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Universidade Vale Do Paraíba Instituto De Pesquisa E Desenvolvimento UNIVERSIDADE VALE DO PARAÍBA INSTITUTO DE PESQUISA E DESENVOLVIMENTO Claudinéia Aparecida da Silva Araujo TOLERÂNCIA DE FUNGOS FILAMENTOSOS AO ESTRESSE OSMÓTICO CAUSADO PELO CLORETO DE POTÁSSIO Orientador: Prof. Dr Drauzio E.N Rangel Co- Orientador: Prof. Dr Newton Soares da Silva São José dos Campos 2016 UNIVERSIDADE VALE DO PARAÍBA INSTITUTO DE PESQUISA E DESENVOLVIMENTO Claudinéia Aparecida da Silva Araujo TOLERÂNCIA DE FUNGOS FILAMENTOSOS AO ESTRESSE OSMÓTICO CAUSADO PELO CLORETO DE POTÁSSIO Dissertação de mestrado apresentado ao Programa de Pós Graduação em Ciências Biológicas da Universidade Vale do Paraiba como complementação dos créditos para obtenção do grau de Mestre em Ciências Biológicas Orientador: Prof. Dr Drauzio E.N Rangel Co- Orientador: Prof. Dr Newton Soares da Silva São José dos Campos 2016 FICHA Banca DEDICATÓRIA Eu dedico este trabalho a Deus que tornou possível esse grande sonho e ao meu pai que sempre deu o seu melhor para que eu chegasse até aqui, meu verdadeiro referencial de vida e a minha mãe que foi tantas vezes o acalanto quando tudo parecia perdido, ao meu amor pela sua compreensão e pela sua paciência. AGRADECIMENTOS A Deus, que se mostrou criador, que foi criativo. Seu fôlego de vida em mim me foi sustento e me deu coragem para questionar realidades e propor sempre um novo mundo de possibilidades. À minha família, por sua capacidade de acreditar em mim e investir em mim. Mãe, seu cuidado e dedicação foi que me deu, em alguns momentos, a esperança para seguir. Pai, sua presença significou segurança e certeza de que não estou sozinha nessa caminhada. Aos meus amigos, Luciana, Paulo Cesár, Breno, Flaviane quantas histórias escrevemos nos livro da vida, quantas conquistas, quanto aprendizado quantas lágrimas derramadas. E hoje estou, dividindo essa grande conquista em minha vida, e trilharemos daqui para frente a nossa nova história e assim vamos vivendo escrevendo cada página onde vocês serão sempre personagens essenciais nessa história da vida, e por vocês; Com vocês, as pausas entre um parágrafo e outro de produção melhora tudo o que tenho produzido na vida. Ao Prof.ª Drauzio pela sua delicadeza, paciência e inteligência, que soube orientar e acreditar na funcionalidade desse trabalho e, que soube passar o seu ensinamento, com leveza e alegria. Ao professor Newton, professora Flavia, a professora Nádia e todos os professores e servidores do programa de Pós Graduação que a mim repassaram seus conhecimentos, fazendo que meu desenvolvimento fosse o melhor possível. A coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), a Fundação de Amparo á Pesquisa do Estado de São Paulo pelo incentivo financeiro para a realização desse projeto. Ao meu amor, amigo, companheiro que sempre me apoiou e me deu força nos momentos de dificuldades e sempre acreditou em mim, muito mais do que eu mesma acreditava. A todos dos Luz do amanhecer que de alguma forma estiveram e estão próximos a mim, fazendo esta vida valer cada vez mais a pena RESUMO Os fungos enfrentam durante todo seu ciclo de vida, fatores de estresse, entre eles o estresse osmotico e a redução da disponibilidade de água no seu ambiente. O cloreto de potassio (KCl) é um estressor muito comum encontrado no habitat de fungos, tornando se ideal para experimentos laboratoriais. Fungos patógenos de insetos são conhecidos como importantes agentes de controle biológico. Os fungos fitopatogênicos são responsaveis por infectarem culturas de importãncia econômica. Os fungos antagonistas são uma opção de biocontrole, para combater os fungos fitopatogênicos.Os fungos pátogenos de insetos estão sujeitos ao estresse osmótico, quando estão crescendo na hemolinfa do inseto. Os fungos fitopatogênicos e antagonistas também são submetidos a esse tipo de estresse quando são expostos a ambientes com escassez hidrica. Este estudo avaliou a capacidade de germinação de várias espécies de fungos a diferentes concentrações de KCl. A germinação foi avaliada, em meio PDA (controle) ou PDA suplementado com KCl (concentrações de KCl 0,1 a 3,0 M para patógenos de insetos, concentrações de KCl 0,1 a 2,8 M para patógenos de plantas e concentrações de 0 a 2,0 para fungos antagonistas) a germinação foi avaliada após 24 horas de incubação a 26C. Entre fungos entomopatogênicos o Trichothecium roseum (ARSEF 1212) (LC90 = 3.350) foi o mais tolerante seguido por Lecanicillium aphanocladii, Simplicillium lanosoniveum, Quatro espécies de fungos mostraram osmotolerancia moderada, incluindo Mariannaea pruinosa, Torrubiella homopterorum, Isaria fumosorosea, Lecanicillium aphanocladii (LC90 de 1.9 á 2.1). Dos 18 isolados do gênero Metarhizium todos apresentaram uma moderada tolerância. Onze isolados foram mais sensiveis ao estresse osmotico, (LC90 de 1.0 á 1.6), entre eles, os fungos Tolypocladium extinguens e