Universidade Federal de Minas Gerais Instituto de Ciências Biológicas Departamento de microbiologia Tese de doutorado Diversidade e aplicações biotecnológicas de fungos endofíticos associados à espécies da sub- família Mirtoideae (Myrtaceae) presentes em ecossistemas do Brasil, Argentina e Espanha Aline Bruna Martins Vaz BELO HORIZONTE 2012 i Aline Bruna Martins Vaz Diversidade e aplicações biotecnológicas de fungos endofíticos associados à espécies da sub- família Mirtoideae (Myrtaceae) presentes em ecossistemas do Brasil, Argentina e Espanha Tese de Doutorado apresentado ao programa de Pós-gradução em Microbiologia do Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais, como requisito para a obtenção do título de Doutor em Microbiologia Orientador: Prof. Dr. Carlos Augusto Rosa Co-orientadores: Luiz Henrique Rosa – Laboratório de Ecologia e Biotecnologia de Leveduras - UFMG Dr. Diego Libkind Frati e Dra Sonia Fontenla - Universidad Nacional del Comahue / CONICET - Centro Regional Universitaio Bariloche - Patagônia, Argentina Colaboradores: Profa. Paula Benevides de Morais e Prof. Raphael Sanzio Pimenta - Universidade Federal do Tocantins Prof. Juan Antonio Ocampo – Estación Experimental Del Zaidín – CSIC (Centro superior de investigaciones científicas) – Granada, Espanha Belo Horizonte Instituto de Ciências Biológicas Universidade Federal de Minas Gerais 2012 ii Agradecimentos “Aqueles que passam por nós, não vão sós, não nos deixam sós. Deixam um pouco de si e levam um pouco de nós” Pequeno príncipe. Saint Exupéry Muitas pessoas participaram da realização deste trabalho e seria realmente impossível descrever a importância de cada um. Alguns me acompanharam durante todo este período, outros, uma parte pequena ou grande, alguns somente passaram. Mas todos conseguiram modificar minha vida e meu coração. Aliás, não posso deixar de agradecer a todos os brasileiros que financiaram todos esses anos de pesquisa pelo pagamento dos seus impostos. Obrigada ao governo brasileiro por investir em pesquisa e desenvolvimento, pois somente assim sairemos do subdesenvolvimento. Gostaria de agradecer especialmente: A Deus, que sempre esteve ao meu lado, me ajudou e me confortou nas horas mais duras de toda a minha vida. Aos meus amigos espirituais que com muito amor me auxiliaram durante todo este período. Ao Prof. Carlos A. Rosa, pela orientação e preciosos ensinamentos. Sempre serei grata às oportunidades a mim dadas durante este período de trabalho. Foi um privilégio desenvolver esta tese sob sua orientação e integrar seu grupo de pesquisa. Aos meus co-orientadores Luis H. Rosa, Sonia Fontenla e Diego Libkind (Argentina), pelas discussões e sugestões. Seu apoio, confiança e amizade foram fundamentais para o bom desempenho deste trabalho. Ao colaborador Juan Antonio Ocampo (Espanha) por ter aberto as portas do seu laboratório. Seus emsinamentos, apoio e humanidade ajudaram muito no meu progresso científico. Aos meus pais, Cida e Waldemar por sempre acreditarem em mim e terem me passado ensinamentos valiosos de união, amor, amizade e educação. Aos meus irmãos, Alexandre e André, pela união, compreensão e amor. Seus exemplos de luta, persistência, honestidade, respeito e resignação sempre foram muito importantes para a construção da minha personalidade. Ao Helindo, pelo amor e companheirismo. Obrigada pela paciência nos momentos difíceis, por acreditar no meu potencial e me dar suporte em todos os aspectos da minha vida. Seu amor, otimismo e apoio foram extremamente importantes para mim. iii Aos amigos do laboratório de Ecologia e Biotecnologia de Leveduras pelos bons momentos que passamos nestes dois anos: Adriana, César, Fernanda, Gabriela, Luciana, Mariana, Natália, Raquel, Renata, Vivian. As minhas estagiárias Mariane e Cintia. Acredito muito no sucesso profissional de vocês. Muito obrigada pela ajuda, pontualidade, compromisso. Aos amigos do laboratório de microbiologia em San Carlos de Bariloche (Argentina), pelo companheirismo, paciência e ajuda durante o período que permaneci com vocês. Aos amigos do laboratório de micro-organismos promotores do crescimento vegetal (Espanha), pela ajuda científica, pessoal e companheirismo: Patricia Godoy, Nuria Rosales, Maria Angeles, Antonio Ilana, Jose Angel, María Isabel, Elizabeth Aranda, Mercedez García, Rafael Morcillo, Rodolfo Torres. Em especial gostaria de agradecer à Inmacula Sampedro, Jose Siles, Julia Martin, Rocío Reina pela amizade e ajuda. A toda minha família, avós, tios e primos pelas comemorações, companheirismo e amizade. Aos parentes e amigos que nos deixaram. As minhas amigas tão queridas, Cíntia Passos, Valéria Carolina, Valéria Santana, Cristiane Ireno, Tânia Apolinário pela amizade verdadeira. A minha nova e querida amiga Sara. Obrigada pelo companheirismo e conselhos durante minha estadia na Espanha, você se mostrou uma amiga extraordinária. Ao pessoal do Laboratório de Biodiversidade e Evolução Molecular pela preciosa ajuda nas técnicas de biologia molecular e nas análises das sequências dos fungos. Aos funcionários da secretaria de Pós-graduação do departamento de Microbiologia, Douglas, Fatinha, Iracema, Patricia, Gina. A todos os professores e colegas do curso de Pós-graduação em Microbiologia, pelo incentivo e convivência agradável. Aos membros da banca examinadora, pelas sugestões que com certeza serão essenciais para o resultado final desta tese. A CAPES por ter me dado a possibilidade de realizar um estagio de doutorado sanduiche no exterior por meio de uma bolsa de estudos. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pelo apoio financeiro e ao Programa de Pós-graduação em Microbiologia, pela oportunidade. 1 Sumário Resumo ............................................................................................................................................ 2 Abstract ........................................................................................................................................... 4 Relevância e justificativa ................................................................................................................ 5 Formatação da tese .......................................................................................................................... 7 Capítulo 1 ....................................................................................................................................... 8 Revisão bibliográfica ................................................................................................................... 8 1. Micro-organismos endofíticos ............................................................................................. 9 2. Interação fungo endofítico – planta hospedeira ................................................................ 12 3. Biogeografia de fungos ..................................................................................................... 13 4. Substâncias bioativas produzidas por fungos endofíticos ................................................. 15 1 Objetivos ............................................................................................................................... 18 1.1 Geral .............................................................................................................................. 18 1.2 Específicos .................................................................................................................... 18 Capítulo 2 ..................................................................................................................................... 19 Diversity and antimicrobial activity of fungal endophyte communities associated with plants of Brazilian savanna ecosystems ............................................................................................... 19 Capítulo 3 ..................................................................................................................................... 33 Fungal endophyte β-diversity associated with Myrtaceae species in an Andean Patagonian forest (Argentina) and an Atlantic forest (Brazil) ..................................................................... 33 Capítulo 4 ..................................................................................................................................... 64 Arbuscular mycorrhizal colonization of Sorghum vulgare in presence of root endophytic fungi of Myrtus communis ........................................................................................................ 64 Capítulo 5 ..................................................................................................................................... 72 Diversity of fungal endophytes in leaves of Myrtus communis studied by traditional isolation and cultivation-independent DNA-based method ..................................................................... 72 Capítulo 6 ..................................................................................................................................... 92 Discussão integrada ................................................................................................................... 92 Capítulo 7 ..................................................................................................................................... 96 Conclusões ................................................................................................................................ 96 Referências bibliográficas ............................................................................................................. 98 Anexos ......................................................................................................................................... 107 Produção científica durante o doutorado ................................................................................. 107 2 Resumo Fungos endofíticos são aqueles que, pelo menos durante parte do ciclo de vida, habitam tecidos vegetais vivos sem causar sintomas aparentes de doença ou efeitos negativos ao hospedeiro. Nos últimos anos, plantas com histórico etnobotânico vêm sendo alvos constantes de estudos de bioprospecção, pois acredita-se que algumas propriedades medicinais atribuídas ao vegetal também possam estar relacionadas à produção de metabólitos secundários por seus fungos endofíticos. O primeiro capítulo deste trabalho teve o objetivo de caracterizar a diversidade de fungos endofíticos associados às plantas medicinais Myrciaria floribunda, Eugenia aff. neomyrtifolia e Alchornea castaneifolia e avaliar seu potencial quanto à produção de metabólitos bioativos. Um total de 93 fungos filamentosos foram isolados e identificados por meio do sequenciamento da região espaçadora transcrita interna (ITS) do rRNA. Trinta e oito extratos fúngicos apresentaram atividade contra pelo menos um dos micro-organismos alvo avaliados. Estudos sobre a biogeografia de micro-organismos são escassos e para elucidar se os padrões de diversidade endofítica são correlacionados à filogenia dos seus hospedeiros ou são influenciados pelas variáveis ambientais ou distância foi avaliada a distribuição da comunidade de fungos endofíticos associados à hospedeiros filogeneticamente relacionados de plantas da tribo Myrtae. O estudo foi realizado seguindo-se três escalas espaciais: regional, 101-5.000 km; local, 0-100 km e microescala, 0-1 km. Um total de 960 isolados foi obtido, agrupados e identificados em 52 espécies distintas. A similaridade da comunidade de fungos endofíticos diminuiu com o aumento da distância geográfica, o que foi observado pela análise de decaimento da distância. A análise de escalonamento metrico não dimensional (NMDS) mostrou que os fungos endofíticos agruparam-se a um nível de escala espacial local. Além disso, a análise de regressão múltipla de matrizes (MRM) sugeriu que a similaridade da comunidade endofítica depende da escala espacial analisada. Em outro trabalho realizado foi mostrado que os fungos endofíticos também podem interagir com fungos micorrízicos arbusculares melhorando a adapatabilidade das plantas por meio da promoção do crescimento vegetal. Neste trabalho, cento e cinquenta isolados de fungos endofíticos foram isolados das raízes de M. communis e oito diferentes grupos foram selecionados para o estudo com Sorghum vulgare. Os fungos não foram capazes de colonizar as raízes de sorgo, porém apresentaram efeitos significativos no crescimento e colonização por micorrizas arbusculares. A quarta parte deste trabalho teve como finalidade avaliar a diversidade de fungos endofíticos associados à Myrtus communis utilizando-se técnicas tradicionais de isolamento e por meio de eletrofose em gel de gradiente desnaturante (DGGE). Um total de 113 isolados de fungos endofíticos foi obtido pela técnica de isolamento em meio de cultura em ágar batata dextrosado (BDA). A similaridade da comunidade endofítica não diminuiu com o 3 aumento da distância geográfica. Os resultados do presente trabalho sugerem uma elevada diversidade de fungos endofíticos associados a hospedeiros presentes em diversos ecossistemas. Além disso, nossos resultados também mostraram o potencial biotecnológico destes fungos como produtores de substâncias antimicrobianas e como promotores do crescimento vegetal. 4 Abstract Endophytic fungi inhabit healthy plant tissues during at least one stage of their life cycle without causing any apparent symptom of disease or negative effects on the hosts. In the last years, the endophytic fungal mycota associated with popular medicinal plants have been perfomed because some medicinal properties have been associated to secondary metabolities production by their endophytic fungi associated. Fungal endophyte communities associated with leaves of Myrciaria floribunda, Alchornea castaneifolia, and Eugenia aff. bimarginata were examined, collected from Brazilian Cerrado ecosystems, and studied for their ability to produce antimicrobial activity. A total of 93 isolates of endophytic fungi were obtained and identified by sequencing of internal transcribed spacer (ITS) regions of the rRNA gene. Thirty-eight fungal extracts presented antimicrobial activity against at least one of the different target microorganisms tested. The biogeography of microorganisms is poorly understood and to to address whether patterns of endophyte diversity are correlated with host phylogeny, environmental variables and distance we designed a geographic survey of related host tree of Myrtae tribe. The study was performed at the following spatial scales: regional, 101-5,000 km; local: 0-100 km and microscale: 0-1 km. A total of 960 isolates of endophytic fungi were obtained from 3,000 leaf fragment samples. These isolates were grouped and identified into 52 species. We found that the similarity of endophytic fungal communities decreases with increasing geographical distance, showed by the distance decay analysis. The nonmetric multidimensional scaling (NMDS) analysis showed that the fungal endophytes communities were grouped at a local scale. The multiple regression on matrices (MRM) suggested that the fungal endophytic community similarity depends on the scale sampled used. Other study showed that endophytic fungi can interact with arbuscular micorryzal, increasing the growth plant. A total of 150 isolates of endophytic fungi were obtained from roots of Myrtus communis and seven groups were selected to interactions study with Sorghum vulgare. None of the root endophytic fungi tested colonized the root of sorghum, but significantly increased the growth and arbuscular mycorrhizal colonization of sorghum. The fourth part of this work evaluated the endophytic community diversity of Myrtus communis by classical isolation from leaves fragments and denaturing gel gradient electrophoresis (DGGE). A total of 113 fungal isolates were obtained and identified by classical isolation in potato dextrose agar. It was found that the similarity of endophytic fungal communities did not decrease with increasing geographical distance. The results obtained in this work suggest a high endophytic fungal diversity associated with Myrtaceae host tree presents indifferent ecossistems. Moreover, these micro-organismos represents a potencial source of substances that can be used to development of new antimicrobial drugs and as a growth promoting fungi. 