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A study on cuticle-degrading enzymes (CDE) of three hyphomycete entomopathogens has produced information on enzyme types, levels, characteristics, mode of action, regulation, sequence of production, cellular localisation and production during host penetration. This is the first critical work on CDE of any entomopathogen. Several pathogenic isolates of Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii when grown in buffered liquid cultures containing comminuted locust cuticle as sole carbon source (good growth occurred on most monomeric and polymeric cuticular constituents), produced a variety of extracellular and bound enzymes corresponding to the major components of insect cuticle e.g. 3 endo-proteases, aminopeptidase, carboxypeptidase A, lipase, esterase, chitinase and N-acetylglucosaminidase. Considerable variations occurred in levels of production between spp. and even within a sp., but endo-proteases were exceptional in being produced in large amounts by all the isolates. CDE were produced rapidly and sequentially in culture. The first activities to appear (< 24 h) were those of the proteolytic complex, chitinases were always produced substantially later. Properties of CDE were investigated in terms of pH and tem- erature optima, substrate specificity, molecular weight, iso-electric point, mechanism of substrate degradation and the effect of specific inhibitors. Studies with culture filtrates and purified CDE revealed that substrates in intact cuticles are amenable to degradation but the prior action of protease is necessary for significant degradation of the chitin. Staining of chitin by a fluorescent lectin (FITC-WGA) and calcofluor only in cuticles from which protein has been removed (by protease or KOH) also suggests initial masking of chitin. This and determination of amino acid composition of peptides solubilised by endo-protease revealed the potential of CDE in studying the physicochemical structure of insect cuticles. The apparently localised action of CDE during host penetration may result from molecular sieving, binding to fungal walls or binding to cuticle. The first possibility is lessened by the small size of the endo-enzymes (<34 K daltons) which could allow diffusion via the various canals which traverse cuticle. However, cuticle effectively binds (ionically) CDE, and also activities of several CDE remain partly bound in various ways to hyphae and conidia (by ionic binding to walls, by disulphide bonds, and on or within membrane structures). The involvement of proteolytic enzymes in infection was suggested by their presence in conidia, penetration structures, and infected cuticle (detected histochemically and following extraction from cuticles). Also the constitutive production of endo- and exo-proteases lends weight to their possible significance in parasitism as synthesis will be subject only to catabolite repression. Chitinase is induced by N-acetylglucosamine and was not detected in infected cuticle. Possible mechanisms and significance of enzymic degradation of cuticle during infection are discussed, particularly in comparison to host penetration by phytopathogenic fungi.

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The aim of this study was to investigate plant species to develop a product with the potential of protecting plants or plant products against plant fungal pathogens. Hexane, dichloromethane, acetone, and methanol leaf extracts of six plant species (Bucida buceras, Breonadia salicina, Harpephyllum caffrum, Olinia ventosa, Vangueria infausta and Xylotheca kraussiana) were evaluated for antifungal activity against seven plant fungal pathogens (Aspergillus niger, A. parasiticus, Colletotrichum gloeosporioides, Penicillium janthinellum, P. expansum, Trichoderma harzianum and Fusarium oxysporum). These plant species were selected from more than 400 plant species evaluated in the Phytomedicine Programme that had good activity against two animal fungal pathogens. All the leaf extracts were active against at least one or more of the phytopathogenic fungi in a serial microdilution assay. Of the six plant species, B. buceras had the best antifungal activity against four of the fungi, with MIC values as low as 0.02 mg/ml and 0.08 mg/ml against Penicillium expansum, P. janthinellum, Trichoderma harzianum and Fusarium oxysporum. The number of active compounds in the plant extracts was determined using bioautography with the above-mentioned plant pathogens. No active compounds were observed in some plant extracts against the fungal plant pathogens indicating possible synergism between metabolites responsible for the antifungal activity of the extract. B. salicina and O. ventosa were the most promising plant species, with at least three antifungal compounds. The antioxidant activities of plant extracts were determined using the qualitative method by spraying TLC chromatograms developed in three eluent systems BEA, CEF and EMW with 1, l-diphenyl -2 picrylhydrazyl (DPPH). The plant extracts of five of these species did not have a strong antioxidant activity. The methanol extract of X. kraussiana was the most active radical scavenger in the DPPH assay amongst the six medicinal plants screened. Based on good activity against Aspergillus niger and A. parasiticus, leaf extracts of the six plant species were also tested for antifungal activity against A. fumigatus, a very important animal fungal pathogen. The acetone extracts of B. buceras, B. salicina, V. infausta and X. kraussina had good antifungal activity against the animal pathogens, with MIC values ranging between 0.02 and 0.08 mg/ml. This indicates that crude extracts of these species may be more valuable in combating Aspergillus infections in animals than in humans. Based on the results discussed above, B. salicina was selected for in-depth study. Serial exhaustive extraction was used to extract plant material with solvents of increasing polarities namely, hexane, chloroform, acetone and MeOH. Amongst the four extractants, MeOH extracted the largest quantity of plant material 12.3% (61.5g), followed by acetone 5.6% (27.8 g), hexane 2.6% (12.8 g) and chloroform 2.1% (10.3 g). The chloroform fraction was selected for further work because it had the best antifungal activity against A. niger, C. gloeosporioides, P. janthinellum and T. harzianum and the bioautography assay showed the presence of several antifungal compounds in the chloroform fraction. Column chromatography was used in a bio-assay guided fractionation and led to isolation of four compounds. The antimicrobial activity was determined against seven plant pathogenic fungi and three bacteria, including the Gram-positive Staphylococcus aureus (ATCC 29213) and the Gram-negative Escherichia coli (ATCC 25922) and Pseudomonas aureus (ATCC 27853). The isolated compounds had good antifungal activity against A. parasiticus with an MIC of 10 μg/ml, while in other cases it ranged from 20 to 250 μg/ml. Amongst the four compounds tested, only