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Agradecemos a compreensão. on 2016-06-08T17:11:23Z (GMT) / Submitted by ISABELLA DE CENÇO LOPES null (isalops@hotmail.com) on 2016-06-09T11:31:06Z No. of bitstreams: 1 Isabella de Cenço Lopes.pdf: 907430 bytes, checksum: fce337e1df2e1a5dfa90bd270c59e5d4 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-06-09T17:59:14Z (GMT) No. of bitstreams: 1 lopes_ic_me_sjrp.pdf: 907430 bytes, checksum: fce337e1df2e1a5dfa90bd270c59e5d4 (MD5) / Made available in DSpace on 2016-06-09T17:59:14Z (GMT). No. of bitstreams: 1 lopes_ic_me_sjrp.pdf: 907430 bytes, checksum: fce337e1df2e1a5dfa90bd270c59e5d4 (MD5) Previous issue date: 2016-05-31 / O objetivo deste trabalho foi de aumentar a produção de conídios de Metarhizium anisopliae através de alterações do substrato e das condições de cultivo e produzi-lo em biorreator de bandeja. Os substratos testados foram arroz tipo 1, quirera de arroz e farelo de arroz, sendo o cultivo realizado em embalagens plásticas contendo 10 g de substrato seco. Inicialmente, foi empregado arroz tipo 1 como substrato, variando-se as formas de umidificação no seu preparo, sendo o cultivo realizado em embalagens plásticas contendo 10g de substrato seco. Determinada a condição adequada de umidificação, os substratos arroz tipo 1 e quirera de arroz foram submetidos a diferentes condições de fotoperíodo: escuto contínuo, claro contínuo e escuro e claro alternados. O farelo de arroz foi adicionado a bagaço de cana-de-açúcar de modo a estruturar fisicamente o meio de cultivo. Os melhores resultados foram obtidos com arroz e quirera de arroz. A primeira alternativa de ampliação de escala foi realizada em embalagens plásticas, utilizando arroz tipo 1 e quirera com 500g de substrato seco. A etapa seguinte da ampliação de escala foi em um biorreator de bandeja com aeração realizada sobre a camada de substrato, sendo os testes realizados com arroz tipo 1, e duas espessuras de camada partículas, 2 e 4 cm. Em todos os ensaios, a resposta observada foi a concentração final de conídios. Foi testada ainda a virulência dos conídios produzidos em relação a lagartas de Diatraea flavipennella nas diferentes condições de produção. De acordo com os resultados apresentados, o farelo de arroz não é uma boa opção de substrato devido a sua pouca praticidade de manipulação e a quirera apresentou resultados satisfatórios nos ensaios, podendo ser considerada uma opção viável para utilização industrial devido ao seu baixo custo. O biorreator de bandeja elaborado apresentou bons resultados de produção de conídios em relação a produção em embalagem plástica de maior capacidade com o substrato arroz tipo 1, a qual, no entanto, apresentou produção de esporos inferior à da embalagem de menor capacidade. Os testes de virulência comprovaram a eficiência dos conídios em todos os ensaios com pequenas variações no tempo de mortalidade. / The work targeted the increase of the production of spores of Metarhizium anisopliae through modifications of the substrate and of the cultivation conditions, and produce such spores in a tray bioreactor. Type 1 rice, broken rice and rice bran were tested as substrate, which were cultivated in plastic bags containing 10 g of dry substrate. Alternatives of humidification were tested with type 1 rice. For the best alternative of humidification, type 1 rice and broken rice were submitted to alternatives of different exposures to light, provided by a fluorescent lamp: continuous dark, continuous light, alternation between dark and light. To the bran was added sugar cane bagasse in order to provide physical structure to the culture medium. The best results were obtained with rice and broken rice and the illumination regime does not influence on the spore production. The first attempt of scaling-up the spore production was the use of plastic bags containing 500 g of dry substrate, either rice or broken rice. The next scaling-up step was in a tray bioreactor aerated flowing air parallel to the top of the cultivation medium, using type 1 rice as substrate in two different thickness of medium, 2 and 4 cm. In every experiments, the response variable was the conidia concentration. The virulence against Diatraea flavipennella caterpillars was also tested for spores produced in the different experimental conditions. The results showed that rice bran is not an efficient alternative due to its difficult manipulation, while broken rice produce conidia concentration similar to type 1 rice and can be considered a cheap alternative for industrial production of spores. The tray bioreactor presented similar results to the large capacity plastic bags, which presented lower conidia concentrations than the small capacity container. The virulence experiments showed high efficiency of the conidia in all tested samples, with little variation in the time of lethality.

