coupled with colorimetric detection, the
sensitivity observed is at least 100-times greater than that achieved with RT-PCR,
and is similar to that of nested RT-PCR. Co-PCR requires only one reaction,
minimizing manipulation and reducing risk of contamination. However, the small
volume of reagents could increase susceptibility to inhibitors, requiring a previous
RNA extraction to reach a good sensitivity in detection (Olmos et al. 2002). The
technique was first developed and used successfully for the detection of plant RNA
5.4 Viral Nucleic Acid Based Tests 277

viruses, such as CTV, PPV, Cucumber mosaic virus (CMV), Cherry leaf roll virus
(CLRV) and Strawberry latent ring spot virus (SLRSV) (Olmos et al. 2002).

(i) Continuous-flow PCR (CF-PCR)

It is a modified method of PCR which could be used to differentiate different
strains of a virus where 50 fluorescent dye labeled oligos are used for amplification.
Upon obtaining the amplicon, the dye fluoresces only in a double stranded hybrid.
This technique is used chiefly to differentiate the viruses with divergence in 30 end
nucleotide sequences and also used to differentiate the multiple strains of the
Potato virus Y (Walsh et al. 2001; Webster et al. 2004).
It is a variation of the above technique. It is used to simultaneously to differ-
entiate between virus strains and multiple virus infections. Several primer sets,
each labeled with a different fluorescent marker, are added to the reaction mixture.
Virus strains are differentiated with primers that differ only at the 3 end, com-
plementary to a nucleotide position that is polymorphic between strains. Extension
occurs only where the 30 nucleotide is complementary. Only primers that generate
amplicons fluorescence and the wavelength emitted identifies the primers that have
been extended. Potatoes infected with multiple strains of Potato virus Y were
identified using this method.

(j) PCR-ELISA
PCR-ELISA assay enables immuno enzymatic determination of PCR products in
the liquid phase without the need for electrophoresis, thereby simplifying the
analysis of the amplified products. These highly sensitive assays have been used
for the diagnosis of PPV-D and PPV-M isolates in plum trees and tobacco (Poggi
Pollini et al. 1997). The usefulness of PCR-ELISA has been demonstrated for the
detection of Citrus tristeza virus and Rupestris stem pitting-associated virus
(Nolasco et al. 2002). The usefulness of PCR-ELISA has been demonstrated for
the detection of Citrus tristeza virus and Rupestris stem pitting-associated virus
(Nolasco et al. 2002). Simultaneous detection and identification of Prunus necrotic
ringspot and Apple mosaic viruses was done by Candresse et al. (1998c). When
serial dilutions of infected plant extracts were assayed, PCR-ELISA was found to
be 100-times more sensitive than conventional IC-PCR (Olmos et al. 1997). The
PCR-ELISA is simple to use, capable of processing large sample numbers, and
eliminates the use of hazardous chemicals (e.g., ethidium bromide) during elec-
trophoretic procedures, especially if restriction fragment length polymorphism
analysis of the amplified products is necessary. To make the PCR-ELISA to be
further simple and effective with wider application, Nolasco et al. (2002) have
modified and simplified the procedure by using asymmetric PCR. This eliminated
the need to denature and neutralize samples prior to hybridization. It also increased
the relative concentration of the target DNA species, making PCR ELISA more
sensitive than TaqManTM, a fluorescence-based detection method. Reducing the
reaction volumes to half and the concentration of the dNTPs and the digoxigenin
label by tenfold significantly reduced the costs of PCR ELISA without reducing its
sensitivity.
278 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

(k) Detecting immobilized, amplified products in a one-phase system

(DIAPOPS)
It is another technique often used for the detection of Potato virus Y (PVY), where
Nucleo Link TM strips (NUNC A/S, Roskilde, Denmark) are covalently coated
with an oligonucleotide which serves as one of the two primers in the following
PCR (RT-DIAPOPS) (Nicolaisen et al. 2001). During PCR, amplicons are cova-
lently attached to the micro well surface which serves as targets for a biotin-
labelled probe subsequently detected in ELISA reader after adding suitable
streptavidin-enzyme/substrate combination. To detect plant pathogenic virus, an
RT step prior to DIAPOPS is required.

(l) Nucleic acid sequence based amplification (NASBA)

NASBA is an isothermal amplification method that can be used to detect RNA
targets. The reaction requires the use of three enzymes, AMV-RT for reverse
transcription and to obtain double stranded cDNA, RNase H to hydrolize the RNA
fragment of the hybrid molecule DNA-RNA and T7 RNA polymerase to produce a
large amount of anti-sense, single strand RNA transcripts corresponding to the
original RNA target. It can be achieved by using two specific primers (one of them
include the T7 promoter at 50 end), NTPs and also dNTPs. The entire NASBA
process is performed at 41 C for 60 min and the typical level of amplification is at
least a factor of 109. The detection of NASBA products can be assessed by chemi-
luminescent or colorimetric detection using an internal specific probe Digoxigenin
(DIG) labelled or in a real-time assay using molecular beacons (Amplified RNA)
(van Beckhoven et al. 2002). NASBA-beacon assay yields results in less than 1 h
(Robert and Kerst 2001), and offer the advantages that no contaminating DNA is
amplified. It is performed at 41 C without the need of a thermal cycler, and
requires only 60 min reaction affording high levels of sensitivity, superior in some
cases to real-time PCR (Scuderi et al. 2007). NASBA technique was applied for
the detection of Strawberry vein banding virus (Vaskova et al. 2004).
Kim et al. (2006) have modified NASBA technique by including electro-
chemiluminescence (ECL) and named the technique as (NASBA-ECL). This
method was faster and hundredfold more sensitive than reverse transcription-
polymerase chain reaction (RT-PCR) for detection of viroid diseases like Apple
scar skin viroid (ASSVd) in apple. The viroid was easily detected by this tech-
nique in leaves, stems, fruit skin and seed coat of ASSVd infected apple. This
technique also helps in identification of seed-borne nature of viroid diseaes.
Another improvement made in virus detection by NASBA is the molecular
beacons combined with NASBA for sensitive detection of Sugarcane yellow leaf
virus (Goncalves et al. 2002). In this technique the AmpliDet RNA consists of
nucleic acid sequence-based amplification (NASBA) of the target RNA with
specific primers and simultaneous real-time detection of the amplification products
with molecular beacons. The results showed that the system produced a detection
level of at least 100fg of purified virus. Sugarcane yellow leaf virus (ScYLV) was
5.4 Viral Nucleic Acid Based Tests 279

readily detected in sugarcane plant tissues with low levels of infection (without the
need of previous RNA extraction) and in the hemolymph of aphids. The method
showed to be virus-specific, testing negative for other species of the Luteoviridae
and the system has potential to become a diagnostic method for the detection of
sugarcane viruses.

(m) Loop-mediated isothermal amplification (LAMP)

Loop-mediated isothermal amplification (LAMP) is another type of isothermal
amplification that it is being increasingly used in the diagnostic field offering
sensitivity and economic costs (Notomi et al. 2000). It requires a set of four
specifically designed primers that recognize six distinct sequences of the target and
a DNA polymerase with strand displacement activity. The amplification products
are stem-loop DNA structures with several inverted repeats of the target and
cauliflower-like structures with multiple loops, yielding [500 mg/ml. The LAMP
reaction was enhanced by the addition of loop primers (Nagamine et al. 2002),
reducing time and increasing sensitivity. The amplification takes place at
60–65 C for 60 min. Although it was initially developed for DNA, it can be
adapted to amplify RNA (RT-LAMP) (Fukuta et al. 2003a).
The LAMP reaction is performed by a set of two specially designed inner
primers (FIP and BIP) and outer primers (F3 and b3). FIP and BIP primers contain
two distinct regions, respectively. FIP primer has the complementary sequence of
F1 region followed by the sequence of F2 region. BIP primer has the sequence of
B2 region. The LAMP reaction is started by the hybridization of FIP primer to F2
region and the complementary strand is synthesized. The strand extended from the
outer primer (F3 or B3) replaces this strand by Bst DNA polymerase with strand
displacement activity. This released strand forms a looped out structure, and acts
as a template for BIP primer. Consequently, a dumb-bell form DNA is produced.
This serves as the material for the LAMP cycling amplification step. The final
products are a mixture of stem–loop DNAs with various lengths.
The LAMP method has been used for the detection of viruses (Notomi et al.
2000; Fukuta et al. 2003b) by amplification of specific sequence of each genomic
DNA. However, most plant pathogenic viruses have RNA genomes. The reverse
transcription-LAMP reaction for RNA templates has been reported for detection of
Japanese yam mosaic virus (JYMV) (Fukuta et al. 2003a).
The method has been applied to the detection of plant viruses such as PPV, with
a sensitivity level similar to that obtained by real-time PCR (Varga and James
2006). Fukuta et al. (2003b) have applied this technique for the detection of
Tomato yellow leaf curl virus in tomato.
The genomic DNA molecule of Tomato yellow leaf curl virus (TYLCV), a
whitefly-transmitted Begomovirus, was amplified from total DNA extracts of
TYLCV-infected tomato by the use of LAMP. The procedure was also used to
amplify TYLCV DNA from total DNA extracts of individual whiteflies (Bemisia
tabaci) that had fed on TYLCV-infected plants. One of the characteristics of the
280 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

LAMP method is its ability to synthesize an extremely large amount of DNA.

Accordingly, a large amount of bye-product, pyrophosphate ion, is produced
yielding a white precipitate of magnesium pyrophosphate in the reaction mixture.
The presence or absence of this white precipitate allows easy detection of
amplification of TYLCV genomic DNA without gel electrophoresis. Nie (2005)
has detected Potato virus Y by following reverse transcription loop-mediated
isothermal amplification of DNA. Kuan et al. (2010) have detected Squash leaf
curl virus (SLRV) by this technique. Although both the LAMP and the PCR
methods were capable of detecting SLCV in infected tissues of squash and melon,
the LAMP method would be more useful than the PCR method for detection of
SLCV infection in cucurbitaceous plants because it is more rapid, simple, accurate
and sensitive. The LAMP reaction is a very efficient and specific method for the
amplification of DNA templates (Notomi et al. 2000; Mori et al. 2001).

(n) Reverse Transcription-Loop-mediated Isothermal Amplification (RT-

LAMP)
In the RT-LAMP method, TSWV genomic RNA could be amplified under iso-
thermal (65 C) conditions within 1 h. The resulting amplicons were detected by
the measurement or observation of the turbidity of the reaction mixture without gel
electrophoresis. Studies on the application of RT-LAMP have shown its applica-
bility in the detection of plant viroid diseases like Potato spindle tuber viroid in
potato (Tsutsumi et al. 2010) and the details are given in the Fig. 5.5.

Fig. 5.5 Detection of PSTVd with the designed RT-LAMP primer. a Measurement result of
turbidity by real-time turbidity meter (LA200, Teramecs). Turbidity of the RT-LAMP reaction at
65 C from the total RNA extracted from the potato leaves (filled circle PSTVd-S, filled triangle
PSTVd-M, and filled square healthy potato) and the tomato leaves (open circle PSTVd-S, open
triangle PSTVd-M, open diamond TCDVd, and open square healthy tomato). b Agarose gel
electrophoresis of the specific amplification products from the total RNA extracted from the
tomato leaves by RT-LAMP. Lane 1 PSTVd-S, 2 PSTVD-M, 3 TCDVd, and 4 healthy tomato.
M:DNA size marker (100 bp ladder). c Confirmation of the amplification by the white precipitate
in the RT-LAMP reaction mixtures. 1 potato leaf infected PSTVd-S, 2 potato leaf infected PSTV-
M, and 3 Healthy potato. Courtesy Tsutsumi et al. 2010
5.4 Viral Nucleic Acid Based Tests 281

5.4.4 Real Time Quantitative PCR

The research workers have welcomed the ability to visualize the progress of
amplification in a quantitative manner. This approach has provided insight into the
kinetics of the PCR reaction and it is the basis of ‘‘real time’’ PCR. The monitoring
of accumulating amplicons in real time has been possible by the labeling of
primers, probes or amplicons with fluorogenic molecules. The increased speed of
real time PCR is largely due to reduced cycle times, removal of post-PCR
detection procedures and the use of fluorogenic labels and sensitive methods of
detecting their emissions. The reduction in amplicon size generally recommended
by the inventors of commercial real-time assays may also play a role in this speed,
but decreased product size does not necessarily improve PCR efficiency. Quanti-
tative real-time PCR is based on detection of a fluorescent signal produced pro-
portionally during the amplification of a PCR product. A probe (e.g. TaqMan) is
designed to anneal to the target sequence between the traditional forward and
reverse primers. The probe is labeled at the 50 end with a reporter fluorochrome
and a quencher fluorochrome added at the 30 end. The probe is designed to have a
higher Tm than the primers, and during the extension phase, the probe must be
100 % hybridized for success of the assay. As long as fluorochromes are on the
probe, the quencher molecule stops all fluorescence by the reporter. However, as
Taq polymerase extends the primer, the intrinsic 50 to 30 nuclease activity of Taq
degrades the probe, releasing the reporter fluorochrome. The amount of fluores-
cence released during the amplification cycle is proportional to the amount of
product generated in each cycle. Similar to the conventional PCR, in case of RNA
viruses, amplification can be measured after extraction of total RNA and prepa-
ration of a cDNA by a reverse transcription (RT) step. Real time PCR has proven
increasingly valuable diagnostic tool for plant viruses. For example, Sweet potato
viruses in sweet potatoes (Kokkinos and Clark 2006), Potato viruses in potato
tubers (Boonham et al. 2009), Plum pox viruses in stone fruits (Jarosova et al.
2010) and grapevine viruses in grapevine (Osman et al. 2012) were identified by
this technique. However, it requires an initial high capital investment to acquire
the needed equipment, as compared to other techniques. In real-time PCR, as well
as for isothermal amplifications the selection of small fragments for amplification
is recommended. For this purpose, software packages with different primer and
probe design are available (Primer Express, Applied Biosystems; Light Cycler
Probe Design, Roche; Primer Explorer, Eiken Chemical Co.;RNA fold Vienna
Package, http://www.rna.tbi.univie.ac.at/cgibin/RNAfold.cgi).
Most of the limitations of conventional PCR mentioned above can be overcome
by using a real-time PCR detection system. Real-time PCR, which detects PCR
products while the reaction is going on, has been available for the last 10 years,
but it has shown a dramatic increase in use in the last 6 years.
282 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

(a) Real time RT-PCR

Possibilities of use of real-time-RT-PCR assays for detection of a large number of
plant viruses have been extensively reviewed in many publications (Schoen et al.
1996; Boonham et al. 2002a; Eun and Wong 2000; Korimbocus et al. 2002;
Chalam et al. 2005b). Detection of Tomato spotted wilt virus (TSWV) in single
vector thrips (Boonham et al. 2002) and Plum pox potyvirus (PPV) in the aphid
vectors have been possible by the technique. It is a highly sensitive technique,
detects the pathogen well before the appearance of symptoms which can help in
disease forecasting. There are also reports available where simultaneous detection
of eight stone fruit viruses has been done by one-step RT-PCR (Sanchez-Navarro
et al. 2005).

