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.

1. The hyphae of three basidiomycetes, <I>Coprinus cinereus</I>, <I>Coriolus versicolor</I> and <I>Gloeophyllum trabeum</I>, were stained with DAPI to ascertain their karyotic state. The process of clamp formation was observed in these fungi. 2. Hyphae and clamp connections were treated with CFW to show areas of chitin synthesis. Clamp connections show the same general pattern of chitin deposition as hyphae. Individual hyphae and clamps were treated with nikkomycin, a drug which inhibits chitin synthesis. Again, clamps behaved in a manner similar to hyphae. 3. Genomic DNA was isolated from <I>C. cinereus</I> and used in polymerase chain reactions to try to identify chitin synthase genes. A product was identified, but it is unclear as to whether this is a true chitin synthase gene. 4. Fungi were treated with the chitinase inhibiting drug, allosamidin. The position of branches and clamps relative to the hyphal apex was altered by this antibiotic indicating apical extension was disrupted. Allosamidin did not affect the specific growth rate of fungi in submerged liquid culture. 5. Fungi were grown under conditions of restricted calcium ion supply. With reduced calcium concentration hyphal branch frequency increased as did clamp frequency. Although the pattern of growth was affected, specific growth rate remained constant in cultures grown in submerged liquid culture. 6. The galvanotropic response of fungi was investigated. The magnitude of the response of hyphae to an electric field was dependent upon field strength, length of exposure to the field, pH and calcium ion concentration.

1. Culture media and conditions were developed to maximize the sexual development of <I>Nannizzia incurvata</I> using liquid culture and solid media, modified Sabouraud's 1/6 salts + hair medium being the most suitable. 2. Strong evidence for the existence of two sex hormones which regulate sexual differentiation was found. It is proposed that these hormones are named (+)-factor (which is produced by (+)-cells and induce a sexual response in (-)-cells) and (-)-factor (which is produced by (-)-cells and induce a sexual response in (+)-cells). 3. Addition of active extracts of concentrated mated culture filtrates caused a switch from asexual to sexual development and the formation of the characteristic sexual cleistothecia. The hormones present are probably extracellular, as mycelial extracts do not cause this response. 4. Further evidence for the existence of these sex hormones was achieved by separation of compatible strains by a semi-permeable membrane, cellophane, which allowed diffusion of the hormones through the medium yet prevented physical, hyphal interactions between the two strains. Sexual morphogenesis occurred in both strains. 5. Both (+)-factor and (-)-factor were shown to be non-volatile by further experiments separating colonies with cellophane membranes. 6. (-)-Factor was solvent extractable using diethyl ether or ethyl acetate, while (+)-factor remained in the aqueous phase. Thus, (-)-factor is more hydrophobic yet less polar and (+)-factor is more hydrophilic yet more polar.

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.

Tsang Yiu-Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 198-218). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgments --- p.v / Table of contents --- p.vi / List of figures --- p.xi / List of tables --- p.xvi / Abbreviations --- p.xviii / Chapter 1. --- Introduction / Chapter 1.1 --- Organic pollutant and environment --- p.1 / Chapter 1.2 --- Polycyclic aromatic hydrocarbon --- p.3 / Chapter 1.2.1 --- Distributions and treatment standards of two target PAHs --- p.5 / Chapter 1.3 --- Pentachlorophenol --- p.8 / Chapter 1.3.1 --- Distribution and treatment standard of PCP --- p.10 / Chapter 1.4 --- Dichlorodiphenyltrichloroethane --- p.12 / Chapter 1.4.1 --- Distribution and treatment standard of DDT --- p.13 / Chapter 1.5 --- Indigo carmine --- p.15 / Chapter 1.6 --- Cleanup technologies towards organopollutants --- p.16 / Chapter 1.6.1 --- Treatment methods for organopollutants --- p.16 / Chapter 1.6.2 --- Enzyme technology on environmental cleanup --- p.18 / Chapter 1.6.3 --- Oxidoreductase --- p.19 / Chapter 1.6.4 --- Enzyme preparation --- p.20 / Chapter 1.6.5 --- Spent mushroom compost --- p.21 / Chapter 1.6.5.1 --- Laccase --- p.22 / Chapter 1.6.5.2 --- Catalytic cycle of laccase --- p.23 / Chapter 1.6.5.3 --- Lignin peroxidase --- p.25 / Chapter 1.6.5.4 --- Catalytic cycle of LiP --- p.26 / Chapter 1.6.5.5. --- Manganese peroxidase --- p.27 / Chapter 1.6.5.6 --- Catalytic cycle of MnP --- p.28 / Chapter 1.6.6 --- Limitations on enzyme technology --- p.29 / Chapter 1.6.7 --- Enhancement of laccase activity and/or catalytic lifetime --- p.30 / Chapter 1.6.8 --- Enhancement of MnP activity and/or catalytic lifetime --- p.32 / Chapter 1.6.9 --- Other general approaches to maintain enzyme activity --- p.34 / Chapter 1.7 --- Aims of my study --- p.35 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Production of spent mushroom compost (SMC) --- p.36 / Chapter 2.2 --- Effect of age and batches of SMCs on enzyme qualities --- p.37 / Chapter 2.3 --- Maximization of enzymes extracted from SMC --- p.38 / Chapter 2.3.1 --- Effect of extraction solution type --- p.38 / Chapter 2.3.2 --- Effect of extraction volume --- p.39 / Chapter 2.3.3 --- Effect of extraction time --- p.39 / Chapter 2.3.4 --- Effect of rotation speed --- p.39 / Chapter 2.4 --- Enzyme and protein quality --- p.39 / Chapter 2.4.1 --- Protein assay --- p.39 / Chapter 2.4.2 --- Laccase assay --- p.40 / Chapter 2.4.3 --- Manganese peroxidase assay --- p.40 / Chapter 2.4.4 --- Lignin peroxidase assay --- p.41 / Chapter 2.4.5 --- p-glucanase assay --- p.41 / Chapter 2.4.6 --- Carboxymethylcellulase assay --- p.42 / Chapter 2.4.7 --- Xylanase assay --- p.42 / Chapter 2.4.8 --- Lipase assay --- p.43 / Chapter 2.4.9 --- Protease assay --- p.43 / Chapter 2.5 --- Freeze-drying on crude enzyme preparation --- p.44 / Chapter 2.5.1 --- Effect of freeze-drying --- p.44 / Chapter 2.6 --- Partial purification on crude enzyme preparation --- p.44 / Chapter 2.6.1 --- PAGE analyses on Pleurotus SMC's laccase and MnP --- p.44 / Chapter 2.6.2 --- Effect of dialysis --- p.45 / Chapter 2.7 --- Characterization of crude enzyme powder --- p.46 / Chapter 2.7.1 --- Metal analysis --- p.46 / Chapter 2.7.2 --- Anion contents --- p.47 / Chapter 2.7.3 --- H202 content --- p.47 / Chapter 2.8 --- Stability of crude enzyme at storage --- p.48 / Chapter 2.9 --- Optimization of crude enzyme activities --- p.48 / Chapter 2.9.1 --- Ligninolytic enzyme --- p.48 / Chapter 2.9.1.1 --- Crude enzyme amount --- p.48 / Chapter 2.9.1.2 --- pH effect --- p.49 / Chapter 2.9.1.3 --- Temperature effect --- p.49 / Chapter 2.9.1.4 --- EDTA addition --- p.49 / Chapter 2.9.1.5 --- Copper ion addition --- p.49 / Chapter 2.9.1.6 --- Manganese ion addition --- p.50 / Chapter 2.9.1.7 --- Hydrogen peroxide addition --- p.50 / Chapter 2.9.1.8 --- Malonic acid addition --- p.50 / Chapter 2.9.2 --- "Other enzymes (beta-glucanase, carboxymethylcellulase and xylanase)" --- p.51 / Chapter 2.9.2.1 --- Temperature effect --- p.51 / Chapter 2.9.2.2 --- pH effect --- p.51 / Chapter 2.10 --- Studies on the degradation ability of crude enzyme towards organopollutants --- p.51 / Chapter 2.10.1 --- Removal of PAH (naphthalene and phenanthrene) --- p.52 / Chapter 2.10.1.1 --- Experimental setup --- p.52 / Chapter 2.10.1.2 --- Effect of PAH concentration --- p.53 / Chapter 2.10.1.3 --- Effect of ABTS addition --- p.54 / Chapter 2.10.1.4 --- Effect of incubation time --- p.54 / Chapter 2.10.1.5 --- Putative identification and quantification of PAHs --- p.54 / Chapter 2.10.2 --- Removal of pentachlorophenol --- p.56 / Chapter 2.10.2.1 --- Experimental setup --- p.56 / Chapter 2.10.2.2 --- Effect of PCP concentration --- p.57 / Chapter 2.10.2.3 --- Effect ofABTS addition --- p.57 / Chapter 2.10.2.4 --- Effect of incubation time --- p.57 / Chapter 2.10.2.5 --- Putative identification and quantification of PCP --- p.57 / Chapter 2.10.3 --- "Removal of 4,4´ة-DDT" --- p.58 / Chapter 2.10.3.1 --- Experimental setup --- p.58 / Chapter 2.10.3.2 --- Effect of DDT concentration --- p.59 / Chapter 2.10.3.3 --- Effect ofABTS addition --- p.59 / Chapter 2.10.3.4 --- Effect of incubation time --- p.59 / Chapter 2.10.3.5 --- Putative identification and quantification of DDT --- p.60 / Chapter 2.10.4 --- Removal of dye ´ؤ Indigo carmine --- p.61 / Chapter 2.10.4.1 --- Experimental setup --- p.61 / Chapter 2.10.4.2 --- Effect of dye concentration --- p.62 / Chapter 2.10.4.3 --- Effect of ABTS