O objetivo deste estudo foi caracterizar a diversidade de Metarhizium em um plantio de cana-de-açúcar e monitorar a eficácia e persistência do isolado ESALQ 1604 de M. anisopliae, após aplicação em campo, através de técnicas moleculares. Isolados foram recuperados de amostras de solo, raiz e cigarrinha-das-raízes infectadas de duas áreas de um canavial em Iracemápolis-SP, uma com aplicação do fungo (isolado ESALQ 5310) e colheita mecânica e outra sem aplicação e colheita manual antecedida por queimada. Os isolados de insetos foram recuperados de ninfas e adultos coletados 51, 42 e 1 dia (s) antes da aplicação do fungo e 7, 30, 60 e 90 dias após a aplicação. Isolados de solo e raiz foram obtidos 90 dias pré-aplicação do fungo e 30 e 90 dias pós-aplicação. A aplicação de M. anisopliae foi realizada em 09/01/15 com suspensão de 3,72x106 conídios viáveis/mL numa vazão de 150L/ha. Nas análises moleculares também foram incluídos 22 isolados provenientes das mesmas áreas coletados em 18/12/2012. A diversidade haplotípica de Metarhizium foi obtida pela genotipagem de 10 marcadores microssatélites para 213 isolados provenientes de amostras de solos, 22 de raízes e 73 de cigarrinha-das-raízes. A identificação específica dos fungos foi obtida pelo sequenciamento do gene 5\'-TEF de 57 isolados provenientes de solos, 6 de raízes e 14 de cigarrinha-das-raízes selecionados dentre os diferentes haplótipos gerados pelas análises dos marcadores microssatélites. Dentre os 310 isolados genotipados, foram obtidos 156 haplótipos do fungo, destes, 132 provenientes de isolados de solo, 17 de raízes e 20 de cigarrinha-das-raízes. A maior diversidade haplotípica foi encontrada nos solos h=0,989 e a menor em insetos h=0,779. A divergência genética entre isolados provenientes de insetos, solos e raízes foi significativa (ΦST =0.303), sendo a maior proporção da variação dentro (69,6 %) do que entre (30,3 %) esses grupos. A mortalidade de cigarrinha-das-raízes por M. anisopliae antes da aplicação variou de 0 a 14,8% para ninfas e 7,3-18,1% para adultos. Sete dias após a aplicação, observou-se 50% de mortalidade confirmada de ninfas e 10,7% de adultos. O isolado aplicado em campo foi recuperado somente em cigarrinha-das-raízes e nas coletas 7, 30 e 60 dias pós-aplicação, compreendendo 50%, 50% e 70,5% de todos os insetos mortos por M. anisopliae, respectivamente. A população da praga reduziu após 90 dias e não foram observadas cigarrinhas infectadas nesta amostragem. No solo, foram encontradas as espécies M. robertsii (clados Mrob 1, Mrob 2 e Mrob 4), M. anisopliae (Mani 1 e Mani 2) e M. brunneum. Em raízes foram encontradas as espécies M. brunneum e M. anisopliae (Mani 2). Em adultos e ninfas de cigarrinha-das-raízes foram encontrados somente haplótipos de um único clado (Mani 2) de M. anisopliae, e o isolado ESALQ 5310 aplicado nos anos anteriores na área não foi recuperado. Os resultados obtidos neste trabalho revelam a grande diversidade haplotípica de Metarhizium spp. e o impacto das populações locais de M. anisopliae na regulação de cigarrinhas em cana-de-açúcar. / The aim of this study was to characterize the diversity of Metarhizium in a sugarcane plantation and to monitor the efficacy and persistence of the isolate ESALQ 1604 of M. anisopliae after field application, by molecular techniques. Isolates were recovered from samples of soil, root and infected spittlebugs of two areas in Iracemápolis-SP: one with application of the fungus (isolate ESALQ 5310) and mechanical harvesting and another without fungal application and manual harvest preceded by burning. The isolates from insects were recovered from nymphs and adults collected 51, 42 and 1 day (s) before application of the fungus and 7, 30, 60 and 90 days after application. Isolates of soil and root were obtained 90 days pre-application of the fungus and 30 and 90 days post-application. The application of M. anisopliae was carried out in 9/Jan/15 with suspension of 3,72 x 106 viable conidia/mL at a volume rate of 150 l/ha. In the molecular analyses, 22 isolates from the same areas collected in 