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Andreia Filipa Lages Cerqueira Effects of Phosphite in Pinus Radiata

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Andreia Filipa Lages Cerqueira Effects of Phosphite in Pinus Radiata Universidade de Aveiro Departamento de Biologia Ano 2016 Andreia Filipa Lages Effects of phosphite in Pinus radiata-Fusarium Cerqueira circinatum interaction Efeitos do fosfito na interação Pinus radiata- Fusarium circinatum DECLARAÇÃO Declaro que este relatório é integralmente da minha autoria, estando devidamente referenciadas as fontes e obras consultadas, bem como identificadas de modo claro as citações dessas obras. Não contém, por isso, qualquer tipo de plágio quer de textos publicados, qualquer que seja o meio dessa publicação, incluindo meios eletrónicos, quer de trabalhos académicos. Universidade de Aveiro Departamento de Biologia Ano 2016 Andreia Filipa Lages Effects of phosphite in Pinus radiata-Fusarium Cerqueira circinatum interaction Efeitos do fosfito na interação Pinus radiata- Fusarium circinatum Tese apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biologia Molecular e Celular, realizada sob a orientação científica da Doutora Glória Catarina Cintra da Costa Pinto, Professora auxiliar convidada do Departamento de Biologia da Universidade de Aveiro, e coorientação do Doutor Artur Jorge da Costa Peixoto Alves, Investigador principal do Departamento de Biologia da Universidade de Aveiro. Apoio financeiro da Comissão Diretiva do Apoio financeiro da Fundação para a Programa Operacional Competitividade e Ciência e Tecnologia (FCT) no Internacionalização (POCI-01-0145- âmbito do projeto PTDC/AGR- FEDER-016785) FOR/2768/2014 "Don’t be afraid. There are exquisite things in store for you. This is merely the beginning." Oscar Wilde, The Picture of Dorian Gray o júri presidente Professora Doutora Maria de Lourdes Pereira Professora Associada com Agregação do Departamento de Biologia da Universidade de Aveiro arguente principal Doutora Lia-Tânia Rosa Dinis Investigadora de Pós-Doutoramento do Departamento de Biologia e Ambiente da Universidade de Trás-os-Montes e Alto Douro orientadora Doutora Glória Catarina Cintra da Costa Pinto Professora Auxiliar convidada do Departamento de Biologia da Universidade de Aveiro agradecimentos Finda mais uma etapa da minha vida, quero agradecer a toda a gente que, de uma forma ou de outra, esteve lá para mim. À Doutora Glória Pinto e ao Doutor Artur Alves, obrigada por me terem acolhido no vosso laboratório, pela generosidade e confiança depositadas em mim e por tudo o que me ensinaram. À Barbara, obrigada por tudo o que me ensinaste e pela paciência em responder a todas as minhas questões. Ao Pedro e ao Helder, por me ajudarem sempre que precisei. Aos amigos de sempre, que mesmo longe estão sempre por perto. Em particular, à Rita obrigada por me ouvires a falar de biologia horas e horas, mesmo sem gostar, e por me animares sempre que tudo parecia perdido. À Leandra, obrigada pelos 14 anos de amizade e por estares presente em todas as ocasiões, do menos bom ao bom. Ao melhor grupo de amigas que podia pedir - à Bárbara, à Cláudia, à Filipa, à Gracinha, à Natacha e à Vanessa. Obrigada pelos almoços no CUA e pelas bifanas, pelos dias passados na biblioteca, pelos risos e ombro amigo, por ouvirem os meus desabafos e por terem sido a minha família longe de casa. Às outras pessoas importantes que Aveiro me trouxe - ao João, por teres tornado 4 destes 5 anos melhores, por te preocupares e por me ensinares o que é lutar pela felicidade. À Ana Rita, por me compreenderes mesmo sem palavras e por teres sido a melhor companheira de quarto/casa/aventuras. À Sara, por partilhares comigo o amor às coisas belas da vida, como a música, os céus e Coura. Ao Alexandre, à Liane, à Diana e à Maria João por tornarem a minha passagem por Aveiro ainda mais especial. E por fim, aos meus pais e irmão. Obrigada pelos sacríficos, ao longo desde 5 anos, e pelo amor, carinho e apoio ao longo de toda a minha vida. A vocês tudo devo e tudo o que faço é por vocês e para vocês. palavras-chave Fusarium circinatum, Pinus radiata, fosfito, indução de resistência, fisiologia resumo O cancro resinoso, provocado pelo fungo Fusarium circinatum, é uma doença que afeta Pinus spp. e Pseudotsuga menziesii em todo o mundo e está sujeita a medidas de quarentena. Caracteriza-se pela formação de grandes cancros resinosos que rodeiam rebentos, ramos e troncos e levam à morte do hospedeiro. Até à data não existem meios para o controlo da doença e, com a crescente necessidade de reduzir o uso de fungicidas, outras abordagens devem ser estudadas. Um método para o controlo de doenças fitopatogénicas passa pela indução da resistência do hospedeiro, através do pré-tratamento de plantas com compostos químicos ou de origem biológica que estimulam as defesas. O fosfito (Phi) é um sal inorgânico que apresenta