Studies on the seasonal changes in the moisture content of western hemlock Tsuga heterophylla (Raf.) Sargent, demonstrated two moisture minima, one in the spring and one in the fall, and two maxima, one in the spring and the second in the winter. Significant differences were noted for both bark and wood moistures between good and poor sites, between seasons of the year and for the interaction between site and seasons of the year. The relative turgidity of bark from the good site was below the 80 per cent level for 95 days through an entire year. On the poor site it remained under this critical value for 123 days of the 8 months observation period. Dwarf mistletoe did not appear to affect the water economy of the portion of branches that had not been invaded by the endophytic system. However the parasite produced moisture stress in the bark at the central area of infections. An investigation of the fungi that were associated with cankered areas of dwarf mistletoe infections revealed the occurrence of twelve different species of fungi. Nine of these were Ascomycetes and three were Fungi Imperfecti. There were two undescribed species, and one species, Mytilidion decipiens Karst. had not been reported previously for North America. Preliminary tests of the parasitism of seven species indicated that one, a member of the Fungi Imperfecti, was mildly parasitic. / Science, Faculty of / Botany, Department of / Zoology, Department of / Graduate

Aspergillus fumigatus is the major etiology of invasive aspergillosis, a leading cause of death in immunocompromised patients. Septins are conserved GTPases that could be mediating host-spore interactions in A. fumigatus. To test this hypothesis, we are using a combination of microscopy techniques and cell culture-based methods. Using Atomic Force Microscopy, we found that spores from the ∆aspB strain have a disorganized rodlet layer compared to the parent strain. Disorganized rodlet layer can lead to an increase in immune recognition of common pathogen associated molecular patterns (PAMPs), chitin and β-glucan. It is unknown whether the increase in immune recognition of ∆aspB spores is due to more exposure to the host or if they total quantity of PAMPs has increased. To test this, we used several staining methods to compare total and exposed levels of PAMPs in the conidia. Our work shows that there is an increase in exposed chitin and total levels of chitin and β-glucan. To evaluate how this plays a role in vitro, we first exposed macrophage-like cells (J744.1) to septin deletion strain’s spores and measured the TNF-a to determine spore immunogenicity using an ELISA. As expected, only the deletions that abolish all septin complexes had a significant increase in TNF-a. Then, we determine how effective macrophages were at killing the spores. We found that the deletion strains were more susceptible to killing by the macrophages. A murine study revealed that ∆aspB infection results in more lung inflammation than the wildtype. Taken together, these results suggest the septin cytoskeleton is involved in A. fumigatus spore cell wall organization and immune evasion.

