Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-18T10:55:48Z No. of bitstreams: 1 Dissertação - Juliana Camila Lopes Cavaion - 2012.pdf: 2928983 bytes, checksum: 908363ad8b87e3a821dd54fe80dcb4ce (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-18T14:24:31Z (GMT) No. of bitstreams: 1 Dissertação - Juliana Camila Lopes Cavaion - 2012.pdf: 2928983 bytes, checksum: 908363ad8b87e3a821dd54fe80dcb4ce (MD5) / Made available in DSpace on 2014-12-18T14:24:32Z (GMT). No. of bitstreams: 1 Dissertação - Juliana Camila Lopes Cavaion - 2012.pdf: 2928983 bytes, checksum: 908363ad8b87e3a821dd54fe80dcb4ce (MD5) Previous issue date: 2012-06-04 / Filamentous fungi have cytochrome P450 enzyme system similar to mammals whose application allows catalyze phase I oxidative reactions of metabolism. Obtaining functionalized derivatives by these microorganisms was the main purpose of this dissertation. Studies in silico to predict metabolism in the programs MetaPrint2D and SMARTCyp indicated S-oxidation, aromatic hydroxylation and dealkylation reactions, as the most likely S-oxidation in thiomorpholine ring of 2-[4-(1,4-thiazinan-4-ylsulfonyl)phenylcarbamoyl]benzoic acid (LASSBio-596). The filamentous fungi Absidia blakesleana ATCC 26617, Aspergillus candidus ATCC 1009, Beauveria bassiana ATCC 7159, Cunninghamella echinulata ATCC 9244, Cunninghamella echinulata ATCC 9245, Cunninghamella elegans ATCC 36112, Fusarium roseum ATCC 14717 and Streptomyces vendulae ATCC 8664 were used for biotransformation of LASSBio-596. In the reaction conditions applied, substrate dissolved in methanol with concentration of 0.25 mg/mL added to liquid culture medium PDSM and incubated at 27 °C with 200 rpm for 96 hours, the fungus Beauveria bassiana ATCC 7159 performed S-oxidation in the thiomorpholine ring resulting in sulfone derivative of LASSBio-596, called LaBioCon 214, yield of 5.84 %, and it was found the hydrolysis of amide reaction obtaining the compound 4-(thiomorpholinosulfonyl)aniline, LaBioCon 202, yield of 13.8 %. It was noted the solvent used affects the biotransformation process. The monitoring of the reactions and purification of the derivatives obtained were performed by high performance liquid chromatography with ultraviolet detector at 267 nm and preparative chromatography with column Microsorb 100-5 C18 (250 x 10 mm, 5 μm). Subsequently, sulfone derivative was obtained, called LaBioCon 223, by synthesis. Structural characterization was made by classical methods such as 1H NMR and mass spectrometry. / Os fungos filamentosos possuem o sistema enzimático citocromo P450 semelhante ao de mamíferos, cuja aplicação permite catalisar reações oxidativas de fase I do metabolismo. A obtenção de derivados funcionalizados por estes microrganismos foi o objetivo principal deste trabalho. Estudos in silico de previsão do metabolismo nos programas MetaPrint2D e SMARTCyp indicaram reações de S-oxidação, hidroxilação aromática e desalquilação, sendo mais provável a S-oxidação no anel tiomorfolina do ácido 2-[4-(1,4-tiazinan-4-ilsulfonil)fenilcarbamoil]benzóico (LASSBio-596). Os fungos filamentosos Absidia blakesleana ATCC 26617, Aspergillus candidus ATCC 1009, Beauveria bassiana ATCC 7159, Cunninghamella echinulata ATCC 9244, Cunninghamella echinulata ATCC 9245, Cunninghamella elegans ATCC 36112, Fusarium roseum ATCC 14717 e Streptomyces vendulae ATCC 8664 foram empregados para biotransformação do LASSBio-596. Nas condições reacionais aplicadas, substrato solubilizado em metanol com concentração de 0,25 mg/mL adicionado ao meio de cultura líquido PDSM e incubado a 27 °C com 200 rpm por 96 horas, a Beauveria bassiana ATCC 7159 promoveu a S-oxidação no anel tiomorfolina resultando no derivado sulfona do LASSBio-596, denominado LaBioCon 214, com rendimento de 5,84 %, e constatou-se a reação de hidrólise da amida obtendo o composto 4-(tiomorfolina-sulfonil)anilina, LaBioCon 202, com rendimento de 13,8 %. Verificou-se que o solvente utilizado interfere no processo de biotransformação. O monitoramento das reações e a purificação dos derivados obtidos foram realizados por cromatografia líquida de alta eficiência com detector ultravioleta em 267 nm e cromatografia preparativa com coluna Microsorb 100-5 C18 (250 x 10 mm, 5 μm). Posteriormente, foi obtido o derivado sulfona, LaBioCon 223, por via sintética. A caracterização estrutural foi realizada por métodos clássicos como a espectroscopia de ressonância magnética nuclear de hidrogênio e espectrometria de massas.

