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Clostridium difficile is a Gram positive pathogen of significant importance in the UK, Europe and the USA. No vaccine has been developed and current treatments are focused on hospital management and the use of antibiotics. The disease is spread in hospitals in the spore form and the role of spores in C. difficile infecton is poorly understood. In this project spores of C. difficile have been characterised. The proteins from the outermost layers of the spore were identified and the genes cloned. Three of these surface proteins have unique enzymatic properties that maybe important for symptoms of disease. The ability of C. difficile spores to adhere to intestinal cells was found to be far greater than with live cells and through this we have identified that the spore may play an important role in colonisation. The regulation of spore coat gene expression during sporulation was also examined and temporal phases of genes expression identified. A major part of this project was to develop a mucosal vaccine to C. difficile. The approach used was to clone the C-terminus of toxin A onto the surface of Bacillus subtilis spores and use these recombinant spores to immunise mice and hamsters. We found that oral delivery of these spores conferred 75% protection to C. difficile infection in a hamster model of infection. Further, parenteral immunisation of the same antigens (toxin A and B) failed to generate mucosal responses and this showed that mucosal immunisation is critical for good protection. Finally, we found that antibodies to the C-terminus of toxin A were cross reactive to the C-terminus of toxin B. This showed that mucosal delivery of just the C-terminus of toxin A is sufficient to confer protection in an animal model of infection. The outcome of this work is that we have shown the parameters for successful immunisation and vaccination against C. difficile.

A Podridão Floral dos Citros é uma doença que tem limitado a produção citrícola em várias regiões produtoras do mundo. A sintomatologia esta associada principalmente quando coincide alta pluviosidade e intensa florada. O fungo se encontra na forma quiescente em órgãos vegetais e na presença de nutrientes exógenos reinicia o processo de infecção. Os objetivos do trabalho foram: (i) avaliar o efeito das águas de lavagem bruta (ALB) obtidas a partir de flores, botões e folhas de laranjeira Valência, sobre o crescimento micelial, germinação e formação de apressórios por C. acutatum; (ii) avaliar o efeito dos compostos voláteis liberados pelas ALB e as frações produzidas a partir das águas de lavagem de flores e botões sobre a germinação e formação de apressórios; (iii) estabelecer uma metodologia para observação de estruturas de infecção em folhas de citros. Foram realizados dois bioensaios de germinação e formação de apressórios e crescimento micelial para as águas de lavagem bruta. O primeiro representado pelos tratamentos: testemunha (T), água de lavagem das flores (F), botões (B), folhas novas (FN), folhas velhas (FV) e a diluição da água das flores em 1:10, 1:100, 1:1000 e 1:10000 (v/v). O segundo por testemunha (T), água de lavagem das flores (F), botões (B), folhas novas (FN), folhas velhas (FV) e a autoclavagem destes mesmos tratamentos. Os voláteis liberados pelas águas de lavagem de flores e botões primeiramente foram identificados por SPME-CG-MS e logo após as frações foram obtidas de acordo com o tempo de retenção de cada pico encontrado, totalizando 5 frações para cada amostra. Realizou-se um bioensaio de germinação e formação de apressórios com as frações obtidas em contato direto com os esporos. Para a observação de estruturas de infecção, discos foliares de plantas cítricas foram inoculados, submetido a clareamento e posterior coloração para a observação ao microscópio. Os resultados das ALB mostraram que todos os tratamentos estimularam a germinação e formação de apressórios por C. acutatum. Os voláteis não apresentaram efeito indutor sobre os esporos, porém quando fracionados e colocados em contato direto com as estruturas, as frações botão apresentaram uma maior atividade indutora quando comparada a testemunha. Nenhum tratamento alterou o crescimento micelial do fungo. A melhor visualização dos esporos nas folhas ocorreu 48 horas após a inoculação. Com base nesses resultados, pode-se concluir que as águas de lavagem estimulam a germinação e formação