5 Relevância e justificativa Micro-organismos 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 ao hospedeiro. Estima-se que existam cerca de 300 mil espécies de plantas nos diferentes biomas do planeta e cada planta pode ser hospedeira de um ou mais micro-organismo endofítico, sendo que pesquisas sugerem que a maioria das plantas, senão todas, em ecossistemas naturais formam associações simbióticas com fungos endofíticos. Estes micro-organismos apresentam uma grande diversidade genética e desempenham importantes funções na manutenção dos ecossistemas. Vários trabalhos sugerem que este grupo de micro-organismos é uma fonte promissora de metabólitos secundários de interesse econômico. Além disso, os fungos endofíticos podem interagir com outros micro-organismos promovendo o crescimento vegetal de plantas de interesse agronômico. Os padrões sobre a distribuição dos endofíticos também vem sendo realizados a fim de determinar a influência das características ambientais na distribuição da comunidade de fungos. Na interação entre fungos endofíticos e planta os fungos endofíticos são beneficiados pelos fotossintatos produzidos e proteção conferida pelo hospedeiro; em troca, garantem maior adaptabilidade ecológica ao hospedeiro, como tolerância ao estresse ambiental, resistência à fitopatógenos e herbívoros, entre outros. Neste tipo de interação os fungos endofíticos podem produzir metabólitos secundários, os quais podem apresentar diferentes atividades biológicas. Os metabólitos secundários, ou produtos naturais, podem ser definidos como substâncias de baixo peso molecular, normalmente produzidos em resposta a condições ambientais específicas. Entre as atividades biológicas envolvendo metabólitos produzidos por micro-organismos endofíticos pode-se mencionar a ação antibacteriana, antifúngica, antioxidante, antiviral, antiparasitária e inseticida. Plantas com histórico etnobotânico são um dos alvos para estudos de bioprospecção, pois acredita-se que algumas propriedades medicinais atribuídas ao vegetal também possam estar relacionadas à produção de metabólitos secundários por seus fungos associados. Neste contexto, os fungos endofíticos associados às medicinais Eugenia aff. neomyrtifolia, Myrciaria floribunda e Alchornea castaneifolia presentes em ecossistemas brasileiros foram avaliadas com respeito a atividade antimicrobiana contra micro-organismos de interesse clínico. Relatos fósseis indicam que a interação fungo endofítico-planta iniciou-se há milhões de anos, quando vegetais superiores surgiram no planeta pela primeira vez. Sabe-se que muitos micro-organismos exibem padrões de diferenciação genética e molecular que podem estar relacionados com a distribuição dos seus hospedeiros. Nesse sentido, no presente trabalho foi avaliado se a comunidade de fungos endofíticos varia ao longo de um gradiente geográfico ou se 6 os fungos endofíticos apresentam correlação filogenética com seus hospedeiros. A tribo Myrtae (família Myrtaceae) foi escolhida para este objetivo por formar um grupo monofilético e por existirem diversos estudos sobre a sua filogenia. Outro aspecto importante da biologia de fungos endofiticos é que estes micro-organismos também podem interagir com fungos micorrízicos arbusculares melhorando a adapatabilidade das plantas por meio da promoção do crescimento vegetal, proteção contra doenças ou aumentando a absorção de fósforo. Desta forma, os fungos endofíticos associados a raízes de Myrtus communis foram isolados e os mais frequentes foram inoculados na rizosfera de raízes de Sorghum vulgare. O sorgo foi escolhido como um modelo de planta de interesse agronômico mundial e constitui o quinto cereal mais consumido no mundo. O estudo da micota endofítica associada às diversas plantas avaliadas neste trabalho será de grande importância para o conhecimento micológico. Os estudos sobre o potencial biotecnológico dos fungos pode levar ao descobrimento futuro de processos metabólicos utilizados por estes micro-organismos úteis para utilização biotecnológica. Além disso, os estudos biogeográficos são fundamentais para melhor entender os padrões de distribuição da comunidade endofítica a fim de determinar a influência das variáveis ambientais ou históricas na distribuição atual da micota endofítica. 7 Formatação da tese Esta tese é apresentada em sete capítulos. O capítulo um refere-se à revisão bibliográfica sobre o tema proposto. O capítulo de 2 trata do estudo da diversidade e da atividade antimicrobiana de fungos do cerrado do Tocantins, Norte do Brasil. Este trabalho foi aceito para publicação na revista African Journal of Microbiology Research. O capítulo 3 fala sobre a diversidade e biogeografia de fungos endofíticos associados espécies vegetais pertencentes à família Myrtaceae presentes em ecossistemas brasileiros e argentinos. O capítulo 4 é sobre as interações de fungos endofiticos com micorrizas arbusculares, e este trabalho foi aceito para publicação na revista Soil Applied Ecology. O capítulo 5 é sobre a diversidade de fungos endofíticos associados a folhas de Myrtus communis estudada por métodos tradicionais de isolamento de fungos e por eletroforese em gel de gradiente desnaturante (DGGE). O capítulo seis aborda uma discussão integrada dos resultados obtidos e o capítulo sete as conclusões do trabalho. 8 Capítulo 1 Revisão bibliográfica 9 1. Micro-organismos endofíticos Os micro-organismos endofíticos foram mencionados pela primeira vez no início do século XIX, mas foi De Bary (1866) quem primeiro delineou a diferença entre eles e os patógenos de plantas. Os primeiros estudos com endófitos foram realizados com plantas da família das gramíneas. No final do século XIX, vários pesquisadores descreveram a presença de micélio fúngico nos carpelos e sementes de plantas sadias de Lolium arvense, L. linicolum, L. remotum e L. temulentum (Guerin, 1898; Hanausek, 1898; Vogl, 1898; Rodriguez et al. 2009). Na década de 1930 pesquisas demonstraram a relação entre fungos endofíticos e a toxicidade de L. temulentum para o gado (Kingsbury, 1964). Entretanto os estudos sobre os fungos endofíticos presentes em outras famílias de plantas, que não as gramíneas, intensificaram-se somente a partir de 1970, quando foi verificado que estes micro-organismos apresentam interações simbióticas com os hospedeiros, protegendo as plantas dos ataques de insetos, de doenças e de mamíferos herbívoros (Azevedo, 1999). Desde então vários trabalhos mostraram a presença de espécies fúngicas em plantas pertencentes a grupos como briófitas (U’Ren et al., 2010), pteridofitas (Petrini et al., 1992), gimnospermas (Soca-Chafre et al., 2011), angiospermas monocotiledôneas (Pinruan et al., 2010) e dicotiledôneas (Vaz et al., 2009; Vaz et al. 2012 a,b; Vieira et al., 2012). Webber (1981) foi provavelmente o primeiro pesquisador a relatar um exemplo de proteção à planta conferida pelo fungo endofítico Phomopsis oblonga, que reduzia a dispersão de Ceratocystis ulmi, fungo causador da doença do olmo, pelo controle de seu vetor, o besouro Physocnemum brevilineum. O autor associou o efeito repelente contra os insetos à compostos tóxicos produzidos pelos fungos. Isto foi confirmado posteriormente por Claydon et al. (1985), os quais mostraram que fungos endofíticos pertencentes à família Xylariaceae, associados à espécies de plantas do gênero Fagus, sintetizavam metabólitos secundários que afetavam larvas de besouros. Desde então vários pesquisadores apresentaram definições para o termo endofítico. Uma das definições mais aceita infere que os micro-organismos 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 (Bacon & White, 2000). Coletivamente, mais de cem anos de pesquisa sugere que a maioria das plantas, senão todas, em ecossistemas naturais formam associações simbióticas com fungos endofíticos (Petrini et al. 1986; Rodriguez et al., 2009). Rodriguez et al. (2009) propõe um divisão dos fungos endofíticos em quatro classes. A primeira é formada pelos fungos clavicipitaceos (endofíticos-C), os quais represetam um pequeno número de espécies de fungos que formam infecções sistêmicas em gramíneas. A maioria destes fungos pertence aos gêneros Epiclhoë e Balansia, e seus anamorfos 10 Neotyphodium e Ephelis, respectivamente, ambos da família Clavicipitaceae (Hypocreales, Ascomycota). Os benefícios conhecidos desta interação para a gramínea hospedeira incluem o aumento da tolerância a metais pesados; aumento da resistência à dessecação; redução no herbivorismo e resistência contra patógenos. A proteção contra herbivoria ocorre por meio de metabólitos secundários bioativos produzidos pelos fungos, como a peramina e lolina (atividade inseticida), e lolitrem B e ergovalina (atividade citotóxica) (Alexopoulos et al., 1996; Schulz & Boyle, 2005). O fungo endofítico beneficia-se dos nutrientes presentes no espaço apoplástico do tecido vegetal, da proteção contra estresses abióticos e da competição com micro-organismos epifíticos (Saikkonen et al., 1998). Os fungos não-clavicipitaceos (endofíticos-NC) apresentam uma alta diversidade de fungos e são representados por três grupos funcionais distintos baseados em características de história de vida e sua significância ecológica (Rodriguez et al., 2009; Saikkonen et al., 1998). Nesta interação os fungos endofíticos são beneficiados pelos fotossintatos produzidos e proteção conferida pelo hospedeiro; em troca, garantem maior adaptabilidade ecológica ao hospedeiro, como tolerância ao estresse ambiental, resistência à fitopatógenos e herbívoros, entre outros (Tan & Zou, 2001; Petrini et al., 1992; Saikkonen et al., 1998; Strobel & Dayse 2003; Linnakoski et al., 2011). A classe dois é formada principalmente por fungos pertencentes ao filo Ascomycota, subfilo pezizomicotina e em minoria também são encontrados fungos do filo Basidiomycota, subfilo Agaricomycotina e Pucciniomycotina. Estes fungos colonizam raízes, caules e folhas; são capazes de formar infecções extensas nas plantas; sua trasmissão pode ocorrer tanto horizontalmente quanto verticalmente; e, apresentam uma alta taxa de infecção (90-100%) em plantas presentes em habitats sob condições de estresse. Um exemplo seria a alga marrom Ascophyllum nodosum que necessita do fungo endofítico Mycophycia ascophylli para crescer e desenvolver (Garbary & Mcdonald, 1995). Quando o fungo Curvularia protuberata e a planta Dichanthelium lanuginosum encontram-se associados ambos são capazes de tolerar temperaturas de ate 65ºC, enquanto em condições não simbióticas esta tolerância não passaria de 40ºC (Márquez et al., 2007). Outra característica seria a tolerância a estresse salino. A associação do fungo Fusarium culmorum com tecidos de Leymus mollis (Poaceae) eleva a tolerância de ambos a níveis de salinidade entre 300 e 500 mM de NaCl (Rodriguez et al., 2008). A terceira classe é composta de fungos que infectam principalmente tecidos aéreos, que se transmitem horizontalmente e a sua colonização nos tecidos vegetais é normalmente localizada. Neste grupo incluem-se a maioria dos fungos endofíticos obtidos de hospedeiros tropicais (Arnold et al., 2001; Gamboa & Bayman, 2001; U'Ren et al., 2009; Vaz et al., 2009; Vaz et al., 11 2012a; Vieira et al., 2012) e temperados (Higgins et al., 2007; Murali et al., 2007; Davis & Shaw 2008). A maioria dos fungos deste grupo pertencem principalmente ao filo Ascomycota e em menor frequência ao Basidiomycota (Rodriguez et al., 2009) A frequência de isolamento, riqueza e diversidade destes fungos geralmente seguem forte gradiente latitudinal com os maiores valores encontrados em ambientes tropicais quando comparados com ambientes temperados (Arnold & Lutzoni 2007). A ocorrência de fungos endofíticos varia com o habitat e alguns gêneros são comuns tanto em vegetais de ecossistemas tropicais quanto temperados, tais como Fusarium, Phomopsis e Phoma. Já em ecossistemas tropicais, os gêneros comumente encontrados são Cladosporium, Corynespora, Colleotrichum, Guinardia, Lasiplodia, Phyllostica, Pestalotiopsis, Sporormiella e Xylaria (Rodrigues & Samuels, 1999; Suryanarayanan et al., 2002; Schulz & Boyle, 2005; Joshee et al., 2009; Gazis et al., 2010; Veja et al., 2010). A classe quatro é formada por fungos septados negros (“Dark spetate endophyte”, DSE), os quais normalmente colonizam tecidos da raiz, sua transmissão ocorre horizontalmente e colonizam os tecidos vegetais extensivamente (Rodriguez et al., 2009). Estes fungos são pertencentes ao filo Ascomycota e formam estruturas com presença de melanina tais como hifas inter e intracelulares e microesclerocios nas raízes de plantas. Os fungos DSE provavelmente constituem os fungos mais abundantemente encontrados em associação com raízes em paralelo com os fungos micorrízicos arbusculares (Mandyam & Jumpponen, 2005; Aveskamp et al., 2009). Além disso, a maioria destes fungos não apresenta especificidade de hospedeiros. Estudos demonstraram que os fungos septados negros, Chloridium paucisporum, Leptodontidium orchidicola, Phialocephala dimorphosphora, Phialocephala fortinii e Phialocephala Finlândia são capazes de colonizar uma ampla faixa de hospedeiros, sendo P. fortinii capaz de colonizar mais de 20 hospedeiros vegetais. Rodriguez et al. (2009) não inclui nesta classificação os fungos micorrízicos arbusculares (MA). Esta associação constitue o sistema radicular absortivo de aproximadamente 90% das plantas terrestes (Smith & Read 2008) e podem ser classificadas em sete tipos (Harley & Smith 1983, Peterson et al., 2004), sendo as micorrizas arbusculares (MA), o tipo mais abundantemente encontrado na natureza. Esta simbiose propicia o melhor desenvolvimento do vegetal devido ao aumento na absorção de água e nutrientes, principalmente fósforo, pelas hifas do fungo MA (Smith e Read, 2008). Apesar dos fungos micorrízicos também serem endofíticos muitos autores os consideram em um grupo separado por ser uma simbiose altamente especializada e bem caracterizada (Stone et al., 2000). 12 Estima-se que cerca de 99% dos micro-organismos presentes na natureza não são cultiváveis utilizando-se as metodologias padrões de isolamento (Amann et al., 1995). Os métodos dependentes de cultivo são os mais utilizados para o estudo da comunidade de fungos endofíticos (Arnold et al., 2001; Bussaban et al., 2001; Wilberforce et al., 2003). Estes métodos apresentam alguns incovenientes, como por exemplo, fungos que apresentam crescimento rápido são capazes de crescer mais rapidamente nos meios de cultura tradicionalmente utilizados impedindo o crescimento de fungos de crescimento lento (Hyde & Soytong 2008). Além disso, fungos em estágios latente, quiescente ou que necessitam de alguns requerimentos nutricionais frequentemente não são isolados por estas metodologias (Götz et al., 2006). Sendo assim, métodos independentes de cultivo, como eletroforese em gradiente de gel desnaturante (Denaturing Gradient Gel Electrophoresis, DGGE; Muyzer et al., 1993), podem fornecer dados mais completos sobre a biodiversidade de fungos endofíticos. A técnica de DGGE tem sido utilizada com sucesso em estudos de ecologia de comunidades fúngicas (Götz et al., 2006; Vainio et al., 2005; Duong et al., 2006). Esta técnica permite o isolamento tanto de fungos endofíticos já isolados por meio de cultivo quanto de espécies não-cultiváveis (Duong et al., 2006). Além disso, os perfis moleculares gerados pela técnica de DGGE possibilitam a análise de várias amostras ambientais simultaneamente, sendo bastante útil para o monitoramento e compreensão de variações temporais e espaciais de comunidade microbianas (Zilli et al., 2003). Isto mostra a importância do uso destas técnicas para melhor caracterizar os padrões de distribuição da comunidade de fungos endofíticos. 2. Interação fungo endofítico – planta hospedeira Apesar da natureza assintomática da ocupação fúngica no tecido vegetal induzir à classificação de um relacionamento simbiótico, a diversidade de fungos endofíticos sugere que estes possam ser sapróbios ou patógenos oportunistas (Strobel & Daisy, 2003). De fato, alguns fitopatógenos têm origem endofítica, pois são micro-organismos oportunistas que podem causar infecções sintomáticas na planta quando esta se encontra em condições de estresse ou senescência. Alguns fungos podem ser oportunistas acidentais, os quais são normalmente encontrados em outros substratos e não são especificamente adaptados aos seus hospedeiros, tais como algumas espécies de fungos coprófilos detectadas nos tecidos vegetais (Schulz & Boyle, 2005). Comensalismo e mutualismo requerem um sofisticado balanço entre as respostas de defesa da planta e a demanda de nutrientes pelo endofítico. Desse modo, a interação mutualística não significa ausência de defesa da planta (Kogel, 2006). Promputtha et al. (2007) avaliaram que fungos colonizadores primários e secundários de folhas deterioradas têm origem endofítica. 