Formulações a base de dispersões oleosas de conídios aéreos de fungos entomopatogênicos apresentam diversas vantagens em comparação à aplicação de produtos não formulados; entretanto, a complexidade e a falta de compreensão dos fatores que influenciam a persistência desse tipo de formulação fazem com que ainda seja pouco utilizada. O efeito de diferentes níveis de secagem de conídios, tipo de óleo, métodos de extração, estabilidade de blastosporos, compatibilidade de surfactantes e adição de aditivos foram avaliados para o desenvolvimento de micopesticidas formulados em óleo. Os melhores resultados obtidos com os conídios em óleo foram avaliados quanto à proteção conferida à radiação UV e a eficiência das formulações na mortalidade de Tenebrio molitor. Zeolita foi a argila mais eficiente na manutenção da viabilidade de blastosporos de M. anisopliae e B. bassiana, e apesar de resultados promissores a estabilidade desses propágulos foi muito inferior a de conídios aéreos. Vinte surfactantes foram avaliados quanto a inocuidade aos conídios, em dois ensaios, culminando com a seleção dos quatro mais compatíveis, dois deles E5 e E7, avaliados nos experimentos posteriores demonstraram bom potencial para compor a formulação como agentes emulsionantes. Dois aditivos foram avaliados na formulação e respostas distintas foram observadas. O A1 foi altamente incompatível, enquanto diferentes concentrações de A2 (1, 3 e 5%) incrementaram a sobrevivência dos conídios em até sete vezes. As vantagens da presença do A2 ficaram evidentes também em combinações de óleo com os quatro emulsionantes selecionados. As dispersões oleosas proporcionaram sobrevivência até sete vezes superior para conídios expostos a radiação UV em relação a conídios não formulados e dois experimentos comprovaram que as formulações oleosas testadas foram mais eficientes no controle de insetos, com reduções de até 2,3 vezes no tempo de sobrevivência de insetos. Os 12 bioensaios com mais de 20.000 leituras de viabilidade descritos neste trabalho permitiram um melhor entendimento dos fatores críticos que afetam a vida de prateleira de conídios, e indicam procedimentos que podem ser utilizados para melhorar a estabilidade de micopesticidas formulados em óleo. / Oil-based formulations of aerial conidia of entomopatogenic fungi have several advantages compared to the use of unformulated products; however, the complexity and lack of understanding of the factors that influence the persistence of this type of formulation make it still not widely used. The effect of different levels of drying conidia, oil type, extraction methods, blastospores stability, compatibility of surfactants and addition of additives were evaluated for the development of a mycopesticide oil formulation. The best results obtained with aerial conidia were evaluated regarding the protection against UV radiation and the efficacy of the formulations to control Tenebrio molitor. Zeolita was the most compatible clay in maintaining the viability of blastospores of M. anisopliae and B. bassiana, and despite promising results in the stability, the shelf life of these propagules was much lower than aerial conidia. Twenty surfactants were evaluated for safety to conidia in two trials resulting in the selection of the four most compatible, two of them E5 and E8, were evaluated in subsequent experiments demonstrating good potential as emulsifiers in oil-based formulations. Two additives were evaluated in the formulation and distinct responses were observed. A1 was highly toxic to the fungi, while different concentrations of A2 increased the survival of the conidia up to seven times. The advantages of A2 were evident also in combinations of oil with the four selected emulsifiers. Oil-based formulation provided survival up to seven times higher for conidia exposed to simulated UV radiation compared to unformulated conidia, and two experiments showed that oil formulations were more efficient in the insect control, with reductions of up to 2.3 times on the survival time of insects. Twelve bioassays and over 20.000 viability readings described in this work, allowed a better understanding of the critical factors that affect the shelf-life of conidia, and indicate methods that can be used to improve the stability of mycopesticides in oil-based formulations.

