Detection and Identification of Plant Viruses in Quarantine

Plant VirusesNucleic acid with a protein coatRNA/ DNASystemic infectionMode of transmissionVectorsMechanicalSeeds

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)

Dry Seed ExaminationHealthyMottlingMottlingHealthyTennis ball-likeSplit seed coatHealthy

Processing of Legume Germplasm in PEQ Greenhouses and Containment Facility

Symptoms only serve as a guide but indispensableSimilar symptoms can be produced by different virusesSymptoms may be extremely variable; the same virus can produce a range of symptomsLack of symptoms does not necessarily mean that no viruses are present. Mixed infection result in more severe symptomsSymptoms are only indicative, not confirmatory

Reduction in growthColour deviationNecrosisMalformation

Symptoms of Plant Viruses

Symptoms of Plant Viruses

Symptoms of PPV

Symptom of MDMVSymptoms of HPV

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

Infectivity Assay- Mechanical Inoculation

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

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

Infectivity Test

Necrotic Local Lesions by BCMV on Chenopodium amaranticolor

Systemic Infection of SMV on Nicotiana tabacum xanthi Infectivity Assay Contd

Biochemical TechniqueStaining of inclusion bodies observation under light microscopeinclusion bodies are viral aggregates or proteins induced in cytoplasm or nucleus staining by Azure A and O-G combinationstCPMVCMVBYMVTMV

Cell Inclusion Bodies Observed with an Electron Microscope

Physical- Electron Microscopy

Reveals shape and size of the particleGives idea of the group to which the virus belongsVery expensive equipment, often not available

Physical Transmission Electron Microscopy Contd

Serological TechniquesBased on antigen - antibody reactionAntigen: a protein or polysachharide which induces the formation of antibodies when injected into a warm-blooded animalAntibody: a specific protein formed in the blood of warm-blooded animals in response to injection of a protein or polysaccharideAntiserum: blood serum containing antibodies

Serological Contd..Conventional methods ImmunodiffusionImmunoprecipitationEnzyme-linked Immunosorbent Assay (ELISA)Dot Immunobinding Assay (DIBA)

Tissue Blotting Immunoassay

Lateral flow strips/ Immunostrips

Different Classes of ImmunoglobulinsIgMIgGIgAIgEIgD

Structural Elements of an IgG molecule

Monoclonal Antibody reacts with a single epitopePolyclonal Antibodyreacts with more than one epitope

(epitopes - antigenic sites)

Polyclonal Antibody ProductionPreparation of antigenImmunization of animalsIntervenous / IntramuscularImmunization schedule:IntervenousAfter 3 weeks, intramuscularAfter 2 weeks, intramuscularCollection of antiserumClarification of antiserum and storage

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

Advantages of Monoclonal AntibodiesUnlimited quantities of the same antibody in a reproducible mannerAbility to produce MAbs for indefinite time period by cryopreservation of hybridomas for unlimited periods

Double Gel Diffusion TechniqueCourtesy: Dr Robert Martin, Corvallis, USA

Variants of ELISA

Requirements for ELISAAntibodiesPositive controlNegative controlBuffer controlEach sample in duplicate wells

ELISA Reader

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

MechanizationSample preparation, tissue grinders of various sorts for handling a few samples to 1,00,000 samplesPlate 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 GrinderPlate WasherCourtesy: Dr Robert Martin, Corvallis, USA

Advantages of ELISAReasonably sensitiveLess susceptible to false positivesLow per sample costHandles large number of samplesCan be subjected to automationDetection kits available commerciallyA boon for technicians

Dot Immunobinding Assay (DIBA)A variant of ELISANitrocellulose membrane as solid supportCrude antisera can be usedStains used for revealing the reactionVery useful for field work

Tissue Blotting Immunoassay (=Tissue Print Immunoassay, Tissue Print Immunoblotting)

Similar to DIBAReactants can be reusedEasily applicable for field samplingSamples can be prepared with virtually no equipment almost anywhereQualitative test

