Recurrent Airway Obstruction (RAO)-affected horses experience bronchoconstriction and airway inflammation in response to inhalation of irritants including hay molds. Steaming hay reduces fungal content, but the effect on the antigenic potential has not been investigated. We tested the hypothesis that RAO-affected horses develop less severe clinical disease when fed steamed versus non-steamed hay and this reduction coincides with decreased hay fungal content. Six RAO-affected horses in clinical remission were divided in two groups and fed steamed or non-steamed hay for 10 days using a two-way cross-over design. Hay was steamed using a commercial hay-steamer. Clinical assessment was performed daily. Full assessment, including airway endoscopy, tracheal mucous scores and maximal change in pleural pressure, was performed on days 1, 5, and 10. Bronchial fluid sampling and cytology were performed on days 1 and 10. Hay core samples were collected pre- and post-steaming and cultured to determine fungal and bacterial concentrations. Statistical analysis was based on data distribution and quantity and performed using SAS®. P-value <0.05 was significant. Steaming significantly decreased the number of bacterial and fungal colony-forming-units in hay. Horses fed non-steamed hay experienced a significant increase in clinical score and a trend towards airway neutrophilia, while parameters were unchanged in horses fed steamed hay. Only horses fed non-steamed hay experienced a significant increase in tracheal mucous score. Horses fed steamed hay gained significantly more weight compared to horses fed non-steamed hay, even though the amount of hay consumed not greater on a dry matter basis. These results indicate that steaming reduces the RAO-affected horse's response to hay which coincides with a reduction in viable fungal content of hay. / Master of Science

No / Eleven species of fungi and one slime-mould are added to the previous list of fungi from Indian Ocean islands made by the authors in 2004. Two other species in the area are confirmed. Our knowledge of fungal distributions in these remote islands is extended and comments are made on some immature collections. This small collection does, however, indicate a palaeotropical element to the mycota with bias towards species found in Southeast Asia, although some have a worldwide distribution.

Thesis (Ph. D.)--University of Tennessee, Knoxville, 2008. / Title from title page screen (viewed on Mar. 10, 2009). Thesis advisor: Kurt H. Lamour. Vita. Includes bibliographical references.

Thesis (M.S.)--Oregon State University, 1993. / Typescript (photocopy). Includes bibliographical references (leaves 40-50). Also available on the World Wide Web.

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

Doctor of Philosophy / Department of Entomology / Raymond A. Cloyd / The life history of the rove beetle, Atheta coriaria (Kraatz) (Coleoptera:Staphylinidae), predation against the fungus gnat Bradysia sp. nr. coprophila (Lintner) and compatibility with pesticides and plant growth regulators was investigated under laboratory conditions using Sunshine LC1 Professional Growing Mix as a substrate. Duration of life stages was 2.2, 7.1, and 7.8 days for egg, larva and pupa respectively, at 26°C, whereas total development time from egg to adult was 17.0 days. In addition, A. coriaria male and female adult longevity was 60.3 and 47.8 days. Average fecundity was 90.2 eggs per female and the number of adults produced per female was 69.1. There were no significant differences in prey consumption when using second and third instar fungus gnat larvae as prey and starved and non-starved rove beetles. Overall, predation efficacy in Petri dishes was high (70 to 80%) as fungus gnat larval density increased with 3.9, 7.0, 11.1, and 15.3 larvae consumed in 24 hours after exposure of 5, 10, 15 and 20 fungus gnat larvae to one rove beetle adult. However, lower predation rates were found at different predator:prey ratios when using 1 to 5 rove beetles and growing medium as a substrate. The direct and indirect effects of pesticides and plant growth regulators on A. coriaria were investigated under laboratory conditions. Rove beetle survival was consistently higher when adults were released 24 hours after rather than before applying pesticides. Acetamiprid, lambda-cyhalothrin, and cyfluthrin were directly harmful to rove beetle adults, whereas Beauveria bassiana, azadirachtin and organic oils were compatible with A. coriaria. Similarly, the plant growth regulators acymidol, paclobutrazol and uniconazole were not harmful to rove beetle adults. In addition, Beauveria bassiana, azadirachtin, kinoprene, organic oils, and the plant growth regulators did not negatively affect A. coriaria development. However, Beauveria bassiana did negatively affect rove beetle prey consumption. This study demonstrated that A. coriaria is not compatible with the pesticides acetamiprid, lambda-cyhalothrin and cyfluthrin, whereas there is compatibility with organic oils, Beauveria bassiana, azadirachtin, and the plant growth regulators. As such, these compounds may be used in combination with A. coriaria in greenhouse production systems.