18/12/2012 were also included. The haplotype diversity of Metarhizium was obtained by genotyping 10 microsatellite markers of 213 isolates from soil samples, 22 from roots and 73 from spittlebugs. The specific identification of fungi was obtained by sequencing the gene 5\'- TEF of 61 isolated from soil, 6 from root and 13 from spittlebug selected among the different haplotypes generated by analysis of microsatellite markers. Among 310 isolates genotyped, 156 haplotypes of the fungus were obtained, of these, 132 from soil isolates, 17 of root and 20 of spittlebug. The highest haplotype diversity was found in soil h=0.989 and the smallest in spittlebug h = 0.779. The genetic divergence among isolates from insect, soil and root was significant (ΦST = 0.303), being the largest proportion of the variation within (69.6%) than among (30.3%) these groups. The mortality of spittlebugs by M. anisopliae before application ranged from 0 to 14.8% for nymphs and 7.3-18.1% for adults. Seven days after application, 50% of nymph mortality and 10.7% of adult mortality was observed. The isolate applied on the field was recovered only in spittlebugs and only 7, 30 and 60 days, post-application, and accounted for 50%, 50% and 70.5% of all insects killed by M. anisopliae, respectively. The pest population reduced after 90 days and no spittlebug infected was observed in this sample date. The species and clades found in isolates from soil were M. robertsii (clades Mrob 1, Mrob 2 and Mrob 4), M. anisopliae (Mani 1 and Mani 2) and M. brunneum. In roots the species found were M. brunneum and M. anisopliae (Mani 2). In adults and nymphs of spittlebug only haplotypes of a single clade (Mani 2) of M. anisopliae were found. The isolate ESALQ 5310 applied in previous years in the area was never recovered. The results obtained in this work revelead the great diversity of haplotype Metarhizium spp. and the impact of local populations of M. anisopliae on population regulation of spittlebugs in sugarcane.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-03-31T10:25:55Z No. of bitstreams: 2 Dissertação - Daniela Cristina da Silva - 2017.pdf: 3223777 bytes, checksum: 67c2832449d10a3a2590e8a834f55b10 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-31T10:44:27Z (GMT) No. of bitstreams: 2 Dissertação - Daniela Cristina da Silva - 2017.pdf: 3223777 bytes, checksum: 67c2832449d10a3a2590e8a834f55b10 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-31T10:44:27Z (GMT). No. of bitstreams: 2 Dissertação - Daniela Cristina da Silva - 2017.pdf: 3223777 bytes, checksum: 67c2832449d10a3a2590e8a834f55b10 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-09 / Entomopathogenic fungi combined with plant extracts may have their development in vitro and their insecticidal activity modified. There are many reports on the activity of entomopathogenic fungi and extracts in insects, but few studies on fungi combined with plant extracts. The objective of this study was to evaluate in vitro the effect of crude ethanol extracts of Artemisia annua, Andrographis paniculata, Curcuma zedoaria, Ginkgo biloba and Rosmarinus officinalis on the Metarhizium anisopliae s.l. IP 46 by the diffusion disc test, to evaluate in vivo susceptibility of Aedes aegypti larvae to extracts by determination the cumulative emergence of adults, development and mortality of larvae for 15 days, then to select the extracts and combine them with IP 46 at 3.3 x 105 conidia.mL-1. There was no significant inhibition germination of IP 46. On the fifteenth day there was a significant effect on cumulative emergence of adults and development of larvae for A. annua at 10 ppm, 33 ppm and 1000 ppm and C. zedoaria at 100 ppm and 333 ppm. There was also a significant effect on accumulated mortality of larvae exposed to G. biloba at 10000 ppm.These extracts with in these concentrations were selected and combined with IP 46 at 3.3 x 105 conidia.mL-1, minus G. biloba which was combined at 5000 ppm after the LC50 and LC90 calculations. These combinations, except for A. annua at 1000 ppm, exhibited a significant