addition --- p.62 / Chapter 2.10.4.4 --- Effect of incubation time --- p.62 / Chapter 2.11 --- Assessment criteria --- p.62 / Chapter 2.11.1 --- Degradation ability --- p.62 / Chapter 2.11.2 --- Toxicity of treated samples (Microtox® test) --- p.63 / Chapter 2.12 --- Statistical analysis --- p.64 / Chapter 3. --- Results / Chapter 3.1 --- The best SMC for enzyme preparation --- p.65 / Chapter 3.2 --- Maximization of enzymes extracted from SMC --- p.72 / Chapter 3.2.1 --- Effect of extraction solution type and volume on crude enzyme recovery --- p.72 / Chapter 3.2.2 --- Effect of extraction time on crude enzyme recovery --- p.79 / Chapter 3.2.3 --- Effect of rotation speed on crude enzyme recovery --- p.79 / Chapter 3.3 --- Effect of dialysis on crude enzyme preparation --- p.82 / Chapter 3.4 --- Freeze-drying on crude enzyme preparation --- p.82 / Chapter 3.5 --- Characterization of crude enzyme powder --- p.86 / Chapter 3.6 --- Optimization of crude enzyme activities --- p.87 / Chapter 3.7 --- Storage stability of crude enzyme in powder form and liquid form --- p.115 / Chapter 3.8 --- Studies on degradation ability of crude enzyme towards organopollutants --- p.135 / Chapter 3.8.1 --- Degradation of naphthalene (NAP) by crude enzyme solution --- p.135 / Chapter 3.8.2 --- Degradation of phenanthrene (PHE) by crude enzyme solution. --- p.141 / Chapter 3.8.3 --- Degradation of pentachlorphenol (PCP) by crude enzyme solution --- p.147 / Chapter 3.8.4 --- "Degradation of 4,4´ة-DDT by crude enzyme solution" --- p.152 / Chapter 3.8.5 --- Degradation of Indigo carmine by crude enzyme solution --- p.158 / Chapter 4. --- Discussion / Chapter 4.1 --- The best SMC for enzyme preparation --- p.163 / Chapter 4.2 --- Maximization of ligninolytic enzymes extracted from SMC --- p.168 / Chapter 4.2.1 --- Effect of extraction solution type and volume on crude enzyme recovery --- p.168 / Chapter 4.2.2 --- Effect of extraction time on crude enzyme recovery --- p.169 / Chapter 4.2.3 --- Effect of rotation speed on crude enzyme recovery --- p.169 / Chapter 4.3 --- Effect of dialysis on crude enzyme extract --- p.171 / Chapter 4.4 --- Freeze-drying on crude enzyme extract --- p.171 / Chapter 4.5 --- Characterization of crude enzyme powder --- p.172 / Chapter 4.6 --- Optimization of crude enzyme activities --- p.173 / Chapter 4.6.1 --- Effect of crude enzyme amount --- p.173 / Chapter 4.6.2 --- Effect of incubation pH --- p.174 / Chapter 4.6.3 --- Effect of incubation temperature --- p.176 / Chapter 4.6.4 --- Effect of EDTA addition --- p.177 / Chapter 4.6.5 --- Effect of copper and manganese ion addition --- p.177 / Chapter 4.6.6 --- Effect of hydrogen peroxide addition --- p.179 / Chapter 4.6.7 --- Effect of malonic acid on maintaining enzyme activities --- p.180 / Chapter 4.6.8 --- Activities and stabilities of ligninolytic enzymes under the combined optimal conditions --- p.181 / Chapter 4.7 --- Storage stability of crude enzyme in powder form and liquid form --- p.182 / Chapter 4.7.1 --- "β-glucanase, carboxymethylcellulase (CMCase) and xylanase activities" --- p.182 / Chapter 4.7.2 --- Protein content --- p.182 / Chapter 4.7.3 --- Laccase activity --- p.183 / Chapter 4.7.4 --- MnP activity --- p.183 / Chapter 4.8 --- Studies on the degradation ability of crude enzyme towards organopollutants --- p.185 / Chapter 4.8.1 --- Degradation of naphthalene (NAP) by crude enzyme solution --- p.185 / Chapter 4.8.2 --- Degradation of phenanthrene (PHE) by crude enzyme solution. --- p.187 / Chapter 4.8.3 --- Degradation of pentachlorophenol (PCP) by crude enzyme solution --- p.189 / Chapter 4.8.4 --- "Degradation of 4,4-DDT by crude enzyme solution" --- p.190 / Chapter 4.8.5 --- Degradation of Indigo carmine by crude enzyme solution --- p.191 / Chapter 4.9 --- Prospect for SMC as a source of organopollutant-degrading enzyme --- p.193 / Chapter 5. --- Conclusions --- p.195 / Chapter 6. --- Further Investigation --- p.197 / Chapter 7. --- References --- p.198

