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Title: Detection and Discrimination of Pratylenchus neglectus and P. thornei in DNA Extracts from Soil

item Okubara, Patricia
item Skantar, Andrea

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/20/2008
Publication Date: 11/1/2008
Citation: Yan, G., Smiley, R., Okubara, P.A., Skantar, A.M., Easley, S.A., Sheedy, J.G., Thompson, A.L. 2008. Detection and Discrimination of Pratylenchus neglectus and P. thornei in DNA Extracts from Soil. Plant Disease. 92(11):1480-1487.

Interpretive Summary: The root lesion nematodes Pratylenchus neglectus and P. thornei are parasites of wheat roots, are well-known in Oregon, Idaho and Montana, and are emerging problems in Washington state. Current diagnosis of P. neglectus and P. thornei is based on morphological characteristics that require an expert eye and is unreliable because the species exhibit overlap in the range of characteristics, requiring measurements from many individuals. Furthermore, diagnosis is limited to mature adult females. We developed a rapid, specific and sensitive PCR-based diagnostic method for identifying P. neglectus and P. thornei directly from soil. Species-specific primers were designed, a protocol for extracting PCR-ready total DNA from soil was developed, PCR amplification was optimized, and detection sensitivity was determined. The PCR assays were validated using soils naturally infested with either P. neglectus and P. thornei or both. The assays were found to be applicable to a wide range of nematode population densities and soil types.

Technical Abstract: A species-specific polymerase chain reaction (PCR) method was developed to detect and identify the root-lesion nematodes Pratylenchus neglectus and P. thornei in soil. A primer set was designed from Pratylenchus 26S rRNA gene sequences of the D3 expansion domain and their specificity was confirmed with plant-parasitic and non-parasitic nematodes typically present in the soil communities, and with six fungal species commonly associated with wheat root rot. DNA obtained using a commercially available kit and an inexpensive method developed in our laboratory gave comparable amplification. Optimized PCR conditions were established and the two species were differentiated by PCR products of 144 bp for P. neglectus and 288 bp for P. thornei. With this assay we were able to detect a single juvenile in 1 gram of sterilized, inoculated soil. Examination of 30 field soil samples revealed that this method was applicable to a range of soils naturally infested with these two pathogens in Oregon. This new PCR-based method is rapid, efficient, and reliable, does not require expertise in nematode taxonomy compared to commonly used nematode-extraction and morphological identification techniques, and can be used as a rapid diagnostic tool in commercial and research applications for disease forecast and management.