All living organisms require food for growth and survival. Heterotrophs depend on autotrophs such as green plants which can synthesize their own food unlike heterotrophic animals. Among heterotrophs, only humans and some insects have the remarkable ability to cultivate crops for food. While humans cultivate plants, three insect lineages—ants, termites, and beetles—cultivate fungi inside their nests in obligate mutualistic exo-symbioses. Interestingly, just like human agriculture, insect fungus farms are also threatened by weeds and pests, e.g. the farms of fungus-growing termites which cultivate Termitomyces fungi can be overgrown by weeds such as the parasitic fungus Pseudoxylaria. Studies on ant and beetle fungus-farming systems have uncovered the important role of chemicals and behaviour in helping these insects to protect their crops from parasitic fungi. On the other hand, studies on the termite system till now, have only revealed the presence of antifungal compounds and actinobacteria which are largely non-specific and inhibitory to the mutualistic crop fungi. Antifungal behavioural mechanisms, if present, are yet to be discovered. Therefore, this thesis focuses on different anti-Pseudoxylaria mechanisms employed by fungus-growing termites, viz. role of nest abiotic factors, mechanism of fungal recognition by termite hosts, behavioural response of termite to Pseudoxylaria presence and coupling of this behaviour to anti-Pseudoxylaria activity. The present thesis has been divided into six chapters. CHAPTER 1 gives a brief literature review on fungus-farming insects and the different mechanisms which insects employ in order to keep their fungal farms safe from growth of parasitic fungi with specific reference to fungus-growing termites. The obligate mutualistic interaction between termites and the Termitomyces fungus is 19–49 My-old and is, therefore, a very ancient agriculture system. The mutualistic fungus is cultivated on partially digested plant matter called fungus comb inside the nest and harvested by termites for nutrition. At the same time, the weedy fungal parasite Pseudoxylaria can compete with the mutualistic fungus for nutrition leading to negative effects on the fungal farms. Termite hosts are believed to use abiotic factors, antibiotics and hygienic behaviours to keep their fungal gardens free from parasitic fungi such as Pseudoxylaria. However, the actual mechanisms used by termites against parasitic fungi are unclear. Unravelling the proximate mechanisms used in fungal cultivar protection is central to understanding the evolutionary stability of these farming mutualisms. CHAPTER 2 examines the diversity and population genetic structure of Termitomyces and Pseudoxylaria strains associated with the fungus-growing termite Odontotermes obesus. Genetic diversity of cultivar and parasite could have important implications for the stability of the mutualistic interaction, e.g. genetic clonality arising from monoculture is generally thought to make populations more prone to infection by parasites. Using molecular phylogenetic tools, within-nest genetic homogeneity was found in Termitomyces species but not in Pseudoxylaria species. Lower OTU but higher genotypic diversity (within the most abundant OTU) was found in the genus Termitomyces compared to Pseudoxylaria. Additionally, population genetics methods suggested a sexual population structure for Termitomyces and clonal propagation for Pseudoxylaria species. This is the first study to investigate the population genetics of the symbiotic fungi associated with the termite genus Odontotermes or any other termite species from India. In CHAPTER 3, the effect of nest micro-environment alone on the growth of the parasitic fungus Pseudoxylaria was examined. For this, seasonal changes in nest xiii temperature and CO2 were recorded and in situ and ex situ growth experiments were performed on Pseudoxylaria. The monthly pattern of mound temperatures was found to be similar to the outside—cycling from highs in summer to lows in winter—but characterised by dampened variation compared to high daily fluctuations outside. Moreover, the mound CO2 levels were found to be orders of magnitude above atmospheric levels and, unlike the outside, were characterised by daily and monthly fluctuations. With in situ experiments during summer and winter, the effect of these dissimilar conditions—inside and outside mounds—was examined on Pseudoxylaria growth. The growth of the parasite was found to be greater inside than outside the mound. Following this, the growth of different parasite isolates under controlled ex situ conditions was examined—spanning the variation in environmental conditions that mounds exhibit daily and seasonally. High CO2 levels decreased parasitic fungal growth in general but temperature had an isolate-dependent effect. Taken together, these results suggested that the parasite is adapted to survive in the mound. However, mound environmental conditions still seemed to exert a negative effect on parasite growth, even if they cannot inhibit Pseudoxylaria completely. These results shed light on the possible new role of termite-engineered structures in impacting parasitic fungus ecology, independent of any direct role of termites in suppressing parasite growth. This is the first study to investigate the effect of abiotic factors on Pseudoxylaria growth. In CHAPTER 4, whether termites can differentiate between Termitomyces and Pseudoxylaria was investigated. In a novel, laboratory-based choice assay, termites displayed a differential response towards the two fungi by burying the Pseudoxylaria with agar. Also, termites were found to be able to differentiate between the fungi using olfactory cues, i.e. smell, alone, for this task. The mutualistic and parasitic fungi were found to emit unique volatile bouquets which could help termites to distinguish between them. This is important because, whether termites use antifungal compounds or hygienic behaviours, it is crucial that they are able to differentiate between the parasitic and mutualistic fungi so that they can selectively use antifungal mechanisms—whether chemical or behavioural—against Pseudoxylaria. This is of special significance because, many actinobacteria and anti-Pseudoxylaria compounds isolated from this system till now, lack specificity and inhibit the mutualistic Termitomyces as well. Also, fungal grooming and weeding behaviours as displayed by fungus-growing ants have not yet been reported in termites. This is the first study to show that termites have the behavioural capacity to differentiate between the mutualistic and parasitic fungi in an ecologically relevant setting. In CHAPTER 5, whether the burying of Pseudoxylaria could affect its growth was investigated. It was found that termites can utilise agar, glass beads and soil for deposition over the offered fungal plugs but the use of agar and glass beads did not inhibit Pseudoxylaria growth effectively. On the other hand, soil deposition was found to decrease growth of both Pseudoxylaria and Termitomyces fungi post-burial. However, Pseudoxylaria was found to be affected more strongly than Termitomyces. Further, hypoxia acting alone seemed to decrease only Pseudoxylaria survival without any apparent effect on Termitomyces. Therefore, hypoxia induced by soil deposition may be the reason behind the decrease in Pseudoxylaria survival. However, presence of antifungal compounds can not be ruled out and they may be selectively applied in larger quantities on Pseudoxylaria with soil deposition. This study demonstrates an anti-Pseudoxylaria activity of this insect behaviour, unique to termites among fungus-farming insects, to the presence of the parasitic fungus. CHAPTER 6 concludes the findings of this thesis and suggests a working model for the mechanism of growth suppression of Pseudoxylaria inside a termite nest. In particular, focus is on the important role of abiotic factors when combined with termite behaviour in the apparent absence of Pseudoxylaria from termite nests. These results not only shed new light on how the ecology of these fungi is affected by their termite host but also reveal the mechanistic bases that may contribute fundamentally to the evolutionary stability of this ancient mutualism.

