Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 91-95).

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Under storage conditions of ambient temperature and relative humidity in South Africa, seed-associated mycoflora proliferates. Fusarium moniliforme is ubiquitous in newly-harvested maize, persisting for variable periods in storage, while Aspergillus flavus may represent the final group of species in the succession of aspergilli after grain storage under high temperature and/or high humidity. Many strains of these fungi produce toxigenic secondary metabolites (mycotoxins) under local storage conditions. Since pathogenic fungi may be present within the tissues of stored seeds, these contaminants will not be eradicated by external fungicide treatment, therefore a possible alternative is biological control. The aim of the present investigation was to ascertain whether certain strains and/or species of Trichoderma have potential as biocontrol agents against the seed-associated pathogenic fungi, Aspergillus flavus and Fusarium moniliforme. A study of the fungal growth in dual cultures revealed that from nine isolates of Trichoderma spp. (T harzianum and T viride), four had a noticeable inhibitory effect on the growth of the pathogenic fungi. Scanning electron microscopical investigation of fungal interaction demonstrated no obvious hyphal penetration by - Trichoderma spp. In addition, significant alteration of Fusarium hyphae, with pronounced collapse and loss of turgor, and production of aberrant conidial heads and microheads by A. flavus were observed. Evidence derived from some biochemical studies revealed that antibiosis (by production of extracellular enzymes, volatile compounds and possible antibiotics) is probably the mechanism involved in the antagonistic effect of the four aggressive Trichoderma spp. The in vitro studies demonstrated that the use of Trichoderma spp. as biocontrol agents against A. flavus and F. moniliforme appears promising. / Thesis (M.Sc.)-University of Natal, 1995.

The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are well known for their ability to produce a diverse array of antimicrobial compounds. Ofparticular interest is the ability of certain strains to produce antifungal compounds. Such organisms have the potential for application in agriculture where they can be used as biocontrol agents against selected plant pathogenic fungi. A study was undertaken to further characterize selected Bacillus isolates that exhibit broad spectrum antifungal activity. Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying degrees. Two isolates, R29 and B81, were selected for further testing and characterization. Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media composition showed significant influences on the antifungal activities of the isolates tested, but that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found to give rise to the largest inhibition zones. Both isolates were tentatively identified using standard biochemical and morphological tests. Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for homology testing against the GeneBank database. Homology testing confirmed that both isolates were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment used for sequencing proved to be too small to give conclusive identification of the isolates. Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve studies using a defined synthetic medium showed that antifungal activity arose during the stationary phase and appeared to be closely linked to sporulation. The antifungal component of cell free culture supematant was extracted using various methods including thin layer chromatography, acid precipitation, hydrophobic interaction chromatography and methanol extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating that at least two antifungal compounds were produced. Methanol extracted samples produced the cleanest sample extract but only revealed one active peak from the HPLC fraction . Nuclear magnetic resonance analysis of purified samples indicated that the antifungal compound/s have aromatic complex and peptide structures. The extracted antifungal compounds were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and, were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar properties to known antifungallipopeptides i.e. iturin A and fengycin A and B. Further characterization and identification of the active compounds is recommended usmg methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption ionisation time-of- flight. The results presented in this dissertation provide a basis from which antifungal compounds produced by strains ofBacillus can be further characterized. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Thesis (Ph. D.)--University of Adelaide, Dept. of Plant Science, 1996? / Includes bibliographical references (leaves 307-329).

M.Sc. / Polygalacturonase-inhibiting proteins (PGIPs) are cell wall-associated plant proteins that inhibit endopolygalacturonases from phytopathogenic fungi. It has been proposed that pgip encoding genes could be utilised for engineering increased resistance in transgenic crops against important fungal pathogens such as Botrytis cinerea. During this study a pgip gene from Malus domestica cv Granny Smith apple fruit was cloned by the degenerate and inverse polymerase chain reaction (PCR) techniques. An alignment of the pear and bean PGIP sequences was used to design degenerate PCR primers in highly conserved regions. Degenerate PCR allowed the amplification of a 351bp internal fragment of the pgip gene, termed ipgip. The DNA sequence of ipgip was used to design inverse PCR primers. A Southern blot of apple genomic DNA probed with the ipgip fragment was used to identify restriction enzyme sites for inverse PCR. Inverse PCR enabled cloning of the remainder of the gene, from which a composite pgip gene sequence was constructed. The composite apple pgip gene comprised an open reading frame of 990bp that is predicted to encode a 330 amino acid polypeptide. The polypeptide contains a putative 24 amino acid N-terminal leader sequence that may function as a signal peptide for secretion. The deduced apple PGIP contains nine cysteine residues and seven potential N-linked glycosylation sites. Ten loosely conserved leucine-rich repeat motifs characteristic of PG1Ps were identified in the apple PGIP sequence. The apple PGIP showed 97% and 55% amino acid identity to the pear and bean PGIPs, respectively. The full-length apple pgip gene was re-isolated from genomic DNA by PCR using primers designed to the 5' and 3' ends of the composite pgip gene. The apple pgip gene was cloned into a plant transformation vector and transformed into tobacco by Agrobacterium-mediated transformation. Phenotypically normal transgenic tobacco plants were produced. Stable transgene insertion into the transgenic tobacco genomes was verified by PCR and Southern blot analyses. Sequence analysis of the pgip construct used for transformation revealed two potential mutations in the deduced amino acid sequence. The substitutions of Asp residues with Asn and Tyr at positions 43 and 196, respectively, could interfere with the secondary structure of the expressed transgene protein. To test whether the apple PGIP was effective against Botrytis cinerea, protein extracts were prepared from apple fruit and transgenic tobacco and tested for inhibitory activity against B. cinerea polygalacturonases. Biochemical assays showed that a heat-denaturable PGIP extract prepared from apple fruit inhibited the polygalacturonases produced by a virulent isolate of Botrytis cinerea grown on pectin and apple cell walls. Protein extracts prepared from transgenic tobacco did not show any inhibitory activity towards Botrytis polygalacturonases. This suggests the absence of active PGIP in the extracts possibly due to inefficient transcription of the transgene or due to the introduced mutations.

