title: Overexpression of the sterol 14α-demethylase gene (MgCYP51) in Mycosphaerella graminicola isolates confers a novel azole fungicide sensitivity phenotype.
abstract: BACKGROUND
  The recent evolution towards resistance to azole fungicides in European populations of the wheat pathogen Mycosphaerella graminicola has been caused by the progressive accumulation of mutations in MgCYP51 gene, encoding the azole target sterol 14α-demethylase. Particular combinations of mutations have been shown specifically to affect the interaction of the MgCYP51 protein with different members of the azole class. Although additional mechanisms, including increased MgCYP51 expression and enhanced active efflux, have been proposed, the genetic changes underlying these mechanisms are unknown.
  
  
  RESULTS
  Analysis of the azole sensitivities of recent M. graminicola isolates identified a novel phenotype, seemingly independent of changes in MgCYP51 coding sequence. Characterised by a 7-16-fold reduction in in vitro sensitivity to all azoles tested and by growth on seedlings at higher doses of azoles in glasshouse tests compared with isolates carrying the same MgCYP51 variant (L50S, S188N, I381V, ΔY459/G460, N513K), isolates with this phenotype constitutively overexpress MgCYP51 by between 10- and 40-fold compared with the wild type. Analysis of sequences upstream of the predicted MgCYP51 translation start codon identified a novel 120 bp indel, considered to be an insertion, in isolates overexpressing MgCYP51.
  
  
  CONCLUSIONS
  The identification of an insertion in the predicted MgCYP51 promoter in azole-resistant isolates overexpressing MgCYP51 is the first report of a genetic mechanism, other than changes in target-site coding sequence, affecting sensitivity to multiple azoles in field isolates of M. graminicola. The identification of recent isolates overexpressing MgCYP51 confirms the ongoing evolution and diversification of resistance mechanisms in European populations of M. graminicola.
authors.affiliation: Department of Plant Pathology and Microbiology, Rothamsted Research, Harpenden, Hertfordshire, UK. hans.cools@bbsrc.ac.uk
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Cools
authors.forename: Carlos
authors.initials: C
authors.lastname: Bayon
authors.forename: Sarah
authors.initials: S
authors.lastname: Atkins
authors.forename: John A
authors.initials: JA
authors.lastname: Lucas
authors.forename: Bart A
authors.initials: BA
authors.lastname: Fraaije
mesh_terms: D001203:Ascomycota / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D001393:Azoles / Q000633:toxicity*; D001483:Base Sequence; D025141:Drug Resistance, Fungal / Q000235:genetics*; D005659:Fungicides, Industrial / Q000633:toxicity*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D058886:Sterol 14-Demethylase / Q000235:genetics*
chemical_list: D001393:Azoles; D005659:Fungicides, Industrial; D058886:Sterol 14-Demethylase