slide

1. Detection and Identification of
Plant Viruses in Quarantine

2. Plant Viruses
• Nucleic acid with a protein coat
• RNA/ DNA
• Systemic infection
• Mode of transmission
 Vectors
 Mechanical
 Seeds

3. Virus Detection Techniques
• Biological (grow out/ infectivity test)
• Physical (electron microscopy)
• Biochemical (staining of inclusions)
• Serological (ELISA, DIBA, ISEM)
• Molecular (NASH, PCR, RT- PCR, Real-time PCR,
Real-time RT-PCR, FTA, Microarrays)

4. Dry Seed Examination
Healthy
Mottling Mottling Healthy
Tennis ball-like Split seed coat Healthy

5. Processing of Legume Germplasm in PEQ Greenhouses and Containment Facility
Glycine max
Pisum sativum
Containment Facility

6. Symptoms only serve as a guide but
indispensable
• Similar symptoms can be produced by different viruses
• Symptoms may be extremely variable; the same virus can
produce a range of symptoms
• Lack of symptoms does not necessarily mean that no viruses
are present.
• Mixed infection result in more severe symptoms
• Symptoms are only indicative, not confirmatory
• Reduction in growth
• Colour deviation
• Necrosis
• Malformation

7. Symptoms of Plant Viruses

8. Symptoms of Plant Viruses

9. Symptoms of PPV

10. Symptom of MDMV
Symptoms of HPV

11. TMV symptoms in trailing petunia
Viruses may cause abnormal colour breaks
Color break symptoms on flowers of flowering tobacco

12. Infectivity Assay- Mechanical
Inoculation

13. Mechanical Inoculation and Early Stages in the Systemic
Distribution of Viruses in Plants

14. Schematic Representation of the Direction and Rate of
Transmission of a Virus in a Plant

15. Infectivity Test

16. Necrotic Local Lesions by
BCMV on Chenopodium
amaranticolor
Systemic Infection of SMV on
Nicotianatabacumxanthi
Infectivity Assay Contd…

17. Biochemical Technique
Staining of inclusion bodies
• observation under light microscope
• inclusion bodies are viral aggregates or proteins
induced in cytoplasm or nucleus
• staining by Azure A and O-G combinationst
CPMVCMVBYMVTMV

18. Cell Inclusion Bodies Observed with an Electron Microscope

19. • Reveals shape and size of
the particle
• Gives idea of the group to
which the virus belongs
• Very expensive
equipment, often not
available
Physical- Electron Microscopy

20. Physical – Transmission Electron Microscopy Contd…

21. Serological Techniques
•Based on antigen - antibody reaction
• Antigen: a protein or polysachharide which induces
the formation of antibodies when injected into a
warm-blooded animal
• Antibody: a specific protein formed in the blood of
warm-blooded animals in response to injection of a
protein or polysaccharide
• Antiserum: blood serum containing antibodies

22. Serological Contd…..
• Conventional methods
– Immunodiffusion
– Immunoprecipitation
• Enzyme-linked Immunosorbent Assay (ELISA)
• Dot Immunobinding Assay (DIBA)
• Tissue Blotting Immunoassay
• Lateral flow strips/ Immunostrips

23. Different Classes of Immunoglobulins
• IgM
• IgG
• IgA
• IgE
• IgD

24. Structural Elements of an IgG molecule

25. Monoclonal Antibody
reacts withasingleepitope
Polyclonal Antibody
reacts withmorethanoneepitope
(epitopes - antigenic sites)

26. Polyclonal Antibody Production
•Preparation of antigen
•Immunization of animals
•Intervenous / Intramuscular
•Immunization schedule:
•Intervenous
•After 3 weeks, intramuscular
•After 2 weeks, intramuscular
•Collection of antiserum
•Clarification of antiserum and storage

27. Monoclonal Antibody Production
Source: R.T.V. Fox (1993)
HAT (Hypoxanthine,
aminopterin, thymidine

28. Advantages of Monoclonal Antibodies
• Unlimited quantities of the same antibody
in a reproducible manner
• Ability to produce MAbs for indefinite time
period by cryopreservation of hybridomas
for unlimited periods

29. Double Gel Diffusion Technique
Courtesy: Dr Robert Martin, Corvallis, USA

30. DAS - ELISA DAC - ELISA
Indirect DAC - ELISADirect DAS - ELISA
Variants of ELISA

31. Antibody
coated
well
Antigen
binds to
antibody
A second
antibody,
linked to
enzyme,
binds to
immobilized
antigen
Substrate is
added and
converted by
enzyme into
coloured
product; the
rate of colour
formation is
proportional to
the amount of
antigen
E E E
S
SE
Wash WashWash
Double Antibody Sandwich - ELISA

32. Requirements for ELISA
• Antibodies
• Positive control
• Negative control
• Buffer control
• Each sample in duplicate wells

33. ELISA Reader

34. B2, C2: BC; F2, G2: NC; F11, G11: PC

35. Mechanization
• Sample preparation, tissue grinders of various sorts for handling a
few samples to 1,00,000 samples
• Plate readers to quantify the results and make statistical analysis
possible.
• Robotics to wash and load the microtitre plates.
• These developments made ELISA a cost-effective method of
detection
Tissue Grinder Plate Washer
Courtesy: Dr Robert Martin, Corvallis, USA

36. Advantages of ELISA
• Reasonably sensitive
• Less susceptible to ‘false positives’
• Low per sample cost
• Handles large number of samples
• Can be subjected to automation
• Detection kits available commercially
− A boon for technicians

37. Dot Immunobinding Assay (DIBA)
• A variant of ELISA
• Nitrocellulose membrane as solid support
• Crude antisera can be used
• Stains used for revealing the reaction
• Very useful for field work

38. Tissue Blotting Immunoassay
(=Tissue Print Immunoassay, Tissue Print
Immunoblotting)
• Similar to DIBA
• Reactants can be reused
• Easily applicable for field sampling
• Samples can be prepared with virtually no equipment almost anywhere
• Qualitative test
Role Tear Blot
Courtesy: Dr Robert Martin, Corvallis, USA

39. Testing for Multiple Viruses- TIBA
Courtesy: Dr Robert Martin, Corvallis, USA

40. Detection – Immunostrips (Source: Agdia Inc., USA)
1 2
3
4 5

41. We use…
ELISA Diagnostic Kits: virus-specific antisera
(from Agdia/ Bio-Rad/ Bioreba/ Loewe/ Neogen)
– Seed directly used in ELISA for detecting
viruses
– Grow-out tests in a greenhouse, followed
by testing the seedlings by ELISA

42. Immunosorbent Electron Microscopy
Combination of serology and electron
microscopy

43. Nucleic Acid Based Techniques
Detection of existing viral nucleic acid vs amplifying the
target nucleic acid:
– Nucleic acid spot hybridization (NASH) - detects
existing viral nucleic acid
- Polymerase chain reaction (PCR) = amplifies the
target nucliec acid
- FTA Technology
– Microarrays

44. Variants of PCR
•PCR
•Reverse Transcription PCR (RT - PCR)
•Immuno capture RT - PCR (IC – RT - PCR)
•Real-time PCR
•Real-time RT-PCR

45. PCR - Principle
Denaturation
primer annealing
amplification
electrophoresis
508 bp

46. Reverse Transcription PCR
• Isolate total nucleic acids
• Reverse transcribe the target gene
• Amplify the cDNA using PCR
- Single-step RT-PCR
- Two-step RT-PCR

47. Reverse Transcription PCR Contd…
•Electrophoresis
•UV Transilluminator/ Gel documentation system

48. Genome Organization of Potyviruses
• Single-stranded, positive sense RNA, about 10 kb
• Genome is expressed as a single polyprotein
• 3' end of the RNA has a poly (A) tail: about 200 ‘A’ s
• CP ORF is at the 3' end of the RNA; is used for delineating
potyviruses into species
• The conserved poly (A) tail and CP region are widely
used as targets for RT-PCR

49. RT- PCR Using Specific Primers
• Utilize 3'poly A tail of the genome: oligo dT
primer is used for 1st
strand synthesis
• Upstream and downstream primers are specific to
BCMV, BCMNV, PSbMV and SMV
• Design primers based on conserved sequences of
known isolates
• Clone and sequence

50. Details of the Primers Used
Name Sequence Product
size
Specificity
B-V9260
(Upstream)
5'GTG GTA CAA TGC TGT GAA GG3' 800 bp BCMV
B-C10060
(Downstream)
5'GGA ACA ACA ARC ATT GCC GT3' 800 bp BCMV
A-V9144
(Upstream)
5'CTT GGC TCG CTA TGC ATT CG3' 467 bp BCMNV
A-C9611
(Downstream)
5'ATA TTC ATA CCC GCA CCT C3' 467 bp BCMNV
PSbMV-V9350
(Upstream)
5'GGG ATG TGG ACA ATG ATG GA3' 568 bp PSbMV
PSbMV-C9918
(Downstream)
5'TCC AGA AAG CCC TAC TGCC3' 568 bp PSbMV
SMV-V8728
(Upstream)
5'TTT GAC CAC TTG CTT GAG TA3' 544 bp SMV
SMV-C9272
(Downstream)
5'TGC CTT TCA GTA TTT TCG GAG TT3' 544 bp SMV