This dissertation describes efforts to improve the oxidative capacity of n-alkane- induced Beauveria bassiana; a fungus and a versatile whole cell biocatalyst used in the biotransformation of steroids. n-Hexadecane was used as the carbon source during the growth of B. bassiana, presumably to induce the expression of oxidative enzymes, thus enhancing the oxidation of unactivated carbons. Dehydroepiandrosterone (DHEA) is an essential endogenous male-hormone and serves as a metabolic intermediate in the production of more potent androgens. Using DHEA as a substrate also provides the opportunity to study the hydroxylation of an unfunctionalized carbon, an attractive reaction that produces valuable intermediates for chemical synthesis. Results showed that exposing and inducing cells in n-hexadecane improves the synthesis of 11α-hydroxy derivatives. Reactions were carried out with cells grown on n-hexadecane, resulting in 65 ± 6.3 % conversion of DHEA to androstenediol (40.3% mM) and 3β,11∝,17β- trihydroxyandrost-5-ene (22.8% mM), as determined by HPLC, NMR and LCMS analyses. However, experiments with non-induced cells resulted in a poor substrate conversion (17%). To extend use of B. bassiana to pharmaceutical applications, it was necessary to optimize reaction conditions such as biocatalyst preparation, substrate concentration, agitation reaction temperature and pH. Higher substrate conversion, selectivity and yield of desired product were achieved with the reactor arrangement of “Resting Cells”. The apparent rate of reaction fits a Michaelis-Menten kinetic model with a maximum reaction rate of 4.45 mM/day, revealing that the transformation of intermediate androstenediol to desired 3β,11∝,17β-trihydroxyandrost-5-ene is the limiting step in the reaction. Interestingly, when a diluted amount of substrate was used, a higher yield of 11∞-hydroxy steroid was achieved. Also, reactions at 26°C with pH ranges between 6.0 and 7.0, resulted in the highest conversion (70%) and the higher product yield (45.8%). The maximum conversion of DHEA (71%) was achieved in experiments with high biomass loading, and the increment of desired product yield (11∝-hydroxy) was directly proportional to the amount of biomass used. Moreover, a high VMax/KM value was achieved with high biomass yields. Interestingly, the changes in biomass yield did not have a considerable effect on reaction selectivity. The main drawbacks of biocatalysis for production of steroids were addressed and approaches to minimize the drawbacks have been presented. The production of desired product (11∝-DHEA) was significantly improved using cells previously adapted to n-hexadecane.

