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A recent comparative proteomics study identified the short-chain dehydrogenase (Sch1) as being required for asexual sporulation (Tan et al. Eukaryotic Cell 7:1916-1929, 2008). Metabolite profiling was undertaken on the mutant strains of Stagonospora nodorum lacking the Sch1 gene to help elucidate its role. Gas chromatography-mass spectrometry of the polar metabolites in the Sch1 mutants identified a secondary metabolite at a 200-fold greater concentration than observed in the wild-type strains. Comparative analysis of the secondary metabolite and the mycotoxin alternariol using ESI-MS/MS confirmed the identity of the compound as alternariol. This is the first report to confirm the presence of a mycotoxin in S. nodorum and compelling the field to consider the health implication of this disease.

The interaction between Stagonospora nodorum and a susceptible wheat cultivar was investigated using a range of microscopic techniques. Germination of pycnidiospores occurred approximately 3 h after making contact with the leaf surface and was followed by attempted penetration 8-12 h later. Penetration was observed through stomata and also directly through periclinal and anticlinal epidermal cell walls. Penetration down the anticlinal cell walls appeared to occur without a differentiated penetrating structure whilst structures identified as either lateral appressoria or hyphopodia were typically present when penetrating over a periclinal cell wall. Once inside the leaf, the fungus continued to grow for the next 4-5 days colonising all parts of the leaf except the vascular bundles. Only in the later phase of the infection was total host cell collapse apparent. Evidence of polyphenolic compounds was observed. The infection cycle was completed within 7 days as indicated by sporulation on the leaf surface. These results have allowed us to understand how the fungus physically interacts with the leaf and will help the overall understanding of the infection process.

A gene encoding a mitogen-activated protein kinase (MAPK) putatively orthologous to Pmk1 from Magnaporthe grisea was cloned and characterised from the wheat glume blotch pathogen Stagonospora nodorum. Protein sequence alignments showed the cloned gene, Mak2, is closely related to homologues from other dothideomycete fungi. Expression studies revealed Mak2 is up-regulated during in vitro growth upon nitrogen starvation but is not sensitive to carbon starvation or osmotic stress. Transcript analysis in planta showed Mak2 to be expressed throughout infection and up-regulated during the sporulation phase of the infection cycle. Fungal strains harbouring a disrupted Mak2 gene were created by homologous gene recombination. The mutant strains had a severely altered phenotype in vitro with reduced growth rate and failure to sporulate. Further phenotypic analysis revealed that the mutants had near-normal levels of secreted protease activity, were not hypersensitive to osmotic stress and appeared to have melanin synthesis intact. The mak2 strains were essentially non-pathogenic to wheat leaves. No penetration structures formed and although entry was observed through stomates, the infection rarely continued. The results within this study are discussed within the context of the differences in downstream regulation of the Mak2 MAPK pathway and the cAMP signal transduction pathway in S. nodorum; and differences are compared to mak2 mutant strains in other pathogenic fungi.

Phaeosphaeria (anamorph Stagonospora) nodorum is the most serious fungal pathogen of wheat in the West Australian (WA) wheat belt and is a diallelic heterothallic loculoascomycete. Its population genetics has received considerable attention. A recent study, which sampled isolates from diverse locations worldwide, has indicated that the mating-type idiomorph MAT1-1 is considerably more frequent than MAT1-2 in many populations. To investigate this, we developed PCR primers that amplify each idiomorph. In a sample of 23 isolates cultured from ascospores collected in the field, nine amplified DNA with the MAT1-1 primers and 14 amplified DNA with the MAT1-2 primers. The virulence of a MAT1-2 isolate was comparable with MAT1-1 isolates. Although these sample sizes are small, we suggest that this result is consistent with the presence of equal numbers of both mating types in populations of ascospores in WA.

During an expressed sequence tag sequencing project, a gene encoding a methylglyoxal lyase (glyoxalase I) was identified, cloned and characterised from the necrotrophic wheat pathogen Stagonospora nodorum. Sequence analysis identified the gene, named Gox1, as having reasonable identity to GLO1 from yeast and hypothetical proteins identified in fungal genome sequencing projects. Expression analysis in vitro revealed Gox1 to be up-regulated in the presence of methylglyoxal and salt but not affected by starvation conditions. Analysis of Gox1 transcription in planta showed its highest expression was in ungerminated spores and during sporulation, suggesting a role for the glycolytic bypass pathway in sporulation. The gene was inactivated by homologous recombination, resulting in a S. nodorum strain with no detectable glyoxalase I activity. The gox1 mutants exhibited no discernable phenotype, with the exception of being more sensitive to the presence of methylglyoxal. Infection assays demonstrated the mutants retained full pathogenicity and sporulation was unaffected. This is the first report describing the characterisation of a glyoxalase I from a pathogen of any description. The gene has been sequenced, functionally characterised and shown not to be required for the infection of wheat by S. nodorum.