Biscogniauxia Mediterranea) in Cork Oak Decline in Portugal
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CHARCOAL CANKER (BISCOGNIAUXIA MEDITERRANEA) IN CORK OAK DECLINE IN PORTUGAL TESE APRESENTADA PARA OBTENÇÃO DO GRAU DE DOUTOR EM ENGENHARIA FLORESTAL E DOS RECURSOS NATURAIS JOANA MARGARIDA CORDEIRO HENRIQUES ORIENTADOR: Doutor Arlindo Lima COORIENTADORES: Doutor Edmundo Manuel Rodrigues de Sousa Doutora Maria Filomena de Sousa Nóbrega, Investigadora Auxiliar do Instituto Nacional de Investigação Agrária e Veterinária, IP JÚRI: Presidente: Reitor da Universidade de Lisboa Vogais: Doutora Maria Ivone Esteves da Clara, Professora Catedrática da Universidade de Évora Doutora Maria Helena Mendes da Costa Ferreira Correia de Oliveira, Professora Associada do Instituto Superior de Agronomia da Universidade de Lisboa Doutor Arlindo Lima, Professor Auxiliar do Instituto Superior de Agronomia da Universidade de Lisboa Doutora Ana Paula Ferreira Ramos, Professor Auxiliar do Instituto Superior de Agronomia da Universidade de Lisboa Doutor Edmundo Manuel Rodrigues de Sousa, Investigador Auxiliar do Instituto Nacional de Investigação Agrária e Veterinária, IP Doutora Maria Helena Neves Machado, Investigadora Auxiliar do Instituto Nacional de Investigação Agrária e Veterinária, IP LISBOA 2015 TABLE OF CONTENTS TABLE OF CONTENTS i ACKNOWLEDGMENTS iii ABREVIATIONS LIST v ABSTRACT vii RESUMO ix RESUMO ALARGADO xi 1. INTRODUCTION 1 OBJECTIVES 4 THESIS STRUCTURE 5 1.1. CORK OAK - DISTRIBUTION, SOCIO-ECONOMIC AND ENVIRONMENTAL IMPACTS 7 1.1.1. CORK OAK DECLINE 9 CORK OAK PESTS AND DISEASES 11 1.2. CHARCOAL CANKER, A CORK OAK DISEASE CAUSED BY BISCOGNIAUXIA MEDITERRANEA 13 1.2.1. THE SPECIES BISCOGNIAUXIA MEDITERRANEA 13 THE XYLAREACEAE 13 THE TELEOMORPH 14 THE ANAMORPH 15 1.2.2. THE DEVELOPMENT OF THE DISEASE 17 DISTRIBUTION AND INCIDENCE 17 DISEASE EFFECTS 18 ENDOPHYTIC FUNGI 20 FAVORABLE FACTORS 22 CONTROL MEASURES 26 LABORATORIAL DETECTION METHODS 27 2. BISCOGNIAUXIA MEDITERRANEA IN CORK OAK STANDS 29 2.1. NEW OUTBREAKS OF CHARCOAL CANKER ON YOUNG CORK OAK TREES IN PORTUGAL 31 HENRIQUES J, INÁCIO ML, LIMA A, SOUSA E. 2012. IOBC/WPRS BULLETIN 76: 85-88 2.2. FACTORS AFFECTING THE DISPERSION OF BISCOGNIAUXIA MEDITERRANEA IN 35 PORTUGUESE CORK OAK STANDS HENRIQUES J, BARRENTO MJ, BONIFÁCIO L, GOMES AA, LIMA A, SOUSA E. 2014. SILVA LUSITANA 22 (1): 83-97 i 3. DIVERSITY OF BISCOGNIAUXIA MEDITERRANEA ASSOCIATED TO CORK OAK 51 3.1. DIVERSITY OF BISCOGNIAUXIA MEDITERRANEA WITHIN SINGLE STROMATA ON CORK OAK 53 HENRIQUES J, NÓBREGA F, SOUSA E, LIMA A. 2014. JOURNAL OF MYCOLOGY, ARTICLE ID 324349, 5P. 3.2. ANALYSIS OF THE GENETIC DIVERSITY AND PHYLOGENETIC RELATIONSHIPS OF 59 BISCOGNIAUXIA MEDITERRANEA ISOLATES ASSOCIATED WITH CORK OAK HENRIQUES J, NÓBREGA F, SOUSA E, LIMA A. FOREST PATHOLOGY (SUBMITTED) 3.3. MORPHO-CULTURAL AND PHYSIOLOGICAL FEATURES OF BISCOGNIAUXIA MEDITERRANEA 79 ISOLATES ASSOCIATED WITH CORK OAK HENRIQUES J, NÓBREGA F, SOUSA E, LIMA A (UNPUBLISHED) 4. GENERAL DISCUSSION 103 5. CONCLUDING REMARKS 111 REFERENCES 115 ii ACKNOWLEDGMENTS To all who contributed in some way to this work, I would like to express my deepest gratitude! To Doctor Edmundo Sousa, who made this work possible, for allowing me to do in what I like, for believing in me and for all the fresh ideas that make me learn and grow up. To Doctor Arlindo Lima for all the understanding and encouragement throughout all the work, and for the valuable suggestions and corrections of the thesis. To Doctor Filomena Nóbrega for having me “adopted” and for sharing all her knowledge of molecular biology, a world that really delighted me! To all the co-authors of the articles, for all their help and valuable contributions. To all who contributed to the collection of isolates used throughout the work. The entire team of the project “Study and demonstration of management practices for cork- oak montado recuperation”, for all the enthusiasm and the good times of field work. To Doctor Lourdes Santos for her essential instructions and help with the acorns and seedlings processing. To Master Sofia Trindade and Doctor Teresa Valdiviesso for their valuable help in making the histological sections. To Doctor Paula Ramos and Eng. Filomena Caetano for all the willingness to help and the long day of cultures characterization. To Eng. Cidália Vila Verde and Eng. Nuno Calado, from UNAC, for the interest and support in data and material collection. To Doctor Natércia Santos, who started to study this disease in the former Estação Florestal Nacional, for all the encouragement to continue this work line. To everyone at INIAV who always expressed their interested and encouraged throughout the work, in particular the closest colleagues: the fungi staff, Helena Machado, Helena Bragança, Maria de Lurdes Inácio and Eugénio Diogo, for all