Márcia Filipa Lopes Rosendo de Castro Silva MSc Studies on the Eucalyptus Leaf Disease Complex in Portugal Dissertação para obtenção do Grau de Doutor em Biologia (especialidade em Microbiologia) Orientador: Doutor Alan John Lander Phillips, FCT/UNL Co-orientador: Doutora Maria Helena Neves Machado, INIAV Outubro 2015 Márcia Filipa Lopes Rosendo de Castro Silva MSc Studies on the Eucalyptus Leaf Disease Complex in Portugal Dissertação para obtenção do Grau de Doutor em Biologia (especialidade em Microbiologia) Orientador: Doutor Alan John Lander Phillips, FCT/UNL Co-orientador: Doutora Maria Helena Neves Machado, INIAV Outubro 2015 Para os meus meninos To my little boys “In God’s garden of grace, even a broken tree can bear fruit.” Rick Warren V “Copyright” Márcia Filipa Lopes Rosendo de Castro Silva FCT/UNL e da UNL A Faculdade de Ciências e Tecnologia e a Universidade Nova de Lisboa têm o direito, perpétuo e sem limites geográficos, de arquivar e publicar esta dissertação através de exemplares impressos reproduzidos em papel ou de forma digital, ou por qualquer outro meio conhecido ou que venha a ser inventado, e de a divulgar através de repositórios científicos e de admitir a sua cópia e distribuição com objetivos educacionais ou de investigação, não comerciais, desde que seja dado crédito ao autor e editor. Excetuando, os capítulos referentes a artigos científicos os quais só podem ser reproduzidos sob a permissão dos editores originais e sujeitos às restrições de cópia impostas pelos mesmos, mais concretamente os capítulos 1, 2, 3, 4, 5 e 6. VII Esta dissertação foi financiada pela Fundação para a Ciência e Tecnologia através da bolsa de doutoramento SFRH/BD/40784/2007. IX Acknowledgements To all who have contributed in some way to this thesis, I would like to express my deepest gratitude. I am grateful to my supervisor, Prof. Dr. Alan Phillips, for his support during this thesis. His knowledge, experience and guidance were crucial for the success of the preparation of the manuscripts. I am thankful to my co-supervisor, Dr. Helena Machado for her support throughout almost a decade, for her critical opinion and experience along with the valuable discussions, all so helpful. To Prof. Dr. Mike Wingfield and Prof. Dr. Pedro Crous for their advice in some aspects of the thesis. I also acknowledge the Portuguese Foundation for Science and Technology, which provided financial support through the grant SFRH/BD/40784/2007. As for the financial support for laboratory work and most of the biological material provided by experimental plantations supported by technical assistance protocol of Instituto RAIZ/AltriFlorestal/INRB. I am also grateful to Eng. Carlos Valente, Dr. Clara Araújo and Dr. Lucinda Neves for their support in the fieldwork and the financial support for part of the fieldwork and laboratory work that was provided by Instituto RAIZ/AltriFlorestal/INIAV Protocol. To Dr. William Quaedvlieg and Dr. Ewald Groenewald who kindly supplied some sequences. To PhD Committee, Prof. Dr. Álvaro Fonseca and Prof. Dr. Octávio Paulo who gave some suggestions during the work and to PhD Coordinator, Prof. Dr. Isabel Sá-Nogueira who was always available for me. To Unit for Research and Services on Agricultural and Forestry Systems and Plant Health of INIA for all facilities directed by Dr. Amélia Lopes and coordinated by Dr. Eugénia de Andrade. To Dr. Teresa David (Coordinator of Forestry Systems) for her encouragement and friendship. To Dr. Edmundo de Sousa as Coordenador of Unit of Forestry Research for the enthusiasm and support with some of the equipment. To the Head of Lab of Molecular Genetics, Dr. Filomena Nóbrega for lab equipment and for her friendship. To the Head of Lab of Seed Lab, Eng. Lourdes Santos for lab equipment, especially for plant climatic chambers. To the Head of Lab of Mycology, Dr. Helena Bragança, lab staff and Eugénio Diogo, and all “ex-Forest Protection Department” staff Adérito, Florinda, Joana, Lurdes, Luís, Margarida Fontes, Margarida Vieira, Maria João, Marina, Pedro, Sofia, Victor and the “outsider” Joaquim, for their support and friendship. To Belén, Eng. Clara Fernandes, Dr. Paula Sá-Pereira for their friendship and help. XI Acknowledgements (Cont.) To my Friends Rosemary Gale and Vitaliy Kolesov who encouraged me in the worst days. Ângela, Clara, Eduardo and Inês for the friendship, confidence and help given. To Augusta for her companionship, encouragement and inspiration during the difficult days. All of my friends for their important companionship and invaluable support throughout this journey. I would like to express my deepest gratitude to Salomé (I will not forget), and Rute for our conversations. To my family My parents for their return in the last difficult two months. My