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ARS Home » Pacific West Area » Corvallis, Oregon » Horticultural Crops Research Unit » Research » Publications at this Location » Publication #243966

Title: The Use of Collagenase to Improve the Detection of Plant Viruses in Vector Nematodes by RT/PCR

Author
item Martin, Robert
item Pinkerton, John
item Kraus, Jennifer

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/7/2008
Publication Date: 1/5/2009
Citation: Martin, R.R., Pinkerton, J.M., Kraus, J.E. 2009. The use of collagenase to improve the detection of plant viruses in vector nematodes by RT/PCR. Journal of Virological Methods. 155:91-95.

Interpretive Summary: Nematodes are important vectors of plant viruses and especially so in perennial crops where a planting is maintained over many years. Nematodes move very short distances laterally in a field but can be moved over longer distances in soil on equipment. As a general practice, growers carry out nematode tests on a site prior to establishing a perennial crop. If Xiphinema species are present, the normal practice would be to fumigate the plot prior to planting. In many crops, the Xiphinema nematodes are not damaging by themselves but rather their greatest impact is caused by the transmission of viruses. In this study, we developed a method for detection of virus in nematodes. With the technique, Tomato ringspot virus and Tobacco ringspot virus were detected in single individuals of Xiphinema americanum nematodes and Tobacco rattle virus was detected in Paratrichodorus allius nematodes. Detection of virus in nematodes by nested RT-PCR was as efficient as biological indexing where nematodes are collected from a test site and allowed to feed on cucumber seedlings that are then tested for the presence of virus. With the test, viruses could be detected in nematodes throughout the year.

Technical Abstract: Tomato ringspot virus (ToRSV) Tobacco ringspot virus (TRSV) and Tobacco rattle virus (TRV) are transmitted to healthy plants by viruliferous nematodes in the soil. We developed a method for extraction of genomic viral RNA from virus particles carried within nematodes and a sensitive nested RT/PCR detection assay. The procedure has been adapted to micro scale for handling multiple samples. This assay is effective for detection of ToRSV or TRSV in Xiphinema americanum or TRV in Paratrichodorus allius. With this method, viruses can be detected in nematodes fed on infected plants or from field collected nematodes where the percentage of viruliferous nematodes is unknown. Soil samples from four red raspberry fields infected with ToRSV were collected in 2003 and 2004. Nematodes isolated from these samples were assayed for ToRSV by RT/PCR and compared to cucumber baiting bioassay for virus transmission from the same soil samples. ToRSV was detected in nematodes throughout the season with with similar frequencies in both the RT-PCR assay and in the transmission bioassay.