51. Gel Electrophoretic Analysis of RT-PCR of
BCMV, BCMNV, PSbMV and SMV
BCMV BCMNV
PSbMV SMV

52. Singleplex RT-PCRSingleplex RT-PCR
((Five Viruses of Quarantine Significance for
India))
ArMV
519 bp
203 bp
BPMV and GFLV
203 bp GFLV
61 bp BPMV
CLRV
283 bp
ToRSV
330 bp
Multiplex RT-PCRMultiplex RT-PCR
(Viruses of Quarantine Significance
for India)
283 bp CLRV
203 bp ArMV
330 bp ToRSV
ArMV, CLRV, ToRSV
CLRV, GFLV, ToRSV
283 bp CLRV
203 bp GFLV
330 bp ToRSV

53. Combination of Serology and PCR Immuno Capture
RT-PCR (IC-RT-PCR)
• one of the biggest problems with PCR assays from plant
tissue is inhibitors.
• immunocapture can be used to remove inhibitors.
• antibodies trap the virus
• detergent decapsidates it
• cDNA synthesized
• scope for routine use
• no RNA extraction

54. Advantages of PCR
• Highly sensitive (can detect picogram quantities of
target nucleic acid)
• Process is automated: very rapid, it takes 2 hrs or less
for the test
• Versatile: can be used for detecting RNA or DNA
• Very useful where ELISA is not effective (viroids,
geminiviruses)

55. Real-time PCR
Real-time PCR monitors the fluorescence emitted during
the reaction as an indicator of amplicon production at
each PCR cycle (in real time) as opposed to the end point
detection

56. Nigel Walker, NIEHS (www)

57. * based on the detection and quantitation of
a fluorescent reporter
* the first significant increase in the amount of PCR product
(CT
- threshold cycle) correlates to the initial amount of
target template
Real-time PCR Principles

59. ABI
Strategene
Roche Roche
Bio-Rad
Popular Real-Time PCR Systems

60. 17w w w .biorad.com
2a. excitation
filters
2b. emission
filters
1. halogen
tungsten lamp
4. sample
plate
3.
intensifier 5. ccd
detecto
r
350,00
0 pixels

61. Three general methods for the quantitative detection:
1. DNA-binding agents (SYBR Green)
2. Hydrolysis probes (TaqMan, Beacons, Scorpions)
3. Hybridisation probes (Light Cycler)
Real-time Principles

62. • Emits a strong fluorescent signal upon binding to
double-stranded DNA.
• During the extension phase, more and more SYBR
Green will bind to the PCR product, resulting in an
increased fluorescence.
• Consequently, during each subsequent PCR cycle
more fluorescence signal will be detected.
I. SYBR Green
(double-stranded DNA binding dye)

63. SYBR Green
SYBR Green
Taq DNA Polymerase
Nucleotides ©
Roche

64. SYBR Green Contd…
SYBR Green
Taq DNA Polymerase
Nucleotides ©
Roche

65. SYBR Green Contd…
SYBR Green
Taq DNA Polymerase
Nucleotides ©
Roche

66. SYBR Green Contd…
SYBR Green
Taq DNA Polymerase
Nucleotides ©
Roche

67. SYBR Green Contd…
SYBR Green
Taq DNA Polymerase
Nucleotides ©
Roche

68. • Blue LED Light Excites the SYBR Dye
• End of Extension
SYBR Green Contd…
©
Roche

69. • SYBR dye emits Fluorescence
• Fluorescence measurement is taken
SYBR Green Contd…
©
Roche

70. • More dye is incorporated
• Fluorescent signal is proportional to DNA yield
SYBR Green Contd…
©
Roche

71. Signal detected at each thermocycle to build
the amplification curve
Amplification Curve
©
Roche

72. •nonspecific binding
* multiplexing???
SYBR Green Disadvantages

73. II. Hydrolysis Probe Chemistry
• Hydrolysis probe is conjugated with a quencher
fluorochrome, which absorbs the fluorescence of the
reporter fluorochrome as long as the probe is intact.
• However, upon amplification of the target sequence, the
hydrolysis probe is displaced and subsequently hydrolyzed
by the Taq polymerase.
• This results in the separation of the reporter and quencher
fluorochrome and consequently the fluorescence of the
reporter fluorochrome becomes detectable.
• During each consecutive PCR cycle this fluorescence will
further increase because of the progressive and
accumulation of free reporter fluorochromes.

