Fungal Pigment Formation in Wood Substrate by Daniela Tudor A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Faculty of Forestry University of Toronto © Copyright by Daniela Tudor 2013 ii Fungal Pigment Formation in Wood Substrate Daniela Tudor Doctor of Philosophy Faculty of Forestry University of Toronto 2013 Abstract A number of fungi produce spalted wood, which is characterized by accumulation of black pigment in fine demarcation lines, often accompanied by discoloration or staining on the wood fibers. Specific spalting fungi were identified by molecular analysis. From a total of 19 isolates and 140 clones studied, 11 fungal species were identified. The two Chlorociboria species from North America were investigated and their anamorphs were unambiguously identified for the first time. Fungal pigment formation under the influence of moisture content and pH variation was investigated in sugar maple, American beech and agar inoculated with spalting fungi. Maximum pigment production occurred at treatment with pH 4.5 for sugar maple and beech inoculated with Trametes versicolor. Xylaria polymorpha produced external pigmentation in beech treated with buffer at pH 5 and sugar maple at pH 4.5. Fungal pigmentation by Trametes versicolor and Xylaria polymorpha was stimulated at low moisture content in both wood species tested. Melanin production by Inonotus hispidus and Polyporus squamosus was stimulated above 22-28% and 34-38% moisture content in iii beech and in sugar maple respectively. Fomes fomentarius and Polyporus brumalis produced maximum pigmentation in beech at 26 - 41% and in sugar maple at 59 - 96% moisture content. The variation of the moisture content and pH values of wood substrates can stimulate the intensity of pigmentation of specific fungi in wood. To investigate melanin synthesis from a variety of melanin precursors, experimental research on three spalting fungi tested their reaction to catechol and L-Dopa melanin precursors in wood and agar substrate. The results indicate multiple biosynthesis pathways for melanin assembly in Trametes versicolor, Xylaria polymorha and Inonotus hispidus, and catechol produced most pigmentation in all spalting fungi investigated. Microscopic analysis by light, fluorescence, electron and confocal microscopy also indicates a bi- or multi-modal activity of melanin production and assembly by several spalting fungi. Possible variations of melanin assembly were identified based on fungal and wood species. Immunofluorescence and immunogold labelling with Mab 6D2 melanin antibody confirmed the melanin nature of the pigments produced by Oxyporus populinus, Trametes versicolor, Xylaria polymorpha, Fomes fomentarius, and Inonotus hispidus. iv Acknowledgments I would like to express my deepest gratitude to my supervisor Dr. Paul Cooper, who first of all gave me the opportunity to aspire to a PhD degree, and afterward guided me mercifully throughout the whole process during those six years we worked together. I thank him for his trust in the choices I made, for his wisdom, for the right amount of confidence he stirred in me when I needed most, and for being the ideal supervisor. I am also deeply thankful to my co-supervisor Sally Krigstin, she was often the fixed point in space when it came to reality-check situations. I thank her for her sharp clarity, good advice and for her friendship. I also have to express my deepest gratitude to my supervisory committee, for the feedback and direction they provided for this project. I sincerely thank Martin Hubbes for always providing his positive support; I thank Jean Marc Moncalvo for his patience, encouragement and for intellectually stimulating discussions. I also have to express my deepest appreciation to Tammy Sage, for her kind guidance and for understanding my personal and technical challenges. I am also very grateful to Sara Robinson for all her technical assistance, and for sharing her passion for spalting with me, to Thierry Koumbi Mounanga for technical assistance. I am extremely thankful to Tony Ung for his help and kind offering of all technical support, to Henry Hong and Kathy Sault for technical support and stimulating conversations, and to Simona Margaritescu for her kind patience, technical support, for her friendship and lastly but not least important, for the good laughs. I thank to Michael Butler for the exciting discussions on melanin, and to Arturo Casadevall for immediate support in providing with melanin antibody. v I am thankful for the generous sharing of their academic knowledge to Sean Thomas, especially for his early contribution to my research, and to Mohini Sain for stimulating and nurturing my interest in wood chemistry. I am also grateful for the help of Deborah Paes, Mary Rose Naudi, John