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Aim: 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)., Methods and 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., Conclusion: This high-throughput assay can play an important role in predicting the risk of resistance breakdown in crops., Significance and Impact of the Study: 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., (© 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

The ascomycete Sclerotinia sclerotiorum is a plant pathogen with a very broad host range. In order to identify and characterize genes involved in S. sclerotiorum infection of Brassica napus (canola), expressed sequence tags (ESTs) were examined from libraries prepared from three tissues: complex appressorium (infection cushions), mycelia grown on agar and lesions formed on leaves of B. napus. A high proportion of genes (68%) had not been previously reported for S. sclerotiorum in public gene or EST databases. The types of novel genes identified in the infection cushion library highlights the functional specificity of these structures and similarities to appressoria in other fungal pathogens. Quantitative real-time PCR was used to analyse tissue specificity and timing of transcription of genes with best matches to MAS3 (appressoria-associated protein from Magnaporthe grisea), cellobiohydrolase I, oxaloacetate acetylhydrolase, metallothionein, pisatin demethylase, and an unknown gene with orthologs in fungal pathogens but not in saprophytic fungi.

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic animal pathogen Aspergillus fumigatus. It is a member of the epipolythiodioxopiperazine (ETP) class of toxins characterised by a disulphide bridged cyclic dipeptide. A putative cluster of 12 genes involved in gliotoxin biosynthesis has been identified in A. fumigatus by a comparative genomics approach based on homology to genes from the sirodesmin (another ETP) biosynthetic gene cluster of Leptosphaeria maculans. The physical limits of the cluster in A. fumigatus have been defined by bioinformatics and by identifying the genes that are co-regulated and whose timing of expression correlates with the production of gliotoxin in culture.

Two genes whose derived amino acid sequences closely resemble the ergosterol biosynthetic enzymes, sterol Delta(5,6)-desaturase (erg3) and sterol 14alpha-demethylase (erg11), were cloned from the plant pathogenic fungus Leptosphaeria maculans. Transcript levels of both these genes increased following exposure of L. maculans to the triazole fungicide, flutriafol, which specifically inhibits the ergosterol biosynthetic pathway. This induction may be due to a decrease in ergosterol content or to abnormal levels of the ergosterol precursor, 24-methylene dihydrolanosterol.