Detection and characterization of difenoconazole resistance in Stagonosporopsis citrulli from watermelon and muskmelon in Zhejiang Province of China

The watermelon and muskmelon productions are important agriculture pillar industries of Zhejiang Province in China. Difenoconazole is an imidazole-class sterol, 14-demethylase inhibitor (DMI), which has been in use for several years to control gummy stem blight (GSB) caused by Stagonosporopsis species. However, the detection and characterization of difenoconazole resistance in GSB have not been clarified. In this study, a total of 112 isolates were collected from samples of GSB on watermelon and muskmelon in five locations of Zhejiang Province, China. All of the isolates were identified as Stagonosporopsis citrulli through morphology and multiplex PCR analysis. The determination of their resistance to difenoconazole via the discriminatory dosage method showed that the total resistance frequency was 89.3%. Among the resistant sub-population, 36.6% had high-level resistance to difenoconazole (Dif HR ), while 46.4% and 6.3% had low- (Dif LR ) and moderate-level resistance (Dif MR ), respectively. Additionally, the difenoconazole showed a positive cross-resistance with four DMIs, i.e., tebuconazole, prochloraz, metconazole, and mefentrifluconazole, but not hexaconazole. The phenotypic analysis found that the difenoconazole resistant (Dif R ) isolates demonstrated attenuated ability in both the mycelial growth and sporulation compared with the difenoconazole sensitive (Dif S ) isolates, while there was no significant difference in pathogenicity on watermelon leaves between the Dif R and Dif S isolates. Further exploration of the mechanism related to difenoconazole resistance of S. citrulli isolates revealed that the resistance to difenoconazole involved four types of mutations in CYP51, i.e., G463S for Dif LR , I444M, Y446H, and A464G for Dif HR . No over-expression of the cyp51 gene was found in the tested Dif R isolates. Furthermore, it was found that 5% of the Dif R isolates were significantly more sensitive to difenoconazole after being treated with 20 μg/mL chlorpromazine hydrochloride, indicating that the efflux mechanism may be involved in these difenoconazole-resistant isolates. Together, our study results suggested that S. citrulli had a strong resistance to difenoconazole on watermelon and muskmelon, and the mutations in cyp51 and changes in fungicide efflux were responsible for the emergence of difenoconazole resistance in S. citrulli .