13 Evidências filogenéticas obtidas por estes autores indicam que alguns fungos, por exemplo, espécies de Colletotrichum, Fusarium e Phomopsis, ocorrem como endofíticos e sapróbios. Portanto, algumas espécies de endofíticos podem mudar sua estratégia ecológica e adotar um estilo de vida saprofítico. Apesar das especulações a respeito da definição do tipo de interação, diferentes autores citam que a distinção entre endofíticos, epifíticos (aqueles que vivem na superfície de plantas) e fitopatógenos (que causam doenças em plantas) é meramente didática. Não existe um claro limite entre os grupos e sim um gradiente entre eles (Azevedo, 1999; Saikkonen et al., 1998). Saikkonen et al. (2004) propuseram que a relação dos fungos endofíticos com a planta hospedeira é bem mais complexa, envolvendo interações entre as espécies e também a influência de fatores bióticos e abióticos. Um fungo endofítico, por exemplo, pode tornar-se um patógeno conforme as condições de ambiente ou equilíbrio com outros endofíticos; um micro-organismo epifítico pode, eventualmente, entrar em uma planta, permanecendo por um determinado período, causando ou não danos à mesma. Pesquisas mostram que a mudança da interação de mutualismo para parasitismo pode ocorrer por mutação em um único gene do genoma microbiano (Kogel, 2006). Tanaka et al. (2006) sugerem um mecanismo de simbiose no qual a produção de espécies reativas de oxigênio (ROS) por Epiclhoë festucae NoxA in planta regula negativamente o desenvolvimento do fungo e o crescimento da hifa, prevenindo assim a colonização excessiva do tecido da planta. O gene NoxA codifica uma NADPH oxidase que cataliza a produção de superóxido pela transferência de elétrons de NADPH para o oxigênio molecular, com geração secundária de H2O2. A inativação do gene NoxA em E. festucae muda a interação do endofítico com o hospedeiro de mutualismo para parasitismo. Sendo assim, interações mutualísticas entre fungos invasores e planta hospedeira são decifradas como um balanço, sobre controle ambiental, fisiológico e genético, que resulta nos benefícios de adaptabilidade para ambos os parceiros (Saikkonen et al., 1998; Azevedo, 1999). 3. Biogeografia de fungos Estima-se que a interação fungo endofítico-planta iniciou-se há milhões de anos, quando vegetais surgiram no planeta pela primeira vez. Foram encontradas evidências da associação planta-endofítico em tecidos vegetais fossilizados presentes em Rhynie Chert, material fossilífero encontrado no interior da Inglaterra. Rhynie Chert possui a primeira evidência fóssil das plantas terrestres, datadas do Devoniano, com cerca de 396 milhões de anos (Krings et al., 2007). A partir dessas evidências, supõe-se que diversos tipos de interações entre vegetais e endófitos podem ter sido estabelecidas ao longo dos anos, incluindo-se relações de 14 especificidade entre os micro-organismos e a planta hospedeira (Tan & Zou, 2001; Strobel, 2002) e que podem estar relacionadas com a distribuição dos hospedeiros (Arnold et al., 2010). Estes simbiontes fúngicos apresentam profundos efeitos na ecologia, adaptação e evolução das plantas (Brundrett, 2006; Arnold et al., 2010), porém existem poucos trabalhos que abordam a biogeografia de micro-organismos (Fierer & Jackson, 2006; Martiny et al., 2006). Biogeografia é o estudo da distribuição da biodiversidade ao longo do espaço e tempo (Martiny et al., 2006). Desde o século dezoito os biólogos têm investigado a biogeografia e diversidade de plantas e animais e mais recentemente os micro-organismos têm sido inseridos nestes estudos (Martiny et al., 2006). Micro-organismos foram considerados como cosmopolitas por muitos anos por possuírem tempos de geração curtos, grandes populações e terem a capacidade de serem dispersos por longas distâncias (Fenchel & Finlay, 2004; Queloz et al., 2011), o que levou a elaboração da teoria de Baas Beckin: “tudo esta em toda parte, mas o ambiente seleciona” (“everything is everywhere, but the environment selects”) também conhecida como hipótese EisE (O´Malley, 2007). Entretanto esta hipótese tem sido muito discutida após o advendo das metodologias moleculares para a taxonomia de micro-organismos, o que permitiu a identificação das espécies de forma mais precisa. Muitos estudos encontraram correlações entre a composição de micro-organismos e características ambientais e geográficas, tais como salinidade, profundidade e latitude (Martiny et al., 2006). Fungos endofíticos já foram investigados em várias escalas espaciais, que vão desde pequenos estudos sobre os padrões de distribuição em uma única folha à aqueles em níveis geográficos (Carrol et al., 1995). Estudos em nível geográfico comparam fungos endofíticos de hospedeiros semelhantes ou relacionados em diferentes áreas geográficas. Estes trabalhos podem considerar hospedeiros em uma distribuição contínua ou disjunta. Hospedeiros em distribuição disjunta são aqueles que são relacionados, mas separados geograficamente (Carrol et al., 1995). Sabe-se que muitos micro-organismos endofíticos exibem padrões de diferenciação genética, morfológica e funcional que estão relacionados à distribuição dos seus hospedeiros (Martiny et. al., 2006). Porém poucos estudos têm comparado os endófitos associados a hospedeiros em ambientes tropicais e temperados para determinar os possíveis efeitos climáticos que poderiam causar diferenças na população de fungos endofíticos (Taylor et al., 1999). Taylor et al. (1999) compararam a população de fungos endofíticos associados à palmeiras presentes em regiões temperadas e tropicais. Foram coletadas folhas da palmeira Trachycarpus fortunei na China (dois locais), Austrália e Suíça. Houve uma grande sobreposição entre as comunidades de fungos endofíticos encontradas. A maior foi entre os fungos isolados das palmeiras na China, com 14 gêneros sendo encontrados nos dois locais de coleta. A sobreposição entre os gêneros 15 encontrados na China, Austrália e Suíça foi menor que 10 gêneros para cada local. Oito gêneros encontrados na Austrália também ocorreram na Suíça. Esta diferença na composição de fungos endofíticos parece ser influenciada por fatores climáticos. Fisher et al. (1995), por exemplo, isolaram fungos endofíticos de Gynoxis oleifolia no Equador. Quando coletadas amostras dentro de latitudes tropicais, mas em habitat temperado (por exemplo, altas altitudes), a composição de endofíticos obtidos foi análoga a aqueles de hospedeiros em habitats temperados. Vujanovic et al. (2006) avaliaram a distribuição de espécies de Fusarium associadas à Asparagus officinalis L. (Aspargos) presentes em 52 plantações no Canadá. Estas plantações estavam incluídas em cinco áreas climáticas. Amostras de plantas saudáveis, doentes e do solo foram coletadas. Observou-se que a abundância de fungos filamentosos tendia a diminuir ao longo de um gradiente de temperatura, das regiões mais quentes do sul para as áreas mais frias a nordeste. Além disso, foi observado que espécies pertencentes ao complexo de Fusarium “vermelhos” eram mais adaptadas ao clima da região Nordeste. Estes autores concluíram que espécies de Fusarium podem flutuar dentro e entre cultivares depedendo da estação climática. Tais flutuações podem variar de acordo com a localização geográfica, como observado quando comparado com plantações de Aspargos norte-americanos e de outros continentes. A fim de estudar os padrões de distribuição biogeográfica de fungos endofíticos foram selecionados hospedeiros relacionados filogeneticamente pertencentes à tribo Myrtae (Myrtaceae). Este grupo foi escolhido por formar um grupo filogeneticamente coeso (Wilson et al., 2001), desta forma seria possível determinar se a distribuição fungos endofíticos era devido a condições ambientais contemporâneas ou relacionadas a efeitos de contingência histórica (Martiny et al., 2006). Análises filogenéticas, moleculares e morfológicas sugerem a origem da família Myrtaceae na Gondwana, sendo que a tribo Myrtae provavelmente se originou e diversificou na Australasia, entre 77 e 56 milhões de anos atrás, quando a Australia estava conectada à América do sul por uma ponte de terra formada pela Antartica (Lucas et al., 2007). A tribo Myrtae representa um grupo monofilético e acredita-se que os eventos de especiação são rápidos e recentes, sendo os movimentos de longa distância a mais provável explicação para explicar movimentos intercontinentais ao invés de eventos de vicariância (Lucas et al., 2007). 4. Substâncias bioativas produzidas por fungos endofíticos A natureza tem sido fonte de agentes medicinais por milhares de anos e continua sendo uma abundante fonte de novos modelos estruturais para o desenvolvimento de diferentes substâncias de interesse medicinal. Produtos naturais ou metabólitos secundários são definidos como substâncias de baixo peso molecular que, aparentemente, não são necessários para o 16 crescimento do micro-organismo e são produzidos em resposta a condições ambientais específicas (Demain, 1981). Estes produtos exercem um enorme impacto na medicina moderna, uma vez que 40% dos fármacos prescritos são baseados neles. Além disso, de acordo com o “Food and Drug Administration” (USA FDA), órgão americano de controle e registro de fármacos, 49% das novas substâncias químicas bioativas registradas são produtos naturais ou seus derivados (Brewer, 2000). Antibióticos são produtos naturais orgânicos de baixo peso molecular produzidos por micro-organismos e que são ativos em baixas concentrações contra outros micro-organismos (Demain, 1981). A introdução dos antibióticos sulfonamida (1930) e da penicilina (1940) revolucionaram a prática médica pela diminuição das taxas de mortalidade relacionadas às doenças infecciosas (Butler & Buss, 2006). Isto despertou o interesse da utilização de micro- organismos como fonte para a descoberta de metabólitos secundários com atividade contra patógenos humanos e de plantas (Strobel & Daisy, 2003). A história científica mostra que as substâncias isoladas dos micro-organismos renderam algumas das drogas mais importantes utilizadas na clínica. Entre estas se destacam as penicilinas, cefalosporinas, aminoglicosídeos, tetraciclinas; ciclosporina, FK506 e rapamicina; agentes que diminuem o colesterol, como mevastatina e lovastatina; drogas antiparasitárias e antihelmínticas, tais como a ivermectina (Cragg et al., 2005). Além disso, vários metabólitos com atividade quimioterápica também foram isolados a partir de micro-organismos, como antraciclina e bleomicina. A partir destes fatos e da descoberta que o fungo endofítico Taxomyces andreanae, isolado do vegetal Taxus brevifolia, produz o diterpeno paclitaxel, um dos agentes anticâncer mais utilizado na clínica atualmente, aumentaram substancialmente os estudos dos fungos endofíticos como fonte de substâncias bioativas (Stierle et al., 1995). Os produtos naturais são metabólitos de grande importância nas interações entre o endofítico e a planta hospedeira e podem atuar em processos de sinalização, defesa e regulação da simbiose (Schulz & Boyle, 2005). Fungos endofíticos estão em constante interação com a planta hospedeira e são tidos como excelentes fontes de produtos naturais bioativos, uma vez que podem ocupar nichos biológicos únicos e crescem em diversos ecossistemas (Schulz et al., 2002). De fato, vários trabalhos demonstraram o potencial dos fungos endofíticos como produtores de substâncias bioativas (Strobel et al., 2002; Bills et al., 2002) ou como promissores para futuros estudos de novas substancias bioativas, avaliados por trabalhos de triagem antimicrobiana contra micro-organismos de interesse clínico (Huang et al., 2001; Peláez et al., 1998; Souza et al., 2004; Phongpaichit et al., 2006; Sette et al., 2006; Vaz et al., 2009, Vieira et al., 2012). 17 A pesquisa por novos antibióticos declinou significativamente nos anos de 1990. Este declínio foi consequência de diversos fatores, dentre os quais incluem principalmente a dificuldade de isolamento e elucidação estrutural de alguns produtos naturais, a falta de interesse das grandes indústrias farmacêuticas e a forte competição com as tecnologias de síntese química combinatorial associada à triagem de alto fluxo e tecnologias mais rápidas para programas de bioprospecção (Peláez, 2006). Como consequência, nos últimos 20 anos, houve um declínio em 56% no número de antibióticos aprovados pelo “Food and Drug administration” (FDA) (Butler & Buss, 2006), sendo que a maioria dos novos fármacos lançados no mercado durante esse período pertencia a antigas classes de antibióticos, como os β-lactâmicos e macrolídeos (Peláez, 2006). Diferentes estratégias são adotadas para a seleção do grupo vegetal para o isolamento de fungos endofíticos produtores de substâncias bioativas. Dentre elas, o estudo de plantas com caráter etnobotânico, endêmicas, presentes em ambientes com elevada diversidade e inexplorados são bastante promissores em estudos de bioprospecção (Strobel & Daisy, 2003). O conhecimento da existência de micro-organismos endofíticos levou ao desenvolvimento de novos programas de triagem para descoberta de novas drogas. Identificar micro-organismos endofíticos produtores da mesma substância bioativa produzida pelos vegetais eliminaria as etapas de plantio, colheita e extração de plantas raras ou de crescimento lento. Além disso, o preço do produto seria reduzido, uma vez que poderia ser produzido por meio de processos fermentativos (Strobel, 2002). No presente trabalho foram selecionados hospedeiros vegetais presentes no ecossistema Cerrado, o qual abriga uma diversa população de fungos endofíticos (Furlanetto & Dianese, 1997). Os hospedeiros selecionados apresentavam histórico etnobotânico, ou seja, plantas que são utilizadas tradicionalmente como medicamento por tribos, grupos étnicos e pela população de um modo geral. Neste contexto, é razoável supor que a atividade farmacológica atribuída a algumas espécies vegetais possa estar relacionada, de alguma forma, às substâncias produzidas por micro-organismos endofíticos ou pela planta (Arnold et al., 2001; Ferrara, 2006). Cabe ressaltar ainda que grande parte das substâncias bioativas obtidas a partir de extratos fúngicos podem ser empregadas diretamente como fármacos, ou mesmo serem modificadas de modo a se obter uma nova molécula com diferentes propriedades, tais como o aumento da atividade, toxicidade seletiva e a diminuição de efeitos colaterais indesejáveis. Mesmo quando estas substâncias não apresentam a atividade esperada, elas podem servir como protótipos para o planejamento e desenvolvimento de novas moléculas, o que evidencia ainda mais a importânciado estudo de fungos endofíticos como fontes de novas substâncias. 18 1 Objetivos 1.1 Geral Caracterizar a diversidade de fungos endofíticos associados à Myrtaceae presentes em ecossistemas do Brasil, Argentina e Espanha. Avaliar o potencial destes fungos como produtores de substâncias antimicrobianas de interesse biotecnológico e estudar o seu papel como promotores do crescimento vegetal. 1.2 Específicos  Isolar e caracterizar as comunidades de fungos endofíticos provenientes de ecossistemas de Mata Atlântica, Mata Amazônica, Cerrado, Andino patagônico e Mediterrâneo;  Identificar os fungos filamentosos endofíticos por meio do sequenciamento da região ITS do gene do rRNA.  Verificar atividade antimicrobiana dos extratos metanólicos dos fungos isolados de plantas de ecossistemas de Mata Atlântica, Mata Amazônica, Cerrado brasileiros sobre bactérias e leveduras de interesse clínico;  Avaliar o padrão de distribuição de fungos endofíticos em plantas filogeneticamente relacionadas da família Myrtaceae presentes em ecossitemas de Mata Atlântica (Brasil) e Andino patagônico (Argentina).  Estudar o papel de fungos endofíticos associados à raízes de Myrtus communis na colonização e peso seco de raízes de plantas de sorgo (Sorghum vulgare) e avaliar seu efeito na simbiose com micorrizas arbusculares. 