In 2007, Kock and co-workers published the Acetylsalicylic acid (ASA) Antifungal Hypothesis that indicated a definite link between oxylipin production [3-hydroxy (OH) oxylipins], mitochondrial activity [mitochondrial transmembrane potential (ÎÏm)] and ASA sensitivity in respiring as well as non-respiring yeasts. Here an increase in mitochondrial activity was observed in the sexual phase where an increase in energy production is probably needed for multiple ascospore development. This hypothesis has since been expanded to also include various anti-mitochondrial drugs as well as the fungi and fungi-like organisms Aspergillus, Rhizopus and Mucor. In this study the yeast Nadsonia fulvescens as well as some species of the notorious plant pathogen Phytophthora was evaluated for their ability to fit the expanded hypothesis now called the Anti-mitochondrial Antifungal Hypothesis. The yeast N. fulvescens is characterized by a unique life cycle. After conjugation between the parent cell and the first bud, the zygote moves into a second bud formed at the opposite end of the parent cell. This second bud is then delimited by a septum and becomes the ascus according to literature. Usually one, rarely two spherical, brownish, spiny to warty ascospores are formed within the ascus giving rise to brown coloured colonies. In this study the parent cell with attached first bud showed increased mitochondrial activity when compared to the ascus. When anti-mitochondrial compounds were added, the mitochondrial activity was inhibited in the parent cell with attached first bud followed by the formation of less asci with ascospores (many not fully developed and white coloured). It is suggested that sufficient mitochondrial activity in the parent cell and first bud is necessary to produce enough energy for the formation of a proper ascus with brown coloured ascospore(s). If the parent cell and first bud is regarded as part of the yeast sexual phase, then N. fulvescens also fits the hypothesis. Further investigations were conducted to study the asci of the yeast N. fulvescens containing mature and fluconazole-treated malformed ascospores using nano scanning Auger microscopy (NanoSAM) in combination with transmission electron microscopy (TEM). This is the first application of NanoSAM to biological material. Transmission electron microscopy exposed a variety of malformed ascospores in asci treated with fluconazole. Here some ascospores produced degenerated spiky protuberances with relatively large inclusions carried inside wrinkled asci. Other ascospores contained no walls or protuberances and were enclosed within smooth spherical shaped asci. The majority of ascospores contained less dense hollow areas surrounded by cytoplasmic material. Nano scanning Auger microscopy studies on these asci corroborate the TEM results although less structural detail was obtained. Nano scanning Auger microscopy showed a decrease in elemental intensities during etching which assisted structural analysis of ascospore less dense hollow areas. In this study it is shown that the oomycete, Phytophthora nicotianae also fits the hypothesis. Fruiting structures (zoosporangia) of this oomycete showed increased beta (β)-oxidation when probing levels of 3-OH oxylipins with specific polyclonal antibodies. In addition increased mitochondrial activity was also observed in the zoosporangia when the ÎÏm probe, Rhodamine 123 was added to the culture. This indicates increased mitochondrial activity in the zoosporangia when compared to the hyphae. When the anti-mitochondrial ASA was added to cultures of this oomycete, the zoosporangia were, as expected most susceptible and were drastically inhibited in the presence of 1 mM of this compound. Similar ASA inhibition results were recorded for P. citrophthora. Anti-mitochondrial compounds may now find application in combating these devastating plant pathogens and urgent further research is needed in this direction.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-11112011-122247 |
| Date | 11 November 2011 |
| Creators | Swart, Chantel W |
| Contributors | Dr C-H Pohl-Albertyn, Prof PWJ van Wyk, Prof JLF Kock |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-11112011-122247/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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