My work focuses on the community ecology of symbioses, specifically of fungal endophytes and their hosts. This thesis describes how plant defense compounds and a seed endophyte influence community structure of maize fungal endophytes. Maize produces benzoxazinoids (BXs), compounds toxic to microbes and insects. I assessed the influence of three factors on endophyte communities: host BX production, host neighbor identity and presence of a BX-detoxifying endophyte, Fusarium verticillioides (FV). To determine the influence of BXs on communities, two BX-producing (BX+) and one BX-nonproducing (BX–) genotype were planted in Ridgetown and Harrow, Ontario (triculture). Fungi were isolated and tested for tolerance to 2-benzoxazolinone (BOA), a toxic BX byproduct. Species and functional diversity (community distribution of BOA tolerance levels) was calculated. In seedling roots and mature leaves, the community proportion with low BOA tolerance was greater in BX– than BX+ plants. Fusarium abundance was up to 35 times higher in mature leaves of BX+ than BX– plants. Next, to assess the effect of host neighbor identity on communities, BX– monocultures were planted, and communities from BX– plants in monoculture and triculture compared. Monoculture root communities had higher species diversity than those in triculture. In vitro experiments were conducted to evaluate the influence of BOA on endophyte species interactions. FV facilitated species with lower BOA tolerance in the presence of BOA. Finally, fields were planted with a BX+ and BX– genotype in Ontario, Canada and Georgia, USA. Seed was inoculated with FV (FV+) or sterilized (FV–). FV abundance was highest in BX+FV+ plants, and Fusarium abundance was greater in BX+ than BX– plants in mature leaves. In Georgia, BX+FV+ communities in below ground tissue had lower abundance of BOA sensitive species than BX+FV–. Overall, results suggest that BXs are a habitat filter that increased colonization by horizontally transmitted and seed-born Fusarium species. This invokes the hypothesis that selective breeding for enhanced BX concentrations increased abundance of Fusarium species in maize. The in vitro study indicated that FV could facilitate other species. In contrast, field results suggest that FV interacts competitively with community members, a trait enhanced in the presence of BXs.

My work focuses on the community ecology of symbioses, specifically of fungal endophytes and their hosts. This thesis describes how plant defense compounds and a seed endophyte influence community structure of maize fungal endophytes. Maize produces benzoxazinoids (BXs), compounds toxic to microbes and insects. I assessed the influence of three factors on endophyte communities: host BX production, host neighbor identity and presence of a BX-detoxifying endophyte, Fusarium verticillioides (FV). To determine the influence of BXs on communities, two BX-producing (BX+) and one BX-nonproducing (BX–) genotype were planted in Ridgetown and Harrow, Ontario (triculture). Fungi were isolated and tested for tolerance to 2-benzoxazolinone (BOA), a toxic BX byproduct. Species and functional diversity (community distribution of BOA tolerance levels) was calculated. In seedling roots and mature leaves, the community proportion with low BOA tolerance was greater in BX– than BX+ plants. Fusarium abundance was up to 35 times higher in mature leaves of BX+ than BX– plants. Next, to assess the effect of host neighbor identity on communities, BX– monocultures were planted, and communities from BX– plants in monoculture and triculture compared. Monoculture root communities had higher species diversity than those in triculture. In vitro experiments were conducted to evaluate the influence of BOA on endophyte species interactions. FV facilitated species with lower BOA tolerance in the presence of BOA. Finally, fields were planted with a BX+ and BX– genotype in Ontario, Canada and Georgia, USA. Seed was inoculated with FV (FV+) or sterilized (FV–). FV abundance was highest in BX+FV+ plants, and Fusarium abundance was greater in BX+ than BX– plants in mature leaves. In Georgia, BX+FV+ communities in below ground tissue had lower abundance of BOA sensitive species than BX+FV–. Overall, results suggest that BXs are a habitat filter that increased colonization by horizontally transmitted and seed-born Fusarium species. This invokes the hypothesis that selective breeding for enhanced BX concentrations increased abundance of Fusarium species in maize. The in vitro study indicated that FV could facilitate other species. In contrast, field results suggest that FV interacts competitively with community members, a trait enhanced in the presence of BXs.