THE USE OF ALLELE-SPECIFIC OLIGONUCLEOTIDE PROBES TO CHARACTERIZE RESISTANCE TO BENOMYL IN FIELD STRAINS OF VENTURIA-INAEQUALIS

A procedure was developed for detecting point mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalis, using the polymerase chain reaction (PCR) in combination with allele-specific oligonucleotide (ASO) analysis. PCR was used to amplify a specific 1,191-bp DNA sequence of the beta-tubulin gene in DNA extracts from axenically grown mycelium or individual apple scab lesions. The amplified DNA sequence was then probed with 18-mer end-labeled oligonucleotides specific for the sensitive phenotype or for three benomyl-resistant phenotypes in strains of V. inaequalis. The point mutations, converting codon 198 from glutamic acid in the sensitive strain to lysine or alanine, were detected by ASO analysis in highly resistant and very highly resistant strains, respectively. A point mutation, converting codon 200 for phenylalanine in sensitive strains to tyrosine, was detected by ASO analysis in all medium resistant strains but one from Chile, isolate CHILE 24B. Sequence analysis revealed that an alternate codon for tyrosine was present in the Chilean isolate. Therefore, the mutation in isolate CHILE 24B was not detected by any of the four ASO probes used in this study. ASO analysis was a useful tool for detecting and characterizing benomyl-resistant strains of V. inaequalis and could be expanded to other plant pathogenic fungi.