Title: Detection and Quantification of Pratylenchus thornei in DNA Extracted from Soil Using Real-Time PCR Authors
|Smiley, Richard -|
|Yan, Guiping -|
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 23, 2011
Publication Date: January 1, 2012
Citation: Smiley, R.W., Yan, G., Okubara, P.A. 2012. Detection and Quantification of Pratylenchus thornei in DNA Extracted from Soil Using Real-Time PCR. Phytopathology. 102(1):14-22. Interpretive Summary: Pratylenchus thornei is one of four major nematode pests of wheat in the Pacific Northwest, USA. This manuscript describes a rapid, sensitive and specific diagnostic assay for detection and quantification of P. thornei in soil samples, as a replacement for laborious microscopy-based diagnostics. The assay is applicable to risk assessment for growers and for distinguishing resistant wheat plants from susceptible plants in greenhouse screens.
Technical Abstract: 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 directly from DNA extracts of soil. A primer set was designed from the internal transcribed spacer region (ITS1) of rDNA. The primer set was highly specific and did not amplify DNA from 27 isolates of other Pratylenchus species, other nematodes or six fungal species present in PNW wheat fields. A standard curve was generated from artificially infested soils relating threshold cycles (Ct) and log values of nematode number. The standard curve was verified by high correlation between the numbers of P. thornei added to soil and the numbers by 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. This real-time PCR has the potential to eliminate time-consuming nematode extraction, microscopic identification, and counting of this species from field and greenhouse soils.