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It is estimated that fungal diseases affect over a billion people and kill more than 1.5 million people annually. Pulmonary fungal infections (PFIs) such as chronic pulmonary aspergillosis (CPA) and pulmonary cryptococcosis are a major cause of morbidity and mortality globally. However, PFIs are still under-diagnosed particularly in resource-limited settings despite risk factors for PFIs being common in the population. The aim of this research was to estimate the burden of disease for CPA and pulmonary cryptococcosis in Indonesia and to evaluate the outcomes of CPA surgery in a cohort of patients in the UK. The first study aimed to establish the cutoffs for two serological assays for the diagnosis of CPA. Patients (n=203) finishing their treatment for pulmonary tuberculosis (TB) and healthy controls (n=90) were recruited and serum from 100 diseased controls were also used as controls. The recommended cutoff for Aspergillus-specific IgG and anti-galactomannan IgG was established to be 11.5 mg/l (sensitivity: 88.5%, specificity: 87.8%) and 106.8 AU/ml (sensitivity: 82.6%, specificity: 68.9%), respectively. The second study was a prospective multi-centre cohort study aiming to establish the prevalence of CPA in patients undergoing treatment for TB in Indonesia. We applied the cutoffs from the first study to 216 patients at the start of TB therapy (0-8 weeks, baseline), 128 patients at the end of TB therapy (5-6 months), and 67 patients post TB therapy (12-14 months). At baseline, probable CPA was diagnosed in 6% (n=12) patients, 8% (n=10) patients at the end of therapy, 10% (n=7) patients at post TB therapy. The mean total St. George's Respiratory Questionnaire score in CPA patients was significantly higher than in non-CPA patients in all aspects (23.3 vs 3, p=0.002) indicating that the symptoms were persistent in CPA patients but resolved in those with TB only. Two patients were diagnosed with concomitant CPA and TB. The third study retrospectively evaluated the surgical outcomes of 61 CPA patients in the National Aspergillosis Centre, UK. Relapse occurred in 25 patients (41%) at mean 26 months after surgery. In 12 patients with data available at the end of follow-up time, the median Aspergillus-IgG level was lower than at the time of relapse (67 mg/L vs 126 mg/L) (p=0.016). Antifungal therapy before or after surgery were found to be protective against relapse (P≤0.005). The fourth study aimed to survey the prevalence of pulmonary cryptococcosis in patients presenting with community-acquired pneumonia (CAP) in Indonesia. Of the 17 patients recruited, 5 (29%) had findings suggestive of pulmonary cryptococcosis. The mean duration of cough in pulmonary cryptococcosis was significantly longer (12 vs 5 weeks) in pulmonary cryptococcosis patients than in non-pulmonary cryptococcosis (p=0.015). The latex agglutination tests were more sensitive than the lateral-flow device in the antigen detection for the diagnosis of cryptococcal pneumonia. In conclusion, CPA and pulmonary cryptococcosis were diagnosed at a considerable rate among risk populations in Indonesia. Most pulmonary TB patients show clinical progression after 6 months of therapy unless there is another concomitant disease such as CPA. Monitoring for CPA is required also after surgery, as the relapse rate is high.

