The Combined Application of Fludioxonil and Cyclobutrifluram Delays the Development of Fungicide Resistance in <i>Fusarium fujikuroi</i>

Cyclobutrifluram and fludioxonil are commonly used fungicides for controlling rice bakanae disease caused by <i>Fusarium fujikuroi</i>. This study evaluated their combined effect on fungicide resistance development in <i>F. fujikuroi</i>. Cyclobutrifluram-resistant (Cyc^R) and fludioxonil-resistant (Flu^R) mutants emerged within 10-30 days under single fungicide exposure, whereas populations exposed to the mixture showed little sensitivity change. Cyc^SFlu^R and dual resistant Cyc^RFlu^R mutants appeared after 64 and 151 days of mixture treatment, demonstrating the combination delays resistance. Flu^R, Cyc^SFlu^R, and Cyc^RFlu^R mutants possessed comparable or impaired fitness compared to parental isolates. Known cyclobutrifluram resistance-conferring mutations FfSdhB-H248Y/D and FfSdhC₂-A83 V were found in Cyc^R mutants. Six distinct FfOs1 variations─T454A, N840 K, 757IVN insertion, N760I, Y1114 V (frameshifts), and 864stop─were identified in resistant mutants; transformation assays confirmed all conferred fludioxonil resistance. Notably, FfSdhC₂-A83 V and FfOs1-T454, as well as FfSdhB-H248Y and the insertion of FfOs1-757IVN, could confer dual resistance to cyclobutrifluram and fludioxonil in <i>F. fujikuroi</i>.