de apressórios, e apenas os voláteis presentes nas frações botão estimularam a germinação de C. acutatum. / The Post Bloom Fruit Drop is a limiting disease in citrus production around the world and the symptoms are associated to high rainfall and intense blooming. The fungus is found in a quiescent state in plant organs and under the presence of exogenous nutrients it restarts the infection process. Thus, the objectives of the work were: (i) evaluate the direct effect of washing crude water (WCW) obtained from flowers, flower buds and leaves of Valencia orange trees on the mycelial growth, germination and appressorium formation by C. acutatum; ii) evaluate the effect of volatile compounds released by the WCW and the fractions obtained from the washing water from flower and flower buds in the germination and appressorium formation, and (iii) establish a methodology for observing structures of fungal infection in citrus leaves. Two bioassays were carried out to evaluate germination, appressorium formation and mycelial growth in the presence of WCW. The first one was represented by the treatments: control (C), washing water from flowers (F), flowers buds (FB), young leaves (YL), old leaves (OL), and dilution (1:10, 1:100 , 1:1000 and 1:10000; v/v ) from the flower washing water. The second one included control (C), washing water from flowers (F), flowers buds (FB), young leaves (YL), old leaves (OL) and all of washings autoclaved. The volatiles released by the washing water from flowers and flowers buds were first separated and identified by SPME-GC-MS and then fractions were obtained according to the retention time of each peak found, making up five fractions for each sample. A bioassay for germination and appressorium formation was carried out by using the fractions obtained and placed into direct contact with the spores. To observe the fungal infection structures, leaf discs from citrus plants were inoculated, subject to bleaching and then stained for observation under a microscope. The results obtained by using the WCW samples showed that all treatments stimulated spore germination and appressorium formation by C. acutatum. The volatiles did not show any inductive effect on the spores, but when fractionated and placed into direct contact with the fungal structures, the fractions from the flowers buds showed a higher inducing activity when compared to the control. None treatment changed the in vitro mycelial growth of the fungus and the best visualization of spores in the leaves occurred 48 hours after inoculation. Based upon on these results, it can be concluded that the crude washing water stimulated germination and appressorium formation, and only the volatiles present in the fraction flowers buds were able to stimulate the germination of C. acutatum spores.

A ferrugem da videira, causada pelo fungo Phakopsora euvitis, é uma doença importante para a viticultura brasileira por causar desfolha precoce e, consequentemente, prejudicar a maturação dos frutos e comprometer as safras seguintes. Por ser um patógeno biotrófico, a preservação de urediniósporos para estudos com o patógeno, se faz em folhas do hospedeiro vivo. Em outras espécies de ferrugens é possível conservar os esporos em condições controladas por longos períodos de armazenamento. Outro ponto importante, para auxiliar na compreensão da epidemiologia do patossistema, está relacionado com as condições ambientais favoráveis na germinação de urediniósporos e na patogenicidade do fungo em folhas de videira. Com o objetivo de preservar o patógeno e determinar as condições ambientais favoráveis para a doença, foram avaliados: (1) O efeito da temperatura e desidratação na manutenção da viabilidade dos esporos; (2) O efeito da temperatura e do período de molhamento na germinação de urediniósporos e na patogenicidade em mudas de \"Niágara Rosada\" inoculadas. Os resultados obtidos mostraram que a desidratação dos esporos proporcionou maior viabilidade destes ao longo do tempo, para todas as condições de armazenamento testadas. A desidratação seguida pelo armazenamento a -80°C conseguiu manter os esporos viáveis, com uma alta porcentagem de germinação, por até 150 dias de armazenamento. Os urediniósporos armazenados no ambiente, independente do processo de desidratação, não conseguiram manter sua viabilidade por um período superior a 15 dias. A faixa de temperatura para germinação de urediniósporos foi ampla, entre 