Development of Procymidone and Difenoconazole Resistance in , the Causal Agent of Kiwifruit Brown Spot Disease.

Brown spot, caused by , is the most important leaf fungal disease threatening kiwifruit production in China, and it is typically controlled through the application of fungicides, such as procymidone and difenoconazole. To date, fungicide resistance development has not yet been systematically reported for the pathogen of kiwifruit. A total of 135 single-conidium isolates were collected from different cities in Zhejiang Province, China. developed prevailing resistance to procymidone and initial resistance to difenoconazole, with resistance frequencies of 60.7 and 13.3%, respectively. Positive cross-resistance was observed between procymidone and iprodione but not between procymidone and difenoconazole, tebuconazole, prochloraz, pydiflumetofen, pyraclostrobin, or thiophanate-methyl. Moreover, no cross-resistance was observed between difenoconazole and all other tested fungicides, including the two other demethylation inhibitors, tebuconazole and prochloraz. A fitness penalty was not detected in procymidone-resistant (Pro) or difenoconazole-resistant (Dif) isolates. However, double-resistant (Pro Dif) isolates had a fitness penalty, showing significantly decreased sporulation, germination, and pathogenicity. The P894L single point mutation, caused by the change from CCA to CTA at the 894th codon of , was detected in Pro isolates. Molecular dynamic simulation showed that the P894L mutation significantly decreased the inhibitory activity of procymidone against AaOs1 in . These results provide insight into the development and characteristics of fungicide resistance, offering guidance for the study and management of kiwifruit diseases.