RoleTearBlotCourtesy: Dr Robert Martin, Corvallis, USA

Testing for Multiple Viruses- TIBACourtesy: Dr Robert Martin, Corvallis, USA

Detection Immunostrips (Source: Agdia Inc., USA)12345

We useELISA Diagnostic Kits: virus-specific antisera (from Agdia/ Bio-Rad/ Bioreba/ Loewe/ Neogen) Seed directly used in ELISA for detecting virusesGrow-out tests in a greenhouse, followed by testing the seedlings by ELISA

Immunosorbent Electron MicroscopyCombination of serology and electron microscopy

Nucleic Acid Based TechniquesDetection 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 TechnologyMicroarrays

Variants of PCRPCRReverse Transcription PCR (RT - PCR)Immuno capture RT - PCR (IC RT - PCR)Real-time PCRReal-time RT-PCR

Reverse Transcription PCRIsolate total nucleic acidsReverse transcribe the target geneAmplify the cDNA using PCR- Single-step RT-PCR- Two-step RT-PCR

Reverse Transcription PCRContdElectrophoresisUV Transilluminator/ Gel documentation system

Genome Organization of Potyviruses

Single-stranded, positive sense RNA, about 10 kbGenome is expressed as a single polyprotein3' end of the RNA has a poly (A) tail: about 200 A sCP ORF is at the 3' end of the RNA; is used for delineating potyviruses into speciesThe conserved poly (A) tail and CP region are widely

used as targets for RT-PCR

RT- PCR Using Specific PrimersUtilize 3'poly A tail of the genome: oligo dT primer is used for 1st strand synthesisUpstream and downstream primers are specific to BCMV, BCMNV, PSbMV and SMVDesign primers based on conserved sequences of known isolatesClone and sequence

Details of the Primers Used

Sheet1

NameSequenceProduct sizeSpecificity

B-V9260 (Upstream)5'GTG GTA CAA TGC TGT GAA GG3'800 bpBCMV

B-C10060 (Downstream)5'GGA ACA ACA ARC ATT GCC GT3'800 bpBCMV

A-V9144 (Upstream)5'CTT GGC TCG CTA TGC ATT CG3'467 bpBCMNV

A-C9611 (Downstream)5'ATA TTC ATA CCC GCA CCT C3'467 bpBCMNV

PSbMV-V9350 (Upstream)5'GGG ATG TGG ACA ATG ATG GA3'568 bpPSbMV

PSbMV-C9918 (Downstream)5'TCC AGA AAG CCC TAC TGCC3'568 bpPSbMV

SMV-V8728 (Upstream)5'TTT GAC CAC TTG CTT GAG TA3'544 bpSMV

SMV-C9272 (Downstream)5'TGC CTT TCA GTA TTT TCG GAG TT3'544 bpSMV

Sheet2

Sheet3

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

Singleplex RT-PCR (Five Viruses of Quarantine Significance for India)Multiplex RT-PCR (Viruses of Quarantine Significance for India)

CLRV, GFLV, ToRSV

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

Advantages of PCRHighly sensitive (can detect picogram quantities of target nucleic acid)Process is automated: very rapid, it takes 2 hrs or less for the testVersatile: can be used for detecting RNA or DNAVery useful where ELISA is not effective (viroids, geminiviruses)

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

Nigel Walker, NIEHS (www)

* 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

Popular Real-Time PCR Systems

Three general methods for the quantitative detection: DNA-binding agents (SYBR Green)Hydrolysis probes (TaqMan, Beacons, Scorpions) Hybridisation probes (Light Cycler)

Real-time Principles

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)

SYBR Green

Roche

This slide starts a series of illustrations that are useful to explain the SYBR green detection chemistry. Do not belabor each slide. Go through them rapidly, with brief comments as required to clarify the activity of each molecule.

In this slide, Taq has bound to template DNA and is synthesizing a new strand. SYBR Green will bind selectively to the double-stranded DNA.

The larger, yellow sphere is Taq polymeraseThe green hexagon is SYBR GreenThe multi-colored sticks are nucleotides

SYBR Green Contd

Roche

As new DNA is made, SYBR Green begins to bind to the double-stranded DNA.

