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.
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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.
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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.
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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
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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
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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
4.2 Materials and methods.…………………………………………………………...55
4.2.1 Specimen examined ………………………………………………………... 55
4.2.2 Molecular analysis …………………………………………………………. 55
4.2.3 Cultures……………………………………………………………………... 56
4.3 Results and discussion ……………………………………………………………57
4.3.1 Morphological description………………………………………...…………57
4.3.1.1. Chlorociboria aeruginosa (Oeder) Seaver ex C.S. Ramamurthi, Korf & L.R. Batra…………………………………………………………………….…57
4.3.1.2. Chlorociboria aeruginascens (Nyl.) Kanouse ex C.S. Ramamurthi,
Korf & L.R. Batra ……………………………………………...………………58
4.3.2 Analyses of ITS sequences…………………………………………………. 64
4.3.3 Laboratory culture and pigmentation ……………………………….………68
4.4 Conclusions ………………………………………………………………………69
Chapter 5 - The influence of moisture content variation on fungal pigment formation in spalted wood …………………………………………………………………………….70
5.1 Introduction……………………………………………………………………….70
5.2 Material and methods ………………………………………………………….....72
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5.2.1 Wood and fungal species selection……………………………………….….72
5.2.2 Moisture content test preparation………………………………………....…74
5.2.3 Inoculation and incubation……………………………………………….….74
5.2.4 Mass loss and pigment assessment………………………………………… 75
5.2.5 Estimation of initial conditions and changes of substrate moisture content ..76
5.3 Results ……………………………………………………………………...…….76
5.3.1 Initial conditions………………………………………………………….….76
5.3.2 Mass loss ……………………………………………………………………79
5.3.3 Influence of induced condition on pigment formation ……………………...79
5.4 Discussion ………………………………………………………………………..85
5.5 Conclusions ………………………………………………………………………88
Chapter 6 - The influence of pH on pigment formation by lignicolous fungi …………..89
6.1 Introduction ………………………………………………………………………89
6.2 Materials and methods …………………………………………………………...90
6.2.1. Wood and fungi species selection ………………………………………….90
6.2.2. Test procedure ……………………………………………………………...91
6.2.2.1 pH test preparation ……………………………………………….…….91
6.2.2.2 Inoculation and incubation …………………………………………….92
6.2.2.3 Pigment assessment ……………………………………………………92
6.2.2.4. Estimation of initial conditions and changes of substrate pH ………...93
6.3 Results ……………………………………………………………………………93
6.3.1 Initial conditions …………………………………………………………….93
6.3.2 Influence of induced condition on decayed wood …………………………..94
6.3.2.1 pH changes ……………………………………………………………..94
6.3.2.2. Moisture content and mass loss ……………………………………….96
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6.3.2.3. Pigment formation …………………………………………………….97
6.3.3 Influence of pH variation in pigment formation on agar …………………...99
6.4 Discussion ………………………………………………………………………101
6.5 Conclusions ……………………………………………………………………. 103
Chapter 7 - Fungal melanin formation from catechol and L-Dopa precursors ….…… 104
7.1 Introduction and background ………………………………………………...…104
7.2 Material and Methods …………………………………………………………..106
7.2.1 Wood and fungal species selection ………………………………………. 106
7.2.2 Test procedure ……………………………………………………………. 106
7.2.2.1 Test preparation, inoculation and incubation ……………………….. 106
7.2.2.2 Pigment assessment …………………………………………………. 107
7.2.2.3 Tricyclazole test ……………………………………………………... 107
7.3 Results …………………………………………………………………………. 108
7.3.1 Fungal reaction to catechol and L- Dopa in agar substrate …..…………... 108
7.3.2 Pigment formation in wood substrate …………………………………….. 113
7.3.3 Fungal reaction to tricyclazole, an inhibitor to DHN melanin …………….117
7.4 Discussion ………………………………………………………………………119
7.5 Conclusions ……………………………………………………………………..122
Chapter 8 - Microscopic investigation of fungal pigment formation and its morphology in wood substrates ………………………………………………………………………...123
8.1 Introduction and background …………………………………………………...123
8.1.1 Melanins …………………………………………………………………...123
8.1.2 Staining fungi ……………………………………………………………...127
8.2 Materials and methods ………………………………………………………….128
8.2.1 Wood and fungal species ………………………………………………….128
8.2.2 Chemical fixation for copper sulfide-silver staining technique …………...129
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8.2.3 Chemical fixation for SEM ………………………………………………..129
8.2.4 Chemical fixation for TEM and LM ………………………………………130
8.2.5 Immunolocalization procedure for TEM …………………………………..130
8.2.6 Immunolocalization procedure for SEM …………………………………..131
8.2.7 Immunofluorescence analyses of spalted wood …………………………...131
8.3 Results …………………………………………………………………………..132
8.3.1 Melanin detection with the copper sulfide - silver staining techniques …...132
8.3.2 Immunofluorescence and immunogold analysis of melanin ………………133
8.3.3 Staining pigments ………………………………………………………….142
8.4 Discussion ………………………………………………………………………144
8.5 Conclusions ……………………………………………………………………..148
Chapter 9 - Synthesis and Conclusions ……………………………………………….. 149
9.1 Major findings and relevance ………………………………………………….. 149
9.1.1 Fungal communities involved in spalted wood ………………………...... 149
9.1.2 The influence of environmental conditions on fungal pigmentation
activity………………………………………………………………………...…150
9.1.3 The influence of wood substrate composition ……………………………. 151
9.2 Significance of original contribution ……………………………………..…….151
9.2.1 Original scientific contribution ………………………………………..…..151
9.2.2 Contribution to original applications in developing and applying research..152
9.3 Future directions and concluding remarks …………………………………….. 154
References ……………………………………………………………………………..154
Appendix ……………………………………………………………………….……..187
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List of Tables
Table 2.1 Fungal species that produce pigmentation in wood…………………………. 10
Table 3.1 Top BLAST hits of fungi isolated from spalted wood samples.…………….. 31
Table 4.1. Material examined in this study and Genbank accession numbers of newly produced ITS sequences………………………………………………………..………..64
Table 4.2 Genbank sequences used in this study………………………………………...65
Table 4.3. Dot plot of mean percentage pigmentation versus growth for cultures of: A – C. aeruginosa UAMH 11657, B - C.aeruginascens UAMH 11655, and C - C aeruginascens UAMH 11656, after eight weeks of incubation…………………………………..…..….68
Table 5.1 Wood and fungal species selection……………………………………………73
Table 5.2 Estimated moisture content (%) values for each treatment of the wood species tested, based on the final moisture content of wood conditioned for eight weeks in sterile conditions……………………………………………………………………………….. 78
Table 5.3 Final moisture content in wood samples (five replicates per set) incubated with various fungal species …………………………………………………………..……… 78
Table 6.1. pH variation in beech and sugar maple samples after treatment with phosphate buffer at different pH-values.………………………………………………………….…94
Table 6.2 Fungal pigment formation in MEA at various pH values.. ……………….....100
Table 7.1 Substrate and fungal species selection for catechol and L-Dopa test …….... 106
Table 7.2 Summary of data for untransformed mass loss for tested fungi in sugar maple and beech. Data shown are the means of nine replicates .…………………………….. 115
Table 7.3 Wood and fungal species selection for tricyclazole test ..………………….. 118
Table 8.1 Substrate and fungal species selection for microscopy analysis. …….…..…129
Table 8.2 Summary of the microscopy methods and results ………………………..…145
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List of Figures
Figure 2.1 Spalted wood produced in sugar maple by Trametes versicolor (A), in poplar by Inonotus hispidus and Scytalidium cuboideum (B) and in sugar maple by Chlorociboria aeruginascens (C)…………………………………………………………8
Figure 2.2 Melanin precursors: A – catechol; B – dihydroxyphenylalanine (Dopa); C – y- glutaminil-3,4-dihydroxybenzene (GDHB); D – 1, 8 - dihydroxynaphthalene (DHN)....22
Figure 3.1 – Phylogenetic tree generated in UNITE by galaxieBLAST method for w1 isolate...... …………………………………………………………………………… 32
Figure 3.2 Phylogenetic tree generated in UNITE by galaxieBLAST method for w2 isolate..………………………………………………………………………………….. 33
Figure 3.3 Phylogenetic tree generated in UNITE by galaxieBLAST method for ITS sequence of w3 isolate. ………………………………………………………………… 34
Figure 3.4 Phylogenetic tree of the Bayesian analysis of w4, w8 and w9 ITS sequences. Bootstrap values of the parsimony analysis are given…………………………………. 34
Figure 3.5 Phylogenetic tree generated in UNITE by galaxieBLAST method for w5 isolate. ………………………………………………………………………………….. 35
Figure 3.6 Consensus tree of the Bayesian analysis for w7, w13, w15, w16 and w17 ITS sequences, highlighted within the Hypsizygus clade. Bootstrap values of the parsimony analysis are given……………………………………………………………………….. 37
Figure 3.7 Phylogenetic tree generated in UNITE by galaxieBLAST method for ITS sequence of w11 isolate. ………..……………………………………………………… 38
Figure 3.8 Phylogenetic tree of the Bayesian analysis of w12 ITS sequence. Bootstrap values are indicated for the parsimony analysis ……………………………………….. 38
Figure 3.9 Phylogenetic tree generated in UNITE by galaxieBLAST method for ITS sequence of w19 isolate. ……….. ……………………………………………………... 39
Figure 3.10 Phylogenetic tree generated in UNITE by galaxieBLAST method for ITS sequence of w18 isolate. ………... …………………………………………………….. 40
Figure 3.11 – Spalted wood sample investigated for fungal identification: (A) burl of Box elder; (B) burled Buckeye tree harvested from California-USA; (C) Sugar maple from Toronto area; (D) Birch twig from Algonquin Park – Ontario; (E), (F) Sugar maple samples harvested from Toronto area…………………………………………………... 41
Figure 3.12 Light microscope imaging of spalted wood ………………………….…... 42
Figure 4.1 Chlorociboria aeruginosa TRTC 167753 ...……………..…………………. 60
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Figure 4.2 Chlorociboria aeruginascens: TRTC167754 (teleomorph) and TRTC167755 (anamorph).……………………………………………………………………………... 61
Figure 4.3 Culture of Chlorociboria in 2% MEA (A-C) and 2% MEA with sugar maple saw dust (D-F): C. aeruginascens UAMH 11655 isolated from single ascospore – A, D; C. aeruginascens UAMH11656 isolated from single conidiospore – B, E; C. aeruginosa UAMH11657 isolated from single ascospore – C, F .………………………………….. 63
Figure 4.4 Growth assessment in various culture media recorded over a period of five weeks, of C. aeruginosa UAMH11657 (A), C. aeruginascens UAMH11655 (B), and C aeruginascens UAMH11656 (C)…………………………………………………...... ….63
Figure 4.5 Bayesian 50% majority-rule consensus tree of ITS sequences. ……………..67
Figure 5.1 Pigment formations in beech by T. versicolor and X. polymorpha...... 75
Figure 5.2 Initial moisture content in beech (A) and sugar maple (B) incubated in culture condition without fungal inoculation, measured over eight weeks period……………....77
Figure 5.3 Initial moisture content at various water level additions in beech (A) and sugar maple (B) incubated in sterile and unsterile condition without fungal inoculation, measured over eight weeks period .………………………………………………..…… 77
Ficure 5.4 Mass loss of beech (a) and sugar maple (b) wood samples incubated with various fungal species ………………………………………………………………….. 80
Figure 5.5 Pigment production in beech (b) and sugar maple (sm) by Trametes versicolor (average of three strains) at various moisture content values ..………………………….81
Figure 5.6 Pigment production in beech (b) and sugar maple (sm) by Xylaria polymorpha (average of three strains) at various moisture content values ..………………………….81