A number of experiments were carried out to study the potential of denitrifying bacteria and reduced nitrogen compounds for control of soil-borne damping-off pathogens. Measurement of the rhizosphere pH of growing soybean roots was carried out in soil adjusted to different pH states and packed into sheet microcosms. The results showed that the rhizosphere pH of soybean was lower than the bulk soil. Nitrate reductase activity and nitrite production was then characterised for the rhizosphere of intact 14 day-old soybean roots that were incubated in nitrate substrates adjusted to different pH values under water-logged conditions. The results showed that the rate and the quantity of nitrite production increased with increasing nitrate concentration and pH in the solution. A growth room experiment was carried out to determine root colonization by denitrifying bacteria in relation to disease caused by soil-borne pathogens, which are favoured by high soil moisture (approximately -5 KPa) and low oxygen levels. Nitrite producing bacteria were isolated from soybean roots grown in Grampian (Insch) soils which had not been cropped with soybean and Thai (Phitsanulok) soils which previously had been cropped with soybean. In the first pot experiment, the nitrite producing bacteria were isolated from different root sections of 12 and 19 day-old soybean plants after 8 weeks of continuous cropping of soils with soybean (a new crop was planted every week), and using different isolation media in order to determine the genus/species composition of the denitrifying bacteria on the rhizoplane. The results showed that continuous cropping of Thai soil and Insch soil with soybean increased pre-emergence damping-off disease and decreased fresh weight yields in seedlings that did emerge. ANOVA showed significant differences between root sections for most bacterial groups monitored {Bacillus spp., Pseudomonas and Enterobacteriaceae), with regression analysis generally showing densities increasing with root age or toward the shoot base. All nitrite producing bacterial isolates were screened for antifungal activity against Macrophomina phaseolina on agar plates and between 10 and 25% of nitrite producing bacteria were found to show in vitro antagonism. In a second pot experiment, the nitrite producing bacteria were isolated from root tissue below the crown (5 cm in length) every 2 weeks of continuous cropping of soils with soybean (a new crop was planted every 2 weeks). Plate-counting was carried out to determine the population of nitrite producing bacteria while a liquid culture MPN method was used for determination of NO, N2O and N2 producing bacteria. Linear regression analysis of the incidence of pre-emergence damping-off and soybean yields in seedling that did emerge showed a highly significant negative correlation between these parameters for both soils. ANOVA showed that there was a significant difference between soil type, with the Thai soil showing higher population densities of antagonistic bacteria on soybean roots. All nitrite producing bacterial isolates were screened for antifungal activity, but the plant pathogenic fungus, Pythium ultimum, was used in this experiment. The results showed that between 10 and 40% of nitrite producing bacteria showed in vitro antagonism. However, regression analysis showed that there was no significant increase or decrease in the nitrite producing antagonistic bacterial population with continuous soybean cropping. All 900 isolates of nitrite producing bacteria isolated from the soybean rhizoplane were screened for antagonistic activity towards Pythium ultimum based on a pot trial assay in the greenhouse. As expected, very low numbers of nitrite producing bacteria showed activity against P. ultimum and only one isolate gave a significant reduction in disease incidence in pot trials. The interactive effects of nitrite producing antagonist and an Arbuscular Mycorrhizal fungus (Glomus mosseae) and Bradryrhizobium japonicum, on control of the fungal pathogens, P. ultimum or M. phaseolina were investigated in the greenhouse. The results showed that improved plant growth was obtained with certain combined inocula involving nitrite producing bacterial antagonists, Glomus mosseae and Bradryrhizobium japonicum.

Recently fungal communities have been observed on wetland and freshwater reptiles in the United States including Ophidiomycosis, or Snake Fungal Disease. Examination of fungal and bacterial communities present on spotted and painted turtles (Chrysemys picta) in Clark County, Ohio was done following the discovery of potential necrosis on spotted turtles (Clemmys guttata). The presence of a specific pathogenic fungal population may account for the decline in population of spotted turtles. Additional analysis of fungal and bacterial populations in common mud turtles (Kinosternon steindachneri) and smooth earthsnakes (Virginia valeriae) in Lafayette County, Mississippi was also conducted. The bacterial and fungal communities found on the snake and turtle populations may be found to be similar between the species and further exploration and research should be done to determine which communities are present in these wetland habitats.