SYBR Green Contd

Roche

SYBR Green Contd

Roche

SYBR Green Contd

Roche

Dye molecules are bound to the length of the DNA molecule and are ready to generate a fluorescent signal.

SYBR Green Contd

Blue LED Light Excites the SYBR Dye

End of Extension

Roche

For SYBR-Green, the opportunity for detection comes at the end of the extension step. Blue light excites the dye and raises it to a higher energy level.

SYBR Green Contd

SYBR dye emits Fluorescence Fluorescence measurement is taken

Roche

When the dye molecules drop to a lower energy level, they release the energy as fluorescent light.

SYBR Green Contd

More dye is incorporated Fluorescent signal is proportional to DNA yield

Roche

When the dye molecules drop to a lower energy level, they release the energy as fluorescent light.

Signal detected at each thermocycle to build the amplification curveAmplification CurveRoche

nonspecific binding

* multiplexing???SYBR Green Disadvantages

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.

TaqMan Probes

FRETDNA 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' endABI Primer Express Software Tutorial (www)

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

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

Hybridisation Probe Chemistry by C Wittwer (www)

Fluorescence Resonance Energy Transfer (FRET)

FRETRoche

Before we can understand the next two detection chemistries, we need to understand a concept called FRET, which stands for Fluorescence Resonance Energy Transfer.Big Picture: with FRET a fluorescent signal becomes possible when two different dye molecules are in close proximity. When one dye becomes excited by a light source it can transfer this excitation energy to a second dye causing it to raise to a higher energy level. When the second dye drops to a lower energy level, it gives off fluorescent light.

FRET Contd..

Donor

FRETDonor Fluor is excitedRoche

A blue light source excites the green dye, raising it to a higher energy level.

FRET Contd..

DonorFRETEnergy RESONATES to excite a nearby acceptorRoche

When the dye molecule drops to a lower energy level, it releases energy, which is absorbed by the second dye molecule.

FRET Contd..

EFRETThe acceptor fluor is excited asenergy is TRANSFERREDRoche

The red dye is now raised to a higher energy level.. When it drops to a lower energy level it will give off light.

FRET Contd..

EFRETThe acceptor emits energy that is detected at a different wavelengthRoche

The red dye emits a red fluorescent signal.

Monitoring with FRET ProbesRoche

Annealing StageHybridization Probe ChemistryRoche

Hybridization probe chemistry for the LightCycler depends on FRET. A pair of hybridization probes are designed to bind to adjacent sequences on the target sequence. One member of the pair is labeled on the 3-end with a green dye. The other member of the pair is labeled on the 5-end with a red dye. When the pair of probes anneals to the target sequence...

Hybridization Probe Chemistry Contd.Roche

the two green and red dyes are adjacent to each other. As long as the two probe molecules remain bound next to each other, FRET can happen. If one or both of the probe molecules becomes unbound, FRET cannot happen.

Hybridization Probe Chemistry Contd.Roche

When both of the hybridization probes are bound, FRET occurs exactly as described in previous series of slides. Here a blue light provides energy to excite the green dye.

EHybridization Probe Chemistry Contd.Roche

As described previously

EHybridization Probe Chemistry Contd.Roche

As described previously.

ESignal detected

Detection at the Annealing StageRocheHybridization Probe Chemistry Contd.

As described previously

Extension StageRocheHybridization Probe Chemistry Contd.

It is important to note, that during the PCR process,the hybridization probes are reused from one cycle to the next. During the extension step, Taq polymerase synthesizes new DNA. When it bumps up against a pair of hybridization probes, the probes are displaced.

RocheHybridization Probe Chemistry Contd.

Taq has now displaced the second hybridization probe. The red and green dyes are now separated. FRET is no longer possible until the probes hybridize during the next annealing step.

More probe-pairs bind Red fluor signal is proportional to DNA yield

RocheHybridization Probe Chemistry Contd.

As described previously

Nigel Walker, NIEHS (www)

Amplification CurveSignal detected at each thermocycle to build the amplification curve

Roche

Quantification - Standard Curve

Starting concentrationLowMediumHighCycle numberYield104 copies103 102

Roche

The LightCycler determines the cycle number where each reaction begins to enter the log-linear phase and matches this value up with the fluorescence data for that sample at that cycle number.The relationship between these two values is used to calculate how much DNA was made for each reaction.