Figure 5.7 Pigment production in beech (b) and sugar maple (sm) by Inonotus hispidus at various moisture content values ……………………….…………………………...……82
Figure 5.8 Pigment production in beech (b) and sugar maple (sm) by Polyporus squamosus at various moisture content values ………………………………………….83
Figure 5.9 Pigment production in beech (b) and sugar maple (sm) by Fomes fomentarius at various moisture content values ………………….…………………………………...83
Figure 5.10 Pigment production in beech (b) and sugar maple (sm) by Polyporus brumalis at various moisture content values …………………….……………………....84
Figure 5.11 Pigment production in beech (b) and sugar maple (sm) by Scytallidium cuboideum at various moisture content values ………………………………………….84
Figure 6.1 pH value of MEA measured after settling of media treated with phosphate
xv buffer for pH adjustment…………………………………………………………..….….94
Figure 6.2 Final pH value in beech (b) and sugar maple (sm) sample treated with phosphate buffer, decayed by T. versicolor, strains: tv1 - Mad 697, tv2 - R105, tv3 - UAMH 11521. Error bars represent one standard deviation…………………………….95
Figure 6.3 Final pH value in beech (b) and sugar maple (sm) sample treated with phosphate buffer, decayed by X. polymorpha, strains: x1 – UAMH 11518, x2 – UAMH 11519, x3 – UAMH 11520. Error bars represent one standard deviation…………….…95
Figure 6.4 Mass loss (A, C) and moisture content (B, D) of decayed sugar maple (sm) and beech (b) samples by T. versicolor (tv) and X. polymorpha (xp). Error bars represent one standard deviation .…………………………………………………………………...... 97
Figure 6.5 Pigment formation in beech and sugar maple treated with phosphate buffer at various pH values, inoculated with T. versicolor (tv) and X. polymorpha (xp). ………..98
Figure 7.1 The effect of catechol on pigmentation (A) and growth (B) of Trametes versicolor culture in 1% agar. Data shown are the means of five replicates………….. 109
Figure 7.2 Trametes versicolor colonies in 1% agar: 1a,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm catechol, C-10ppm catechol, D- 100ppm catechol on agar……………………………………… 109
Figure 7.3 The effect of L- Dopa on pigmentation (A) and growth (B) of Trametes versicolor culture in 1% agar. Data shown are the means of five replicates. ………… 109
Figure 7.4 Trametes versicolor colonies in 1% agar: 1A,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm L- Dopa, c- 10ppm L-Dopa, d- 100ppm L-Dopa on agar………………………………………….. 110
Figure 7.5 The effect of catechol on pigmentation (A) and growth (B) of Xylaria polymorpha culture in 1% agar. Data shown are the means of five replicates …..….... 110
Figure 7.6 Xylaria polymorpha colonies in 1% agar: 1A,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm catechol, C-10ppm catechol, D- 100ppm catechol on agar ……………………………………... 111
Figure 7.7 The effect of L- Dopa on pigmentation (A) and growth (B) of Xylaria polymorpha culture in 1% agar. Data shown are the means of five replicates ..……… 111
Figure 7.8 Xylaria polymorpha colonies in 1% agar: 1A,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm L- Dopa, C-10ppm L- Dopa, D- 100ppm L- Dopa on agar…………………………………...... 111
Figure 7.9 The effect of catechol on pigmentation (A) and growth (B) of Inonotus hispidus culture in 1% agar. Data shown are the means of five replicates ……...……..112
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Figure 7.10 Inonotus hispidus colonies in 1% agar: 1A,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm catechol, C-10ppm catechol, D- 100ppm catechol on agar ……………………………………... 112
Figure 7.11 The effect of L- DOPA on pigmentation (A) and growth (B) of Inonotus hispidus culture in 1% agar. Data shown are the means of five replicates……………. 113
Figure 7.12 Inonotus hispidus colonies in 1% agar: 1A,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm L- Dopa, C-10ppm L- Dopa, D- 100ppm L- Dopa on agar …………………………………….. 113
Figure 7.13 Pigmentation and zone line formation by T. versicolor in wood substrate treated with catechol and L-Dopa …………………………………………...…………114
Figure 7.14 Pigmentation and zone line formation by X. polymorpha in wood substrate treated with catechol and L-Dopa …………………………………………...…………116
Figure 7.15 Pigmentation and zone line formation by Inonotus hispidus in wood substrate treated with catechol and L-Dopa ………………………………………….. …………117
Figure 8.1 Acer saccharum with zone lines formations by Oxyporus populinus …….. 134
Figure 8.2 Immunofluorescence of fungal melanin in cross section of parenchyma rays of Acer saccharum with melanized mycelium of Oxyporus populinus ..………………....135
Figure 8.3 Imaging of Acer saccharum ray parenchyma with melanized mycelium of Oxyporus populinus in cross section, obtained by confocal microscopy……………....136
Figure 8.4 Imaging by TEM and immuno-FL of Acer saccharum with melanin produced by T. versicolor ……………………………………………………………………..….137
Figure 8.5 Imaging by TEM and immuno-FL of Fagus grandifolia with melanin produced by T. versicolor ……………………………………………………………...138
Figure 8.6 Imaging of Acer saccharum and Fagus grandifolia produced by X. polymorpha …..………………………………………………………………………...140
Figure 8.7 Imaging of natural melanin produced by Fomes fomentarius in Betula alleghaniensis……………………………..……………………………………………141
Figure 8.8 Immunolabeling of melanin produced by I. hispidus in Acer saccharum. …141
Figure 8.9 TEM imaging of pigments produced by S. cuboideum in cross section of Acer saccharum, Fagus grandifolia and Populus sp...... ………………………….142
Figure 8.10 TEM imaging of tracheids in longitudinal section, with green pigment produced by C. aeruginascens (A) - in monoculture inoculated in Populus sp., and (B) – pretreated with T. versicolor in Acer saccharum………………………………………143
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List of Abbreviations ANOVA Analysis of Variance AWPA American Wood Protection Association BLAST Basic Local Alignment Search Tool BSA Bovine serum albumin CM Confocal microscopy CODIT Compartmentalization of decay in trees DNA Deoxyribonucleic acid Dopa dihydroxyphenylalanine D-Dopa D-3,4-dihydroxyphenylalanine DHN 1, 8 - dihydroxynaphthalene ELISA Enzyme-linked immunosorbent assay EM Electron microscopy FL Fluorescence microscopy HMDS Hexamethyldisiloxane FSP Fiber saturation point FTIR Fourier transformed infrared GDHB y-glutaminil-3,4-dihydroxybenzene GTR-CAT Generalized time-reversible Categorization GXM Glucuronoxylomannan ICN International Code of Nomenclature for Algae, Fungi, and Plants ITS Internal transcribed spacer L-Dopa L-dihydroxyphenylalanine LM Light microscopy MAb Melanin antibodies MC Moisture content MCMC Markov chain Monte Carlo MEA Malt extract agar NIR Near infrared NMR Nuclear magnetic resonance PBS Phosphate buffered saline PCR Polymerase chain reaction PDA Potato dextrose agar PLS Partial least squares
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RMSEP Root-Mean-Square Error of Prediction RNA Ribonucleic acid ROS Reactive oxygen species SEM Scanning electron microscopy SG Specific gravity TEM Transmission electron microscopy UV Ultraviolet WHC Water holding capacity
1 Chapter 1 Introduction
1.1 Motivation and Significance
The demand for natural wood material with certain aesthetic character has lately increased in the furniture industry and the uniqueness of spalted wood determines the high value and demand for this type of product (Donovan and Nichols 2003). The natural occurrence of spalted wood does not satisfy the demand on the market, due to the difficulty of finding sound spalted hardwood logs. The shortage of supply has resulted in investigations and research into reliable methods for commercial production.
Spalted wood is a specific form of decay that involves wood pigmentation due to fungal activity (Robinson et al. 2007), and fungal pigment production in wood substrate is one of the most important characteristics of spalted wood. The understanding and characterization of this phenomenon might lead to the development of commercial production of spalted wood, as a value added process. Moreover, any new information of fungal pigmentation biology and biochemistry contributes to the understanding of fungal behavior and reaction to various conditions and changes that might occur in the wood substrate.
There are a few known species that produce black melanin pigments in thin dense layers (“zone lines”) that delimitate the outer boundaries of each fungal colony in a given wood segment. As a result, the sectioned wood has a marbled appearance, with fine irregular demarcation lines, that form unique designs. Zone line formations were characterized in wood colonized by Xylaria polymorpha (Campbell 1933), Armillaria mellea (Campbell 1934) and Polyporus squamosus (Campbell and Munson 1936). Trametes versicolor, Bjerkandera adusta, Stereum hirsutum, Armillaria bulbosa, Phanetochaete velutina, Phlebia radiata, Piptoporus betulinus and Xylaria hypoxylon fungi were also studied in the context of demarcation line formation and for their inter- and intra- specific reactions (Rayner and Todd 1977, Rayner and Todd 1979, Boddy 2000, Coates and Rayner 1985a, Coates and Rayner 1985b, Lopez-Real 1975, Boddy and Rayner 1983, Rayner and Boddy 1988, Robinson et al. 2007). Spalted wood value is also enhanced by the presence of staining fungi that colour the wood substrate by producing blue, green, red, brown, purple and yellow pigments, primarily by colonizing the wood parenchyma cells (Seifert 1964).
2 The fungus Chlorociboria aeruginascens was identified in green stained veneer used in intarsia art works (Blanchette et al. 1992), while Scytallidium cuboideum is known for staining wood a red colour (Golinski et al. 1995, Robinson et al. 2011). The identification of pigmented fungi in wood was traditionally achieved by morphological observation of the fruiting bodies grown directly on wood and of mycelium formation of isolated fungi in culture. The subsequent research on pigmentation patterns and spalting formation was carried out with fungi already known for their spalting capacity. The method of DNA identification of fungi from spalted wood with ITS markers has the potential to determine fungal species previously not associated with spalting. From a safety perspective of the woodworkers that process spalted wood, it is also important to verify if any fungi with pathogenic effects are present in this type of wood.
The environmental conditions to which the host wood substrate is exposed during the incipient decay and through the entire fungal incubation period influence the decay and pigmentation patterns. Fungi are designed by evolution to occupy specific niches within the wood decomposition process (Boddy et al. 1989); therefore differences among fungi in the optimal conditions for development are anticipated. It is known that decomposition activity by fungi can alter the moisture content as well as the pH of the colonized wood substrate (Miller 1932, Lopez-Real and Swift 1975, Dix 1985, Chapela and Boddy 1988, Boddy et al. 1989, Heilmann- Clausen 2001, Pearce 1991, Humar et al. 2001, Schmidt 2006, Boddy and Heilmann-Clausen 2008). The delimitation zone lines also have the function to maintain the optimal moisture content required by the colonizer fungus. While fungi from Xylaria species have the tendency to maintain wood drier than ambient conditions (Boddy et al. 1989; Heilmann-Clausen 2001), Armillaria species retain higher moisture content than normal (Lopez-Real and Swift 1975; Chapela and Boddy 1988). It is known that the hydrogen ion concentration of the wood substrate could vary within the tree along and across the stem, and this factor influences the enzymatic activity of wood-degrading fungi during wood degradation (Pearce 1991; Schmidt 2006). While basidiomycete fungi prefer a slightly acidic environment, ascomycetes can tolerate a more alkaline substrate (Humar et al. 2001). All these aspects of pH variation are reflected in fungal diversity and distribution of species succession on wood.