American leaf spot disease, known in Latin America as ojo de gallo, is a coffee fungal disease caused by the fungal agent Mycena citricolor. Recent increase in the disease and its ability to cause severe yield losses in coffee raised questions about our understanding of the disease dynamics in coffee farms, and our ability to predict and prepare for possible outbreaks and avoid the economic losses similar to those caused by recent outbreaks of the coffee leaf rust across Latin America. In this thesis, I investigate some of the factors that might impact patterns of American leaf spot disease in an organic shaded coffee farm in Chiapas, Mexico. In chapter 1, I assess the co-occurrence pattern of American leaf spot (ALS) disease with the coffee leaf rust, another fungal disease caused by Hemileia vastatrix, which caused severe outbreaks in Latin America since 2012/2013. Based on seven months of sampling of ALS and coffee leaf rust incidence and intensity in an organic coffee farm in Chiapas, Mexico, results from chi-squared analyses of contingency tables of disease presence suggest that the incidences of the two diseases are not independent. Significantly fewer coffee plants are infected with both diseases than expected at random, while significantly higher number of coffee plants are infected with only one disease than expected at random. The patterns show evidence that there may be trade-offs between the control of the two pathogens. Specifically, the planting of coffee leaf rust resistant varieties may be more susceptible to American leaf spot disease and may lead to an outbreak of this fungal disease. In chapter 2, I explore the possible effects of shade trees and a dominant ant on ALS. As nitrogen fixing trees can usually add nutrients to the soil and possibly improve plant health, I proposed that coffee plants under nitrogen fixing shade trees will have a lower incidence and intensity of ALS. In addition, due to the complex ecological interactions that the dominant ant, Azteca sericeasur, can have with many organisms in the system and maintaining stability through biocontrol, I hypothesized that trees with A. sericeasur nests will have a negative impact on ALS disease. Using data of American leaf spot incidence and intensity collected in Finca Irlanda, Chiapas, Mexico, along with shade tree types and Azteca ant nest presence, I ran chi-squared analyses, Kruskal-Wallis rank sum test, and t-tests to understand the disease patterns with respect to three shade tree types (Inga spp [a n-fixing genus in the family Fabacea], Alchornea latifolia [the second most common species after Inga], and all other trees). I found that shade tree types do not significantly impact American leaf spot incidence and intensity in the same way. Coffee plants around Inga spp., the nitrogen fixing trees in the coffee farm, have significantly higher disease intensities than other shade trees but are not different from Alchornea. The presence of Azteca nests in shade trees nearby coffee bushes did not have a significant effect on the incidence of ALS. The results suggest that shade trees have complex impact on American leaf spot disease, and multiple factors, including nutrients, shade level and microclimate, and possible ecological interactions can have a combined effect. Future research focusing on independent factors associated to shade trees can further improve our understanding of American leaf spot disease patterns.

Cowpea (Vigna unguiculata) is a legume crop which is grown in many warm regions around the world. Genomic resources have been developed for cowpea which has enabled the identification of QTL and candidate genes which can be utilized in trait improvement.Fungal diseases cause significant constraints to cowpea yield. Fusarium oxysporum f.sp. tracheiphilum (Fot) race 3 and race 4 cause vascular wilt disease and are problematic in California. Genetic mapping identified the Fot3-1 locus which confers resistance to Fot race 3 in the CB27 x 24-125B-1 population. Fot3-1 was identified on BAC clone CH093L18, which carries leucine-rich repeat serine/threonine protein kinases. QTLs were identified which confer resistance against Fot race 4. Fot4-1 was identified in the IT93K-503-1 x CB46 population and Fot4-2 was identified in the CB27 x 24-125B-1 and CB27 x IT82E-18(Big Buff) populations. The syntenic loci for Fot4-1 and Fot4-2 were examined with Glycine max, where several disease resistance candidate genes were identified. Macrophomina phaseolina is a fungal pathogen which causes diseases under high temperatures and drought-stress. QTLs, Mac-10, Mac-11, Mac-12 and Mac-13, were identified in the Sanzi x Vita 7 population. The Mac-11 locus was positioned within BAC clone CH038D17 where an auxin response factor was present. Mac-13 was identified within BAC clones CH062O11 and CH069K06, where an auxin-responsive GH3 family protein was present. Leaf morphology was studied in the cowpea RIL population, Sanzi x Vita 7, in which a QTL was identified for leaf shape, Hls. High co-linearity was observed for the syntenic Hls region in Medicago truncatula and Glycine max where EZA1/SWINGER genes were present. Heat-induced browning of seed coats is caused by high temperatures which discolors the seed coats of cowpea. Three QTL, Hbs-1, Hbs-2, and Hbs-3, were identified using cowpea RIL populations IT93K-503-1 x CB46 and IT84S-2246 x TVu14676. Hbs-1 was identified in BAC clone CM018C23 where ethylene forming enzymes were present. Hbs-3 was identified in BAC clones CH047M01 and CM014K16 where ACC synthase genes were present. Practical outcomes from these studies are the identification of molecular markers which can be used in a Marker Assisted Selection breeding scheme, which should expedite variety development for cowpea.

