A Rapid Genomic DNA Extraction Method for Direct Quantitative Polymerase Chain Reaction-Based On-Site Detection of...

Soil-borne phytopathogenic fungi cause root rot, wilt, and damping-off in crops, leading to major yield losses worldwide. Because symptoms appear only after underground infection progresses, early detection is crucial. Here, a rapid 20-min genomic DNA extraction method was developed for eight pathogens-Alternaria tenuissima, Botryosphaeria dothidea, Fusarium oxysporum, Glomerella cingulata, Phytophthora cactorum, Rosellinia necatrix, Sclerotium rolfsii, and Sclerotinia sclerotiorum. The protocol uses a cetyltrimethylammonium bromide-based buffer, steel and glass beads, brief heating (95°C, 1 min), vortexing, and sequential purification with Q-Sepharose and magnetic beads. All pathogens were detected within 30 quantitative polymerase chain reaction cycles, while soil-only controls exceeded 30 Cq. Sclerotial DNA of S. rolfsii (Cq ≈ 25) was also detected, confirming applicability for overwintering inocula. This simple and low-cost protocol enables rapid, reliable detection of multiple soil-borne fungi directly from soil, providing a practical tool for on-site disease diagnosis and management.