a capacidade de indução da resistência e uma potencial estratégia mais amiga do ambiente. Neste estudo, a utilização do fosfito de potássio (KPhi) na redução do desenvolvimento dos sintomas da doença do cancro resinoso, assim como os seus efeitos no crescimento do fungo, foram estudados em diferentes concentrações. Numa primeira fase, colónias de F. circinatum foram crescidas em PDA suplementado com Phi (0%, 1% e 4%) para avaliação do seu efeito no crescimento radial. Posteriormente foram estudados os efeitos da aplicação foliar de Phi (0%, 1% e 4%) em plântulas de Pinus radiata, inoculadas e não inoculadas. A taxa de sobrevivência e a performance fisiológica (potencial hídrico, trocas gasosas e performance fotoquímica, pigmentos, peroxidação lipídica, libertação de eletrólitos, prolina e carbohidratos) foram avaliados. Os resultados mostram que aplicação de Phi atrasa o desenvolvimento dos sintomas de doença numa forma dependente da concentração de Phi, em semelhança ao observado relativamente à inibição do crescimento do micélio in vitro. Alterações fisiológicas ao nível da prolina e carbohidratos, peroxidação lipídica e trocas gasosas foram observadas. A aplicação de Phi apresenta-se como uma potencial alternativa viável na gestão da doença do cancro resinoso. keywords Fusarium circinatum, Pinus radiata, phosphite, induction of resistance, physiology abstract The pitch canker, caused by the fungus Fusarium circinatum, is a disease under quarantine measures affecting Pinus spp. and Pseudotsuga menziesii worldwide. Characterized by the formation of large resinous cankers that girdle shoots, branches, and trunks, leads to the death of the host. To date, there are no means for the control of the pitch canker and, with the growing need to reduce the use of fungicides, another approaches must be studied. A method for the control of phytopathogenic diseases is the enhancement of host resistance, through pre-treatment of seedlings with chemicals or biologically derived compounds that stimulate defense responses. Phosphite (Phi) is an inorganic salt with the capability of inducing host resistance and presents an approach more environmentally friendly. In this study, the ability of potassium phosphite (KPhi) in delaying the pitch canker symptom development, as well as its effects in fungal growth, were studied at different concentrations. In a first phase, F. circinatum colonies were grown in PDA medium supplemented with Phi (0%, 1% and 4%) to evaluation in the radial growth of the fungus. Posteriorly, were studied the effects of foliar application of Phi (0%, 1% and 4%) in Pinus radiata seedlings, inoculated and non-inoculated. Survival and physiological performance (water potential, gas exchange and photochemical performance, pigments, lipid peroxidation, electrolyte leakage, proline and carbohydrates) were assessed. Results showed that Phi application delayed disease symptoms in a dose dependent manner similarly to what was observed in mycelial growth inhibition during in vitro assays. Physiological alterations in proline, carbohydrates, lipid peroxidation and gas exchange parameters were observed. Thus, Phi application presents a potential viable alternative to the management of the pitch canker disease. Index Part I 1 The genus Pinus: a brief description of Pinus radiata 1 Fusarium circinatum, the causal agent of the pitch canker 2 Morphological identification 3 Biology and epidemiology 3 Infection process and symptoms 4 Pathogen classification 5 Host-pathogen interaction 6 Plant physiological responses upon pathogenic attack 7 Disease management 9 The role of phosphites 10 Thesis main purposes 12 Part II 13 Phosphite delays the progression of Fusarium circinatum on Pinus radiata: a physiological approach 13 Abstract 13 Introduction 14 Materials and Methods 16 Results 20 Discussion 31 Conclusion 35 References 35 Part III 45 Future perspectives 45 References 47 Part I The genus Pinus: a brief description of Pinus radiata Considered to be the most significant tree genus in the world, the genus Pinus represents an outstanding group of gymnosperms in the Plant Kingdom and is found in almost any terrestrial habitat (Richardson & Rundel 2000; Ioannou et al. 2014). It is composed of a total of 111 species that have the ability to spread into a remarkably wide range of environments and grow continually throughout the year (Ghildiyal et al. 2010; Gernandt et al. 2015). These plants grow naturally or are introduced in both hemispheres, being essentially distributed over the Northern hemisphere but also occurring in subtropical and tropical regions of Central America and Asia (Ioannou et al. 2014). In Europe, pine forests cover an area of more than 50 million hectares. In Portugal, Pinus is the second most planted genus, representing approximately 29% of the Portuguese forest (ICNF 2013b),
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