title: Alterations in the predicted regulatory and coding regions of the sterol 14α-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae.
abstract: The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops are increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivity of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37:erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to subinhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting that PbCYP51 evolution is ongoing and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.
authors.affiliation: Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK.
authors.forename: Helen E
authors.initials: HE
authors.lastname: Carter
authors.forename: Bart A
authors.initials: BA
authors.lastname: Fraaije
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: West
authors.forename: Steven L
authors.initials: SL
authors.lastname: Kelly
authors.forename: Andreas
authors.initials: A
authors.lastname: Mehl
authors.forename: Michael W
authors.initials: MW
authors.lastname: Shaw
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Cools
mesh_terms: D001203:Ascomycota / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001393:Azoles / Q000494:pharmacology*; D025141:Drug Resistance, Fungal / Q000235:genetics / Q000502:physiology; D005659:Fungicides, Industrial / Q000494:pharmacology*
chemical_list: D001393:Azoles; D005659:Fungicides, Industrial
keywords: P450; demethylation inhibitors; fungicide resistance; light leaf spot; target site mutation