19 Capítulo 2 Diversity and antimicrobial activity of fungal endophyte communities associated with plants of Brazilian savanna ecosystems Este capítulo foi publicado na revista African Journal of Microbiological research (2012) DOI: 10.5897/AJMR 11.1359 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Capítulo 3 Fungal endophyte β-diversity associated with Myrtaceae species in an Andean Patagonian forest (Argentina) and an Atlantic forest (Brazil) 34 Fungal endophyte β-diversity associated with Myrtaceae species in an Andean Patagonian forest (Argentina) and an Atlantic forest (Brazil) Aline B. M. Vaz 1* , Sonia Fontenla 2 , Fernando S. Rocha 3 , Luciana R. Brandão 1 , Mariana L. A.Vieira 1 , Virginia de Garcia 2 , Carlos A. Rosa 1* 1 Departamento de Microbiologia, ICB, C. P. 486, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901, Minas Gerais, Brazil. 2 Laboratório de Microbiología Aplicada y Biotecnología, Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, INIBIOMA-CONICET, Argentina. 3 Departamento de Fitotecnia, Universidade Federal de Santa Catarina, Florianópolis, 88034 - 001, Santa Catarina, Brazil. *Corresponding author: Aline B. M. Vaz and Carlos A.Rosa. Departamento de Microbiologia, ICB, C. P. 486, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901, Minas Gerais, Brazil. Telephone: 55 (31) 34092751; Fax number: 55 (31) 34092730; Email address: alinebmv@hotmail.br. 35 Summary The biogeography of microorganisms is poorly understood, and there is an open debate regarding how microbial diversity is structured. We examined the community similarity of the fungal endophytes of phylogenetically related Myrtaceae trees in a latitudinal gradient. A total of 951 isolates were obtained and identified into 55 distinct OTUs based on the sequencing of ITS regions of the rRNA gene. The study was performed at the following spatial scales: regional, 101-5,000 km; local, 0-100 km; and microscale, 0-1 km. Similarity of fungal endophyte communities decreased with increasing geographical distance. At regional scales, environmental factors were also important for explaining the fungal community similarity. Moreover, dispersal limitation was significant at all of the analysed scales, and there was no relationship between the fungal community similarity and the host tree proximity. We conclude that fungal endophytes display a biogeographic pattern and that considering multiple scales is important for understanding fungal biogeography. Keywords: Biogeography/ Distance decay/Fungal endophyte/ Myrtaceae/β-diversity 36 Introduction Biogeography is the study of the distribution of biodiversity over space and time (Martiny et al., 2006), and its study can illuminate the mechanisms that generate and maintain diversity, such as dispersion, speciation, extinction, and species interactions (Brown et al., 1998). The community similarity between two groups often decreases as the distance between them increases, a pattern observed in communities from all domains of life and known as distance decay or β-diversity (Nekola and White, 1999). Two primary explanations for this pattern have been proposed. Niche theory predicts that community similarity decreases with environmental distance, irrespective of geographic proximity, as a result of species differences along environmental gradients (Tilman, 1982). Neutral theory, in contrast, predicts that the decay of community similarity is caused by spatially limited dispersal, independent of environmental differences between sites (Hubbell, 2001). The past several years have witnessed an increase in debates about the biogeographic patterns of microorganisms and whether these patterns are similar to those observed in macroorganisms. Microorganisms have long been regarded as cosmopolitan because they exhibit short generation times and large population sizes and because they disperse over long distances (Fenchel and Finlay, 2004), prompted Baas Becking’s hypothesis that “everything is everywhere, but, the environment selects” (the EisE hypothesis) (Baas Becking, 1934; Green & Bohannan, 2006). The distance decay relationship is used to demonstrate how the processes of selection, drift, dispersal, and mutation shape biogeographic patterns (Hanson et al., 2012). When considering the EisE hypothesis, the distance decay curve would be due to a gradient of selective factors, which are thus spatially autocorrelated (Martiny et al., 2011; Hanson et al., 2012). Therefore, organisms with different niche preferences are selected from the available pool of taxa as the environment changes with distance (Martiny et al., 2011). Distance decay patterns, and therefore β-diversity, can also be influenced by dispersal limitation. This effect should reflect the influence of historical processes on the current biogeographic patterns (Martiny et al., 2011). Microbial biogeography studies were challenged following the advent of the molecular taxonomy of microorganisms, which allowed species to be distinguished more accurately (Fierer, 2008). The internal transcribed spacer (ITS1 and ITS2) and 5.8S regions of the nuclear ribosomal repeat unit are the most widely used molecular markers in fungal endophyte diversity studies (Promputtha et al., 2007; U’ren et al., 2010) and have been used as a primary fungal barcode marker (Schoch et al., 2012). The main advantage of ITS is the ease with which it can be amplified from all lineages of fungi using universal primers (Nilsson et al., 2008) and the 37 large size of the available ITS sequence database, such as GenBank and EMBL (Vilgalys, 2003). Fungal diversity remains relatively poorly explored, as evidenced by the small proportion of fungi that have been identified to date. Recent estimates have suggested that as many as 5.1 million fungal species exist (Blackwell, 2011), making fungi among the most diverse groups of organisms on our planet. Fungal endophytes inhabit healthy plant tissues during at least one stage of their life cycles without causing any apparent symptoms of disease or negative effects on the host (Petrini et al., 1992, Arnold et al., 2001). Endophytes have been isolated from all studied plant groups, including bryophytes (U’Ren et al., 2010), pteridophytes (Petrini et al., 1992), gymnosperms (Soca-Chafre et al., 2011), and both monocotyledonous (Pinruan et al., 2010) and dicotyledonous angiosperms (Vaz et al., 2009; Vieira et al., 2012). Many studies have documented remarkable endophyte richness in tropical plants (Saikkonen et al., 1998, Arnold & Lutzoni, 2007; Vaz et al., 2009, 2012; Vieira et al., 2012). Similar tendencies have been observed in temperate environments, where a host tree may harbour dozens of fungal endophytes (Saikkonen et al., 1998; Stone et al., 2000). Little is known about how fungal endophyte communities vary across a geographic range or whether endophytes show phylogenetic correlations with their hosts. Many host-associated microorganisms exhibit patterns of genetic, morphological and functional differentiation that are related to the distribution of their hosts (Papke et al., 2004). Myrtaceae species are found in diverse habitats, and a robust phylogeny exists for this group, making them ideal subjects for comparisons of endophyte community diversity. Phylogenetic analyses of morphological and molecular data have suggested a Gondwanan origin of Myrtaceae, with the Myrtae tribe originating and diversifying in Australasia between 77-56 ma, when Australia was connected to South America via warm-temperate Antarctic land bridges (Lucas et al., 2007). Tribe Myrtae DC. is a monophyletic group of plants that probably has experienced a recent and rapid speciation, with long-distance dispersals more likely than vicariance to explain at least some of the intercontinental movements (Lucas et al., 2007). We studied a phylogenetically related Myrtae (Myrtaceae) host species to determine the β- diversity, and therefore the distance decay patterns, of its fungal endophyte communities. We developed a phylogenetic hypothesis for the relationships among the fungal endophytes associated with the Myrtaceae host samples. It was considered Luma apiculata and Myrceugenia ovata var. nanophylla which constitute a single clade. We included one Myrtaceae species not present in this clade, Eugenia neomyrtifolia, as an “outgroup”. The objectives of the present study were the following: (1) to determine whether the fungal endophyte β-diversity, specifically the slope of distance decay curve, varies over regional, local or microscale scales; and (2) to 38 determine whether environmental variation or dispersal limitation explains the fungal community assemblage diversity along latitudinal gradients. Experimental procedures Study areas Three different sites in Patagonia, Argentina were studied. These sites were located in the Andean Patagonian region, near the city of San Carlos de Bariloche, which is situated within Nahuel Huapi National Park. This region is characterised by native forests, which are dominated by Nothofagus species or native conifers, such as Austrocedrus chilensis, Araucaria araucana, Fitzroya cupressoides and Pilgerodendrum uviferum (Donoso, 2006). In Argentina, Luma apiculata was collected in two different sites, the Arrayanes Forest and Puerto Blest; Myrceugenia ovata var. nanophylla was collected in one site, Espejo Lake. In Brazil, the study was conducted at the Centro de Pesquisas e Conservação da Natureza Pró-Mata of Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), in São Francisco de Paula, Rio Grande do Sul state. This area is a confluence of three phytoecological regions, including Araucaria forest, Atlantic rainforest and an herbaceous-bushy formation known regionally as “hill-top fields”. Myrceugenia ovata var. nanophylla and Eugenia neomyrtifolia were collected in this Atlantic rainforest site (Figure 1). All the sampled sites were characterised by low antropic impact and minimal atmospheric pollution. Fungal endophyte isolation Five apparently healthy leaves were collected from each of 20 individuals of all Myrtaceae species that occur in the studied sites. The trees were spaced approximately 5 m apart. All the leaves were stored in sterile plastic bags, and fungal isolation was performed on the same day of the collection. The leaves were surface-sterilised via successive dipping in 70% ethanol (1 min) and 2% sodium hypochlorite (3 min), followed by washing with sterile distilled water (2 min). After the leaf surface sterilisation, six fragments (approximately 4 mm 2 ) were cut from each leaf: one from the base, two from the middle vein, one from the left margin, one from the right margin and one from the tip (6 leaf fragments/leaf; 30 leaf fragments/tree; 600 leaf fragments/site; 3,000 leaf fragments overall) . All the fragments were plated onto potato dextrose agar (PDA, Difco, USA) supplemented with 100-µg/ml chloramphenicol (Collado et al., 1996). The plates were incubated at 15C for up to 60 days. To test the effectiveness of the surface sterilisation, 100 µl 39 of the water used during the final rinse was plated on PDA to test for epiphytic microbial contaminants. Individual colonies were purified on PDA, and their morphologies were documented and photographed. For filamentous fungi, the long-term preservation of mycelial samples was performed in sterile distilled water at room temperature. All the fungal isolates were deposited in the Culture Collection of Microorganisms and Cells of the Universidade Federal of Minas Gerais. Fungal identification Pure cultures of the fungal isolates were grouped based on their morphological characteristics, including aerial mycelium formation, colony colour, surface texture and margin characters. At least fifty percent of the fungal isolates of each morphospecies were identified by directly extracting their total genomic DNA and sequencing the ITS region of the rRNA gene. The extraction of DNA from filamentous fungi was performed according to Rosa et al. (2009). The ITS domains of the rRNA gene were amplified using the universal primers ITS1 (5´- TCCGTAGGTGAACCTGCGG-3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC-3´), as described by White et al. (1990). The amplification of ITS and the sequencing were performed as described by Vaz et al. (2009). Analysis of ecological data The diversity was estimated using the Shannon (H`) index (H = -Σ ni/n ln (ni/n), where ni is the number of individuals in the taxon i and n is the total number of individuals). Values of the Shannon index usually lie between 1.5 and 3.5, with 1.5 representing the lowest diversity and 3.5 the highest (Gazis & Chaverri, 2010). Species accumulation curves were used to determine whether a sufficient number of samples had been obtained from each study site and were generated for each host tree species in each site using EstimateS, version 8.0 (Colwell, 2005). For calculations and statistical analyses, each individual fragment was considered a sample unit; a total of 3,000 sample units were evaluated. To visualise the trends and groupings of the fungal endophytes, Nonmetric Multidimensional Scaling (NMDS) analyses were conducted. The Jaccard distance was used in these analyses due to its simplicity, widespread use and more conservative measurement of community similarity than distances based on species abundance data, which are more sensitive to disturbances and local environmental differences (Nekola & White, 1999). To reduce the influence of the most abundant species, the data were square-root transformed prior to the NMDS analysis (Clarke & Warwick, 2001; Joshee et al., 2009). 40 The rate of distance decay of the fungal endophyte communities was calculated according to Nekola and White (1999), with the assumption that community similarities decrease with increasing geographical distance. The distance decay relationship was calculated as the slope of a least-squares linear regression on the relationship between the (ln-transformed) geographic distance and the fungal endophyte community similarity measured by the Jaccard index. The slope of the distance-similarity relationship is one of the most common measures of β-diversity in ecological studies (Nekola & White, 1999). We chose to transform the geographic distance because of our sampling scheme, which purposely sampled over many orders of magnitude; otherwise, the data points would have been highly skewed (Martiny et al., 2011). In addition, we tested whether the slope of the distance decay curve of each collection site was significantly different from zero using a randomisation procedure with 1,000 iterations. To create geographic distance matrices between all the sampling points, we recorded the location and compass direction of each sampling transect with a handheld GPS unit (Martiny et al., 2011). We used the GPS points, bearing angle, and sample spacing along each transect to calculate the geographic distance between samples within each collection site. To investigate the relationships among the fungal endophyte community similarity, geographic distance and environmental characteristics across all the spatial scales, we first used the ecodist R package (R Development Core Team, 2005; Goslee & Urban, 2007; Martiny et al., 2011) to apply a ranked partial Mantel test (which assumes a monotonic, but not linear, relationship). We performed a principal components analysis (PCA) of several environmental variables (water precipitation, altitude and temperature) and then computed the dissimilarities for the first component. To compare additional variables, we created a distance matrix that considered geographic distances. Correlations were examined with the Spearman correction, and the p values were based on 10,000 permutations. To tease apart the relative importance of the environmental variables on fungal endophyte community similarity, we used multiple regression on matrices (MRM) (Goslee & Urban, 2007). To reduce the effects of spurious relationships between variables, we performed the MRM test, removed the nonsignificant variables, and then repeated the test (Harrel, 2001, Martiny et al., 2011). The model results reported here are from the second performance of the test. To further examine the relative importance of each predictor variable at the three spatial scales (regional: 101-5,000 km, local: 0-100 km and microscale, 0-1 km), we investigated scale-specific MRM models. We tested the significance of each model by performing 10,000 permutations. All the analyses were performed using the R program (R Development Core Team, 2005). 