The ability to manipulate fungal reactions through moisture content and pH changes in wood offers a chance to enhance the pigmentation intensity and patterns currently available with spalted woods. Research under laboratory conditions on the specific spalting fungi is limited,
3 and a more elaborate and consistent investigation is necessary to determine the optimal conditions for pigmentation for different wood and fungal species utilized in spalting.
Fungi produce melanins by different biosynthesis pathways (Wheeler 1983, Bell and Wheeler 1986, Fogarty and Tobin 1996, Butler and Day 1998, Henson et al. 1999, Jacobson 2000, Butler et al. 2001). However, the general understanding is that ascomycetes fungi produce melanin from 1,8 dihydroxynaphthalene (DHN), while basidiomycetes fungi usually produce catechol melanin from γ-glutaminyl-3,4 – dihydroxybenzene precursor, also known as GDHB melanin, and more rarely from L-dihydroxyphenylalanine (L-Dopa) (Turner 1971, Wheeler and Bell 1985). The fungal metabolism is affected by the nutrient availability and chemical composition of the wood substrate (Schmidt 2006). The black pigmentation, also known as melanin, is known to be of phenolic nature (Hamilton and Gomez 2002); therefore the production of those pigments might be directly influenced by the nature of the substrate and the available phenolic precursors synthesized within the substrate.
The ultrastructure of melanin formation within the fungal cells was investigated in many fungi of interest due to their plant pathogenic activity. The melanin appears to be confined to the fungal cell wall region, either externally or within the cell walls of various structures like sclerotial formations, aged hyphae, spores, and pigmented hyphae due to wounding or as result of light exposure and extreme environmental conditions (Campbell 1933, Campbell 1934, Campbell and Munson 1936, Ellis and Griffiths 1974, Wheeler et al. 1976, Bell et al. 1976, Hegnauer et al. 1985, Bell and Wheeler 1986, Wheeler and Bell 1988). Several methodologies for the localization of melanin synthesis at the cell level have been evaluated. A quantitative assay of cell wall melanin using the dye Azure A for DHN and Dopa melanin was developed (Nicolaus et al. 1964, Bull 1970, Butler and Lachance 1987). A copper sulfide-silver staining technique for fungal melanin detection in electron microscopy was also investigated by Butler et al. (2005), based on Dancher’s method (1981), and it proved to be more efficient in melanin labeling. However, innovative research on serological methods and phage display techniques led to the generation of a series of melanin antibodies that were used in electron microscopy as well as for labeling and visualization by fluorescence microscopy (Polacheck and Kwon-Chung 1988, Casadevall and Scharff 1991, Kammeyer et al.1992, Liu and Jimbow 1993, Cherniak and Sundstrom 1994, Nosanchuck and Casadevall 1997, Nosanchuck et al. 1998, Nosanchuck et al. 1999, Carzaniga et al. 2002). The main studied organisms were plant pathogens like
4 Gaeumannomyces graminis var. graminis (Sacc.) Arx & D.L. Olivier and human pathogens like Cryptoccocus neoformans (San Felice) Vuill. Research on fungal pigment deposition within the wood cells by various spalting fungi is limited, and a comparative study of in vitro and in vivo pigmentation morphology should contribute to the general understanding of decay patterns and could determine the morphological differentiation of various types of melanin.
1.2 Scope
This work develops an essential part of a larger study aimed at producing spalted wood utilizing a variety of identified fungal species, and using the manipulation of environmental conditions described previously. The scope of this study was to investigate the specific fungal biodiversity in natural occurring spalted wood, and to determine the influence of moisture content and pH of the wood substrate on fungal pigmentation. Also, the experimental and microscopic investigation of melanin biosynthesis induced by synthetic precursors, is used to explain the complexity and dynamics of pigment production based on available phenolic resources, specific to each fungal species. Since the study of the various biosynthetic pathways is ongoing, the information elucidated in this research can give insights related to the melanin formation process. The motivation of this work in particular, is derived from the lack of general analysis of the native melanin produced by spalting fungi, characterized in wood substrate without chemical extraction.
1.3 Research Hypotheses
Ø Molecular analysis can be used to provide significant information concerning the array of pigment producing fungi involved in spalting. Ø The moisture content and pH of the wood substrate influence fungal pigment formation. Ø White rot fungi can individually synthesize multiple types of melanins. Ø The morphology of pigmentation is affected by fungal species, wood substrate and availability of phenolic precursors.
5 1.4 Objectives
The overall objectives of this study are to explore fungal diversity in spalting, to investigate the environmental factors that influence the spalting process, and to characterize fungal pigmentation of various fungi in Acer saccharum and Fagus grandifolia by microscopic analysis. The specific objectives of this study are:
1. Identification of fungal species in natural spalted wood using molecular analysis. 2. Determine effects of pH and moisture content values induced in Acer saccharum and Fagus grandifolia wood substrates, on spalting ability of different fungi. 3. Investigate the pigmentation reaction of spalting fungi to catechol and L-dopa precursors introduced in wood and agar, as well as effect of a known melanin inhibitor, tricyclazole. 4. Investigation of the fungal pigment deposition patterns within the wood cells by various spalting fungi, and the immuno-labelling of melanin by available primary monoclonal melanin antibody.
1.5 Thesis overview
This dissertation uses a combination of experimental data and lab analytical techniques, in conjunction with molecular and multivariate analyses to meet the research objectives. Consequently, in addition to the intoductory, literature review and concluding chapters, this dissertation has been organized into six individual chapters, each addressing unique research questions.
A short introduction and overview of the spalting formation and spalting production as value added process, research scope, hypotheses and objectives are presented in chapter one. Chapter two provides the background and literature review of morphology and conditions of various fungal pigment formation, and function and chemical characterization of several fungal pigments in wood substrate. In chapter three, fungal diversity in natural spalted wood is investigated by DNA isolation and analysis, and chapter four characterizes two Chlorociboria species, of importance for their green-staining pigment. This chapter includes molecular analysis and morphological descriptions that make the connection of these fungi with their
6 anamorphs. Chapter five provides information on the influence of moisture content of the substrate on fungal pigmentation, and chapter six investigates the influence of wood substrate pH value on pigment stimulation. Chapter seven studies the effect of treatment of the substrate with melanin precursors on fungal melanin formation. Chapter eight describes the morphology of pigment formation in wood using microscopic analysis.
Chapter nine, the last chapter, presents the research findings, highlights the research limitations and provides recommendations for the application of environmental conditions and substrate modification for spalting production.
7
Chapter 2
Spalting Fungi: the Study Organisms and the Pigment Formation – a Literature Review
Wood decomposer fungi are an important component in the carbon cycle in nature. The biological decomposition of wood is a complex phenomenon, highly orchestrated by a more or less specialized group of fungi that populate wood in successional stages, engaging in various strategies of deterioration and degradation. Wood’s moderate to high resistance to decay is dictated mainly by the amounts and chemical compositions of the extractives, lignin and hemicelluloses of various wood species, influencing greatly the diversity of successional fungal species that can access and transform wood substrate (Rayner 1977, Rayner 1977a, Rayner 1978, Rayner and Todd 1979, Rayner and Todd 1982, Rayner and Boddy 1988a, Boddy 2000). Primary colonizers usually feed on readily available sugars in wood cells, and deteriorate wood by discoloration and altering its permeability without affecting its structure. They easily invade wood cells in search of new food sources. Secondary colonizers, white, brown or soft rot fungi, deteriorate wood by decomposition, degrading lignin, cellulose and hemicellulose. They convert those wood cell wall complex biopolymers into much shorter molecules by enzymatic digestion. Following degradation, wood chemical composition is progressively changed along with the loss of wood strength (Rayner and Boddy 1988a, Boddy et al. 1989).
A fairly common phenomenon in nature, spalting refers to any coloration and random patterns of black demarcation lines (Robinson et al. 2007), in wood (Figure 1.1). It is produced by an ensemble of wood inhabiting fungi that come into action at once or in succession when the wood substrate is more favorable to their decomposing ability. One interesting aspect of spalting degradation pattern is the lack of wood cells decomposition within the area of black pigment accumulation (Bell and Wheeler 1986).
8
1 cm 1 cm
A B
10 mm
C
Figure 2.1 Spalted wood produced in sugar maple by Trametes versicolor (A), in poplar by Inonotus hispidus and Scytalidium cuboideum (B) and in sugar maple by Chlorociboria aeruginascens (C).
2.1 Fungi responsible of spalted wood
There are several hardwood species known to develop spalting, especially sugar maple (Acer saccharum Marshall) and trembling aspen (Populus tremuloides Michx.) as well as other wood species like oak, beech, and walnut. Based on physical observation, a number of fungal species are associated with spalting. Two main categories of fungi are involved in spalting formation: white rot and staining fungi (Robinson et al. 2007). White rot fungi primarily decompose lignin, consequently whitening the wood fiber and creating three-dimensional black demarcation lines; staining fungi invade wood cells with pigmented hyphae, without extensive damage of wood integrity (Schwarze et al.
9 2000). The black demarcation lines are comprised from sclerotial mycelium with small black granules deposited along the outside layer of the fungal cell (Bell and Wheeler 1986). The black pigment, melanin, is a complex chemically undetermined biopolymer resistant to degradation, that provides structural strength and protection in a challenging environment (Henson et al. 1999). There are more species of white rot fungi than there are of brown rot fungi, and in the northern hemisphere many of the brown rot fungi are specific to conifers. The simultaneous demand for environmental resources is less critical compared with white rot fungi. This aspect, along with their ability to decompose only cellulose and hemicellulose, and their incapacity to utilize phenolic compounds from lignin and wood extractives might explain that few brown rot fungi produce zonation.
There are approximately 1,600 wood decay species (Bennet et al. 2002), and identifying decay fungi from wood is difficult and laborious. Rayner and Todd (1979) studied fungal populations in dead stumps by direct methods of sampling and analysis. However, determination at the microscopic level requires substantial expertise and is sometimes difficult to carry out in early stages of decay (Schmidt and Moreth 1999, Kim et al. 2005). Zone line formations were mostly studied as decay patterns, and in connection with pathogenicity. Several fungal species are known to produce zone lines in wood substrate, such as Xylaria polymorpha (Pers.) Grev (Campbell 1933), Armillaria mellea (Vahl) P. Kumm. (Campbell 1934), Polyporus squamosus (Huds.) Fr (Campbell and Munson 1936), Xylaria hypoxylon (L.) Grev (Rayner and Boddy 1988 a,b), Trametes versicolor (L.) Lloyd and Bjerkandera adusta (Willd.) P. Karst (Rayner and Todd 1979). Wood staining fungi like Chlorociboria (Blanchette et al. 1992) and Scytalidium cuboideum (Sacc. & Ellis) Sigler & Kang (Golinski et al. 1995) were previously identified in spalted wood. Hedgeock (1906) reported Penicillium aureum Hedgc. and two other species of Penicillium as capable of staining pine wood an orange red color, and Fusarium roseum Kalchbr. as causing pink, red and violet coloration in pine lumber. Scheffer and Lindgren (1932) identified Fusarium moniliforme J. Sheld. in pink stains in southern pine.
