MUTATIONS LEADING TO SUBSTITUTIONS AT AMINO-ACIDS 198 AND 200 OF BETA-TUBULIN THAT CORRELATE WITH BENOMYL-RESISTANCE PHENOTYPES OF FIELD STRAINS OF BOTRYTIS-CINEREA

Three phenotypes were identified among benomyl-resistant strains of Botrytis cinerea in Israel when tested for sensitivity to carbendazim (M BC) and diethofencarb (NPC): Ben(HR)NPC(S) = highly resistant to MBC (50% effective concentration [EC(50)] > 50 mu g/ml) and sensitive to 0.5 mu g/ml NPC; Ben(MR)NPC(R) = moderately resistant to MBC (10 less than or equal to EC(50) < 20 mu g/ml) and resistant to 10 mu g/ml NPC; and Ben(HR)NPC(R) = highly resistant to MBC and resistant to NPC. A 1-kb fragment of the wild-type gene encoding for beta-tubulin (designated benA) in B. cinerea was cloned and sequenced. The deduced partial amino acid sequence of the B. cinerea beta-tubulin showed a high degree of similarity to beta-tubulins of other filamentous fungi. A polymerase chain reaction approach was used to amplify and sequence 992-bp benA fragments from strains representing the three phenotypes. in the eight Ben(R) strains analyzed, three single base-pair mutations were identified and found to correlate with the different phenotypes: codon 198, encoding glutamic acid in the wild type, was changed to an alanine codon in the Ben(HR)NPC(S) phenotype or to a lysine codon in the Ben(HR)NPC(R) phenotype; codon 200, encoding phenylalanine, was changed to a tyrosine codon in the Ben(MR)NPC(R) phenotype. These mutations were similar to those identified in benomyl-resistant field strains of other phytopathogenic fungi.