Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid

A total of 618 isolates of corynespora leaf spot fungus (Corynespora cassiicola) collected from 24 commercial cucumber greenhouses in 12 cities in Ibaraki Prefecture, Japan, were tested for their sensitivity to boscalid. Boscalid-resistant isolates were detected in 17 out of 19 greenhouses with a history of use of this fungicide and detection frequencies of the resistant isolates exceeded 47% in nine greenhouses. Frequencies of very highly resistant (VHR) isolates with 50% effective concentration (EC50) values of boscalid exceeding 30 mu g mL(-1) were higher than those of moderately resistant (MR) isolates with EC50 ranging from 2.0 to 5.9 mu g mL(-1) in 11 greenhouses. Additionally, highly resistant (HR) isolates with EC50 from 8.9 to 10.7 mu g mL(-1) were first detected. Furthermore, molecular characterization of genes encoding succinate dehydrogenase (SDH) subunits (SdhA, SdhB, SdhC and SdhD) was carried out to elucidate the amino acid substitution responsible for the resistance to boscalid. All 23 VHR isolates had the same mutation from CAC to TAC in the SdhB gene leading to the substitution of histidine with tyrosine at amino acid position 278 (B-H278Y). At the same position, the substitution to arginine conferred by a mutation to CGC (B-H278R) was detected in all four HR isolates. Some MR isolates showed a substitution from serine to proline at position 73 in SdhC (C-S73P), from serine to proline or from glycine to valine at position 89 (D-S89P) and 109 (D-G109V), respectively, in SdhD. There was no common mutation in SDH genes of all MR isolates.