By: Charlie TaMentor: Dr. Inga Zasada

United States Department of Agriculture: Agricultural Research Services

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

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

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

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.

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Steps:Steps:

Reverse Transcriptase -

Quantitative Polymerase

Chain Reaction (RT-qPCR)

Enzyme-Linked Immunosorbent Assay

(ELISA)

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

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

I. Develop and optimize the efficiency of a RT-qPCR to detect ToRSV

II. Quantify acquisition and saturation level of ToRSV in X. americanum

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 nematodes

Objective I: Development

IPC unsuccessful Individual genetic

diversity in the group X. americanum

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

10-110-2

10-510-3

10-4 10-710-610-8 10-9 10-10

A 1 to 10 dilution series of ToRSV from leaves. 10-1 to 10-10 all

amplified

Objective I: Efficiency

Detection of ToRSV in roots 11, 9, 6, and 5

weeks

Threshold values of ToRSV from amplification plot

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

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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

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

Dr. Inga ZasadaAmy PeetzDr. Bob MartinKaren KellerNola MosierRuth PriceDr. Kevin AhernHoward Hughes Medical InstituteCripps Scholarship