As formigas cortadeiras (Hymenoptera-Formicidae) estão distribuídas desde o sul dos Estados Unidos até a Argentina. São herbívoros comuns de florestas que coletam material vegetal para cultivar um fungo mutualista específico. São conhecidas pelo seu papel ecológico na aeração do solo, na infiltração da água e na ciclagem de nutrientes. Atividades humanas, como o desmatamento e a agricultura, afetam o ambiente, alterando também o comportamento das saúvas, que acabam atacando os espécimes cultivados. Devido aos prejuízos causados à agricultura, novos inseticidas específicos são necessários. Muitos trabalhos têm sido desenvolvidos usando metabólitos secundários para essa finalidade. Este trabalho tem como objetivo avaliar o efeito da cafeína na mortalidade de Atta sexdens rubropilosa (Forel, 1908) e no crescimento in vitro de seu fungo mutualístico Leucoagaricus gongylophorus (Möller) Singer (Leucocoprineae: Agaricaceae), obtidos de sauveiros mantidos em laboratório. Foram utilizadas quatro concentrações de cafeína, 0,01%, 0,05%, 0,10% e 0,50%. Mortalidade das formigas foi avaliada pelo ensaio de ingestão, acrescentando a cafeína a dietas artificiais sólidas. A cafeína foi incorporada ao meio de cultura para medir a sua influência no crescimento in vitro. Independente das concentrações de cafeína, esse metabólito parece atuar como repelente para a saúvas. A respeito do fungo, quanto maior a concentração de cafeína, menor o crescimento in vitro. Inibição do crescimento foi observada em 0,10% e 0,50% e morte do fungo foi observada em algumas amostras Em conclusão, os resultados sugerem que a cafeína pode ser usada como fungicida, sendo adicionada a iscas que serão coletadas pelas formigas e carregada aos ninhos, causando a redução do fungo e/ou a morte e consequentemente, a morte das formigas. / The leaf-cutting ants (Hymenoptera-Formicidae) are found from south of United States to Argentina. They are common florest herbivorous which collect plant material to feed a specific mutualist fungus. These insects are known by their ecological role at soil aeration, water permeation and nutrient cycling. Human activities, like deforestation and agriculture, affect the environment, affecting the behavior of leaf-cutting ants, which start to attack the crops. Due to crops lost, new specific pesticides are needed. Several researches have been developed using secondary metabolites for this purpose. The present work aimed to evaluate the effect of caffeine at Atta sexdens rubropilosa (Forel, 1908) mortality, and at in vitro growth of the mutualist fungus Leucoagaricus gongylophorus (Möller) Singer (Leucocoprineae: Agaricaceae), collected from laboratory nests. Four caffeine concentrations were tested: 0.01%, 0.05%, 0.10% and 0.50%. Ant\'s mortality was evaluated by ingestion assay, adding caffeine to artificial diets. Caffeine was added to culture medium, to measure its influence on in vitro fungus growth. Despite caffeine concentrations, this compound seems to act as repellent to ants. Concerning to the fungus, the higher the caffeine concentration, the lower the in vitro fungus growth. Growth inhibition was observed at both 0.10% and 0.50% concentrations and death of fungus was observed in some samples. In conclusion, the results suggest that caffeine could be used as fungicide, being added to baits which could be collected by ants and carried to the nests, causing fungus reduction and/or death and consequently, the death of the nests.

