IMPACT OF 0 TUBULIN ISOFORMS ON DIAGNOSIS OF BENZIMIDAZOLE RESISTANCE IN RHYNCOSPORIUM SECALIS
J BUTTERS and DW HOLLOMON IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, BS18 9AF, UK. Background Benzimidazole fungicides bind to 0-tubulin and affect microtubule function. Resistance to this group of fungicides in pathogen populations has been limited to mutations at codons 198 and 200 of the 0-tubulin (benA) gene, although amino acid substitutions conferring benzimidazole-resistance exist at another seven codon positions in laboratory induced mutants[l]. In monitoring studies of field populations of the barley pathogen Rhynchosporium secalis we have identified two mutations linked to benzimidazole resistance; at codon 198 the change of GAG to GGG substitutes glycine for glutamic acid, and at codon 200 the change of TTC to TAC substitutes tyrosine for phenylaianine. An aliele-specific DNA based diagnostic was designed to identify these mutations. Results and conclusions Experiments to study the effect of specific mutations on pathogenicity by transformation of a wild-type benzimidazole-sensitive strain with an altered (GGG198) gene produced two highly benzimidazole-resistant mutants. Results from southern blot hybridisation to the benA gene of R.secalis indicated not only was the resident wild-type gene deleted, but that the transforming BENA was also absent. This was confirmed by PCR using primers specific for the R.secalis benA gene. Instead, a second 0-tubulin isoform was present which supported normal growth and pathogenicity. Sequence information showed that this second 0-tubulin contained both GLU198 and PHE200 and should show wild-type (benA) binding to benzimidazole and diethiofencarb fungicides, however the mutants are resistant to both of these fungicides. These results show that benzimidazole-resistance can be caused through substitution of the normal wild-type 0-tubulin (benA) gene with a second isoform (tubB). This has a serious implication on the design of diagnostic kits for the detection of benzimidazole resistance in Rhynchosporium secalis as this tubB isoform does not impair pathogenicity. References 1. Davidse LC, ]shii H, 1995. In Modern Selective Fungicides, 2nd Edition. Ed. H. Lyr, Springer-Veriag, Jena, 305-322 2. Butters J, Kendall SJ., Wheeler 1, Hollomon DW, 1994. Proceedings of Fourth International Conference on Plant Diseases, pp.981-985.