Plant fungal pathogens are a major threat to food security worldwide. The most important method of protecting plants against fungal attack is the use of fungicides, but the development of resistance towards synthetic fungicides is of great concern. Moreover, the health risks associated with the use of chemical fungicides increase the need to search for safe, efficacious and environmentally friendly fungicides. Plants produce antifungal agents by secondary metabolism to protect themselves from fungal attack, and therefore many plant species have substantial antifungal activity. The use of plant extracts could enable the development of inexpensive and environmentally acceptable fungicides based on locally available natural products. This study was undertaken to investigate weedy and invasive plant species for antifungal activity against plant pathogens in order to develop a useful product using a widely available resource. Acetone leaf extracts of seven invasive species (Chromoleana odorata, Ipomoea alba, Tecoma stans, Passiflora suberosa, Passiflora subpeltata, Aristolochia sp, Solanum seaforthianum) were screened against eight plant fungal pathogens viz Rhizoctonia solani, Fusarium oxysporium, Penicillum janthinellum, Penicillum expansum, Aspergillus parasiticus, Aspergillus niger, Pythium ultimum and Phytophthora nicotiana, using microdilution assay and bioautography. The acetone extract of Tecoma stans had reasonable antifungal activity with an average minimal inhibitory concentration (MIC) value against all the fungi of 550 ìg/ml and clear zones on bioautograms indicating inhibition of fungal growth of a compounds with an Rf of 0.082 in BEA against several of the fungal pathogens. Due to the clear compound on bioautography and availability of Tecoma stans, this species was selected for further work. Bioassay-guided fractionation of the leaves of the Tecoma stans dichloromethane (DCM) extract obtained from solvent-solvent fractionation resulted in one major compound, oleanolic acid. The isolated compound had antifungal activity with an average MIC value of 130 ìg/ml against the 10 plant pathogenic fungi and clear bands with an Rf value of 0.082 on bioautograms, indicating fungal growth inhibition. It was surprising that the MIC value of the crude DCM extract was as high as that of the only compound with antifungal activity based on bioautography. These results clearly indicated the possibility of synergisms especially since the average total activity of the extract was nearly 6.5 times higher than that of oleanolic acid with total activity values of 60154 ml for the extract and 9262 ml for oleanolic acid. Cellular cytotoxicity of DCM extract and oleanolic acid was investigated using tetrazoliumbased colorimetric assay (MTT) on Vero monkey kidney cells. The toxicity of the extract and oleanolic acid was determined by LC50 values. The DCM extract and oleanolic acid were toxic with and LC50 of 0.413 mg/ml and 0.129 mg/ml respectively, lower than that of berberine the toxic compound used as control. However therapeutic index which can be defined here as the LC50 in (ìg/ml)/MIC in (ìg/ml), indicated that though the extract and oleanolic acid were toxic, they could be used under controlled conditions against infections of certain of the fungal pathogens. The crude extract had a high therapeutic index value of 21 against microorganisms T. harzianum, R. solani, F. oxysporium and P. expansum; and oleanolic acid had high therapeutic index values of 16 and 64 of against T. harzianum and R.solani respectively. This high therapeutic index value of crude extract and oleanolic acid means that, crude extract and oleanolic acid may be used for treatment of infections by these tested fungi with very little toxicity under controlled conditions. Oleanolic acid had very low antibacterial activity (MIC >250 ìg/ml). against two Grampositive (Staphylococcus aureus, ATCC 29213 and Enterococcus faecalis, ATCC 29212) and two Gram-negative bacteria (Escherichia coli, ATCC 27853 and Pseudomonas aeruginosa, ATCC 25922). Animal pathogenic fungi were more resistant than the plant fungal pathogens. Based on the good activity of the DCM crude extract, the surprising selectivity in activity against different fungi coupled with reasonably good therapeutic indexes and the wide availability of T stans leaves opens up the possibility that a commercial product to protect plants against certain pathogens may be developed from T. stans leaves. Copyright / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2008. / Paraclinical Sciences / unrestricted

The ability to sense and respond to environmental stimuli is essential for the survival of all living things. As a result, nature has evolved an uncountable number of ways to detect environmental signals. At the cellular level, G protein-coupled receptors (GPCRs) are used by eukaryotes, including fungi and humans, to convert extracellular molecular binding events into intracellular responses. Recently, synthetic biologists have shown that biological sensing systems can be repurposed to suit human needs, developing tools such as diagnostic devices and drug screening platforms. In this thesis, I present work exploring the potential of fungal GPCRs to be used as sensing elements in yeast-based biosensors. Chapter 1 gives background information related to synthetic biology, biosensors, and yeast signaling pathways. Chapter 2 describes the development of the baker's yeast Saccharomyces cerevisiae into a diagnostic device for detection of fungal pathogens, using fungal GPCRs. In Chapter 3 I demonstrate that the substrate specificity of fungal GPCRs can be altered using directed evolution. Chapter 4 describes experiments further probing the native binding abilities of fungal GPCRs, specifically examining protein ligands. Finally, in Chapter 5 we move beyond fungal GPCRs and engineer yeast to detect other stimuli, in the context of an engineered living material.

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