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Detection and Quantification of Pratylenchus thornei in DNA Extracted from Soil Using Real-Time PCR

January 2012 , Volume 102 , Number  1
Pages  14 - 22

Guiping Yan, Richard W. Smiley, and Patricia A. Okubara

First and second authors: Oregon State University, Columbia Basin Agricultural Research Center, P.O. Box 370, Pendleton 97801; and third author: United States Department of Agriculture–Agricultural Research Service, Root Disease and Biological Control Research Unit, Pullman, WA 99164-6430.

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Accepted for publication 23 August 2011.

The root-lesion nematode Pratylenchus thornei is one of the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). It is laborious and difficult to use microscopy to count and identify the nematodes in soils. A SYBR Green I-based real-time polymerase chain reaction (PCR) assay was developed to detect and quantify this species from DNA extracts of soil. A primer set, designed from the internal transcribed spacer region (ITS1) of rDNA, was highly specific to P. thornei and did not amplify DNA from 27 isolates of other Pratylenchus spp., other nematodes, and six fungal species present in PNW wheat fields. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. The standard curve was supported by a high correlation between the numbers of P. thornei added to soil and the numbers quantified using real-time PCR. Examination of 15 PNW dryland field soils and 20 greenhouse samples revealed significant positive correlations between the numbers determined by real-time PCR and by the Whitehead tray and microscopic method. Real-time PCR is a rapid, sensitive alternative to time-consuming nematode extractions, microscopic identification, and counting of P. thornei from field and greenhouse soils.

Additional keywords: in silico analysis, root disease.

© 2012 The American Phytopathological Society