โครงสร้างสังคมของราเอคโตไมคอร์ไรซาและการประยุกต์เพื่อการปลูกป่าไม้วงศ์ไม้ยาง นางสาวสุนัดดา โยมญาติ วิทยานิพนธ์นี้เป็นส่วนหนึ่งของการศึกษาตามหลักสูตรปริญญาวิทยาศาสตรดุษฎีบัณฑิต สาขาวิชาเทคโนโลยีชีวภาพ คณะวิทยาศาสตร์ จุฬาลงกรณ์มหาวิทยาลัย ปีการศึกษา 2551 ลิขสิทธิ์ของจุฬาลงกรณ์มหาวิทยาลัย COMMUNITY STRUCTURE OF ECTOMYCORRHIZAL FUNGI AND REFORESTATION APPLICATION IN DIPTEROCARPACEAE Miss Sunadda Yomyart A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Program in Biotechnology Faculty of Science Chulalongkorn University Academic year 2008 Copyright of Chulalongkorn University vi ACKNOWLEDGMENTS I would like to express my greatest appreciation to my thesis advisor, Associate Professor Prakitsin Sihanonth, Ph.D. and my thesis coadvisor, Assistant Professor Jittra Piapukiew, Ph.D., Professor Roy Watling, Ph.D. and Professor Anthony J.S. Whalley, Ph.D. for their valuable advice and encouragement throughout this study. My appreciation is also expressed to Associate Professor Suthep Thaniyavarn, Ph.D., Associate Professor Sirirat Rengpipat, Ph.D. and Associate Professor Songsri Kulpreecha, Ph.D. for their kindness and helpful suggesting for the complements of this and serving as thesis committee. I would like to express my sincere gratitude and thanks to Mr. Aniwat Chalermpongse for valuable suggestions throughout my study and serving as external thesis committee. I also wish to express my appreciation to the Program in Biotechnology and Department of Microbiology, Faculty of Science, Chulalongkorn University for providing facilities during my study. My sincere gratitude is also extended to Professor Taizo Hogetsu and Dr. Bingyun Wu, Laboratory of Forest Botany, Graduate School of Agricultural and Life Science, The University of Tokyo, Japan for laboratory facilities. The financial supports from The Royal Golden Jubilee Ph.D. Program and the Graduate school, Chulalongkorn University are also gratefully acknowledged. I also thank members of Room 401, Department of Microbiology for their friendship, help and kindness. Finally, the greatest gratitude is expressed to my parents for their true loves and continuing support throughout this study. CONTENTS Page ABSTRACT (IN THAI). ............................................................................................. iv ABSTRACT (IN ENGLISH)........................................................................................ v ACKNOWLEDGEMENTS.......................................................................................... vi CONTENTS………………………………………………...……………………………… vii LIST OF TABLES...................................................................................................... xi LIST OF FIGURES........................................................................................... ......... xii LIST OF ABBREVIATIONS…………………………………………………….………… xv CHAPTER I : INTRODUCTION................................................................................. 1 CHAPTER II : LITERATURE REVIEW....................................................................... 3 2.1 Ectomycorrhiza........................................................................................ 3 2.1.1 Morphological and anatomical features of ectomycorrhizas...… 3 2.1.2 Role of ectomycorrhizal symbiosis.............................................. 5 2.1.3 Mycorrhizal associations of different tree species...................... 5 2.2 Taxonomically the ectomycorrhizas forming fungi………..……………… 6 2.2.1 Diversity and Community of ECM fungi....................................... 6 2.3 Community structure of ECM fungi........................................................... 7 2.3.1 Molecular methodologies for analyzing ECM fungal communities 9 2.3.2 Effect of biotic and abiotic factors to ECM community structure 10 2.4 Observation of ectomycorrhizas in tropical ecosystem………………….. 12 2.5 Application of ECM fungi…………………………………………..………… 16 2.5.1 Reforestation and afforestation of degraded areas in tropical forests……………………………………………………………………… 16 2.5.2 Effect of ECM fungi associated with dipterocarps...................... 17 2.5.2.1 Tissue weight ……………………………………………… 18 2.5.2.2 Plant height ................................................................... 18 2.5.2.3 Stem diameter ……………………………..……………… 18 2.5.2.4 Shoot volume ................................................................ 18 2.5.2.5 Leaf area and number …………………………………… 18 viii Page 2.5.2.6 Root colonization ........................................................... 19 2.5.2.7 Transplant survival …………………………………..…… 19 2.5.3 Use of ectomycorrhizal symbiosis for plantation forestry………. 20 2.5.4 Production of ectomycorrhizal fungi inoculums........................... 23 2.5.4.1 Soil inoculum……………………………………………..