41 Results Diversity of fungal endophytes A total of 951 fungal endophyte isolates were obtained from 3,000 leaf fragments. Fifty- five distinct operational taxonomic units (OTUs) were identified based on the sequencing of the ITS region of rRNA. Twenty-three taxa were identified at the species level. Thirty-two OTUs exhibited a high divergence in the ITS region, with nucleotide differences from the other fungal sequences deposited in GenBank that ranged from 4 to 11 % (Supplementary table 1). All the taxa belong to the Ascomycota phylum, except Trametes, which belongs to the Basidiomycota phylum. The ascomycetous fungi were identified as members of the Sordariomycetes, Dothideomycetes, Leotiomycetes, Euromycetes and Pezizomycetes classes. Pseudocercospora basintrucata and Xylaria sp.1 were the most frequent taxa isolated from the L. apiculata present at the Arrayanes Forest and Puerto Blest collection sites (Andean Patagonian forest), respectively. Mycosphaerella sp. and Xylaria enteroleuca were most frequently isolated from M. ovata var. nanophylla from Espejo Lake (Andean Patagonian forest, Argentina) and the Atlantic rainforest (Brazil), respectively. The most frequent fungal species isolated from E. neomyrtifolia (Atlantic rainforest, Brazil) was Colletotrichum sp. Only species of the genus Xylaria were isolated simultaneously from all the host plants studied. The accumulation curve reached an asymptote when the singletons were removed, which indicates that the sampling effort had sufficient statistical power to capture the total species richness of the cultivable fungal endophytes (Figure 2). The results of the bootstrap analysis of the observed species richness fell within the 95% confidence interval, which indicated that the sampling of fungal endophytes was statistically complete (data not shown). The diversity indexes are shown in Supplementary table 2. The host trees present in Atlantic rainforest ecosystem in Brazil, M. ovata and E. neomyrtifolia, displayed the highest Shannon values. In contrast, the L. apiculata host trees present in the Andean Patagonian forests exhibited low Shannon index values. The M. ovata that grew in the Atlantic rainforest (Brazil) presented greater diversity than the same species collected in the Andean Patagonian forest (Argentina). This species did not share any fungal endophyte species between the two sampled sites. The Luma apiculata collected in two different Argentinean sampling sites shared six fungal endophyte species. The M. ovata present in the Atlantic forest shared three fungal endophyte species with the E. neomyrtifolia present at the same site. The NMDS plots revealed a clear separation between the groups found at the three Andean Patagonian forest sites (Argentina) and those at the Atlantic rainforest site (Brazil) (Figure 3). A plot of the fungal endophyte community similarity versus the distance for each pairwise set of 42 samples revealed a significant, negative distance decay curve for the fungal endophyte community (slope = - 0.01, P < 0.0001) (Figure 4). Furthermore, a microscale analysis of the slope of this curve revealed significant variation between the host tree transects. There were significantly negative distance decay slopes for M. ovata (slope = - 0.02, P = 0.03) from Puerto Blest in the Andean Patagonian forest and for L. apiculata (slope = - 0.01, P = 0.01), M. ovata (slope = - 0.11, P < 0.0001) and E. neomyrtifolia (slope = - 0.03, P = 0.002) from the Atlantic rainforest in Brazil. However, the distance decay slope for L. apiculata in the Arrayanes Forest did not differ significantly from zero (P = 0.447). Over a regional scale, geographic distance appears to influence the fungal endophyte community β-diversity. The ranked partial Mantel tests revealed that the dissimilarity in the fungal community was highly correlated with geographic distance (ρ = 0.27, P < 0.05), but not with environmental distance (P = 0.9) (Table 1). MRM were used to investigate the relative importance of the factors contributing to these correlations. Over a regional scale, the MRM model explained a low proportion of the variability in the fungal endophyte community similarity (R 2 = 5%, P = 0.05; Table 2). The geographical distance (β = 0.95, P = 0.0001), altitude (β = - 0.27, P = 0.05) and air temperature (β = - 0.75, P = 0.001) significantly contributed to the partial regression coefficients. Water precipitation did not play a significant role (P = 0.957) in describing the model. Geographic distance had a strong effect on community similarity when considered at a local scale; i.e., neither the two different ecosystems (the Andean Patagonian and Atlantic rainforests) nor the environmental variables were significant in explaining the model. Geographic distance did not influence the similarity of the fungal endophyte community to that of L. apiculata, which were spaced approximately 24 km apart. However, the geographic distance (approximately 2,300 km) between the M. ovata samples from Argentina and Brazil was statistically significant (R 2 = 0.11, P = 0.0001; ρ = 0.34, P = 0.0001). Discussion We used a culture-based approach and the sequencing of ITS region of the rRNA gene to assess the diversity and taxon composition of fungal endophytes associated with phylogenetically related Myrtaceae species. The ITS region was chosen because this region has the highest probability of allowing the successful identification of a broad range of fungi (Schoch et al., 2012). The diversity patterns of the host trees in an Atlantic forest in Brazil (Supplementary table 2) were similar to those determined by extensive studies of foliar fungal endophytes of Podocarpaceae in New Zealand (Joshee et al., 2009); Euphorbiaceae in Peru 43 (Gazis & Chaverri, 2010); Lycopodiaceae, Rosacea and Pinaceae in Canada (Higgins et al., 2007); and Cupressaceae in the USA (Hoffman & Arnold, 2008). However, this pattern was not present in the host trees sampled in the Andean Patagonian forest (Argentina) in the present work, which exhibited less diversity than that observed in temperate ecosystems (Higgins et al., 2007). The sampling of 3,000 leaf fragments was sufficient to adequately capture the richness of the cultivable endophytes, which was confirmed by a species accumulation curve analysis. The curves of all the host tree species reached a plateau, indicating that our sampling strategies allowed a robust estimate of the fungal endophyte species number and abundance. Both the number and size of the sampled fragments have important effects on the number of species isolated: when the size of the leaf fragments is reduced while their number is increased, the number of isolated fungal species increases (Gamboa et al., 2002). The number of leaf fragments sampled in this work was greater than those of other studies of fungal endophytes (Higgins et al., 2007; Vega et al., 2010), making the diversity pattern obtained here more likely to be genuine. This reliability is confirmed by the low number of singletons obtained in the present study when compared with studies of tropical and temperate angiosperms (Arnold et al., 2001; Higgins et al., 2007). There is an ongoing debate as to whether microorganisms exhibit biogeographical patterns similar to those of macroorganisms or whether such patterns are obscured by the population sizes and dispersal capabilities of microorganisms (Martiny et al., 2011). This work compared the spatial variations in fungal endophyte communities across a latitudinal gradient and with phylogenetically related host tree species. The distance decay analysis showed that the fungal endophyte similarity decayed with increasing distance when analysed at regional and local scales. However, the slope of the distance decay curve varied in the microscale analysis. The only non-significant distance decay slope was observed for the L. apiculata collected in the Arrayanes Forest, which is dominated by this species. In wood plants, non-systemic endophytes are horizontally transmitted by spores from plant to plant (Saikkonen et al., 1998), and infection by airborne inoculum likely plays an additional role (Carroll et al., 1977; Bertoni & Cabral, 1988). These modes of transmission may explain the absence of a geographic distance effect on the distance decay in this study and suggest that the mycota associated with the forest trees surrounding are probably responsible for the fungal endophytes associated with L. apiculata. Therefore, fungal dispersal counteracts the distance decay relationship at the microscale level (Hanson et al., 2012). The NMDS analysis suggested that the fungal endophyte assemblages were shaped at a local scale (Figure 3). This fact is further indicated by the clear separation of groups between the 44 three Andean Patagonian forest sites (Argentina) and the Atlantic rainforest site (Brazil). The partial Mantel test showed that dispersal limitation significantly influenced the fungal endophyte community β-diversity at a regional scale (ρ = 0.27, P < 0.05). The MRM analysis allowed us to evaluate the independent contributions of geographic distance and environmental variables to the fungal endophyte community distribution. The varying importance of geographic distance and environmental parameters at different spatial scales likely reflects differences in their underlying variability at these scales (Martiny et al., 2011). The MRM analysis indicated that at a regional scale, the measured variables could not explain most of the variability in fungal endophyte community similarity, which was reflected in an unexplained variance of 95% (Table 1). We may have missed spatially autocorrelated abiotic or biotic factors with strong influences on the fungal endophyte communities. However, geographic distance and environmental variables (altitude and temperature) were found to influence the fungal endophyte community at different scales (Table 1). This fact likely reflects the large environmental differences between the ecosystems sampled in Argentina and Brazil because these parameters were not significant when separately analysed in each region (at a local scale) (Table 1). The distance (2,324 km) between these two ecosystems encompasses a latitudinal gradient on the east to the southwest of South America, which results in a high amount of variability in environmental distance. Previous studies have shown that the fungal endophyte community assemblage is affected by weather, with temperature and moisture being the most important variables for explaining fungal diversity (Talley et al., 2002). This pattern was also observed by Martiny et al. (2011) in an analysis of the distribution of Nitrosomadales bacteria along a large transect in North America. It has been suggested that in some systems, both deterministic (niche-based) and stochastic (neutral-based) processes are responsible for structuring ecological communities (Chave, 2004). A literature review performed by Hanson et al. (2012) indicated that both contemporary selection and historical processes shape microbial biogeographic patterns. Furthermore, that work revealed that most studies have found that at least one environmental factor or the distance effects, including dispersal limitation, influence microbial composition. The results of the present work suggested that the distance and environmental influence will influence depend on the scale sampled used. When considered at local scales, only geographic distance was significant in explaining the β-diversity (Table 1). The MRM model was exceptionally robust in explaining the local-scale variation in the fungal endophyte community similarity at the Argentinean collection sites (the Andean Patagonia ecosystem) (R 2 = 0.86, P = 0.0001). Furthermore, our results suggest that the primary control of fungal endophyte diversity is exerted by dispersal limitation, independent of 45 environmental differences between sites in the same ecosystem. When the M. ovata var. nanophylla samples present in the Andean Patagonian forest (Argentina) and the Atlantic rainforest (Brazil) were compared, only geographic distance was significant in explaining the fungal endophyte community similarity. Therefore, there was no relationship between fungal endophyte community similarity and the phylogenetic relationship of the host tree species. We conclude that fungal endophytes may show distribution patterns that are not profoundly different from those of higher organisms (Astorga et al., 2012). Conclusion It has long been assumed that microorganisms have cosmopolitan distributions however the results of this work show that fungal endophytes exhibit a biogeographic pattern, corroborating some previous studies which show that microorganism are not randomly distributed. We conclude that fungal endophyte communities are influenced by both environmental factors and geographic distance and that these effects depend on the spatial scale analysed. Overall, our study shows that there is no relationship between fungal community similarity and the host tree phylogenetic proximity. Acknowledgements This work was funded by the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento do Ensino Superior (CAPES) and the Fundação do Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG). We thank the authorities of the Administración de Parques Nacionales (Argentina) for their courtesy and cooperation. 46 Figure 1. Map of the geographical distribution of the host tree species. 47 Figure 2. Accumulation curves for the fungal endophytes of Luma apiculata (Arrayanes forest) (L1), Luma apiculata (Puerto Blest) (L2), Myrceugenia ovata var. nanophylla (Argentina) (M1), Myrceugenia ovata var. nanophylla (Brazil) (M2), and Eugenia neomyrtifolia (Brazil) (E2). Singleton fungal species were not included in the analysis. 48 Figure 3. Nonmetric multidimensional scaling (NMDS) plots based on the Jaccard index that show the differences between fungal endophyte community compositions. Each symbol represents a single host tree species. Argentina: the squares represent Luma apiculata from Puerto Blest, the crosses represent Luma apiculata from the Arrayanes Forest, and the circles represent Myrceugenia ovata var. nanophylla from Lago Espejo. Brazil: the rectangles represent Myrceugenia ovata var. nanophylla, and the stars represent Eugenia neomyrtifolia. The R 2 values between the ordination distance and the distance in the original space are 0.23 and 0.15 for axis 1 and axis 2, respectively. 49 Figure 4. Distance decay relationship for fungal endophyte communities from an Andean Patagonia forest (Argentina): Luma apiculata (Arrayanes Forest), L. apiculata (Puerto Blest) and Myrceugenia ovata var. nanophylla (Espejo Lake) and from an Atlantic forest (Brazil): Myrceugenia ovata var. nanophylla and Eugenia neomyrtifolia. Pairwise community similarities were calculated using the Jaccard index and plotted against the natural logarithms of the distances among the study sites. The solid black line denotes the linear regression across all the spatial scales, and the slope is significantly smaller than zero (slope = - 0.01, P < 0.0001). 50 Table 1. Comparison of the fungal endophyte partial Mantel test results, where Spearman ρ is the correlation between the fungal endophyte community dissimilarity and either geographic distance or environmental distance. Correlation between fungal endophyte community dissimilarity and: Controlling for: Continental scales ρ P Geographic distance Environmental distance 0.27 < 0.05 Environmental distance Geographic distance - 0.21 0.99 The environmental variables (Environment) were first examined using a principal components analysis (PCA) that considered the elevation, pluviomietry and temperature of each collection site. The P values are one-tailed and based on 10,000 permutations. 