74. TaqMan Probes
FRET
DNA Polymerase 5' exonuclease activity
* Tm value 100
C higher than primers
* runs of identical nucleotides (no consecutive Gs)
* G+C content 30-80%
* more Cs than Gs
* no G at the 5' end
ABI Primer Express Software Tutorial (www)

75. Mocellin et al. Trends Mol Med 2003 (www)
DNA Polymerase 5' Exonuclease Activity

76. Mocellin et al. Trends Mol Med 2003 (www)
Molecular Beacons

77. Hybridisation Probe Chemistry by C Wittwer (www)

78. Fluorescence Resonance Energy Transfer
(FRET)
FRE
T
©
Roche

79. Donor
FRE
T
Donor Fluor is excited
FRET Contd…..
©
Roche

80. Donor
FRE
T
Energy RESONATES to excite a
nearby acceptor
Acceptor
E
FRET Contd…..
©
Roche

81. E
FRE
T
The acceptor fluor is excited as
energy is TRANSFERRED
FRET Contd…..
©
Roche

82. E
FRE
T
The acceptor emits energy that is
detected at a different wavelength
FRET Contd…..
©
Roche

83. Monitoring with FRET Probes
©
Roche

84. Annealing Stage
Hybridization Probe Chemistry
©
Roche

85. Hybridization Probe Chemistry Contd….
©
Roche

86. Hybridization Probe Chemistry Contd….
©
Roche

87. E
Hybridization Probe Chemistry Contd….
©
Roche

88. E
Hybridization Probe Chemistry Contd….
©
Roche

89. E Signal detected
Detection at the Annealing Stage
©
Roche
Hybridization Probe Chemistry Contd….

90. Extension Stage
©
Roche
Hybridization Probe Chemistry Contd….

91. ©
Roche
Hybridization Probe Chemistry Contd….

92. • More probe-pairs bind
• Red fluor signal is proportional to DNA yield
©
Roche
Hybridization Probe Chemistry Contd….

93. Nigel Walker, NIEHS (www)

94. Amplification Curve
Signal detected at each thermocycle to build
the amplification curve
©
Roche

95. Quantification - Standard Curve
Starting concentration
Low
Medium
High
Cycle number
Yield
10
4 copies
10
3
10
2
Crossing Points/
Threshold Cycle (CT)
©
Roche

96. Log of concentration
CrossingPoint
101
102
103
Cycle number
Yield
Unknown
©
Roche
Quantification - Standard Curve

97. Online, Real-time Fluorescent Monitoring
Online Monitoring
Constant feedback
for evaluation
Real-time Detection
Once-per-cycle
detection for analysis
©
Roche

100. Detection by Real-time RT-PCR
• Extract total nucleic acids
• Copy into cDNA (Reverse transcriptase)
• Do real-time PCR
• Analyse results

101. Detection of BCMV in French Bean Seed Using
Real-time RT-PCR
Real-time RT-PCR Profile of Amplification
of BCMV from Single Seed

102. Detection of PSbMV in Pea Seed Using
Real-time RT-PCR
Real-time RT-PCR Profile of Amplification of
PSbMV from Single Seed

103. Detection of BPMV (Not Reported from India)
Real-time RT-PCR

104. * General screening prior to moving to probe based assays
* When the PCR system is fully optimized -no primer
dimers or non-specific amplicons, e.g. from genomic DNA
When to Choose SYBR Green

105. * no post-PCR processing of products
(high throughput, low contamination risk)
*not influenced by non-specific amplification
* confirmation of specific amplification by melting point analysis
•amplification can be monitored real-time
* most specific, sensitive and reproducible
* * ultra-rapid cycling (30 minutes to 2 hours)
Real-time PCR Advantages

106. * not ideal for multiplexing
* setting up requires high technical skill and support
* high equipment cost
Real-time PCR Disadvantages

107. FTA Technology
• FTA (Flinders Technology Associates) is a trademark of
Whattman Inc. and is patented in the U.S.
• Cotton-based cellulose membrane containing lyophilized
chemicals that lyses many bacteria and viruses.
• Chemical treatment , unique to whattman that allows for the
rapid isolation and protection of nucleic acid at room
temperature.
• Used for efficient sampling and recovery of viral pathogens
from infected leaf tissue and their-subsequent molecular
analysis for geminiviruses in maize, cassava, tomato, and also
in TMV, PVY and TEV.

