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Developing a Real-Time PCR Assay for Detection and Quantification of Pratylenchus neglectus in Soil

Yan Guiping, Smiley Richard W., Okubara Patricia A., Skantar Andrea M., Reardon Catherine L.
Plant disease 2013 v.97 no.6 pp. 757-764
DNA primers, Pratylenchus neglectus, bioassays, crop rotation, cultivars, detection, internal transcribed spacers, microscopy, plant parasitic nematodes, polymerase chain reaction, quantitative analysis, ribosomal DNA, roots, soil, soil sterilization, wheat, Pacific States
Pratylenchus neglectus is one of the most widespread and economically important nematodes that invades plant roots and restricts wheat productivity in the Pacific Northwest. It is challenging to quantify P. neglectus using microscopic methods for studies that require large-scale sampling, such as assessment of rotation crops, wheat cultivars, and other management practices. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. neglectus from DNA extracts of soil. The primers, designed from the internal transcribed spacer region of rDNA, showed high specificity with a single melt curve peak to DNA from eight isolates of P. neglectus but did not amplify DNA from 28 isolates of other plant-parasitic and non-plant-parasitic nematodes. A standard curve (R² = 0.96; P < 0.001) was generated by amplifying DNA extracted from soil to which nematodes were added. The soil standard curve was validated using sterilized soil inoculated with lower numbers of P. neglectus. A significant positive relationship (R² = 0.66; P < 0.001) was observed for nematode numbers quantified from 15 field soils using qPCR and the Whitehead tray and microscopic method but the qPCR generally tended to provide higher estimates. Real-time PCR potentially provides a useful platform for efficient detection and quantification of P. neglectus directly from field soils.