Development and application of a multiplex TaqMan real-time PCR assay for detecting pathogenic fungi causing root rot in red kidney bean

<i>Fusarium</i> root rot poses a significant threat to red kidney bean (<i>Phaseolus vulgaris</i>) production in Shanxi Province, China, and is primarily caused by <i>Fusarium oxysporum</i>, <i>Fusarium tricinctum</i>, and <i>Fusarium solani</i>. Currently, no method exists for the rapid and simultaneous detection of these three pathogens. In this study, we developed a multiplex TaqMan real-time PCR assay targeting the translation elongation factor 1-alpha (TEF-1α) gene. Specific primers and probes were designed based on conserved regions within TEF-1α. The assay demonstrated high specificity and sensitivity, with detection limits of 1.46 × 10³, 7.17 × 10³, and 1.76 × 10² copies·μL^-1 for <i>F. oxysporum</i>, <i>F. tricinctum</i>, and <i>F. solani</i>, respectively. Intra- and inter-assay variability tests showed high reproducibility, with coefficients of variation below 2%. Field surveys conducted in Shanxi's main production areas assessed the root rot disease index, and corresponding soil samples were collected. A logistic regression model was established to predict disease index based on the total DNA copy number of the three pathogens per gram of soil: y= 100-96.670/ [1 + (x/7.253)^9.350] (R² = 0.927). This multiplex TaqMan real-time PCR assay provides a rapid, specific, and reproducible tool for simultaneous detection of these <i>Fusarium</i> pathogens, supporting early disease intervention and reducing crop losses in red kidney bean.