P-436 - Developing a Real-time Quantitative PCR Assay for Direct Detection of the New Root-lesion Nematode Pratylenchus dakotaensis from Soybean Roots

Root-lesion nematodes (Pratylenchus spp.) pose a significant threat to soybean production.  Pratylenchus dakotaensis, a recently named root-lesion nematode species from North Dakota, exhibits high infection and reproduction in soybean roots. Early and rapid detection of P. dakotaensis is critical for effective management, but traditional identification methods are time-consuming and labor-intensive. Thus, the objective of this research is to develop a SYBR Green-based real-time quantitative PCR (qPCR) assay for direct and rapid detection of P. dakotaensis in DNA extracted from soybean roots. The species-specific primers previously designed in our lab were evaluated and they were specific, detecting only P. dakotaensis DNA when co-extracted with root extracts. PCR inhibitors were found present in soybean root extracts and subsequently neutralized. Sensitivity analysis revealed that the assay could detect as low as 1/128^th of a single nematode in 0.2 g of roots. A standard curve was generated, showing high amplification efficiency and coefficient of determination. The qPCR assay was further tested with DNA extracts from artificially-inoculated root samples and infected soybean roots grown in infested soil. qPCR estimates from the standard curve for both types of samples had strong, positive correlations with ­P. dakotaensis densities determined by traditional methods. The developed assay provides a rapid and efficient molecular tool for detecting P. dakotaensis directly from DNA extracts of infected soybean roots, facilitating timely management of this new nematode pest.