Quantification - Standard Curve

Log of concentration101102103Roche

The LightCycler determines the cycle number where each reaction begins to enter the log-linear phase and matches this value up with the fluorescence data for that sample at that cycle number.The relationship between these two values is used to calculate how much DNA was made for each reaction.

Online, Real-time Fluorescent MonitoringOnline Monitoring Constant feedback for evaluationReal-time Detection Once-per-cycle detection for analysisRoche

As mentioned earlier, one of the key LC features is that it is an on-line, real-time instrument. This provides the benefit of:constant feedback of reaction dataallowing a retrospective data analysis

Detection by Real-time RT-PCR Extract total nucleic acids

Copy into cDNA (Reverse transcriptase)

Do real-time PCR

Analyse results

Detection of BCMV in French Bean Seed Using Real-time RT-PCR

Real-time RT-PCR Profile of Amplification of BCMV from Single Seed

Detection of PSbMV in Pea Seed Using Real-time RT-PCR

Real-time RT-PCR Profile of Amplification of PSbMV from Single Seed

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

* 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 DNAWhen to Choose SYBR Green

* no post-PCR processing of products (high throughput, low contamination risk)

*not influenced by non-specific amplification * confirmation of specific amplification by melting point analysisamplification can be monitored real-time

* most specific, sensitive and reproducible* * ultra-rapid cycling (30 minutes to 2 hours)Real-time PCR Advantages

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

FTA TechnologyFTA (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.

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?

Advantages of FTA CardsCaptures 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.

FTA Cards in Areas Other than VirologyTransgenicsGenomicsDiagnosticsAnimal identificationPlasmids screeningDrug discoveryForensic sciences Transfusion medicine

DNA MicroarraysSynonymsGene ChipDNA MicroarrayGene CardBio ChipDNA Chip

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

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.

DurableLow background noiseMany probes can be labeled with different fluorescesWashing -- improve reproducibilityFlatness, rigidity and transparency -- improve image acquisition and image processingReusable

Advantages of Glass Slides

Microarrays for Detecting Viruses

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

1113.bin

Eppendorf Biochip Systems

Helicase Dependent Amplification (HDA)

No denaturation, helicase does the jobStrands of double stranded DNA are separated by a DNA helicase Entire reaction at 65oC for 11/2 hrPrimer temperature and annealing temperature are sameThermal cycler/ water bath/ incubatorKit: Biohelix

Detection of BPMV using HDABean pod mottle virus, not reported from India

Loop Mediated Isothermal Amplification (LAMP)

Detection of HPV using LAMPHigh plains virus, not reported from IndiaFour primers recognizing 6 distinct regions on the targetOnly one enzyme, BST DNA polymeraseBST DNA polymerase has strand displacement activityReaction under isothermal condition, 60-65oC for 30-60 minutesTerminate reaction by incubating at 80oC for 5 min. or 95oC for 2 min. Thermal Cycler (Heat block)/ Incubator with hot bonnetTurbidity of Magnesium pyrophosphate (by product) changes after amplificationTurbidimeter/ visibleKit: EIKEN, Chemical Co. Ltd., Japan

Serology after PCRThirty 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.

Pest Diagnosis - Use the Best Tool for the Job

Germplasm Collections Infected with Seed-transmitted Viruses

Guidelines for Safe Movement of Germplasm by Bioversity International (formerly IPGRI)Aromatic Plants VanillaCereals 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/

Analysis of Risk of Introducing Plant Viruses along with the GermplasmPlant Quarantine Order (Regulation of Import into India) 2003 - Schedule IV, V, VICheck-list of Seed-transmitted Viruses of LegumesPotential Quarantine Pests of CerealsPotential Quarantine Pests of Legumes being editedCheck-list of Seed-transmitted Viruses of Non-legumesCrop Protection compendium by CAB InternationalPlant 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)