increase in larval mortality and/ or showed an effect on development, indicating that combined methods may be more effective in controlling A. aegypti than isolated methods. / Fungos entomopatogênicos combinados a extratos de plantas podem ter o seu desenvolvimento in vitro e sua atividade inseticida in vivo modificada. Existem muitos relatos sobre a atividade de fungos entomopatogênicos e extratos em insetos, porém são poucos os estudos sobre fungos combinados a extratos de plantas. O objetivo do trabalho foi avaliar in vitro o efeito de extratos brutos etanólicos de Artemisia annua, Andrographis paniculata, Curcuma zedoaria, Ginkgo biloba e Rosmarinus officinalis em isolado de Metarhizium anisopliae s.l. IP 46 por meio do teste de disco difusão, avaliar in vivo a suscetibilidade de larvas de Aedes aegypti expostas a esses extratos por meio da avaliação da emergência acumulada de adultos, do desenvolvimento e da mortalidade de larvas, durante 15 dias, para então selecionar os extratos e combiná-los com IP 46 a 3,3 x 105 conídios.mL-1. Observou-se que não houve inibição significativa de IP 46 pelos extratos. No décimo quinto dia houve efeito significativo na emergência acumulada de adultos e no desenvolvimento de larvas expostas a A. annua a 10 ppm, 33 ppm e 1000 ppm e de C. zedoaria a 100ppm e 333 ppm. Também houve efeito significativo na mortalidade acumulada de larvas exposta a G. biloba a 10000 ppm. Esses extratos nessas concentrações foram selecionados e combinados com IP 46 a 3,3 x 105 conídios.mL-1, menos o de G. biloba que foi combinado a 5000 ppm após cálculos da CL50 e CL90. Essas combinações, exceto para A. annua a 1000 ppm, exibiram aumento significativo na mortalidade larval e/ou apresentararam efeito sobre o desenvolvimento, indicando que métodos combinados podem ser mais efetivos no controle de A. aegypti do que métodos isolados.

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.

Diversos estudos evidenciam dezenas de espécies de fungos de diferentes gêneros nas colônias de formigas-cortadeiras. No entanto, a procedência desses microrganismos ainda é incerta. Alguns desses podem ser endofíticos oriundos do material vegetal transportado para o ninho. Assim sendo, determinados fungos endofíticos possuem a capacidade de contaminar a colônia das cortadeiras ou influenciar no seu microbioma simbiótico. Com isso, os endófitos fúngicos têm potencial no controle de formigas-cortadeiras como parceiros mutualistas das plantas. Esse sistema simbiótico pode se tornar uma estratégia no manejo dessa importante praga dentro do ecossistema florestal. O objetivo deste trabalho foi avaliar o potencial de colonização endofítica de mudas de Eucalyptus urophylla pelos fungos Escovopsis sp., Metarhizium anisopliae e Trichoderma strigosellum, e analisar, por meio da avaliação de características biométricas, a influência da inoculação desses microrganismos sobre o desenvolvimento das plantas. O delineamento adotado foi o inteiramente casualizado, usandose o esquema fatorial 4 x 3, com 15 repetições, sendo cada parcela constituída por uma muda. Os fatores foram: três espécies de fungos (Escovopsis sp., M. anisopliae, T. strigosellum) mais um controle e três métodos de inoculação (inoculação via foliar, inoculação via solo e inoculação via plântula). Para avaliação da colonização endofítica, utilizaram-se dez mudas, sendo cinco destinadas para avaliação das características biométricas. No método de inoculação via plântula, o fungo T. strigosellum foi isolado apenas nas raízes. Contudo, no método de inoculação via solo, além das raízes, esse fungo colonizou também o caule. As plantas de E. urophylla não foram colonizadas endofiticamente quando inoculadas pelo método de inoculação via foliar. As plantas inoculadas com o fungo T. strigosellum, pelo método de inoculação via plântula, apresentaram maiores valores nas características altura de plantas, número de folhas, massa seca da parte aérea e massa seca total quando comparado com os outros métodos de inoculação. Houve incremento também, por esse método, para a variável altura de