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This thesis tests the hypothesis that the influence of fungal pathogens and arbuscular mycorrhizal (AM) fungi varies in response to proximity to parent trees and light environment to affect seedling establishment in two tree species, Milicia regia and Antiaris toxicaria, in Ghanaian tropical rain forest. Both species are from the same family (Moraceae) yet they differed markedly in their degree of susceptibility to disease and AM infection. Milicia regia seedlings were highly vulnerable to disease (caused mainly by Oomycetes, particularly Phytophthora species) and exhibited high levels of mycorrhizal colonisation, whereas Antiaris toxicaria seedlings suffered no mortality due to disease and had very low levels of AM colonisation in all forest situations. This species was, however, more susceptible to predation, the intensity of which depended on proximity to conspecifics. Spatial patterns of disease were evident in Milicia regia: seedlings had a higher probability of incurring pathogen-induced mortality close to female trees than under male trees or at a distance from parents, and were less susceptible to disease in light gap conditions. It is possible that spatial patterns in seedling disease were due to negative feedback resulting in higher inoculum loads of soil-borne Oomycetes under female trees. Differences in disease occurrence in contrasting light conditions were due to increased resistance of the host in gaps as opposed to differential pathogen activity. AM colonisation also varied spatially, with seedlings displaying host-specific inoculum preference. Moreover, AM colonisation also varied in response to light environment, being higher in gap conditions. It is proposed that a continuum of mycorrhizal benefit exists for Milicia regia - from improved nutrition to protection against pathogens, with the type of benefit being highly dependent on light environment. Milicia regia regeneration is thus subject to complex interactions between pathogens, mycorrhizal fungi and light environment which vary spatially around conspecifics, whereas these processes have limited influence on Antiaris toxicaria seedlings. Implications for patterns of seedling regeneration and the maintenance of tropical forest species diversity are discussed.

Two main characteristics make the fungal cell wall a good target for the development of antifungal antibiotics: its integrity is essential for the survival of the fungus, and it is mainly composed of polysaccharides which are not found in animal cells (Cabib et al., 1988). As chitin is the second major component in the cell wall of the pathogenic yeast form in P. brasiliensis and its content is almost three times higher than in the mycelial cell wall (Kanetsuna et al, 1969; San-Blas, 1985), its biosynthesis is an attractive target for the use of antifungal antibiotics against this medically important fungus in Central and South America. In the present work, a first step has been taken in trying to understand the biosynthesis of chitin in P. brasiliensis through the identification and cloning of fungal genes. Five different chitin synthase genes namely PbCHSl, PbCHS2, PbCHS3, PbCHS4 and PbCHS5, were identified during the course of the present work in the dimorphic fungal human pathogen P. brasiliensis, strain IVIC Pb73. The identification of these genes was accomplished through PCR amplification by using primers designed on regions of high homology amongst fungal chitin synthases (Bowen et al., 1992; Mellado et al., 1995). The complete nucleotide sequence was obtained for PbCHS2. Analysis of the expression of PbCHSl, PbCHSl, PbCHS4 and PbCHSS by northern hybridisation suggested that these genes may be regulated during dimorphism, and appear to be preferentially expressed in the mycelial form of the fungus. The use of RFLP analysis as a typing method for P. brasiliensis strains presented 100% typeability, 100% in vitro reproducibility and a discriminatory power between 0.972 and 1, depending on the restriction enzyme used. Computational analysis by using evolutionary distances of the RPLP patterns obtained for 10 P. brasiliensis isolates from different endemic areas suggested a relationship between genetic identity of the isolates and the endemic area from where they were isolated.

Fusarium head blight is one of the devastating diseases of cereal crops caused by Fusarium graminearum. This fungal pathogen produces mycotoxins like deoxynivalenol (DON), depositing it in wheat kernels and making them unfit for consumption. In addition, it causes decreases in the nutritional content of the wheat. Since wheat contaminated with DON above permissible levels must be discarded, it also leads to huge economic losses to the farmers. Fungi have a complex network of hyphae that lets them sense their surroundings. These are advantageous for nutrition and pathogenesis needs. Fungi have evolved nuanced mechanisms to orient hyphae towards external cues. Through our studies described in Chapter 2, we have elucidated the role of Ste3 GPCR in mediating fungal chemotropism towards peroxidases (previouly shown for Ste2). Both of these receptors were shown to activate the CWI-MAPK pathway in response to peroxidases. In addition, pathology assays on germinating wheat coleoptiles and detached Arabidopsis leaves showed that a Ste3 knockout strain was significantly compromised in its' ability to cause lesion development. In Chapter 3, we investigated the heterodimerization between FgSte3 and FgSte2 in response to peroxidase and a potential HRP-derived ligand of fungal origin. BRET and pull-down experiments confirmed the interaction. Chapter 4 consists of ongoing projects that go beyond the scope of the timeline for this dissertation. This includes establishing an Sf21 insect cell expression platform for the expression and purification of full length FgSte3 with a goal to elucidate the structure of the protein. Together these studies enhance our understanding of the mechanistic aspects of fungal pathogenesis and represent a step forward toward the development of novel anti-fungal compounds.

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