Detection of SdhB mutations in Botrytis cinerea isolates from strawberry using a High-Resolution Melting (HRM) assay

Botrytis cinerea, the causal agent of Botrytis fruit rot (BFR), is one of the most destructive pathogens of strawberry. In Florida, BFR management relies heavily on succinate dehydrogenase inhibitor (SDHI) fungicides; however, resistance associated with mutations in the SdhB subunit has increasingly been reported. This study developed and validated a high-resolution melting (HRM) assay for rapid detection of SdhB mutations directly from B. cinerea spores, and evaluated resistance frequencies to fluopyram, isofetamid, and pydiflumetofen across Florida strawberry fields. Two HRM primer sets successfully differentiated SdhB mutations H272R or Y, N230I, and P225F based on distinct melting profiles. The assay detected mutations from spore concentrations as low as 10⁵ spores/ml, without requiring DNA extraction or post-PCR processing. Sanger sequencing subsequently confirmed complete agreement with HRM genotyping results. Among 133 field isolates collected from 2017 to 2022, N230I and P225F were the most frequent mutations, correlating with moderate to high resistance to fluopyram and isofetamid. Resistance monitoring of 1,496 isolates over eight strawberry seasons revealed increasing resistance to fluopyram (44.9-99%) and isofetamid (≤57%), whereas pydiflumetofen resistance remained below 15%. Sequencing of SdhC and SdhD subunits revealed minimal variability and no new target-site mutations. Detached fruit assays confirmed reduced efficacy of isofetamid against isolates harboring N230I. The HRM assay developed here provides a rapid, accurate, and cost-effective tool for detecting and monitoring SDHI resistance in B. cinerea populations in strawberry production systems.