(b) Real-time PCR involving fluorescence methods

With real-time PCR, there are currently three different fluorescence methods
available for detecting the production of PCR amplicons (Mackay et al. 2002): Taq
Man probes, Fluorescent Resonance Energy Transfer (FRET) probes, and molec-
ular beacons. All of these methods are based upon the hybridization of fluorescently
labeled oligonucleotide probe sequences to a specific region within the target
amplicon that is amplified using traditional forward and reverse PCR primers.
Real-time PCR monitoring with specific instruments and fluorescent probes offer
the advantage of combining the amplification, detection and quantification of the
target molecule in a single step. The chemistries (http://www.eurogentec.com/code/
en/catalogues.htm#top) most commonly used with real-time PCR can be divided
into non-specific and specific methods. Non-specific methods use a dye (e.g. SYBR
green I Morrison et al. 1998) emitting fluorescent light when intercalated into
double-stranded DNA (dsDNA). In solution, unbound dye exhibits very little
fluorescence but when the dye is bound to DNA, fluorescence is greatly enhanced
and is proportional to the amount of total dsDNA in the reaction. Since these dyes
do not discriminate between the different dsDNA molecules, synthesis of non-
specific amplicons, as well as of dimers, must be prevented by accurate primer
design and optimization of conditions. Specific methods are based on the use of
oligonucleotide probes labeled with a donor fluorophore and an acceptor dye
(quencher) (Whitcombe et al. 1999) that generate a light signal according to
Fluorescence Resonance Energy Transfer (FRET) chemistry. The advantage of
fluorogenic probes over DNA binding dyes is that specific hybridization between
the probe and the target DNA sequence is required to generate a fluorescent signal;
so that non-specific amplifications do not generate a signal. Furthermore, fluoro-
genic probes can be labeled with different distinguishable reporter dyes to amplify
and detect two or more distinct sequences in a single PCR reaction tube, without
melting curve analysis (multiplex PCR). The specific method includes TaqMan
(Livak et al. 1995), molecular beacons (Tyagi and Kramer 1996), and scorpion PCR
(Whitcombe et al. 1999). The fluorescent probes and the instrumentation required
are still very expensive, although there is a less expensive alternative: portable rapid
cycling real-time PCR platforms (e.g., Smart Cycler, International Laboratory),
which allow multiple sample analysis and can be used for on-site (field) detection.
5.4 Viral Nucleic Acid Based Tests 283

(c) Fluorescence RT-PCR by using Taqman technology

In the Taqman system, an oligonucleotide probe sequence of approximately 25–30
nucleotides in length is labeled at the 50 end (Holland et al. 1991) with a fluoro-
chrome (Lee et al. 1993), usually 6-carboxy fluoresce in-(6-FAM) and a quencher
fluorochrome, usually 6-carboxy tetramethyl-rhodamine (TAMRA), at the 30 end.
The taqman probe is degraded by the 50 –30 exonuclease activity of the Taq
polymerase as it extends the primer during each PCR amplification cycle and the
fluorescent chromophore is released. Two primers flank the sequence of interest
and a third fluorescently labeled primer anneals between them. As the flanking
primers extend, the labelled primer is released and fluorescence occurs. The
amount of fluorescence is monitored during each amplification cycle and is pro-
portional to the amount of PCR product generated. The first TaqMan assays
described was for the Potato leaf roll virus (Schoen et al. 1996). TaqMan assays
have been described for Tomato spotted wilt virus detection in thrips vector
(Boonham et al. 2002) and in plants (Roberts et al. 2000).Additional assays have
been described for Sugar cane yellow leaf virus in sugarcane plants (Korimbocus
et al. 2002), Potato mop-top and Tobacco mottle virus in potato tubers (Mumford
et al. 2000b), and two orchid viruses, Cymbidium mosaic potyvirus (CymMV) and
Odontoglossum ringspot tobamovirus (ORSV) (Eun et al. 2000). Probes were
designed that were specific to the RNA-dependent RNA polymerase gene and the
coat protein genes. As little as 5 fg each of CymMV and ORSV was detected in
diseased flower tissues. Similar to the fungal real-time PCR assays, which require
efficient DNA extraction methods, viral assays require RNA to be extracted from
samples prior to setting up the assay. In addition, a reverse transcription (RT)
reaction needs to be incorporated in the assay, since a majority of plant viruses are
RNA-containing viruses. By following TaqMan real-time RT-PCR Agindotan
et al. (2007) simultaneously detected potato viruses, viz., PLRV, PVA, PVX and
PVY from dormant potato tubers.
The advantages of this method are that no post-reaction processing is required
to detect the reaction product and that it is quantitative. However, unless large-
scale testing is envisaged, the cost of a Taqman ABI Prism 7700 Sequence
Detection System and the labeled primers may be prohibitive. A Taqman assay to
detect Potato spindle tuber viroid, was 1000-times more sensitive than a chemi-
luminescent assay. The thrips vector of Tomato spotted wilt virus was successfully
screened for the viruses by this method (Roberts et al. 2000; Boonham et al. 2002).
It is also successfully established that this technique is a rapid method for the on
site detection of Western flower thirips (Frankliniella occidentalis), the vector of
Tospoviruses, at the port of entry in plant quarantine (Huang et al. 2010).

(d) Molecular beacons and their use in plant virus detection

Fluorescent hair pin shaped oligonucleotide probes where the loop portion of the
probe contains nucleotide sequences complimentary to the target amplicon are
termed as molecular beacons. Molecular beacons are fluorescent oligonucleotide
probes that are designed to include stem-loop folding. These nucleotide sequences
are complementary to the target amplicon. A fluorescent chromophore is attached
284 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

at the 50 end of the probe and a quencher molecule is attached at the 30 end. A stem
structure is formed by annealing of the complementary arm sequences that are
added on both sides of the probe sequence. When a stem structure is formed, the
fluorophore transfers energy to the quencher, and no fluorescence is emitted.
However, when the probe hybridizes to the target amplicon during PCR amplifi-
cation, the fluorophore and quencher become separated from each other and
fluorescence can be detected (Didenko 2001; Cockerill and Smith 2002). This
mechanism has been used successfully in the detection of viruses.
A novel fluorescence-based nucleic acid detection technique was developed by
Tyagi and Kramer (1996). Molecular beacons are single-stranded nucleic acid
molecules with a stem-loop conformation. The stem portion consists of comple-
mentary sequences at the 50 and 30 terminals of the molecule, while the loop
portion consists of probe sequences that are complementary to the target sequences
of choice. A fluorescent moiety is attached to one end, while a quenching moiety is
attached to the opposite end. Reverse transcription-polymerase chain reactions are
carried out with primers that amplify specific genome sequences of interest,
yielding targets complementary to their respective molecular beacons for sub-
sequent detection. From Singapore, Eun and Wong (2000) have designed four
molecular beacons specific to the RNA-dependent RNA polymerase and coat
protein genes of two orchid viruses, namely Cymbidium mosaic virus (CymMV)
and Odontoglossum ringspot virus (ORSV). This technology is successfully
applied to detect as little as 0.5 ng of viral RNA of both orchid viruses simulta-
neously in 100 mg of coinfected Oncidium orchid leaves. This rapid and specific
technique is applicable to the orchid industry, which routinely carries out virus
indexing and screening for virus-resistant cultivars. It is expected that use of this
molecular beacon approach can be extended to the detection of multiple plant
viruses in various crops.

5.4.5 PCR-RFLP for Detection of Plant Virus Diseases

The RFLP analysis is used in combination with PCR to identify differences

between viruses based on the presence or absence of restriction enzyme recog-
nition sites. After PCR amplification, the amplicon is digested with a restriction
enzyme(s) and the fragment sizes analysed by gel electrophoresis. RFLP is a
method that can be used to differentiate isolates of viruses without the expenses of
cloning and sequencing. Its effectiveness relies on polymorphisms within restric-
tion enzyme recognition sites. RFLP was used to show that only members of
subgroup 2 of Cucumber mosaic virus were present in Western Australian lupin
crops (Wylie et al. 1993).
A particular format of PCR, PCR-RFLP (RT-PCR-RFLP for ssRNA viruses), is
a powerful tool to study plant virus evolution, a subject that, viewed from a
molecular stand point, is known as molecular epidemiology which has been
reviewed by Garcia-Arenal et al. (2001). PCR-RFLP can carry to typify a great
5.4 Viral Nucleic Acid Based Tests 285

number of isolates and characters using hot spots provided by sequence data to
detect variations in the resident virus population and can predict the emergence of
resistance-breaking pathotypes as in the reported cases of Tomato spotted wilt
virus (Hooftman et al. 2001; Aramburu et al. 2002; Finetti-Sialer et al. 2002). It
can also be used to assess the risks of new control strategies such as those
involving the use of virus-resistant transgenic plants.

(a) In situ PCR (IS-PCR)

This technique allows specific nucleic acid sequences to be detected in intact cells
and tissues. It is based on a reaction performed on fixed whole cells or tissue
sections, to identify amplicons at the site where they are produced (Nuovo 1992).
When applied to plant viruses, it is an excellent approach to localize virus and
virus-related sequences in infected cells. Similar results can in principle be
obtained with the In situ Hybridization (ISH) described above. However, ISH not
only requires hundreds of target molecules per cell for a reliable signal, but
detection of the hybrid molecule often involves autoradiography or immuno
detection. Although ISH has been successfully applied to plant viruses, there are
cases in which virus and virus-related sequences are below detection level either
because they occur in extremely low amounts, or are restricted to certain tissues
(e.g. phloem) or are erratically distributed. This problem can be circumvented with
a two step protocol using in situ PCR to increase the amount of the target mole-
cules, and ISH to detect the amplicon.

5.4.6 PCR Application for the Detection of Viruses

in the Vectors

In addition to diagnosis of viruses and viroids in the plant material, PCR technique
is quite useful for detection of plant viruses in the vectors. For example, TSWV
was detected in individual thrips vectors by reverse transcription-PCR (Tsuda et al.
1994). Similarly PCR was applied in the identification of Rice tungro bacilliform
virus in the leaf hopper vectors (Varma et al. 1999), Rice stripe virus (RSV) in the
brown plant hopper (Lijun et al. 2003), begomoviruses in whiteflies (Navot et al.
1992; Deng et al. 1994; Mehta et al. 1994; Atzmon et al. 1998; Leamkhang et al.
2005; Mason et al. 2007); Lettuce mosaic virus, Citrus tristeza virus and Banana
bunchy top virus in aphids (Hu et al. 1996; Mehta et al. 1997; Moreno et al. 2007;
Selvarajan and Balasubramanian 2008). Other viruses like Grapevine virus A and
B, Grapevine leaf roll virus, Barley yellow dwarf virus, Potato leaf roll virus,
Plum pox virus, and Cauliflower mosaic virus were also detected in their insect
vectors (Lopez-Moya et al. 1992; Hadidi et al. 1993; Levy and Hadidi 1994;
Minafra and Hadidi 1994; Singh et al. 1995; Cannning et al. 1996; Hu et al. 1997).
Tomato spotted wilt virus (TSWV) was also detected in single thrips vector by
Boonham et al. (2000). The information of weather insect vectors are carrying
virus or not, would be helpful primarily in epidemiological studies.
286 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

(a) PCR application for viroid disease diagnosis

In recent years diagnostic techniques such as nucleic acid hybridization and
reverse transcription coupled with polymerase chain reaction (RT-PCR) have
become popular for diagnosis of plant viroid diseases because of their relatively
simplicity and high sensitivity (Shamloul et al. 1995; Mumford et al. 2000a;
Bernard et al. 2006; Hassan et al. 2008b). RT-PCR was successfully applied in the
detection of viroids from Potato (Boonham et al. 2004); pome (Hadidi and Yang
1990) and stone fruits (Shamloul et al. 1995; Hadidi et al. 1992, 1997), citrus
(Yang et al. 1992), grapevine (Puchta and Sanger 1989; Rezaian et al. 1992; Staub
et al. 1995; Wah and Symons 1997), avocado (Schnell et al. 1997), Apricot (Pallas
et al. 2003). While studying Apple scar skin viroid detection by RT-PCR,
Sipathioglu et al. (2007) reported that out of the three conventional extraction
methods, silica-capture RNA extraction method was found to be superior for total
RNA extraction as compared to citric acid buffer and lithium chloride methods.
From Iran, Bagherian et al. (2009) have developed a technique (RT-PCR-DBH)
by incorporating RT-PCR and dot blot hybridization (DBH) for the detection of
hop stunt and exocortis viroid diseases in citrus. In this method, instead of using
nucleic acid extracted directly from the plants, RT-PCR products are subjected to
dot-blot hybridization. This method was about 1000-times more sensitive than
Southern blot and 100-times more sensitive than PCR in detecting hop stunt viroid
in citrus. It avoids the use of ethidium bromide, a carcinogenic dye used in
electrophoresis of PCR products. Potential misinterpretation of results using
polyacrylamide or agarose gel electrophoresis analysis is completely avoided by
RT-PCR-DBH. Pallas et al. (2003) in their review article described the application
of RT-PCR-DBH in detection of Hop stunt viroid in apricot as shown in schematic
diagram Fig. 5.6. Earlier Shamloul et al. (2002) have developed a novel multiplex
RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection
of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid and Posp-
iviroid. The diagnosis of viroid and virus diseases of plants in different crops is
provided in Table 5.4. Majority of the viroid diseases are seed transmitted and
sometimes distributed through the commercial seeds. For example, Singh et al.
(2009) have detected the presence of Citrus exocortis viroid (CEVd) in the seeds
as well as the seedlings that developed from infected seeds of Impatiens walle-
riana and Verbina x hybridia by RT-PCR assay. Seed-borne viroids can also be
identified by R-PAGE as seen in the case of Potato spindle tuber viroid (PSTVd)
infected true potato seeds (TPS) and also viroid infected dormant potato tubers
(Singh et al. 1988). Hence, R-PAGE can be used to assess the PSTVd content of
various potato seed lots before planting either from seeds or from In vitro seed-
lings, when germplasm is valuable and available in small quantities.
Conclusions: Many techniques which are based on protein and nucleic acid of
viruses have been discussed in this chapter which are used for rapid, specific, and
sensitive detection of plant pathogenic viruses has much improved in the last few
years. The signal amplification through chemical, molecular or electronic methods
has increased, becoming more and more independent from visible disease symp-
toms. As new genomic and proteomic data have become available, techniques with
5.4 Viral Nucleic Acid Based Tests 287