A castanheira (Bertholletia excelsa Humb. & Bonpl.) é uma árvore nativa da região Amazônica muito valorizada por suas sementes, as castanhas-do-Brasil, que apresentam alto valor nutritivo e são uma rica fonte de selênio, um agente antioxidante. O Brasil está entre os países que mais produzem e exportam estas castanhas. As condições climáticas da região Amazônica, assim como as demais etapas da cadeia produtiva, podem favorecer a infecção fúngica neste substrato, principalmente por Aspergillus nomius, espécie extremamente relacionadas à produção de aflatoxinas. Esta micotoxina está associada ao desenvolvimento de tumores, imunossupressão e alterações hepáticas tornando-a um risco para a saúde pública. Sendo assim, a realização de estudos que forneçam informações adequadas sobre o comportamento de A. nomius é de extrema relevância, pois contribuem no conhecimento das condições propícias para a produção de aflatoxinas. Neste contexto, o objetivo deste estudo foi avaliar a resposta ecofisiológica de cepas de A. nomius isoladas de castanhas-do-Brasil (crescimento micelial, expressão gênica e produção de aflatoxinas) em diferentes temperaturas (25, 30 e 35 °C). O crescimento micelial foi mensurado diariamente a partir da inoculação de 8 cepas em ágar coco, mantidas no escuro até 7 dias. A partir destas colônias foi feita análise da expressão dos genes aflR, aflD e aflQ, envolvidos na biossíntese das aflatoxinas, com utilização de PCR em Tempo Real. Com as mesmas colônias também foi feita análise do potencial aflatoxigênico (B1, B2, G1 e G2) qualitativo (Cromatografia em Camada Delgada) e quantitativo (Cromatografia Líquida de Alta Eficiência). A temperatura ideal para crescimento micelial das cepas de A. nomius foi 30 °C. Esta condição foi a melhor para a expressão dos genes aflR, aflD e aflQ. Contudo, o gene aflQ também apresentou alta expressão a 25 °C e foi o gene mais expresso em todas as temperaturas avaliadas. Em relação ao potencial toxigênico das cepas, a maior produção ocorreu a 25 °C. Em todas as temperaturas avaliadas houve maior produção de aflatoxinas do grupo B do que do grupo G. Pôde-se observar que a temperatura que propiciou a maior produção destas toxinas coincide com as da região Amazônica, território nativo das castanheiras. Com base nos resultados reportados, este estudo poderá servir como ferramenta na elaboração de eficientes estratégias para o controle de A. nomius e aflatoxinas em castanhas-doBrasil. / The Brazil nut tree (Bertholletia excelsa Humb. & Bonpl.) is an Amazonian native species that produce seeds with high nutritional value and rich source of selenium, an antioxidant agent. Brazil is one of the major producers and exporters of these nuts. The Amazon weather conditions in the production area and also on the productive chain play a critical role in the fungal infection, specially by Aspergillus nomius, important species associated to the aflatoxin contamination. This mycotoxin is related to the development of tumors, immunosuppression and liver alterations that becomes a risk to public health. Therefore, studies that provides adequate informations about the behavior of A. nomius are extremely relevant, contributing to better understand the favorable conditions to the aflatoxins production. The main goal of the present study was to evaluate the ecophysiological response of A. nomius strains isolated from Brazil nuts (mycelial growth, gene expression and aflatoxin production) at different temperatures (25 °C, 30 °C, and 35 °C). The mycelial growth of 8 strains was measured daily for 7 days in coconut agar. From these colonies, the expression of aflR, aflD, and aflQ genes, that are involved in aflatoxin biosynthesis was analyzed by using Real Time PCR. From the same colonies, the aflatoxigenic potential (B1, B2 , G1 and G2 ) were analyzed qualitative and quantitative by Thin Layer Chromatography and High Performance Liquid Chromatography, respectively. Mycelial growth assessment revealed that the optimal temperature for the radial growth rate and the average of final growth was at 30 °C. This was also the best condition for the expression of aflR, aflD, and aflQ genes. However, the aflQ also showed high expression at 25 °C and was the most expressed gene at all evaluated temperatures. The highest aflatoxin production occurred at 25 °C, with higher toxins production on group B than group G. It was possible to notice that the optimum temperature to aflatoxin production coincides with those in Amazon region, the most important producing area. These results also may contribute to enhance the management strategies of aflatroxin control in Brazil nuts.