Numerous researchers studied pigment-production in wood, and the most important fungi associated with zone line formation or staining in wood are summarized in Table 2.1.
10
Table 2.1 Fungal species that produce pigmentation in wood. Spalting fungi Types of Fungi Reference Pigment color
Heterobasidion annosum (Fr.) Bref. Basidiomycetes, White Ikediugwu et al. Black demarcation lines rot, (1970) Armillaria mellea (Vahl) P. Kumm. Basidiomyc etes , White rot Campbell (1934) Black demarcation lines
Polyporus squamosus (Huds.) Fr Basidiomyc etes , White rot Campbell and Black demarcation lines Munson (1936) Xylaria hypoxylon (L.) Grev Ascomyc etes , White rot Rayner and Boddy Black demarcation lines (1988a,b) Trametes versicolor (L.) Lloyd Basidiomyc etes , White rot Rayner and Todd Black demarcation lines (1979) Bjerkandera adusta (Willd.) P. Karst Basidiomycetes, White rot Rayner and Todd Black demarcation lines (1979) Fomitopsis cajanderi (P. Karst.) Basidiomycetes, Brown Adams and Roth Black demarcation lines Kotl. & Pouzar rot (1967) Stereum hirsutum (Willd.) Pers. Basidiomycetes, White rot Coates and Rayner Black demarcation lines (1985b) Chondrostereum purpureum (Pers.) Basidiomycetes, White rot Coates and Rayner Black demarcation lines Pouzar (1985b) Hypholoma fasciculare (Huds.) P. Basidiomycetes, White rot Coates and Rayner Black demarcation lines Kumm (1985b) Ophiostoma piceae (Münch) Syd. & Ascomycetes, staining Harrington (1987) Blue stain P. Syd. fungus Chlorociboria sp. Ascomycetes, staining Blanchette et al. Green fungus (1992) Scytalidium cuboideum (Sacc. & Ascomycetes, staining Golinski et al. Pink, blue Ellis) Sigler & Kang fungus (1995)
Penicillium aureum Hedgc Ascomycetes, staining Hedgeock (1906) Orange red in pine fungus wood Fusarium roseum Kalchbr. Ascomycetes, staining Hedgeock (1906) Pink, red and violet in fungus pine Fusarium moniliforme J. Sheld. Ascomycetes, staining Scheffer and Pink in southern pine. fungus Lindgren (1932) Scytalidium lignicola Pesante Ascomycetes, staining Wang and Zabel Blue stain fungus (1999)
2.2 Fungal reaction to substrate conditions
There are many species of wood inhabiting fungi, which have various effects on wood, based on the process of the transformation of the wood matter into nutrients. Among those, decay fungi modify the wood character and composition, until complete decomposition. They are normally classified into three categories, white rot fungi, that remove all wood components, brown rot fungi, that mainly degrade cellulose and hemicelluloses, and soft rot fungi, that attack wood much slower and don’t usually have a dramatic effect on wood structure.
11 Studies on populations and communities of wood-decomposing basidiomycetes provide us with information on genetic recombination, patterns of reproduction, distribution and propagation, as well as evolutionary and ecological information.
2.2.1 Nutrients
One of the most important factors in establishing the nature of fungal interactions is the competition for nutritional resources that ensure survival and perpetuation of fungal species. The general definition of competition given by Keddy (1989) implies negative effects by one organism on another by consuming or controlling access to a limited resource. Harper (1961) observes that, from an ecological perspective, the word “interference” is preferably to “competition”, since the main concern deals with “those hardships which are caused by the proximity of neighbors”. The succession and relationships of fungal communities are diverse and very complex, and many causalities and effects are yet to be discovered. To retain the perspective of their interconnectedness, we are more comfortable referring to them as “interactions”. Groups of fungi are specialized in utilizing various sources of carbon, from the simple to the most complicated molecule available in wood. Their nutritional preferences influence the dynamics of fungal succession, and their colony distribution at a given time, and may trigger antagonistic interactions as a reflex of protecting food source to ensure colony survival for as long as possible (Boddy et al.1989). In the case of spalting fungi, the antagonistic reactions have as effect the production of black demarcation lines and other staining pigments.
In forest ecosystems, total decomposition and redistribution of nutrients rely mostly on fungal succession, in a process involving complex interactions among fungal populations that have genetically co-evolved to efficiently decompose organic matter. Many fungi evolved high specificity in organic matter decomposition, while other more aggressive fungi have a high degree of adaptability. The population and community analysis of a decayed wood segment at a given moment, gives information on the critical reciprocal action or influence that determines the formation and dynamics of the decay pattern. According to Wicklow (1992), fungal antagonism is the mechanism that permits early colonizers to gain exclusive access to a given section of wood substrate by inhibiting or obstructing the invasion of other fungi. The inhibition model applies to early colonizers, by impeding the establishment of other fungi or by suppressing the growth of those already established by release of toxic metabolites –antibiotics
12 or mycotoxins. Melanin producing fungi react to the presence of those toxic metabolites by producing the dense layer of black pigment that protects the colony.
Later successional species can occupy the substrate only after the dominating species release the resource (Connel and Slayter 1977). Antagonistic interactions among late colonizers are the subject of many research studies. Horn (1977) established that the succession of saprophytic colonizers takes place based on a competitive hierarchy based on increasingly aggressive antagonistic reactions and resistance to the toxic exudates released by earlier colonizers.
The released antibiotics have immediate action, resulting in lysis of the competitive species’ hyphae or inhibition of spore germination (Stanley and English 1965). The end of antibiotic activity is induced by enzymatic degradation (Brian 1957). The production of fungal antibiotics and growth are in inverse proportion, although they are not mutually exclusive processes. The release of mycotoxins can be a reaction to an imminent attack by another fungus, but it cannot take place unless the substrate provides enough levels of energy (Bu’Lock et al. 1974). However, at any stage of wood degradation, there is also the possibility of exploitation competition and symbiotic coexistence, when two or more fungi can feed on the same resource without securing space (McNaughton and Wolf 1973).
In natural conditions, based on the local variety in fungal species of a given space, a wood source is subject to multiple fungal infections that develop in nutritional based successional colonies all at once. Rayner and Todd (1979) and Henningson (1967) found that decay fungi that are predominant in later stages of decay are most competitive in agar tests. In vitro experiments on agar by Lundborg (1988) indicates that cellulolysis is either promoted or inhibited in contact zones, depending on the fungal species involved in antagonism. The interaction of white rot Heterobasidion araucariae P.K. Buchanan with the brown rot Fomitopsis pinicola (Sw.) P. Karst indicated an increase of cellulolysis and hydrogen peroxidase production. However, pairing between white rot fungi indicated phenoloxidase activities, which promoted melanin formation, accompanied by inhibition of cellulose degradation even though relatively high amounts of cellulases were detected.
Several studies indicate that the same species of decay fungi isolated from differed sources develop a barrier zone or zone lines at the contact between their colonies when paired on agar substrate. Adams and Roth (1967) proved that the intraspecific antagonism between isolates of
13 Fomitopsis cajanderi (P. Karst.) Kotl. & Pouzar is a result of pairing dikaryons, and the zone line formation is an indicator of genetic differences between dikaryons. Barrett and Uscuplic (1971) confirmed this behavior for Phaeolus schweinitzii (Fr.) Pat. In natural condition, zone lines were mentioned by Brodie (1935) for Exidiopsis calcea (Pers.) K. Wells, and later on, Adams and Roth (1969) inoculated wood samples and incubated them in plastic bags for five months and demonstrated that the antagonistic reaction between dicaryotic isolates of F. cajanderi had the same activity in nature. At the end of the incubation period, spatial patterns of genetically different dicaryons of the same fungal species were separated by demarcation lines. Normal growth of a singular colony in wood, accompanied by demarcation lines was reported by Campbell (1933,1934), Campbell and Manson (1936) and Lopez-Real (1975), as characteristic behavior for Armillaria mellea (Vahl) P. Kumm., Stereum hirsutum (Willd.) Pers,, Polyporus squamosus (Huds.) Fr. and Xylaria polymorpha (Pers.) Grev. Rayner (1976) reported narrow zone lines on rather undecayed wood between colonies of white rot basidiomycetes Trametes versicolor (L.) Lloyd, Bjerkandera adusta (Willd.) P. Karst. and Stereum hirsutum (Willd.) Pers, as a result of antagonistic reactions that were reproduced also in agar cultures. Investigation of the population structure of T. versicolor on a Betula stump, showed that dikariotic isolates from distinct colonies were consistently antagonistic in culture, while those from the same colony easily fused. Monokaryons from fruiting bodies corresponding to distinct colonies were able to interbreed, forming dicaryons when paired in culture. The same results were obtained from population analysis of Bjerkandera adusta (Willd.) P. Karst, Piptoporus betulinus (Bull.) P. Karst, Hypholoma acutum (Sacc.) E. Horak, Stereum hirsutum (Willd.) Pers, and Phlebia radiata Fr. (Rayner and Todd 1977, Todd and Rayner 1978, Rayner and Todd 1979). According to Rayner (1970), zone lines pigmentation intensity produced by fungi varies with the intensity of the interaction, from sepia by T. versicolor, brown-yellow by B. adusta, brick-red by P. radiata and, shades of yellow by S. hirsutum.
Intraspecific and interspecific antagonism, resulting in hard barriers of sclerotic mycelium that delimit longitudinal decay columns, is often associated with undecayed wood substrate (Rayner and Todd 1979). The zone lines formation should not be confused with the reaction of the living tree to fungal infection. “Compartmentalization of decay in trees” (CODIT) host reactions are usually characterized by thicker light to brown colored zone lines (Rayner and Todd 1979). The zone reaction depends on the host resistance and is specific to tree species. At microscopic level
14 it can be observed that the zone lines are formed by tyloses and wound gum deposition that filled wood cells to limit fungal access to expand into wood.
2.2.2 Moisture content
Reactions to intolerable environmental conditions like desiccation can influence fungal succession, distribution and therefore zone line formation (Lopez-Real and Swift 1975, 1977). The moisture content (MC) available in wood substrate varies greatly with seasonal periods and within the host perimeter. It is established that optimal fungal growth is achieved at 35–50 % MC on a dry weight basis, with a minimum required of 20–30 % necessary for fungal development; the values vary for different fungal species and inhabited wood substrates (Cartwright and Findlay 1958; Rayner and Todd 1979). If desiccation occurs after the colonies are well established, the survival rate varies from one fungal species to another (Theden 1961). Moisture content of the wood substrate above 40 % modifies the quantity of oxygen available; therefore high MC also inhibits fungal development (Cartwright and Findlay 1958; Boddy 1983a,b). Analysis of species distribution on standing spruce poles in soil by Käärik (1974) indicated three distinctive groups that colonize wood based on moisture content preference: fungi that occupy wood below ground at 37-47% MC, fungi that prefer the aerial portion of the wood poles of 12-15% MC, and fungi that had no restriction in terms of water availability.
