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Development of a real-time PCR assay for direct detection and quantification of the new root-lesion nematode species, Pratylenchus dakotaensis from soybean roots

Siddanta Ranabhat,Guiping Yan

Published 2025 in Nematology

ABSTRACT

Root-lesion nematodes (Pratylenchus spp.) pose a significant threat to soybean productivity by attacking and infecting plant roots. Pratylenchus dakotaensis, a newly named root-lesion nematode species characterised in North Dakota, was found to exhibit high infection and reproduction in soybean. Early rapid detection of nematodes is critical for effective management, but traditional methods for nematode identification and quantification are labour-intensive and time-consuming. Thus, this research aimed to develop a SYBR Green-based real-time quantitative PCR (qPCR) assay for direct, rapid detection and quantification of P. dakotaensis in soybean roots. The primer pair previously designed in our lab was found specific to P. dakotaensis DNA when co-extracted with soybean root DNA. PCR inhibitors were identified in the DNA extracted using a commercial DNA extraction kit and were neutralised. Sensitivity tests revealed the assay could detect as low as 1/128th of a single nematode per 0.2 g of roots. A standard curve with high amplification efficiency (%) and coefficient of determination () was generated. The assay was validated using DNA extracts from artificially-inoculated root samples and infected soybean roots. qPCR estimates from both types of samples showed strong, positive correlations with P. dakotaensis densities ( and 0.80, respectively). This is the first report of developing a qPCR assay to detect and quantify P. dakotaensis directly from plant roots. This assay provides a rapid and efficient molecular tool for detecting and quantifying P. dakotaensis directly from soybean roots, thereby facilitating monitoring and timely management of this new nematode pest.

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