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Abstract

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—superfine. 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.

Item Type:	Thesis - PhD
Authors/Creators:	Cruickshank, Ralph Hayes
Keywords:	Fungi
Copyright Holders:	The Author
Copyright Information:	Copyright 1988 the Author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).
Additional Information:	Thesis (Ph.D.)--University of Tasmania, 1989. 8 journal articles in pocket at back of vol. Includes bibliographies
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