DEPARTAMENTO DE CIÊNCIAS DA VIDA 2010 FACULDADE DE CIÊNCIAS E TECNOLOGIA UNIVERSIDADE DE COIMBRA Azores: assessing e Arbuscular Mycorrhizal Fungal diversity and ion in pastures of th of ion in pastures nt practices t e composition in pasttures of the Azores: assessing the impact of management practices iversity and composi iversity and impact of managem impact of the Mycorrhizal Fungi d r Arbuscula Catarina Alexandra Drumonde Melo 2010 Catarina Melo DEPARTAMENTO DE CIÊNCIAS DA VIDA FACULDADE DE CIÊNCIAS E TECNOLOGIA UNIVERSIDADE DE COIMBRA Arbuscular Mycorrhizal Fungal diversity and composition in pastures of the Azores: assessing the impact of management practices Tese apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra para obtenção do grau de Doutor em Biologia, na especialidade de Ecologia, realizada sob a orientação científica da Professora Doutora Helena Freitas (Universidade de Coimbra) e do Professor Doutor Paulo A. V. Borges (Universidade dos Açores) Catarina Alexandra Drumonde Melo 2010 Acknowledgements I wish to thank my supervisors Prof. Dr. Helena Freitas and Prof. Dr. Paulo Borges for their encouragement, patient supervision and constructive criticisms. I will always be grateful to Prof. Dr. Paulo Borges, for being the person who most contributed to my scientific training. Special thanks to Prof. Dr. Raimundo Cabrera and Dr. Maria Jaizme-Vega for their friendship, help, advice, knowledge that provided a stimulating, productive and enjoyable work environment during part of my study period in ULL (Tenerife). I would like to thank to the Instituto Canario de Investigaciones Agrarias (ICIA) for logistical support to my experiences in Tenerife, especially to Mirriam and Paulito. I also thank to Susana for her advice, knowledge and suggestions with molecular techniques and helpful discussions, and for providing to me friendly working conditions in Coimbra Lab during part of my study period. I am grateful to Dr. C. Walker for his comments and help in spores’ identification and description. Special thanks to my dearest friend Clara Gaspar, for her friendship, advices, help with statistical analysis and suggestions. I would also like to thank to all members of Centro de Biotecnologia dos Açores (CBA) for their friendship and understanding, especially to Prof. Dr. Artur Machado for his encouragement during the last step of my PhD. I gratefully acknowledge financial support for this research that was provided by the Portuguese Fundação para a Ciência e a Tecnologia to my PhD grant (SFRH/BD/18355/2004). To my parents for keeping me motivated and for believing in me, especially to my mother for travelling with me during my study periods in Tenerife and Coimbra, and for taking care of my son Gustavo. Special thanks to my husband Vasco for helping in all the field work, for his patience, understanding, love and encouragement during this task. Finally, and “because the last are the first” to my sons, Martim and Gustavo for all the love and affection that helped me recover in the most difficult times of this task, and to Gustavo for all the companionship and sharing over the past 5 years. Aos meus filhos…… TABLE OF CONTENTS Page Abstract .................................................................................................................. 13 Resumo .................................................................................................................. 15 Chapter I ................................................................................................................. 19 General Introduction ............................................................................................... 21 1. The importance of soil microorganisms .............................................................. 21 2. Mycorrhizal fungi ................................................................................................ 22 3. Arbuscular mycorrhizal fungi .............................................................................. 23 3.1. The AMF life cycle ........................................................................................... 25 3.2. Paris- and Arum- type colonisation .................................................................. 26 3.3. Ecology of Arbuscular Mycorrhizae ................................................................. 28 3.3.1. Host specificity .............................................................................................. 28 3.3.2. Plant succession and mycorrhizal colonisation ............................................ 29 3.4. Genetics of AMF .............................................................................................. 30 4. Arbuscular mycorrhiza ........................................................................................ 32 4.1. Nutritional benefits ........................................................................................... 32 4.2. Other beneficial features ................................................................................. 34 5. Identification, systematic, phylogeny and diversity of the Glomeromycota………35 5.1. Systematics and phylogeny of the Glomeromycota………………………………35 6. Methods for AMF identification ........................................................................... 39 6.1. Morphological methods ................................................................................... 39 6.2. Molecular methods .......................................................................................... 41 6.2.1. Marker genes for AMF community studies ................................................... 42 7. AMF and Agriculture ........................................................................................... 43 8. Study areas ......................................................................................................... 45 8.1.1. Island overview ............................................................................................. 45 8.1.2. Studied pastures ........................................................................................... 46 8.2. Land use history and soil management ........................................................... 48 8.2.1. Land use in past ............................................................................................ 48 8.2.2. Land use today ............................................................................................. 50 9. Aims of this dissertation ...................................................................................... 52 Chapter II Species composition of arbuscular mycorrhizal fungi in semi-natural and intensive pastures from Terceira Island (Azores) ................................................................... 53 1. Introduction ......................................................................................................... 55 2. Materials and methods ........................................................................................ 59 2.1. Study sites ....................................................................................................... 59 2.2. Sample collection ............................................................................................. 61 2.3. Study plant ....................................................................................................... 61 2.4. Soil analyses .................................................................................................... 62 2.5. Establishment of a trap culture ......................................................................... 62 2.6. AMF spore extraction and identification ........................................................... 63 2.7. Data analysis ................................................................................................... 65 3. Results ................................................................................................................ 66 3.1. AMF species recorded ..................................................................................... 66 3.2. Species descriptions ........................................................................................ 68 3.3. AMF species composition ................................................................................ 77 3.4. Soil nutrient parameters .................................................................................. 83 3.5. AMF spore density ........................................................................................... 84 4. Discussion .......................................................................................................... 86 Chapter III Seasonal dynamics of arbuscular mycorrhizal fungal communities in roots of Holcus lanatus L. in semi-natural and intensive pastures in Terceira island (Azores) ........ 93 1 .Introduction ......................................................................................................... 95 2. Materials and methods ....................................................................................... 99 2.1. Experimental design ........................................................................................ 99 2.2. Site description ................................................................................................ 99 2.3. Study plant ...................................................................................................... 101 2.4. Rhizosphere extraction ...................................................................................