MOLECULAR STRATEGIES FOR DETECTION AND IDENTIFICATION OF NEMATODES INFECTING FORAGE LEGUMES AND DEVELOPMENT OF HOST RESISTANCE
Location: Nematology Laboratory
Title: Developing a real-time PCR assay for detection and quantification of Pratylenchus neglectus in soil
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 26, 2012
Publication Date: May 15, 2013
Citation: Yan, G., Smiley, R.W., Okubara, P.A., Skantar, A.M., Reardon, C.L. 2013. Developing a real-time PCR assay for detection and quantification of Pratylenchus neglectus in soil. Plant Disease. 97(6):757-764.
Interpretive Summary: Plant-parasitic nematodes are microscopic worms that attack plant roots and cause an estimated ten billion dollars of crop loss each year in the United States and 100 billion dollars globally. The root-lesion nematode Pratylenchus is one of the most widespread and economically important nematodes that invade plant roots and restrict wheat productivity in the Pacific Northwest. This nematode is difficult to quantify by microscopic methods for studies that require large-scale sampling, such as evaluation of wheat cultivars, rotation crops, and other management practices. In the present study, scientists from Oregon State University and ARS scientists from Beltsville, MD, Pullman, WA, and Pendleton, OR developed a highly sensitive molecular diagnostic assay to detect P. neglectus using DNA extracted directly from soil samples. The results are significant because the assay can be used to quantify P. neglectus in soil and distinguish this species from other lesion nematodes. Commercial diagnostic laboratories, other scientists, action agencies, and extension agencies engaged in nematode research and control will use this research.
Pratylenchus neglectus is one of the most widespread and economically important nematodes that invade plant roots and restrict 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 quantitative real-time 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 a single peak and high specificity 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 was generated from artificially infested soil showing a significant negative correlation (R2 = 0.96) between cycle threshold and log value of nematode numbers. The soil standard curve was validated using sterilized, non-infested soil inoculated with lower numbers of P. neglectus. A significant positive correlation (R2 = 0.66) was observed for nematode numbers quantified from fifteen field soils using qPCR and the Whitehead tray and microscopic method. Real-time PCR provides a useful platform for efficient detection and quantification of P. neglectus directly from field soils.