Fig. 5.6 Schematic diagram of the non-organic sample processing procedure used for viroid
detection. Courtesy Vicente Pallas; CIHEAM—Options Mediterraneennes

increased sensitivity and specificity will probably be developed and adapted for the
simultaneous and realtime detection of viruses and other plant pathogens using hot
spots in their genetic profile (Table 5.5).
Control of plant pathogenic viruses and viroids is difficult and hence preventive
measures are essential to minimize the losses they cause in various crops. In this
context, rapid and accurate methods for detection and diagnosis of these pathogens
Table 5.4 Application of PCR and its variants in the detection of viruses and viroids in seed and vegetative propagules of certain tropical crops
288

Plant Virus/viroid References

Vegetative propagules
Apple Apple mosaic virus Choi and Ryu (2003), Lakshmi et al. (2011)
Apple scar skin viroid Sipathioglu et al. (2007)
Apple stem pitting virus Ito et al. (2002), Kundu and Yoshikawa (2008), Hassan et al. (2008a)
Apple chlorotic leaf spot virus Candresse et al. (1995a, b)
Apple stem grooving virus Nemchinov et al. ( 1995b), Kinard et al. (1996), Nickel et al. (2004), Hassan et al. (2008a)
Apricot Hop stunt viroid Pallas et al. (2003)
Avocado Avocado sunblotch viroid Schnell et al. (1997), Lutting and Manicom (1999)
Banana Banana bunchy top virus Karan et al. (1994), Shamloul et al. (1995), Wanitchakorn et al. (1997), Sharman et al. (2000), Anandhi et al.
(2007), Galal (2007), Selvarajan et al. (2007), Prakash et al. (2010)
Banana bract mosaic virus Thomas et al. (1997), Sharman et al. (2000), Dassanayake (2001), Selvarajan and Bala Subramanian (2008)
Cucumber mosaic virus Hu et al. (1995), Singh et al. (1995), Dietzgen et al. (1999), Aglave et al. (2007)
Banana streak virus Harper et al. (2002), Anita et al. (2004), James et al. (2004), Le Provost et al. (2006), Prakash et al. (2010),
James et al. (2011a, b), Singh et al. (2011), Manoranjitham et al. (2012)
Black pepper Cucumber mosaic virus Bhat and Siju (2007), Siju et al. (2007)
Piper yellow mottle virus Bhat and Siju (2007), Hareesh and Bhat (2010)
Carrot Carrot mottley dwarf virus Vercruysse et al. (2000)
Cassava African cassava mosaic virus Alibi et al. (2008)
Cassava brown streak virus Rwegasira et al. (2011)
Cassava mosaic disease Berrie et al. (1997), Harrison et al. (1997), Rothenstein et al. (2006)
East African cassava mosaic cameron virus Alibi et al. (2008)
Indian cassava mosaic virus Kumar et al. (2005)
Chrysanthemum Cucumber mosaic virus Kumar et al. (2005)
Citrus Citrus ring spot virus Hoa and Ahlawat (2004)
Citrus yellow mosaic virus Ahlawat et al. (1996), Baranwal et al. (2003, 2005), Borah et al. (2008), Borah et al. (2009)
Citrus exocortis viroid Duran vila et al. (1988), Ben-Shaul et al. (1995), Ramachandran et al. (2003), Bernard et al. (2006),
Bagherian et al. (2009), Fisher et al. (2011)
Citrus mosaic virus Ahlawat et al. (1996), Baranwal et al. (2003)
Citrus tristeza virus Yokomi et al. (2010), Fisher et al. (2011)
Indian citrus ring spot virus Rustici et al. (2000 a,b)
Colocasia (taro) Konjac mosaic virus Padmavathi et al. (2011)
5 Diagnosis and Detection of Plant Virus and Viroid Diseases

(continued)
Table 5.4 (continued)
Plant Virus/viroid References
Elephant foot yam Dasheen mosaic virus Babu et al. (2011)
Garlic Leek yellow stripe virus Leisova-Svobodova and Karlova-Smekalova (2011)
Onion yellow dwarf virus Takaichi et al. (1998, 2001), Leisova-Svobodova and Karlova-Smekalova (2011)
Shallot latent virus Meenakshi et al. (2009), Leisova-svobodova and Karlova-Smekalova (2011)
Gladiolus Bean yellow mosaic virus Katoch et al. (2002)
Grapevine Arabis mosaic virus Ipach et al. (1992)
Bois noir Marzachi et al. (2003)
Fan leaf virus Rowhani et al. (1993), Wetzel et al. (2002), Digiaro et al. (2007), Blahova and Pidra (2009)
Flavescence doree Palermo et al. (2007), Margaria et al. (2007, 2009), Gori et al. (2007), Hren et al. (2007)
Grapevine fleck complex virus El Beaino et al. (2001), Kopecky et al. (2004)
5.4 Viral Nucleic Acid Based Tests

Grapevine leaf roll clostero virus
Grapevine viroid
Grapevine Virus-A
Yellow speckle viroid
Onion Iris yellow spot virus
Onion yellow dwarf virus
Tobacco streak ilarvirus
Pepper Pepper yellow mottle virus
Potato Potato viruses
Potato spindle tuber viroid
Strawberry Strawberry mild yellow edge potexvirus
Sugar beet Beet mosaic virus
Beet necrotic yellow vein virus
Beet mild curly top virus
Routh et al. (1998), Acheche et al. (1999), Ling et al. (2001), Bertazzon and Angelini (2004), Niu et al.
(2004), Faggioli and La Starza (2006), Osman et al. (2007), Ling et al. (2008), Tsai et al. (2008),
Margaria et al. (2009)
Rezain et al. (1988), Staub et al. (1995), Wah and Symons (1997)
Pacifico et al. (2009)
Nakaune and Nakano (2006)
Bulajic et al. (2009), Sivamani et al. (2009)
Arya et al. (2006)
Sivaprasad et al. (2010)
de Silva et al. (2002)
Barker et al. (1993), Hadidi et al. (1993), Spiegel and Martin (1993), Singh and Singh (1996), Singh et al.
(1996), Schoen et al. (1996), Verma et al. (2003), Agindotan et al. (2007), Gawande et al. (2007),
Kaushal et al. (2007), Boonham et al. (2009), Khan et al. (2009), Latvala-Kilby et al. (2009), Awan et al.
2010, Crosslin and Hamlin (2011), Gawande et al. (2011), Lee et al. (2011a, b)
Shamloul et al. (1997), Singh et al. (2003), Boonham et al. (2004), Singh et al. (2006), Khan et al. (2009),
Crosslin and Hamlin (2011)
Hadidi et al. (1991), Kreuziger et al. (1995),
Nemchinov et al. (2004)
Kruse et al. (1994)
Chen et al. (2008)

(continued)
289
Table 5.4 (continued)
Plant Virus/viroid
Sugarcane Sugarcane Fiji disease virus
Sugarcane mosaic virus
Sugarcane streak mosaic virus
Sugarcane yellow leaf virus
Sweet orange Citrus tristeza virus
Sweet potato Sweet potato viruses
Taro Taro bacilliform virus
Yam Cucumber mosaic virus
Yam mosaic virus
True seed transmitted viruses and viroids
Avacado Avocado sunblotch viriod
Cocoa Cocoa swollen shoot virus
Cowpea Bean common mosaic virus
Cowpea aphid borne mosaic virus
Cowpea mottle virus
Cucumber mosaic virus
Cucumber Cucumber mosaic virus
Common bean/French Bean common mosaic virus
bean Tomato yellow leafcurl virus
Lettuce Lettuce mosaic virus
Lupin Cucumber mosaic virus
Maize Sugarcane mosaic virus
Mung bean Bean common mosaic virus (Pst strain)
Pea Pea seed-borne mosaic virus
290
References
Smith and Van de Verde (1994), James et al.(2001)
Smith and Van de Verde (1994), Yang and Mirkov (1997), Gaur et al. (2003), Balamuralikrishnan et al.
(2004), Rao et al. (2006), Zhang et al. (2008), Viswanathan et al. (2010), Subba Reddy et al. (2011)
Smith and Van de Verde (1994), Hema et al. (1999, 2003, 2008), Rao et al. (2006), Van Antwerpen and
Rutherford (2008), Damayanthi et al. (2010), Viswanathan et al. (2010), Subba Reddy et al. (2011)
Viswanathan et al. (2009), Goncalves et al. (2012)
Cambra et al. (2000)
Mukasa et al. (2003), Li et al. (2004), Kokkinos and Clark (2006), Aritua et al. (2009), Kashif et al. (2012)
Harding (2008)
Eni et al. ( 2008b)
Eni et al. (2008a)
Lutting and Manicom (1999)
Muller et al. (2001), Quainoo et al. (2008)
Hao et al. (2001)
Udayashankar et al. (2010)
Akinjogunla et al. (2008), Udayashankar et al. (2009)
Salem et al. (2010), Amayo et al. (2012)
Gillaspie et al. (2000)
Gillaspie et al. (1998a), Abdullahi et al. (2001),
Salem et al. (2010), Amayo et al. (2012)
Berniak et al. (2009)
Saiz et al. (1994)
Lapidot (2002)
Soleimani et al. (2011)
Wylie et al. (1993)
Li et al. (2007)
Choi et al. (2006)
Kohnen et al. (1992)
Phan et al. (1997)
5 Diagnosis and Detection of Plant Virus and Viroid Diseases
(continued)
Table 5.4 (continued)
Plant Virus/viroid
Peanut Cowpea aphid-borne mosaic virus
Cucumber mosaic virus
Indian peanut clump virus
Peanut clump virus
Peanut mottle virus
Peanut stripe virus
Peanut stunt virus
Tomato spotted wilt virus
Pepper Cucumber mosaic virus
Pepper chat fruit viroid
Pumpkin Cucumber mosaic virus
Cucumber mosaic virus
Zucchini yellow mosaic virus
Squash Zucchini yellow mosaic virus
Tomato Pelargonium zonate spot virus
Tomato, Bell pepper Tobacco mosaic virus
Wheat Wheat streak mosaic virus
Non seed transmitted viruses
Barley Barley mild mosaic virus
Bittergourd Pepper leaf curl Bangladesh virus
Squash vein yellowing virus
Blackgram Tobacco streak virus
Capsicum Capsicum chlorosis virus
Tomato leaf curl virus
Peanut bud necrosis virus
References
Gillaspie et al. (2001)
Salem et al. (2010)
Dietzgen et al. (2001)
Miller et al. (1996), Naidu et al. (2000)
Lee et al. (2004)
Gillaspie et al. (1994, 2001),
Dietzgen et al. (1994, 2001)
Gillaspie et al. (1994, 2000),
Dietzgen et al. (2001)
Dietzgen et al. (2001)
Jain et al. (1998b)
5.4 Viral Nucleic Acid Based Tests
de Silva et al. (2002)
Verhoeven et al. (2009)
Tobias et al. (2008)
Berniak et al. (2009)
Tobias et al. (2008)
Hosseini et al. (2007), Simmons et al. (2011)
Lapidot et al. (2010)
Vinayarani et al. (2011)
Jones et al. (2005)
Vaianopoulos et al. (2003)
Raj et al. (2010)a
Adkins et al. (2008)
Ladha Lakshmi et al. (2005)
Krishnareddy et al. (2008)
Tsai et al. (2011)
Damayanti and Naidu (2009)
(continued)
291
Table 5.4 (continued)
292

Plant Virus/viroid References

Cardamom Cardamom mosaic virus Biju et al. (2010)
Chickpea Chickpea stunt virus Naidu et al. (1997)
Cucurbits Cucumber vein yellowing virus Gil-Salas et al. (2009)
Cucurbit yellow stunting disorder virus Papayiannis et al. (2010)
Tobacco streak virus Sarovar et al. (2010)
Tomato yellow leaf curl virus Anfoka et al. (2009)
Faba bean Tobacco streak virus Ali et al. (2008)
Lettuce Tomato chlorotic spot virus Colariccio et al. (2003)
Maize Maize streak virus Rybicki and Hughes (1990)
Okra Tobacco streak virus Krishnareddy et al. (2003)
Papaya Papaya ring spot virus Jain et al. (1998c), Sreenivasulu and Saigopal (2010)
Peanut Groundnut rosette virus Naidu et al. (1998)
Tomato spotted wilt virus Jain et al. (1997)
Rice Rice tungro viruses Varma et al. (1999), Joshi et al. (2003), Niazi et al. (2005), Periasamy et al. (2006), Mangrauthia et al. (2010)
Soybean Tobacco streak virus Arun Kumar et al. (2008)
Soybean mosaic virus Omunyin et al. (1996)
Sunflower Tobacco streak virus Ravi et al. (2001), Bhat et al. (2002), Karunakaran et al. (2008), Sharman et al. (2008), Sarovar et al. (2010)
Tobacco Tobacco viruses Jin et al. (2012)
Tomato spotted wilt virus Mumford et al. (1996), Pappu et al. (1998)
Tomato Peanut bud necrosis virus Manjunatha (2008)
Tomato leaf curl virus Martinez-Culebras et al. (2001), Tsai et al. (2011), Thakuria et al. (2012)
Tomato spotted wilt virus Garland et al. (2005), Dietzgen et al. (2005)
Tomato yellow leaf curl virus Atzmon et al. (1998), Leam Khang et al. (2005), Mason et al. (2007)
5 Diagnosis and Detection of Plant Virus and Viroid Diseases
5.4 Viral Nucleic Acid Based Tests 293

