Cyclobutrifluram Resistance in Alternaria alternata: Molecular Mechanisms and Detection Strategies

The resistance risk and mechanisms of cyclobutrifluram in Alternaria alternata are unclear. The baseline sensitivity of 111 A. alternata isolates to cyclobutrifluram was 0.10 ± 0.07 μg/mL. Eight cyclobutrifluram-resistant mutants of A. alternata were generated in the laboratory, exhibiting high-level and relative stable resistance. Cross-resistance was found between cyclobutrifluram and boscalid or pydiflumetofen, yet no cross-resistance was observed between cyclobutrifluram and iprodione, azoxystrobin, mefentrifluconazole, or difenoconazole. Each resistant mutant exhibited a reduced compound fitness index (CFI) compared to their parental isolates. An S73L substitution in AaSdhC or a P113T, H134N, or D145N mutation in AaSdhD conferred cyclobutrifluram resistance. Allele-specific PCR (AS-PCR) methods for the detection of mutations AaSdhC^S73L, AaSdhD^P113T, AaSdhD^H134N, and AaSdhD^D145N were successfully developed. In summary, A. alternata exhibits a moderate risk for developing resistance to cyclobutrifluram, attributed to the S73L substitution in AaSdhC or the P113T, H134N, or D145N mutations in AaSdhD, which are detectable using specific AS-PCR methods.