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Endophytic Fungi of Olive Tree and Their Exploitation in the Biological

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Endophytic Fungi of Olive Tree and Their Exploitation in the Biological ESCUELA SUPERIOR Y TÉCNICA DE INGENIERÍA AGRARIA INGENIERÍA DE BIOSISTEMAS Endophytic fungi of olive tree and their exploitation in the biological control of olive anthracnose Doctoral Thesis Maria de Fátima Tomé Martins Director: Professora Doutora Paula Cristina Santos Baptista León 2020 ESCUELA SUPERIOR Y TÉCNICA DE INGENIERÍA AGRARIA INGENIERÍA DE BIOSISTEMAS Hongos endofíticos del olivo y su aprovechamiento para el control biológico de la antracnosis del olivo Tesis Doctoral Maria de Fátima Tomé Martins Director: Profesora Doctora Paula Cristina Santos Baptista León 2020 This research was supported by FEDER funds through the COMPETE (Operational Programme for Competitiveness Factors) and by the Foundation for Science and Technology (FCT, Portugal) within the POCI-01-0145-FEDER-031133 project and FCT/MCTES to CIMO (UIDB/00690/2020), as well as the Horizon 2020, the European Union’s Framework Programme for Research and Innovation, for financial support the project PRIMA/0002/2018. Fátima Martins also thanks the individual research grant ref. SFRH / BD / 112234/2015 award by FCT. The studies presented in this thesis were performed at the AgroBioTechnology Laboratory, at the Mountain Research Centre (CIMO), School of Agriculture Polytechnic Institute of Bragança. Às minhas filhas Acknowledgements Neste momento, é com enorme gosto e satisfação que agradeço a todos aqueles que direta ou indiretamente contribuíram para a realização e conclusão deste trabalho. Em primeiro lugar gostaria de agradecer em especial à minha orientadora. À Professora Doutora Paula Cristina dos Santos Baptista, da Escola Superior Agrária, por toda a orientação prestada durante a realização do trabalho ao nível laboratorial e escrito, bem como, por todo o conhecimento transmitido, paciência, disponibilidade e acima de tudo pela amizade e carinho demonstrados ao longo destes anos. Por me ajudar a crescer como pessoa e como profissional, pelo apoio incondicional prestado mesmo nas horas menos fáceis, por acreditar nas minhas capacidades e me incentivar sempre. Muito obrigada! Ao professor José Alberto pelo suporte de apoio nos trabalhos realizados, pelos conselhos sempre sábios na orientação das amostragens de campo, pela confiança demonstrada. Muito obrigada! O meu agradecimento aos amigos e colegas de laboratório que estiveram diariamente presentes na constante boa disposição, companheirismo, pela partilha de ideias e angústias que me acompanharam ao longo deste percurso, agradeço ao Diogo Mina, Cristina Cameirão, Lara Pinheiro, Rosalina Marrão, Ricardo Malheiro, Nuno Rodrigues, Jacinto Benhadi-Marín, Maria Villa, Teresa Gomes, Luana Fernandes. Agradeço também a todos aqueles que estiveram só de passagem, mas onde deixaram a sua marca. Agradeço à Maria Villa pela ajuda prestada com a tradução do espanhol. Quero agradecer também às técnicas de laboratório, D. Isabel, D. Fátima, D. Arminda e à D. Céu Fidalgo pelo apoio e auxílio prestado. Agradeço ao Professor Doutor Pedro Casquero, da Escuela Superior y Técnica de Ingeniería Agraria da Universidad de León, pelo apoio prestado ao longo deste percurso. Por fim quero agradecer aqueles que são o meu suporte incondicional, que estão sempre presentes, aconteça o que acontecer: Aos meus pais, a quem devo tudo o que sou aos meus irmãos e sobrinhos pelo amor e carinho, incentivo, apoio incondicional. A ti, Paulo, por estares ao meu lado pela paciência, compreensão e companheirismo constante. Ao meu maior bem da vida, a quem me enche o coração todos os dias de amor e carinho, às minhas filhas. Grata por tudo! Table of contents Abstract…………………………………………………………………………….... i Resumen ……………………………………………………………………………. ii List of Abbreviations………………………….………………………………...xxviii Chapter 1 - General Introduction ................................................................................. 1 1.1 Framework and objectives ...................................................................................... 2 1.2 Olive Anthracnose: A general overview ................................................................. 4 1.3 Anthracnose is caused by a complex of Colletotrichum species ............................ 5 1.4 Epidemiology and life cycle ................................................................................... 6 1.5 Management strategies for olive anthracnose ......................................................... 8 1.6 Fungal endophytes and their potential as biocontrol agents against Colletotrichum spp. ............................................................................................................................ 11 1.7. Fungal endophytes on the control of olive anthracnose....................................... 15 1.8 Conclusions ........................................................................................................... 17 1.9 References ............................................................................................................. 17 Chapter 2 - Endophytic fungal community succession in reproductive organs of two olive tree cultivars ......................................................................................................... 23 2.1 Abstract ................................................................................................................. 25 2.2 Introduction ........................................................................................................... 25 2.3 Materials and methods .......................................................................................... 27 2.3.1 Samples collection.......................................................................................... 27 2.3.2 Isolation of endophytic fungi ......................................................................... 28 2.3.3 Identification of fungal isolates ...................................................................... 29 2.3.4 Abundance and diversity of fungal endophytes ............................................. 30 2.3.5 Data analysis .................................................................................................. 30 2.4 Results ................................................................................................................... 32 2.4.1 Diversity and composition of endophytes differ between cultivars ............... 32 2.4.2 Diversity and composition of endophytes in reproductive organs over different phenological stages ................................................................................................. 33 2.4.3 Plant organ and phenological stage are the most important factors for shaping the fungal community.............................................................................................. 38 2.4.4 Cultivar- and phenological stage preference of endophytic fungi ................. 38 2.5 Discussion ............................................................................................................. 41 2.5.1 Contribution of plant organ and host genotype on endophytic fungal assembly ................................................................................................................................. 42 2.5.2 Patterns of plant-endophyte association over different time points of inflorescence and fruit development ....................................................................... 44 2.5.3 Can differences in susceptibility of the cultivars to anthracnose be related to the endophytic community naturally present in inflorescence and fruit? ............... 45 2.6 References ............................................................................................................. 46 2.7 Supporting Information ......................................................................................... 54 Chapter 3 - Endophytic fungal community structure in olive orchards with high and low incidence of olive anthracnose .............................................................................. 60 3.1 Abstract ................................................................................................................. 62 3.2 Introduction ........................................................................................................... 62 3.3 Materials and methods .......................................................................................... 64 3.3.1 Study area and plant material collection ........................................................ 64 3.3.2 Anthracnose incidence and severity ............................................................... 66 3.3.3 Endophytic fungi isolation ............................................................................. 66 3.3.4 Identification of endophytic fungi .................................................................. 67 3.3.5 Diversity and structure of fungal community ................................................ 67 3.3.6 Factors driving fungal community structure .................................................. 68 3.3.7 Identification of fungal OTUs associated with each orchard or plant organ . 69 3.4 Results ................................................................................................................... 69 3.4.1 Anthracnose incidence and Colletotrichum abundance ................................. 69 3.4.2 Fungal community comparison: High versus low anthracnose incidence ..... 70 3.4.3 Contribution of different drivers for fungal community shaping ................... 75 3.4.4 OTUs associated with each olive orchard and plant organ ...........................
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