108. • Retaining integrity of viral pathogens within the
sampled plant tissues is often a limiting factor,
especially when sample size is large and when
working in regions remote from laboratory
facilities.
Why FTA Technology?

109. Advantages of FTA Cards
• Captures nucleic acid in one easy step.
• Nucleic acid collected on FTA-cards are stable for an year at room
temp.
• Do not require organic solvents in extraction of nucleic acids.
Involves non-organic chemicals in further process of nucleic acid
extraction.
• Do not require refrigeration and centrifugation facility during
complete process.
• Available in variety of configuration to meet application
requirement.
• Suitable for virtually any cell type like blood, culture cells, plant
material, bacteria, plasmids, virus particle, M13 plaque, solid
tissues.
• Total cost of one reaction is approximately 0.75 US$, thus, it is
cost effective technology.

110. FTA Cards in Areas Other than Virology
• Transgenics
• Genomics
• Diagnostics
• Animal identification
• Plasmids screening
• Drug discovery
• Forensic sciences
• Transfusion medicine

111. DNA Microarrays
Synonyms
•Gene Chip
•DNA Microarray
•Gene Card
•Bio Chip
•DNA Chip

112. • DNA Microarrays are small, solid supports onto which the
sequences from thousands of different genes are immobilized/
attached, at fixed locations.
• The supports themselves are usually glass microscope slides
(organo-functional alkoxysilane), the size of two side-by-side
fingers
• but can also be silicon chips or nylon membranes.
• This electronic device is able to map entire genetic material and
can scrutinize tens of thousands of genes at once.
• The DNA is printed, spotted, or actually synthesized directly onto
the support.
DNA Microarrays

113. DNA Microarrays Contd…
• Each spot on an array is associated with a
particular virus/ fungus/bacterium.
• Each color in an array represents either healthy
(control) or diseased (sample) tissue.
• Depending on the type of array used, the
location and intensity of a color will tell us
whether the virus is present in either the
control/ sample DNA.

114. DOT-BLOT
ssDNA on membrane
Hybridized with labeled probe
Autoradiography
DNA-CHIPS
Oligonucleotides on the chip
Tag with fluorescent
dye/radiolabeled
Hybridized with test DNA sample
Check the hybridization for
fluorescence & scanned on
computer/Autoradiography

115. • Durable
• Low background noise
• Many probes can be labeled with different
fluoresces
• Washing -- improve reproducibility
• Flatness, rigidity and transparency -- improve
image acquisition and image processing
• Reusable
Advantages of Glass Slides

116. Microarrays for Detecting Viruses

117. Gene Chip Instrument System Provides a Complete Solution for
the Analysis of Complex Genetic Information
GeneChip Hybridization
Oven 320
GeneChip Fluidics Station 400 Hewlett-Packard
GeneArray Scanner

118. Eppendorf Biochip Systems

119. Helicase Dependent Amplification (HDA)
61 bp BPMV
• No denaturation, helicase does
the job
• Strands of double stranded DNA
are separated by a DNA helicase
• Entire reaction at 65o
C for 11/2
hr
• Primer temperature and
annealing temperature are same
• Thermal cycler/ water bath/
incubator
• Kit: Biohelix
Detection of BPMV using HDA
Bean pod mottle virus, not reported from India

120. Loop Mediated Isothermal Amplification
(LAMP)
Detection of HPV using LAMP
High plains virus, not reported from India
• Four primers recognizing 6 distinct regions
on the target
• Only one enzyme, BST DNA polymerase
• BST DNA polymerase has strand
displacement activity
• Reaction under isothermal condition, 60-
65o
C for 30-60 minutes
• Terminate reaction by incubating at 80o
C for
5 min. or 95o
C for 2 min.
• Thermal Cycler (Heat block)/ Incubator with
hot bonnet
• Turbidity of Magnesium pyrophosphate (by
product) changes after amplification
• Turbidimeter/ visible
• Kit: EIKEN, Chemical Co. Ltd., Japan

121. Serology after PCR
• Thirty years later, the use of serology for detection
assays is still increasing for disease management
applications.
• Increases in the number of assays, formats and the
diversity of pathogens being detected.
• Formats are available for the scientists, farmers.

