title: Estimating frequencies of virulent isolates in field populations of a plant pathogenic fungus, Leptosphaeria maculans, using high-throughput pyrosequencing.
abstract: OBJECTIVE
  To develop a pyrosequencing assay to monitor the frequency of alleles of an avirulence gene, AvrLm4, in populations of sexual spores of Leptosphaeria maculans, a fungal pathogen of canola (Brassica napus).
  
  
  RESULTS
  The predominant mutation in AvrLm4 responsible for virulence to the corresponding resistance gene, Rlm4, is a single nucleotide polymorphism (SNP) at base 358. Pyrosequencing primers were designed to amplify a 90-bp region that included this SNP. The assay was developed and validated by analysing the frequency of AvrLm4 in isolate mixtures of different proportions. Furthermore, the frequency of avrLm4 (virulence allele) determined by pyrosequencing of populations of sexual spores was consistent with the frequency of avrLm4 determined by Sanger sequencing of the entire AvrLm4 gene from single isolates cultured from the same stubble.
  
  
  CONCLUSIONS
  This high-throughput assay can play an important role in predicting the risk of resistance breakdown in crops.
  
  
  CONCLUSIONS
  Similar assays can be applied to monitor frequencies of fungicide resistance in pathogens of crops and to assay diversity in microbial soil communities such as in soil samples from bat caves where white-nose syndrome has been detected.
authors: School of Botany, University of Melbourne, Melbourne, Vic., Australia. apvdw2@unimelb.edu.au
authors: A P
authors: AP
authors: Van de Wouw
authors: B J
authors: BJ
authors: Howlett
mesh_terms: D000483:Alleles; D001203:Ascomycota / Q000235:genetics* / Q000472:pathogenicity; D029688:Brassica napus / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005787:Gene Frequency; D059014:High-Throughput Nucleotide Sequencing; D009154:Mutation; D010935:Plant Diseases / Q000382:microbiology*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013172:Spores, Fungal / Q000235:genetics; D014774:Virulence / Q000235:genetics
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal