Detection and Molecular Characterization of Boscalid-Resistant Botrytis cinerea Isolates from Strawberry

Abstract

Botrytis cinerea isolates (n = 122) were collected from strawberry fields located in northern Greece during a 3-year period (2008-10) and tested for their sensitivity to the succinate dehydrogenase inhibitor boscalid. Sensitivity measurements showed three distinct phenotypes consisting of isolates highly sensitive (fungicide concentration causing inhibition of germ tube growth by 50% [EC₅₀ values] of 0.05 to 0.21 μg ml^-1), moderately resistant (EC₅₀ values of 1.37 to 7.79 μg ml^-1), or highly resistant (EC₅₀ values of >50 μg ml^-1) to boscalid. Sequence analysis of the sdhB gene revealed five mutations leading to amino acid substitutions in the SdhB subunit in isolates moderately resistant and highly resistant to boscalid. Three moderately resistant isolates showed a nucleotide change from A to T at codon 230, resulting in an asparagine to isoleucine (N230I) substitution. Several moderately resistant isolates showed a nucleotide change from C to T at codon 272, resulting in a substitution from histidine to arginine (H272R) whereas, in another set of isolates, a nucleotide change from A to G was found at the same codon, leading to a substitution from histidine to tyrosine (H272Y). One highly resistant isolate had a nucleotide change from A to T at codon 272, leading to a substitution from histidine to leucine (H272L), whereas, in three other highly resistant isolates, a double nucleotide change from CC to TT was observed at codon 225, resulting in a substitution from proline to phenylalanine (P225F). To facilitate rapid detection of these mutations associated with resistance to boscalid, a primer-introduced restriction analysis polymerase chain reaction was developed. The method was successfully applied to the moderately and highly resistant subpopulations and showed that the H272R mutation was predominant with relative frequencies of 28.5, 37.5, and 30% during 2008, 2009, and 2010, respectively. In contrast, the H272L mutation was detected at a frequency of 2.5% only in the 2009 population, whereas the P225F mutation was detected at a frequency of 7.5% only in the 2010 population.