Table 5.5 Comparison of ISEM, ELISA, traditional and real time RT-PCR on the cost, easiness,
sensitivity, specificity and quantification of these methods
Methods Cost Easiness Sensitivity Specificity Quantification
ISEM +++ + + ++ +
ELISA + +++ + ++ +++
RT-PCR ++ ++ +++ +++ +
Realtime RT-PCR +++ ++ ++++ ++++ ++++
Source Rao and Singh (2008)

are required to apply. Plant viruses are generally detected and identified by particle
morphology under electron microscope, host range and the serological assays.
Electron microscopy is most convenient approach of direct detection of viruses but
it is generally not used for routine diagnostic purpose. Moreover, the negative
results in electron microscopy does not necessarily mean the absence of viral
pathogens as it is quite likely the tissue used for electron microscopy may not have
virus particles. Host range studies or biological indexing though, useful but it is
basically a time consuming procedure and requires a well-equipped glass house
and long-term maintenance of test host. Serological techniques such as ELISA and
its variants are used in most cases for detection of viruses and are sensitive for
most viruses where titre of antibodies is higher enough for ELISA testing. Cross-
reactivity of antisera raised against viruses from different groups has frequently
been used for detection and establishment of taxonomic relationships. However,
nucleic acid sequence data are accumulating rapidly and allow more accurate
relationship to be established between the individual members of virus groups than
serological methods do. Invention of nucleic acid hybridization and PCR tech-
niques revolutionized the detection and diagnosis of virus and viroid infections in
the plants. They are around 100 to 1000-fold more sensitive than serological
assays such as ELISA for plant virus detection. Furthermore, PCR has been greatly
improved by the introduction of the second generation PCR, known as the real
time PCR where closed-tube fluorescence detection and quantification during PCR
amplification (in real time) is possible, eliminating the need for laborious post-
PCR sample processing steps which greatly reduces the risk of carryover con-
tamination. Using real time PCR, it is possible not only to detect the presence or
absence of the target pathogen, but it is also possible to quantify the amount
present in the sample allowing the quantitative assessment of the pathogen in the
sample. PCR and nucleic acid hybridization techniques can be applied for
detection diagnosis and characterization of many plant viruses and viroids
occurring in low concentration in plant tissues and also for viruses that are poor
immunogens. The nucleic acid hybridization and PCR based tests are particularly
effective in the detection of viroids and other sub viral agents that lack coat
protein. The current trend in protocols for the detection of plant viruses and
subviral agents is to combine conventional (biological), serological and molecular
techniques in integrated approaches. The successful application of PCR and its
variants in diagnosis of plant virus and viroid diseases in different crop plants is
provided in Table 5.4.
294 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

Plants are infected by a wide range of viruses and viroids. Many cause dev-
astation of plants and crops resulting in significant economic losses and threats to
the viability of certain horticultural and agricultural industries. Resources available
for routine detection of plant viruses tend to be limited. This means that techniques
adopted for routine diagnosis must be of low cost, yet sensitive and reliable.
Approaches that allow simultaneous detection of multiple plant viruses (multi-
plexing) reduce the number of tests required, reagent usage, time for analysis, and
consequently, the cost. Multiplex PCR, polyvalent PCR, non-isotopic molecular
hybridization techniques, real-time PCR, and array technologies allow simulta-
neous detection of multiple plant viruses. The increased sensitivity achieved with
some techniques, such as real-time PCR, permits the use of simple, low-cost target
isolation methods such as direct binding, tissue printing, or immunocapture. These
result in reduced overall cost. Multiplexing techniques have the capacity for
simultaneous broad-spectrum and specific identification by combining primers and
(or) probes that target various taxonomic levels such as family, genus, and species.
Polyvalent PCR and broad-spectrum probes have the potential to detect unknown
or uncharacterized viruses, improving our ability to monitor and successfully
control these pathogens. Techniques such as microarray analysis offer the potential
for development of a single biochip that may facilitate detection of all viruses
affecting a particular crop (e.g., a cucurbit or potato biochip). This may be
expanded in time to the detection of every pathogen, including viruses and viroids,
affecting a particular plant. With even more advances in molecular biology and
immunology, scientists and farmers alike will be able to improve plant disease
diagnosis. Efforts are already underway to produce better diagnostic kits to detect
pathogens in crops important to developing countries. For instance, the Depart-
ment of Biotechnology of India’s Ministry of Science and Technology is devel-
oping diagnostic kits to detect viruses in fruits, ornamentals, spices, and plantation
crops. The Genetic Engineering Services Unit of Egypt’s Agricultural Genetic
Engineering Research Institute has developed diagnostic kits and testing services
to detect viruses in crop plants.

5.5 Recombinant DNA Technology

Recombinant DNA (rDNA) technology has proved to be very reliable and sensi-
tive technique in plant virus diagnosis. This technology also facilitated the gen-
eration of transgenic crops with new or improved traits and the development of
newer, accurate and sensitive diagnostics of plant pathogens. For example, viral
genome based diagnostics (probes, primers, nucleic acid hybridization, PCR, DNA
microarrays) have wide applications (Webster et al. 2004; Boonham et al. 2007;
Rao et al. 2008). The following section described the types of rAbs, their
advantages, and some applications.
5.5 Recombinant DNA Technology 295

(a) Production of polyclonal antibodies based on recombinant protein

expressed in heterologous system
Traditionally purified virus preparations or isolated capsid proteins from purified
viruses are generally used as immunogens to immunize laboratory animals for
production of PAbs or MAbs (Matthews 1993). But this approach is not suitable
for certain viruses as they are difficult to purify to the required quality and quantity
necessary for conventional PAbs production. To overcome the limitations of PAbs
production to certain plant viruses, rDNA technology and molecular immunology
based approaches have been used to produce antibodies useful for detection and
diagnosis of plant viruses in recent times (Helias et al. 2003; Abou-Jwadah et al.
2004; Cotillon et al. 2005). The genomes of several plant viruses have been either
partially or completely sequenced and deposited in public databases (Gen Bank,
EMBL, and DDBJ). This data is useful to exploit plant viral genes for several
purposes. For example, specific viral genes can be amplified, cloned into a vector
for expression in heterologous host systems and the expressed proteins are purified
and used as immunogen for production of polyclonal antibodies (Jagadish et al.
1991; Joseph and Savithri 1999; Chatchen et al. 2006). These antibodies have been
used in tests like ELISA, DBIA and IC-RT-PCR for the detection of several plant
viruses viz., Watermelon bud necrosis in watermelon (Jain et al. 1998a); Faba
bean necrotic yellows virus (Kumari et al. 2001); Sugarcane streak virus in sug-
arcane (Hema et al. 2003); Citrus yellow mosaic badnavirus (Anthony Johnson
and Saigopal 2012); Cucurbit yellow stunting disorder in cucurbits (Cotillon et al.
2005); Groundnut bud necrosis virus in tomato (Hemalatha et al. 2008); and in
groundnut and watermelon (Jain et al. 2005) and Potato mop top virus in potato
(Helias et al. 2003) have been identified by using antibodies based on recombinant
coat protein expressed in E. coli.

(b) Production of PAbs and MAbs in heterologous systems based on antibody

engineering
Synthetic antibodies called recombinant antibodies (rAbs) can be created using
antibody genes made in a laboratory or taken from human cells, completely
eliminating animals from the antibody-production process. rAbs can be used in all
applications in which traditional mAbs are used and have inherent advantages over
their animal-derived counterparts as well.

5.5.1 Production of Recombinant Antibodies by Phage

Display Technology

Recombinant antibody fragments are produced from a heterologous source using

rDNA technology. Enlightening of molecular structure of immunoglobulins and
sequence data made it possible to develop immunoglobulin-specific oligonucleo-
tide primers and to use them in conjunction with PCR techniques to clone antibody
gene fragments for generating recombinant antibodies.
296 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

5.5.1.1 Phage Display

Phage display, refers to the display of functional peptides, proteins or antibody

fragments on the surface of bacteriophage (Smith 1985; Clackson et al. 1991). This
is accomplished by fusion of the DNA coding sequences of the protein to be
displayed into phage genome to the gene (e.g., gene III, gene VIII in M13 phage)
encoding one of the phage structural protein (Gao et al. 1999).
Surface display of the antibodies allows affinity selection by the antigen
in vitro, an analogue of selection by an antigen in natural immunity (Patrenko and
Vodyanoy 2003). The captured phage particles eluted from the antigen, amplified
by infecting E. coli host cells and used in a subsequent round of affinity selection.
During the repetitive rounds of affinity selection binding, washing, elution and
amplification lead to selection of specific clone of phage that has high affinity to
the target molecule. After the final round of selections, phage particles are
amplified to prepare and characterize their displayed antibodies individually.
Finally, the monoclonal phage population with the desired binding specificities can
be isolated (Willates 2002).
A modified version of phage display technology is the selectively infective
phages (SIP) technique (Spada and Pluckthun 1997). The advantage of this method
is that only those phages that combine with ligand (e.g., antigen) are capable of
infecting E. coli. scFv-ALP fusion protein reagents are also produced for some
viruses like Beet necrotic yellow vein virus (BNYVV), Citrus tristeza virus (CTV)
and Plum pox virus (PPV). For detection of CTV, the scFv-ALP fusion proteins
were reported to be excellent substitutes for conjugates in DAS-ELISA (Terrada
et al. 2000).

5.5.2 Single Chain Variable Fragment Antibody (scFv)

It is a man-made product resulting from the development of the biotechnology and

antibody engineering. scFv molecules are the smallest antibody fragments of
26–27 kDa in size. They contain the complete binding site consisting of individual
heavy and light chain V domain (12–14 kDa each) and/or linked to a single protein
by a 15 amino acid long hydrophilic and flexible polypeptide linker which can
additionally also has His tag (an immuno-detection epitope) and a protease specific
cleavage site. The linker must be long enough (*3.5 nm) to connect C-terminus
of one domain to the N-terminus of second domain. Suitable linking of domain is
critical for correct conformation and expression and for proteolytic stability issues.
The resulting scFv facilitates equal expression of both Fv fragments in heterolo-
gous microorganisms, mammalian cells and plants.
5.5 Recombinant DNA Technology 297

5.5.2.1 Different forms of scFv

(a) Soluble scFv antibody: The molecule is produced by a single polypeptide, the
most popular form of scFv antibody and it shares all advantages of the MAbs. e.g.,
Citrus tristeza virus (Terrada et al. 2000).
(b) Recombinant phage display scFv antibody: The scFv genes are fused to one
of the capsid proteins of the filamentous bacteriophage (e.g.: M13). This leads to
expression of scFv on the surface of the phage. An advantage of this form of scFv
is that the supernatant of the phage infected bacterial culture can be directly used
in ELISA. This is useful for screening specific scFv to target antigens from a scFv
library through 3–4 rounds of selection.
(c) Dimeric forms of scFv or miniantibody: Such molecules preserve the
bivalency of native antibody molecules. scFv fragments can be linked by a small
modular dimerization domain in the form of one or two amphipathic helices. In
terms of avidity, the miniantibodies are indistinguishable from a native antibody
(Pluckthun and Pack 1997).
(d) Fusions of scFv with diagnostic enzymes: A common fusion is with alkaline
phosphatase (ALP). This enables the production of antibody conjugates in bac-
teria, which can decrease the cost of ELISA reagents (Suzuki et al. 1997).
(e) Bi-specific scFv (diabody): These antibodies have been used to redirect the
T. lymphocytes against defined antigens on tumor cells (Mack et al. 1997).

5.5.3 Plantibodies

A plantibody (derived from plant and antibody) is a special type of antibody

created from genetically altered crops. The term plantibody as well as the concept
is trade marked by the company Biolex, although plants do not have an immune
system of their own, and do not normally make antibodies, plantibodies have been
shown to function in the same way as normal antibodies.
A number of plant species like alfalfa, pea, soybean, rice, wheat, barley and
maize are being experimented with the production and cultivation of plantibodies.
Plants represent a cost-effective, convenient and safe alternative production system
and are slowly gaining acceptance. Five plant-derived therapeutic recombinant
antibodies (plantibodies) are undergoing clinical evaluation, three of which can be
used as prophylactics.
These plantibodies are formed by various methods like conventional method,
cell tissue culture method, breeding and sexual crossing, transgenic seeds, tar-
geting and compartmentalizing. These are further purified by various methods like
filtration, chromatography, diafiltration, immunofluorescence, polymer fusion and
further evaluated by RIA (Radioimmunoassay), ELISA, immunofluorescence,
southern blot analysis, western blot analysis, northern blot analysis.
One of the several methods for synthesizing plantibody is conventional method
which uses transformation and transient expression to introduce new genes into a
298 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

host cell. The transformant cell is then introduced into the plant embryo, propagation
of plant in open field allow large-scale production of antibodies. Plant tissue culture
is the most economic and time saving method for production of antibodies from
plants.
Both Agrobacterium-mediated transformation and particle bombardment have
been used to introduce antibody genes into plants. Particle bombardment allows
the simultaneous introduction of multiple constructs, thereby expediting the
recovery of transgenic lines expressing multimeric antibodies such as secretory
immunoglobulin A (sIgA).
Transient expression systems involving viral vectors or agroinfiltration are
effective means for obtaining moderate quantities of recombinant product within a
very short time frame. Such systems may prove to have advantages compared with
routine small-scale bacterial expression systems for obtaining correctly folded,
soluble proteins.
Furthermore, the importance of antibodies as an in vitro research tool has been
extended to in vivo applications in functional studies of proteins and other com-
pounds. Although some plant-derived antibody products have successfully com-
pleted early phase clinical trials, several issues including regulatory guidelines and
public acceptance must still be resolved. Long-term targets for plant bioreactors
may therefore encompass high-volume, low-cost antibodies, which do not require
extensive purification.
At different labs of the research institutions and universities, researches are
being carried out on the application of plantibodies on plant virus inhibition and
virus management. For example, plantibody mediated inhibition of Potato leaf roll
virus without genetic alteration of viral genome was reported for the first time by
Nickel et al. (2008).
Over the past few years, a range of different plant systems has been developed for
the large-scale production of recombinant proteins, including rAbs. The choice of
system depends on many factors, but the intrinsic efficiency and the suitability for
scale-up, storage and downstream processing are particularly important. Planti-
bodies inhibited the replication of Artichoke mottled crinkle virus (Tavladoraki et al.
1993) and Tobacco mosaic virus (Voss et al. 1995). For more details on plantibodies,
can be had from the review articles of Rybicki (2008) and Thanavala et al. (2006).