122. Pest Diagnosis - Use the Best Tool for the Job

123. Germplasm Collections Infected with Seed-
transmitted Viruses
IndiaV. radiataULCV
IndiaV. mungo
USAGlycine maxSMV
India (ICRISAT)Arachis hypogaeaPeMoV
USALens culinaris
Canada, France, India, New
Zealand, USA, UK,
Pisum sativumPSbMV
IranV. unguiculataCABMV
IndiaVigna mungo
USAPhaseolus vulgarisBCMV
CountryGermplasmVirus

124. Guidelines for Safe Movement of Germplasm
by Bioversity International (formerly IPGRI)
• Aromatic Plants – Vanilla
• Cereals – Small Grain Temperate Cereals
• Industrial Crops – Sugarcane
• Legumes – Legume
• Roots, Tubers and Aroids – Cassava, Edible Aroid, Potato, Sweet
Potato, Yam
• Temperate Fruits – Grapevine, Small Fruit, Temperate Fruits
• Tree Species – Acacia spp., Eucalyptus spp., Pinus spp.
• Tropical Fruits – Cacao, Citrus, Coconut, Musa spp.
• Vegetables - Allium spp.
Source: http://www.bioversityinternational.org/scientific_information/themes/germplasm_health/

125. Analysis of Risk of Introducing Plant Viruses
along with the Germplasm
• Plant Quarantine Order (Regulation of Import into India) 2003
- Schedule IV, V, VI
• Check-list of Seed-transmitted Viruses of Legumes
• Potential Quarantine Pests of Cereals
• Potential Quarantine Pests of Legumes – being edited
• Check-list of Seed-transmitted Viruses of Non-legumes
• Crop Protection compendium by CAB International
• Plant Viruses Online (http://image.fs.uidaho.edu/vide/refs.htm)
• Descriptions of Plant Viruses (http://www.dpvweb.net/)
• ICTV dB Descriptions
(http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/index.htm)

126. Seed-transmitted Viruses of Legumes Not Known to
Occur in India
1. Artichoke yellow ring spot
virus
12. Lucerne Australian latent virus
2. Bean mild mosaic virus 13. Mulberry ringspot virus
3. Bean pod mottle virus 14. Pea early browning virus
4. Broad bean mottle virus 15. Pea enation mosaic virus
5. Braod bean stain virus 16. Peanut stunt virus
6. Broad bean true mosaic virus 17. Raspberry ring spot virus
7. Cherry leaf roll virus 18. Red clover mosaic virus
8. Clover yellow mosaic virus 19. Red clover vein mosaic virus
9. Cocoa necrosis virus 20. Satsuma dwarf virus
10. Cowpea mottle virus 21. Tomato ring spot virus
11. Cowpea severe mosaic virus 22. Vicia cryptic virus

127. Seed-transmitted Viruses of French Bean
Virus reported world over India On French bean in India
Alfalfa mosaic virus + -
Artichoke yellow ringspot virus - -
Bean common mosaic virus + +
Bean common mosaic necrosis virus + +
Bean mild mosaic viris - -
Bean pod mottle virus - -
Bean yellow mosaic virus + +
Broad bean wilt virus + -
Cacao necrosis virus - -
Cherry leaf roll virus - -
Cowpea mild mottle virus + -
Cowpea severe mosaic virus - -
Cucumber mosaic virus + +

128. Seed-transmitted Viruses of French Bean Contd..
Virus reported world over India On French bean in India
Pea early-browning virus - -
Peanut mottle virus + -
Red clover vein mosaic virus - -
Satsuma dwarf virus - -
Southern bean mosaic virus + -
Soybean mosaic virus + -
Tobacco necrosis virus + -
Tobacco rattle virus + -
Tobacco streak virus + -
Tomato aspermy virus + -
Tomato black ring virus + -
Urd bean leaf crinkle virus + +?
Total 9 (not
present)
20 (not present)

129. Growing-on Test of Legume Germplasm in
Post-entry Quarantine Greenhouse

130. Crop No. of
Samples
Crop No. of
Samples
Glycine spp. 2,268 V. mungo 9
Lathyrus spp. 15 V. radiata 383
Phaseolus spp. 1,599 V. unguiculata 645
Pisum sativum 542 Vicia spp. 117
Vigna spp. 36 V. faba 828
Total 6,442
Legumes Processed against
Seed-transmitted Viruses (2000-2012)

