Molecular characterization of benzimidazole-resistant B. cinerea field isolates with reduced or enhanced sensitivity to zoxamide and diethofencarb

Sensitivity profiles of Botrytis cinerea field isolates to zoxamide and the molecular basis of the resistance mechanism involved in cross-resistance relationships between benzamides, benzimidazoles and N-phenylcarbamates were investigated. B. cinerea isolates collected from southern, central and northern Greece were characterized based on their sensitivity to zoxamide, the benzimidazole carbendazim and the N-phenylcarbamate diethofencarb. Isolates exhibiting baseline sensitivity to carbendazim and zoxamide but no sensitivity to diethofencarb were considered wild type (S phenotype) and accounted for 44% of the total strains sampled. Thirty-three percent of the isolates had increased sensitivity (HS phenotype) to zoxamide and diethofencarb and were highly resistant to carbendazim compared to S isolates. Eight percent of the sample was highly resistant (HR phenotype) to all anti-tubulin agents studied. The rest of the isolates were moderately resistant to zoxamide (MR phenotype) and equally sensitive to benzimidazoles and N-phenylcarbamates compared to isolates of the S phenotype. Fungitoxicity tests with botrycides belonging to other chemical classes revealed no cross-resistance relationships between zoxamide and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the hydroxyanilide fenhexamid, the anilinopyrimidine cyprodinil, the carboxamide boscalid and the strobilurin-type fungicide pyraclostrobin. Study of fitness characteristics did not show any significant difference between zoxamide resistant and sensitive isolates with respect to the parameters tested. PCR-RFLP analysis of a part of the β-tubulin gene sequence detected mutations in position 198 for both HS and HR zoxamide-sensitivity phenotypes. DNA sequence analysis of the B. cinerea β-tubulin gene revealed two previously described benzimidazole-resistance-conferring mutations. The first one was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), which was identified in all HS isolates. The second mutation (E198K) was a GAG-to-AAG substitution resulting in the replacement of glutamic acid with lysine present in all B. cinerea isolates highly resistant to all three anti-tubulin classes of fungicides. A number of mutations in other positions of the β-tubulin gene were detected in the moderately zoxamide-resistance phenotype.