title: Detection ofsdhB Gene Mutations in SDHI-Resistant Isolates ofBotrytis cinereaUsing High Resolution Melting (HRM) Analysis.
abstract: Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen' resistance to SDHIs is associated with several mutations insdhgene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence ofsdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant's DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence ofsdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of severalsdhB mutations inB. cinereaand it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides.
authors.affiliation: Faculty of Agriculture, Forestry and Natural Environment, Laboratory of Plant Pathology, Aristotelian University of Thessaloniki Thessaloniki, Greece.
authors.forename: Anastasios
authors.initials: A
authors.lastname: Samaras
authors.affiliation: Institute of Applied Biosciences, Centre for Research and Technology Hellas Thessaloniki, Greece.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Madesis
authors.affiliation: Faculty of Agriculture, Forestry and Natural Environment, Laboratory of Plant Pathology, Aristotelian University of Thessaloniki Thessaloniki, Greece.
authors.forename: George S
authors.initials: GS
authors.lastname: Karaoglanidis
keywords: boscalid; fungicide resistance; gray mold; strawberry; succinate dehydrogenase gene