Echinacea purpurea is a native herbaceous perennial with substantial economic value for its medicinal and ornamental qualities. Arbuscular mycorrhizae are symbiotic fungi that form relationships with plant roots and are known to enhance growth in the host. Mycorrhizae and other fungal endophytes often affect stress resistance and secondary metabolism in the host, as well as the ecology of other endophytes in the plant. A newly emerging paradigm in sustainable biotechnique is the targeted use of fungal endophytes to enhance growth and secondary metabolism in crops. Many of the therapeutic compounds in E. purpurea could be affected by fungal colonization. In this research the effects of inoculation of Echinacea purpurea with two classes of fungal endophytes: the arbuscular mycorrhizal fungi Glomus intraradices and Gigaspora margarita and the entomopathogenic endophyte Beauveria bassiana were evaluated . Endophyte colonization and impacts on plant growth and phytochemistry were tested in multiple greenhouse experiments. Arbuscular mycorrhizae and B. bassiana effectively colonized E. purpurea with some significant interactive effects. Consistent, substantial, and significant increases in all growth parameters were observed in mycorrhizal plants; mycorrhizal plants produced up to four times the biomass of controls in 12 weeks. Broad spectrum changes in fertilization were necessary to produce mycorrhizal and nonmycorrhizal samples of equal size, and severely nutrient-limited mycorrhizal E. purpurea seedlings maintained growth rates comparable to well fertilized samples. Treatment with B. bassiana had minor and inconsistent effects on some plant growth parameters, and there were significant interactive effects with arbuscular mycorrhizae. Phytochemical concentrations in all metabolite classes tested responded significantly to inoculation with both classes of fungal endophytes. Changes were observed in various pigments, caffeic acid derivatives, alkylamides, and terpenes. Many of the affected compounds have important roles in metabolism or have bioactive value as natural products. When considered from a net production perspective (concentration X dry weight), compared to controls, plants inoculated with endophytes produced as much as 30 times the content of some compounds in 12 weeks. This work effectively demonstrates that fungal endophytes can enhance the bioactivity of plant tissues and the production of natural products in E. purpurea.

Echinacea purpurea is a native herbaceous perennial with substantial economic value for its medicinal and ornamental qualities. Arbuscular mycorrhizae are symbiotic fungi that form relationships with plant roots and are known to enhance growth in the host. Mycorrhizae and other fungal endophytes often affect stress resistance and secondary metabolism in the host, as well as the ecology of other endophytes in the plant. A newly emerging paradigm in sustainable biotechnique is the targeted use of fungal endophytes to enhance growth and secondary metabolism in crops. Many of the therapeutic compounds in E. purpurea could be affected by fungal colonization. In this research the effects of inoculation of Echinacea purpurea with two classes of fungal endophytes: the arbuscular mycorrhizal fungi Glomus intraradices and Gigaspora margarita and the entomopathogenic endophyte Beauveria bassiana were evaluated . Endophyte colonization and impacts on plant growth and phytochemistry were tested in multiple greenhouse experiments. Arbuscular mycorrhizae and B. bassiana effectively colonized E. purpurea with some significant interactive effects. Consistent, substantial, and significant increases in all growth parameters were observed in mycorrhizal plants; mycorrhizal plants produced up to four times the biomass of controls in 12 weeks. Broad spectrum changes in fertilization were necessary to produce mycorrhizal and nonmycorrhizal samples of equal size, and severely nutrient-limited mycorrhizal E. purpurea seedlings maintained growth rates comparable to well fertilized samples. Treatment with B. bassiana had minor and inconsistent effects on some plant growth parameters, and there were significant interactive effects with arbuscular mycorrhizae. Phytochemical concentrations in all metabolite classes tested responded significantly to inoculation with both classes of fungal endophytes. Changes were observed in various pigments, caffeic acid derivatives, alkylamides, and terpenes. Many of the affected compounds have important roles in metabolism or have bioactive value as natural products. When considered from a net production perspective (concentration X dry weight), compared to controls, plants inoculated with endophytes produced as much as 30 times the content of some compounds in 12 weeks. This work effectively demonstrates that fungal endophytes can enhance the bioactivity of plant tissues and the production of natural products in E. purpurea.

No description available.

