High-resolution melting analysis for rapid detection and characterization of Botrytis cinerea phenotypes resistant to fenhexamid and boscalid

A novel, high-resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty-six single-spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high-resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B.cinerea populations.