51 Table 2. Results of the multiple regressions on matrices (MRM) analysis of the fungal endophytes by spatial scale. Regional scale R 2 = 0.05 * Local scale Argentina R 2 = 0.86 *** Brazil R 2 = 0.32 *** Ln (geographic distance) 0.95 *** 0.93 *** 0.56 *** Altitude - 0.27 * Temperature - 0.75 ** The variation (R 2 ) of the community dissimilarity that is explained by the remaining variables and the partial regression coefficients (β) of the final model are shown. Where a partial regression is shown, its significance level (via one-way tests) is < 0.005. * P ≤ 0.01, ** P ≤ 0.001, *** P ≤ 0.0001. The water precipitation, altitude and temperature were measured at each collection site. The water precipitation was not significant at any scale and is not shown in the table. 52 Table 3. Comparison of the fungal endophyte partial Mantel test results, where Spearman ρ is the correlation between the fungal endophyte community dissimilarity and either geographic distance or environmental distance. Correlation 1 between fungal endophyte community dissimilarity and: Controlling for: Continental scales ρ P Geographic distance Environmental distance 0.27 < 0.05 Environmental distance Geographic distance - 0.21 0.99 1 The environmental variables (Environment) were first examined using a principal components analysis (PCA) that considered the elevation, pluviomietry and temperature of each collection site. The P values are one-tailed and based on 10,000 permutations. 53 Table 4. Results of the multiple regressions on matrices (MRM) analysis of the fungal endophytes by spatial scale. The variation (R 2 ) of the community dissimilarity that is explained by the remaining variables and the partial regression coefficients (β) of the final model are shown. Where a partial regression is shown, its significance level (via one-way tests) is < 0.005. Regional scale 1 R 2 = 0.05 2 Local scale Argentina R 2 = 0.86 3 Brazil R 2 = 0.32 3 Ln (geographic distance) 0.95 3 0.93 3 0.56 3 Altitude - 0.27 2 Temperature - 0.75 4 1 The water precipitation, altitude and temperature were measured at each collection site. The water precipitation was not significant at any scale and is not shown in the table. 2 P ≤ 0.01, 3P ≤ 0.0001. 4P ≤ 0.001. 54 Supplementay table 1. Closest related fungal species according to the percent similarities between the ITS regions of the rRNA gene revealed by alignment with sequences from related species retrieved from the GenBank database and the number of fungal endophyte isolates obtained from Luma apiculata, Myrceugenia ovata var. nanophylla and Eugenia neomyrtifolia from an Andean Patagonian forest (Argentina) and an Atlantic forest (Brazil). Closest related species UFMGCB Identification Pb Similarity % L1* L2* M1* M2* E1* Sordariomycetes, Coniochaetales Coniochaeta velutina STE-U [GQ154545] 3827 Coniochaeta velutina [JQ346221] 706 100 5 Sordariomycetes, Diaphortales Amphilogia gyrosa [EF026147] 3826 Amphilogia sp. [JQ346197] 616 93 6 Diaporthe australafricana [AF230760] 3841 Diaphorte sp.1 [JQ327869] 577 96 2 2 Diaporthe helianthi [AJ312356] 5242 D. helianthi [JQ346194] 478 99 4 Diaporthe phaseolorum [EU272530] 5311 Diaphorte sp.2 [JQ327871] 434 90 2 Diaporthe phaseolorum [EU272530] 5212 Diaphorte sp.3 [JQ327870] 498 93 3 7 Diaporthe phaseolorum [EU272530] 5173 Diaphorte sp.4 [JQ327872] 484 95 12 Diaporthe phaseolorum [EU272530] 5211 Diaphorte sp.5 [JQ327871] 464 96 10 Diaporthe phaseolorum [EU272530] 3855 D. phaseolorum [JQ327873] 467 97 5 Diaporthe phaseolorum [EU272530] 5203 Diaphorte sp.6 [JQ327874] 485 94 16 8 Diaporthe stewartii [FJ889448] 5264 Diaporthe stewartii 329 99 6 Greeneria uvicola [GU907101] 3949 Greeneria sp.1 [JQ346195] 599 89 34 1 55 Greeneria uvicola [GU907101] 5274 Greeneria sp.2 453 90 1 Sordariomycetes, Glomerellales Colletotrichum boninense [AB042313] 5233 Colletotrichum sp.1 [JQ346206] 531 98 23 Colletotrichum boninense [AB042313] 5230 Colletotrichum boninense [JQ346207] 532 99 5 Colletotrichum boninense [AB042313] 5253 Colletotrichum sp. 2 [JQ346208] 507 98 7 Colletotrichum boninense [AB042313] 5201 Colletotrichum sp.3 [JQ346209] 520 97 9 Colletotrichum boninense [AB042313] 5197 Colletotrichum sp.4 [JQ346210] 467 98 20 Colletotrichum boninense [AB042313] 5259 Colletotrichum sp.5 [JQ346211] 471 98 5 Colletotrichum boninense [AB042313] 5177 Colletotrichum sp.6 [JQ346212] 484 97 22 Colletotrichum boninense [AB042313] 5171 Colletotrichum sp.7 [JQ346213] 526 13 Colletotrichum boninense [AB042313] 5215 Colletotrichum sp.8 402 96 3 Sordariomycetes, Hypocreales Cephalosporium curtipes var. uredinicola CBS 650.85 [AJ292405] 3956 Cephalosporium sp. [JQ346222] 557 89 6 Sordariomycetes, Xylariales Annulohypoxylon bovei [JN225906] 3885 Annulohypoxylon sp.3 [JQ327866] 567 92 2 Annulohypoxylon squamulosum [EF026139] 3852 Annulohypoxylon sp.1[JQ327864] 547 92 3 Annulohypoxylon squamulosum [EF026139] 5249 Annulohypoxylon sp.2 [JQ327865] 485 82 13 Annulohypoxylon squamulosum [EF026139] 3946 Annulohypoxylon sp.4 [JQ327867] 560 92 1 Biscogniauxia cylindrispora [EF026133] 3834 Biscogniauxia sp.1 [JQ327868] 706 85 1 Nemania aenea [AF201704] 3884 Nemania aenea [JQ327863] 588 97 56 Nemania illita [026122] 3918 Nemania sp. [JQ327862] 481 94 4 27 Xylaria berteri [GU324750] 5268 Xylaria berteri [JQ327861] 577 99 34 17 Xylaria catorea [GU324751] 3880 Xylaria castorea [JQ327858] 574 99 2 5 Xylaria catorea [GU324751] 3872 Xylaria sp.1 [JQ327859] 581 94 9 112 11 Xylaria enteroleuca [163033] 5292 Xylaria enteroleuca [JQ327860] 494 98 46 Dothideomycetes, Botryosphaeriales Guignardia sp. MUCC 0441 [AB454355] 3916 Guinardia sp. 1 [JQ346219] 561 92 1 Guignardia mangiferae CBS 356.52 [FJ538342] 5169 Guinardia mangiferae 513 99 37 21 Dothideomycetes, Dothideales Dothiora cannabinae [DQ470984] 3894 Dothiora cannabinae [JQ346227] 556 99 4 Dothideomycetes, Capnodiales Cladosporium cf. subtilissimum CBS 172.52 [EF679390] 3843 Cladosporium subtilissimum [JQ346203] 548 99 2 6 Cladosporium colombiae [FJ936159] 3901 Cladosporium colombiae [JQ346204] 544 99 1 2 Mycosphaerella brassicicola CBS 174.88 [EU167607] 3928 Mycosphaerella sp. [JQ346202] 528 89 191 Pseudocercospora basitruncata CBS 114664 [DQ303071] 3898 Pseudocercospora basintrucata [JQ346205] 534 97 72 18 1 Dothideomycetes, Pleosporales Camarosporium brabeji CBS 123026 [EU552105] 3848 Camarosporium brabeji [JQ346215] 538 99 2 Didymella phacae strain CBS 184.55 [EU167570] 3925 Didymella sp. [JQ346225] 415 94 1 Lewia infectoria ATCC 12054 [AF229480] 3824 Lewia infectoria [JQ346214] 566 99 8 Microsphaeropsis olivacea CBS 432.71 [GU237863] 3886 Microsphaeropsis olivacea [JQ346217] 444 98 1 57 Paraconiothyrium sporulosum [GU566257] 3837 Paraconiothyrium sp. [JQ346216] 510 96 1 2 Leotiomycetes, Helotiales Cryptosporiopsis actinidiae [EU482298] 3887 Cryptosporiopsis actinidiae [JQ346199] 554 98 1 1 Mollisia cinerea [DQ491498] 3832 Mollisia sp. [JQ346200] 554 94 1 Mollisia cinerea [DQ491498] 3900 Mollisia cinerea [JQ346201] 554 99 1 Pezicula corylina [AF141176] 3840 Pezicula corylina [JQ346198] 554 97 3 34 Eurotiomycetes; Chaetothyriales; Exophiala capensis CBS 128771[JF499861] LAB 2IIIB4 Exophiala capensis [JQ346226] 448 97 Eurotiomycetes; Eurotiales Penicillium restrictum NRRL 25744 [AF033459] 3803 Penicillium restrictum [JQ346224] 510 99 1 Pezizomycetes; Pezizales Peziza varia [AF491570] 3892 Peziza sp. [JQ346218] 579 94 2 Fungi incertae sedis; Mortierellales Mortierella sclerotiella CBS 529.68 [HQ630310] 3802 Mortierella sclerotiella [JQ346223] 582 98 3 Badisiomycete Trametes hirsuta LE 231668 [HQ435841] 5251 Trametes hirsute [JQ346220] 486 97 15 L1: Luma apiculata (Arrayanes forest); L2: Luma apiculata (Puerto Blest); M1: Myrceugenia ovata (Espejo lake, Argentina); M2: Myrceugenia ovata (Brazil); E1: Eugenia neomyrtifolia (Brazil). UFMGCB: Culture collection of the Universidade Federal of Minas Gerais. * Number of segments plated per plant species: 600. 58 Supplementary table 2. Study site and fungal endophyte community characteristics for each collection site in Argentina and Brazil. Luma apiculata Myrceugenia ovata var. nanophylla Eugenia neomyrtifolia Country Argentina Argentina Argentina Brazil Brazil Collection area Arrayanes Forest Puerto Blest Espejo lake PUCRS PUCRS Coordinates 40º 51´ S 71º 36´ W 41º 01´ S 71º 48´ W 40º 47´ S 71º 40´ W 29º 27 ´ S 50º 0.6´ W 29º 27 ´ S 50º 0.6´ W Alt a 850 850 970 912 905 ºC b 10 9 10 15 15 mm c 1500 3000 1800 2252 2252 Number of isolates obtained 106 159 336 225 134 Number of fungal species 12 15 19 15 14 Singletons 1 6 8 3 1 Shannon diversity index 1.29 1.04 1.48 2.24 2.36 Number of leaves sampled: 100; number of segments plated per plant species: 600. a Alt: altitude above sea level. b Annual mean temperature in ºC. c Annual precipitation in mm. The calculation of the diversity indexes excluded singleton fungal species. 59 References Arnold EA, Maynard Z, Gilbert G, 2001. Fungal endophytes in dicotyledonous neotropical trees: patterns of abundance and diversity. Mycological Research 105: 1502-1507. 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San Diego, California, USA: Academic Press, pp 315-322. 64 Capítulo 4 Arbuscular mycorrhizal colonization of Sorghum vulgare in presence of root endophytic fungi of Myrtus communis Artigo publicado na Applied Soil Ecology DOI 10.1016/j.apsoil.2011.10.017 65 66 67 68 69 70 71 72 Capítulo 5 Diversity of fungal endophytes in leaves of Myrtus communis studied by traditional isolation and cultivation-independent DNA-based method Artigo em fase final de preparação 73 Diversity of fungal endophytes in leaves of Myrtus communis studied by traditional isolation technique and by denaturing gel gradient electrophoresis A. B. M. Vaz a,b , I. Sampedro c , J.A. Siles c , I. García-Romera c , C. A. Rosa a , J. A. Ocampo c a Departamento de Microbiologia, ICB, C. P. 486, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901, Minas Gerais, Brazil. alinebmv@hotmail.com, carlrosa@icb.ufmg.br b Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Ministério da Educação do Brasil, Caixa Postal 250, Brasília – DF 70040-020, Brazil; c Department of Microbiology, Estación Experimental del Zaidín, C.S.I.C., Profesor Albareda, 1, E-18008, Granada, Spain. sampedro@eez.csic.es; jose.siles@eez.csic.es; inmaculada.garcia@eez.csic.es; juanantonio.ocampo@eez.csic.es; 74 Abstract The diversity of endophytic community of Myrtus communis in a Mediterranean ecosystem in south Spain has been evaluated by classical isolation from leaves fragments and denaturing gel gradient electrophoresis (DGGE). A total of 113 fungal isolates were obtained and identified by sequencing the ITS region of the rRNA gene. Of these, four genotypes were represented by only one sequence (singletons). Molecular identification revealed seven different endophytic taxa; all belonged to the phylum Ascomycota. The most frequent endophytic fungal genus isolated from M. communis was Mycosphaerella. We used the distance decay approach to analyze and compare the biogeography of endophytic fungal community. It was found that the similarity of endophytic fungal communities did not decrease with increasing geographical distance (p=0.974). The results obtained in this work suggests that the biogeographical patterns observed in endophytic fungi communities are fundamentally different from those observed in well studied plant and animal communities that have provided the foundation for biogeographical theory to date. Keywords: Fungal endophytes, DGGE, Distance-decay. 75 Introduction Fungal endophytes inhabit healthy plant tissues during at least one stage of their life cycle without causing any apparent symptom of disease or negative effects on their host plants (Petrini, 1992). Endophytes have been isolated from all plants previously studied, including bryophytes (U’Ren et al., 2010), pteridophytes (Petrini et al., 1992), gymnosperms (Soca-Chafre et al., 2011), and both monocotyledonous (Pinruan et al., 2010) and dicotyledonous angiosperms (Vaz et al., 2009). These fungi colonise on all available tissues, such as roots, stems, leaves, bark, fruits, seeds, and floral organs (Petrini et al., 1992; Rodriguez et al., 2009; Vaz et al., 2009). Most endophytic fungi isolated are ascomycetous, and Sordariomycetes and Dothideomycetes classes contain the majority of foliar fungal endophyte species (Arnold and Lutzoni, 2007). Several studies have studied the endophytic fungal diversity from angiosperms from temperate zones (Saikkonen et al., 1998; Stone et al., 2000) and these hosts may harbor dozens of endophytic fungi (Saikkonen et al., 1998, Higgins et al., 2007, Arnold & Lutzoni 2007). However, the biodiversity of fungi remains far little explored considering the small proportion of fungi that have been identified to date, and more recent estimates has been suggested that as many as 5.1 million fungal species exist (Blackwell 2011), which makes the fungi among the most diverse groups of organisms in our planet. It is estimated that 99% of microorganisms observable in nature typically are not cultivated using cultivation-dependent methods (Amann et al. 1995), which have been traditionally used for the analysis of the community structure of endophytic fungi (Arnold et al., 2001; Bussaban et al., 2001; Wilberforce et al., 2003). These methods have many inherent problems and the endophytic fungal communities will be biased towards faster growing fungi that are capable of growing rapidly on the media used (Hyde & Soytong 2008). Moreover, fungi in a latent/quiescent stage or with special growth requirements are often not recovered (Götz et al., 2006). Then, these methods may provide incomplete data concerning endophyte biodiversity and molecular-based techniques, such as denaturing gradient gel electrophoresis (DGGE), may provide new insights into the endophytic fungal diversity (Muyzer et al., 1993; Vainio et al., 2005). DGGE has been successfully applied to document fungal communities (Götz et al., 2006; Vainio et al., 2005; Duong et al., 2006) and is able to detect known and abundant fungi as well as unknown endophytic fungi (Duong et al., 2006). Furthermore, we used the results obtained by cultivation and molecular-based techniques to study the distribution of fungal endophytes across a host tree transect. The similarity between two observations often decreases as the distance between them increase, a pattern long observed in communities from all domains of life and once called “the first law of biogeography” (Nekola 76 & White, 1999; Green et al., 2004). Although microorganisms are perhaps the most diverse and abundant types of organisms on Earth the distribution of microbial diversity is poorly understood (Fierer & Jackson, 2006). Little is known about how endophyte communities vary across a geographic range. In the present study, we combined cultivationdependent methods, DGGE and phylogenetic analysis to investigate the fungal endophyte communities of leaves of Myrtus communis. These results were used to compare the endophytic fungal diversity along a south to north transect of twenty host trees of Myrtus communis in a Mediterranean ecosystem. In addition we investigate the fungal endophyte diversity composition in different fragments leaves of M. communis. Material and Methods Site description and fungal isolation The study site was in Sierra Tejeda, Almijara y Alhama Natural Park (36º51´N, 3º41´W, and elevation of around 360 m above sea level). The Park is located in the south of Andalucia, nearby Mediterranean Sea, between Malaga and Granada and consists of several dolomitic mountain ranges. Five apparently healthy leaves were collected from each of 20 individuals of all Myrtaceae species that occur in the studied sites. The trees were spaced approximately 5 m apart. All the leaves were stored in sterile plastic bags, and fungal isolation was performed on the same day of the collection. The leaves were surface-sterilised via successive dipping in 70% ethanol (1 min) and 2% sodium hypochlorite (3 min), followed by washing with sterile distilled water (2 min). After the leaf surface sterilisation, six fragments (approximately 4 mm 2 ) were cut from each leaf: one from the base, two from the middle vein, one from the left margin, one from the right margin and one from the tip (6 leaf fragments/leaf; 30 leaf fragments/tree; 600 leaf fragments/site; 3,000 leaf fragments overall) . All the fragments were plated onto potato dextrose agar (PDA, Difco, USA) supplemented with 100-µg/ml chloramphenicol (Collado et al., 1996). The plates were incubated at 15C for up to 60 days. To test the effectiveness of the surface sterilisation, 100 µl of the water used during the final rinse was plated on PDA to test for epiphytic microbial contaminants. Individual colonies were purified on PDA, and their morphologies were documented and photographed. For filamentous fungi, long-term preservation of mycelial samples was carried out in sterile distilled water at room temperature and in 30% sterile aqueous glycerol solution at –80°C. All fungal isolates were deposited in the Culture Collection of Microorganisms and Cells of the Universidade Federal of Minas Gerais. 