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Virus vectoring ability of a Xiphinema americanum population

By: Charlie Ta Mentor: Dr. Inga Zasada United States Department of Agriculture: Agricultural Research Services. Virus vectoring ability of a Xiphinema americanum population. Justification.

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Virus vectoring ability of a Xiphinema americanum population

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  1. By: Charlie Ta Mentor: Dr. Inga Zasada United States Department of Agriculture: Agricultural Research Services Virus vectoring ability of a Xiphinema americanum population

  2. 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

  3. 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 odontostyle head tail

  4. 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 molt odontostyle

  5. Molecular Protocols Reverse Transcriptase -Quantitative Polymerase Chain Reaction (RT-qPCR) Enzyme-Linked Immunosorbent Assay (ELISA) The coloration occurs due to adding p-nitrophenyl phosphate. • 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. Steps: 1 2 3 4 5 http://homepage.usask.ca/~vim458/virology/studpages2007/Maura_Tim/For%20Maura%20-%20Virology%20website%20assignment/elisa.jpg

  6. Hypothesis • Could a RT-qPCR method be developed to enable detection of small concentrations of ToRSV?

  7. 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

  8. Objectives Probe: ACA AAT AAA AGG CAC TGC GTG TCT CG • Develop and optimize the efficiency of a RT-qPCR to detect ToRSV • Quantify acquisition and saturation level of ToRSV in X. americanum Forward: GGA GAC GAT AAA TCC TAT GTG GG Reverse: TCG AAT TTA AAT GCA GAC TCA AGA TAT G

  9. Methodology Objective I: Development • 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 nematodes

  10. Data • IPC unsuccessful • Individual genetic diversity in the group X. americanum

  11. 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

  12. Data Objective I: Efficiency • A 1 to 10 dilution series of ToRSV from leaves. • 10-1 to 10-10 all amplified 10-1 10-2 10-9 10-8 10-10 10-3 10-7 10-4 10-6 10-5

  13. Data • Threshold values of ToRSV from amplification plot • Detection of ToRSV in roots • 11, 9, 6, and 5 weeks

  14. Data • Dilution series of ToRSV in Roots • Detection of ToRSV in roots was lower than ToRSV in leaves • 10-1 to 10-4 amplified 10-1 10-2 10-3 10-4

  15. Objective II: http://mexicanmortarandpestle.net/images/large%20mortar%20and%20pestle.jpg http://www.tari.gov.tw/tarie/photos/introduction/introduction_PPD_17.jpg http://www.medicine.virginia.edu/research/cores/biomolec/images/rt-pcr.jpg http://2010.igem.org/wiki/images/thumb/c/cb/IC_Assay_3_sept.jpg/300px-IC_Assay_3_sept.jpg http://image.made-in-china.com/2f0j00dCLaFpKzrDos/96-Well-Cell-Culture-Plate.jpg

  16. 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

  17. 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

  18. Acknowledgements • Dr. Inga Zasada • Amy Peetz • Dr. Bob Martin • Karen Keller • Nola Mosier • Ruth Price • Dr. Kevin Ahern • Howard Hughes Medical Institute • Cripps Scholarship

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