three had a clear band, indicating that the growth of the pathogenic fungi was inhibited in the bioautography assay. Nuclear magnetic resonance spectroscopy (NMR) and mass spectroscopy (MS) were used for identification of isolated compounds. Only one compound was identified as the triterpenoid ursolic acid. Ursolic acid has been isolated from several plant species and has antifungal activity against Candida albicans (Shai et al. 2008). This is the first report on the isolation of antifungal compounds from leaves of Breonadia salicina. The other compounds isolated appeared to be mixtures of fatty acids based on mass spectroscopy and the structures were not elucidated. The cytotoxicity of acetone extracts and the four isolated compounds were determined against Vero cells using a tetrazolium-based colorimetric (MTT) assay. The acetone extract was selected based on good in vitro antifungal activity and was used in an in vivo fruit experiment. The acetone extract was less toxic toward the Vero cells with an LC50 of 82 μg/ml than ursolic acid and compound 4 which had LC50 values of 25 and 36 μg/ml respectively. Compounds 2 and 3 had low toxicity against the cells with LC50 values greater than 200 μg/ml. The potential use of the extract or isolated compound(s) against three plant fungal pathogens Penicillium expansum and P. janthinellum as well as P. digitatum (isolated from infected oranges) were tested after treating the oranges with the extract and ursolic acid. The model used gave good reproducible results. The concentration that inhibited growth correlated reasonably well with MIC values determined by serial microplate dilution. There were substantial differences in the susceptibility of the different isolates tested. The activity of ursolic acid was in the same order as that of the crude acetone leaf extract of B. salicina. The LC50 of the extract varied from 1 to 1.8 mg/ml. Penicillium digitatum was more resistant to amphotericin B in comparison to other Penicillium species. It has been reported that the fungus was resistant to the three fungicides: sodium ï-phenylphenate (ï-phenylphenol), imazalil, and thiabendazole used commercially in the fruit industry to reduce postharvest decay (Holmes and Eckert 1999). The toxicity of the extract to Vero cells was in the order of 10 times lower than the LC50 of the extracts to the fungal pathogens. Although much work still has to be done, there is good potential that a commercial product can be developed from an acetone leaf extract of B.salicina leaves, especially if the activity of this extract can be improved by removing inactive compounds. The results confirm the traditional use of B. salicina and demonstrate the potential value of developing biopesticides from plants. / Thesis (PhD)--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted

There are many examples of associations between insects and fungi. Where the fungi involved are pathogens, such associations may be of economic importance. Insects of no economic concern alone can also become important pests because of their association with fungal pathogens. Insects may assist in the spread of pathogens by carrying them on or in their bodies. Insects may also predispose plants to infection by creating wounds during feeding, oviposition or other behavioural activities. Knowledge of associations between insects and fungal pathogens often form a crucial component in the management strategy of these pathogens. The pitch canker fungus, Fusarium circinatum, causes severe disease symptoms on mature pines in the USA. Various insects have been implicated as vectors of this disease. In South Africa, F. circinatum is reported only to cause disease on pine seedlings, where it results in severe losses in nurseries. Various insects are present in the nursery that could possibly be associated with the spread of the fungus or the infection of its hosts. Amongst these insects, fungus gnats are the prime suspects due to their history of association with fungal pathogens in other nurseries. The presence of fungus gnats in South African pine nurseries and their possible association with F. circinatum and other pathogens has never been investigated critically. The objective of this study was to expand the base of knowledge of fungus gnats in South African pine nurseries, and to consider their possible association with F. circinatum and their population structure within and between nurseries. The literature review provides a summary of fungus gnats in the nursery environment. This includes their description, biology and association with fungal pathogens. Information from these studies is used to evaluate the possible association between fungus gnats and F. circinatum in South African pine nurseries. In nurseries around the world where fungus gnats are considered pests, various control options have been used, and these are further discussed. The first research aim of this study was to determine whether fungus gnats are present in the major pine nurseries of South Africa. Thus, in Chapter 2, surveys were undertaken in four of the major pine nurseries. All fungus gnats collected were identified to species level. Other diptera collected were identified to family level. Furthermore, all diptera collected were isolated on general and selective growing medium to examine for the presence of F. circinatum. Results from Chapter 2 showed that only one species of fungus gnats was present in the nurseries and it was present in all four of the nurseries surveyed. This raised interesting questions regarding the phylogeographic structure of these populations and the diversity within and between populations. These questions are addressed in Chapter 3 using analysis of mitochondrial COI sequence data from fungus gnats collected in the four nurseries. Of particular importance was the interpretation of these results as it pertains to the movement of fungus gnats between populations, together with their associated fungi. Using general and specific growing medium to isolate fungal pathogens from insects is not necessarily an accurate method. Pathogens may be overgrown by faster growing fungi before they are noticed, especially if they are present only in small amounts. Chapter 4 examined the use of DNA-based methods as a tool to detect fungal pathogens on fungus gnats. Fungus gnats were collected from the same four nurseries as in Chapter 2. Species-specific primers for F. circinatum and Botrytis cinerea were used to detect these fungi. Dilution series were done to examine the sensitivity of the primers. General primers were used to detect other fungi. This dissertation includes some of the first studies ever undertaken on fungus gnats in South African pine nurseries. Their association with the very virulent pitch canker fungus is also considered in some detail. It is my hope that these studies will form a foundation for future research on fungus gnats in South Africa. Copyright / Dissertation (MSc)--University of Pretoria, 2006. / Zoology and Entomology / unrestricted