plantas, quando comparadas às plantas controle e às inoculadas pelos fungos Escovopsis sp. e M. anisopliae. Dentre os fungos estudados, o isolado T. strigosellum colonizou endofiticamente o E. urophylla e influenciou positivamente no seu desenvolvimento, quando inoculado via plântula. / Several studies evidenced dozens of fungus species of different genera in the colonies of leafcutting ants. However, the origin of these microorganisms is still uncertain. Some of these may be endophytes from plant material transported to the nest. Thus, certain endophytic fungi have the ability to contaminate the leaf-cutting ant colony or influence its symbiotic microbiome. Thus, fungal endophytes have the potential to control leaf-cutting ants as mutual plant partners. This symbiotic system could become a strategy in the management of this important forest pest. The objective of this work was to evaluate the potential of endophytic colonization of Eucalyptus urophylla plants by fungi Escovopsis sp., Metarhizium anisopliae e Trichoderma strigosellum, and to analyze, by means of the evaluation of biometric characteristics, the influence of the inoculation of these microorganisms on the plant development. Experimental design was completely randomized, using the factorial scheme 4 x 3, with 15 replicates, each plot consisting of one plant. The factors were: three fungi species (Escovopsis sp., M. anisopliae, T. strigosellum) plus one control and three methods of inoculation (foliar inoculation, inoculation via soil and seedling inoculation). For the evaluation of endophytic colonization, ten plants were used; five of them were destined to evaluate the biometric characteristics. In the seedling inoculation method, the T. strigosellum fungus was isolated only in the roots. However, in the soil inoculation method, besides the roots, this fungus also colonized the stem. E. urophylla plants were not colonized endophiatically when inoculated by foliar inoculation method. The plants inoculated with T. strigosellum by the seedling inoculation method had higher values in the characteristics of plant height, number of leaves, root dry mass and total dry mass when compared to the other inoculation methods. There was also an increase, by this method, for the plant height variable, when compared to the control plants and those inoculated by the fungi Escovopsis sp. and M. anisopliae. Among the fungi studied, the isolate T. strigosellum endofitically colonized E. urophylla and positively influenced its development when inoculated via seedling.

Orientador: João Cláudio Thoméo / Banca: Márcia Luiza Rizzatto / Banca: Roberto da Silva / Resumo: Este trabalho teve como objetivo produzir enzimas lipolíticas por fermentação em estado sólido em um biorreator de leito fixo, empregando para isso o fungo mesofílico Metarhizium anisopliae ICB 421 e o fungo termofílico Thermomucor indicae-seudaticae N31, utilizando como substrato bagaço de cana de açúcar (BC), farelo de trigo (FT) e grãos de soja moída (S). As enzimas produzidas podem ser utilizadas para diversos fins industriais, entre eles a produção de biodiesel. Os testes para produção de enzimas foram realizados inicialmente em sacos de polipropileno e apresentaram para M. anisopliae atividade lipolítica de 13,71 U/gss a 28 °C, em meio composto por 50% BC, 25% FT e 25% S, com umidade de 70% e cultivado por 5 dias; para T. indicae-seudaticae atividade lipolítica de 2,81 U/gss a 45 °C, em meio composto por 50% BC, 25% FT e 25% S, com umidade de 70% e cultivado por 2 dias. Nestes ensaios, as variáveis testadas, composição do meio, umidade inicial, substituição do óleo de soja por farelo de soja e tempo de fermentação, afetaram a atividade das enzimas obtidas. Na sequência, como parte do aumento de escala, os ensaios foram conduzidos em dois biorreator de leito fixo, tendo-se como variáveis a vazão de ar e a umidade inicial do substrato, sendo que ambas afetaram a atividade enzimática, que atingiu 5,61 U/gss para M. anisopliae a 28 °C, em meio composto por 50% BC, 25% FT e 25% S, com umidade de 70%, vazão ar 180 