Chaofan Jiang, Lin Zhou, Weifeng Cheng, Wei He, Yue Yang, Kaidi Cui, Leiming He
Abstract BACKGROUND Peanut stem rot, a destructive soil‐borne disease caused by the multinucleate fungus Sclerotium rolfsii , is primarily controlled using the succinate dehydrogenase inhibitor (SDHI) fungicide thifluzamide. However, the sensitivity and resistance mechanisms of S. rolfsii to thifluzamide remain unclear. RESULTS This study determined that thifluzamide exhibits strong inhibitory activity against S. rolfsii with a mean half‐maximal effective concentration (EC 50 ) value of 0.046 ± 0.021 μg/mL. Nine stable thifluzamide‐resistant mutants were generated through laboratory resistance domestication. Mutants exhibited similar or reduced fitness compared with the parental isolate. Nuclear counts did not differ significantly between resistant mutants and the parental isolate. Gene sequencing, molecular docking, and protoplast transformation analyses revealed that a heterozygous mutation in the SdhD subunit (SdhD H103Y ) confers resistance to thifluzamide and other SDHI fungicides in S. rolfsii . CONCLUSION These findings demonstrate that thifluzamide resistance in S. rolfsii associated with the target mutation SdhD H103Y , rather than with variation in nuclear number. © 2025 Society of Chemical Industry.