By producing zonation barriers of compact melanin pigments, Xylaria species seem to maintain wood drier than ambient conditions (Boddy et al. 1989; Heilmann-Clausen 2001), while Armillaria species occupy wood wetter than ambient (Lopez-Real and Swift 1975, Chapela and Boddy 1988). Fungi are capable to regulate the moisture content of the substrate based on the environmental conditions to which the substrate is exposed. This characteristic is a vital property and ensures the water availability necessary for survival, by regulating the level of water availability either by wood decomposition to improve moisture content, or by extraction of water surplus into aerial mycelium (Miller 1932). Differences in wood moisture content are also observed in neighboring fungal colonies, and this strategy might be involved in antagonistic reactions, to limit the access of fungal competitors (Boddy and Heilmann-Clausen 2008). Fungal melanin production and deposition barrier demarcation zones could also be triggered by limited water availability (Hubert 1924). The anticipation of desiccation of Xylaria polymorpha (Pers.) Grev., Bjerkandera adusta (Willd.) P. Karst., Phellinus igniarius (L.) Quél. and Porodaedalea pini (Brot.) Murrill determined the development of an effective strategy to ensure the survival of
15 the colonies. Fungi produce the melanin-type pigment blocking the water exchange within the wood substrate. In the case of Armillaria mellea and S. hirsutum, the variation of moisture content does not influence the production of melanized mycelium, which occurs at any moisture content when growth is possible, except for S. hirsutum at low humidity (< 35% MC), when pigmentation is inhibited at early stages of growth (Lopez-Real and Swift 1975, 1977).
2.2.3 Temperature
The optimal temperature for fungal growth varies with fungal species and evidently with their geographical distribution. Their tolerance to extreme condition is a mark of genetic evolution that permits fungal species to survive and occupy certain environmental niches (Boddy 1983a). In general the interval of optimal fungal growth for most fungi is between 0 and 45 0C (Zabel and Morrell 1992), as many fungal species developed tolerance to extreme temperature. The minimum and maximum temperature tolerance characteristic to each fungus can determine fungal colonies distribution within the wood substrate, among other factors like wood substrate composition, moisture content, and air flow. However the time temperature combination of lethal exposure to prolonged extreme temperature varies from one species to another (Chidester 1939).
For wood inhabiting fungi, the variation of temperature within the wood substrate will be attenuated by the wood’s insulating property, ensuring a more stabile and favorable condition for fungal growth and implicit decay activity, and most wood decay fungi have optimal growth between 15 to 40 0C.
The temperature directly influences the fungal metabolic activities regulated by enzymatic reactions. At minimum temperature levels, enzymatic activity is limited, and increases with the increase of temperature. After passing the optimal range, the enzymes reactions are maintained until the maximum tolerated temperature is reached, after which the metabolic reactions are completely ceased (Zabel and Morrell 1992). At this stage, the survival of the fungus is conditioned by the duration of the extreme temperature exposure. The fungal enzymatic activity in wood is also influenced by the moisture content of the substrate. However, given the higher thermal capacity of water, the wood moisture content will condition and further influence the temperature of the wood substrate (Rayner and Boddy 1988).
16 2.2.4 pH
The variation of hydrogen ion concentration within the wood substrate influences the enzymatic activity of wood inhabiting fungi, with direct implication in metabolism and wood degradation. (Pearce 1991, Schmidt 2006). While basidiomycete fungi prefer a slightly acidic environment, the ascomycetes tolerate a more alkaline substrate. However, fungi are capable of modifying the pH of the substrate to more suitable values, through metabolic regulation activity (Humar et al. 2001). The aspects of pH variation may play an important role in fungal biodiversity distribution and species succession on wood debris.
The pigmented substances produced by staining fungi are secondary metabolites that act as physical and chemical barriers in the wood substrate. S. cuboideum produces extracellular pigmentation, which plays an important role in controlling the growth of other fungi. The toxic property of the diffusible pigment is maintained even after autoclaving the wood, and is active at about pH 5, however, it seems to be altered at pH 7 (Brian, 1957). S. cuboideum can colonize and red stain many deciduous and coniferous woods without causing significant degradation of the wood structure (Schmidt and Diez, 1985), and can change from red to blue when dissolved in solvents with higher polarity, and in basic solution (Golinski et al 1995).
Those substrate conditions are undoubtedly important factors that influence fungal pigment production. However, there is no research known to investigate pigmentation in spalted wood in different wood species and composition, and at various moisture content levels and pH values.
2.3 The morphology of pigment formation by spalting fungi in wood substrate
Microscopic studies are often involved in pigment characterization of its formation and ultrastructure, in analysis of the effect of growing condition, gene modification and inhibitory effects. Within the variety of fungal pigmentation, zone lines formations were of tremendous interest to many researchers, due to their specific structure assembly and properties. While those hard and resistant black zone are formed at the peripheries of the colonies into coherent structures to delimitate the occupied wood substrate, the staining pigments produced mostly by ascomycetes fungi are fluidly dispersed within the wood substrate. Those mycotoxic pigments
17 are usually water soluble for rapid dispersion, and often preceed fungal colonization, to ensure the wood invasion before mycelium propagation.
2.3.1 Melanin in zone lines formation
Fungal melanins have been described in many developmental stages, such as sclerotial formation, aging of hyphae, sporogenesis, and hyphal pigmentation due to wounding or as a result of light exposure and extreme environmental conditions.
It was demonstrated that the peripheral demarcation lines formed by fungi like Xylaria polymorpha, Armillaria mellea and Polyporus squamosus consist of compacted black bladder- like hyphae found in the lumina of wood cells that are practically unaltered. The matrix of bladder hyphae forms an amorphous black matter (Campbell 1933, Campbell 1934, Campbell and Munson 1936). Detailed research on fungal melanin produced by Verticillium dahliae Kleb., Humicola grisea Traaen, Epicoccum nigrum Link, Colletotrichum coccodes (Wallr.) S. Hughes and Amorphotheca resinae Parbery revealed that fungal melanin appears to be confined to the fungus cell wall region, either externally or within the cell walls of various structures. In some cases accumulation of electron-opaque granules of melanin externally secreted by hyphae, formed vesicle-like conglomerates with rough surfaces and 30 to 200 nm in size (Ellis and Griffiths 1974).
Comparative microscopic studies of the of melanin formation ultrastructure of V. dahliae in a wild type isolate and albino mutant concluded that scytalone is a natural precursor of melanin, (Wheeler et al. 1976, Bell et al. 1976a,b). Granular melanin formed in the sclerotial cell walls of the wild type, was similar with the melanin formed by the albino mutant when treated with scytalone, and differed from the melanin of the same albino mutant treated with catechol, DOPA, and other phenols.
Melanin biofilm that protects the spores of Agaricus bisporus (J.E. Lange) Imbach were studied by electron microscopy (Hegnauer et al.1985). The results indicated the presence of two types of melanin structure: partly amorphous and partly granular plate-like particles 50-100 nm in diameter, and electron-dense round particles of 30 to 200 nm. However, the evidence of this study remained inconclusive, due to harsh chemical treatments applied for melanin extraction.
Wheeler and Bell (1988) describe wall-bound and extracellular melanins formed by many fungi
18 regardless of the nature of the phenolic precursors used for melanin biosynthesis. A third type, the less common cytoplasmic melanins, was identified in Aspergillus niger Tiegh. The evidence that the electron-dense materials (appearing dark in electron micrographs) in fungal cell walls were melanin pigments was reported by Bell and Wheeler (1986). The inhibition of melanin synthesis by tricyclazole, which affected the electron-dense granule formation in several fungal species tested, indicates 1,8 dihydroxynaphthalene (DHN) melanin production, mostly characteristic to ascomycetes fungi (Bell and Wheeler 1986).
How melanization affects extracellular protein production is not known, but it was hypothesized that constitutive melanization limits secretion of lytic enzymes necessary for host tissue degradation and subsequent pathogenesis (Henson et al. 1999). The cell wall structure could be affected by the amount of melanin deposited in the wall, and may occlude protein secretion. The extracellular proteins could be either bound by melanized cell walls or inhibited by melanin or melanin precursors. It was argued that limitation of protein secretion as a response to environmental stress could conserve cellular resources and influence transcription of gene encoding of extracellular protein (Henson et al. 1999).
The high density of melanin biopolymer, as a result of the parallel stacking of adjacent planar units, gives a high contrast of melanin in electron microscopy of even unstained biological samples (Hegnauer et al. 1985). However, the labeling of melanin particles within the fungal cell was mandatory to determine the mechanism of melanin formation. A quantitative assay of cell wall melanin was developed by Butler and Lachance (1987), based on the high affinity of dye Azure A for DHN and L-Dopa melanin, as previously described by Bull (1970) and Nicolaus et al. (1964). The localization of melanin at the cell level was possible with the development of a sulfide-silver staining technique, based on the binding of CuS to the melanin granules, which further precipitates silver particle visible in electron microscopy (Danscher 1981, Caesar-Tonthat et al. 1995, Butler et al. 2005). Specific silver precipitations indicate that melanin formed in hyphal septa, and did not occur near hyphal tips nor in tricyclazole treated hyphae or their septa. Immuno-labelling techniques using monoclonal antibodies were developed more recently, and led to essential studies of melanin formation in fungal pathogens like Cryptoccocus neoformans (San Felice) Vuill, Gaeumannomyces graminis (Sacc.)Arx et D.L. Olivier and Alternaria alternata (Fr.) Keissl. (Kammeyer et al. 1992, Liu and Jimbow 1993, Casadevall and Scharff 1991, Nosanchuck and Casadevall 1997, Nosanchuck et al. 1998,
19 Nosanchuck et al. 1999, Rosas et al. 2000, Casadeval et al. 2000, Carzaniga et al. 2002, Rosas et al. 2002, Dadakova and Casadevall 2005, Dadakova et al. 2007).
However, studies on the morphology of melanin formation by decay fungi within the wood tissue are scarce, and those techniques might be difficult to apply due to the similar properties of fungal melanin and lignin biopolymers from wood.
2.3.2 Staining pigments
Fungal pigments formations by staining fungi were studied mostly from fungal cultures grown on various agar media in petri dishes. The challenges of the research on pigmentation in wood substrate are determined by the solubility of most of the pigments produced by staining fungi in water, acetone and alcohol, which might cause impediments in preparing samples for imaging with electron microscopy in such a manner that the pigmentation would be preserved in its natural state.
Red stain pigments
The pigments produced by Scytalidium cuboideum diffuse into wood or agar substrate to precede hyphae invasion. The fungus grows rapidly in culture, producing numerous clusters of spores, at first ivory – yellow in color, to become pink and later, dark spots of tyrian blue pigmentation appear in the aged cultures (Chidester 1940). Due to this characteristic, they were often classified as blue stain fungi. Research of Golinski et al. (1995) described the change in color of the red pigment to blue when dissolved in alkaline solutions.
Scytalidium cuboideum pigments have antifungal properties and are known to produce antagonistic reactions. They diffuse easily in wood tissue, ensuring the integrity of fungal colonies, food resources, and space sequestration (Brian 1957). The pigmentation becomes visible within three weeks after inoculation in pine wood samples and is more concentrated around resin accumulations, and at higher moisture contents of the substrate. In vitro experiments indicate that S. cuboideum has little decay potential in either soft- or hardwood species (Schmidt and Diez 1985, Robinson et al. 2011c). However, the fungus was described as strongly cellulolytic (Sigler and Carmichael 1976, Sigler and Carmichael 1983). Scytalidium cuboideum was categorized as a soft rot type 1, due to the presence of diffuse cavities in the S2 layer of the pine wood cell wall, while the S3 layer remains intact (Anagnost et al. 1994).