L/h, cultivado por 5 dias, e 2,48 U/gss para T. indicae-seudaticae a 45°C, em meio composto por 50% BC, 25% FT e 25% S, com umidade de 70%, vazão ar 80 L/h, cultivado por 2 dias. Das enzimas produzidas, a de M. anisopliae apresentou valores muito baixos de transesterificação, descartando-a para a aplicação na obtenção de biodiesel, mas a de T. indicae-seudaticae mostrou-se promissora para esta aplicação / Abstract: This study aimed to produce lipolytic enzymes through solid state fermentation (SSF) in a fixed-bed bioreactor, using Metarhirium anisopliae ICB 421 mesophilic fungi and Thermomucor indicae-seudaticae N31 thermophilic fungi, and sugar cane bagasse, wheat, and soybeans as substrate. The enzyme produced can be used for various industrial purposes, including biodiesel production. Tests for enzyme production initially performed in polypropylene and showed to M. anisopliae lipolytic activity of 13.71 U / gds at 28 ° C in medium composed of 50% BC, and 25% FT 25% S, with 70% humidity and cultivated for 5 days for T. indicae seudaticae lipolytic activity of 2.81 U / gds at 45 ° C in medium composed of 50% BC, and 25% FT 25% S, with 70% humidity and cultivated for 2 days. In these assays, the variables tested, medium composition, initial moisture, replacement of soybean oil by soybean meal and fermentation time, affected the activity of the enzymes obtained. Furthermore, as part of scaling, tests were conducted in a fixed bed bioreactor, testing again the process conditions for optimizing the production of lipase; the variables used in this step were air flow, and initial substrate moisture, both of which affect the enzymatic activity, that reached 5.61 U/gds to M. anisopliae at 28°C in medium composed of 50% BC, and 25% FT 25% S, with 70% humidity, air flow 180 L/h, for 5 days, and 2.48 U/gds for T. indicae-seudaticae at 45°C in medium composed of 50% BC, and 25% FT 25% S, with 70% humidity, air flow 80 L/h, for 2 days. . Enzymes produced, M. anisopliae showed very low transesterification, dismissing the application for obtaining biodiesel, but for T. indicae-seudaticae has shown promise for this application / Mestre

Orientador: Antonio Batista Filho / Banca: Wilson Badiali Crocomo / Banca: José Eduardo Marcondes Almeida / Resumo: Neste trabalho estudou-se a patogenicidade de alguns isolados de Metarhizium anisopliae à cigarra do cafeeiro Fidicinoides pronoe. Além disso, utilizou-se o isolado IBCB 348 para determinação da CL90 e avaliação da compatibilidade do fungo com produtos fitossanitários empregados na cultura do café. Todos os isolados de M. anisopliae testados em cigarras do café foram patogênicos, causando mortalidades confirmadas de 50 %, com exceção dos isolados IBCB 410 e IBCB 348, que atingiram mortalidade de 60 %. Observou-se que a mortalidade foi maior nos 5 primeiros dias, havendo diminuição da mesma a partir do 6o dia. No teste de compatibilidade com produtos fitossanitários verificou-se que o fungicida oxicloreto de cobre, nas duas doses avaliadas (5.000g/ha e 2.000g/ha), resultou em maior produção de conídios e redução do crescimento vegetativo. O thiabendazole (35g/ha concentração mínima) e o hidróxido de cobre (5.000g/ha concentração máxima) não diferiram da testemunha quanto à produção de conídios. Já os produtos thiabendazole (1.500g/ha concentração máxima), benomyl (1.000g/ha), tebuconazole (100mL/ha), cyproconazole (200mL/ha) e mancozeb (5.000g/ha e 3.500g/ha) foram estatisticamente iguais entre si, mas apresentaram menor produção de conídios do que a testemunha. Através da fórmula de T, que avalia a compatibilidade dos produtos e sua toxicidade, o oxicloreto de cobre (2.000g/ha) e o thiabendazole (35g/ha) foram considerados compatíveis com M. anisopliae. Os demais fungicidas avaliados foram classificados como muito tóxicos e não apresentaram produção de conídios e crescimento vegetativo, com exceção de thiabendazole (1.500g/ha), que apresentou crescimento vegetativo. Quanto aos herbicidas avaliados, todos os produtos foram classificados como muito tóxicos nas concentrações testadas, afetando o crescimento vegetativo e a produção de conídios... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This paper deals with the pathogenicity of some isolates of Metarhizium anisopliae to the coffee cicada, Fidicinoides pronoe. Also, the isolate IBCB 348 were used to determine the LC90 and to evaluate the compatibility of this fungus with the pesticides used on the coffee crop. All M. anisopliae isolates were pathogenic to the coffee cicada, causing at least 50 % of confirmed mortality. The virulence of the fungus was higher in the first five days, decreasing after the sixth day. The fungicide copper oxicloreto, in both concentrations, resulted in higher production of conidia and reduction of vegetative growth. Thiabendazole (minimum concentration) and copper hydroxide (maximum concentration) led to conidia production similar to the control. Thiabendazole (maximum concentration), benomyl, tebuconazole, cyproconazole and mancozeb (both concentrations) showed lower production of conidia than the control. By using the T formula, which evaluate the compatibility of the pesticides and their toxicity, copper oxicloreto and thiabendazole were considered compatibles with M. anisopliae. The remaining fungicides were classified as very toxic and showed neither production of conidia nor vegetative growth, excepting thiabendazole (maximum concentration), which resulted in vegetative growth. All herbicides were very toxic to the fungus. Among the insecticides, thiamethoxam were considered compatible to M. anisopliae; the other products resulted in lower production of conidia and were considered from moderately to very toxic. / Mestre

Made available in DSpace on 2016-04-28T20:15:25Z (GMT). No. of bitstreams: 1 2007-Ana Paula Rodrigues Moraes.pdf: 1193160 bytes, checksum: 61d4adc426c0fdbeab75966756651ece (MD5) Previous issue date: 2007-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Stomoxys calcitrans is a hematophagous fly that is related to reduction in animal production due to the stress and dissemination of pathogens. The knowledge in plague control, using entomopathogenic fungi, has been growing year by year, but few works have been done to control S. calcitrans. The aims of this study were rearing a colony of S. calcitrans to make a bioassay of microbial control, verify the pathogenic potential of Metarhizium anisopliae in eggs, larvae and pupae and access three methods of egg s exposition to the studied fungi. After methodologies adaptation of rearing S. calcitrans that are described in literature, the colony was maintained in the Laborat?rio de D?pteros Hemat?fagos - UFRRJ. The bioassays were done in the Laborat?rio de Controle Microbiano de Atr?podes de Import?ncia Veterin?ria - UFRRJ, using the strain 959 of M. anisopliae in a water suspension. In the first method, eggs were immersed in the suspension (2.2 ? 108 conidia /ml and dilutions) and transferred to Petri plates with hydrophilic cotton. In the second methods (2.3 ? 108 conidia/ml and dilutions), eggs were immersed and transferred to the larval rearding medium. In the third method (2.1 ? 108 conidia/ml and dilutions), eggs were put in the larval rearding medium surface and one aliquot of fungus suspension were added. Larvae with nine days of development were immersed in suspension (2.1 ? 108 conidia/ml and dilutions) and after that were transferred to the larval rearding medium while pupae with 16 days of development were immersed in suspension (2.3 ? 108 conidia/ml and dilutions) and were held in Petri plates. Experiments were carried out with 107, 106 and 105 concentrations of conidia/ml and positive and negative controls. The mortality was assessed on the 10th day after fungus exposure. It was verified high mortality in all treated and control groups in the first method of eggs exposure. In the other two methods the eggs exposed to 108 conidia/ml concentration were 100% unviable. In the second method the eggs was 92.5% unviable and the third method was 53.33% both in 107 conidia/ml concentration. The subsequent concentrations did not show significant difference when