Development of a Real-Time Quantitative PCR Assay for Direct Detection and Quantification of the Root-Lesion Nematode...

The root-lesion nematode, Pratylenchus penetrans, is a migratory endoparasite that attacks potato roots, causing necrotic lesions and yield losses of up to 73%. Traditional detection and quantification methods are labor-intensive, time-consuming, and require prior nematode extraction and taxonomic expertise. This study aimed to develop a SYBR Green-based real-time quantitative PCR (qPCR) assay for detecting and quantifying P. penetrans directly from potato root DNA extracts. Bovine serum albumin (BSA) was tested to neutralize qPCR inhibitors in root DNA extracts. The assay showed high specificity and sensitivity to P. penetrans, detecting 1.56 × 10^-2 of a single nematode in 0.2 g of roots. A standard curve based on artificial nematode inoculations demonstrated a strong linear relationship between Cq values and log-transformed nematode numbers (R² = 0.993) with high amplification efficiency. Assessment using root samples from two greenhouse experiments involving five potato cultivars showed strong correlations (r = 0.902 and 0.887) between qPCR estimates and microscopic nematode counts. This study presents a new qPCR assay specifically optimized for direct detection and quantification of P. penetrans within potato root tissues, offering enhanced sensitivity and applicability for rapid in planta diagnostics to facilitate effective management strategies.