Some of the approaches in rDNA technology of plant virology are:

(a) Cloning and expression of specific plant virus gene (e.g., coat protein gene) in
bacteria or yeast system and use of purified recombinant proteins as an antigen to
raise PAbs/MAbs in laboratory animals.
(b) Cloning of plant virus antigen encoding gene into the mammalian expression
vector and immunizing the laboratory animal with recombinant vector DNA to
induce PAbs production (scFv).
(c) Production of recombinant antibodies to plant viruses by cloning and
expression of antibody genes on the capsid surface to bacteriophages (phage
display).
(d) Plantibodies production by using recombinant technology.
5.5 Recombinant DNA Technology 299

5.5.4 Induction of Polyclonal Antibodies (PAbs) by rDNA

Based Immunization

Hinrichs et al. (1997) have studied the induction of antibodies to the TMV CP and
PVY non-structural protein P1 by cloning genes encoding these two viral proteins
in the mammalian expressions vector (pSG5) and separately injecting intramus-
cularly into New Zealand white rabbits. Specific immune response is detected
against CP of TMV.
Matic et al. (2009) have studied three strategies for generating specific anti-
bodies against Little cherry virus 1 (LChV-1, an unassigned member of the family
Closteroviridae) CP by immunizing with: (1) Partially purified virus particles, (2)
Recombinant bacterially expressed CP and (3) DNA prime-protein boost. They
have amplified the CP gene from total nucleic acid extract of virus infected tissues
by RT-PCR.

(a) Applications and other advantages

Recombinant antibodies obtained from antibody gene libraries can be used in all
applications in which traditional MAbs are used (e.g., Western blotting, immuno
histochemistry, fluorescence activated cell sorting and immunofluorescence; they
have several more advantages over their traditional animal based counter parts as
well.
In future, this approach of raising antibodies to plant viruses is an alternative to
conventional method of producing PAbs in view of the following advantages:
(a) Elimination of time consuming and technically demanding steps of antigen
isolation and purification.
(b) The virus protein purification can result in changes in protein conformation
and the loss of epitopes. This problem is probably not encountered during in vivo
expression of the antigens after DNA-based immunization.
(c) Less distress for the animal because the administration of vector DNA does
not require any adjuvants that may induce local inflammation.

5.6 Array Technologies

Array technology has revolutionized the world of viral diagnosis because of its
efficiency in screening a large number of field samples in a single array plate or
reaction. 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 thus has been used
in variant pathogen diagnosis. There are mainly two types of arrays: (1) Macro-
arrays and (2) microarrays based on the volume of the sample and the droplet size
used for the analysis.
300 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

5.6.1 Macroarray

Macroarray is a technique, where the amount of sample used is higher than that of
microarrays and the droplet size is more than 200 lm 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 eleven potato viruses and a viroid (Agindotan and Perry 2008).

5.6.2 Microarray

Arrays, both microarrays and macroarrays, have been used for some years as a tool
for visualizing relative changes in global expression levels of mRNAs, as well as
single nucleotide polymorphism typing and host–pathogen interactions. A number
of groups have extended its use to include diagnosis and genotyping of human
pathogens, including viruses (Wang et al. 2002). ssDNA probes are irreversibly
fixed as an array of discrete spots to a surface of glass, membrane or polymer.
Microarrays are high-density arrays with spot sizes smaller than 150 microns. A
typical microarray slide can contain up to 30,000 spots. Arrays printed with probes
corresponding to a large number of virus species (or indeed, any type of pathogen)
can be utilized to simultaneously detect all those viruses within the tissue of an
infected host. Viral nucleic acids are extracted from the host, reverse-transcribed
and amplified where appropriate, then labeled with a probe-either radioactive or
fluorescently tagged nucleotides such as fluorescin, Cy3 or Cy5 during the reverse
transcription reaction. The labeled target molecule is denatured and allowed to
hybridize with the arrayed probes. Excess target is washed from the surface and
spots where labelled target molecules have bound, become fluorescent under
appropriate lighting conditions. The position of a visible spot corresponds to the
presence of a particular virus in the plant sample (Boonham et al. 2007).
Since the development of microarray technology for gene expression studies
(Kato-maeda et al. 2001; Wang et al. 2002; Lareu et al. 2003), new approaches are
extending their application to the detection of pathogens. Microarrays are gener-
ally composed of thousands of specific probes spotted on to a solid surface (usually
nylon or glass). Each probe is complementary to a specific DNA sequence (genes,
ITS, ribosomal DNA) and hybridisation with the labeled complementary sequence
provides a signal that can be detected and analysed. There is great potential for
microarray technology in the diagnosis of plant diseases, the practical develop-
ment of this application is seen in the diagnosis of number of plant viruses. For
example, following the methodology utilised for genetic analysis (Brown and
Botstein 1999) large numbers of DNA probes used in two-dimensional arrays have
allowed thousands of hybridisation reactions to be analysed at the same time
5.6 Array Technologies 301

(Hadidi et al. 2004). The microarray technology focuses its use in multiplex format
of similar or very different pathogens, taking advantage of the number of probes
that can be employed in one chip (Bonants et al. 2002, 2005; Schoen et al. 2002,
2003; Fessehaie et al. 2003; Franke-Whittle et al. 2005; Boonham et al. 2007; van
Doorn et al. 2007; Pasquini et al. 2008). With the availability of genomic
sequences of plant viruses and the rapid development of microarray technology, as
well as a renewed emphasis on detection and characterization of quarantine-plant
viruses, there is a rush in the European Union to set up this technology and apply it
to detection. Several international projects have developed diagnostic microarrays
for plant viruses and are being used for detection. For example detection and
differentiation of four cucurbit infecting Tobamoviruses (Lee et al. 2003) and four
potato viruses (Boonham et al. 2003) were assayed by microarray methodology
and was proved to be potential in viral diagnostics. Since this method is com-
pletely generic, it can be used to detect all viruses whose sequence is currently
available, but its cost is very high. Consequently, it is still far from common in
routine detection, but it is being increasingly used in functional genomics studies.
The probes can be prepared in at least three basic formats:
(a) PCR fragments arrayed on nylon membranes, hybridised against cDNA
samples radioactively labeled, called macroarrays (Richmond et al. 1999);
(b) PCR products spotted onto glass slides and DNA labeled with fluorescent dyes
(Richmond et al. 1999; Zimmer et al. 2000; Wei et al. 2001);
(c) Oligonucleotides of different length (from 18 to 70 bp) arrayed and hybridised
with the same type of labeled DNA material (Lockhart et al. 1996; Loy et al.
2002, 2005; Fessehaie et al. 2003; Peplies et al. 2003).

Further advancement in the microarray technology is the membrane (Nylon)-

based microarray which is inexpensive and has potential to detect hundreds of
pathogens in a single assay. In recent years, cheap and disposable cartirages or in
high throughput microfluidic devices (lab on a chip) are being used. For this, no
specialized instrumentation or facilities are required, materials and reagents are
relatively inexpensive, probes can be easily spotted on to small pilot arrays and
evaluated for effectiveness and membranes can be reused for multiple times. Based
on this technique, Agindotan and Perry (2007) have detected PVY and PLRV
either singly or in mixed infection. Even Plumpox virus in stonefruit trees was
detected by this technique (Pasquini et al. 2008; Barba and Hadidi 2012). The
sensitivity of this described microarray was comparable with that of DAS-ELISA,
with conventional RT-PCR and real time RT-PCR being 103 and 105 times more
sensitive, respectively. More information on microarray application can be
obtained from the review articles of Hadidi et al. (2004), Boonham et al. (2007),
and Hadidi and Barba (2008).

(a) DNA Microarrays

The principle of microarray involves hybridization of fluorescently labelled
sequences (targets) to their complimentary sequences spotted on a solid surface
acting as probes. The DNA microarray technology, originally designed to study
302 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

gene expression and generate single nucleotide polymorphism (SNP) profiles, is

currently a new and emerging pathogen diagnostic technology, which in theory,
offers a platform for unlimited multiplexing capability (Hadidi and Barba 2008).
DNA microarrays or biochips are the most recent tool developed for plant virus
detection. A DNA chip allows the simultaneous interrogation of hundreds to
thousands of cDNAs arrayed on a small surface (a microscope slide in the simplest
format) approximately 1 cm2 in size. Each cDNA is located at a specific address
on the surface, called a spot or a feature. Interrogation is carried out by reverse
mixed-phase hybridization format, in which target molecules extracted from the
sample are cDNAs labeled with a specific fluorophore and maintained in solution,
while probes are cDNAs or short oligonucleotides (50 bp) obtained from sequence
data of the pathogen to be detected and arrayed on the support. The detection
system uses one or more fluorophores, which are read with laser technology, while
nucleic acids are extracted from the sample, labelled and hybridized using standard
laboratory techniques. Chip technology can be used to monitor gene expression in
different plant-pathogen combinations.
The general procedure for microarray assay as follows:
(1) From virus infected or uninfected tissue, extract total RNA
(2) cDNA synthesis of total plant RNA
(3) Fluorescent labelling of cDNA with cyanine 3 (greene or cyanine 5 red)
(4) Hybridization of slide printed virus oligonucleotide probes with fluorescent
labelled cDNA
(5) Scanning hybridized slides using a 532 nm laser for cyanine 3 and a 635 nm
laser for cyanine 5
(6) Fluoresence intensities quantification.

The spots are then identified and located on the array. Measurements of fluo-
rescence on local background fluorescence for each DNA spot are recorded.
Signals are considered positive if at least 5 fold above the local background.

(b) Probes for microarrays

Because the employment of array technology for plant virus detection is recent,
commercial plant virus arrays are available to a limited extent and therefore must
be made individually. Of primary importance in making an array is probe design.
Probes determine the sensitivity of the array and the amount of information that
they provide. Access to sequence databases and powerful sequence alignment
software is therefore essential. Of importance is a knowledge of the genomic
strategy of each target species (whether it is single-stranded or double-stranded,
RNA or DNA, positive or negative sense). Two probe types can be used to con-
struct arrays, cDNAs and oligonucleotides. cDNA probes are denatured PCR
amplicons derived from the virus of interest. Both strands of the amplicon are fixed
to the membrane. The advantage of this strategy is that long probes (100–500
bases in length) can be synthesized more cheaply by PCR than by oligonucleotide
synthesis. However, there are a number of draw backs to this method over syn-
thesis of oligonucleotide probes. The probe must be purified from other amplicons,
5.6 Array Technologies 303

Fig. 5.7 Schematic view of microarray technology principle for detection of pathogens. Source
Alberts et. al. 1994; Photos courtesy of http://www.msu.edu

nucleotides and enzymes. It is important to determine the sequence of the

amplicon to ensure that it is not an unintended amplification product of PCR.
There is little flexibility in its use for differentiating strains. Access to the virus is
essential in order to amplify the probe. Oligonucleotide probes are synthesized
single-stranded DNA fragments of 20–70 nucleotides. Unlike cDNA probes, only
one strand is present; so it is important that the probe corresponds to the coding
strand of RNA viruses in order to hybridize to the labeled anticoding cDNA strand
of the virus. Specificity is easier to achieve with shorter probes than longer cDNA
probes. The main limitation of arrays, especially microarrays, is the high cost of
both the spotting and detection equipment and the labeled nucleotides, and the
need for dust-free rooms (work station) (Fig. 5.7).

(c) cDNA chip technology

A plant virus cDNA chip was developed by Lee et al. (2003) by using viral cDNA
clones and microarray technology. The cDNA chip was designed for detection and
differentiation of the four species of selected cucurbit-infecting Tobamoviruses.
Targetted viruses were: Cucumber green mottle mosaic virus (CGMMV),
Cucumber fruit mottle mosaic virus (CFMMV), Kyuri green mottle mosaic virus
304 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

(KGMMV), and Zucchini green mottle mosaic virus (ZGMMV). The chip con-
sisted of cDNA clones of the four cucurbit-infecting Tobamoviruses, two target-
related Tobamoviruses, and another three unrelated plant viruses. The PCR
products were amplified from the selected cDNA clones and arrayed onto slide
glass. The cDNA chip, which was called cucurbit-virus chip, detected successfully
specific target viruses. When applied to probes made from ZGMMV-infected
samples, ZGMMV reacted strongly with its homologous cDNA and moderately
reacted with KGMMV and CFMMV, while it did not react with CGMMV on the
same chip. CGMMV probe gave strong signal intensity to its homologous cDNA
spot and weakly reacted with ZGMMV, KGMMV, and CFMMV. The signal
intensity of all combinations of probe and target was correlated significantly with
nucleotide sequence identities between the probes and target viruses. The signals
could be made as image files for specific virus detection, and this could be useful
for virus identification and differentiation (Lee et al. 2003).
Another type of microarray is called the nanochip (Sosnowski et al. 1997;
Nanogen, Inc., San Diego, CA 92121, USA) based on an electronically address-
able electrode array that provides direct electric field control over the transport of
charged molecules to selected micro locations and concentration over an immo-
bilized substrate. A particular feature of this system is that biotinylated immobi-
lised molecules can be either oligo capture probes or amplified PCR samples.
Hybridisation is detected and analysed by fluorescent oligo probes. By regulating
the electric-field strength, hybridisation stringency can be adjusted for homologous
interactions. Nanochips have shown high specificity and accuracy to diagnose
plant viral and virus-like pathogens affecting potato, due to their ability to dis-
criminate single nucleotide changes (Ruiz-Garcia et al. 2004). The potential of
microarray technology in the detection and diagnosis of plant diseases incited by
plant viruses and viroids is very high, due to the multiplex capabilities of the
system. Moreover, it can be coupled with other systems, i.e., to perform nucleic
acid extraction on the chip (Liu et al. 2007), achieve PCR reactions and their
detection on the same device (van Doorn et al. 2007) or even mix all the systems in
one (Lee et al. 2006), provides the possibility of automation that can be of great
importance and utility. This possibility, with the of coupling with previous steps of
the analyses (extraction, PCR, detection) promises a wider use in future protocols
(Bonants et al. 2005; Lee et al. 2006; Boonham et al. 2007; van Doorn et al. 2007;
Liu et al. 2007).

5.7 Rolling Circle Amplification (RCA) in Plant Virus

Diagnosis

RCA is based on the rolling replication of short, single stranded DNA circles by
certain DNA polymerases at constant temperature. It is isothermal in vitro method
for the hybridization-triggered enzymatic synthesis of hundreds to billions of
5.7 Rolling Circle Amplification (RCA) 305

linear copies of small-single stranded circular DNA probe. RCA method has the
possibility to improve DNA cloning techniques that have been restricted by the
limitation of the PCR method or by the host cells.
A sequence-nonspecific, rolling-circle amplification (RCA) technique was
developed by James et al. (2011a) for Banana streak virus (BSV) in Banana
wherein, they have showed that discrimination between integrated and episomal
BSV DNA, specifically detecting the latter in several banana cultivars known to
contain episomal or integrated sequences of Banana streak Mysore virus
(BSMyV), Banana streak OL virus (BSOLV), and Banana streak GF virus
(BSGFV). Using RCA, the presence of BSMyV and BSOLV was confirmed in
Australia, while BSOLV, BSGFV, Banana streak Uganda I virus (BSUgIV),
Banana streak Uganda L virus (BSUgLV), and Banana streak Uganda M virus
(BSUgMV) were detected in Uganda. This is the first confirmed report of epi-
somally-derived BSUglV, BSUgLV, and BSUgMV in Uganda; and as well as its
ability to detect BSV. RCA was shown to detect two other Pararetroviruses,
Sugarcane bacilliform virus in sugarcane and Cauliflower mosaic virus in turnip
(James et al. 2011b). In Pakistan Bashir et al. (2012) have identified the unknown
components of Banana bunchy top virus by this method. It was also established
that the rolling circle amplification revolutionizes diagnosis and genomics of
geminiviruses (Haible et al. 2006).

