Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by browse.metadata.advisor "Cruywagen, C. W."

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    Interactions of the lignicolous fungus Coniochaeta pulveracea
    (Stellenbosch : Stellenbosch University, 2015-12) Van Heerden, Andrea; Botha, Alfred ; Van Zyl, Willem Heber ; Cruywagen, C. W.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Coniochaeta pulveracea is a microcyclic dimorphic lignicolous fungus that has mostly been isolated from the woody cauloplane. Since the majority of studies conducted on this fungus were taxonomic investigations that focused on macro- and microscopical characterization little is known about their eco-physiology. The latter include morphogenesis as a result of changing environmental conditions, as well as interactions with other fungi, such as yeasts, also occurring on the woody cauloplane. As such, the overall aim of this project was to study the role of Coniochaeta pulveracea’s physiology and morphogenesis in relation to its interactions on the woody cauloplane, including symbioses with yeasts originating from this environment. A representative of the microcyclic fungus C. pulveracea was isolated from a decaying Acacia tree and subsequently identified using morphology, as well as sequence analysis of the D1/D2 (600 – 650 bp) region of the large subunit ribosomal deoxyribonucleic acid gene complex. Plate assays for extracellular hydrolytic enzyme activity indicated that this C. pulveracea isolate, as well as representatives of its closest relatives including Coniochaeta boothii, Coniochaeta rhopalochaeta, and Coniochaeta subcorticalis, were able to produce cellulase and xylanase. All representatives were also able to degrade birchwood toothpicks during a 50-day incubation period at 30°C. To test the ability of these fungi and their enzymes to release simple sugars from complex cellulosic substrates, filtrates obtained from liquid cultures of Coniochaeta, cultivated on carboxymethyl cellulose (CMC) as sole carbon source, were analyzed using high-performance liquid chromatography analysis. Consequently, the presence of mono- and disaccharides such as glucose and cellobiose was confirmed in these culture filtrates. Two subsequent experiments were conducted to determine whether these simple sugars released from woody material by filamentous growth of Coniochaeta may enhance growth of yeasts previously isolated from wood degrading fungi or decaying wood. In the first experiment, representatives of Coniochaeta were co-cultured with selected yeasts suspended in agar plates containing birchwood toothpicks, followed by examination of plates for colony formation. Results indicated that Coniochaeta growth on the toothpicks enhanced growth of nearby yeast colonies in the agar plates. In the second experiment, representatives of selected yeasts and Coniochaeta species were co-cultured on CMC and xylan containing plates where after yeast colony formation was recorded on the plates. Saccharomyces cerevisiae strains, engineered to utilize specific wood degradation products, i.e., cellobiose or xylose, as sole carbon source were used as positive controls. While it was found that cellobiose released from CMC was assimilated by the yeasts, no evidence could be obtained that xylose released from xylan was used as carbon source by the yeasts. These ambiguous results could be ascribed to secretion of nutritious metabolic end products, other than the products of fungal xylanases. Since the above-mentioned interactions were studied whilst the Coniochaeta strains were mostly in their filamentous growth stage, microcycle conidiation of C. pulveracea strains and representatives of the closely related species, C. boothii and C. subcorticalis was also studied under different environmental conditions. The strains were found to exhibit hyphal growth on solid substrates and underwent a dimorphic switch to produce microcycle conidiation upon transfer to a liquid medium which differed in physico-chemical composition compared to the original solid medium. Factors that were found to contribute to this dimorphic switch were temperature, pH and the presence of complex nitrogen sources such as casamino acids and peptone in the medium. However, C. pulveracea showed intraspecific differences with regard to its response to changes in the physico-chemical environment. The interactions of microcyclic Coniochaeta strains with selected yeasts, such as representatives of Meyerozyma guilliermondii and Cryptococcus neoformans, were also studied in complex liquid media and it was found that, depending on medium composition, the microcyclic Coniochaeta exerted different effects on the different yeasts strains. In some co-cultures a positive effect on yeast growth was observed, while in other cases microcyclic Coniochaeta inhibited yeast growth.