20 Blue stain pigments
The blue stain of wood is associated mostly with fungi like Ophiostoma sp., Ceratocystis coerulescens (Münch) B.K. Bakshi, Scytalidium sp. and Alternaria alternata. It was demonstrated by Zink and Fengel (1988) that the nature of pigment in dark hyphae of those fungi belongs to a group of melanins, and is associated with carbohydrates and protinaceous constituents. The pigment is produced in the fungal cell wall in the form of deposition of small globules during aging, in three distinctive layers (Zink and Fengel 1989, 1990). Wood cell wall penetration by blue stain fungi is possible due to mechanical power achieved by appresorial formation or by intensive localized enzymatic attack (Wilcox 1973)
Grey stain pigments
Research by Encinas and Daniel (1996, 1997) showed that Lasiodiplodia theobromae (Pat.) Griffon & Maubl. stains wood as a result of pigmented hyphae in wood cells, as well as from the extracellular secretion of pigments deposited along the luminal cell wall of colonized wood cells. The colonization pathway in hardwood species begins with parenchyma cells and vessels, while coniferous species are colonized through longitudinal and radial resin canals and ray parenchyma cells. The fungus is specialized in degradation of the secondary cell wall, beginning in early wood.
Green stain pigments
The green staining fungus Chlorociboria aeruginascens Kanouse ex C.S. Ramamurthi, Korf & L.R. Batra has no apparent degradation capacity of cellulose and lignin (Robinson and Laks 2010b). The green appearance of wood is generated by the presence of pigmented mycelium within the wood cells, and is more accentuated in advanced stages of colonization, when the pigment seem to be diffused into wood fibers. The patterns of wood colonization by Chlorociboria were studied by Blanchette et al. (1992), and the light and electron microscopy analysis show deposits of dark-green and yellowish pigments, located within the wood cells, more predominantly in ray parenchyma cells. TEM imaging reveals that hyphae displayed accumulation of pigmented substances around the outer cell walls. The pigmented hyphae were present in unaltered wood cells, as well as in cells with erosion of the secondary wall, typical of white rot degradation that could have been the result of decay activity by previous wood inhabiting white rot fungi.
21 2.4 Chemical characteristics of fungal pigments
2.4.1 Melanins –properties and biosynthesis
The most common black or dark brown pigment produced in nature, is a chemically undetermined and complex biopolymer termed “melanins”. Besides fungi, many organisms such as plants, animals, insects and other microorganisms also produce melanins (Henson et al. 1999).
Melanins are known to enhance virulence in pathogenicity and protection for fungal hyphae (Wheeler and Bell 1985, Butler and Day 1998a, Henson et al.1999, Pearce 1991, Campbell 1934). It provides structural strength and resistance to most forms of degradation (Piattelli et al. 1965, Selvakumar et al. 2008), and also increases fungal resistance to toxic levels of metals, due to its high capacity to bind with metal ions (Fogarty and Tobin 1996). Butler et al. (2001) noted that melanins are able to absorb sound and electromagnetic energy, all wavelengths of light, from visible light to gamma rays, X-rays, ultraviolet and infrared light, transferring the energy at the cell level, ensuring fungal protection to most wavelengths of light. Although these pigments are not essential for growth and development, they enhance the longevity and competitive abilities of species in a hostile environment. Melanized fungi can survive harsh cold climates and contamination from nuclear reactors (Rosa et al. 2010; Zhdanova et al. 2000). Moreover, melanized fungi can even survive treatment with heat and detergents (Zalar et al. 2011). Melanin production is critical to host invasion in plant pathogens like Colletotrichum kahawae J.M. Waller & Bridge and Magnaporthe grisea (T.T. Hebert) M.E. Barr that depend on appressoria formation, to penetrate plant tissue (Chen et al. 2004, Howard and Valent 1996). Pihet et al. (2009) showed that melanin can increase the negative charge and hydrophobicity of the fungal cells.
Chemically, melanin polymers may have similar structures to lignin, but contain, in addition, unpaired electrons as stable free radicals, which can react further with metal ions or make a chemical bond with some proteins that may also be part of the melanin structure itself (Enochs et al. 1993).
The structure of melanin polymer is not entirely known, but it was established that it is black in color, insoluble in water and organic solvents, soluble in hot alkali solution, resistant to
22 degradation by hot or cold concentrated acids and degradation by oxidizing agents (Bell and Wheeler 1986, Butler and Day 1998b). Melanins can also be enzymatically degraded by ligninase enzymes (peroxidases and laccases) produced by white rot fungi (Butler and Day 1998b, Ratto et al. 2001)
Fungi are known to synthesize different types of melanin (Rizner and Wheeler 2003). Most ascomycetes synthesize 1,8-dihydroxynaphtalene (DHN) (Henson et al. 1999, Butler et al. 2001) and basidiomycetes are known to synthesize in their cell walls melanin derived from y- glutaminil-3,4-dihydroxybenzene (GDHB) or catechol, as the immediate phenolic precursor of the melanin polymer. Some mushrooms were thought to synthesize Dopa melanin (similar with human melanin) but ultimately that hypothesis was reconsidered and the melanin considered to be in fact GDHB in nature (Bell and Wheeler 1986) (Figure 2.2). The heterogeneous melanins result from the action of secreted fungal tyrosinase, laccase, or peroxidase on plant phenols that oxidize the phenolic metabolites into new polymers. The phenolic precursors derived from lignin and wood extractives that are oxidized into black fungal pigments in wood include catechol, Dopa, dopamine, tannic acid, gallic acid and quinol (Haars and Huttermann 1980, Martin and Haider 1980, Martin et al. 1972, Martin et al. 1979). Other similar brown pigments have been referred to as fungal humic acids or humic melanins (Haider and Martin 1967, Schnitzer and Neyround 1975). These pigments are derived from various phenols, amino acids, proteins, carbohydrates, and lipids (Martin and Haider 1980, Schnitzer and Neyround 1975). Synthesis of this type of melanin requires secretion of phenols into the medium, and the pigmentation is usually triggered by an increase in pH into the alkaline range, which allows autoxidation of phenols and interference of oxidation products with other phenols and compounds such as proteins in the culture media.
A B C D Figure 2.2 Melanin precursors: A – catechol; B – dihydroxyphenylalanine (Dopa); C – y-glutaminil-3,4-dihydroxybenzene (GDHB) (Weijn et al. 2013).; D – 1, 8 - dihydroxynaphthalene (DHN) (Tsai et al. 2001);
23 Progress on elucidation of melanin structure has been made with the use of solid state Nuclear Magnetic Resonance (NMR) spectroscopy. Research on purified “melanin ghosts” of Cryptococcus neoformans (San Felice) Vuill. showed that melanin is an integrated component of the fungal cell walls, which is covalently cross-linked with polysaccharide components especially those containing mannose (Zhong et al. 2008). It was inferred that the granular appearance of melanin deposits could be explained by the presence of chitin as a “scaffold” for melanin in fungal cells walls. Chitin polymer subunits joined in antiparallel chains by hydrogen bonding produce strong microfibrils, which might cross-link to other cell wall polysaccharides and proteins to form up to 40% of the fungal cell wall (Banks et al. 2005, Walton et al. 2005, Baker et al. 2007).
It was also demonstrated that C. neoformans is able to use a wide array of substrates to produce melanin, maximizing its ability to survive. Several substrate precursors were identified as catecholamines as well as D-3,4-dihydroxyphenylalanine (D-DOPA) (Eisenman et al. 2007), plant derived substances including flavonoids (Fowler et al. 2011), caffeic acid (Vidotto et al. 2004) and bacterially-derived homogentisic acid (Frases et al. 2007). Although the fungus produces many type of melanins, their properties might differ (Garcia-Rivera et al. 2005).
Melanin inhibitors were used to identify the type of melanin biosynthesis in fungal culture in vitro. Compounds like tricyclazole, pyroquilone, pthalide, and chlobenthiazone inhibit the enzymatic reduction of hydroxynaphtalene compounds to scytalone and vermelone precursors of DHN melanin, but not of Dopa melanin, which is inhibited by tropolone, kojic acid and, 2- mercaptobenzimidazole and diethyldithiocarbamate (Elliot 1995, Henson et al. 1999).
The black color of melanin pigments is a result of their property to absorb light energy at all visible wavelengths. The percentage of absorption of black melanin pigments is generally greatest in the UV region and decreases progressively as the wavelength is increased to that of the far-red region. Often this decrease in absorbance is nearly linear with increasing wavelength (Ellis and Griffiths 1974).
In a study on synthetic eumelanin, an important conclusion was that the electrostatic interaction of charged groups on the surface of melanin polymer plays an important role in organization of the granule; this mechanism of counter ion condensation on a melanin granule surface is a very effective modifier of melanin polymerization degree (Matuszak and Wasiliewska-Radwanska
24 2006). It was determined that at basic pH, the synthetic DOPA melanin structure is softened and polymer breaks and dissolves into small particles; but when pH decreases towards acidic pH, the polymer reorganizes to dense and larger particles (Felix et al. 1978).
Some melanins of microscopic soil fungi, such as Epicoccum nigrum Link, Stachybotrys chartarum (Ehrenb.) S. Hughes, Hendersonula toruloidea Nattrass and others resembled humic acids, with respect to certain chemical properties and the IR-spectra also indicated similarities in several functional groups. The studied model polymers from specific mixtures of phenols together with peptones and prepared either by oxidation with phenolase or by autoxidation, closely resembled the fungal melanins. This represents strong evidence for a similarity in the structural units and their linkages in both the melanins and the model polymers, and indicates that phenols and amino-acid compounds are constituent elements of the fungal pigments (Filip et al. 1974).
2.4.2 Staining pigments
Red stain
Some red staining fungi from the Scytalidium genus, often classified as blue stain fungi, are known to produce antagonistic reaction, and more important to retain antifungal properties. It was observed by Klingström and Johansson (1973) that in culture media, the peripheral hyphae of the rival wood decay fungi were disintegrated by a yellow-orange pigmentation produced extracellularly by Scytalidium hyphae, and diffused in front of the advancing mycelium. The lytic enzymes can digest glucan and chitin that forms the cell walls of wood decay fungi. The diffusible toxic factor seems to be altered at pH 7, restored when readjusted to pH 5, and remained active even after the collapse of fungi by autoclaving.