Seed-transmitted Viruses of Legumes Not Known to Occur in India

1.Artichoke yellow ring spot virus12.Lucerne Australian latent virus

2.Bean mild mosaic virus13.Mulberry ringspot virus3.Bean pod mottle virus14.Pea early browning virus4.Broad bean mottle virus15.Pea enation mosaic virus5.Braod bean stain virus16.Peanut stunt virus6.Broad bean true mosaic virus17.Raspberry ring spot virus7.Cherry leaf roll virus18.Red clover mosaic virus8.Clover yellow mosaic virus19.Red clover vein mosaic virus9.Cocoa necrosis virus20.Satsuma dwarf virus10.Cowpea mottle virus21.Tomato ring spot virus11.Cowpea severe mosaic virus22.Vicia cryptic virus

Seed-transmitted Viruses of French Bean

Virus reported world overIndiaOn French bean in IndiaAlfalfa 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++

Seed-transmitted Viruses of French Bean Contd..

Virus reported world overIndiaOn French bean in IndiaPea 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++?Total9 (not present)20 (not present)

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

Legumes Processed against Seed-transmitted Viruses (2000-2012)

CropNo. of SamplesCropNo. of SamplesGlycine spp.2,268V. mungo9Lathyrus spp.15V. radiata383Phaseolus spp.1,599V. unguiculata645Pisum sativum542Vicia spp.117Vigna spp.36V. faba828Total6,442

Detection of Plant Viruses in Exotic Germplasm Imported into India (2000-2012) Virus present in India but not recorded on the host on which intercepted

VirusCropSource of ImportAlfalfa mosaic virusGlycine maxAVRDC (Taiwan), IITA (Nigeria), Brazil, Myanmar, Sri Lanka, USAPhaseolus vulgarisCIAT (Colombia), Canada, Kenya, USAPisum sativumUSAVigna radiataJapanV. unguiculataCIAT (Colombia), IITA (Nigeria), USABean common mosaic virusG. maxAVRDC (Taiwan), IITA (Nigeria), Brazil, Thailand, USA P. vulgarisCIAT (Colombia), CIS Hungary, Kenya, USAV. radiataAVRDC (Taiwan), Japan, USAV. subterraneaGhanaBean common mosaic necrosis virusP. vulgarisCIAT (Colombia), Kenya, Russia

*Virus not yet reported from India; Virus present in India but not recorded on the host on which interceptedDetection of Plant Viruses Contd.

VirusCropSource of ImportBean yellow mosaic virusGlycine maxIITA (Nigeria), Myanmar, USAPhaseolus vulgarisCIAT (Colombia)Pisum sativumUSAVicia fabaICARDA (Syria), Bulgaria, SpainBlackeye cowpea mosaic virus, now a strain of BCMVVigna subterraneaGhanaBroad bean stain virus*Vicia fabaICARDA (Syria), BulgariaBrod bean wilt virusV. fabaICARDA (Syria)P. sativumUSACherry leaf roll virus*G. maxAVRDC, Sri Lanka, Thailand, USAP. vulgarisCIAT (Colombia), Sri LankaCowpea aphid-borne mosaic virusGlycine maxAVRDC (Taiwan), IITA (Nigeria), Myanmar, Sri Lanka, Thailand, USAVigna radiataAVRDC (Taiwan)V. unguiculataIITA (Nigeria), Eritrea, Guyana, Philippines, USA

Detection of Plant Viruses Contd.*Virus not yet reported from India; Virus present in India but not recorded on the host on which intercepted

VirusCropSource of ImportCowpea mosaic virusVigna radiataUSAV. unguiculataIITA (Nigeria)Cowpea mottle virusV. unguiculataPhilippinesV. subterraneaGhanaCucumber mosaic virusGlycine maxAVRDC (Taiwan), IITA (Nigeria), Brazil, Myanmar, Sri Lanka, USA Phaseolus vulgarisCIAT (Colombia)V. radiataUSAV. unguiculataIITA (Nigeria), USAGrapevine fan leaf virusG. maxAVRDC (Taiwan)Pea seed-borne mosaic virusPisum sativumAVRDC (Taiwan), Australia, Bulgaria, Colombia, Eritrea, Germany, Holland, Nepal, Russia, Syria, USAVicia fabaICARDA (Syria), Bulgaria, SpainRaspberry ring spot virus*G. maxAVRDC (Taiwan), Sri Lanka, Thailand, USASouthern bean mosaic virusG. maxAVRDC, IITA (Nigeria), Sri Lanka, Thailand, USA P. vulgarisCIAT (Colombia)