… 24 2.5.4.2 Spore inoculum………………………………………..…… 24 2.5.4.3 Vegetative inoculum…………………………….………… 25 2.4.5.3.1 Cultivation of ECM fungi in solid substrate............................................................................ 25 2.4.5.3.2 Submerged cultivation of ECM fungi ………..… 25 2.5.5 Selection of effective ECM fungi inocula..................................... 26 CHAPTER III : MATERIALS AND METHODS........................................................... 29 3.1 Chemicals……………………………………………………………………… 29 3.2 Instruments................................................................................................ 29 3.3. Description of study sites…………………………………………………..… 31 3.4 Sampling plots and sampling of soil blocks………………………………… 33 3.4.1 Analysis of physical soil properties…………………………………. 35 3.5 Sampling of sporocarps and ectomycorrhizal root tips from soil blocks.. 35 3.6 Molecular identification of ectomycorrhizal fungi…………………………... 36 3.6.1 DNA preparation and ITS-TRFLP analysis………………………… 36 3.6.2 Identification of underground ECM fungi……………………..….… 37 3.6.3 ITS sequence analysis……………………………………………….. 38 3.4.6 Statistical analysis…………………………………………………….. 39 3.7 Selection of ectomycorrhizal fungi on growth stimulation of seedlings…. 39 3.7.1 Pure culture isolation and inoculum production…………………... 39 3.7.2 Ectomycorrhizal formation test in selected dipterocarp species and experimental design……………………………………………. 40 3.7.3 Plant measurements and harvest………………………………….. 41 3.7.4 Statistics analysis……………………………………………………. 42 ix Page CHAPTER IV : RESULTS AND DISCUSSION............................................................ 43 4.1 Community structure of ECM fungi on a dipterocarp forest and a D. alatus plantation in Thailand……………………………………………… 43 4.1.1 Analysis of soil properties…………………………………………… 43 4.1.2 Analysis of aboverground and underground of ECM community 44 4.1.2.1 Identification of the aboveground ECM sporocarps……. 45 4.1.2.2 Identification of underground ectomycorrhiza…………… 54 4.1.2.3 Underground ECM fungi compared with ECM sporocarps…………………………………………………… 63 4.1.2.4 Dynamic change of ECM community……………………... 65 4.1.2.5 Statistical analysis of ECM communities…………………. 71 4.1.3 Host effected on ECM fungal community: insight from native mixed dipterocarps forest and D. alatus plantation……………… 76 4.2. Selection of ECM fungi on growth stimulation of dipterocarps seedlings................................................................................................ 77 4.2.1 Macroscopic examination of sporocarp of A. ordoratus, 78 A. asiaticus and P. abditus …………………………………..…….. 4.2.2 Scanning electron microscopy examination of basidiospores….. 83 4.2.3 Pure culture isolation and inoculum production…………………... 83 4.2.4 Ectomycorrhizal formation test in selected Dipterocarpaceae species and experimental design………………………..………... 84 4.2.4.1 Preliminary test of ectomycorrhiza formation……………. 84 4.2.4.2 Testing for ectomycorrhizal colonization of dipterocarp seedling roots………………………………………………... 86 4.2.4.3 Measurements and harvest of Dipterocapaceae seedlings…………………………………………………….. 92 CHAPTER V : CONCLUSIONS…………………………………………….……………. 102 REFERENCES……………………………………..……………………….……………… 104 APPENDICES............................................................................................................ 119 x Page APPENDIX A…………………………………………………………………...…… 118 APPENDIX B.................................................................................................... 124 APPENDIX C ………………………………..……………………………………… 125 APPENDIX D.................................................................................................... 126 APPENDIX E.................................................................................................... 128 APPENDIX F.................................................................................................... 132 APPENDIX G.................................................................................................... 135 APPENDIX H.................................................................................................... 146 BIOGRAPHY.............................................................................................................. 160 LIST OF TABLES Table Page 4.1 Sequence affinity of ECM sporocarps and corresponding terminal fragment lengths for the ITS1regions………………………………………………............ 47 4.2 Sequence affinity of ECM sporocarps and corresponding terminal fragment lengths for the ITS1regions………………………………………………………… 51 4.3 Morpho-anatomy describtion of 12 ECM taxa. fragment lengths for the ITS1 regions………………………………….………….. 55 4.4 Sequence affinity of ECM root tips and corresponding terminal fragment lengths for the ITS1 regions…………………………………...……………………