10 e 30°C, com um ótimo em 20°C. A expressão de sintomas em plantas inoculadas foi maior nas temperaturas de 25 e 30°C. O período de molhamento mínimo estimado pelo modelo monomolecular foi de 7 horas para as plantas mantidas a 15 °C e 5 horas para as plantas mantidas a 20, 25 e 30 °C. Para períodos acima de 6 horas de molhamento, a maior severidade da doença ocorreu na temperatura de 30 °C. Não ocorreu sintomas em plantas inoculadas e mantidas a 35°C em nenhum dos períodos de molhamento testados. O período de latência da ferrugem foi de 7 dias para plantas inoculadas e mantidas a 25 e 30°C, estendendo-se para 13 dias quando as plantas foram mantidas a 15°C. / Grapevine rust, caused by Phakopsora euvitis, is an important disease in brazilian viticulture, causing early defoliation and jeopardizing the maturation of fruits and the following crop season. P. euvitis is a biotrophic pathogen, and for research purpose, uredospores are kept through inoculation in grapevine plants. In other species of rust, it is possible to preserve spore under controlled conditions for long periods of storage. Understand the favorable environmental conditions for uredospores germination and the pathogenicity of the fungus on grapevine leaves are important for the understanding of the pathosystem epidemiology. In order to preserve the pathogen and determine the favorable conditions for the disease were evaluated: (1) The effect of temperature and dehydration in maintaining the viability of the spores; (2) The effect of temperature and wetness period on uredospores germination and pathogenicity in \'Niágara Rosada\' inoculated plants. The results showed that spore dehydration maintained high viability, for all the tested storage conditions. Dehydration followed by storage at -80°C kept viable spores, with a high percentage of germination, for 150 days of storage. The uredospores stored in the environment, independent of the dehydration process, could not maintain their viability for a period exceeding 15 days. The temperature range for uredospores germination varied from 10 to 30°C, with an optimal at 20°C. The expression of symptoms in inoculated plants was higher at 25°C and 30°C temperatures. The minimum wetness period estimated by the monomolecular model was 7 hours for plants kept at 15 °C and 5 hours for plants kept at 20, 25 and 30 ºC. For wetness periods up to 6 hours, the highest disease severity occurred at 30 °C. There were no symptoms on inoculated plants maintained at 35°C in any of the tested wetness periods. The rust latency period was 7 days for inoculated plants kept at 25 and 30°C extending for 13 days when the plants were kept at 15°C.

A paleoecological study was carried out on a 4.7 m peat core from Anderson Fen on central Vancouver Island, using a multi-proxy approach. Pollen, non-pollen palynomorphs, and physicochemical analyses were used to document past vegetation, peatland developmental history, and carbon and nitrogen accumulation rates over the last 14,000 years. Lake sediment and aquatic plant remains at the base of the core indicate a shallow pond was present at the site after deglaciation. By ~11,700 calendar years before present (cal yr BP), the shallow pond became a herb-dominated wetland (marsh) through terrestrialization. Bog formation started around 10,500 cal yr BP with decreasing water levels, as indicated by high C:N, Sphagnum and fungal remains, and testate amoebae such as Archerella flavum and Heleopera. A fen developed by ~9400 cal yr BP with fluctuating water levels through the rest of Holocene. Carbon accumulation rates were highest towards the surface and during the early Holocene warm period, with an overall mean rate of 12.9 g/m2/cal yr, which is low compared to continental and northern peatlands. Pollen analysis reveals that non-arboreal communities dominated by Salix prevailed soon after deglaciation before the expansion of Pinus forests 13,200 cal yr BP. Pseudotsuga menziesii dominated forests between ~10,700 and 8400 cal yr BP under warm and dry conditions. Tsuga heterophylla rainforest was established by ~7000 cal yr BP under increasingly cool and wet conditions. Overall, Anderson fen and the surrounding area experienced major and rapid changes in environmental conditions and vegetation in response to climate change during the late glacial and early Holocene, while mid- to late Holocene changes have been more subtle and relatively gradual. / Graduate / 2020-10-25