Interception of Plant Viruses Contd.*Virus not yet reported from India; Virus present in India but not recorded on the host on which intercepted

Virus CropSource of ImportSoybean mosaic virusGlycine maxAVRDC (Taiwan), IITA (Nigeria), Australia, Brazil, Hungary, Sri Lanka, Thailand, USAPhaseolus vulgarisCIAT (Colombia)Tobacco necrosis virusPisum sativumUSATobacco rattle virusP. vulgarisCIAT (Colombia)Tobacco ring spot virusG. maxIITA (Nigeria), MyanmarTobacco streak virusG. maxAVRDC (Taiwan), Australia, Brazil, Sri Lanka, Thailand, USAP. sativumUSAVigna unguiculataCIAT (Colombia)Tomato aspermy virusP. vulgarisCIAT (Colombia)Tomato black ring virusG. maxAVRDC (Taiwan), Brazil, Sri LankaP. vulgarisCIAT (Colombia)Vigna unguiculataIITA (Nigeria)Tomato ring spot virus*G. maxAVRDC (Taiwan), Sri Lanka, Thailand, USA

IncineratorIf not salvaged, material is incineratedAt NBPGR

Vectors of Plant Viruses Intercepted*Virus Not reported from India

VirusVectorAMV14 aphid species, Myzus persicaeBCMVAcyrthosiphon pisum, Aphis fabae and Myzus persicae, Aphis gossypii, A. medicaginis, A. rumicis, Hyalopterus atriplicis, Macrosiphum ambrosiaeBCMNVAphidsBlCMVAphidsBYMV20 aphid species Acyrthosiphon pisum, Macrosiphum euphorbiae, Myzus persicae and Aphis fabaeBBWVAphidsCABMVAphidsCLRV*Xiphinema coxi, X. diversicaudatum and X. vuitteneziCPMoVBeetlesCMV80 species of aphids

VirusVectorCPMVvarious beetlesCPMoV*Galerucid beetle, Ootheca mutabilis, Paraluperodes lineataGFLVXiphinema index. X. americanum, X. italiaePSbMVAphidsRRSV*Longidorus spp.SBMVLeaf beetlesSMVAphidsTAVAphidsTNVOlpidium brassicaeTRVTrichodorus, ParatrichodorusTRSV*Xiphinema americanumTSVThrips tabaci and F. occidentalisTBRVLongidorusToRSVXiphinema americanum

ENVIRONMENTVIRUSVECTORHOSTCourtesy: Dr. R.K. Khetarpal, NBPGR

Variability in Plant VirusesAlfalfa mosaic virus, Bean yellow mosaic virusNumerous strains knownBean common mosaic virusTen different strains reported Cherry leaf roll virusWide range of serological variants existType (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.

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 reportedPea seed-borne mosaic virusFour pathotypes viz., P1, P2, P3 and P4 known on pea

Variability in Plant Viruses ContdSouthern bean mosaic virusStrain B Severe bean mosaic strain or Mexican strain Resistance-breaking strains Soybean mosaic virusSeven pathotypes representing seven strain groups (G1G7) in the United StatesCN-18, a new strain of SMV was reported from KoreaRaspberry ringspot virusMany minor variants occurThree 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.

