USING ALLELE-SPECIFIC OLIGONUCLEOTIDE PROBES TO CHARACTERIZE BENZIMIDAZOLE RESISTANCE IN RHYNCHOSPORIUM-SECALIS

Benzimidazole fungicides are important mixture components in strategie s to combat fungicide resistance in Rhynchosporium secalis Davis. To m onitor the performance of these strategies, a rapid, accurate assay ha s been developed to detect point mutations in the beta-tubulin gene wh ich confers resistance of benzimidazoles. The beta-tubulin gene of a b enzimidazole-resistant strain of R. secalis has been cloned and sequen ced. Except for the difference in the position of one of its six intro ns, this gene showed a strong homology with other beta-tubulin genes f rom filamentous fungi. Resistance was related to a point mutation in c odon 198 which caused a glutamic acid to glycine change in resistant f ield strains, but glutamic acid to lysine in a laboratory mutant. A DN A fragment surrounding codon 198 was amplified directly from diseased lesions using a 'nested' set of PCR primers. Combining PCR amplificiat ion of a target DNA sequence with hybridization of Allele-Specific Oli gonucleotide probes (ASOs, 15-mers) allowed accurate detection of benz imidazole resistance. Only two probes, one sensitive and one resistant , were sufficient to monitor current field populations. Detection was achieved using either P-32-labelled probe, or non-radioactively using a biotin-labelled probe coupled to streptavidin/alkaline phosphatase. This rapid method using ASOs can detect benzimidazole resistance withi n 48 h compared with 6-8 weeks by conventional assay procedures.