Benzimidazole fungicides are important mixture components in strategies to combat fungicide resistance in Rhynchosporium secalis Davis. To monitor the performance of these strategies, a rapid, accurate assay has been developed to detect point mutations in the β‐tubulin gene which confers resistance of benzimidazoles. The β‐tubulin gene of a benzimidazole‐resistant strain of R. secalis has been cloned and sequenced. Except for the difference in the position of one of its six introns, this gene showed a strong homology with other β‐tubulin genes from filamentous fungi. Resistance was related to a point mutation in codon 198 which caused a glutamic acid to glycine change in resistant field strains, but glutamic acid to lysine in a laboratory mutant. A DNA fragment surrounding codon 198 was amplified directly from diseased lesions using a ‘nested’ set of PCR primers. Combining PCR amplificiation of a target DNA sequence with hybridization of Allele‐Specific Oligonucleotide probes (ASOs, 15‐mers) allowed accurate detection of benzimidazole resistance. Only two probes, one sensitive and one resistant, were sufficient to monitor current field populations. Detection was achieved using either 32P‐labelled probe, or non‐radioactively using a biotin‐labelled probe coupled to streptavidin/alkaline phosphatase. This rapid method using ASOS can detect benzimidazole resistance within 48 h compared with 6–8 weeks by conventional assay procedures.
Benzimidazole‐resistant mutants of Rhynchosporium secalis were easily generated in the laboratory using UV mutagenesis. Three levels of resistance were identified (low, LR; moderate, MR; high, HR), but there was no negative cross‐resistance with N‐phenylcarbamate fungicides. In all cases pathogenicity was reduced, in some cases drastically. Benzimidazole‐resistant field strains were first detected in 1990, some 15 years after this fungicide group was first used in UK barley crops. Unlike laboratory mutants, only HR phenotypes were isolated from the field, and all showed negative cross‐resistance to N‐phenylcarbamates. These field resistant strains were no less pathogenic than wild‐type ones. Carbendazim binding to tubulin‐like protein from HR phenotypes, whether generated in the laboratory or isolated from the field, was reduced.
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