Green stain
The blue-green pigment produced by Chlorociboria fungi has been studied since the 19th century. Greville (1827) suggested that chips of wood are turned green by activity of the fungus, and Gümbel (1858) later named the pigment isoxylin acid. Rommier (1868) extracted a green pigment with alkali solvents, which he named xylindein, and Lieberman (1874) pursued his own investigation, and extracted a crystalline xylindein with a low nitrogen content, insoluble in most solvents, except in concentrated aqueous solution of sulfuric acid and phenol. The
25 complete structure of xylindein - a dimeric naphthoquinone, was independently deduced by Blackburn et al. (1965) and by Edwards and Kale (1965) based on derivative studies. The absolute configuration xylindein was established by Saikawa et al. (2000) by X-ray crystallographic analysis of the natural product extracted from fruiting bodies of Chlorociboria aeruginosa (Oeder) Seaver ex C.S. Ramamurthi, Korf & L.R. Batra, Chlorociboria aeruginascens (Nyl.) Kanouse ex C.S. Ramamurthi, Korf & L.R. Batra and Chlorociboria omnivirens (Berk.) J.R. Dixon. Studies on the synthesis of xylindein carried out more recently by Donner et al. (2012), reported the synthesis of pyranonaphthoquinone corresponding to one half of the xylindein framework, and investigated methods for self-coupling of dihydroxynaphthoquinones to give extended quinones.
2.5 Biotechnological applications of fungal pigment production
From the industrial perspective, fungal pigments were also studied as biological material with various applications in food, textile and pharmaceutical industry and environmental applications such as biological control and soil and water decontamination.
The antibiotic effect, demonstrated in both culture media and in wood substrate, led to numerous studies of Scytalidium fungi as biological agents against other wood decay fungi (Ricard 1966, Bruce and King 1983, Morris et al. 1983).
Other pigments produced by Monascus species are in the carotenoid group, ranging in color from yellow to orange and red (Duran et al. 2002, 2009). Some functional metabolite compounds are known to have antibacterial and antioxidant activity and to inhibit mutagenesis (Juzlova et al. 1996), while others are of great interest as dyes to the food industry (DeCarvalho et al. 2005).
Chlorociboria species produce a green pigment, xylindein. The bright green wood has been used in intarsia works on furniture and wood panels since the 15th century (Blanchette et al. 1992). Although the pigment has been thoroughly studied (Gümbel 1858, Rommier 1868, Blackburn et al. 1962, Saikawa et al. 2000), there have been few known properties and functions assigned to it. Rommier (1868) studied its dyeing properties on silk and wool, while
26 craftsmen from Hanover, Germany and Tunbridge Wells, England, produced green stained wood for their production of intarsia panels and furniture since the 19th century, by allowing mycelium to grow in wood (Hedgeock 1906, Hartig 1882, Berkely 1860). Ramsbottom (1963) also mentions artificial inoculation of wood for spalted wood productions, and even applied for a patent. More recently, Robinson and Laks (2010b) studied the use of C. aeruginascens for spalted wood production. A compound derived from the green pigment produced by Chlorociboria, and assessed as an anti-osteoporosis, antithrombosis, anti-inflammatory, immunosuppressive agent, as well as a diuretic, was the subject of another patent application (Futoshi et al.1996). Other patent applications targeted the ability of xylindein to inhibit plant germination without any hazardous effects on cultivated crops (Shibata et al. 2007), and as an animal cell growth retardant and antioxidant (Shinnosuke et al. 2004). The potential of this pigment is tangible for both craftsmanship and industrial applications, but there has been no significant application to this day, partially because of the lack of readily obtainable natural or synthetic xylindein.
The unique properties of melanin determine the importance and potential of this biopolymer for environmental and medical applications. The ability of melanized fungi to survive and uptake high levels of toxic heavy metals like uranium, iron and copper, and other hazardous chemicals makes it a valuable candidate for biological remediation and soil decontamination (Purvis et al. 2004, Dighton et al. 2008, Turick et al. 2008, Turick et al. 2011, Harms et al. 2011). The study of melanin and other fungal pigment decomposition led to research on dye decontamination of water waste (Heinfling et al. 1998, Rodriguez et al. 1999), while the extraction of specific fungal pigments are of particular interest for the natural food colorant industry, as ecologically friendly pigments (Duran et al. 2002).
The assemblies of biodegradable semiconducting fungal melanin in thin films are studied for their potential application in neural and cardiac tissue engineering, and other applications in material sciences, due to their unique conductive and electrical properties (Bettinger et al. 2009, Abbas et al. 2009). However, the inhibition of melanin formation is another aspect largely studied for their importance in human and plant pathogenicity. Melanin's binding capacity enhances the resistance to treatment of fungal infection (Nosanchuk and Casadevall 2006), and even blocking the drug action (Van de Sande et al. 2007). Melanin producing fungi like Magnaporthe grisea and Sclerotinia sclerotiorum are important fungal pathogens of
27 economically important crops. The inhibition of melanin assembly also reduces considerably their pathogenic activity (Howard and Valent 1996, Butler et al. 2009, Henson et al. 1999).
The stimulation of fungal pigmentation in wood substrate can be utilized as a value added process of wood products in spalting production, and the research presented in this thesis is focused almost entirely on this subject. Previous research by Robinson et al. (2011abc, 2012abc, 2013), determined the wood preference of spalting fungi, studied the red and green pigment stimulation in underutilized American beech (Fagus grandifolia) and other wood species for increase of commercial value and investigated methods for commercial spalting production in logs of various wood species. The efficacy of several commercial wood coatings to prevent fungal pigment degradation by UV light was also investigated. Research by Yang and Gignac (2011) identified 15 fungal species suitable for spalting applications, in sugar maple, yellow birch and white birch. The fungal species have potential for coloring the wood in red, brown, purple, green, grey and black colors.
2.6 Conclusions
Fungal pigmentation has a protective role and ensures fungal survival. Regardless of the research difficulties reflected by the complexity of pigment structure and formation, considerable progress has been made in understanding their biosynthesis, function, degradation, and cell wall assembly. Given the elaborate process of pigment production, and the tremendous potential of the applicability of both pigment stimulation and inhibition, the study of melanins and other pigments offer many opportunities in biological, medical, environmental and industrial research.
28
Chapter 3 Identification of Spalting Fungi from Wood Using ITS Markers
3.1 Introduction
There are several hardwood species known to develop spalting, especially sugar maple (Acer saccharum Marshall) and trembling aspen (Populus tremuloides Michx.). Based on physical and morphological observations, a number of fungal species are associated with spalting, but their diversity and identities have never been investigated by molecular analysis. The spalting might be influenced by antagonistic reaction between the fungal species inhabiting the wood substrate, although, it was established that even the presence of one fungus in wood substrate could generate zone lines (Campbell and Munson 1936, Rayner and Todd 1982, Robinson et al. 2007).
An important characteristic of spalted wood is discoloration as a result of wood colonization and degradation accompanied by demarcation lines produced by fungi, constituted from granules of black melanin deposited within the wood fibers in response to stress (Rayner and Todd 1979). Staining fungi, however, produce pigments with antibiotic properties that often diffuse in wood substrate (Margalith 1992).
Identification of wood decay fungi is difficult and laborious. Previous research by Rayner and Todd (1979) studied fungal populations in dead stumps by direct methods of sampling and analysis. However, determination at the microscopic level requires substantial expertise and is sometimes impossible to carry out in early stages of decay (Schmidt and Moreth 1999, Kim et al. 2005). Molecular methods have an enlarged receptiveness and selectivity, making them a better approach to identify decay fungi. Many taxonomic analyses have focused on ribosomal DNA, employing PCR primer sets to amplify the fungal specific ITS1F plant-exclusive primer, and the general fungal primer ITS4 (White et al. 1990, Gardes and Bruns 1996). Identification of DNA sequences using basic local alignment search tool (BLAST) with similar sequences existing in the GenBank is limited by the existence of many misidentified sequences, and by the lack of full sequence and species coverage in the database. To overcome those limitations, phylogenetic analyses using the maximum-likelihood or parsimony analysis are more reliable in
29 revealing the correct relationships between similar sequences (Nilsson et al. 2004).
In this research, sequence data from the ITS rDNA barcode marker was used to identify fungi responsible for natural spalting of common wood species in Canada/North America using the method described by Schoch et al. (2012). For visualization of pigment formation within the wood cells, the studied samples were also examined by light microscopy (LM).
3.2 Materials and methods 3.2.1. Wood samples
Three wood samples were selected from spalted sugar maple (Acer saccharum Marshall), one sample from box elder (Acer negundo L.) and one sample from birch (Betula L.) harvested from Ontario, Canada; one sample of a burled buckeye tree (Aesculus glabra Willd.) was collected from California. The samples were chosen based on the spalting prevalence in the respective wood species, and based on their high density of zone lines formation. Test pieces were air dried and kept at 21 o C and 40% relative humidity for five months prior to DNA isolation. The specimens were provided by a commercial supplier of spalted wood.
3.2.2. Molecular analysis
Wood fibers were isolated from heavily pigmented zones or from distinct fungal colonies delimited by zone lines. DNA was extracted using E.Z.N.A. Soil DNA Kit from Omega Bio- Tek. A total of 19 isolates taken from six wood samples were investigated. The use of fungal specific primers allowed selective amplification of fungal DNA in preference to plant or other DNA contaminants. The primers ITS1F and ITS4 have specificity for both ascomycetes and basidiomycetes fungi and were previously used to specifically amplify fungi from heterogeneous samples (Manter and Vivanco 2007). Amplification using PCR was carried out in 25 µL reactions containing 10 ng genomic DNA, 10 mM Tris-HCl, 50 mM KCl, 2.5 mM
MgCl2, 0.1% gelatin, 0.8 mg/mL bovine serum albumin, 0.2 mM dNTP, 0.2 µM of each primer and 1U of Platinum® Taq DNA Polymerase (Life Technologies – Invitrogen). The thermal conditions were: 94°C for 2 minutes followed by five cycles at: 94°C for 30 sec., 60°C for 30 sec., 72°C for 1 minute; 30 cycles at: 94°C for 30 sec., 55°C for 30 sec., 72°C for 1 minute and a final cycle at 72°C for 5 min. The PCR products were visualized on 1% agarose gel stained
30 with ethidium bromide. The PCR amplicons were further purified using QIAquick PCR purification kit (QIAGEN). Seven purified PCR amplicons were cloned using QIAGEN PCR cloning kit and the DNA obtained from transformed cells was subsequently amplified with ITS1F and ITS4 primers following the same protocol described before. All purified PCR amplicons were sequenced at both strands using the BigDye Terminator v3.1 Cycle Sequencing Kit (ABI). The sequencing protocol followed closely the ABI protocol. Sequence data analyses were performed on BioEdit Sequence Alignment Editor and Basic Local Alignment Search Tool (BLAST) in GenBank nucleotide collection database. In the case of Fusarium sp., the BLAST of ITS sequences were also carried in the more specific Fusarium-ID and Fusarium MLST (multilocus sequence typing) online databases (Geiser et al. 2004, O'Donnell et al. 2012).
Phylogenetic analysis were first carried within the UNITE database for molecular identification of fungi (Abarenkov et al. 2010) by galaxieBLAST method. For parsimony analysis, the best significantly matching alignments are selected for each input ITS sequence, and the script outputs the corresponding phylogenetic tree; outgroups were decided dynamically as the sequence of the largest distance to the query sequence (Nilsson et al. 2004). The unresolved phylogenetic trees by galaxieBLAST method were further carried out using a Maximum Likelihood (ML) and Bayesian analyses in RAxML v7.0.4 (Stamatakis 2006) with 1000 fast bootstraps reps in RAxML (Stamatakis 2006, Stamatakis et al. 2008) using the GTR-CAT model; representative ITS sequences similar with our sequences from GenBank, were prior aligned in MUSCLE v.3.6 (Edgar 2004) and optimized by eye in SE-AL (Rambaut 2002).
