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Luiz Henrique Rosahttp://lattes.cnpq.br/3197093513022771Carlos Augusto RosaDavid Wedgehttp://lattes.cnpq.br/0146234844897996Camila Rodrigues de Carvalho2022-06-14T12:52:35Z2022-06-14T12:52:35Z2015-04-23http://hdl.handle.net/1843/42494Os fungos endofíticos são aqueles que, pelo menos durante parte do seu ciclo de vida, habitam tecidos vegetais vivos sem causar sintomas aparentes de doença ou efeitos negativos a seus hospedeiros. Estes microrganismos podem ser encontrados em diferentes grupos vegetais e representam fontes promissoras de metabólitos bioativos de interesse farmacológico e na agricultura. Diferentes espécies de plantas medicinais vêm sendo utilizadas ao longo da história da humanidade no mundo. Dentre as espécies medicinais do Brasil, as do gênero Copaifera são utilizadas devido à ação anti-inflamatória e cicatrizante do seu óleo resina; nos Estados Unidos, Equinacea purpurea é amplamente utilizada devido à habilidade de estimular o sistema imunológico. Poucos estudos foram realizados com o objetivo de conhecer a micota endofítica dessas plantas, apesar do seu amplo emprego na medicina popular. Portanto, este estudo teve como objetivo caracterizar a comunidade de fungos endofíticos associados à Copaifera langsdorffii, C. pubiflora e Equinacea purpurea, bem como avaliá-las quanto à produção de metabólitos antimicrobianos. Novecentos e um isolados de fungos endofíticos foram obtidos associados às plantas medicinais C. langsdorffi (308 isolados), C. pubiflora (360 isolados) e E. purpurea (233 isolados). Os gêneros Cladosporium, Colletotrichum, Diaporthe, Fusarium, Guignardia/Phyllosticta e Pestalotiopsis foram compartilhados pelas três plantas hospedeiras estudadas. Os táxons Diaporthe sp. 6, Xylariaceae sp. 1, Diaporthales sp. 1 e Diaporthales sp. 2 foram os dominantes em C. langsdorffi; Diaporthe sp. 5, Guignardia sp. 2, Diaporthales sp., Diaporthe sp. 8 e Guignardia sp. 1 em C. pubiflora; e Alternaria alternata, Colletotrichum dematium e Stagonosporopsis sp. 2 em E. purpurea. Os valores do índice de Fisher α demonstraram que a comunidade endofítica com maior diversidade foi à associada a C. langsdorffii (Fisher α = 16,6), seguida por E. purpurea (Fisher α = 14,96) e C. pubiflora (Fisher α = 7,23). A riqueza da comunidade fúngica de C. langsdorffi foi a mais alta (Margalef = 8,41), seguida por E. purpurea (Margalef = 7,52) e C. pubiflora (Margalef = 4,57). O índice de Simpson (dominância) exibiu valores próximos para as comunidades endofíticas associadas às três plantas hospedeiras (C. langsdorffi = 0,94, C. pubiflora = 0,90, E. purpurea = 0,84). Somente 11 (1,6%) extratos dos fungos associados às espécies de Copaifera apresentaram porcentagem de inibição de ≥70% frente a pelo menos um dos microrganismos alvos, sendo selecionados para determinação da sua concentração inibitória mínima (CIM). Os extratos diclorometânicos de Alternaria daucifolii UFMGCB 7491, Diaporthe sp. 6 UFMGCB 7696 e 7720, Diaporthe sp. 7 UFMGCB 7927 e Diaporthe sp. 8 UFMGCB 7646, 7653, 7701, 7719, 7770, 7772 foram submetidos à espectroscopia de RMN 1H, a qual possibilitou a detecção de sinais referentes a presença majoritária de ácidos graxos nos extratos de A. daucifolii UFMGCB 7491 e Diaporthe sp. 7 UFMGCB 7927. Os ácidos linoléico, palmítico e elaídico foram predominantes no extrato de A. daucifolii UFMGCB 7491, e os ácidos oléico, linoléico, estearidônico e palmítico no extrato de Diaporthe sp. 7 UFMGCB 7927. O fracionamento do extrato de Diaporthe sp. 8 UFMGCB 7719 ocasionou a identificação de citocalasina H e citocalasina J, as quais apresentaram atividade antifúngica frente Phomopsis obscurans e