the patience for discussing my existential doubts; Marina Cardoso for always being the fungi’ tender godmother; Florinda Medeiros for the precious assistance in the laboratory work; Adérito Matos for all the availability and support mainly in the field work; the boys, Luís Bonifácio and Pedro Naves, for all the iii suggestions, cheerful moments and, of course, for all the cookies; Ricardo Petersen for the fellowship in this PhD situation, Maria João Barrento for putting up with my chats in the bureau, to Margarida Fontes, Margarida Vieira, Isabel Evaristo, Vitor Gonçalves, Miguel Pestana and Francisco Martins for the always ongoing encouragement. To my family and friends, for all the support and encouragement throughout this work, and ever since I chose this direction. To Tiago, for the strength and support and, above all, all the patience! iv ABBREVIATIONS LIST ANOVA - Analysis Of Variance AT - Asymptomatic Tree BIC - Bayesian Information Criterion BLAST - Basic Local Alignment Search Tool CBS - Centraalbureau voor Schimmelcultures dH2O - Distilled water DNA - Deoxyribonucleic Acid dNTPs - Deoxynucleotides DT - Declined Tree EMB - Embryo END - Endosperm INIAV – Instituto Nacional de Investigação Agrária e Pescas IPCC - Intergovernmental Panel on Climate Change IS - Inner Seed ISTA - International Seed Testing Association ITS-rDNA - Internal Transcribed Spacers of rDNA LSD - Least Significant Difference MCL - Maximum Composite Likelihood MEAN - Micoteca da Estação Agronómica Nacional MEGA- Molecular Evolutionary Genetic Analysis ML - Maximum Likelihood MSP-PCR - Microsatellite-Primed PCR NCBI - National Center for Biotechnology Information NTSYSpc - Numerical Taxonomy and Multivariate Analysis System - Version 2.02h NUV -near-Ultra Violet light PCR - Polymerase Chain Reaction PDA - Potato Dextrose Agar PDAA - PDA acidified with lactic acid PDYA - PDA supplemented with yeast extract PT - Protection Tissue PWP - predawn leaf water potential RAPD -Random Amplified Polymorphic DNA rDNA - ribosomal DNA RNA - Ribonucleic Acid RT-PCR – Real Time PCR SM - Simple Matching coefficient TBE - Tris-Borate-EDTA (Ethylenediamine tetraacetic acid) TEF- Translation Elongation Factor 1-α TUB1 - β-Tubulin 1 TUB2 - β-Tubulin 2 UPGMA -Unweighted Pair Group Method with Arithmetic Mean USA – United States of America v vi ABSTRACT Charcoal canker is a disease caused by Biscogniauxia mediterranea that affects Quercus suber and other hardwood species. The fungus develops endophytically in the hosts becoming pathogenic under stress conditions. However, it has become more frequent, particularly in young trees expressing atypical symptoms. This work intends to clarify the agent involved in these disease outbreaks and its variability in different situations, as well as dispersion in natural conditions. The fungal anamorph was identified as Nodulisporium sp. in young hosts. In the stands, ascospores airborne dispersal is predominant, occurring after precipitation periods, and vertical contamination of plants doesn’t happen. A collection of isolates obtained from cork oak in Portugal and other Mediterranean countries, from other hosts and from trees with different ages and disease expression were analyzed by microsatellite-primed PCR, multigene sequencing, morpho-cultural characteristics and growth rates at different temperatures. All the approaches revealed high level of intraspecific polymorphism among isolates, not allowing relating the disease development with any considered feature. The results highlighted the variability and adaptation ability of this fungus in special in the Mediterranea region, in the present worrying scenario of climatic change. All the conditions are gathered to favor the aggravation of the disease in cork oak stands. Key-Words: dispersion, intraspecific variability, Mediterranean ecosystem, Nodulisporium, Quercus suber vii viii O carvão do entrecasco (Biscogniauxia mediterranea) no processo de perda de vigor do montado de sobro em Portugal RESUMO O carvão do entrecasco é uma doença causada por Biscogniauxia mediterranea que afeta Quercus suber e outras espécies lenhosas. O fungo desenvolve-se endofiticamente nos hospedeiros tornando-se patogénico sob condições de stresse. Tem-se tornado mais frequente, particularmente em árvores jovens que expressam sintomas atípicos. Este trabalho pretende clarificar o agente envolvido nestes surtos da doença e a sua variabilidade em diferentes situações, assim como a sua dispersão em condições naturais. Identificou-se o anamorfo Nodulisporium sp. em hospedeiros jovens. No campo, a dispersão aérea dos ascósporos é predominante, ocorrendo após períodos de precipitação, e não existe contaminação vertical de plantas. Uma coleção de isolados obtidos a partir de sobreiro em Portugal e outros países mediterrânicos, de outros hospedeiros, árvores