Virus vectoring ability of a Xiphinema americanumOregon State 病毒矢量的剑线虫americanum 俄勒冈.ppt

Virus vectoring ability of a Xiphinema americanum population By: Charlie TaMentor: Dr. Inga ZasadaUnited States Department of Agriculture: Agricultural Research ServicesJustification Plant-parasitic nematodes cause $100 billion in crop loss annually worldwide; $10 billion in the U.S. (blueberries, red raspberries, and wine grape industry)Plants affected by X. americanum or nepoviruses become(s) necrotic, yield is reduced, and plant mortality can occur Currently few methods exist to control nematodes or remediate the diseases they transmitRegulations by the U.S. Environmental Protection Agency will soon limit/ban pre-plant fumigation which has traditionally been used to eradicate virus-transmitting nematodesXiphinema americanum(Dagger Nematode) Microscopic roundworm(s) that parasitize plantsMigratory ectoparasiteAcquire and transmit nepoviruses such as Tomato Ringspot Virus (ToRSV) and Tobacco Ringspot Virus (TRSV) with their odontostyleheadtailNepovirusesNematode-transmitted virus with polyhedral particlesType IV virus under the Baltimore classification system (positive sense single-stranded RNA that directly translates into protein)Acquisition of virus occurs during feeding and binds to the surface ofthe odontostyleViruses are lost when nematodes moltodontostyleMolecular ProtocolsA RT-qPCR can be used for the detection of ToRSV in X. americanum at low concentration levels.Virus detection using RT- qPCR allows for a detailed study of nematode-virus interactions.The coloration occurs due to adding p-nitrophenyl phosphate.http://homepage.usask.ca/~vim458/virology/studpages2007/Maura_Tim/For%20Maura%20-%20Virology%20website%20assignment/elisa.jpg12345Steps:Reverse Transcriptase -Quantitative Polymerase Chain Reaction (RT-qPCR)Enzyme-Linked Immunosorbent Assay (ELISA)HypothesisCould a RT-qPCR method be developed to enable detection of small concentrations of ToRSV?PredictionA RT-qPCR method will be proficient in detecting low concentrations of viruses.X. americanum acquires ToRSV within a week of feeding on a virus infected host.This time period allows for additional virus particles to be acquired by the nematodeObjectivesI.Develop and optimize the efficiency of a RT-qPCR to detect ToRSVII.Quantify acquisition and saturation level of ToRSV in X. americanumProbe: Reverse: Forward: MethodologyDevelop an internal positive control (IPC) for RT-qPCR by examining homogeneity of the internal transcribed spacer (ITS) region 1 of X. americanumDesign IPC to similar length as the ToRSV primer/probe set for multiplex purposesAnalyze the two sets for cross reaction and non target RNA with each other. Examine the thermodynamic compatibility using hybridization software and cross referencing sequence data available on GenbankValidate RT-qPCR method with known virus infected samples.Ensures that our samples have nematodesObjective I: DevelopmentDataIPC unsuccessfulIndividual genetic diversity in the group X. americanumDataChromatograph illustrating the heterogeneity within the ITS1 region of rDNA for a single individual X. americanumA single signal becomes multiple signals; We observed this with individuals other than Xiphinema as well Literature suggest phylogenetic studies on nematodes is a common problem Data10-110-210-510-310-410-710-610-810-910-10A 1 to 10 dilution series of ToRSV from leaves.10-1 to 10-10 all amplifiedObjective I: EfficiencyDataDetection of ToRSV in roots 11, 9, 6, and 5 weeksThreshold values of ToRSV from amplification plotInoculation DateRNA6/23 A6/23 B7/26 A7/26 B7/26 C7/8 A7/8 B7/8 3C7/8 D7/8 E7/8 F7/8 G7/8 H7/8 I8/5 A8/5 B8/5 C8/5 D8/5 E8/5 F8/5 G051015202530Ct Mean of ToRSV in Roots DataDilution series of ToRSV in RootsDetection of ToRSV in roots was lower than ToRSV in leaves10-1 to 10-4 amplified10-110-210-310-4Objective II:http://image.made-in-ObstaclesX. americanumHave low fecundityDelicate and sensitive to disturbancesInoculation and recovery of nematodes were lowRNA extraction was poorIPC did not workFuture Studies Develop and optimize a RT-qPCR method to detect TRSV in X. americanumDetermine the persistency and duration of ToRSV/TRSV within X. americanum by using the developed primers/probes for RT-qPCRLink the genetic variability of X. americanum populations to virus vectoring capabilities as a means to facilitate the development of diagnostic toolsAcknowledgementsvDr. Inga ZasadavAmy PeetzvDr. Bob MartinvKaren KellervNola MosiervRuth PricevDr. Kevin AhernvHoward Hughes Medical InstitutevCripps Scholarship。