Ecophysiological characteristics of ectomycorrhizal ammonia fungi in Hebelomatoid clade January 2013 Ho Bao Thuy Quyen Graduate School of Horticulture CHIBA UNIVERSITY (千葉大学学位申請論文) 千葉大学学位申請論文) Ecophysiological characteristics of ectomycorrhizal ammonia fungi in Hebelomatoid clade 2013 年 月 千葉大学大学院園芸学研究科 環境園芸学専攻生物資源科学コース 環境園芸学専攻生物資源科学コース Ho Bao Thuy Quyen Contents Page Contents I List of Tables IV List of Figures .V Acknowledgements VII Abstract IX General Introduction Chapter The first record of Hebeloma vinosophyllum (Strophariaceae) in Southeast Asia Introduction Materials and Methods Collection Observation Phylogenetic analysis Mating tests Results 10 Taxonomy 10 Phylogenetic analysis 13 Mating tests 14 Discussions 15 Chapter Ability of ectomycorrhization of late phase fungi in Hebelomatoid clade 17 Introduction 17 Materials and Methods 18 Organisms 18 Mycorrhizal colonization 19 Results 21 Fungal growth and fruiting ability 21 I Growth of seedlings 21 Ectomycorrhization 21 Discussions 27 Chapter Photo-response of fruit body formation in ectomycorrhizal ammonia fungi Alnicola lactariolens and Hebeloma vinosophyllum 28 Introduction 28 Materials and Methods 29 Pre-cultivation 29 Effect of light on fruit body formation of Alnicola lactariolens and Hebeloma vinosophyllum 29 Statistical analysis 30 Results and Discussions 30 Responses of fruit body formation in Alnicola lactariolens to different light intensities 30 Responses of fruit body formation in Hebeloma vinosophyllum to different light intensities 33 Responses of fruit body formation in Alnicola lactariolens and Hebeloma vinosophyllum to different light periods 36 Chapter The effects of ammonium-nitrogen and nitrate-nitrogen concentrations on fruit body formation in ectomycorrhizal ammonia fungi Alnicola lactariolens and Hebeloma vinosophyllum 38 Introduction 38 Materials and Methods 39 Pre-cultivation 39 Effect of ammonium-nitrogen and nitrate-nitrogen concentrations on fruit body formation of Alnicola lactariolens and Hebeloma vinosophyllum 39 Results and Discussions 40 Growth and reproductive responses of Alnicola lactariolens to ammonium-nitrogen and nitrate-nitrogen concentrations 40 II Growth and reproductive responses of Hebeloma vinosophyllum to ammonium-nitrogen and nitrate-nitrogen concentrations 42 Chapter The absorbing abilities of cesium and coexisting elements in an ectomycorrhizal ammonia fungus Hebeloma vinosophyllum 44 Introduction 44 Materials and Methods 45 Results and Discussions 47 The uptake of cesium and coexisting elements by Hebeloma vinosophyllum 47 The effect of NH4+ concentrations on the cesium, potassium and phosphorus uptake by Hebeloma vinosophyllum 50 General Discussions 55 Literature Cites 60 III List of Tables Page Table Collection details of specimens and cultures in the phylogenetic analysis Table Published sequences in the phylogenetic analysis 10 Table Dikaryon-monokaryon mating tests between dikaryotic stock cultures of Japanese Hebeloma vinosophyllum and monokaryotic strains of Vietnamese Hebeloma sp 15 Table Fungal isolates of LP hebelomatoid fungi used to investigate ECM association 19 Table Responses of fruiting in Alnicola lactariolens to different light intensities 31 Table Responses of fruiting in Hebeloma vinosophyllum to different light intensities 34 Table Days required for fruit body formation of Alnicola lactariolens and Hebeloma vinosophyllum to different light exposure periods per day 37 Table Days required for the fruit body initiation of Anicola lactariolens and final pH of media after harvesting fungal biomass 40 Table Days required for the fruit body initiation of Hebeloma vinosophyllum and final pH of media after harvesting fungal biomass 42 Table 10 Concentrations and chemical forms of Cs and coexisting elements (K, Ca, Mg, Zn, Cu, Fe and P) in the medium 46 Table 11 Concentrations of K in media added different concentrations of NH4+ 46 Table 12 Concentration (mg/kg dry sample) of Cs, K, Ca, Mg, Zn, Cu, Fe and P in mycelium and fruit bodies of Hebeloma vinosophyllum 48 Table 13 Translocation of Cs, K, P, Ca, Mg, Fe, Zn and Cu in Hebeloma vinosophyllum from mycelium to fruit bodies 49 IV List of Figures Page Fig Hebeloma vinosophyllum 12 Fig Hebeloma vinosophyllum from the urea plot in Vietnam 12 Fig Biogeographic distribution of Hebeloma vinosophyllum in Japan (Honshu: Saitama, Tochigi, Tokyo, Chiba, Shiga, Shizuoka, Kyoto, Tottori; Shikoku: Kochi and Kyushu: Oita, Miyazaki), South Korea, China (Fujian Province) and Vietnam (Da Lat) 13 Fig The maximum likelihood phylogenetic tree based on ITS data set (A) and LSU data set (B) of Hebeloma vinosophyllum and allied species 14 Fig The fruit body formation of LP hebelomatoid fungi in the association with Pinus densiflora 22 Fig The fruit body formation of LP hebelomatoid fungi in the association with Quercus serrata 22 Fig The hyphal colonization of LP hebelomatoid fungi in roots of Pinus densiflora 23 Fig ECM between Alnicola lactariolens and Quercus serrata 