5.8 Metagenomics in Plant Viral Diagnosis

Metagenomics is an approach for the study for virus and virus-like pathogen
populations in a sample by analyzing the nucleotide sequence content. This
method has been also applied to a wide range of samples, including bacterial
metagenomes from deep mines (Edwards et al. 2006), and the sea (Sogin et al.
2006), viral metagenomes from the human gut (Zhang et al. 2006), sea water
(Angly et al. 2006) and fresh water (Breitbart et al. 2009). Diagnosis through
metagenomic procedure offers the possibility of overcoming certain limitations of
pathogen detection associated with traditional detection procedure. Sequence
produced from an infected plant will include sequence from any pathogen(s)
present in the plant. The extraction of RNA from infected plant, production of
cDNA with a random priming method and sequencing will produce data for a large
range of potential pathogens. RNA viruses, viroids and the RNA stages of actively
replicating DNA viruses can be directly screened.
The metagenomic diagnostic procedure utilizing next-generation sequencing
has been applied for the detection of Pepino mosaic virus (PepMV) infecting
tomato plants and an uncharacterized Gay feather mild mottle virus (GMMV)
infecting Liatris spicata. A subtractive hybridization method was followed to
enrich viral cDNA. From the 71,146 fragments of sequence generated, 29,095
were identified as having similarity to published viruses based on BLAST
searching. Cluster analysis from an alignment of 1a replicase protein sequence
306 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

from the Bromoviridae family reliably placed the new virus within the Cucumo-
virus genus and the whole-genome comparisons showed that the most related virus
was Tomato aspermy virus (TAV). During 2010 Beatrix Coetzee, has successfully
applied this approach for a viral profiling in South African vineyards. However, for
most diagnostic requirements a full genome sequence is not a necessity. This
method expedites the entire process of novel virus discovery, identification, viral
genome sequencing and subsequently the development of more routine assays for
new viral pathogens. Although identification of the new virus using this approach
is extremely rapid, the analysis costs (approximately 1000 Euros per sample) are
highly prohibitive (Adams et al. 2009). Unless the testing cost is brought down to a
level affordable to the growers, this approach may be only in the realm of aca-
demic interest, than of practical utility.

5.9 Biosensors

A biosensor can be defined as a device that consists of a biological recognition

system, often called a bioreceptor and a transducer. The interaction of the analyte
with the bioreceptor is designed to produce an effect measured by the transducer,
which converts the information into a measurable effect such as an electrical
signal. Biosensors that include transducers based on integrated circuit microchips
are often referred to as biochips. In general, a biochip consists of an array of
individual biosensors that can be individually monitored and generally are used for
the analysis of multiple analytes.
Biosensors can play an important role to biosecurity, homeland security, food
safety, environmental monitoring and medical diagnostics primarily in health
applications. Indeed, the current success of biosensors is attributed to the
extraordinary demands of disease diagnoses and control, as well as the ability of
biosensors to offer a convenient, hygienic, rapid, and compact method for personal
monitoring.

(a) Role of biosensors in plant virus detection

The powerful tools of biotechnology are replacing the guesswork of early twen-
tieth Century medicine with twenty-first Century diagnostic skills that increasingly
rely on knowledge of physiology at the molecular level. In the last two decades,
viral diagnostics have been shaped by breakthroughs in immunology fueled
development of ELISA, PCR and other tests that use antibodies and chemical tags
to find evidence of plant viruses and viroids in diagnostic samples. Although they
are used as standard routine assays, these methods have a relative reliability, a
moderate sensitivity and expensive laboratory infrastructure.
Biosensors can also play an important role in diagnosis of plant virus and viroid
disease detection. Biosensors are devices with qualities quoted as rapidity, sensi-
tivity and specificity. Biosensors based on nanotechnologies are now widely used to
5.9 Biosensors 307

identify different types of biological objects. In this direction, the porous silicon is
now widely considered as a candidate for the biosensors (De Stefano et al. 2007).

(b) Mesoporous silicon sensors

The Mesoporous silicon sensors can effectively select viruses with sizes mostly
less than 50 nm among huge number of known viruses. Vashpanov et al. (2008)
observed the changes in electric parameters of mesoporous silicon treated by
plasma chemical etching with fluorine and hydrogen ions under the adsorption of
plant viruses such as Tomato ringspot virus (ToRSV), Grapevine fan leaf virus
(GFLV) and protein macromolecule from ToRSV particles. The current response
to the applied voltage is measured for each virus particle to investigate the material
parameters which are sensitive to the adsorbed particles. The peculiar behaviors of
the response are modeled by the current–voltage relationship in a metal-oxide
semiconductor field emission transistor (MOSFET). This model explains the
behavior and the double gate model of the MOSFET informs that the mesoporous
silicon is a highly sensitive means of detecting the viruses in the size range less
than 50 nm.
Biosensors based on living cells are characterized by high sensitivity, selec-
tivity and rapid response times. Perdikaris et al. (2011) have developed a novel
portable cell biosensor system for the detection of plant viruses, based on
immobilized cells carrying on their membrane virus specific antibodies, was
developed and designated as High Through Put Bioelectric Recognition Assay
(BERA-HTP) and tested for the detection of purified Potato virus Y (PVY) and
Tobacco rattle virus (TRV) in single as well as in mixed infections in two different
plant host species. The sensor was based on live, mammalian cells, the membrane
of which has been artificially saturated with antibodies specific to different plant
viruses. The attachment of PVY, CMV or TRV viral particles to the homologous
electro-inserted antibodies caused a virus specific change of the cell membrane
electric potential that was not observed with virus free samples or with heterol-
ogous viruses. Fluorescence microscopy observations showed that attachment of
virus particles to the cell membrane bearing the homologous antibody was asso-
ciated with a decrease of [Ca2+]cyt. The perspectives for the development of
BERA-HTP as a portable, reliable and rapid test have been studied (Perdikaris
et al. 2011).

(c) Quartz crystal microbalance based DNA

Recently, Quartz crystal microbalance based DNA biosensors (QCM), which is a
sensitive mass measuring device consisting of a quartz crystal wafer sandwiched
between two metal electrodes which are connected to an external oscillator circuit
that records the resonant frequency.
Quartz crystal microbalance (QCM) method was used by Eun et al. (2002) to
detect plant viruses based on DNA-RNA hybridization. Oligonucleotide probes
were immobilized on a QCM which bound to complementary target viral RNA of
two orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum
Ringspot Virus (ORSV) present within the crude sap of infected orchids.
308 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

QCM biosensors offer a more economical means of plant virus detection

compared with other techniques. The entire set up of the detection system is
estimated to be less than US$2,000 (Eun et al. 2002), with most of the equipment
and raw materials readily available commercially. The relative cost per sample
analyzed is low because these biosensors do not require the use of expensive
labeling reagents for example radioactive isotopes, enzymes and primers fluores-
cent probes. Upon optimization of the nucleic acid immobilization techniques and
hybridization times, QCM biosensors require minimal sample preparation and data
is rapidly collected. Leaf crude saps can be used directly for the tests, purification
of viral RNA is not necessary. The nucleic acid based CymMV and ORSV QCM
biosensors are more sensitive than the corresponding antibody based ones (Eun
et al. 2002). This could be due to the mechanism of probe and target recognition:
QCM immuno-biosensors possess immobilized antibodies which recognize the
epitopes on the viral coat proteins whereas QCM DNA biosensors possess
immobilized nucleic acid sequences which hybridize with their complementary
virus CP gene sequences. This method will be a viable alternative for plant
diagnostic clinics and field experimental stations during large scale plant screening
for virus free certification, quarantine verification, germplasm collection, and
selection of disease resistant plants.

(d) Antibody-Based Surface Plasmon Resonance (SPR) Detection of Intact

Viral Pathogen
The Surface Plasmon Resonance (SPR) technique has been used to investigate
primarily the binding of macromolecules with surface-bound ligands (e.g., pro-
teins). The effort has been recently extended to large size analytes such as viral and
bacterial pathogens (Boltovets et al. 2004; Fratamico et al. 1998; Koubova et al.
2001), and cells (Hide et al. 2002; Quinn et al. 2000). Major advantages of the SPR
technique (Homola 2003), such as rapid, real-time, and non-labeling analysis and
miniaturization for portable applications, support the potential development of
pathogen detection biosensors. However, in many cases, the SPR applications in
virology have focused on indirect detection of viral sub-elements such as viral
peptides rather than an entire form of intact viruses (Wittekindt et al. 2000). The
analysis of intact viruses should be considered to develop a pathogen detection
biosensor for exposure in a natural environment where biochemical treatment is
undesirable, and for real-time medical diagnostics.
Boltovets et al. (2004) investigated an intact plant virus, Tobacco mosaic virus
(TMV), by using the SPR. Through a pre-incubation step, they prepared an anti-
viral IgG–TMV complex and then detected specific binding between the complex
and a protein A (IgG-specific) immobilized on a sensor substrate.

(e) Biochip detects apple virus

A direct method of detecting plant viruses using an aptamer based biochip has
been proposed by Gyurcsanyi, Budapest University of Technology and Economics
and Meszaros, Semmelweis University, Budapest for the detection of apple viruses
(Anon 2010). Apple stem pitting virus (ASPV) is a worldwide virus that has been
5.9 Biosensors 309

Fig. 5.8 Planar gold sensor

chip for the detection of
Apple stem pitting virus.
Courtesy Bonne 2010

associated with complex growth disorders in fruits. Routine methods to detect

ASPV use biological indexing by grafting infected tissue onto indicator hosts,
which can be complex and costly. Now a biosensor that can detect a characteristic
protein of the virus in a fast, cheap and reproducible way has been developed.
The infection can be diagnosed by identifying the unique proteins of the virus
explained by Gyurcsanyi. They designed DNA aptamers, which they attached to
gold surface plasmon resonance (SPR) chips, to target and bind to an ASPV
specific protein. Surface Plasmon resonance (SPR) imaging monitors the direct
interaction of these aptamers with the protein and indicates the presence of the
disease (Fig. 5.8). The combination of aptamer receptors and imaging SPR offers a
novel sensing platform that meets the demand for fast and cost effective detection.

(f) Quartz crystal microbalance (QCM) immunosensors

In this novel technique for plant virus detection, a quartz crystal disk is coated with
virus specific antibodies. Voltage is applied across the disk, making the disk warm
slightly via a piezoelectric effect. Adsorption of virus particles to the crystal surface
changes its resonance oscillation frequency in a concentration dependent manner
hence it is therefore qualitative and quantitative technique for plant virus detection.

5.10 DNA Barcodes Use as Genetic Markers

for Identifying Pathogens

DNA barcode is essentially a short stretch of nucleotide sequence that aid in the
specific identification of species, strains or sub-strains. They are used to resolve
pathogen taxonomy, phylogeny and for identifying pathogens and pests (Kumar and
Sharma 2010). DNA barcodes are also known as DNA markers or DNA finger prints.
310 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

The plant virus and viroids are characterized by sequencing the specific genes
and the data generated is used to interpret origin and spread of the pathogen,
taxonomy and phylogeny. For diversity assessment, gene targets are selected based
on the pathogen that comprise, ribosomal Internal Transcribed Sequence (ITS),
mitochondrial cytochrome oxidase sub unit (COI), histone, virus coat protein etc.,
The concept is simple and a sample of the specimen is processed to produce the
barcode. This is then matched against a library of known barcodes and in this way
the specimen is identified. To do this, the barcode data base must first be con-
structed. The DNA barcode libraries are under construction for the medicinal
plants of several nations including South Africa and Nigeria.
The approach has been used at IITA (Nigeria) for assessing the diversity of
Cassava brown streak virus, Banana bunchy top virus and several other pathogens
including fungi like Colletotrichum, Pythium and Cercospora species. Information
generated from these studies have proved valuable clues to understand the origin
and drivers of spread, identification of previously uncharacterised pathogens (Alibi
et al. 2008;Kumar and Sharma 2010).
This technique has been used in the diagnosis of Alstroemeria yellow spot
Tospovirus, Prunus necrotic ring spot virus, Potato yellow vein virus etc. The
DNA barcoding offers accurate identification of plant virus and virus like diseases
and focuses on strengthening the link between traditional and molecular
taxonomy.

