Mechanism of prothioconazole resistance in the multinucleate fungus Sclerotium rolfsii

Peanut stem rot, caused by Sclerotium rolfsii, is a soil-borne disease, which severely threatens global peanut production. At present, the control of this disease mainly depends on the application of chemical fungicides. Despite the recent registration of the DMI fungicide prothioconazole in China, field resistance has rapidly emerged. Evaluating 51 field isolates (with mean EC₅₀ = 1.88 ± 6.13 μg/mL), we found that less sensitive isolates incur fitness costs in mycelial growth but exhibit markedly enhanced sclerotial production. To elucidate the underlying mechanisms, we systematically investigated target-site and non-target-site factors. Gene expression analysis revealed that prothioconazole exposure triggers a dramatic upregulation of CYP51 in less sensitive isolates. Target-site mutations (despite the identification of an I96V mutation in CYP51), altered nuclear counts, and enhanced efflux pump activity (atrB, atrD, and MFS1) may not be the major resistance drivers. These findings demonstrate that CYP51 overexpression is the primary molecular basis for prothioconazole resistance in S. rolfsii. A significant positive correlation was observed between sensitivity to prothioconazole and the DMI fungicides difenoconazole and tebuconazole. Therefore, it is recommended to use prothioconazole in alternation with fungicides that have different modes of action, such as thifluzamide and isopyrazam.