131. Detection of Plant Viruses in Exotic Germplasm Imported
into India (2000-2012)
Virus Crop Source of Import
Alfalfa mosaic
virus
Glycine max AVRDC (Taiwan), IITA (Nigeria), Brazil,
Myanmar, Sri Lanka, USA
Phaseolus vulgaris♣
CIAT (Colombia), Canada, Kenya, USA
Pisum sativum♣
USA
Vigna radiata♣
Japan
V. unguiculata CIAT (Colombia), IITA (Nigeria), USA
Bean common
mosaic virus
G. max♣
AVRDC (Taiwan), IITA (Nigeria), Brazil,
Thailand, USA
P. vulgaris CIAT (Colombia), CIS Hungary, Kenya,
USA
V. radiata AVRDC (Taiwan), Japan, USA
V. subterranea Ghana
Bean common
mosaic necrosis
virus
P. vulgaris CIAT (Colombia), Kenya, Russia
♣
Virus present in India but not recorded on the host on which intercepted

132. Virus Crop Source of Import
Bean yellow mosaic virus Glycine max IITA (Nigeria), Myanmar, USA
Phaseolus
vulgaris
CIAT (Colombia)
Pisum sativum USA
Vicia faba ICARDA (Syria), Bulgaria, Spain
Blackeye cowpea mosaic
virus, now a strain of
BCMV
Vigna
subterranea
Ghana
Broad bean stain virus* Vicia faba ICARDA (Syria), Bulgaria
Brod bean wilt virus V. faba ICARDA (Syria)
P. sativum USA
Cherry leaf roll virus* G. max AVRDC, Sri Lanka, Thailand, USA
P. vulgaris CIAT (Colombia), Sri Lanka
Cowpea aphid-borne
mosaic virus
Glycine max♣
AVRDC (Taiwan), IITA (Nigeria),
Myanmar, Sri Lanka, Thailand, USA
Vigna radiata♣
AVRDC (Taiwan)
V. unguiculata IITA (Nigeria), Eritrea, Guyana,
Philippines, USA
*Virus not yet reported from India; ♣
Virus present in India but not recorded on the host on which intercepted
Detection of Plant Viruses Contd….

133. Detection of Plant Viruses Contd….
Virus Crop Source of Import
Cowpea mosaic virus Vigna radiata♣
USA
V. unguiculata IITA (Nigeria)
Cowpea mottle virus V. unguiculata Philippines
V. subterranea Ghana
Cucumber mosaic virus Glycine max AVRDC (Taiwan), IITA (Nigeria), Brazil,
Myanmar, Sri Lanka, USA
Phaseolus
vulgaris
CIAT (Colombia)
V. radiata USA
V. unguiculata IITA (Nigeria), USA
Grapevine fan leaf virus G. max AVRDC (Taiwan)
Pea seed-borne mosaic
virus
Pisum sativum AVRDC (Taiwan), Australia, Bulgaria, Colombia,
Eritrea, Germany, Holland, Nepal, Russia, Syria,
USA
Vicia faba♣
ICARDA (Syria), Bulgaria, Spain
Raspberry ring spot
virus*
G. max AVRDC (Taiwan), Sri Lanka, Thailand, USA
Southern bean mosaic
virus
G. max♣
AVRDC, IITA (Nigeria), Sri Lanka, Thailand, USA
P. vulgaris♣
CIAT (Colombia)
*Virus not yet reported from India; ♣
Virus present in India but not recorded on the host on which intercepted

134. Interception of Plant Viruses Contd….
Virus Crop Source of Import
Soybean mosaic virus Glycine max AVRDC (Taiwan), IITA (Nigeria), Australia, Brazil,
Hungary, Sri Lanka, Thailand, USA
Phaseolus vulgaris♣
CIAT (Colombia)
Tobacco necrosis virus Pisum sativum♣
USA
Tobacco rattle virus P. vulgaris CIAT (Colombia)
Tobacco ring spot virus G. max IITA (Nigeria), Myanmar
Tobacco streak virus G. max♣
AVRDC (Taiwan), Australia, Brazil, Sri Lanka,
Thailand, USA
P. sativum♣
USA
Vigna unguiculata♣
CIAT (Colombia)
Tomato aspermy virus P. vulgaris CIAT (Colombia)
Tomato black ring virus G. max♣
AVRDC (Taiwan), Brazil, Sri Lanka
P. vulgaris♣
CIAT (Colombia)
Vigna unguiculata♣
IITA (Nigeria)
Tomato ring spot virus* G. max AVRDC (Taiwan), Sri Lanka, Thailand, USA
*Virus not yet reported from India; ♣
Virus present in India but not recorded on the host on which intercepted