24 Fig ECM between Hebeloma vinosophyllum and Quercus serrata 25 Fig 10 ECM between Hebeloma aminophilum and Quercus serrata 26 Fig 11 Effect of light intensity on the morphology of mature fruit bodies in Alnicola lactariolens 32 Fig 12 Effect of light intensities on fruit body formation in Alnicola lactariolens 33 Fig 13 Effect of light intensity on the morphology of mature fruit bodies in Hebeloma vinosophyllum 35 Fig 14 Effect of light intensities on fruit body formation in Hebeloma vinosophyllum 36 V Fig 15 Effect of different concentrations of diammonium sulfate on vegetative growth and fruit body formation of Alnicola lactariolens 41 Fig 16 Effect of different concentration of potassium nitrate on vegetative growth and fruit body formation of Anicola lactariolens 41 Fig 17 Effect of different concentration of diammonium sulfate on vegetative growth and fruit body formation of Hebeloma vinosophyllum 43 Fig 18 Effect of different concentration of potassium nitrate on vegetative growth and fruit body formation of Hebeloma vinosophyllum 43 Fig 19 The transfer factors of Cs, K, P, Ca, Mg, Fe, Zn and Cu in Hebeloma vinosophyllum biomass (mycelium and fruit bodies) 48 Fig 20 The transfer factors of Cs, K and P in Hebeloma vinosophyllum biomass (mycelium and fruit bodies) under different NH4+ concentrations 50 Fig 21 Fruit body formation of Hebeloma vinosophyllum in different NH4+ concentrations 51 Fig 22 Dry biomas of Hebeloma vinosophyllum (mycelium and fruit bodies) in different NH4+ concentrations 52 Fig 23 The ratios of Cs and K concentrations in mycelium and fruit bodies of Hebeloma vinosophyllum in different NH4+concentrations 53 Fig 24 The supposed successive occurrence in the field of LP hebelomatoid fungi 57 Fig 25 The potential application of Hebeloma vinosophyllum as bioremediation in the control of radiocessium in forest systems 58 VI Acknowledgements I would like to send the deepest gratitude to Prof Akira Suzuki, Graduate School of Horticulture, Chiba University, Chiba, Japan, my supervisor, for the best support to my scientific ideas and works He and his wife, Ms Tomoko Suzuki, also helped me so much for adapting with life in Japan I would like to send a deep gratitude to • Dr Kiminori Shimizu (Medical Mycology Research Center, Chiba University, Chiba, Japan) who is always kind with me and taught a lot of knowledge about biomolecular works to me • Dr Satoshi Yoshida (National Institute of Radiological Sciences, Chiba, Japan) who provided the facilities and taught methods of element measurement in mushroom samples to me • Dr Toshimitsu Fukiharu (Natural History Museum and Institute, Chiba, Japan) who helped and taught the fungal morphology in this research to me • Dr Takashi Yamanaka (Forest and Forest Products Research Institute, Tsukuba, Japan) who provided seeds and seedlings of oaks • Dr Hisayasu Kobayashi (Ibaraki Prefectural Forestry Research Institute, Ibaraki, Japan) who provided seeds of pines • Dr Naohiko Sagara (Prof Emeritus of Kyoto University, Kyoto, Japan) who provided the fungal isolate The other deep gratitude would like to be sent to • Medical Mycology Research Center, Chiba University, Chiba, Japan • National Institute of Radiological Sciences, Chiba, Japan • Natural History Museum and Institute, Chiba, Japan • Forest and Forest Products Research Institute, Tsukuba, Japan and • Prof Susumu Kawamoto, Medical Mycology Research Center, Chiba University, Chiba, Japan • Prof Ken-ichi Tozaki, Faculty of Education, Chiba University, Chiba, Japan VII • Prof Hiromi Hamada, Faculty of Education, Chiba University, Chiba, Japan • Prof Chihiro Tanaka, Kyoto University, Kyoto, Japan for the help to develop my research in doctoral dissertation I also want to send the thanks to Prof Tatsuaki Kobayashi, Prof Kazunori Sakamoto, Dr Seigo Amachi for their suggestions to improve my doctoral dissertation My doctoral dissertation was also supported by the below budgets • The doctoral fellowship of Ministry of Education and Training, Vietnamese Government, Vietnam in fiscal year 2009 – 2012 • The AGGST program for students, Chiba University, Chiba, Japan in fiscal year 2011 – 2012 and fiscal year 2012 – 2013 for expanding my research In the end, I want to send the deepest gratitude to my parents, my husband, my son, my great family and all of my friends in Vietnam, in America and in Japan who always was behind and encouraged me in any time VIII Literature Cites Boyle H, Zimdars B, Renker C and Buscot F 2006 A molecular phylogeny of Hebeloma species from Europe Mycological Research 110: 369-380 Bresadola J 1981 Iconographia Mycologia, Vol I Mediolani MCMLXXXI, Italia, pp 718 Brückmann A and Wolters V 1994 Microbial immobilization and recycling of 137Cs in the organics layers of forest ecosystems: relationship to environmental conditions, humidification and invertebrate Science of the Total Environment 157: 249-256 Byrne A R 1988 Radioactivity in fungi in 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