In rural inland, south-eastern Australia, allergy to the fungus Alternaria is prevalent and an important risk factor for asthma. The aim of the thesis was to investigate the distribution and factors influencing allergens of Alternaria in the air. As airborne allergenic spores were thought to arise from harvesting of nearby crops, two towns with different agricultural practices were studied. Moree has two crop harvesting periods in summer and autumn whilst Wagga Wagga has one harvesting period in summer. Over two years, air was sampled daily in Wagga Wagga and Moree using Burkard traps. The reliability of measurements from a single site to represent the distribution of airborne concentrations of spores across each town was examined using data from three traps simultaneously, sited 2.0 to 4.9 km apart, over four weeks. Substantial intra-class correlation coefficients (ICC) were observed between the three sampling sites across both towns (ICC=0.52, 95% CI 0.30-0.71 to 0.76, 95% CI 0.61-0.87) when counts of Alternaria spores were relatively high. The correlation was poor when counts were low. Of more than 365 trap tapes examined, the two microscopic traverses strongly correlated for counts of Alternaria spores (ICC=0.95, 95% CI 0.94-0.96). Alternaria was detected in both towns throughout the two year period with peaks in spore concentrations reflecting the season of crop harvesting in each region. Individual exposure to spores was examined. Thirty three subjects (adults and children from nine families) wore nasal air samplers and personal air samplers both inside and outside their homes. The effects of activity, location, age on the inhalation of Alternaria spores and variation between individuals in the same environment were determined. Every subject inhaled Alternaria spores. Personal exposure to Alternaria in the home environment varied substantially between subjects. Levels of fungal spores inhaled were higher during periods of activity than during rest, and higher while subjects were outdoors than indoors. During outdoor activity, the number of Alternaria spores inhaled ranged from 4 to 794 (median 11) spores/hr. Sources of airborne spores was investigated by sampling air above wheat and cotton crops near the towns during harvesting and non-harvesting periods, in a grain and cotton seed storage shed, and a cotton gin. Substantially higher concentrations were detected above crops during harvesting periods compared to non-harvesting periods. Peaks were associated with harvesting and other activities where plants were manipulated. By regression analysis spore concentrations in both towns were modelled against those detected above crops and with weather variables. Only one crop sampling period (cotton harvest) independently correlated with concentrations in town. Analysis combining all data showed concentrations of spores above crops correlated with spore concentrations in the town when lagged by one day. Variables of rainfall and maximum temperature influenced concentrations in both towns, and wind direction in Wagga Wagga alone. Parents of asthmatic children were asked by questionnaire in which locations symptoms were provoked. Asthma was reported to be exacerbated at grain farms and with disturbance of local vegetation in town and home gardens. Nasal sampling confirmed that activities that disturbed dust or vegetation increased the inhalation of spores. The factors that release allergen from spores were determined in a modified Halogen immunoassay. Approximately 60% of spores released allergen, and the proportion was influenced by isolate, nutrient availability, viability, and not influenced by sunlight or culture age up to 21 days. Germinating the spores significantly increased the proportion that released total allergen and Alt a 1 (p<0.0001). Alt a 1 appears to be a minor contributor to the total allergen released from spores except when spores have germinated. Conclusions: People living in inland rural regions of Australia are exposed to substantial quantities of allergenic spores of Alternaria. Exposure is a highly personal event and is largely determined by disturbance of local vegetation releasing spores such as from nearby crops by wind, harvesting, slashing, transport and processing of produce, and from within town and home gardens. Most spores inhaled are likely to be allergenic, with potency potentially increasing with viability.