A degradação enzimática rápida, eficiente e robusta de polissacarídeos derivados de biomassa lignocelulósica é atualmente um grande desafio na produção de biocombustíveis e considerada uma alternativa viável e promissora para se enfrentar a crise energética mundial e diminuir a dependência das fontes fósseis de energia. O bagaço de cana-de-açúcar no Brasil é a principal matéria lignocelulósica sustentável de grande potencial para a produção do etanol de 2ª geração. O principal requisito para a consolidação dessa abordagem é a disponibilidade de enzimas que hidrolisam a celulose, hemicelulose e outros polissacarídeos em açúcares fermentescíveis e em condições adequadas para a utilização industrial. O presente estudo visou à caracterização molecular, estrutural e funcional da endoglucanase GH12 do fungo Aspergillus terreus (AtGH12) por diferentes técnicas. O gene que codifica para essa enzima foi clonado e expressado no fungo filamentoso A. nidulans linhagem A773. A cepa com maior secreção foi selecionada e a sequência da enzima confirmada por espectrometria de massas MALDI TOF MS. Posteriormente, através de estudos funcionais de parametrização enzimática como pH e temperatura ótimos, estabilidade térmica, efeitos supressores e potencializadores de aditivos, a enzima AtGH12 foi caracterizada bioquímica e fisicamente. A espectrometria de massas do substrato hidrolisado pela catálise enzimática foi tomada como uma forma de investigar o padrão de clivagem da hidrólise e estudo do reconhecimento enzima/substrato para a AtGH12. As caracterizações estruturais das enzimas recombinantes obtidas utilizando as técnicas de espalhamento dinâmico de luz, dicroísmo circular, espalhamento de raios X a baixo ângulo e gel nativo serviram para determinação do enovelamento e estado oligomérico em solução da AtGH12. Com o intuito de fornecer subsídios para o desenvolvimento de coquetéis enzimáticos mais eficazes para hidrólise da biomassa lignocelulósica, a atividade da AtGH12 foi avaliada frente ao bagaço de cana-de-açúcar pré-tratados pelos processos hidrotérmicos e organossolve. Posteriormente, o seu grau de sinergismo nesse tipo de substrato foi determinado com o coquetel enzimático comercial Acellerase&reg. / Fast, more efficient and robust enzymatic degradation of lignocellulosic biomassderived polysaccharides is currently a major challenge in the production of biofuels and considered a feasible and promising alternative to confront the global energy crisis and reduce the dependence on fossil energy resources. The sugarcane bagasse in Brazil is the most abundant and sustainable lignocellulosic material for the production of 2nd generation ethanol. The main requirement for the consolidation of this approach is the availability of enzymes that hydrolyze cellulose, hemicelluloses and other polysaccharides into fermentable sugars suitable for industrial use. The present study was aimed at molecular, structural and functional characterization of an endoglucanase from the fungus Aspergillus terreus (AtGH12) using different techniques. The gene encoding this enzyme has been cloned and expressed in the filamentous fungus Aspergillus nidulans strain A773. The strain with increased secretion was selected and the enzyme sequence was confirmed by mass spectroscopy MALDI TOF MS. Later, functional studies such as analysis of optimal pH and temperature, thermal stability, suppression and enhance effects of additives were applied to the AtGH12 characterization. The mass spectrometry of hydrolyzed substrate from the enzyme catalysis was acquired as a way to investigate the cleavage pattern of hydrolysis and the study of the enzyme/substrate interaction. Structural characterization of the recombinant enzymes was obtained using techniques such as dynamic light scattering, circular dichroism as well as small angle X-ray scattering and native gel, aided to determine the folding and oligomeric state of AtGH12 in solution. In order to provide support for the development of more effective enzyme cocktails for hydrolysis of lignocellulosic biomass, the activity of AtGH12 was evaluated using sugarcane bagasse pretreated by hydrothermal and organosolv processes. Subsequently, the degree of synergism in this type of substrate was measured using a commercial enzyme cocktail Acellerase&reg.

