Zymograms as criteria in the taxonomy of fungi

A sensitive and efficient electrophoretic technique for the examination of fungal pectic enzymes was developed. This involved electrophoresis of culture fluids in polyacrylamide slab gels containing pectin. Gels were soaked in malic acid solution which gradually reduced the gel pH from 8.7 to 3.0 and allowed the enzymes to act on the incorporated pectin when the pH was suitable. Ruthenium red treatment stained the unaltered pectin revealing unstained zones due to polygalacturonase activity and zones with intensified staining where pectinesterases had converted the pectin to pectic acid. Results were recorded as photograms. Other enzyme systems were investigated. Zymogram clarity was improved by reduction of gel thickness from 3.5 to 2.0 mm and sample wells from 15 to 4.5 ul. Curvature of buffer fronts was eliminated by efficient gel cooling. Tailing was reduced and isozyme band clarity was improved by loading samples as a slurry with Sephadex G150‚ÄövÑvÆsuperfine. Thorough gel polymerization before gel moulds were opened was found to be essential for migration characteristics to be uniform through the depth of the gels. The technique was applied to the enzymes of Botrytis and Penicillium species and the Rhizoctonia complex. The influences of culture medium composition were examined for each genus and media suitable for pectic enzyme production were developed. Potent yields of polygalacturonase were obtained from Botrytis species in the absence of pectin. Zymograms from these differed from those produced from pectin media and both were used to advantage. Distinctive and characteristic zymograms were obtained from Botrytis species and provided an accurate means for their identification, and indications of synonymy. An extensive study of B. cinerea detected nine pectic enzyme genotypes. One of these was principally associated with Vitis and may constitute B. fuckeliana. The use of zymograms as taxonomic criteria in Penicillium provided a means for the accurate identification of species in this notoriously difficult genus. The extensive synonymy in subgenus Penicillium was examined by zymograms and 80 taxa were assigned to 21 species, with significant changes in assignments to P. aurantiogriseum and P. commune. The latter was shown to be the previously unknown ancestor of the domesticated P. camembertii. Zymogram studies revealed the presence of five zymogram groups in Thanatephorus, five in Ceratobasidium and one in Waitea, from Rhizoctonia isolates provided from the grain belt of Western Australia. A similar result was obtained from isolates provided from South Australia and a link was shown between grouping of isolates by anastomosis behaviour and by zymograms in the case of four anastomosis groups available from that State. Other anastomosis groups and species in the Ceratobasidiaceae were examined and the correlations found between zymogram patterns and anastomosis groups and subgroups gave promise of aiding the practical recognition of species in the complex.