Identification of a novel point mutation in the <Emphasis Type="Italic">β</Emphasis>-tubulin gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay |
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| Authors: | Basil N Ziogas Dimitra Nikou Anastasios N Markoglou Anastasios A Malandrakis John Vontas |
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| Institution: | (1) Laboratory of Pesticide Science, Agricultural University of Athens, Votanikos, 118 55, Athens, Greece |
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| Abstract: | The molecular basis of resistance to benzimidazole fungicides with laboratory and field mutant isolates of Botrytis cinerea was investigated. After chemical mutagenesis with N-methyl-N-nitrosogouanidine (NMNG) two different benzimidazole-resistant phenotypes were isolated on media containing carbendazim or
a mixture of carbendazim and diethofencarb. The mutant isolates from the fungicide-mixture-containing medium were moderately
resistant to carbendazim with wild-type tolerance to diethofencarb while mutant isolates from carbendazim-containing medium
were highly resistant to carbendazim but sensitive to diethofencarb. The studied field isolates were highly resistant to benzimidazoles
and sensitive to diethofencarb. Study of fitness characteristics of benzimidazole highly-resistant isolates showed that the
resistance mutation(s) had no apparent effect on fitness-determining parameters. Contrary to this, the moderately benzimidazole-resistant
strains, with no increased diethofencarb sensitivity, had a significant reduction in certain ecological fitness-determining
characteristics. Analysis of the sequence of the β-tubulin gene revealed two amino acid replacements in the highly benzimidazole-resistant mutants compared to that of the wild-type
parent strain. One was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), identified in both laboratory
and field highly benzimidazole-resistant isolates, a mutation previously implicated in benzimidazole resistance. The second
was a novel benzimidazole resistance mutation of glutamic acid (GAG) to glycine (GGG) substitution at the same position 198
(E198G), identified in a highly benzimidazole-resistant laboratory mutant strain. Molecular analysis of the moderately benzimidazole-resistant
strains revealed no mutations at the β-tubulin gene. A novel diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole-sensitive (E198) but absent in both resistant genotypes (E198G and E198A)
was developed for the detection of both amino acid replacements at the β-tubulin gene. |
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| Keywords: | β -tubulin mutations Molecular diagnostic assay Diethofencarb sensitivity Monitoring resistance |
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