P. viticola. Dos extratos dos endofíticos associados a E. purpurea, oito foram selecionados para análise química, sendo que os espectros de RMN 1H de Colletotrichum sp. 4 EPU8FB, Fusarium sp. EPU9CB, Nigrospora oryzae EPU13CA, Colletotrichum sp. 4 EPU30CA, Fusarium sporotrichioides EPU36CA e Sordariomycetes sp. 2 EPU40CE exibiram sinais referentes a presença majoritária de ácidos graxos. Posteriormente, a presença de uma mistura de 16 ácidos graxos foi confirmada nos extratos destes fungos. O extrato diclorometânico de Biscognauxia mediterranea EPU38CA foi submetido ao fracionamento biomonitorado e isoladas duas substâncias, a (-)-5-metilmeleína e (- )(3R)-8-hidróxi-6-metóxi-3,5-dimetil-3,4-diidroisocumarina. (-)-5-metilmeleína apresentou atividade antifúngica frente a P. obscurans e P. viticola, e com exceção de C. gloeosporioides, (- )(3R)-8-hidróxi-6-metóxi-3,5-dimetil-3,4-diidroisocumarina apresentou atividade frente a todos os demais microrganismos alvos, em pelo menos uma concentração e intervalo de tempo avaliados. Os efeitos antifúngicos observados nos extratos e frações selecionados, assim como a produção das citocalasinas H e J, (-)-5-metilmeleína e (-)(3R)-8-hidróxi-6-metóxi-3,5-dimetil 3,4-diidroisocumarina, reforçam a hipótese que a simbiose entre fungos endofíticos e plantas hospedeiras pode produzir substâncias com atividade frente fitopatógenos e predadores das plantas em geral. Os resultados obtidos demostraram que as plantas medicinais podem representar um promissor reservatório de fungos endofíticos, dentre eles táxons produtores de metabólitos bioativos.Endophytic fungi are microorganisms which live inside plant tissues or organs, without causing them any harmful symptoms. These microorganisms may be found in several species of plants and represent a promising source of bioactive metabolites for pharmacological and agricultural area. The medicinal plants have been used throughout human history in the world. Specifically in Brazil, the Copaifera genus are frequently used due its anti-inflammatory effects and wound healing of their oleoresin. In the United States, Echinacea purpurea is commonly used because it may stimulate the immune system. However, despite its ethnobotany use, few studies have been conducted in order to know their endophytic fungi communities. The aim of this study were to characterize the diversity of the community of endophytic fungi associated with Copaifera langsdorffii, C. pubiflora, Equinacea purpurea and evaluate it as a source of bioactive metabolites as well. Nine hundred-one endophytic fungal isolates were obtained in association with the medicinal plant C. langsdorffi (308 isolates), C. pubiflora (360 isolates) and E. purpurea (233 isolates). Cladosporium, Colletotrichum, Diaporthe, Fusarium, Guignardia/Phyllosticta and Pestalotiopsis genera were shared among the three host plants. Diaporthe sp. 6, Xylariaceae sp. 1, Diaporthales sp. 1 e Diaporthales sp. 2 were the dominant species associated wih C. langsdorffi; Diaporthe sp. 5, Guignardia sp. 2, Diaporthales sp., Diaporthe sp. 8 e Guignardia sp. 1 with C. pubiflora; and Alternaria alternata, Colletotrichum dematium and Stagonosporopsis sp. 2 with E. purpurea. The fungal community associated with C. langsdorffii showed the highest diversity (Fisher α = 16.6), followed by E. purpurea (Fisher α = 14.96) and C. pubiflora (Fisher α = 7.23). The fungal community of C. lansdorffii showed the highest species richness (Margalef = 8.41), followed by E. purpurea (Margalef = 7,52) and C. pubiflora (Margalef = 4,57). The Simpson index (dominance) showed close values for the three communities (C. langsdorffi = 0.94, C. pubiflora = 0.90, E. purpurea = 0.84). Only 