© University of Pretoria DECLARATION I, Angelica Marsberg declare that the thesis, which I hereby submit for the degree Magister Scientiae at the University of Pretoria, is my own work and has not previously been submitted by me for a degree at this or any other tertiary institution. ___________________ Angelica Marsberg June 2012 2 © University of Pretoria TABLE OF CONTENTS Acknowledgements ....................................................................................................... i Preface ......................................................................................................................... ii CHAPTER 1 Factors affecting endophyte-host interactions ........................................................ 1 Abstract ......................................................................................................................... 2 1. Introduction: Old term, new concept .................................................................. 3 2. The evolution of endophyte-host interactions ..................................................... 5 3. Direct effects of endophytes on their hosts ........................................................ 8 4. Complexity of the endophyte infection process .................................................. 12 5. Factors affecting endophyte communities on host plants ................................... 13 6. Conclusion ......................................................................................................... 16 7. References ......................................................................................................... 18 CHAPTER 2 Environmental barcoding of the endophytic fungi associated with a healthy Syzygium cordatum tree in South Africa ................................................................................... 28 Abstract ......................................................................................................................... 29 1. Introduction ........................................................................................................ 30 2. Materials and Methods ....................................................................................... 34 3. Results ............................................................................................................... 40 4. Discussion .......................................................................................................... 44 5. References ......................................................................................................... 50 Figures and Tables .................................................................................................. 62 3 © University of Pretoria CHAPTER 3 Unique, dominant and diverse assemblages of Mycosphaerellaceae, Teratosphaeriaceae and Cladosporiaceae endophytes from Myrtaceae in South Africa............................ 77 Abstract ......................................................................................................................... 78 1. Introduction ........................................................................................................ 79 2. Materials and Methods ....................................................................................... 81 3. Results ............................................................................................................... 85 4. Discussion .......................................................................................................... 88 5. References ......................................................................................................... 91 Figures and Tables .................................................................................................. 97 Summary ...................................................................................................................... 106 4 © University of Pretoria ACKNOWLEDGEMENTS The writing of a thesis is never the work of one individual alone. A number of people have contributed to this body of work in their separate ways and I would like to thank the following especially: First and foremost I would like to thank God for His wisdom and guidance during the writing of this thesis as well as during the many paths my life has taken. I would like to thank my parents, Graham and Maria Marsberg, for their love and support throughout my schooling and tertiary education. Without them I would never have been able to further my career and this thesis would never have been written. I would also like to thank my brother, Justin, for his tolerance and for being an amicable roommate. To my fiancé, Stephan de Villiers, I express my thanks for his love and support and for being my rock through the stressful times during the writing of this thesis. I would next like to thank my supervisors, Dr. Marieka Gryzenhout, Prof. Bernard Slippers and Prof. Mike Wingfield. Without their leadership this thesis would not have transpired in the manner that it has. I thank them for the many discussions that have broadened my knowledge and understanding of many different topics. I would like to thank Dr. Jeff Garnas who helped with the statistical analyses as well as Dr. Seonju Marincowitz for her help with the descriptions of the new species. I also thank Kerry- Anne Pillay for providing some of the Eucalyptus isolates and sequence data used in the study as well as for her help during the analysis of some of the results. Finally I would like to thank the Thutuka Funding programme of the National Research Foundation (NRF) as well as members of the Tree Protection Co-operative Programme (TPCP) for their financial support. i © University of Pretoria PREFACE Fungal endophytes are capable of infecting their host without any visible signs of disease. These diverse organisms have been isolated from all plants studied to date. Some species have undergone co-evolution with their host plants and offer benefits to their hosts. Others are often seen to be saprophytes or latent pathogens whose effects become noticeable when the host is stressed or dying. In South Africa, very few studies have been done on endophytes, an exception being the endophytic canker forming Botryosphaeriaceae pathogens. Syzygium cordatum is a native South African tree that occurs along the East Coast of KwaZulu Natal. No studies have yet investigated the fungal diversity that may be associated with S. cordatum, unlike Eucalyptus trees which are well-studied. The aim of this study will be to determine the endophytic diversity of S. cordatum and to determine if the pathogenic fungi that occur on Eucalyptus trees are also associated with S. cordatum. This becomes problematic when a pathogen is introduced into a new environment and adapts to infect a new host with potentially damaging consequences to economically important plantations and natural ecosystems alike. Chapter 1 of this thesis focuses on the literature available on fungal endophytes. The specific aim of the review was to gauge the complexity of these interactions and to try and understand the importance of these organisms in their environment, endophyte-host interactions, modes of infection, and the influence that endophytes have on their hosts as well as the co-evolution of life-history traits. ii © University of Pretoria In Chapter 2, the diversity of fungal endophytes present in S. cordatum will be assessed. Two complimentary methods, namely isolate-based barcoding and environmental barcoding, will be used in combination to assess the diversity of fungal endophytes present. With culture-based methods, many fungal species may be missed as they do not grow in culture or grow so slowly that other fast growing species outgrow them. Culture-independent methods such as environmental barcoding make it possible to characterise multiple samples and species directly from the environment, thus increasing the probability that a greater diversity of endophytes will be detected. The latter technique is largely driven by next generation sequencing techniques such as 454 GS FLX Titanium pyrosequencing. Chapter 3 aims to properly define a group of endophytes that belong to the Cladosporiaceae, Mycosphaerellaceae and Teratosphaeriaceae using multi-locus identification. Endophytes that were identified as belonging to the Cladosporiaceae, Mycosphaerellaceae and Teratosphaeriaceae in Chapter 2, and in a similar study done on a Eucalyptus grandis x E. camaldulensis GC540 hybrid clone, were subjected to multi-locus sequence analysis in order to establish their identities. These fungi are well-known leaf and shoot pathogens of Eucalyptus species and collectively cause the disease known as Mycosphaerella leaf blotch. The endophytic fungi that will be identified in this study are expected to be native as they were isolated from a native South African tree. As S. cordatum and Eucalyptus species have a tendency to occur in the same environments, one will also expect that some fungal species will occur on both hosts. This study is expected to provide new insight into the potential diversity of fungi in southern Africa. iii © University of Pretoria Factors affecting endophyte-host interactions 1 © University of Pretoria Abstract Endophytes are organisms that inhabit plant organs and colonise plant tissues without causing obvious signs of disease. Much research has been done to elucidate the effects that endophytes have on their hosts, but the majority of these studies have been done on grass species that are considered model organisms.