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Fungos Endofíticos De Folhas E Caule De Lippia Sidoides Cham

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Fungos Endofíticos De Folhas E Caule De Lippia Sidoides Cham UNIVERSIDADE FEDERAL DE PERNAMBUCO CENTRO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE MICOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA DE FUNGOS Virgínia Medeiros de Siqueira Fungos endofíticos de folhas e caule de Lippia sidoides Cham. e avaliação da atividade antimicrobiana Recife-PE 2008 UNIVERSIDADE FEDERAL DE PERNAMBUCO CENTRO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE MICOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA DE FUNGOS Virgínia Medeiros de Siqueira Fungos endofíticos de folhas e caule de Lippia sidoides Cham. e avaliação da atividade antimicrobiana Dissertação de Mestrado submetida ao Programa de Pós-graduação em Biologia de Fungos do Departamento de Micologia do Centro de Ciências Biológicas da Universidade Federal de Pernambuco em cumprimento da exigência para obtenção do grau de Mestre em Biologia de Fungos. Orientadora: Profa Dra Cristina Maria Souza Motta Co-orientadora: Profa Dra Janete Magali de Araújo Recife-PE 2008 Siqueira, Virgínia Medeiros de Fungos endofíticos de folhas e caule de Lippia sidoides Cham. e avaliação da atividade antimicrobiana./ Virgínia Medeiros de Siqueira. – Recife: A Autora, 2008. 94 fls. .: il. Dissertação (Mestrado: Biologia de Fungos) – UFPE. CCB 1.Fungos endofíticos 2.Lippia sidoides 3.Atividade antimicrobiana I.Título 582.28 CDU (2ª. Ed.) UFPE 579 CDD (22ª. Ed.) CCB – 2008 – 06 O objetivo da vida não é ser feliz. É ser útil, honrado, compassivo, fazendo com que nossa vida, bem vivida, faça alguma diferença. Dedico à Kátia, Érika e Murilo. AGRADECIMENTOS À minha mãe, Kátia Maria Medeiros de Siqueira, por ser amorosa, honesta, serena e compreensiva. É meu porto seguro e minha referência de vida. Obrigada pelo amor, mimos, confiança e compreensão. Ao meu pai, Raimundo Nonato, por sempre me incentivar a estudar. Aos meus irmãos, Érika e Murilo, pela amizade, pelos momentos de descontração e pelo apoio em todas as horas. Em especial para Érika, além de irmã, uma verdadeira amiga. Quando temos irmãos ao nosso lado, tudo fica mais fácil. À minha avó Gilda Medeiros de Albuquerque (in memorian) pelo acolhimento cheio de mimos e preocupações desde minha chegada em Recife e à minha tia Magnólia Medeiros (in memorian), ser humano de alma superior e de coração único. Obrigada pelos momentos de suas vidas dedicados a mim. A Bidu (in memorian) e Nzinga, pelo amor incondicional indispensável ao meu bem estar. À professora Dra. Cristina Maria Souza Motta, minha orientadora, pelos conhecimentos transmitidos e pela confiança depositada em mim. À professora Dra. Janete Magali, minha co-orientadora, pelos conhecimentos transmitidos e pelo acolhimento sem restrições no Departamento de Antibióticos. Às professoras Débora Maria Massa Lima e Maria José dos Santos Fernandes, pela colaboração na identificação de muitos dos meus fungos isolados. Ao professor Dr. Uwe Braun, da Universidade Martin Luther, Alemanha, pela co- autoria de um dos artigos que compõe esta dissertação. Obrigada pela sua colaboração e disponibilidade. Às minha amigas, Ludmila Lima, Evanilza Moura, Ana Josephina, Lucimary Araújo e Pamela Simoa, pelos grandes momentos de amizade vividos juntos, desde nossa infância até os tempos de hoje. A nossa verdadeira amizade eternizou o amor que tenho por vocês. À Judith Advíncula e Alexandra Muniz, pelos conselhos, pelos momentos de estudos e pelas muitas alegrias compartilhadas. A todos os meus colegas do X Curso de Especialização em Micologia, onde dei início aos meus estudos micológicos, pelos ótimos momentos, aulas, trabalhos e seminários. Nesta turma não havia lugar para vaidade ou competição e sim para algo precioso: a simplicidade. Como foi bom conviver com vocês. À Adriana, Ivana e Raphael, um muito obrigado pelo apoio dado em todos os momentos. A companhia de vocês foi essencial para a mim. Vocês são tão especiais que se tornaram minha segunda família, além de serem amigos extraordinários. Obrigado também a todos os meus colegas do Departamento de Antibióticos: a disposição que cada um tem de ajudar o outro é uma riqueza única. Ao CNPq, pela bolsa de estudos concedida durante os dois anos dos meus estudos e pelo apoio financeiro essencial para a realização deste trabalho através do projeto RENNEBRA. A todos que direta ou indiretamente contribuíram de alguma forma para a realização deste trabalho. Toda a nossa ciência, comparada com a realidade, é primitiva e infantil – e, no entanto, é a coisa mais preciosa que temos. Albert Einstein (1879-1955) RESUMO Fungos endofíticos vivem por todo ou uma parte do seu ciclo de vida no interior de tecidos vegetais, sem causar sintomas de doença. Desde que foram devidamente estudados despertaram grande interesse biotecnológico em virtude