title: Development of a two-step high-resolution melting (HRM) analysis for screening sequence variants associated with resistance to the QoIs, benzimidazoles and dicarboximides in airborne inoculum of Botrytis cinerea.
abstract: A rapid, high-resolution melting (HRM) analysis protocol was developed to detect sequence variations associated with resistance to the QoIs, benzimidazoles and dicarboximides in Botrytis cinerea airborne inoculum. HRM analysis was applied directly in fungal DNA collected from air samplers with selective medium. Three and five different genotypes were detected and classified according to their melting profiles in BenA and bos1 genes associated with resistance to benzimidazoles and dicarboximides, respectively. The sensitivity of the methodology was evident in the case of the QoIs, where genotypes varying either by a single nucleotide polymorphism or an additional 1205-bp intron were separated accurately with a single pair of primers. The developed two-step protocol was completed in 82 min and showed reduced variation in the melting curves' formation. HRM analysis rapidly detected the major mutations found in greenhouse strains providing accurate data for successfully controlling grey mould.
authors.affiliation: Department of Agriculture, Crop Production and Rural Environment, Laboratory of Plant Pathology, University of Thessaly, Volos, Greece.
authors.forename: Michael
authors.initials: M
authors.lastname: Chatzidimopoulos
authors.forename: Ioannis
authors.initials: I
authors.lastname: Ganopoulos
authors.forename: Evangelos
authors.initials: E
authors.lastname: Vellios
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Madesis
authors.forename: Athanasios
authors.initials: A
authors.lastname: Tsaftaris
authors.forename: Athanassios C
authors.initials: AC
authors.lastname: Pappas
mesh_terms: D000391:Air Microbiology; D000935:Antifungal Agents / Q000494:pharmacology*; D001562:Benzimidazoles / Q000494:pharmacology*; D020171:Botrytis / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D025141:Drug Resistance, Fungal; D005800:Genes, Fungal; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D007094:Imides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D016533:Mycological Typing Techniques; D011809:Quinones / Q000037:antagonists & inhibitors*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013997:Time Factors; D044366:Transition Temperature
chemical_list: D000935:Antifungal Agents; D001562:Benzimidazoles; D004271:DNA, Fungal; D007094:Imides; D011809:Quinones
keywords: Botrytis cinerea; HRM; QoIs; benzimidazoles; dicarboximides; melting curve