77 DNA extraction and fungal identification of cultured fungi The protocol for DNA extraction of filamentous fungi was adapted from Girlanda et al. (2002). Approximately 500 mg of mycelia of 7-day-old cultures were frozen in liquid nitrogen, ground with a prechilled mortar and pestle, transferred to 2-ml eppendorf tubes containing 600 μl of cetyltrimethylammonium bromide buffer [2% CTAB, 0.1 mM Tris-HCl pH 9, 1.4 M NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA) pH 8, 0.2% β-mercaptoethanol, 1% polyvinylpyrrolidone]. The tubes were vortex-mixed and incubated at 60ºC for 60 min. After a 10-min centrifugation at 19.000 g, the aqueous phase was removed to a new tube and extracted twice with one volume of chloroform: isoamyl alcohol (24:1) and after maintained 30 min at 0ºC. Nucleic acids were precipited from the aqueous phase by addition of one volume of cold isopropanol and overnight incubated at - 4ºC. DNAs were collected by centrifugation at 19000 g for 20 min and the pellets were washed with 70% ethanol, dried briefly and resuspended in 50 μL of sterile water. The DNA extractions were diluted 1:150 and used as template for PCR amplification. The internal transcribed space (ITS) domains of rRNA gene were amplified using the universal primers ITS1 (5´- TCCGTAGGTGAACCTGCGG-3´) and ITS4 (5´- TCCTCCGCTTATTGATATGC-3´). Amplification of ITS and sequencing protocols were performed as described to Vaz et al. (2009) and subsequently sequenced using an automatic DNA sequencer (ABI-PRISM 310; Applied Biosystem, Inc.) DNA extraction and fungal identification of uncultured fungi Three leaf of each individual host tree was surface sterilized, grind in liquid nitrogen and 100 mg were used to DNA extraction with DNeasy Plant mini kit (Qiagen) following the manufacturer´s instruction. The primer pair NS1 (GTAGTCATATGCTTGTCTC) and GCfung (ATTCCCCGTTACCCGTTC) was used to amplify the variable V1 and V2 regions of 18S rDNA of fungi (Das et al., 2007). NS1 had at its 5´ end a GC-rich clamp, the sequence of which has been reported by May et al. (2001). The total reaction mixture of the PCR consisted of 25 μl with the following ingredients: 2 μl of extracted DNA (approx. 10 ng), 2 μM primer NS1, 2 μM primer GCfung, 25 mM of MgCl2, 2 mM of each dNTP, 1 U of Taq DNA polymerase (BIOLINE), 5X of PCR buffer (160 mM (NH4)2 SO4, 670 mM Tris-HCl) and sterile Mili-Q water to a final volume. The PCR conditions were: initial denaturation at 95 ºC for 5 min; 35 cycles of amplification, each at 94 ºC for 30 s, then 55 ºC for 30 s and 72 ºC for 1 min, followed by a final extension step at 72 ºC for 5 min. PCR products from three parallel amplifications were pooled, separated in 1.5% (w/v) agarose gel and stained with ethidium bromide. 78 DGGE analysis The INGENYphorU-2 system for DGGE (Ingeny International BV, Goes, NL) was used. Samples were loaded onto 8% polyacrylamide–bisacrylamide (37.5:1) gels. For the separation of amplified products from 18S, denaturing gradients were from 20% to 35%, [where 100% corresponds to 7 M urea and 40% (v/v) deionized formamide]. Gels were run at 85 V in 0.5x TAE (20 mM Tris acetate, 10 mM sodium acetate, 0.5 mM Na-EDTA at pH 7.4) at 60º C for 16 h, then stained with SYBR Gold (Invitrogen, Carlsbad, CA) in 1x TAE for 45 min at room temperature and visualized under UV illumination. DGGE banding patterns were digitized and processed using the InfoQuestTM FP (version 4.5; Bio-Rad Laboratories, Hercules, CA) and manually corrected for further analyses. An unweighed pair group method with arithmetic means (UPGMA) dendrogram was generated from a similarity matrix based on common band positions between lanes and calculated using the Jaccard coefficient (Li & Moe, 2004). Analysis of ecological data The isolation rate of the fungal endophytes was calculated by the following formula: total number of isolates/ total number of samples in a trial X 100, and expressed as percentage. The rates of colonization were calculated as follows: Total number of samples ≥1/ total number of samples in a trial. The data in most cases did not fit the assumptions for parametric statistics and this was confirmed by a Shapiro-Wilk test in our study. Therefore, non-parametric statistical tests were used throughout. A Kruskal-Wallis test was performed to evaluate whether the numbers of endophytic fungal species were statistically different among the host tree species. A Tukey type multiple comparison “Nemenyi” test was used to evaluate if there were the statistically significances between each two host tree species. These tests were done using the software package R (R Development Core Team, 2005). The diversity was estimated by Shannon (H`) index (H = -Σ ni/n ln (ni/n), where ni is number of individuals of the taxon i and n is the total number of individuals). The Shannon index is usually between 1.5 and 3.5, 1.5 representing the lowest diversity and 3.5 the highest (Maggurran, 2004; Gazis & Chaverri, 2010). The species accumulation curves were used to determine whether enough samples were taken of each study site. Species accumulation curves were generated for each host tree species using EstimateS, version 8.0 (Colwell 2005). For calculation purposes and statistical analysis, each individual fragment was considered a sample unit and a total of 600 sample units were evaluated, 100 from each fragment. We used the distance decay approach to analyze and compare the biogeography of endophytic fungal community. The rate of distance decay of the endophytic fungal 79 communitiesassumes that community similarities will decrease with increasing geographical distance and was calculated according to Nekola & White (1999). The dissimilarities among endophytic communities was calculated by using the Jaccard distance, which was used due to its simplicity, its widespread use and represents a more conservative measure of community similarity than ones based on species abundance data which are more sensitive to disturbance and local environmental differences (Nekola & White, 1999). Community dissimilarity were calculated and compared with pairwise distances among individual host tree using the function “mantel” in the Vegan R package (R development core team, 2010). Linear regression was used to calculate the effect of distance in endophytic fungi community composition. These tests were done using the software package R (R development core team, 2010). Results A total of 600 fragment samples were analysed and were obtained 113 endophytic fungal isolates in culture-dependent technique. The overall colonization rate (%) and isolation rate for the assemblages of endophytes recovered was 0.10 and 44.5%, respectively. The samples do not fit the assumptions of a normal distribution, which could be confirmed by the low P value in the Shapiro-Wilk test (P = 2.2 10-16). The Kruskall-Wallis test showed that there was no significant difference in the number of endophytic fungal species isolated from each individual host tree species (χ2 =12, P = 0.44). The accumulation curves of endophytic fungal assemblage of cultured endophytes did not reach an asymptote which means the sampling effort was no statistically sufficient to capture the total species richness of cultivable endophytic fungi associated with leaves of M. communis (Figure 1). The Shannon index obtained from cultivable endophytic fungi was 2.5. A total of 113 endophytic fungal isolates were isolated, grouped and 30% of representative fungal isolates were identified by sequencing the ITS region of the rRNA gene. Of these, four genotypes were represented by only one sequence (singletons). Molecular identification revealed thirteen different operational taxonomic units (OTUs); all belonged to the phylum Ascomycota (Table 1).The genera of fungi associated with M. communis levaes were: Alternaria, Aspergillus, Ascochyta, Cladosporium, Diaporthe, Gnomoniopsis, Mycosphaerella, Neofusicoccum, Neonectria, Phaeosphaeria, Pseudocercospora. The most frequent endophytic fungus genus isolated from M. communis was Mycosphaerella. A phylogenetic tree was constructed with Mycosphaerella because many isolates obtained in this work presented 99% similarity with different species of Mycosphaerella deposited in GenBank. The manually adjusted ITS alignment contained 53 sequences (including out group sequence) and 572 characters. Of the 572 80 characters used in the phylogenetic analysis, 132 were parsimony-informative, 113 were variable and parsimony-uninformative and 327 were constant. Neighbour-joining analysis using two substitution models (Maximum likelihood and Kimura-2) on the sequence alignment yielded trees with identical topologies to one another and support the same clades as obtained from the parsimony analysis (Figure 2). For the parsimony analysis, only the first 10 000 equally most parsimonious trees were retained, the first of which is shown in Figure 1 (TL = 500, CI = 0.72, RI = 0.91, RC = 0.65). The endophytic Mycosphaerella isolates obtained in this work did not produced spores when cultured on common mycological media such as potato dextrose, cornmeal, malt extract, potato dextrose, Sabouraud dextrose, and yeast extract sucrose agars. Pure cultures of the fungal isolates were grouped based on morphological characters including aerial mycelium form, colony colours, surface texture and margin characters. The isolates were identified at genus level based on morphological characteristics and phylogenetic analysis (Figure 2) and five Mycospharella groups were defined. We used the distance decay approach to analyze and compare the biogeography of endophytic fungi community. A plot of community similarity versus geographic distance for each pairwise set of samples revealed that the distance decay curve of cultivable endophytic fungi did not differ from zero (P = 0.974) (Figure 3). The DGGE analysis suggested that the is no difference among the Discussion In this study the diversity of endophytic fungi in leaves from M. communis was assessed by means of traditional isolation techniques and cultivation-independent methods. Endophytic fungi were isolated by plating leaf fragments on potato dextrose agar, which is a common method for isolation of endophytic fungi (Vizcaíno et al., 2005; Phongpaichit al., 2006; Vaz et al., 2009). The accumulation curves of cultivable fungi did not reach an asymptote suggesting that further sampling would be needed to obtain an accurate notion of the cultivable endophytic fungal community diversity. The diversity index obtained from cultivable fungi were similar to other works with foliar endophytic fungi in temperate ecosystems, as those obtained to Lycopodiaceae, Rosacea and Pinaceae from Canada (Higgins et al., 2007) and Cupressaceae from USA (Hoffman & Arnold, 2008). Most of the fungal genera obtained as endophytes in M. communis have already been described as endophytes of plants from tropical and temperate ecosystems, e.g., Alternaria, Ascochyta, Aspergillus, Cladosporium, Diaporthe, Mycosphaerella, Neonectria and Phaeosphaeria. The most frequent endophytic fungus genus isolated from M. communis was 81 Mycosphaerella. Mycosphaerella species have been isolated as pathogens, endophytes or saprophytes and are commonly found on the leaves of Myrtaceae, many of which are defoliated by these pathogens (Crous et al., 2007; Carnegie, 2007; Cheewangkoon et al., 2009). While Mycosphaerella species are generally accepted to be host specific, several species now been shown to have wider host ranges than was commonly accepted (Crous et al., 2008). Species of Alternaria and Phaeosphaeria also has been found as pathogens on numerous plants (Simmons 1999; Thomma 2003). Many of the fungi most commonly isolated as endophytes are considered typical epiphytic saprobes, as Cladosporium species, which are considered ubiquitous epiphytic but also are capable of internal colonization of healthy tissue (Stone 2000). Gnomoniopsis idaeicola (anamorph Diaporthe idaeicola) have been associated with necrosis of leaves and chestnut galls (Magro et al., 2010) however there this is the first report of G. idaeicola species as endophyte. Neofusicoccum genera has been isolated from Myrciaria floribunda present in west of the Tocantins state, Brazil, in an ecotone formed by Cerrado, Amazon forest, and Pantanal ecosystems (Vaz et al., 2012). We found that the similarity of endophytic fungal communities did not decrease with increasing geographical distance and this pattern has been found to root-associated fungal (Queloz et al., 2011), soil bacteria (Fierer & Jackson, 2006) and endophytic fungi associated with liverworths (Davis & Shaw 2008). Despite the high taxonomic resolution to identify the endophytic fungi in this work, we found no biogeographical pattern unlike observations for plants and animals (Gaston, 2001). Consequently the results obtained in this work suggests that the biogeographical patterns observed in endophytic fungi communities are fundamentally different from those observed in well studied plant and animal communities that have provided the foundation for biogeographical theory to date. Conclusion The results obtained in this work provided useful information on the taxonomy, diversity and distribution of endophytic fungal communities presents in surface-sterilized leaves of M. communits. Moreover, this work represents the first comprehensive survey of cultivable and unculturable endophytic fungi community of M. communis in Spain. We did not present the DGGE results because we are finishing this analysis. Acknowledgements The authors wish to thank Rafael León Morcillo to take the plants samples. Financial support for this study was provided by the Comision Interministerial de Ciencia y Tecnología (CICYT), 82 project (AGL2008-00572). Aline B. M. Vaz received a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (2010-Processo n◦ 2330-10-5/CAPES) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brazil). 