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

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https://ir.library.oregonstate.edu/concern/articles/v405sb146

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  • The root-lesion nematode Pratylenchus (hornet 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 (ITSI) 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.
  • Keywords: root disease, Pacific Northwest, Potato cyst nematode, Root lesion nematodes, Quantitative PCR, Meloidogyne incognita, in silico analysis, Genus pratylenchus, Polymerase chain reaction, Fragment length polymorphism, Wheat, Identification
  • Keywords: root disease, Pacific Northwest, Potato cyst nematode, Root lesion nematodes, Quantitative PCR, Meloidogyne incognita, in silico analysis, Genus pratylenchus, Polymerase chain reaction, Fragment length polymorphism, Wheat, Identification
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  • Yan, G., Smiley, R., & Okubara, P. (2012). Detection and quantification of pratylenchus thornei in DNA extracted from soil using real-time PCR. Phytopathology, 102(1), 14-22. doi: 10.1094/PHYTO-03-11-0093
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  • 102
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  • 1
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  • This research was supported by the Oregon State University, Agricultural Research Foundation project ARF 7135A, and a subcontract between Oregon State University and the United States Department of Agriculture– Agricultural Research Service (USDA-ARS) (SCA 58-5348-9-100, “Control of Root Diseases of Wheat and Barley”), and USDA-ARS project number 5248-22000-012-00D (P. A. Okubara).
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