Southern corn leaf blight (SCLB) caused by Cochliobolus heterostrophus, is a widespread foliar disease that has a substantial impact on maize yield in the Huang-Huai-Hai Region of China. Pydiflumetofen (Pyd), a new succinate dehydrogenase inhibitor (SDHI), has been found as a promising fungicide for the efficient control of SCLB, however, resistance of C. heterostrophus to Pyd has not been studied well. Here, five Pyd-resistant mutants were generated through fungicide adaptation. Sequence alignment analysis revealed that these mutants primarily mutated in ChSdhB and ChSdhD, with three genotypes: ChSdhB^H277Y, ChSdhB^I279T and ChSdhD^H133Y, exhibiting two distinct categories of resistance: high resistance (HR) and moderate resistance (MR), among which the resistance factors were 214.22 and 44.33–53.67, respectively. These mutants were more pathogenic than the wild-type parental strains, but there was a significant reduction in mycelial growth rate and sporulation in the resistant mutants, indicating a significant fitness cost associated with resistance to Pyd. In addition, this study revealed a positive cross-resistance between Pyd and another SDHI fungicide cyclobutrifluram. However, no cross-resistance was found between Pyd and other classes of fungicides, including prochloraz, fludioxonil, iprodione or pyraclostrobin. Homology modeling and molecular docking further confirmed that point mutations of ChSdhB^H277Y, ChSdhB^I279T, and ChSdhD^H133Y could reduce binding affinity between Pyd and its target subunits from –74.07, –74.07, –152.52 kcal mol^–1 to –3.90, –4.95, –9.93 kcal mol^–1, respectively. These findings not only provided valuable insights for managing SCLB caused by C. heterostrophus, but also enhanced our understanding of molecular mechanism underlying plant pathogen resistance to Pyd.

Rice bakanae disease (RBD) is a devastating plant disease caused by Fusarium fujikuroi. This study aimed to evaluate the potential of cyclobutrifluram, a novel succinate dehydrogenase inhibitor (SDHI), to control RBD, and determine the risk and mechanism of resistance to cyclobutrifluram in F. fujikuroi. In vitro experiments showed that cyclobutrifluram significantly inhibited mycelial growth and spore germination, and altered the morphology of mycelia and conidia. Treatment with cyclobutrifluram significantly decreased mycotoxin production and increased cell membrane permeability in F. fujikuroi. The baseline sensitivity of 72 F. fujikuroi isolates to cyclobutrifluram was determined using mycelial growth and spore germination inhibition assays, which revealed EC₅₀ values of 0.0114–0.1304 and 0.0012–0.016 μg mL^–1, with mean EC₅₀ values of (0.0410±0.0470) and (0.0038±0.0015) μg mL^–1, respectively. Pot experiments demonstrated that the protective effect of cyclobutrifluram against F. fujikuroi was more significant than that of phenamacril and azoxystrobin, indicating that cyclobutrifluram is a promising antifungal agent for the control of RBD. Six cyclobutrifluram-resistant mutants of F. fujikuroi were obtained via fungicide adaptation. Moreover, these mutants exhibited weaker fitness than their parental isolate and positive cross-resistance with other SDHI fungicides, including pydiflumetofen and penflufen; however, no cross-resistance was detected with other classes of fungicides, including phenamacril, fludioxonil, prochloraz, or azoxystrobin. These results indicated that the resistance risk of F. fujikuroi to cyclobutrifluram might be moderate. Sequencing analysis revealed that mutations, including H248D in FfSdhB, A83V in FfSdhC₂, and S106F and E166K in FfSdhD, contributed to resistance, which was confirmed by molecular docking and homologous replacement experiments. The results suggest a high potential for cyclobutrifluram to control RBD and a moderate resistance risk of F. fujikuroi to cyclobutrifluram, which are meaningful findings for the scientific application of cyclobutrifluram.