<p>Coprophilous ascomycetes are a diverse group of saprobes, of which many belong to three families, Delitschiaceae, Phaeotrichaceae and Sporormiaceae, within the large order Pleosporales. The natural relationships and circumscription of these families are unclear, especially within the family Sporormiaceae, where the generic delimitation have been questioned. There is also a need to understand how different ecological processes affect species richness and occurrence of coprophilous ascomycetes in general. The aim of this thesis was therefore to test earlier classifications of coprophilous taxa within Pleosporales, using phylogenetic analyses of DNA sequences; and to study how the habitat, dung type and herbivores´ food choice may affect the species richness and species composition of coprophilous ascomycetes.</p><p>A phylogenetic study shows that coprophilous taxa have arisen several times within Pleosporales. Sporormiaceae and Delitschiaceae are separate monophyletic groups and should continue to be recognized as two distinct families within Pleosporales. Phaeotrichaceae forms a monophyletic group, and is, unexpectedly, a strongly supported sister-group to Venturiaceae, but if they belong to Pleosporales or not, remains unresolved. Testudinaceae and Zopfiaceae, which previously had an unclear position in Ascomycota, are shown to be members of Pleosporales and should be treated as two separate families. The genus <i>Eremodothis</i> is, however, not related to Testudinaceae, but is nested within Sporormiaceae and should be transferred to <i>Westerdykella</i>.</p><p>The natural relationships within Sporormiaceae are still not fully resolved and consequently, I suggest a rather conservative generic classification, accepting <i>Preussia, Sporormia, Westerdykella</i>, as well as <i>Sporormiella</i>, despite that the latter is not conclusively well supported as monophyletic. Characters previously used in the taxonomy and classification of Sporormiaceae, as choice of substrate, presence or absence of an ostiole, presence or absence of germ slits, and spore ornamentation, were all homoplastic and not very useful for circumscribing monophyletic groups.</p><p>Field-studies of moose (<i>Alces alces</i>), mountain hare (<i>Lepus timidus</i>) and roe deer (<i>Capreolus capreolus</i>) dung resulted in several new species records, which suggests that coprophilous ascomycetes in boreal Sweden are poorly known. Fungal species richness and occurrence on moose dung varied significantly between habitats. Species diversity was negatively associated with amount of insect attack, and insects feeding either on the dung and/or the fungi may be an important factor explaining the observed pattern. Species richness of coprophilous fungi varied also significantly between different dung types. A study of moose, mountain hare, and roe deer dung did not show any consistent patterns in respect to the animals´ digestive system. There was, however, a general strong positive relationship between the total number of ascomycete species and the number of plant species foraged by the three herbivores. Fungal species with large spores (≥ 50 µm) were over-represented on roe deer dung, and under-represented on moose dung, while the reverse was found for species with small spores (<10µm). This suggests that the foraging level of the herbivore, which in turn mirrors species-specific differences in spore dispersal of the fungi, may be an important factor in explaining species richness and diversity of the coprophilous community.</p>

Coprophilous ascomycetes are a diverse group of saprobes, of which many belong to three families, Delitschiaceae, Phaeotrichaceae and Sporormiaceae, within the large order Pleosporales. The natural relationships and circumscription of these families are unclear, especially within the family Sporormiaceae, where the generic delimitation have been questioned. There is also a need to understand how different ecological processes affect species richness and occurrence of coprophilous ascomycetes in general. The aim of this thesis was therefore to test earlier classifications of coprophilous taxa within Pleosporales, using phylogenetic analyses of DNA sequences; and to study how the habitat, dung type and herbivores´ food choice may affect the species richness and species composition of coprophilous ascomycetes. A phylogenetic study shows that coprophilous taxa have arisen several times within Pleosporales. Sporormiaceae and Delitschiaceae are separate monophyletic groups and should continue to be recognized as two distinct families within Pleosporales. Phaeotrichaceae forms a monophyletic group, and is, unexpectedly, a strongly supported sister-group to Venturiaceae, but if they belong to Pleosporales or not, remains unresolved. Testudinaceae and Zopfiaceae, which previously had an unclear position in Ascomycota, are shown to be members of Pleosporales and should be treated as two separate families. The genus