High-Resolution Melting Analysis for Differentiation of <i>Sclerotium</i> (= <i>Agroathelia</i> ) <i>rolfsii</i> , <i>S. delphinii</i> , and Two Intraspecific Groups of <i>S. rolfsii</i>

<i>Sclerotium</i> (=<i>Agroathelia</i>) <i>rolfsii</i> and <i>S. delphinii</i> are soilborne fungal pathogens responsible for southern blight and collar rot in numerous crops worldwide. Accurate detection and identification of these related pathogens are critical for disease management and the selection of resistant plant cultivars. In this study, we identified a polymorphic simple sequence repeat (SSR) locus through reference genome sequence analysis and developed a quantitative PCR (qPCR)-based high-resolution melting (HRM) assay to distinguish <i>S. delphinii</i>, <i>S. rolfsii</i>, and two intraspecific groups (G1 and G2) of <i>S. rolfsii</i>. Using a single primer pair in a closed-tube system without probes, HRM analysis differentiated the target groups by melting temperature profiles and detected as little as 1 pg of genomic DNA. The assay was validated on pure cultures, artificially inoculated soil, and rhizosphere soil from diseased tomato plants. This qPCR-HRM method provides a rapid, cost-effective, and robust diagnostic tool for the detection and discrimination of <i>S. rolfsi</i>i G1, <i>S. rolfsii</i> G2, and <i>S. delphinii</i>, complementing conventional PCR and probe-based qPCR diagnostics.