Molecular Characterization of Boscalid Resistance in Field Isolates of Botrytis cinerea from Apple

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to boscalid. Boscalid-resistant (Bos(R)) isolates were classified into four phenotypes based on the levels of the concentration that inhibited fungal growth by 50% relative to control. Of the 220 isolates tested, 42 were resistant to boscalid, with resistant phenotypes ranging from low to very high resistance. There was cross resistance between boscalid and carboxin. Analysis of partial sequences of the iron-sulfur subunit of succinate dehydrogenase gene in B. cinerea (BcSdhB) from 13 Bos(R) and 9 boscalid-sensitive (Bos(S)) isolates showed that point mutations in BcSdhB leading to amino acid substitutions at the codon position 272 from histidine to either tyrosine (H272Y) or arginine (H272R) were correlated with boscalid resistance. Allele-specific polymerase chain reaction (PCR) analysis of 66 Bos(R) isolates (including 24 additional isolates obtained from decayed apple fruit) showed that 19 carried the point mutation H272Y and 46 had the point mutation H272R, but 1 Bos(R) isolate gave no amplification product. Analysis of the BcSdhB sequence of this isolate revealed a different point mutation at codon 225, resulting in a substitution of proline (P) by phenylalanine (F) (P225F). The results indicated that H272R/Y in BcSdhB were the dominant genotypes of mutants in field Bos(R) isolates from apple. A multiplex allele-specific PCR assay was developed to detect point mutations H272R/Y in a single PCR amplification. Levels of boscalid resistance ranged from low to very high within isolates carrying either the H272R or H272Y mutation, indicating that, among Bos(R) isolates, different BosR phenotypes (levels of resistance) were not associated with particular types of point mutations (H272R versus H272Y) in BcSdhB. Analysis of genetic relationships between 39 Bos(R) and 56 Bos(S) isolates based on three microsatellite markers showed that 39 Bos(R) isolates and 30 Bos(S) isolates were clustered into two groups, and the third group consisted of only Bos(S) isolates, suggesting that the development of resistance to boscalid in B. cinerea likely is not totally random, and resistant populations may come from specific genetic groups.