Eremodothis is, however, not related to Testudinaceae, but is nested within Sporormiaceae and should be transferred to Westerdykella. The natural relationships within Sporormiaceae are still not fully resolved and consequently, I suggest a rather conservative generic classification, accepting Preussia, Sporormia, Westerdykella, as well as Sporormiella, despite that the latter is not conclusively well supported as monophyletic. Characters previously used in the taxonomy and classification of Sporormiaceae, as choice of substrate, presence or absence of an ostiole, presence or absence of germ slits, and spore ornamentation, were all homoplastic and not very useful for circumscribing monophyletic groups. Field-studies of moose (Alces alces), mountain hare (Lepus timidus) and roe deer (Capreolus capreolus) dung resulted in several new species records, which suggests that coprophilous ascomycetes in boreal Sweden are poorly known. Fungal species richness and occurrence on moose dung varied significantly between habitats. Species diversity was negatively associated with amount of insect attack, and insects feeding either on the dung and/or the fungi may be an important factor explaining the observed pattern. Species richness of coprophilous fungi varied also significantly between different dung types. A study of moose, mountain hare, and roe deer dung did not show any consistent patterns in respect to the animals´ digestive system. There was, however, a general strong positive relationship between the total number of ascomycete species and the number of plant species foraged by the three herbivores. Fungal species with large spores (≥ 50 µm) were over-represented on roe deer dung, and under-represented on moose dung, while the reverse was found for species with small spores (&lt;10µm). This suggests that the foraging level of the herbivore, which in turn mirrors species-specific differences in spore dispersal of the fungi, may be an important factor in explaining species richness and diversity of the coprophilous community.

À travers le monde, l'asthme ainsi que les maladies allergiques sont considérées comme les pathologies les plus fréquentes et constituent un problème majeur de santé publique. La population vulnérable la plus touchée par l'asthme est celle des enfants, et de plus en plus d'études mettent en cause les spores dans l'exacerbation de la maladie. Depuis quelques décennies, on observe une dégradation de la qualité de l'air en milieu urbain, accentuée par une hausse de la température qui est en partie associée au changement climatique. Ce contexte pourrait favoriser dans le futur la hausse du contenu aéroallergène dans l'air urbain et contribuer à dégrader de façon plus marquée la qualité de l'air. Ces projections inquiétantes sont à la base de cette étude qui vise à évaluer l'effet à court terme des concentrations d'aéroallergènes sur les visites et réadmissions à l'urgence pour asthme chez les enfants montréalais de 0-9 ans, entre 1994 et 2004. Les indicateurs sanitaires proviennent de la Régie d'Assurance Maladie du Québec et les données de concentrations de spores, soient les spores de Basidiomycètes, Deuteromycètes, Cladosporium et Ganoderma, ont été obtenues par l'acquisition de bases de données fournies par le laboratoire de recherche en aérobiologie « Aerobiology Research Laboratory ». Des analyses épidémiologiques écologiques de séries temporelles, basées sur des modèles paramétriques log-linéaires de Poisson, ont été effectuées. Des ajustements ont été nécessaires au niveau des tendances et des cycles temporels, ainsi que pour les variables météorologiques (température, pression) et les polluants chimiques (ozone troposphérique, dioxyde d'azote). De plus, l'effet à court terme des concentrations de spores sur les visites à l'urgence a été vérifié jusqu'à 5 jours après le jour de l'exposition aux aéroallergènes, ceci dans le but de valider le délai de l'impact. Enfin, les analyses statistiques ont été réalisées sur les premières visites ainsi que sur l'ensemble des réadmissions. Les concentrations de spores associées positivement avec les visites à l'urgence pour asthme sont: les Basidiomycètes et les Cladosporium, 4 jours après l'exposition pour les premières visites, respectivement: MPClag4=0.34%; 95% IC: 0.10-0.59%; MPClag4=0.18%; 95% IC: 0.02-0.33%. De faibles associations négatives sont observées concernant les spores de Deutéromycètes et Ganoderma, 2 jours après l'exposition, pour les réadmissions, respectivement: MPClag2=-0.57%; 95% IC : -1.05, -0.09; MPClag2=-0.57; 95% IC : -1.05, 0.09.lLes résultats de l'étude montrent que les spores de Basidiomycètes et de Cladosporium exercent une influence dans l'exacerbation de l'asthme chez les enfants notamment pour les premières visites à l'urgence. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Spores, Asthme pédiatrique, Analyse de séries temporelles, GLM, Changements climatiques, Polluants atmosphériques, Épidémiologie.

