ARS | Publication request: Identification and quantification of Pratelynchus neglectus and P. thornei from soils in the Pacific Northwest using real-time polymerase chain reaction

Submitted to: Society of Nematologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: July 14, 2010
Publication Date: July 14, 2010
Citation: Yan, G., Smiley, R.W., Okubara, P.A. 2010. Identification and quantification of Pratelynchus neglectus and P. thornei from soils in the Pacific Northwest using real-time polymerase chain reaction. Society of Nematologists Proceedings. Annual Meeting of the Society of Nematologists, Boise, ID, July 11-14, 2010, Abstract #134, p.123-124.

Technical Abstract: Root-lesion nematodes, Pratylenchus neglectus and P. thornei, are the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). Pre-plant populations of both species are frequently inversely correlated with wheat yield. It is estimated that these nematodes reduce profitability of farms in Idaho, Oregon and Washington by about $51 million annually. The best approach to control damage from root-lesion nematodes is to grow cultivars that are both resistant and tolerant. Individual wheat cultivars may differ in their ability to resist and tolerate these nematodes. Optimal cultivar selection requires that the lesion nematode species present in each field or region be accurately identified and quantified. It is challenging to discriminate P. neglectus, P. thornei and other closely related species by morphological characters. It is laborious and difficult to use microscopy to count and identify these nematodes in large numbers of field samples in which other nematode species are also present. A SYBR Green I-based real-time quantitative-polymerase chain reaction assay was developed to facilitate the identification and quantification of individual species in soil. A primer set for P. neglectus was designed from the Pratylenchus 28S ribosomal RNA gene sequences of the D3 expansion domain. A primer set for P. thornei was designed from the internal transcribed spacer ITS1 of the ribosomal DNA. Melting curve analysis revealed that the primer sets were highly specific. The P. neglectus-primers did not amplify DNA from 10 isolates of other Pratylenchus species and other species and genera of nematodes typically present in the soil communities, and from six fungal species commonly associated with wheat root rot. The analysis of PCR amplification efficiency (E) indicated that DNA extracted from soil samples by a commercial kit (PowerSoil DNA Isolation Kit) can be directly used in real-time PCR without any additional purification step (E = 96% for P. neglectus, 97% for P. thornei). Standard curves were generated from artificially inoculated soils showing a negative linear regression between threshold cycles (Ct) and Log values of number of nematodes (r2 = 0.92 for P. neglectus, 0.98 for P. thornei). Validation tests using 15 field soil samples were conducted to determine the relationship between nematode numbers detected by the real-time PCR assay and the numbers reported by commercial diagnostic laboratories and our research lab using traditional methods. The real-time PCR has the potential to enable laboratories to avoid time-consuming physical separations, microscopic identifications, and counting of these species from field samples with mixed populations of other plant-parasitic and non-plant-parasitic nematodes.