great-grandmother, avó Maria, my grandparents, avô Mário and avó Inácia that I lost during this work, for their character, unconditional love and support, they were extremely important, even without their physical presence. Mário, blood of my blood, for his values, character, encouragement that has given me strength when I needed the most, unconditional love and support, which were extremely important. To my two sons, Dinis and Francisco, for their sweetness smile, who were born during this work and did not have much of my time, at least as they should have and deserve. To God, for Him, and with His help, I did and can do all things (Philippians 4:13)! ABSTRACT Native from south eastern Australia, Eucalyptus globulus is the main species in eucalypts plantations in Portugal. The most serious foliar disease in eucalypt plantations is linked to Mycosphaerella senso lato, which affects young trees in the juvenile phase foliage causing leaf necrosis. This disease results in reduced growth rate of the host and lower wood volume, thus causing significant productivity losses. The most common name for this disease was Mycosphaerella Leaf Disease that became inappropriate when most of the pathogens on eucalypts were re-distributed into several genera. The term "Eucalyptus Leaf Disease Complex" is now more appropriate. The overall aim of this thesis was to investigate the Eucalyptus Leaf Disease Complex in Portugal, focusing on species diversity, taxonomy and the role played by each species in the disease complex on Eucalyptus globulus. Literature on the Eucalyptus Leaf Disease Complex was reviewed and the species were distributed into several genera. A survey based on symptomatic leaves collected from several Eucalyptus globulus plantations and characterized by morphological and molecular tools provided an overview of species incidence and of the most frequent species in the disease complex. The present work reveals additional species of Mycosphaerella senso lato associated with eucalypt plantations in Portugal. Thus, five new records of Teratosphaeria and phylogenetically related species were added to the Iberian Peninsula, namely, Neodevriesia hilliana, for the first time on Myrtaceae; Quasiteratosphaeria mexicana, Teratosphaericola pseudoafricana, Teratosphaeria pluritubularis and Teratosphaeria lusitanica, a new species. Furthermore, new anamorphic structures were found and two new combinations were made. Regarding other genera, some species were observed for the first time, such as Cladosporium cladosporioides, Fusicladium eucalypti, Mycosphaerella madeirae, in the mainland. In addition to leave diseases, Teratosphaeria gauchensis was found causing a severe stem and trunk canker on Eucalyptus globulus. The aggressiveness of several species was compared to evaluate each species individually in the complex, permitting to distinguish different behaviours, from primary to secondary pathogens. Cladosporium cladosporioides, M. communis and M. lateralis, appeared to be more aggressive than Teratosphaeria nubilosa. In fact, contrary to the prevailing views on this disease complex, Teratosphaeria nubilosa is not the only species responsible for the disease, which clearly involves a complex of species acting together. Keywords: Leaf Disease; Canker; Mycosphaerella; Teratosphaeria; Eucalypts and Eucalyptus globulus. XIII RESUMO Originário do sudoeste da Austrália, Eucalyptus globulus é a principal espécie de eucalipto em plantações em Portugal. A doença mais grave nas folhas de eucalipto está relacionada com espécies de Mycosphaerella senso lato as quais afetam árvores jovens com folhagem na sua fase juvenil, resultando na diminuição da sua taxa de crescimento e do volume de madeira produzido, o que causa perdas significativas de produtividade. O nome mais comum desta doença é Doença das Manchas das Folhas do Eucalipto (Mycosphaerella Leaf Disease) que se tornou inapropriado uma vez que as diversas espécies envolvidas na doença foram reorganizadas em diversos géneros, sendo agora o uso do termo “Complexo da Doença das Folhas do Eucalipto” (Eucalyptus Leaf Disease Complex) o mais adequado. O objetivo geral desta tese é investigar o Complexo da Doença das Folhas do Eucalipto em Portugal, especialmente a diversidade de espécies, a taxonomia e o papel de cada espécie no complexo da doença. O Complexo da Doença das Folhas do Eucalipto foi recentemente revisto e as espécies envolvidas distribuídas em diversos géneros. Efetuou-se a prospeção de folhas sintomáticas em diversas plantações de Eucalyptus globulus e as espécies detetadas foram caracterizadas morfologicamente e com ferramentas moleculares, permitindo uma análise geral das espécies que se encontram no complexo da doença. O presente trabalho indica um aumento do número de espécies