5.11 Conclusions

With even more advances in molecular biology and immunology, scientists and
farmers alike will be able to improve plant disease diagnosis. Efforts are already
underway to produce better diagnostic kits to detect pathogens in crops important
to developing countries. Diagnostic kits are an investment; they may be expensive,
but the costs can be offset by gains, such as reduced crop losses and more envi-
ronment-friendly crop-management practices. Their development should be made
a priority by both the public and private sectors in developing countries. New
problems will continue to arise. Perhaps new diseases will evolve or will spread to
regions where they have previously been absent, and new techniques for studying
these pathogens have to be developed as technologies advance. The plant
pathologist, who understands and keeps abreast of modern concepts about the
nature of plant disease and the ways in which they can be controlled, will be a vital
resource for the agricultural and horticultural crops.
As it has been seen in the last 20 years, plant viruses and viroids are becoming
more widespread and there are real threats of new virus epidemics. It is therefore
essential that the movement of viruses around the world be documented and
quarantine restrictions put in place wherever necessary. Among the methods of
detection discussed above, arrays are capable of detecting a wide range of viruses
and show the most promise of accurately identifying new viruses as they move to
5.11 Conclusions 311

new geographical areas and to new hosts. At present, however, the costs and
technical difficulties of designing, constructing and utilizing microarrays, limited
use in government quarantines, agricultural organizations, and MNC companies.
Hopefully, costs will reduce as chips become available commercially and as
economies of scale are realized. In the mean time, organizations ideally should
utilize more than one diagnostic technique, and they should screen for high-risk
viruses like tospo and Begomoviruses even where they are not known to exist in
the region which would prevent the catastrophic plant virus outbreaks.
The techniques available have evolved significantly in the last few years to
achieve rapid and reliable detection of virus and viroid pathogens, extraction of the
target from the sample being important for optimizing detection. For viruses,
sample preparation has been simplified by imprinting or squashing plant material
or insect vectors onto membranes. Specific polyclonal, monoclonal, and/or
recombinant DNA technology-based antibodies are available for many plant
viruses and have contributed to the specificity of serological detection. Molecular
detection can be optimized through the automatic purification of nucleic acids
from viruses by columns or robotics. New variants of PCR, such as simple or
multiplex nested PCR in a single closed tube, co-operative-PCR and real-time
monitoring of amplicon or quantitative PCR, allow high sensitivity in the detection
of one or several viruses in a single assay. The latest development in the analysis
of nucleic acids is microarray technology, but it requires genomic DNA/RNA
extraction and pre-amplification methods to increase detection sensitivity. The
advances in research that will result from the sequencing of many plant virus
genomes, represent a new source of information for the future development of
sensitive and specific detection techniques for viruses.
The effectiveness of a detection method is highly influenced by the way the
tissue samples were collected, because of its simplicity and possibility of handling
a large number of samples at one time. Recent developments in molecular
detection technology led to the development of more convenient, effective, and
specific assays and permitted the use of these tests for detecting plant pathogens,
including viruses. Such assays will help growers, crop agronomists, and plant-
health professionals not to rely exclusively on symptomatology and/or time-con-
suming diagnostic procedures, and permit early detection of viral and viroid
infections. Above discussed new techniques are effective management tools to be
used in parallel with knowledge of the crop, understanding thebiology of the
pathogen and the ecology of the disease. Thus, these tools can be applied to
determine the point in time at which control measures should be implemented. In
addition, such diagnostic assays are essential tools for programs devised to pro-
duce virus-free plant propagative materials. Viral genome sequence data available
made it very easy to design primers for different uses, for broad or specific
detection of viral pathogens. Similarly, the production of monoclonal antibodies
has helped in the development of immunological tests increased capacity in terms
of specificity, not provided earlier by polyclonal antibodies. Among the variety of
immunological tests now available, TBIA proved to be extremely helpful for large
scale testing at a very low cost without much compromise on sensitivity or
312 5 Diagnosis and Detection of Plant Virus and Viroid Diseases

specificity even at field conditions. It is an essential test to be used in developing

countries where sophisticated equipments are not available and the cost/sample is
a limiting factor for any assay to be adopted for large scale testing.
The use of polyphasic or integrated approaches for detection is advised,
especially when the targets are plant quarantine viruses or viroids (Lopez and
Cambra 1996; Lopez et al. 2003; Alvarez 2004; Janse 2005). The recently pub-
lished new versions of the official EU protocols for certain bacterial and viral
diseases of plants have incorporated PCR as screening test in an integrated pro-
tocol, including serological techniques, isolation and bioassays, for higher accu-
racy of the detection of quarantine pathogens. This approach, not only increases
our ability to detect plant pathogens but also can provide new insights into their
ecology and epidemiology (Martin et al. 2000; Alvarez 2004). The methodology
for selecting and validating a test for routine diagnosis has also been discussed
(Janse 2005).

