With the further application of succinate dehydrogenase inhibitors (SDHI), the resistance caused by double mutations in target gene is gradually becoming a serious problem, leading to a decrease of control efficacy. It is important to assess the sensitivity and fitness of double mutations to SDHI in Corynespora cassiicola and analysis the evolution of double mutations. We confirmed, by site-directed mutagenesis, that all double mutations (B-I280V+D-D95E/D-G109V/D-H105R, B-H278R+D-D95E/D-G109V, B-H278Y+D-D95E/D-G109V) conferred resistance to all SDHI and exhibited the increased resistance to at least one fungicide than single point mutation. Analyses of fitness showed that all double mutations had lower fitness than the wild type; most of double mutations suffered more fitness penalties than the corresponding single mutants. We also further found that double mutations (B-I280V+D-D95E/D-G109V/D-H105R) containing low SDHI-resistant single point mutation (B-I280V) exhibited higher resistance to SDHI and low fitness penalty than double mutations (B-H278Y+D-D95E/D-G109V) containing high SDHI-resistant single mutations (B-H278Y). Therefore, we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI. Taken together, our results provide some important reference for resistance management.

Corynespora leaf spot caused by Corynespora cassiicola is an important foliar disease in cucumber. Succinate dehydrogenase inhibitors were mainly fungicides to control this disease. In recent years, with the application of succinate dehydrogenase inhibitors (SDHIs) in field, boscalid-resistant isolates were continuously detected in field. Resistance monitoring programs were performed to investigate the frequency and genotypes of resistant isolates. In our resistance monitoring, the frequency of resistant isolates rapidly increases from 9.68% to 85.88% in 2005-2020. Nine genotypes conferring SDHIs resistance were found in resistant isolates, with different levels of resistance to SDHIs: B-H278R/L/Y, B-I280V, C-N75S, C-S73P, D-D95E, D-H105R and D-G109V. The first sdh mutation was detected in Hebei Province in China, conferring to an amino acid substitution at codon 278 in sdhB subunit from histidine to tyrosine (B-H278Y), and it was the dominant resistance genotype in 2014-2015. Subsequently, other genotypes were gradually detected in field, and the dominant mutations varied from year to year and between different regions. The newest genotypes (B-H278L) conferring SDHIs resistance was found in 2020. To our best knowledge, it is the first reported in C. cassiicola. To date, multiple resistance to SDHIs, quinone outside inhibitors (QoIs), benzimidazole fungicides (MBCs) and dicarboximide fungicides (DCFs) were detected, accounting for 75.64% in SDHIs-resistant isolates. Therefore, the above four fungicides must be restricted strictly, and further monitoring work in other provinces with more isolates should be performed in the future.