A vinhaça e o bagaço de cana são os principais subprodutos oriundos do processamento da cana-de-açúcar nas indústrias sucroalcooleiras, sendo geradas grandes quantidades dos mesmos. O fungo basidiomiceto Pleurotus ostreatus tem a capacidade de degradar materiais lignocelulolíticos e produzir enzimas lignolíticas de interesse para as indústrias. Com o objetivo de avaliar a produção das enzimas lacases e peroxidase, o fungo Pleurotus ostreatus, foi cultivado em meio contendo bagaço pré-tratado e vinhaça, ou em meio contendo apenas vinhaça, em sistema de fermentação semissólido ou submerso; as enzimas extracelulares foram avaliadas após 7, 10 e 12 dias de cultivo. O bagaço peneirado foi considerado pré-tratado fisicamente (T1); para o pré-tratamento T2 o bagaço umedecido foi submetido a autoclave (121°C e 1 atm por 15 min); nos pré-tratamentos químicos, T3 e T4, o bagaço foi tratado com peróxido de hidrogênio e hidróxido de sódio nas seguintes concentrações: 0,75% H2O2 + 0,75% NaOH (T3) e 0,75% H2O2 + 1% NaOH (T4) na proporção 1:10 (p/v) e, em seguida foram submetidos à autoclave (121°C e 1 atm por 15min). A vinhaça utilizada foi proveniente de uma indústria sucroalcooleira (V1) e outra de destilaria (V2); a composição físico-química mostrou que a primeira possuía os índices de matéria orgânica e fósforo mais elevados que na vinhaça V2, enquanto que a relação C:N foi menor na vinhaça V1. Os extratos enzimáticos foram obtidos após filtração do meio submerso; para o meio semissólido foi necessário a adição de tampão citrato (1:5 p/v) antes da filtração. A atividade de lacasse e peroxidase em meio submerso, nos tratamentos com a vinhaça V1, foi superior ao observado em meio semissólido. A produção das enzimas em fermentação submersa, utilizando a vinhaça V1, apresentou valores de atividade de lacase, no tratamento TL1 e TL2, de 784,9 e 707,5 U.L-1, com atividade específica de 3,04 e 2,86 U.mg-1, respectivamente, e a amostra VL1, contendo apenas vinhaça, de 1,91 U.mg-1, no 12º dia de fermentação. Os valores mais altos de atividade de peroxidase foram obtidos nos tratamentos TL1, TL2, VL1, com 133,1; 131,2 e 126,1 U.L-1, respectivamente, após 12 dias de cultivo. A maior atividade específica obtida foi na VL1 (0,86 U.mg-1) no 7º dia de cultivo. O pré-tratamento físico do bagaço mostrou melhores condições para a produção das enzimas. Para a produção da lacase e da peroxidase é fundamental a composição da vinhaça. / The vinasse and bagasse are the principal by-products derived from the processing of sugarcane in the sugarcane industry, which generated large amounts of them. The basidiomycete fungus Pleurotus ostreatus, has the ability to degrade lignocellulolytic materials and produce lignolíticas enzymes of interest to industry. In order to evaluate the production of laccase and peroxidase enzymes, fungus P. ostreatus was grown in medium containing pre-treated bagasse and vinasse, or in medium containing only vinasse in semi-solid or submerged fermentation system; extracellular enzymes were evaluated after 7, 10 and 12 days of cultivation. The screened bagasse was considered pretreated physically (T1); for the pretreatment T2 moistened residue was subjected to autoclaving (121°C and 1 atm for 15 min). The chemical pretreatments, T3 and T4, the residue was treated with hydrogen peroxide and sodium hydroxide solution in the following concentrations 0,75% H2O2 + 0,75% NaOH (T3) and 0,75% H2O2 + 1% NaOH (T4) 1:10 (w/v) and then underwent autoclaving (121°C and 1 atm for 15 min). Vinasse used was coming from a sugar and alcohol industry (V1) and a distillery (V2); the physico-chemical composition showed that the former had the rates of organic matter and phosphorus higher than in V2 vinasse, whereas the C: N ratio was lower in V1 vinasse. The enzymatic extracts were obtained after filtration medium the submerged; to semisolid medium was necessary the addition of citrate buffer (1:5 w/v) prior to filtration. The activity of peroxidase and lacasse in submerged medium, in the treatments with the V1 vinasse, was higher than observed in semi-solid medium. The production of enzymes by submerged fermentation using the vinasse V1, presented laccase activity values, in the treatment TL1 and TL2, of 784,9 and 707,5 UI.L-1, with specific activity of 3,04 e 2,86 U.mg-1, on the 12th day of fermentation. Higher values peroxidase activity were obtained in the treatments TL1, TL2, VL1, with 133,1; 131,2 and 126,1 UI.L-1, respectively, after 12 days of culture. The highest specific activity was obtained at VL1 (0,86 U.mg-1) on the 7th day of culture. Physical bagasse pretreatment showed better conditions for the production of enzymes. For the production of the laccase and peroxidase is fundamental composition of vinasse.