135. Incinerator
If not salvaged,
material is
incinerated
At NBPGR…………

136. Vectors of Plant Viruses Intercepted
Virus Vector
AMV 14 aphid species, Myzus persicae
BCMV Acyrthosiphon pisum, Aphis fabae and
Myzus persicae, Aphis gossypii, A.
medicaginis, A. rumicis, Hyalopterus
atriplicis, Macrosiphum ambrosiae
BCMNV Aphids
BlCMV Aphids
BYMV 20 aphid species Acyrthosiphon
pisum, Macrosiphum euphorbiae,
Myzus persicae and Aphis fabae
BBWV Aphids
CABMV Aphids
CLRV* Xiphinema coxi, X. diversicaudatum
and X. vuittenezi
CPMoV Beetles
CMV 80 species of aphids
Virus Vector
CPMV various beetles
CPMoV* Galerucid beetle, Ootheca mutabilis,
Paraluperodes lineata
GFLV Xiphinema index. X. americanum, X.
italiae
PSbMV Aphids
RRSV* Longidorus spp.
SBMV Leaf beetles
SMV Aphids
TAV Aphids
TNV Olpidium brassicae
TRV Trichodorus, Paratrichodorus
TRSV* Xiphinema americanum
TSV Thrips tabaci and F. occidentalis
TBRV Longidorus
ToRSV Xiphinema americanum
*Virus Not reported from India

137. ENVIRONMENT
VIRUS VECTOR
HOST
Courtesy: Dr. R.K. Khetarpal, NBPGR

138. Variability in Plant Viruses
Alfalfa mosaic virus, Bean yellow mosaic virus
• Numerous strains known
Bean common mosaic virus
• Ten different strains reported
Cherry leaf roll virus
• Wide range of serological variants exist
• Type (cherry) strain, Elm mosaic strain, Rhubarb strain, Golden
elderberry strain Red elder ringspot strain, Dogwood ringspot
strain, Birch strain, Walnut ringspot strain, walnut yellow vein
strain, Blackberry strain and red raspberry strains.

139. Variability in Plant Viruses Contd……
Cowpea aphid- borne mosaic virus
Strains
• European (type) strain, African (neotype) strain, African mild
strain and African vein-banding strain, South African
Passiflora strain, Zimbabwe strain, Brazilian strain and
Moroccan strain.
Serotypes
• Seven distinct CABMV serotypes reported
Pathotypes
• Considerable evidence of pathogenic variability reported
Pea seed-borne mosaic virus
• Four pathotypes viz., P1, P2, P3 and P4 known on pea

140. Southern bean mosaic virus
• Strain B
• Severe bean mosaic strain or Mexican strain
• Resistance-breaking strains
Soybean mosaic virus
• Seven pathotypes representing seven strain groups (G1–G7) in
the United States
• CN-18, a new strain of SMV was reported from Korea
Raspberry ringspot virus
• Many minor variants occur
• Three important strains: The Scottish strain, the type strain; The
English strain, differs from the Scottish strain serologically and
in vector relations; The Lloyd George yellow blotch (LG) strain
Variability in Plant Viruses Contd……

141. Variability in Plant Viruses Contd…….
Tobacco ringspot virus
• Many variants reported, based primarily on differences in
symptomatology
• Many natural antigenic variants also reported
Tobacco streak virus
• Many variants exist
• Number of strains known in India
• Recently found to infect Bt cotton also

142. Tomato black ring virus
• Tomato black ring strain (The type strain)
• Lettuce ringspot strain
• Potato bouquet strain of Köhler
• Potato pseudo -aucuba strain of Köhler
• Beet ringspot strain
• Celery yellow vein strain
Tomato ringspot virus
• Tobacco strain = tobacco ringspot virus No. 2 (The type strain)
• Peach yellow bud mosaic strain
• Grape yellow vein strain
Variability in Plant Viruses Contd……

143. Through
Exclusion- Quarantine
Prediction ?
Vector Control ?
Seed Certification
Incorporation of Resistance Genes
Management of Plant Viruses

144. Challenges in Virus Diagnosis in
Plant Quarantine
• Sample size
• Detecting an unknown/ exotic virus
• Part of the planting material to be tested
• Availablity of antisera/ primers/ sequences
• Post-entry quarantine
• Urgency of clearance of the sample
• Conformity to International Standards

145. Technique alone is not enough
 We need a strategy covering
 Simultaneous detection of
fungi, bacteria, viruses,
nematodes, insect pests,
weeds……

146. Researchable Issues
• Variability of viruses/ vectors
• Monitoring the vector scenario
• Development of diagnostic kits

147. Pest Diagnosis - Use the Best Tool for the Job