Evaluation of growth media parameters for the cultivation of selected biological control agents.
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Trichoderma harzianum kmd, Gliocladium virens MMI and Colletotrichum gloeosporioides C6 are potential biological control agents. Trichoderma harzianum kmd and and G. virens MMI have been shown to have excellent growth stimulation and disease suppressive characteristics by the Biocontrol for Africa team of researchers at the University of Natal, Pietermaritzburg. Colletotrichum gloeosporioides C6 has been shown to have effective control of the invasive weed, Hakea sericea (Shrad.). The aim of this dissertation was to establish a method which was most effective for the mass production of the biological control agents (BCAs). Various parameters and the impact of carbon-to-nitrogen and total organic carbon (TOC) on the growth of the BCAs were investigated. Fingerprinting and detection of mutations between strains of Colletotrichum gloeosporioides C6 of different ages were attempted using AFLPs for patenting purposes. Pine shavings and molasses were used in the semi-solid fermentation of T harzianum kmd, G. virens MMI and C. gloeosporioides C6. A 70% (v/v) ethanol soak was the most effective pretreatment in the removal of resin off the pine shavings as well as eradication of contamination. Parameters tested were pH, C:N ratios and TOCs. The optimal pH range for T harzianum kmd and C. gloeosporioides C6 was between pH 6.5 and 7. The optimal pH for G. virens MMI was pH6. Various C:N ratios and TOCs produced highly significant differences in spore yield and mycelial biomass (P<O.OOOl). The most prolific spore production of a 11 three fungal cultures occurred at a C:N ratio of 31:1 and TOCs ranging .between 10 g L-1 to 15 g L-1 • Chlamydospores of T harzianum kmd were produced at high C:N ratios of 20:1 to 31:1. The growth of C. gloeosporioides C6 on a novel gel medium using solid-state fermentation was also investigated. Highest spore yield production was achieved at a C:N ratio of 5:1. The addition of basal salts to the gel medium enhanced spore production. The highest C:N ratio of 40:1 produced abundant mycelial growth. This was determined using Environmental Scanning Electron Microscopy. Scoring of the mycelial mats of the unamended gel medium resulted in the production of larger lipid bodies. The spore yield of C. gloeosporioides C6 was substantially decreased when grown on the novel gel as the mycelium integrated itself within the medium. It was concluded that semi-solid fermentation was a more suitable option for the production of C. gloeosporioides C6. Semi-solid state fermentation on pine shavings and molasses holds a lot of promise with regards up-scale of production of the BCAs. The effects of C:N ratios and carbon (C) concentration on growth, conidiation and germination of C. gloeosporioides C6 were investigated. Carbon concentration and C:N ratios produced highly significant differences on all parameters tested (P<0.05). Highest spore yields were obtained at C concentrations of 5 g L-l and 10 g L-l, at a C:N ratio of 15:1. Carbon concentration of 20 g L-l and a C:N of 40: 1 produced the heaviest mycelial biomass. Highest spore germination percentage occurred at a C concentration of 5 g L-l and at a C:N ratio of 20:1. Specific spore yields (Ysp) and specific mycelial biomass (Yms) were indicators used to determine the most economical production of C. gloeosporioides C6. The optimum Ysp and Yms production occurred at a C concentration of 5 g Cl and at a C:N of 15:1. Ysp was not affected by variation of C:N ratios at a C concentration of 10 g L-l. The interactive relationship between C:N ratios and C concentration was significant in the production of Yms (P=0.05) and conidiation (P=O.OOO1). The effect of C concentration and C:N ratios on the ultrastructure of C. gloeosporioides C6 was also investigated. Significant differences in the morphology of the spores at various C:N ratios and C concentrations were observed. Cell wall thickness increased with increasing C concentrations and reached a maximum thickness of 131.94 nrn at a C concentration of 20 g L-1 and a C:N ratio of 40: 1. Larger and fewer mitochondria was produced at the lower C concentrations and C:N ratios. Larger lipid bodies were observed at the higher C concentrations and C:N ratios. The nature of the extracellular matrix changed at various C concentrations and C:N ratios. A globular network was formed at a C concentration of 5 g L-1 and a fine fibrillar network was formed at a C concentration of 40 g L-l. These criteria can be used to assess which spores would have the longest shelf life as well as best efficacy in the field. Amplified fragment length polymorphism (AFLP) was used to detect mutations between the parent strain of C. gloeosporioides C6 and a culture that had been subcultured from the parent strain. The mutations affected both the nature and colour of the mycelial mats formed by the two cultures as well as the spore yield production. The AFLP technique was unsuccessful as various parameters need to be optimized, including the DNA isolation procedure. The work was discontinued due to financial and time constraints.