Tomato (Solanum lycopersicum L.) produces an array of volatile secondary metabolites that act as constitutive and induced defenses against a variety of insect pests and diseases. We studied the effect of beneficial microorganisms, an arbuscular mycorrhiza fungus (AM), Glomus intraradices, an entomopathogenic fungus (Bb), Beauveria bassiana and a combination of both (AM+Bb), as well as, pests, such as rootknot nematode (RKN) (Meloidogyne incognita) and beet armyworm (BAW) (Spodoptera exiguae H.) on the production of volatile compounds in the leaves and roots of tomato. Benzyl alcohol, 3-hexenoic acid, total other compounds and β-myrcene were significantly increased (P<0.05) in the leaves by AM and Bb colonization. Upon herbivory, control plants showed a trend for high volatile contents among all four treatment groups contrary to the trend observed without herbivory, when control showed low volatile contents. Herbivory significantly decreased all the volatiles in the leaves compared to those without herbivory. Pest bioassay revealed that these two fungi together can significantly induce resistance against BAW. In the experiment with RKN, a trend was observed with low production of most volatile compounds in the leaves from the RKN plants. Varying durations of herbivory exposure had significant effects on many leaf volatile compounds compared to those without herbivory. Effect of RKN was significant (P<0.05) on the production of methyl salicylate, and (Z)-geraniol in the roots. Interaction effect of RKN with 18 hours of herbivory was significant for (Z)-geraniol, and with 42 hours of herbivory, it was significant for (Z)-geraniol, benzyl alcohol, and total volatiles in the roots. Beet armyworm preferred RKN plants and caused greater damage to them compared to the control plants. Semi-quantitative RT-PCR showed higher expression of the SlSAMT gene in the roots colonized with RKN, compared to the control roots and those from a resistant line. The experiments demonstrated that interaction with these organisms can change the volatile compounds in the leaves and roots of tomato plant, can alter herbivore preference, and can upregulate defense genes such as SlSAMT.

University, Diss., 2003--Heidelberg.

Made available in DSpace on 2014-06-11T19:28:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-04Bitstream added on 2014-06-13T19:16:46Z : No. of bitstreams: 1 takada_hm_me_botfca.pdf: 587174 bytes, checksum: 68fcfeed820ef9c331817e76b2f9fab3 (MD5) / Com o objetivo de avaliar a patogenicidade e a virulência de fungos entomopatogênicos ao gorgulho aquático do arroz irrigado, Oryzophagus oryzae, foram testados 24 isolados de Metarhizium anisopliae e 2 de Beauveria bassiana. Os bioensaios foram realizados em condições controladas (26+1°C e 12 horas de fotofase). Nos testes para determinar a concentração adequada, a espécie M. anisopliae, isolado E9, apresentou maior patogenicidade quando comparado aos isolados de B. bassiana. A mortalidade confirmada, provocada pelo M. anisopliae, teve início no 4º dia, aumentando em seguida até estabilizar-se no 8º dia. Para a seleção dos isolados de M. anisopliae, adultos dos insetos foram inoculados com 1 mL de suspensão fúngica a 5x108 conídios/mL (1,6x104 conídios/mm2). Todos os isolados foram patogênicos ao inseto, sendo que a mortalidade variou entre 9,3 e 85,3 %. Os isolados de M. anisopliae mais patogênicos foram CB-103, CB-104, CB-233, E9 e CB-10. No teste de produção, os isolados CB-104, CB-233 e CB-103 foram superiores ao padrão E9, apresentando-se promissores para a utilização no controle de Oryzophagus oryzae. / Strains of Metarhizium anisopliae and Beauveria bassiana were evaluated concerning their pathogenicity and virulence for control of the rice water weevil, Oryzophagus oryzae. Bioassay conditions were 26+1ºC and 12 h. light. M. anisopliae (E9 strain) was been more pathogenic than two Beauveria bassiana strains (CB-66 and CB-74). For Metarhizium anisopliae (E9 strain), the mean mortality started at 4th day, increasing soon after until stabilize at 8 th day. A screening of M. anisopliae isolates was done inoculating adults of the insect with 1 mL of spore suspension containing 5x108 conídia/mL (1.6x104 conídia/mm2). All isolates were pathogenic to the insect, causing mortality between 9.3 and 85.3%. The most pathogenic isolates were CB-103, CB-104, CB-233, E9 and CB-10. These 6 isolates were evaluated concerning their spore production on cooked rice. The isolates CB-104, CB-233 and CB-103 afforded the highests spores production, significantly higher compared to E9, showing potential to be used for Oryzophagus oryzae control.