Biological and molecular characterization of laboratory mutants of Cercospora beticola resistant to Qo inhibitors

The resistance to strobilurin-related fungicides and its molecular basis in laboratory mutant isolates of Cercospora beticola was investigated. After ultraviolet mutagenesis, mutants with high, moderate or low resistance levels to pyraclostrobin were isolated from a wild-type strain of C. beticola. Fungitoxicity tests on the response of resistant isolates on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration, indicated that the biochemical mechanism of alternative oxidase was not responsible for the reduced sensitivity to pyraclostrobin for half of the mutants. Cross-resistance studies with other inhibitors of the cytochrome bc ₁ complex of the mitochondrial respiratory chain showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other Qo inhibitors such as azoxystrobin and fenamidone, but not to the Qi inhibitor cyazofamid. No effect of pyraclostrobin-resistant mutation(s) on fungitoxicity of the carboxamide boscalid, the triazoles epoxiconazole and flutriafol and to the benzimidazole benomyl, which affect other cellular pathways or other steps of the respiratory chain, was observed. Study of fitness parameters showed that most mutants had a significant reduction in sporulation and pathogenicity compared to the wild-type parental isolate. However, experiments on the stability of the resistant phenotype did not show a significant reduction of the resistance for half of the mutants when grown for at least four generations on pyraclostrobin-free medium. Molecular analysis of cytochrome b cDNA, isolated from the wild-type and the pyraclostrobin-resistant mutant isolates, revealed two novel amino acid replacements at positions involved in Qo resistance in other species. The glycine (GGT) to serine (AGT) replacement at position 143 (G143S) was found in the isolate with the highly resistant phenotype. The second amino acid change was the replacement of phenylalanine (TTC) by valine (GTC) at position 129 (F129V), which was found in a mutant strain with the moderately resistant phenotype. Four additional mutations located in conserved regions of the mitochondrial cytochrome b gene (I154L, N250D, E256G and V261D) were detected in some mutant isolates of C. beticola but their possible role in Qo-resistance needs further investigation. This is the first study reporting C. beticola strains resistant to Qo inhibitor fungicides due to the biochemical mechanism of target-site modification, resulting from amino acid changes in the mitochondrial cytochrome b␣gene.