Molecular Profiling and Precise Diagnosis of Pratylenchus penetrans Infestation in Soil: A qPCR-Based Molecular Approach.

Abstract

Pratylenchus penetrans, an important soil pathogen, has been reported on various crops in the temperate regions of South Korea. In concern, there is an urgent need for a precise, species-specific quantitative polymerase chain reaction (qPCR) kit to detect and quantify root lesion nematodes for early pest management and to controls yield losses. The present study focuses on D2-D3 region, a known marker for molecular profiling of Pratylenchus sp. A primer set mined from the highly conserved D2-D3 region of P. penetrans was used in a SYBR green based qPCR assay. Initial examination identified P. penetrans from infested soil samples using morphological and phylogenetic analyses. The DPp7F12R primer set demonstrated significant specificity in identifying P. penetrants by both conventional polymerase chain reaction (PCR) and qPCR assays. Linear regression of serially diluted DNA from nematode and nematode inoculated soil revealed a limit of quantification of 2 picograms (r² = 0.984), while also highlighting the impact of soil inhibitors. The qPCR using the DNA from varying densities of P. penetrans inoculated in soil demonstrated a robust correlation (r² = 0.98), indicating the limit of detection down to single nematode. Primer specificity evaluation with field soil sample precisely detected only P. penetrants. Species-specific DPp7F12R facilitate the direct detection of P. penetrants from soil DNA in very shorter time. Reliability of PCR was confirmed using BLAST algorithm, which identified partial sequence of PCR amplicon (300 bp) as P. penetrants. Finally, PCR assay using DPp7F12R is crucial for early detection of P. penetrans infestations, helping improve the plant health.