compared to controls, neither between two methods of fungi exposure. Larvae and pupae exposure did not show statistic difference between treated and controls groups. Metarhizium anisopliae suspensions in high concentrations were capable to make unfeasible eggs of S. calcitrans; however, it did not happen in larvae and pupae experiments, in which fungi did not prevent their development. The second method was the best one to control immature forms of S. calcitrans but the third one resembles the nature conditions, in which has significant result only in the highest fungi concentration. Experimental methodologies that simulate nature conditions may optimize the utilization of fungi in arthropods control, in a way that it does not modify its action when applied in the field. / Stomoxys calcitrans ? uma mosca hemat?faga associada ? redu??o da produtividade, devido ao estresse causado aos animais e a dissemina??o de pat?genos. O conhecimento do controle de pragas, utilizando fungos entomopatog?nicos, vem crescendo no decorrer dos anos, por?m poucos trabalhos t?m sido realizados visando o controle de S. calcitrans. O objetivo deste estudo foi estabelecer uma col?nia de S. calcitrans direcionada ao bioensaio de controle microbiano, verificar o potencial patog?nico de Metarhizium anisopliae sobre ovos, larvas e pupas, e ainda, avaliar tr?s m?todos de exposi??o dos ovos ao fungo estudado. Ap?s adapta??o das metodologias de cria??o de S. calcitrans descritas na literatura, a col?nia foi mantida no Laborat?rio de D?pteros Hemat?fagos UFRRJ. Os bioensaios foram realizados no Laborat?rio de Controle Microbiano de Artr?podes de Import?ncia Veterin?ria UFRRJ, onde foi utilizada a cepa 959 de M. anisopliae para elabora??o das suspens?es aquosas. No primeiro m?todo, os ovos foram imersos na suspens?o (2,2 x 108 con?dios/ml e dilui??es) e transferidos para placa de Petri contendo algod?o hidr?filo. No segundo m?todo (2,3 x 108 con?dios/ml e dilui??es) os ovos foram imersos e transferidos para o meio de desenvolvimento larval. No terceiro m?todo (2,1 x 108 con?dios/ml e dilui??es), os ovos foram depositados sobre a superf?cie do meio de desenvolvimento larval e uma al?quota da suspens?o f?ngica foi adicionada. As larvas com nove dias de desenvolvimento foram imersas na suspens?o (2,1 x 108 con?dios/ml e dilui??es) e transferidas para o meio de desenvolvimento larval, enquanto que as pupas de 16 dias de desenvolvimento foram imersas na suspens?o (2,3 x 108 con?dios/ml e dilui??es) e depositadas em placas de Petri. Foram utilizadas dilui??es na concentra??o de 107, 106 e 105 con?dios/ml e controle positivo e negativo. A mortalidade foi avaliada no d?cimo dia ap?s exposi??o, onde foi observado se ocorria mudan?a de est?gio. Foi verificada alta mortalidade em todos os grupos (tratados e controles) no primeiro m?todo de exposi??o de ovos. Nos outros dois m?todos, foram obtidos 100% de inviabilidade dos ovos expostos ? concentra??o de 108 con?dios/ml. No segundo m?todo, houve 92,5% de inviabilidade dos ovos, enquanto que no terceiro m?todo, a inviabilidade foi de 53,33%, ambos na concentra??o de 107 con?dios/ml. As concentra??es subseq?entes n?o apresentaram diferen?a significativa frente aos controles e nem entre os dois m?todos de exposi??o f?ngica. Nas exposi??es de larvas e pupas n?o ocorreu diferen?a estat?stica entre os tratamentos e os controles. Suspens?es de M. anisopliae em altas concentra??es s?o capazes de inviabilizar ovos de S. calcitrans, entretanto, isto n?o ocorreu em larvas e pupas, pois o fungo n?o impediu o desenvolvimento destes. O m?todo mais efetivo para o controle das formas imaturas de S.calcitrans foi o segundo m?todo, entretanto, o que mais se assemelha ?s condi??es naturais seria o terceiro, onde foi obtido resultado significativo apenas na maior concentra??o f?ngica. A ado??o de metodologias experimentais que simulem condi??es naturais pode otimizar a utiliza??o de fungos no controle de artr?podes, de forma que sua a??o n?o seja comprometida, quando aplicados no campo.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.