Sensitivity and resistance risk analysis of Didymella bryoniae populations to fluopyram

Fluopyram is an succinate dehydrogenase inhibitors （SDHI） fungicide that has been registered in China to control gummy stem blight （GSB） in watermelons for many years.However,whether the field pathogens of GSB are still sensitive to fluopyram or not is unknown.Therefore,we collected 69 Didymella bryoniae isolates from the fields that usually use fluopyram to control GSB to determine the sensitivity change.The EC50 （50%inhibition effect）values of fluopyram against D.bryoniae ranged from 0.0691 to 0.3503μg mL–1 and the variation factor was 5.07.The mean EC50 value was （0.1579±0.0669）μg mL–1 and the curve of sensitivity was unimodal.No resistant strains were found in the isolates,which means that the pathogens were still sensitive to fluopyram.The minimal inhibition concentration （MIC） of fluopyram against D.bryoniae was 3μg mL–1.Four low-resistant mutants and two mediumresistant mutants were obtained using fungicide taming and the resistance of mutants could be inherited stably.The growth rate of mutants decreased significantly compared with that of wild-type strains while the biomass of most mutants was similar to that of wild-type strains.The sensitivity of most resistant mutants to various stresses was increased compared with that of wild-type strains.The virulence of mutants receded except for low-resistant mutant XN51FR-1,which had the same lesion area as XN51 on the watermelon leaves.The results indicated that the fitness of resistant mutants was decreased compared with that of wild-type strains.The cross-resistance assay indicated that fluopyram-resistant mutants were positive cross-resistant to all six SDHI fungicides in this test but were still sensitive to fluazinam and tebuconazole.So the resistance risk of D.bryoniae to fluopyram was moderate.In addition,we found that the SdhB gene of low-resistant mutant XN30FR-1 had three new point mutations at positions K258N,A259P,and H277N.Medium-resistant mutant XN52FR-1 showed a mutation at position H277N and other mutants did not have any point mutation.