Tobacco ringspot virusMany variants reported, based primarily on differences in symptomatologyMany natural antigenic variants also reported

Tobacco streak virusMany variants exist Number of strains known in India Recently found to infect Bt cotton also

Variability in Plant Viruses ContdTomato black ring virusTomato black ring strain (The type strain) Lettuce ringspot strain Potato bouquet strain of Khler Potato pseudo -aucuba strain of KhlerBeet ringspot strain Celery yellow vein strain Tomato ringspot virusTobacco strain = tobacco ringspot virus No. 2 (The type strain) Peach yellow bud mosaic strainGrape yellow vein strain

ThroughExclusion- QuarantinePrediction ? Vector Control ?Seed CertificationIncorporation of Resistance Genes

Management of Plant Viruses

Challenges in Virus Diagnosis in Plant Quarantine

Sample size Detecting an unknown/ exotic virusPart of the planting material to be testedAvailablity of antisera/ primers/ sequencesPost-entry quarantineUrgency of clearance of the sampleConformity to International Standards

Technique alone is not enough We need a strategy covering

Simultaneous detection of fungi, bacteria, viruses, nematodes, insect pests, weeds

Researchable IssuesVariability of viruses/ vectorsMonitoring the vector scenarioDevelopment of diagnostic kits

Pest Diagnosis - Use the Best Tool for the Job

This slide starts a series of illustrations that are useful to explain the SYBR green detection chemistry. Do not belabor each slide. Go through them rapidly, with brief comments as required to clarify the activity of each molecule.

In this slide, Taq has bound to template DNA and is synthesizing a new strand. SYBR Green will bind selectively to the double-stranded DNA.

The larger, yellow sphere is Taq polymeraseThe green hexagon is SYBR GreenThe multi-colored sticks are nucleotides

As new DNA is made, SYBR Green begins to bind to the double-stranded DNA.

Dye molecules are bound to the length of the DNA molecule and are ready to generate a fluorescent signal.For SYBR-Green, the opportunity for detection comes at the end of the extension step. Blue light excites the dye and raises it to a higher energy level. When the dye molecules drop to a lower energy level, they release the energy as fluorescent light.When the dye molecules drop to a lower energy level, they release the energy as fluorescent light.Before we can understand the next two detection chemistries, we need to understand a concept called FRET, which stands for Fluorescence Resonance Energy Transfer.Big Picture: with FRET a fluorescent signal becomes possible when two different dye molecules are in close proximity. When one dye becomes excited by a light source it can transfer this excitation energy to a second dye causing it to raise to a higher energy level. When the second dye drops to a lower energy level, it gives off fluorescent light.A blue light source excites the green dye, raising it to a higher energy level. When the dye molecule drops to a lower energy level, it releases energy, which is absorbed by the second dye molecule.The red dye is now raised to a higher energy level.. When it drops to a lower energy level it will give off light.The red dye emits a red fluorescent signal.Hybridization probe chemistry for the LightCycler depends on FRET. A pair of hybridization probes are designed to bind to adjacent sequences on the target sequence. One member of the pair is labeled on the 3-end with a green dye. The other member of the pair is labeled on the 5-end with a red dye. When the pair of probes anneals to the target sequence...the two green and red dyes are adjacent to each other. As long as the two probe molecules remain bound next to each other, FRET can happen. If one or both of the probe molecules becomes unbound, FRET cannot happen.When both of the hybridization probes are bound, FRET occurs exactly as described in previous series of slides. Here a blue light provides energy to excite the green dye.As described previouslyAs described previously.As described previouslyIt is important to note, that during the PCR process,the hybridization probes are reused from one cycle to the next. During the extension step, Taq polymerase synthesizes new DNA. When it bumps up against a pair of hybridization probes, the probes are displaced.Taq has now displaced the second hybridization probe. The red and green dyes are now separated. FRET is no longer possible until the probes hybridize during the next annealing step.As described previouslyThe LightCycler determines the cycle number where each reaction begins to enter the log-linear phase and matches this value up with the fluorescence data for that sample at that cycle number.The relationship between these two values is used to calculate how much DNA was made for each reaction.The LightCycler determines the cycle number where each reaction begins to enter the log-linear phase and matches this value up with the fluorescence data for that sample at that cycle number.The relationship between these two values is used to calculate how much DNA was made for each reaction.As mentioned earlier, one of the key LC features is that it is an on-line, real-time instrument. This provides the benefit of:constant feedback of reaction dataallowing a retrospective data analysis