Using allele-specific oligonucleotide probes to characterize benzimidazole resistance in Rhynchosporium secalis

A - Papers appearing in refereed journals

Wheeler, J. E., Kendall, S. J., Butters, J., Hollomon, D. W. and Hall, L. 1995. Using allele-specific oligonucleotide probes to characterize benzimidazole resistance in Rhynchosporium secalis. Pesticide Science. 43 (3), pp. 201-209.

AuthorsWheeler, J. E., Kendall, S. J., Butters, J., Hollomon, D. W. and Hall, L.
Abstract

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 beta-tubulin gene which confers resistance of benzimidazoles. The beta-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 beta-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 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 within 48 h compared with 6-8 weeks by conventional assay procedures.

KeywordsAgronomy; Entomology
Year of Publication1995
JournalPesticide Science
Journal citation43 (3), pp. 201-209
Open accessPublished as non-open access
ISSN0031613X
PublisherWiley
Copyright licensePublisher copyright

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