83 Table 1. Fungal endophytes obtained from Myrtus communis and closest related species according to similarity percentage of the ITS region of the rRNA gene by alignment with sequences of related species retrieved from the GenBank database. Closest related species Identification e-value Query cover pb Similarity % Number of isolates Alternaria alternata BASALT5-10 [HQ540552] A. alternata [KC959211] 0 100 502 100 1 Ascochyta obiones CBS 786.68 [GU230753] Ascochyta sp. [KC959210] 9e-157 90 422 93 1 Cladosporium oxysporium CBS 125.80 [AJ300332] C. oxysporium [KC959209] 0 98 532 99 4 Diaporthe cynaroidis CBS 122676[EU552122] D. cynaroidis [KC959207] 0 100 532 99 17 Gnomoniopsis idaeicola [GU320824] G. idaeicola [KC959208] 0 100 538 99 3 Mycosphaerella fragariae CPC 4903 [GU214691] Mycosphaerella fragariae [KC959216] 0 100 493 99 4 Mycosphaerella handelii CBS 113302 [EU167581] Mycosphaerella sp.1 [KC959218] 0 100 484 96 6 Mycosphaerella coacervata [EU167596] Mycosphaerella sp. 2 [KC959219] 0 100 497 100 16 Mycosphaerella buckinghamiae CBS 112175 [EU707856] Mycosphaerella sp. 3 [KC959217] 0 100 495 100 35 Neofusicoccum australe CBS 115185 [FJ150696] Neofusicoccum austral [KC959212] 0 100 546 99 6 Neonectria macrodidyma CBS 112601 [JF268765] Neonectria macrodidyma [KC959213] 0 99 495 99 1 Phaeosphaeria pontiformis CBS 589.86 [AF439499] Phaeosphaeria pontiformis [KC959214] 0 100 494 97 1 Pseudocercospora crousii CBS 119487 [GQ852756] Pseudocercospora sp. [KC959215] 0 100 499 99 12 84 0 4 8 12 16 20 0 5 10 15 20 C u m m u la ti v e n u m b er o f sp ec ie s Number of samples Figure 1. Species accumulation curves for fungal endophytes of leaves of Myrtus communis based on the Mao Tao estimator. Species groups were based on 97% similarity ITS rDNA sequence similarity. Solid black line indicates observed richness and solid gray line indicate 95% CI around the observed richness. Dashed black line indicates bootstrap estimates of total species richness inferred using EstimateS v.8.0. a 85 UFMGCB 252 Pseudocercospora natalensis CBS 111069 [DQ303077 ] Pseudocercospora crousii CBS 119 487 [GQ852756] Pseudocercospora humuli CPC 11358 [GU214676 ] Pseudocercospora pseudoeucalyptorum CPC 13816 [GQ852763] Mycosphaerella gracilis CPC 11181[DQ302962] Mycosphaerella tumulosa NSWF005313 [DQ530218 ] Pseudocercospora cydoniae KACC 42644 [EF535716] Pseudocercospora abelmoschi KACC 42648 [EF535719] Pseudocercospora paraguayensis CBS 111286 [DQ267602] Pseudocercospora lythracearum KACC 42649[EF535720] Pseudocercospora basiramifera [AF309595] UFMGCB 269 UFMGCB 263 UFMGCB 268 UFMGCB 285 UFMGCB 262 Mycosphaerella coavervata CBS 113391 [EU167596] Mycosphaerella linorum CBS 261.39 [EU167590] UFMGCB 284 Mycosphaerella handelii CBS 113302 [EU167581] Mycosphaerella stromatosa CBS10195 [EU167598] Mycosphaerella scytalidii CBS 516.93 [DQ303014] Mycosphaerella gregaria CBS 110501 [EU167580] Mycosphaerella microsora CBS 100352 [EU167599] Mycosphaerella buckinghamiae CBS 112175 [EU707856] UFMGCB 299 UFMGCB 288 UFMGCB 257 UFMGCB 270 Mycosphaerella epplipsoidea CBS 110843 [AY725545] Mycosphaerella africana CBS 680.95 [AY626981] Mycosphaerella aurantia SJ30[EU042175] Ramularia proteae CBS 112161 [EU707899] Ramularia eucalypti CBS 120726 [EF394860] UFMGCB 259 Mycosphaerella fragariae CPC 4903 [GU214691] Ramularia aplospora CBS 545.82[EU040238] Cercospororella centaureicola CBS 120253 [EU019257] Ramularia acroptili CBS120252 [GU214689] Cladoriella eucalypti CBS 115899[EU040224] 88 100 100 100 99 99 95 80 98 69 64 89 85 84 98 80 74 71 0.05 P. natalensis Mycosphaerella sp.1 Mycosphaerella handelli Mycosphaerella sp.2 Mycosphaerella fragariae Figure 2. One of 10 000 equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the ITS sequence alignment using PAUP v.4.0b 10. The scale bar shows 10 changes and bootstrap support values from 1000 fast stepwise replicates are shown at the nodes. The tree was rooted with Cladoriella eucalypti [EU040224] as the out-group. 86 0 20 40 60 80 100 0 .4 0 .5 0 .6 0 .7 0 .8 0 .9 1 .0 Geographic distance between samples (m) C o m m u n it y si m il a ri ty Figure 3. Distance decay relationship for cultivable fungal endophytes community of Myrtus communis. Pairwise community similarities were calculated using Jaccard index and plotted against the distance among study sites. The solid black lines denote the linear regression across all spatial scales and showed that are not statistical significance. 87 References Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. 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No presente trabalho foi obtido um total de 108 espécies de fungos endofíticos, sendo que deste valor 50% podem representar novas espécies sugerindo a importância deste grupo para as estimativas de fungos no planeta. Biogeografia de fungos endofíticos Relatos fósseis indicam que a associação endofítica existe a aproximadamente 400 milhões de anos quando os vegetais surgiram pela primeira vez no planeta (Krings et al., 2007). Desde então, supõe-se que diversos tipos de interações entre vegetais e endófitos podem ter sido estabelecidas ao longo dos anos, incluindo-se relações de especificidade entre os micro- organismos e a planta hospedeira (Tan & Zou, 2001; Strobel, 2002) e que podem estar relacionadas com a distribuição dos hospedeiros (Arnold et al., 2010). Micro-organismos foram considerados como cosmopolitas por muitos anos por possuírem tempos de geração curtos, grandes populações e terem a capacidade de serem dispersos por longas distâncias (Fenchel & Finlay, 2004; Queloz et al., 2011) levando a hipótese de que os micro-organismos são aleatoriamente distribuídos no espaço (Martiny et al., 2006). Entretanto esta hipótese tem sido muito discutida após o advento das metodologias moleculares para a taxonomia de micro- organismos, o que permitiu a identificação das espécies mais efetivamente (Taylor et al., 2000). A primeira lei da biogeografia considera que a similaridade da comunidade frequentemente diminui com o aumento da distância geográfica, e este padrão tem sido amplamente observado para os macroorganismos (Nekola & White, 1999; Green et al., 2004). Este padrão também foi observado neste trabalho, no capítulo 3, cujo objetivo foi avaliar o padrão de distribuição de fungos endofíticos associados a plantas presentes na Argentina e Brasil. A análise de escalonamento métrico não dimensional (NMDS) sugeriu que os fungos endofíticos agrupam-se a nível local. A análise de regressão múltipla de matrizes (MRM) permitiu avaliar a contribuição das variáveis ambientais e da distância na distribuição da comunidade de fungos endofíticos. Os resultados obtidos sugerem que a comunidade de fungos endofíticos é influenciada por fatores ambientais e distância geográfica dependendo da escala espacial utilizada. Além disso, não foi 94 encontrada correlação entre a similaridade da comunidade de fungos endofíticos e a proximidade filogenética entre os hospedeiros. No capítulo 5 o padrão de distribuição da comunidade de fungos endofíticos também foi avaliado, porém em menor escala. Nesta parte, foram amostrados indivíduos de Myrtus communis ao longo de um transecto, sendo cada indivíduo separado do seguinte em uma distância de 5 metros, compreendendo um total de 100 metros de distância entre o primeiro e último indivíduos. Para avaliar a influência da metologia de isolamento na determinação dos padrões de diversidade, foram utilizados métodos dependentes e independentes de cultivo. Entretanto as técnicas independentes de cultivo ainda estão sendo realizadas e, portanto conclusões a este respeito não foram abordadas. Considerando os dados obtidos dos fungos isolados em meio de cultura foi observado que a similaridade na comunidade de endófitos não diminui com o aumento da distância geográfica. Ao contrário dos resultados obtidos para as plantas descritas no capítulo três. Métodos dependentes de cultivo apresentam alguns incovenientes, como por exemplo, fungos que apresentam crescimento rápido são capazes de crescer mais rápido nos meios de cultura tradicionalmente utilizados impedindo o crescimento de fungos de crescimento lento (Hyde & Soytong 2008). Além disso, fungos em estágios latentes, quiescente ou que necessitam de alguns requerimentos nutricionais frequentemente não são isolados por estas metodologias (Götz et al., 2006). Os dados obtidos neste trabalho fornecem dados interessantes sobre a diferença nos padrões de diversidade e distribuição de fungos endofíticos ao longo de diferentes ecossistemas. Potencial biotecnológico dos fungos endofíticos Fungos endofíticos podem apresentar efeitos profundos na ecologia, adaptação e evolução das plantas (Brundrett, 2006) além de poder contribuir para a adaptação das plantas a estresses bióticos e abióticos (Giordano et al. 2009) fato que tem sido correlacionado com a produção de metabólitos naturais pelos fungos associados (Zhang et al. 2006; Suryanarayanan et al., 2009; Aly et al., 2010). Os resultados obtidos no presente trabalho confirmam o potencial na produção de metabólitos secundários pelos fungos, uma vez que 41% dos extratos metanólicos obtidos de fungos endofíticos associados a plantas presentes em ecossitemas de Cerrado e em uma área de ecótono entre Cerrado, Mata Amazônica e Pantanal (Capítulo 2) apresentaram atividade antimicrobiana contra diferentes micro-organismos testados. Os fungos Emericellopsis donezkii and Colletotrichum gloesporioides mostraram os melhores valores de concentração inibitória mínima (CIM), cujos valores foram menores ou similares à valores de CIM de drogas antifúngicas e antibacterianas conhecidas. 95 Outro aspecto importante e útil da utilização dos fungos endofíticos é o seu potencial efeito promotor do crescimento vegetal. No capítulo 4 foi avaliado o potencial da comunidade endofítica de raízes de M. communis na colonização e aumento do peso seco de plantas de S. vulgare. Apesar dos fungos mais frequentemente isolados de M. communis não terem a capacidade de infectar raízes de S. vulgare foi observado que alguns destes fungos aumentaram significativamente o peso seco da parte aérea e a porcentagem de colonização por micorrizas arbusculares, desta forma estes fungos atuaram como micro-organismos promotores do crescimento vegetal. Este trabalho contribui para o conhecimento da riqueza da micota endofítica e o potencial biotecnológico destes micro-organismos. A bioprospecção realizada no capítulo dois mostrou que dois isolados de fungos endofíticos podem ser considerados como promissores em futuros estudos químicos para o isolamento de moléculas bioativas antimicrobianas. O estudo de interação entre micro-organismo e vegetal realizado no capítulo quatro também mostrou o potencial biotecnológico de isolados de fungos endofíticos. Estes fungos podem atuar como promotores do crescimento vegetal de plantas de interesse agronômico. 96 Capítulo 7 Conclusões 97 O estudo da biogeografia de fungos endofíticos associados à hospedeiros presentes em diversos ecossistemas é ainda muito escasso. Além disso, estes fungos constituem uma riqueza em termos de patrimônio genético e podem ser fontes de diferentes substâncias químicas úteis em processos biotecnológicos. Desta forma, considerando os resultados parciais deste trabalho foi possível inferir as sequintes conclusões:  A análise molecular e filogenética dos mostrou que provavelmente 50% das espécies isoladas podem representam novas espécies de fungos. Isto mostra o potencial destas espécies vegetais em abrigar espécies de fungos ainda desconhecidas;  Dois isolados de fungos pertencentes às espécies Emericellopsis donezkii and Colletotrichum gloesporioides podem ser considerados como promissores para estudos detalhados quanto ao isolamento do(s) metabólito(s) ativo(s);  Os resultados obtidos da análise biogeográfica dos fungos endofíticos mostraram que a comunidade endofítica não é aleatoriamente distribuída. A análise de decaimento da distância confirmou que a composição de fungos varia ao longo do gradiente latitudinal estudado. A comunidade de fungos endofíticos é influenciada por fatores ambientais e pela distância geográfica e este efeito depende da escala espacial avaliada;  Fungos endofíticos isolados de raízes de Myrtus communis não infectaram raízes de Sorghum vulgare, porém alguns fungos atuaram como promotores do crescimento vegetal quando inoculados na rizosfera de sorgo. Estudos mais detalhados devem ser feitos para determinar os mecanismos os mecanismos pelos quais estes fungos proporcionaram o aumento do peso seco e porcentagem de micorrização arbuscular de sorgo;  A similaridade da comunidade de fungos endofíticos cultiváveis associados à M. communis não diminuiu com o aumento da distância geográfica;  Este trabalho mostra o potencial biotecnológico dos fungos endofíticos como produtores de substâncias antimicrobianas e seu potencial como promotor do crescimento vegetal em plantas de interesse econômico. 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Diversity and antimicrobial activity of fungal endophyte communities associated with plants of Brazilian savanna ecosystems. African journal of microbiology research, 6: 3173- 3185. Vaz ABM, Sampedro I, Siles JA, Vasquez JA, García-Romera I, Vierheilig H, Rosa CA, Ocampo JA (2012) Arbuscular mycorrhizal colonization of Sorghum vulgare in presence of root endophytic fungi of Myrtus communis. Applied soil ecology, 61: 288-294. Artigos científicos submetidos para publicação Vaz ABM, Fontenla S, Rocha FS, Brandão LR, Vieira MLA, Garcia V, Rosa CA. Diversity and biogeography of endophytic fungi associated with Myrtaceae species present in different ecosystems of Argentina and Brazil. Vaz ABM, Sampedro I, Siles JA, García-Romera I, Rosa CA, Ocampo JA (2011) Diversity of fungal endophytes in leaves of Myrtus communis studied by traditional isolation and cultivation- independent DNA-based method. Trabalho publicado em anais de congresso sob forma de resumo expandido Vaz ABM, Gil VB, Vieira MLA, Mafra M, Sobral M, Pimenta RS, Moraes PB, Rosa LH, Rosa CA. Aplicações biotecnológicas de fungos endofíticos isolados do Cerrado e regiões ecotonais do Estado do Tocantins In: XI Encontro Nacional de Microbiologia Ambiental, 2008, Fortaleza. XI Encontro Nacional de Microbiologia Ambiental, 2008. p. 365 – 366. 109 Apresentação de pôsteres em congressos Apresentação de pôster / Painel no XI Encontro Nacional de Microbiologia Ambiental, 2008. Vaz ABM, Gil VB, Vieira MLA, Mafra M, Sobral M, Pimenta RS, Moraes PB, Rosa LH, Rosa CA. Aplicações biotecnológicas de fungos endofíticos isolados do Cerrado e regiões ecotonais do Estado do Tocantins . In: XI Encontro Nacional de Microbiologia Ambiental, 2008, Fortaleza. p. 365 – 366. Apresentação de Pôster / Painel no (a) IX Encontro de Pesquisa do Instituto de Ciências Biológicas, IV Encontro Anual de Pesquisa em Bioquímica e Imunologia, 2008. Vaz ABM, Gil VB, Vieira MLA, Mafra M, Sobral M, Pimenta RS, Moraes PB, Rosa LH, Rosa CA. Aplicações biotecnológicas de fungos endofíticos isolados do Cerrado e regiões ecotonais do estado do Tocantins. Prêmio Melhor Pôster / Painel no (a) IX Encontro de Pesquisa do Instituto de Ciências Biológicas, IV Encontro Anual de Pesquisa em Bioquímica e Imunologia, 2008. Vaz ABM, Gil VB, Vieira MLA, Mafra M, Sobral M, Pimenta RS, Moraes PB, Rosa LH, Rosa CA. Aplicações biotecnológicas de fungos endofíticos isolados do Cerrado e regiões ecotonais do estado do Tocantins. 110 Produção científica relacionada à trabalhos desenvolvidos em colaboração durante a tese de doutorado Artigos científicos publicados em revistas internacionais Vieira MLA, Hughes AFS, Gil VB, Vaz ABM, Alvez TMA, Zani CL, Rosa CA, Rosa LH (2012). Diversity and antimicrobial activities of the fungal endophyte community associated with the traditional Brazilian medicinal plant Solanum cernuum Vell. (Solanaceae). Canadian Journal of Microbiology, 58: 54-66. Gonçalves, Vívian N.; Vaz, Aline B. M.; Rosa, Carlos A.; Rosa, Luiz H (2012). Diversity and distribution of fungal communities in lakes of Antarctica. FEMS Microbiology Ecology, 82 (2): 459-471. Vaz ABM, Rosa LH, Vieira MLA, Garcia V, Brandão LR, Teixeira LCS, Moliné M, Libkind D, Van Brook M, Rosa CA (2011). The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica. Brazilian Journal of Microbiology, 42: 937-947. Brandão LR, Libkind D, Vaz ABM, Espírito Santo LC, Moliné M, de García V, Van Broock M., Rosa, CA (2011) Yeasts from an oligotrophic lake in Patagonia (Argentina): diversity, distribution and synthesis of photoprotective compounds and extracellular enzymes. FEMS Microbiology Ecology, 76:1:13. VAZ, A. B. M.; Mota, R. C.; Bomfim, Maria Rosa Q.; Vieira, M. L. A.; Zani, C. L.; Rosa, C. A.; Rosa, L. H. (2009) Antimicrobial activity of endophytic fungi associated with Orchidaceae in Brazil. Canadian Journal of Microbiology, 55: 1381-1391. 111 Outras atividades Expedições Participação na Operação Antártica XXVII no período 17/12/2008 a 15/01/2009, exercendo função de pesquisadora do Projeto Vivian, na estação antártica Comandante Ferraz. Cursos Internacionais Curso de especialização em Edafologia e Biologia vegetal oferecido pela UNESCO na Estación Experimental Del Zaidin, Granada, Espanha. 1210 h. 2010-2011. Aplicación de metodos moleculares y herramientas de análisis para el estúdio de la biodiversidad microbiana. Centro Brasileiro Argentino de Biotecnologia, CBAB, Argentina. Ministério Ciência e Tecnologia. 80h. 2010 International Course on Molecular Methods for Diagnosis, genomic analysis and Biotechnological applications of microorganisms. Universidad Nacional del Comahue, UNCo, Argentina. 60 h. 2009 Introducción al Analisis Multivarido. Universidad Nacional del Comahue, UNCo. Argentina. 60h. 2009. Cursos nacionais Modelagem estatística utilizando o programa R. 80h. 2012. ABG consultoria estatística. Biotecnologia de Fungos Endofíticos da Amazônia. Universidade do Amazonas. UEA. Centro Brasileiro Argentino de Biotecnologia, CBAB, Brasil. Ministério Ciência e Tecnologia. 80h. 2009. Treinamento Pré-Antártico. Centro de adestramento da Ilha da Marambaia. Marinha do Brasil, RJ. 40 h. 2007. Estatística Aplicada utilizando o R. 48h. 2010. ABG consultoria estatística.