da aplicabilidade de seus metabólitos secundários na medicina, indústria e agricultura. Lippia sidoides Cham,. planta medicinal popularmente conhecida como alecrim-pimenta, é encontrada no Nordeste brasileiro e utilizada principalmente como antisséptico. Folhas e caule foram superficialmente desinfectados e fragmentados. Os fragmentos dos tecidos vegetais foram distribuídos na superfície dos meios Batata dextrose agar e Sabouraud agar acrescidos de tetraciclina e cloranfenicol (100µL/mL) contidos em placas de Petri e cultivados a 30o C por até 30 dias. Todos os isolados foram submetidos ao ensaio antimicrobiano primário em meio sólido (bloco de gelose) e aqueles que se mostraram ativos a algum microrganismo teste foram então submetidos ao teste de atividade microbiana em meio líquido (fermentação). Os microrganismos teste utilizados foram Staphylococcus aureus (ATCC-6538), Bacillus subtilis (UFPEDA-16), Escherichia coli (ATCC-25922), Klebsiella pneumoniae (ATCC- 29665), Pseudomonas aeruginosa (ATCC-27853), Candida albicans (UFPEDA-1007), Malassezia furfur (URM-5436) e Trichophyton rubrum (URM-4350). Um total de 207 fungos endofíticos pertencentes a quatorze espécies foram isolados: 125 (52%) de folhas e 82 (34,1%) de caule. Colletotrichum gloeosporioides foi a espécie mais freqüente, seguida de Alternaria alternata, Guignardia bidwelli e Phomopsis archeri. No bloco de gelose, 8% (16) se mostraram ativos a pelo menos um dos microrganismos teste. Destes, 10 apresentaram atividade nos ensaios em meio líquido, com os melhores halos de inibição de 25mm, 18mm e 13mm contra S. aureus, B. subtillis e K. pneumonie, respectivamente. Palavras-chave: Fungos endofíticos; Diversidade; Atividade antimicrobiana; Lippia sidoides ABSTRACT Endophytic fungi colonize healthy living tissue of the host plant, typically causing no apparent symptoms of disease. Since they were truly studied they called great attention to the biotechnological interesting because of the applicability of they secondary metabolites in medicine, industry and agricultural. Lippia sidoides Cham., a medicinal plant popularly known as “alecrim-pimenta”, is founded at Brazilian northeast and used mostly as antiseptic. Leaves and stem were superficially disinfected and fragmented. The fragments were placed on the surface of the media Potato dextrose agar and Sabouraud agar supplemented with tetraciclin (100µg/mL) and chloramphenicol (100µg/mL) on Petri dishes and cultivated at 30o C up to 30 days. All the strains were submitted to the agar plug screening assay (“bloco de gelose”) and those who showed antimicrobial activity were submitted to the fermentation assay. The microorganisms test were Staphylococcus aureus (ATCC-6538), Bacillus subtilis (UFPEDA-16), Escherichia coli (ATCC-25922), Klebsiella pneumoniae (ATCC-29665), Pseudomonas aeruginosa (ATCC-27853), Candida albicans (UFPEDA-1007), Malassezia furfur (URM-5436) and Trichophyton rubrum (URM-4350). A total of 207 endophytic fungi were isolated, 125 (52%) from leaves and 82 (34,1%) from stem, belonging to fourteen species. Colletotrichum gloeosporioides was the mostly frequently specie, fallowed by Alternaria alternata, Guignardia bidwelli and Phomopsis archeri. At the agar plug screening assay, 8% (16) endophytic fungi showed antimicrobial activity to at least one microorganism test. From these, ten showed antimicrobial activity during the fermentation. The bests inhibition zone were 25mm, 18mm e 13mm against S. aureus, B. subtillis e K. pneumonie, respectively. Keywords: Endophytic fungi; diversity, antimicrobial activity; Lippia sidoides. LISTA DE FIGURAS Figura 1. Equilíbrio antagônico entre virulência fúngica e defesa da planta. Adaptado de SCHULZ et al. 2002. 05 Pág. Figura 2. Cryptocandin A e Criptocin (LI et al. 2000). 12 Figura 3. Colletotric Acid (ZOU et al. 2000). 13 Figura 4. 2,4-diidroxi-5,6-dimetil benzoato de etila e phomopsilactona (SILVA et al. 2005). 13 Figura 5. Brefeldin A (WANG et al. 2006). 14 Figura 6. Planta medicinal Lippia sidoides Cham. 16 Figura 7. Estrutura Química do Carvacrol (A) e Timol (B) (COSTA et al. 16 2001). Primeiro Capítulo: Fungos Endofíticos de Folhas e Caule de Lippia sidoides Cham. Figura 1. Percentagem de fungos endofíticos de diferentes grupos. 37 Figura 2 Diferenças em número e especificidade de fungos endofíticos de Lippia sidoides nos tecidos vegetais 37 Terceiro Capítulo: Corynespora subcylindrica sp. nov. – uma nova espécie de Hyphomycete do Brasil e uma discussão sobre a taxonomia de gêneros “corynespora- like” Figura 1. Corynespora subcylindrica sp. nov. – Cultura em BDA (Diâmetro da 71 placa de Petri: 90mm). Figura 2. Corynespora subcylindrica sp. nov. Conidióforo surgindo 71 lateralmente de uma hifa em cultura em BDA, com conídio primário (conídio secundário da cadeia já formado). – Bar = 10 µm. Figura 3. Corynespora subcylindrica sp. nov. – A. Hifa. – B. conidioforos.
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