Mutations of the β-tubulin gene associated with different phenotypes of benzimidazole resistance in the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis

Seven phenotypes were identified among field isolates of Tapesia yallundae and Tapesia acuformis when tested for susceptibility to the benzimidazole fungicides carbendazim and thiabendazole and the N-phenylcarbamates diethofencarb, MDPC, and swep. PCR was used to amplify and sequence 627-bp fragments of the beta-tubulin gene from 32 Tapesia spp. strains representing the seven field phenotypes and from six T yallundae laboratory mutants. All benzimidazole-resistant field isolates analyzed had a punctual allelic mutation at codon 198, 200, or 240 of the beta-tubulin gene fragment. Codon 198, which encodes glutamic acid in benzimidazole-sensitive strains (resistant to N-phenylcarbamates), was converted to a codon for alanine, glycine, lysine, or glutamine in benzimidazole-resistant strains exhibiting increased sensitivity toward the N-chlorophenylcarbamates MDPC and swep; the first two allelic mutations (alanine and glycine) also conferred susceptibility to diethofencarb. In T:yallundae, benzimidazole-resistant phenotypes, which remained resistant to all the tested N-phenylcarbamates, had a tyrosine instead of a phenylalanine at codon 200 or a phenylalanine instead of a leucine at codon 240. In T. acuformis, however, the change of a phenylalanine at codon 200 for a tyrosine conferred a weaker susceptibility to MDPC and swep as well as a reduced resistance to benzimidazoles compared to their I: yallundae counterparts. The same molecular analysis was performed with T yallundae laboratory mutants obtained after UV mutagenesis and selection on carbendazim or diethofencarb of a former benzimidazole-sensitive or benzimidazole-resistant field strain. We found in two mutants a punctual change at codon 198, replacing the glutamic acid by a glycine or an aspartic acid, but multiple mutations were observed in the four other mutant strains: a double mutation in codon 198 resulting in the substitution of the glutamic acid by a threonine; a mutation at codon 198 (an alanine instead of a glutamic acid) and a mutation at codon 200 (a serine instead of a phenylalanine); a mutation at codon 198 (an alanine instead of a glutamic acid) and a mutation at codon 250 (a phenylalanine instead of a leucine); and one mutant had four codon changes: at codon 179 (a glycine substituting a valine), at codon 185 (a serine replacing an alanine), at codon 190 (an asparagine replacing a histidine), and at codon 198 (an alanine instead of a glutamic acid). We show here that each different phenotype could be correlated with particular mutations at the beta-tubulin gene level. (C) 1999 Academic Press.