T. geodes foram os menos tolerantes. Entre os patogenos de plantas o Fusarium verticiloides (FGSC 7600) foi o mais tolerante e o menos tolerante foi Colletotrichum gleosporioides. Os isolados SF 134, SF 114, e SF 116 mutantes do isolado Fusarium fujikoroi (FMKC 1995) com alta produção de carotenoide, foram menos resistente que o isolado selvagem. Entre os fungos antagonistas os mais resistentes foram os isolados Clonastachys candelabrum (ARSEF 9499) LC90 = 2.677 e o isolado Clonastachys rosea (ARSEF 7200) LC90 = 2.530 frente à concentração de 1.9 M de KCl. O isolado Trichoderma lignorum (ARSEF 5176) apresentou tolerância moderada 1.6 M de KCl (LC90 = 1.653), e o menos tolerante foi o isolado Gliocladium roseum (ARSEF 3001) com 1.4 M (LC90 = 0.853)..Em geral os fungos patógenos de plantas foram mais resistentes ao cloreto de potássio que os fungos entomopatogênicos e os fungos antagonistas, entretanto os fungos entomopatogênicos Trichothecium roseum e Simplicillium lanosoniveum foram os mais tolerantes de todos os fungos testados. Palavras-chave: Fungos filamentosos, Estresse osmótico, Cloreto de potássio. TOLERANCE OF FILAMENTAL FUNGUS TO THE OSMOTIC STRESS CAUSED BY POTASSIUM CHLORIDE ABSTRACT Fungi face throughout their life cycle stress factors, including osmotic stress and reducing the availability of water in their environment. Potassium chloride (KCl) is a very common stressor found in fungi habitat, making it ideal for laboratory experiments. Pathogenic fungi of insects are known as important biological control agents. Phytopathogenic fungi are responsible for infecting crops of economic importance. The antagonistic fungi are a biocontrol option, to combat phytopathogenic fungi, through competition and myoparasitism. Pathogenic fungi of insects are subject to osmotic stress, when they are growing in the hemolymph of the insect. Phytopathogenic fungi and antagonists are also subjected to this type of stress when exposed to environments with water scarcity. This study evaluated the germination capacity of several fungal species at different concentrations of KCl. Germination was evaluated among PDA (control) or PDA supplemented with KCl (KCl concentrations of 0.1 to 3.0 M to insect pathogens, KCl concentrations from 0.1 to 2.8 M to plant pathogens and concentrations From 0 to 2.0 for antagonistic fungi) the germination was evaluated after 24 hours of incubation at 26 ° C. Among entomopathogenic the Trichothecium roseum (ARSEF 1212) (LC90 = 3.350) was the most tolerant followed by Lecanicillium aphanocladii, Simplicillium lanosoniveum, four species of fungi showed moderate osmotolerancia including Mariannaea pruinosa , homopterorum Torrubiella, Isaria fumosorosea, Lecanicillium aphanocladii (LC90 of 1.9 á 2.1) Of the 18 isolates of the genus Metarhizium all showed a moderate tolerance. Eleven isolates were more sensitive to osmotic stress (LC90 1.0 to 1.6), among them fungi extinguens Tolypocladium geodes . were less tolerant to osmotic stress. Among plant pathogens Fusarium verticiloides (FGSC 7600) was the most tolerant and the least tolerant was Colletotrichum gleosporioides. The isolates SF 134, SF 114, and SF 116 mutants of the isolated Fusarium fujikoroi (FMKC 1995) with high carotenoid production were less resistant than the wild isolate. Among the fungi toughest antagonists were isolated Clonastachys candelabrum (ARSEF 9499) LC90 = 2.677 and isolated Clonastachys rosea (ARSEF 7200) LC90 = 2.530 against the concentration of 1.9 M KCl. The isolated Trichoderma lignorum (ARSEF 5176) showed moderate tolerance 1.6 M KCl (LC90 = 1.653), and was less tolerant isolated Gliocladium roseum (ARSEF 3001) with 1.4 M (LC90 = 0.853) .. In general fungal pathogens plants were more resistant to potassium chloride entomopathogenic fungi and fungal antagonists, however entomopathogenic fungus Trichothecium roseum and Simplicillium lanosoniveum were more tolerant of all tested fungi. Keywords: Fungi, Osmotic stress, Potassium chloride. LISTA DE FIGURAS Figura 1 - Tolerância de fungos patógenos de insetos ao cloreto de potássio com concentrações de 0 á 2.0 M ...................................................................................................................................................... 24 Figura 2 - Tolerância de fungos patógenos de insetos ao cloreto de potássio com concentrações de 0 á 2.0 M ...................................................................................................................................................... 25 Figura 3 - Tolerância de fungos patógenos de insetos ao cloreto de potássio com concentrações de 0 . 26 Figura 4 - Tolerância de fungos patógenos de insetos ao cloreto de potássio com concentrações de 0 á 2.4 M . ...................................................................................................................................................
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