McCarron, and Ian Kennedy at the Faculty of Forestry. My huge appreciation for all the members of the Cooper Lab and for their cooperation, friendship, and good conversations over the years. And most importantly, I am deeply thankful to my family, my mom Paulina and my dad Nicolae for their love and nurturing, and to my sister for making me believe that the sky is not at all the limit. Special thanks to my husband Cata, and my daughter Andra, for their incredible, indescribable tremendous and unconditional support, encouragement, patience and love. There will never be enough words. vi Table of contents Abstract .............................................................................................................................. ii Acknowledgements ............................................................................................................iv Table of Contents………….............................................................................................. vi List of Tables.................................................................................................................... xii List of Figures ................................................................................................................. xiii List of Abbreviations ..................................................................................................... xvii Chapter 1 - Introduction ..................................................................................................... 1 1.1 Motivation and Significance ................................................................................... 1 1.2 Scope ....................................................................................................................... 4 1.3 Research Hypotheses .............................................................................................. 5 1.4 Objectives ............................................................................................................... 5 1.5 Thesis overview ...................................................................................................... 5 Chapter 2 - Spalting Fungi: the Study Organisms and the Pigment formation – a Literature Review ………………………………………..…………................................ 7 2.1 Fungi responsible for spalted wood ……………………………………………… 8 2.2 Fungal reaction to substrate conditions ………………………..………….…...... 10 Nutrients……………………………………………………………….…………... 11 Moisture content …………………………………………………..…...…….....… 14 Temperature……………………………………………………………...….……... 15 pH ………..………………………………………………………………..……….. 16 2.3 The morphology of pigment formation by spalting fungi in wood substrate ……16 2.3.1 Melanin in zone lines formation………………………………….………….17 2.3.2 Staining pigments ……….…………………………………..….…………..19 Red stain pigments...…..……..………………………….……....….……….… 19 vii Blue stain pigments ……………….……………………..……………….…… 20 Grey stain pigments.……………….…………………………………..….……20 Green stain pigments ………………………….………….…….…….………. 20 2.4 Chemical characteristics of fungal pigments ………………………...….……… 21 2.4.1 Melanins –properties and biosynthesis …………………………….………. 21 2.4.2 Staining pigments ……………………………………………..…………… 24 2.5 Biotechnological applications of fungal pigment production ……………...…… 25 2.6 Conclusions …………………………………………………………….………. 27 Chapter 3 - Identification of spalting fungi from wood using ITS markers ………...… 28 3.1 Introduction ……………………………………………………………..........…. 28 3.2 Materials and methods …………………………………………………...……... 29 3.2.1. Wood samples ………………………………………………...….………. 29 3.2.2. Molecular analysis ………………………………..…………..…………... 29 3.2.3. Microscopy ……………………………………………………………...… 30 3.3 Results …………………………………………………….……………..…….... 30 3.3.1 Phylogenetic analysis …………...………………….…………..………….. 32 Fusarium sp. ……………………………….…………………………………... 32 Polyporus squamosus ………………………………………………………….. 33 Botryosphaeria sp. ……………………………………………………………... 33 Oxyporus populinus ……………………………………………………………. 34 Nectriaceae (cf. Fusarium sp.) ……………………………………………….…. 35 Hypsizygus sp. …………………………………………….…………….………. 35 Hypocrea rufa – anamorph Trichoderma viride ……………………….……….. 36 Kretzschmaria sp. ……………………………………………………….………. 36 Lecythophora sp. ………………………………………………………….…….. 39 viii Chlorociboria aeruginosa ………………………………………………………. 39 3.3.2 Fungal species distrubution in wood samples ……………..……………… 40 3.4 Discussion …………………………………………...………………………….. 45 3.4.1. Fungi that produce zone lines ……………………………………..……… 45 3.4.2 Staining fungi …………………………………………………...…………. 49 3.5 Conclusions ……………………………………………………...……….……... 51 Chapter 4 - Morphological and molecular characterization of the two known North American Chlorociboria species and their anamorphs……………………………….….52 4.1 Introduction……………………………………………………………………….52