11 (1.6%) extracts from Copaifera species showed ≥70% inhibition against at least one target microorganism, and those extracts were selected for the minimal inhibitory concentrations determination (MIC). The dichloromethane extracts of Alternaria daucifolii UFMGCB 7491, Diaporthe sp. 6 UFMGCB 7696 and 7720, Diaporthe sp. 7 UFMGCB 7927 and Diaporthe sp. 8 UFMGCB 7646, 7653, 7701, 7719, 7770, 7772 were submitted to 1H NMR spectrometry. The extracts of A. daucifolii UFMGCB 7491 and Diaporthe sp. 7 UFMGCB 7927 were predominately composed of fatty acids. The linoleic,palmitic and elaidic acids were the predominant in the A. daucifolii UFMGCB 7491 extract. The oleic, linoleic, stearidonic and palmitic acids in the Diaporthe sp. 7 UFMGCB 7927 extract. The Diaporthe sp. 8 UFMGCB 7719 extract was fractionated and two pure compounds were identified, cytochalasin H and cytochalasin J. Those compounds showed antifungal activity against Phomopsis obscurans e P. viticola. Eight extracts from endophytic fungi associated to E. purpurea were selected for chemical analysis. The 1H NMR analysis indicated that the extracts of Colletotrichum sp. 4 EPU8FB, Fusarium sp. EPU9CB, Nigrospora oryzae EPU13CA, Colletotrichum sp. 4 EPU30CA, Fusarium sporotrichioides EPU36CA and Sordariomycetes sp. 2 EPU40CE were predominately composed of fatty acids. Further analysis of all of the extracts confirmed the presence of a mixture of 16 fatty acids. Using a bioassay-guided purification approach (-)-5-methylmellein and (-) (3R)-8-Hydroxy-6-methoxy-3,5-dimethyl-3,4- dihydroisocoumarin were obtained from Biscognauxia mediterranea EPU38CA. The (-)-5- methylmellein showed antifungal activity against P. obscurans and P. viticola. The (-) (3R)-8- Hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin was not able to inhibited the C. gloeosporioides growth, however the substance showed activity against all the others microorganism tested in at least one concentration and time of incubation. The antifungal effects observed in extracts and fractions selected, as well as the cytochalasin H, cytochalasin J, (-)-5- methylmellein and (-) (3R)-8-hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin production give evidence that the symbiotic relationship between endophytic fungi and their host plants may be able to produce substances against pathogenic fungi and herbivores animals. The results obtained in this study showed that medicinal plants can represent a reservoir of endophytic fungi promising for bioprospecting.FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas GeraisporUniversidade Federal de Minas GeraisPrograma de Pós-Graduação em MicrobiologiaUFMGBrasilICB - DEPARTAMENTO DE MICROBIOLOGIAMicrobiologiaEndófitosPlantas MedicinaisEchinaceaFabaceaeAntifúngicosFungos EndofíticosPlantas medicinaisCopaiferaEquinacea purpureaEfeitos antifúngicosDiversidade e bioprospecção de fungos endofíticos associados à plantas bioativas do Brasil e Estados Unidosinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMGORIGINALTese_Camila Rodrigues de Carvalho.pdfTese_Camila Rodrigues de Carvalho.pdfapplication/pdf10636870https://repositorio.ufmg.br/bitstream/1843/42494/1/Tese_Camila%20Rodrigues%20de%20Carvalho.pdf4aaa964a6a7f3c187962e62aede9aeceMD51LICENSElicense.txtlicense.txttext/plain; charset=utf-82118https://repositorio.ufmg.br/bitstream/1843/42494/2/license.txtcda590c95a0b51b4d15f60c9642ca272MD521843/424942022-06-14 09:52:36.019oai:repositorio.ufmg.br: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ório InstitucionalPUBhttps://repositorio.ufmg.br/oaiopendoar:2022-06-14T12:52:36Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
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