title: Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates.
abstract: BACKGROUND
  Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies.
  
  
  RESULTS
  P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes.
  
  
  CONCLUSIONS
  The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.
authors.affiliation: Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, Hertfordshire, UK.
authors.forename: Helen E
authors.initials: HE
authors.lastname: Carter
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Cools
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: West
authors.forename: Michael W
authors.initials: MW
authors.lastname: Shaw
authors.forename: Bart A
authors.initials: BA
authors.lastname: Fraaije
mesh_terms: D001203:Ascomycota / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001562:Benzimidazoles / Q000494:pharmacology*; D029741:Brassica rapa / Q000382:microbiology*; D002219:Carbamates / Q000494:pharmacology*; D026141:Drug Resistance, Multiple, Fungal; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005659:Fungicides, Industrial / Q000494:pharmacology*; D009154:Mutation; D048448:Phenylcarbamates / Q000494:pharmacology; D010935:Plant Diseases / Q000382:microbiology*; D014404:Tubulin / Q000737:chemistry / Q000235:genetics*
chemical_list: D001562:Benzimidazoles; D002219:Carbamates; D005656:Fungal Proteins; D005659:Fungicides, Industrial; D048448:Phenylcarbamates; D014404:Tubulin; C070961:diethofencarb; C036868:methyl carbamate; C031000:benzimidazole
keywords: fungicide resistance; light leaf spot; oilseed rape; pyrosequencing; β-tubulin