We have developed a homologous transformation system for the wheat-pathogenic fungus Septoria nodorum based on a benomyl-(MBC-) resistant allele of the beta-tubulin gene. The beta-tubulin gene was isolated by heterologous hybridization from a cosmid library prepared from an MBC-resistant mutant. Cosmids carrying the gene conferred MBC resistance when introduced into a sensitive strain, demonstrating that resistance to MBC fungicides in S. nodorum may be determined by the beta-tubulin gene. This MBC resistant allele of the beta-tubulin gene (tubA(R)) was subcloned into pUC18 and used as a dominant selectable marker for transformation of wild-type sensitive strains. Transformants arose at frequencies of approximately 5 per mu-g of DNA, were integrative in nature and were mitotically stable. Some transformants showed a marked reduction in vigour, both in the presence and absence of MBC; this is thought to arise from overproduction of beta-tubulin. The S. nodorum tubA(R) gene also conferred MBC resistance on the related species Leptosphaeria maculans, a pathogen of Brassica, following its introduction by cotransformation. Probing digested S. nodorum DNA with tubA(R) at low stringency revealed only a single beta-tubulin gene. We anticipate that tubA(R) will prove a useful tool for the investigation of the pathogenicity of S. nodorum and other fungi.

The phytopathogenic fungus Septoria nodorum has been transformed using a plasmid (pAN7-1) containing the Escherichia coli hygromycin phosphotransferase gene (hph). Large, stable hygromycin-resistant transformant colonies appeared at frequencies between 2 and 25 per μg DNA when wheat-adapted and barley-adapted wild type strains were used as recipients. These transformants grew at hygromycin concentrations up to ten times that which inhibits the wild types. A second type of colony also developed on transformation plates. These appeared at higher frequencies, grew less vigorously and could not be subcultured in the presence of hygromycin. They are believed to be abortive transformants. Southern hybridization analyses indicated that transformation takes place via the integration of plasmid DNA into the fungal chromosomal DNA. Multiple integrations occur producing tandemly iterated arrays of plasmid molecules. Some transformants arose as heterokaryons. These could be resolved by propagation through a single spore and transformants purified in this way remained mitotically stable. All of 1,025 transformants tested were unchanged in pathogenicity. Reisolates from leaves retained their hygromycin-resistance, indicating that transformants remain stable during growth in plant tissue. Cotransformation of an unselected plasmid (p3SR2) carrying the Aspergillus nidulans amdS gene occurred at a high frequency.

Methods were established for growth, sporulation and storage of Septoria nodorum . Following mutagenesis with ultraviolet light or nitrosoguanidine, auxotrophic mutants were isolated in two parent strains by screening survivors on minimal and supplemented media. Pairs of phenotypically-different mutants of the same parent strain complemented each other to produce prototrophic mycelia when co-inoculated onto minimal medium. This procedure was used to assign phenotypically-identical mutations to genes. The most extensive of such complementation analyses involved six arginine-requiring mutants which were assigned to five genes. Growth tests on intermediates of arginine biosynthesis with the same mutants gave results consistent with the complementation analysis and revealed three different points of blockage in the arginine biosynthetic pathway. Spontaneous mutants resistant to potassium chlorate were isolated in five strains by plating pycnidiospores onto chlorate-containing medium. Five phenotypes were distinguished amongst these mutants by growth tests on various nitrogen sources; four of these phenotypes were unable to utilize nitrate. Complementation analysis of these nitrate non-utilizing mutants revealed that at least six different genes were involved. The phenotypes and genetic basis of these chlorate-resistant mutants resemble those in Aspergillus nidulans and Neurospora crassa , suggesting that nitrate assimilation in S. nodorum is similar to the well-characterized process in these other fungi. The mutants isolated are being used in investigations of the pathogenicity and genetics of S. nodorum at the molecular, cellular and population levels.

This paper is subtitled “A Cautionary Tale” because it illustrates the risks of contamination where successive cycles of selective propagation are involved. Selective techniques are central to microbial and molecular genetics and are likely to be important in the application of the methodology of these disciplines to the analysis of plant pathogenesis. Where selective procedures are being carried out on plants, control over contamination becomes a major practical consideration.

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