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-29Bitstream added on 2014-06-13T20:44:14Z : No. of bitstreams: 1 mochi_da_dr_jabo.pdf: 1918684 bytes, checksum: f29aa98fefb34604c991782bab720e3b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A mosca-dos-chifres Haematobia irritans é um dos principais ectoparasitos associados a bovinos em pastagens. O presente trabalho objetivou analisar a ação patogênica dos isolados E9, IBCB425 e IBCB159 do fungo Metarhizium anisopliae, JAB06, JAB07 e AM09 de Beauveria bassiana, IBCB133 e CB75 de Isaria fumosorosea (=Paecilomyces fumosoroseus) e CG189 e CG195 de Isaria farinosa (=Paecilomyces farinosus) para a mosca-dos-chifres H. irritans em laboratório e campo. Avaliou-se em laboratório, a ação de todos os isolados dos fungos sobre ovos, larvas, pupas e adultos. Grupos de 30 ovos foram inoculados com suspensões fúngicas, e colocados sobre papel de filtro esterilizado, em 5 g de fezes bovinas frescas, acondicionadas em tubos de plástico. No ensaio com larvas, pedaços de papel filtro esterilizado contendo na superfície grupos de 30 ovos foram colocados sobre 5 g de fezes bovinas frescas, contidas em tubos de plástico, às quais incorporaram-se diversas concentrações de conídios mg fezes-1. No ensaio com ovos e larvas, o término do experimento ocorreu com a emergência dos adultos. Grupos de 20 pupas foram imersas por 60 segundos em suspensão do fungo, e colocadas em placas de Petri. Após a emergência, os adultos foram transferidos para gaiolas de isopor pequenas. Para o ensaio com adultos, grupos de 30 adultos foram separados e pulverizados com 0,3 mL das suspensões de conídios. Doze horas após, as moscas foram transferidas para gaiolas de isopor e avaliadas diariamente até o 15o dia. Para todos os ensaios, foram utilizadas suspensões contendo 106, 107 e 108 conídios mL-1. O isolado que promoveu os melhores resultados nos ensaios anteriores, foi usado em um experimento de campo para avaliar a ação fúngica em larvas e adultos de H. irritans em bovinos naturalmente infestados. Para o controle de larvas, foi oferecido aos bovinos conídios de M. anisopliae isolado... / Horn fly Haematobia irritans is one of the main ectoparasites associated to cattle on pastures. The present work had the objective of analyzing the pathogenic action of the isolates E9, IBCB425 and IBCB159 of the fungus Metarhizium anisopliae, JAB06, JAB07 and AM09 of Beauveria bassiana, IBCB133 and CB75 of Isaria fumosorosea (=Paecilomyces fumosoroseus) and CG189 and CG195 of Isaria farinosa (=Paecilomyces farinosus) for horn fly H. irritans, in laboratory and field conditions. The action of all the fungi isolates over the eggs, larvae, pupae and adults were evaluated in laboratory. Groups of 30 eggs were inoculated with fungal suspensions and placed over sterilized filter paper on top of 5 g of fresh bovine feces, put in plastic tubes. In the larvae assay, peaces of sterilized filter paper, containing groups of 30 eggs on the surface, were placed over 5 g of fresh bovine feces, contained in plastic tubes, in which different concentrations of conidia mg feces-1 were incorporated. In the eggs and larvae assay, the end of the experiment matched the emergence of the adults. Groups of 20 pupae were immersed for 60 seconds in the fungus suspension and placed on Petri dishes. After the emergence, the adults were transferred to small Styrofoam cages. In the adults assay, groups of 30 adults were separated and pulverized with 0,3 ml of the conidia suspensions. After twelve hours, the adults were transferred to Styrofoam cages and the evaluations were done daily until the 15th day. Suspensions containing 106, 107 and 108 conidia ml-1 were used for all the assays. The isolate that promoted best results on the previous assays was used in a field experiment, where the fungal action on larvae and adults of H. irritans, on naturally infested bovines, was evaluated. For the larvae control conidia of the E9 isolate of M. anisopliae, microencapsulated... (Complete abstract click electronic access below)