Outbreaks of pathogens that threaten both human and nature have increased in recent years. Infectious and transmittable diseases, such as chytridiomycosis, which is caused by the emerging pathogen Batrachochytrium dendrobatidis, has been identified as one of the most important drivers of the current declines in amphibian numbers. This pathogen has spread globally and is not only responsible for the declines in amphibian population numbers, but also for the extinction of species in several countries. As part of the Amphibian Conservation Action Plan, the IUCN recommended ex situ breeding of amphibian species to try and stem the global loss of amphibian species. Due to chytridiomycosis being one of the most eminent threats for amphibians, it poses an additional threat for the ex situ breeding plan. There is thus a need for safe and effective measures to treat chytridiomycosis, especially in breeding programs for endangered species. F10 (Health and Hygiene) is a veterinary antiseptic that has shown to be 100% effective in killing B. Dendrobatidis in vitro. Before any chemical treatment can be applied the efficacy and toxicity of F10 has to be determined to establish if F10 can be effectively applied across different amphibian species and across different life stages. We propose to develop a treatment protocol for F10 for the effective treatment of amphibian chytridiomycosis by challenging juveniles of Amietophrynus gutturalis with B. dendrobatidis and subsequently treating the infection with a proposed concentration of F10. The survival of B. dendrobatidis zoospores was also determined in the presence of F10. The results obtained showed survival of tadpoles at a 1:10,000 concentration of F10 for 30min, and juveniles at a concentration of 1:2000 for 15 min. Furthermore the in vitro tests showed that the B. dendrobatidis zoospores died after 10 min at a 1:10,000 concentration and 30 min at a 1:15,000 concentration. The successful treatment of tadpoles as well as juveniles will increase any species chance for survival, especially when treating tadpoles as the pathogen will then be eradicated before the tadpole metamorphoses and reaches the disease-susceptible life stage. By establishing a partnership between the industry, academic and zoo/wildlife communities we hope to maximise the likelihood of implementing this program in the future and thus ensuring long term sustainability. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Outbreaks of pathogens that threaten both human and nature have increased in recent years. Infectious and transmittable diseases, such as chytridiomycosis, which is caused by the emerging pathogen Batrachochytrium dendrobatidis, has been identified as one of the most important drivers of the current declines in amphibian numbers. This pathogen has spread globally and is not only responsible for the declines in amphibian population numbers, but also for the extinction of species in several countries. As part of the Amphibian Conservation Action Plan, the IUCN recommended ex situ breeding of amphibian species to try and stem the global loss of amphibian species. Due to chytridiomycosis being one of the most eminent threats for amphibians, it poses an additional threat for the ex situ breeding plan. There is thus a need for safe and effective measures to treat chytridiomycosis, especially in breeding programs for endangered species. F10 (Health and Hygiene) is a veterinary antiseptic that has shown to be 100% effective in killing B. Dendrobatidis in vitro. Before any chemical treatment can be applied the efficacy and toxicity of F10 has to be determined to establish if F10 can be effectively applied across different amphibian species and across different life stages. We propose to develop a treatment protocol for F10 for the effective treatment of amphibian chytridiomycosis by challenging juveniles of Amietophrynus gutturalis with B. dendrobatidis and subsequently treating the infection with a proposed concentration of F10. The survival of B. dendrobatidis zoospores was also determined in the presence of F10. The results obtained showed survival of tadpoles at a 1:10,000 concentration of F10 for 30min, and juveniles at a concentration of 1:2000 for 15 min. Furthermore the in vitro tests showed that the B. dendrobatidis zoospores died after 10 min at a 1:10,000 concentration and 30 min at a 1:15,000 concentration. The successful treatment of tadpoles as well as juveniles will increase any species chance for survival, especially when treating tadpoles as the pathogen will then be eradicated before the tadpole metamorphoses and reaches the disease-susceptible life stage. By establishing a partnership between the industry, academic and zoo/wildlife communities we hope to maximise the likelihood of implementing this program in the future and thus ensuring long term sustainability. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

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4 pp. / This publication will explain what fungus gnats are and how to manage them.

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