Diversity and distribution patterns of foliar fungal endophytes in Theobroma cacao in Central Sulawesi and interactions between endophytes and host plant Dissertation zur Erlangung des Doktorgrades der Fakultät für Agrarwissenschaften der Georg-August-Universität Göttingen vorgelegt von Carsten Schmidt geboren in Leipzig Göttingen im November 2010 4 __________________________________________________________________________ Table of Contents TABLE OF CONTENTS ............................................................................................. 4 SUMMARY ................................................................................................................. 7 GENERAL INTRODUCTION ...................................................................................... 9 OBJECTIVES ........................................................................................................... 12 CHAPTER 1: THE LOST ENDOPHYTES: ENDOPHYTIC FUNGAL DIVERSITY OF A NEOTROPICAL TREE (THEOBROMA CACAO) IN CENTRAL SULAWESI, INDONESIA .............................................................................................................. 13 Abstract ................................................................................................................................................................ 13 Introduction ......................................................................................................................................................... 14 Material and Methods......................................................................................................................................... 15 Area of investigation......................................................................................................................................... 15 Collection and isolation of endophytes ............................................................................................................. 16 Statistical Analysis............................................................................................................................................ 17 Results .................................................................................................................................................................. 18 Discussion ............................................................................................................................................................. 22 Total diversity ................................................................................................................................................... 22 Diversity Gradient ............................................................................................................................................ 24 Conclusions .......................................................................................................................................................... 25 Acknowledgements .............................................................................................................................................. 26 References ............................................................................................................................................................ 26 Appendix .............................................................................................................................................................. 31 CHAPTER 2: ENDOPHYTES STRESSED OUT: EFFECT OF A SIMULATED ENSO-DROUGHT ON FUNGAL ENDOPHYTE COMMUNITIES IN CACAO TREES ................................................................................................................................. 34 Summary .............................................................................................................................................................. 34 5 __________________________________________________________________________ Introduction ......................................................................................................................................................... 35 Results .................................................................................................................................................................. 36 Discussion ............................................................................................................................................................. 40 Species diversity ............................................................................................................................................... 40 Species composition ......................................................................................................................................... 40 Seasonal changes and fungal species shift ........................................................................................................ 41 Conclusion ........................................................................................................................................................... 42 Experimental Procedures ................................................................................................................................... 43 Study site .......................................................................................................................................................... 43 Artificial drought .............................................................................................................................................. 43 Sample collection.............................................................................................................................................. 44 Fungal endophyte isolation ............................................................................................................................... 44 Statistical analysis ............................................................................................................................................. 45 Acknowledgements .............................................................................................................................................. 46 References ............................................................................................................................................................ 46 CHAPTER 3: DISTRIBUTION AND DIVERSITY OF FUSARIUM SPP. IN CACAO PLANTATIONS OF CENTRAL SULAWESI - INDONESIA ..................................... 52 Abstract ................................................................................................................................................................ 52 Introduction ......................................................................................................................................................... 53 Material and Methods......................................................................................................................................... 54 Investigation area and sampling ....................................................................................................................... 54 Isolation of Fusarium species ........................................................................................................................... 54 Fungal DNA extraction ..................................................................................................................................... 55 Diversity study of selected Fusarium isolates .................................................................................................. 55 Sequence analysis ............................................................................................................................................. 55 Additional statistics .......................................................................................................................................... 56 Results .................................................................................................................................................................. 56 Cluster analysis of the LSU of 172 fungal isolates ........................................................................................... 56 Correlation analysis .......................................................................................................................................... 58 Fusarium diversity based on TEF 1-alpha analysis .......................................................................................... 60 Discussion ............................................................................................................................................................. 62 Phylogenetic identification ............................................................................................................................... 62 6 __________________________________________________________________________ Regional and temporal distribution ................................................................................................................... 63 Environmental gradients ................................................................................................................................... 64 Fusarium, its influence on cacao yield and pathogenicity ................................................................................ 64 Alternative host trees .......................................................................................................................................