Methods for detection of sensitivity of Venturia inaequalis to demethyltion inhibitor (DMI) fungicides were assessed. Three isolates differing in EC50 were tested with myclobutanil. A hyphal growth test was more reliable than either a germ tube growth test or spore germination test. Image processing and analysis could provide rapid and more accurate measurements of the lengths of germ tubes and hyphal growth especially for irregular shapes. However, it was considered relatively time consuming in image processing stage. A hyphal growth test was used to assess sensitivity of 10 isolates of V. inaequalis from main apple growing districts of New Zealand to myclobutanil. The EC₅₀ of isolates ranged from 0.035-0.679 mg/litre. The EC₅₀ value of the most resistant isolate was about 20 times the most sensitive one. Cross resistance was assessed using a colony growth test with 9 isolates and myclobutanil, bitertanol, triforine, flusilazole, penconazole, pyrifenox and fenarimol. All isolates were cross resistance to both trlazole and non-trlazole DMI fungicides. Isolate 5 was the most resistant isolate and had high cross resistance to most products. A leaf disc system was used to estimate the duration of the incubation period, percentage of diseased area and conidial production. Six isolates classed as medium-resistant type (MR), low-resistant type (LR) and sensitive type (S) were tested with 2 levels of inoculum density (5x10³ and 5x10⁴ spores per ml). The higher the inoculums density, the greater was the degree of host tissues colonization and consequently higher conidia produced. By contrast, the higher the inoculum density, the shorter was the duration of the incubation periods. However, none of the fitness parameters were significantly different between resistant and sensitive isolates of V. inaequalis.

Diagnostic medical mycology has been slow to advance due to a lack of species specific antigens in organisms which cause serious diseases in man. Toward this end, an HPLC analysis was done of the following fungal antigens: histoplasmins HKC-43 and H-42, blastomycin KCB-26, an alkali-soluble, water soluble antigen from Blastomyces dermatitidis (b-ASWS), a coccidioidin prepared from a toluene lysate of the mycelial-arthroconidia phase of Coccidioides immitis, and an alkali-soluble, water-soluble antigen from Coccidioides immitis (c-ASWS). The HPLC survey included size-exclusion chromatography (SEC), ion exchange chromatography (HPIEC), and reversephase chromatography (RP). Resolution was poor with both SEC and HPIEC but was excellent with RP chromatography. The use of RP will allow sufficient separation for further antigenic and structural analysis.

Soluble protein samples were prepared from resistant and susceptible lines of the William Massey variety of peas which had previously been inoculated with Fusarium oxyporum f.sp. pisi, a fungal wilt pathogen. These samples were separated chromatographically into protein bands by electrophoresis on gradient polyacrylamide gels, and subsequently protein stained with a variety of staining compounds. Protein spectra of the harvest samples from inoculation at germination until seven weeks from this time were investigated for differences in spectral band composition using amido black, a general protein stain. Differences occurred in the harvests made between one and three weeks, suggesting further investigation in this area was warranted. These samples were subsequently tested for enzymic activity using staining techniques specific for particular enzymes. Some interesting results arose with the enzyme esterase enabling a theory for the possible involvement of this enzyme in resistance to be formulated.