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Index
A Avocado Sunblotch viroid (ASBVd), 45,
Abutilon mosaic virus, 50
Aerial vectors, 193
African cassava mosaic virus, 14, 28, 102,
123, 275
African cereal streak virus, 6
African oil palm ringspot virus, 43, 82
Agalina albidula, 198
Agarose gel electrophoresis, 238, 269, 286
Agglutination Tests, 240
Alfalfa cryptic virus, 59
Alfalfa mosaic virus, 114, 122
Alkaline phosphatase (ALP), 249, 255, 257,
258, 260, 297
Allexivirus, 17, 39
American plum line pattern virus, 45
Amplified fragment length polymorphism
(AFLP), 200
Andean potato latent virus, 192
Antibody-Based Tests, 239
Aphids virus transmission, 19, 78
Apple chlorotic leaf spot virus, 18, 43,
250, 288
Apple dimple fruit viroid, 60
Apple mosaic virus, 129, 192, 256,
266, 277
Apple scar skin viroid (ASSVd), 77, 85, 170,
239, 261, 278, 286
Apple stem grooving virus, 18, 271, 276
Applications of DBH, 265–267, 286
Applications of DIBA, 258, 259
Arabis mosaic virus, 90, 171, 206, 207,
268, 275
Array Technologies, 294, 299
Asparagus bean mosaic virus, 171
Australian grapevine viroid, 60, 170
K. S. Sastry, Plant Virus and Viroid Diseases in the Tropics,
DOI: 10.1007/978-94-007-6524-5, Ó Springer Science?Business Media B.V. 2013
77, 170
Avsunviroidae, 81, 84, 85, 91
Azuki bean mosaic virus, 171
B
Babuvirus, 59, 195, 198
Badnavirus, 18, 21, 163, 195, 204, 205, 295
Banana bract mosaic virus, 6, 102, 127
Banana bunchy top virus (BBTV), 15, 102,
195, 285, 305, 310
Banana mild mosaic virus, 43
Banana streak badna virus (BSV), 15
Barley stripe mosaic virus, 107, 169
Barley yellow dwarf virus, 15, 106–109,
260, 285
Bean common mosaic virus, 109, 114, 162,
195, 246
Bean golden mosaic virus (BGMV), 6, 7, 110,
267, 272
Bean pod mottle virus, 101, 111
Bean urd leaf crinkle virus, 189
Bean yellow mosaic virus, 109, 111–114
Beet cryptic virus, 60
Beet curly top virus, 109, 115, 118, 198, 267
Beetle vectors, 162, 203, 204
Beet soil-borne mosaic virus, 72
Beet western yellows virus (BWYV), 58,
101, 120
Begomovirus, 3, 15, 102, 103, 195, 200, 201,
267, 285, 311
Bemisia tabaci, 15, 199–202, 263, 279
Benyvirus, 16, 18, 72, 207
BERA-HTP, 307
Bhendi yellow vein mosaic virus, 100, 117, 201
355
356
Bioassays using indicator plants, 23
Biological Approaches, 236
Bioterrorism, 28, 29
Bitter gourd yellow vein virus, 50
Bittergourd yellow mosaic virus, 6
Blackberry chlorotic ringspot virus, 45
Blackcurrant reversion virus, 37
Blackeye cowpea mosaic virus, 162, 175, 242
Black gram leaf crinkle virus, 189
Blackgram mottle virus, 175, 248, 251,
252, 262
Brassica chinensis, 189
Bromoviridae, 16, 17, 30, 33, 45, 46, 198, 306
Bromovirus, 17, 45, 203, 237
Bunyaviridae, 16, 18, 26, 47, 199
C Coconut cadang–cadang viroid (CCCVd), 61,
Cacao swollen shoot virus, 6, 14, 27, 102, 104,
134, 204
Cacao yellow mosaic virus, 44
Capsicum chlorosis virus, 6, 103, 202, 253,
254, 291
Cardamom mosaic virus, 62, 250, 292
Cardamom vein clearing virus, 250
Carla virus, 7, 18
Carmovirus, 17, 70, 196, 203, 207, 208
Carnation ringspot virus, 17, 70, 193
Carrot thin leaf virus, 63, 119
Cassava mosaic virus, 29, 102, 104, 123, 124,
200, 201, 246, 272
Cauliflower mosaic virus, 14, 18, 47, 120, 237,
245, 285, 305
Caulimoviridae, 16, 18, 46, 47, 103, 198, 272
cDNA chip technology, 303
Cereal yellow dwarf virus, 58, 90
CGIAR, 9
Chenopodium amaranticolor, 237
Chenopodium murale, 185
Cherry leaf roll virus, 37, 252, 253, 266,
271, 277
Cherry mottle leaf virus, 43, 205, 267, 272
Cherry necrotic rusty mottle virus, 43
Chick pea chlorotic dwarf virus, 6
Chickpea chlorotic stunt virus, 58
Chilli leaf curl virus, 50, 116
Chilli ringspot virus, 63
Chilli veinal mottle virus, 63
Chrysanthemum stunt viroid, 61, 78, 190, 267
Cicadellidae, 138, 198
Citrullus vulgaris, 177, 185
Citrus exocortis viroid, 61, 77, 126, 266,
286, 288
Citrus infectious variegation virus, 6
Index
Citrus leaf rugose virus, 45
Citrus leprosis virus, 72
Citrus mosaic virus, 6, 264, 268, 272, 274
Citrus psorosis virus (CPsV), 6, 18, 59,
126, 266
Citrus ring spot virus, 250, 288
Citrus tristeza virus (CTV), 6, 27, 29, 101,
102, 104, 125, 242, 248, 250, 261, 267,
271, 277
Citrus variegation virus (CVV), 45, 267
Citrus yellow mosaic virus, 47, 272
Classification of plant viruses, 36
Climate and agricultural productivity, 8
Closteroviridae, 17, 48, 49, 198, 202, 299
Cocoa necrosis virus, 37
Cocoa swollen shoot virus (CSSV), 6, 27, 176,
204, 246, 272, 290
78, 85, 137
Coleus blumei viroid, 61, 85
Colocasia bobone disease virus (CBDV),
125, 165
Complementary nucleic acid probes, 264
Construction of a species name, 33
Continuous-flow PCR (CF-PCR), 277
Controversial seed transmission, 162
Co-operational-PCR (Co-PCR), 276
Cotton leaf curl virus, 104, 133, 208, 254
Cowpea aphid borne mosaic virus, 6, 63, 100,
112, 176, 246, 251, 270
Cowpea severe mosaic virus, 6, 36, 177, 251
Crinivirus, 17, 49, 102, 200, 202
Crop losses in biofuel crops, 140
Crop losses in cereals and millets, 104
Crop losses in edible oil seed crops, 136
Crop losses in food legumes, 109
Crop losses in fruit crops, 11, 99, 126
Crop losses in industrial crops, 131, 294
Crop losses in spice crops, 139
Crop losses in tuber crops, 104, 121
Crop losses in vegetables, 11, 99, 104, 115
Crypticviruses, 170
Cucumber chlorotic spot virus, 6
Cucumber green mottle mosaic virus
(CGMMV), 6, 74, 118, 177, 193, 246,
303, 304
Cucumber mosaic virus, 6, 14, 17, 27, 33,
34, 45, 90, 112, 114, 116, 118, 123,
178, 197, 237, 242, 243, 250–253,
257–260, 271, 275–277, 284, 288,
290, 291, 307
Cucumber pale fruit viroid (CPFVd), 190
Cucurbit aphid-borne yellows virus, 58, 252
Curtovirus, 18, 57, 195, 198, 231
Index
Cypripedium virus Y, 64
Cytorhabdovirus, 18, 36, 196, 198
D F
DAC-ELISA, 256
Dahlia mosaic virus, 47
Dalbulus maidis, 108, 198
Dapple apple virus (DAV), 129
DAS-ELISA, 247, 249, 255–257, 263,
296, 301
Dasheen mosaic virus, 6, 64, 125, 165, 289
Defective interfering particles, 90, 267
Delphacidae, 198, 199
Diagnosis and Detection, 263, 293
Diagnosis and detection of virus physical
tests, 236
Diagnosis detecting plant viruses, 241, 266,
268, 308, 311
Dianthovirus, 16, 17, 70, 71
DIAPOPS, 278
Diascorea bacilliform virus (DBV), 47
Direct binding PCR, 274, 294
Disperse dye immunoassay (DDIA), 262
DNA Barcodes, 235, 309, 310
DNA Microarrays, 294, 301
DNA viruses, 15, 16, 18, 27, 102, 103, 200,
205, 266, 272, 286
Dolichos yellow mosaic virus, 6, 51
Dot blot-ELISA, 257, 259
Dot-blot hybridization (DBH), 84, 87,
265–267, 272, 286
Dot-immuno binding assay (DIBA), 258
dsDNA viruses, 27, 272
dsRNA viruses, 16, 272
Duplex PCR, 274, 275
E History of plant viruses, 12, 13
East African cassava mosaic virus, 6, 28, 51,
102, 123, 172, 175, 288
EBRIA, 247
Electroblot immuno assay (EBIA), 258
Electron Microscopy, 236, 244–246, 293
ELISA, 162, 234, 242, 247, 249, 250,
255–259, 261–263, 272–274, 277, 278,
286, 293, 295–297, 301, 306
ELISA for virus detection in insect vectors,
263
ELISA for virus detection in seeds, 262
Enamovirus, 16, 17, 58, 195
Endornaviridae, 16, 18, 27, 49
Enzyme Linked Immunosorbent Assay, 234,
244, 247, 249, 250
357
Equator, 1
Euphorbia leaf curl virus, 51
Fabavirus, 17, 37, 195, 198, 237
Fig mosaic virus, 72
Fiji disease virus, 16, 69
Flexiviridae, 198
Fluorescence RT-PCR, 283
Fluoresence intensities quantification, 302
Foveavirus, 18, 42, 276
Frankliniella occidentalis, 202, 283
Fungal vector, 120, 193
Furovirus, 18, 73, 207, 237
G
Garlic mite-borne filamentous virus, 39
Gel double immuno diffusion, 242
Gel electrophoresis, 84, 89, 234, 238, 239,
261, 280, 286
Geminiviridae, 16, 27, 122, 201, 267, 305
Geminiviruses, 21, 27, 122, 201, 267, 305
Genome structure, 80, 194
Gomphrena globosa, 237
Grape leaf roll clostero virus, 246, 250
Grapevine fan leaf virus (GFLV), 128, 307
Groundnut bud necrosis virus (GBNV/PBNV),
46, 203, 295
Groundnut eye spot virus, 6
Groundnut mottle virus, 251
Groundnut rosette virus, 103
Groundnut yellow spot virus, 46
H
High plains virus, 179, 206, 253
Hop stunt viroid (HSVd), 61, 78, 85, 261, 266,
286, 288
Horsegram yellow mosaic virus, 6, 52
Hosta virus X, 40
Host range and transmission, 78
Hostuviroid, 61, 82, 85, 168, 286
I
ICNV, 30, 32
IC-PCR, 273, 274, 277
IC-RT-PCR, 273, 274, 295
ICTV, 14, 16, 30–35, 76, 92, 102, 134,
136, 201
Immuno Diffusion Tests, 241
358
Immunochromotography, 243
Immuno fluorescent assay (IFA), 248
Immunosorbent electron microscopy (ISEM),
245, 246
In situ hybridization (ISH), 268, 285
In situ PCR (IS-PCR), 285
Indian cassava mosaic virus, 6, 246, 272
Indian citrus ring spot virus, 250, 288
Indian peanut clump virus, 74, 136, 193,
207, 247
Indonesian soybean dwarf virus, 6, 59
Insect vector transmission, 193
International Agricultural Research Centers, 9
Iris yellow spot virus, 7, 139, 203, 263
IUMS, 30, 32
IWGLV, 31
J Necrovirus, 71, 208, 237
Jatropha mosaic virus (JMV), 140
Johnsongrass mosaic virus, 64
L Nineth ICTV report, 35
Labeled Antibody Based Assays, 245
LAMP, 279, 280
Lateral flow test, 244
Latin and binomial nomenclature, 33
Leaf, plant and tree hopper virus
transmission, 198
Lettuce big-vein associated virus, 18, 73
Lettuce mosaic virus, 120, 194, 285
Lettuce necrotic yellows virus, 18, 36
List of ICTV reports, 35
List of seed transmission of plant viroids, 76
List of seed transmission of plant viruses, 162
Lucerne Australian latent virus, 38, 180
Lucerne transient streak virus, 90
Lupinus albus, 184
Luteoviridae, 16, 25, 271, 279
M 260, 292
Maclura mosaic virus, 17, 62
Macroarray, 84, 300
Maize rayado fino virus, 198
Maize streak masterovirus (MSV), 15
Mealybug vectors, 102, 204, 205
Melon aphid-borne yellows virus, 58
Mesoporous silicon sensors, 307
Metagenomics in viral diagnosis, 305
Mirafiori lettuce big-vein virus, 59
Microarray, 84, 235, 257, 294, 299–304, 311
Mirids, 205
Index
Modifications of ELISA, 257
Molecular beacons, 278
Molecular Hybridization, 282–284
Monoclonal antibody (MAb), 256
MOSFET, 307
Multiplex nested-PCR, 276, 311
Multiplex-PCR, 275, 276, 284, 294
Mungbean yellow mosaic virus, 7, 52, 112,
113, 201
N
Nanoviridae, 16, 27, 59
Nanovirus, 16, 18, 21, 27, 59, 163, 196, 198,
269, 272
NASBA, 278
NASBA-ECL, 278
NASH, 264, 265
Nephotettix nigropictus, 198
Nepovirus, 16, 17, 37, 38, 163–167, 169
Nested-PCR, 276
Nilaparvata lugens, 69, 199, 263
Nucleorhabdovirus, 18, 36, 196, 198
O
Okra leaf curl virus, 118, 254
Okra mosaic virus, 7, 44
Okra yellow vein mosaic virus, 53, 200, 254
Olpidium brassicae, 120, 207
Onion yellow dwarf virus, 65, 139,
180, 289
Ophioviridae, 16, 18, 26, 59
Ophiovirus, 18, 53, 208
P
Papaya leaf curl virus, 53, 201
Papaya leaf distortion mosaic virus, 65
Papaya ring spot virus, 128, 180, 259,
Pararetroviruses, 16, 305
Particle morphology, 21, 23, 91, 157, 194,
207, 293
Partitiviridae, 16, 18, 27, 59, 60
PAS-ELISA, 249, 256
Passion fruit woodiness virus, 65, 261
Pathogenecity, 22, 80
PCR application for viroid diagnosis, 286
PCR application for virus diagnosis, 286
PCR-ELISA, 277
PCR-RFLP, 284
Index
PCR variants, 273
Pea early browning virus, 75, 180, 207,
252, 253
Pea leaf roll virus, 7, 110
Pea mosaic virus, 110, 114
Pea seed-borne mosaic virus, 65, 169, 247,
252, 253, 290
Peanut bud necrosis virus, 7, 103, 254,
291, 292
Peanut clump virus, 7, 18, 74, 136, 182, 193,
207, 247, 253, 254
Peanut mottle virus, 65, 101, 111, 136, 182,
251, 253, 261, 291
Peanut stripe virus, 111, 136, 186, 253, 261,
270, 291
Peanut stunt virus, 45, 90, 136, 182, 253, 291
Pecluvirus, 18, 21, 74, 207
Pepino mosaic virus, 40, 162, 183, 192,
253, 305
Pepper chat fruit viroid, 61, 183, 291
Peregrinus maidis, 199
Persea americana, 171
Phase Display Technology, 295, 296
Phyto rhabdoviruses, 21
Phytopathology, 79, 207
Phytoplasma, 11, 14, 85–88, 103, 105, 119,
121, 122, 132, 138, 140, 198, 276
Phytoplasma taxonomy, 87
Picornavirales, 36–39
Pigeonpea sterility mosaic virus, 7
Pineapple mealybug wilt-associated
virus, 48, 131
Pisum sativum, 110, 174, 180–182
Plant Virus Diseases in Tropics, 7
Plant Virus Subcommittee (PVS), 31
Plantibodies, 297, 298
Plant-virus interactions, 27
Plum pox virus (PPV), 14, 266, 268, 271, 296
Polyacrylamide gel electrophoresis (PAGE),
238
Polyclonal Antibodies (PAbs), 299
Polymerase chain reaction (PCR), 235,
268, 269
Polymyxa graminis, 207
Poplar mosaic virus, 42, 192
Pospiviroidae, 60, 61, 81, 82, 84, 85, 91
Potato leaf roll virus, 15, 100, 101, 103, 104,
121, 246, 248, 257, 260, 271, 283,
285, 298
Potato mop-top virus, 18, 74
Potato spindle tuber viroid, 61, 77, 85, 101,
121, 170, 183, 190, 263, 266, 267, 280,
283, 286, 289
359
Potyviridae, 16, 17, 19, 25, 31, 61–68, 102,
138, 198, 207, 271
Potyvirus, 6, 15, 17, 19, 21, 62–68, 102, 135,
163–167, 195, 238, 244, 258, 267
Precipitation Tests, 240, 241
Principles of nomenclature, 32
Probes for microarrays, 302
Prune dwarf virus, 25, 46, 256, 266
Prunus necrotic ringspot virus, 46, 266
PTA-ELISA, 249, 255, 256
Q
Quartz crystal microbalance (QCM), 307, 309
R
Radial immuno diffusion test, 242
Radio immuno sorbent assay (RISA), 247
Rapid immuno filter paper assay (RIPA), 243
Raspberry bushy dwarf virus, 18, 72, 237
Raspberry ringspot virus, 38
R-ELISA, 257, 277, 286
Real Time Quantitative PCR, 281
Real time RT-PCR, 84, 268, 282, 283,
293, 301
Recombinant Antibodies, 257, 295, 297–299
Recombinant DNA Technology, 294, 311
Reoviridae, 16, 18, 27, 69, 272
Rhabdoviridae, 16, 18, 26, 36, 198, 199, 271
Rice dwarf virus, 69
Rice grassy stunt virus, 7, 73, 105
Rice hoja blanca virus, 7, 73, 103–105, 199
Rice stripe virus, 7, 18, 73, 105, 199, 285
Rice transitory yellowing virus, 7
Rice tungro bacilliform virus, 18, 48, 103,
105, 285
Rice tungro spherical virus, 17, 39, 103, 105
Rice yellow mottle sobemovirus, 15
Rolling Circle Amplification (RCA), 304, 305
R-PAGE, 239, 286
RT-LAMP, 279, 280
RT-PCR, 84, 162, 270, 273–276, 278,
282–284, 286, 293, 295, 299, 301
S
Satellite viruses, 16, 89, 91
scFv, 257, 296
Secoviridae, 16, 17, 25, 36–39
Sequivirus, 17, 38, 39, 195, 198
Single-Cell-RT-PCR (SC-RT-PCR), 271
Single diffusion in tubes, 241
360
Sirevirus, 69
Sobemovirus, 15, 18, 72, 73, 165, 196,
202, 238
Soil-borne cereal mosaic virus, 73
Soil-borne wheat mosaic virus, 7, 18, 73, 107,
189, 193, 207
Sorghum chlorotic spot virus, 7, 18, 73, 107,
193, 207, 237
Sorghum mosaic virus, 73
South African cassava mosaic virus, 7, 54, 102
Southern and Northern blot hybridization, 267
Southern bean mosaic virus, 18, 28, 72, 100,
174, 200, 238, 251
Sowbane mosaic virus, 7, 72, 128
Soybean golden yellow mosaic virus, 7
Soybean mosaic virus, 7, 66, 101, 111, 184,
247, 253, 257, 259, 261, 266, 292
Soymovirus, 18, 48
Spiroplasma, 13, 86, 88, 89, 140
Squash leaf curl virus, 54, 267, 280
ssDNA Viruses, 16, 18, 272
ssRNA Viruses, 17, 18, 208, 270, 272, 284
Steps in PCR, 268
Strawberry latent ringspot virus, 39
Subterranean clover mottle virus, 72, 90,
186, 192
Subterranean clover red leaf virus, 114
Sugarbeet yellows virus, xix
Sugarcane bacilliform virus, 7, 204, 305
Sugarcane chlorotic streak virus, 7
Sugarcane fiji disease virus, 7, 16, 290
Sugarcane mosaic virus, 7, 29, 66, 100, 102,
108, 109, 131, 186, 197, 248, 251,
258, 274
Sugarcane streak mosaic virus, 62, 251, 256,
259, 273, 274, 290
Surface Plasmon Resonance (SPR), 308, 309
Sweet potato chlorotic fleck virus, 7, 42, 246
Sweet potato chlorotic stunt virus, 7, 28, 29,
123, 200
Sweet potato feathery mottle potyvirus, 15,
102, 167
Sweet potato leaf curl virus, 7, 55
Sweet potato vein clearing virus, 48
T Transmission Through Contact and
Taqman technology, 283
Taro bacilliform virus, 47, 124, 290
TAS-ELISA, 249, 255, 256
Taxonomy of viroids, 30
Tea phloem necrosis virus, 7
Tenuivirus, 16, 18, 73, 196
Tepovirus, 43
Index
Thrips vectors, 196, 202, 203, 285
Tissue blot immuno binding assay
(TBIA), 260
Tissue print immunoassay (TPIA), 260, 261
Tobacco etch virus, 67, 116, 133
Tobacco leaf curl virus, 7, 116, 133
Tobacco mosaic virus, 13, 14, 18, 30, 33, 34,
74, 100, 115, 128, 133, 162, 193, 236,
248, 266, 298, 308
Tobacco necrosis virus, 13, 17, 21, 71, 110,
128, 193, 207, 237
Tobacco rattle virus, 8, 16, 18, 75, 207, 307
Tobacco ring spot virus, 111, 117–119, 128,
169, 187
Tobacco streak virus, 111, 136, 138, 187, 190,
191, 203, 270, 274, 291, 292
Tobacco stunt virus, 118, 134, 207
Tobacco vein distorting virus, 59
Tobacco vein mottling virus, 67, 133
Tobamovirus, 18, 21, 27, 33, 34, 74, 75, 274,
283, 301, 303, 304
Tobravirus, 16, 18, 21, 75, 197
Tomato aspermy virus, 45, 188, 306
Tomato golden mosaic virus, 8, 55
Tomato infectious chlorosis virus, 49
Tomato leaf curl virus, 56, 90, 115, 200, 201,
263, 291, 292
Tomato mosaic virus, 7, 66, 67, 75, 192,
193, 253
Tomato pseudo-curly top virus, 18, 58, 199
Tomato spotted wilt virus, 14, 18, 30, 46, 103,
110, 115, 136, 196, 202, 253, 270,
282, 285
Tomato torrado virus, 15, 17, 39, 200
Tomato yellow leaf curl virus, 14, 15, 57, 104,
115, 200, 201, 209, 279, 292
Tombusviridae, 16, 17, 25, 70, 72, 208, 271
Tombusvirus, 17, 71, 72, 165, 208, 238
Topocuvirus, 18, 58, 199
Torradovirus, 17, 39
Tospovirus, 6, 15, 16, 18, 21, 29, 46, 103, 196,
202, 203, 238, 256, 271, 310
TPMS, 32
Transmission electron microscopy (TEM), 236
Transmission of Plant Viruses
and Viroids, 161
Mechanical, 191
Transmission Through Pollen, 190
Transmission Through Seed, 162, 170
Transmission Through Vector, 193–206
Transmission Through Vegetative
Propagules, 161
Transmission Through Water, 193
Index
Trichovirus, 18, 43, 163, 165, 195, 204,
238, 272
Tritimovirus, 17, 19, 68
Tropical agriculture, 5, 9
Tropical Countries, 2–4, 6, 9, 78, 87, 102,
124, 199
Tropical Crops, 5, 11, 208, 237, 250, 288
Tropics and Climate, 1
Tropics of cancer, 1, 3
Tropics of capricon, 1, 3
True seed-transmitted viruses, 251
Tulip breaking virus, 67
Tungrovirus, 14, 18, 48, 198
Turnip mosaic virus, 7, 67, 119, 120, 189
Tymovirus, 3, 17, 44, 45, 163, 166, 170,
196, 203
U Wound tumor virus, 18, 69
Ugandan cassava brown streak virus, 62
Umbravirus, 18, 73, 195
Urd bean leaf crinkle virus, 189
V of okra, 117, 118, 200
Vegetative propagules, 161, 246, 263, 288
Velvet tobacco mottle virus, 73, 90, 205
Vicia cryptic virus, 60
Viral genome-based tests, 294
Viroid Classification, 82, 83, 85
Viroids movement, 25, 26, 84
Viroids symptomatology, 79, 311
Virus nomenclature, 32
361
Virus Classification, 30–35
Virus diseases of Tropical Countries, 6–8
Virus Identification Data Exchange project
(VIDE), 31
Virus replication, 25–27
Viruses and Sub-Viral Agents, 11, 32
W
Waikavirus, 17, 39, 195
Watermelon bud necrosis virus, 8, 119, 202
Watermelon mosaic virus, 7, 8, 62, 65, 67, 118,
119, 189
Western blot analysis, 261, 297
Wheat soil borne mosaic virus, 8, 189
Wheat streak mosaic virus, 8, 17, 68, 106, 189,
206, 254, 291
Whitefly vectors, 102, 285
Y
Yam mosaic virus, 68, 125, 251, 256, 290
Yellow vein mosaic virus
Yield Losses in Different Crops, 6, 99, 104
Z
Zucchini green mottle mosaic virus, 75, 304
Zucchini yellow mosaic virus, 8, 68, 189, 247,
270, 291