In rural inland, south-eastern Australia, allergy to the fungus Alternaria is prevalent and an important risk factor for asthma. The aim of the thesis was to investigate the distribution and factors influencing allergens of Alternaria in the air. As airborne allergenic spores were thought to arise from harvesting of nearby crops, two towns with different agricultural practices were studied. Moree has two crop harvesting periods in summer and autumn whilst Wagga Wagga has one harvesting period in summer. Over two years, air was sampled daily in Wagga Wagga and Moree using Burkard traps. The reliability of measurements from a single site to represent the distribution of airborne concentrations of spores across each town was examined using data from three traps simultaneously, sited 2.0 to 4.9 km apart, over four weeks. Substantial intra-class correlation coefficients (ICC) were observed between the three sampling sites across both towns (ICC=0.52, 95% CI 0.30-0.71 to 0.76, 95% CI 0.61-0.87) when counts of Alternaria spores were relatively high. The correlation was poor when counts were low. Of more than 365 trap tapes examined, the two microscopic traverses strongly correlated for counts of Alternaria spores (ICC=0.95, 95% CI 0.94-0.96). Alternaria was detected in both towns throughout the two year period with peaks in spore concentrations reflecting the season of crop harvesting in each region. Individual exposure to spores was examined. Thirty three subjects (adults and children from nine families) wore nasal air samplers and personal air samplers both inside and outside their homes. The effects of activity, location, age on the inhalation of Alternaria spores and variation between individuals in the same environment were determined. Every subject inhaled Alternaria spores. Personal exposure to Alternaria in the home environment varied substantially between subjects. Levels of fungal spores inhaled were higher during periods of activity than during rest, and higher while subjects were outdoors than indoors. During outdoor activity, the number of Alternaria spores inhaled ranged from 4 to 794 (median 11) spores/hr. Sources of airborne spores was investigated by sampling air above wheat and cotton crops near the towns during harvesting and non-harvesting periods, in a grain and cotton seed storage shed, and a cotton gin. Substantially higher concentrations were detected above crops during harvesting periods compared to non-harvesting periods. Peaks were associated with harvesting and other activities where plants were manipulated. By regression analysis spore concentrations in both towns were modelled against those detected above crops and with weather variables. Only one crop sampling period (cotton harvest) independently correlated with concentrations in town. Analysis combining all data showed concentrations of spores above crops correlated with spore concentrations in the town when lagged by one day. Variables of rainfall and maximum temperature influenced concentrations in both towns, and wind direction in Wagga Wagga alone. Parents of asthmatic children were asked by questionnaire in which locations symptoms were provoked. Asthma was reported to be exacerbated at grain farms and with disturbance of local vegetation in town and home gardens. Nasal sampling confirmed that activities that disturbed dust or vegetation increased the inhalation of spores. The factors that release allergen from spores were determined in a modified Halogen immunoassay. Approximately 60% of spores released allergen, and the proportion was influenced by isolate, nutrient availability, viability, and not influenced by sunlight or culture age up to 21 days. Germinating the spores significantly increased the proportion that released total allergen and Alt a 1 (p<0.0001). Alt a 1 appears to be a minor contributor to the total allergen released from spores except when spores have germinated. Conclusions: People living in inland rural regions of Australia are exposed to substantial quantities of allergenic spores of Alternaria. Exposure is a highly personal event and is largely determined by disturbance of local vegetation releasing spores such as from nearby crops by wind, harvesting, slashing, transport and processing of produce, and from within town and home gardens. Most spores inhaled are likely to be allergenic, with potency potentially increasing with viability.