3.2.3. Microscopy
Wood samples with demarcation lines or stained by fungal mycelium were sectioned with a sliding microtome to obtain thick sections of minimum 1 µm. The specimens were stained with Toluidine B (zone lines samples), Lacto Fuchsin or kept unstained (samples with staining fungi) and examined with LEICA DMI300 Invertfluor LM.
3.3 Results
From the six wood samples studied that exhibited demarcation lines, discoloration and/or pigmented areas, three wood staining species of fungi and eight species that produce melanin deposits in black zone lines were identified. The accession numbers for all ITS sequences
31 determined in this study, along with the results of BLAST analysis of obtained sequences compared with similar sequences from GenBank are summarized in Table 3.1.
Table 3.1: Top BLAST hits of fungi isolated from spalted wood samples.
Sample no. Provenance Isolate GenBank Seq. length BLAST Similarity no. no. (*complete ITS) 1. Acer Toronto, w1 JX239628 * 480 Fusarium sp. 475/480 negundo ON, Canada AF178402 98.9% w2 JX239622 * 596 Polyporus squamosus 596/596 FR686579 100% 2. Aesculus CA, USA w3 JX239629 * 484 Botryosphaeria sp. 494/494 glabra GQ471813 100% 3. Acer Toronto, w4 JX239621 * 521 Oxyporus populinus 520/521 saccharum ON, Canada EF011121 99.8 % w5 JX239630 * 464 Atractium crassum 451/464 EU860056 97.1% w6 JX239632 * 465 Unknown 442/465 ascomycota 95% GU055712 w7 JX239623 * 598 Hypsizygus 597/598 marmoreus 99.8 % FJ609279 4.Acer Toronto, w8 JX239635 * 532 Oxyporus populinus 531/532 saccharum ON, Canada EF011121 99.8 % w9 JX239620 * 532 Oxyporus populinus 531/532 EF011121 99.8 % w10 - * 598 Oxyporus populinus 589/598 (bad EF011121 98.4 % sequence) w11 JX239631 368 Hypocrea rufa 100% partial ITS1 JQ272442 w12 JX239627 395 Kretzschmaria sp. 392/395 partial ITS1 HQ846573 99.2 % w13 JX239633 * 598 Hypsizygus 597/598 marmoreus 99.8 % FJ609279 w19 JX239624 * 502 Lecythophora sp. 493/502 HQ660445 98.2% 5. Acer Toronto, w14 - 589 Similar to Oxyporus 492/589 saccharum ON, Canada (bad populinus 83.5% sequence) w15 JX239636 359 Hypsizygus 358/359 partial ITS1 marmoreus 99.7% FJ609280 w16 JX239626 * 598 Hypsizygus 598/598 marmoreus 100 % FJ609268 w17 JX239634 * 598 Hypsizygus 597/598 marmoreus 99.8 % FJ609279 6. Betula spp. Algonquin w18 JX239625 * 473 Chlorociboria 472/473 Park, aeruginosa 99.7% Canada AY755360
32 3.3.1 Phylogenetic analysis
Fusarium sp.
The 100 top BLAST hits for isolate w1 were all sequences of Fusarium species or its Nectria teleomorph (>99% similarity), including sequences labeled Fusarium sp. AF178402 of unknown origin with 99% similarities, and AY633561 isolated from soil. Other sequences such as Nectria haematococca (Berk. & Broome) (anamorph Fusarium solani (Mart.) Sacc.) under GenBank number JX270181, Fusarium ambrosium (Gadd & Loos) Agnihothr. & Nirenberg (teleomorph genus in Gibberella) AF178397, and Fusarium oxysporum (Schlechtend.:Fr.)(teleomorph genus in Gibberella) EU888922 isolated from Chinese red pine, as well as other environmental isolates (JX891686 to JX891784) were similar to w1, with maximum identity of 98.9 %. The most significant phylogenetic analysis of ITS sequence alignments in UNITE, Fusarium MLST database, as well as phylogenetic analysis in RAxML of the w1, identified the isolate only at the genus level (Figure 3.1) due to the complexity of Fusarium species (Nilsson et al. 2004). One isolate of the basidiomycetes Corticium salmonicolor EU435009 was wrongly placed by UNITE within the Fusarium clade, and probably the reported sequence was obtained from a contaminated sample.
Figure 3.1 Phylogenetic tree generated in UNITE by galaxieBLAST method for w1 isolate. The numbers on the branches indicate the number of times the partition of the species into the two sets which are separated by that branch occurred among the trees, out of three trees.
33 Polyporus squamosus
The sequence obtained from w2 isolate had significant alignment with 16 sequences of Polyporus squamosus (Huds.) Fr. (FR686579, AF516572-3, AF516586-9, AF516590, AY218421, JX843717, etc) (99 to 100% identity); the similarity drops to 92% or less with other Polyporaceae (Datronia mollis AF516557, Mycobonia flava AY513570, Ganoderma sp. AJ536663, etc.). Consistent with these findings, phylogenetic analysis in UNITE with dynamically selected outgroups, place isolate w2 in the monophyletic clade established for Polyporus squamosus (Figure 3.2).
Figure 3.2 Phylogenetic tree generated in UNITE by galaxieBLAST method for w2 isolate. The numbers on the branches indicate the number of times the partition of the species into the two sets which are separated by that branch occurred among the trees, out of three trees.
Botryosphaeria sp.
A GenBank search conducted with the ITS sequence of w3 isolate revealed a 100% similarity with four sequences of Botryosphaeria sp. isolated from a twig of Terminalia sambesiaca Engl. & Diels tree from Tanzania (GQ471813, GQ471811, GQ471810, GQ471809), and several Botryosphaeria rhodina (Berk, & Curt.) v. Arx (anamorph Lasiodiplodia theobromae (Pat.) Griffon & Maubl) isolates: FJ 904842 isolated from Melia azedarach (L.), a deciduous tree in the mahogany family Meliaceae from Kenya, FJ 904841 isolated from Grevillea robusta (A.Cunn. ex R.Br.), commonly known as the silky oak from Kenya, FJ904839 isolated from Eucalyptus grandis (W.Hill ex Maiden) also from Kenya, EF 622073 isolated from corn and EF622068 isolated from an avocado tree (Persea americana Mill.) from Tanzania. Phylogenetic
34 analyses carried by galaxieBLAST method in UNITE database placed w3 isolate in the Botryosphaeria genus, and it could not be distinctly identified at the species level (Figure 3.3).
Figure 3.3 Phylogenetic tree generated in UNITE by galaxieBLAST method for ITS sequence of w3 isolate. The numbers on the branches indicate the number of times the partition of the species into the two sets which are separated by that branch occurred among the trees, out of three trees.
Oxyporus populinus
Top BLAST hits for isolates w4, w8, and w9 are four sequences of Oxyporus populinus (EF011121, FN907910) and O. subpopulinus (FJ644281, FJ644282) (99%), and nine sequences of O. corticola (KC176667-76, JQ673195) (96-93%). Two of the latter were used as outgroups for parsimony analysis. The ITS alignment consisted of 9 taxa and was 571 bp in length. Branch-and-bound search retrieved three equally-parsimonious trees (tree length 68, CI = 0.985, RI = 0.981), which firmly confirms the identity of w4, w9, and w8 isolates as O. populinus (Figure 3.4). Isolates w10 and w14 were also similar with O. populinus.
Figure 3.4 Phylogenetic tree of the Bayesian analysis of w4, w8 and w9 ITS sequences. Bootstrap values of the parsimony analysis are given.
35 Nectriaceae (cf. Fusarium sp.)
ITS sequence of w5 isolate matched with 97.1 % similarity Atractium crassum (Wollenw.) Seifert and Gräfenhan (synonym Fusarium merismoides var. crassum Wollenw., teleomorph in Pezizomycotina), GenBank number EU860056. The proximate highest counterpart with 92% similarity corresponded to Neonectia galligena (Bres.) Rossman & Samuels AM419080 isolated from apple tree (Malus domestica Borkh.), and three Neonectria dittisima (Tul. & C. Tul.) Samuels & Rossman strains DQ178169 isolated from an apple tree from the Netherlands, strains DQ178167 and DQ178168 isolated also from apple trees from Slovenia. All these sequences belong to the Hypocreales in the Nectriaceae family.
ITS sequences have been shown to be difficult for distinguishing between adjacent Fusarium species. For w5, parsimony analysis from UNITE, Fusarium MLST database, as well as phylogenetic analysis in RAxML could not convincingly assign a species name to them. However, w5 can be identified as a species of Nectriaceae (Figure 3.5).
Figure 3.5 Phylogenetic tree generated in UNITE by galaxieBLAST method for w5 isolate. The numbers on the branches indicate the number of times the partition of the species into the two sets which are separated by that branch occurred among the trees, out of three trees.
Hypsizygus marmoreus
Top BLAST hits for isolates w7, w13, w15, w16, and w17 were several sequences labeled Hypsizygus marmoreus (Peck) H.E. Bigelow (GenBank number FJ609279, JX046007 to JX046038) (99%); Hypsizygus ulmarius (Bull.:Fr) Redhead (EF421105) and one strain labeled Pleurotus ostreatus (Jacq. ex Fr.) P.Kumm. (HM770897) (96-99%). Next highest (91-92%) hits
36 include sequences from Clitocybe subditopoda Peck (EU852800), Clitocybe dealbata (Sowerby) P. Kumm. (JN021000), Clitocybe vibecina (Fr.) Quél. (JF907821), Clitocybe candicans (Pers.) P. Kumm. (DQ202268), Lyophyllum sykosporum Hongo & Clémençon (AF357051), Lyophyllum semitale (Fr.) Kühner ex Kalamees (HM572552), Lyophyllum gangraenosum (Fr.) Gulden (JF908335), Tephrocybe gibberosa (Jul. Schäff.) P.D. Orton (AF357042), and Tephrocybe anthracophila (Lasch) P.D. Orton AF357054. All these sequences belong to the Lyophylleae (Moncalvo et al. 2002; Hofstetter et al. 2002).
ITS datamatrix included 93 taxa and the sequence alignment was 635 bp in length. The best ML tree was 297 length (CI = 0.758, RI = 0.757). The phylogenetic analysis could not resolve the Hypsizygus marmoreus clade, one isolate of H. ulmarius and one of P. ostreatus were attributed within the polyphyletic clade. However, it can be concluded that isolates w7, w13, w15, w16 and w17 are identified as Hypsizygus marmoreus (Figure 3.6).
Hypocrea rufa – anamorph Trichoderma viride
The first 55 hits are 100% similar with several Hypocrea rufa Pers., and its anamorph Trichoderma viride with the first match JQ272443 isolated from Rhododendron sp. L. roots from USA, JX847765 isolated from commercial soil in UK, and JX501302 isolated from decayed wood in France. Phylogenetic analysis in UNITE also unambiguously identify w11 as Hypocrea rufa (Figure 3.7).
Kretzschmaria sp.
A GenBank search in BLAST conducted with ITS sequence of w12 isolate revealed 99.2 % similarity with Kretzschmaria sp. HQ846573 isolated from Panicum virgatum L. (switchgrass), and 97.8 % similarity with Kretzschmaria deusta (Hoffm.) P.M.D. Martin, GenBank number KC477237 isolated from the hybrid Tilia petiolaris L. from Germany, and six isolates of Kretzschmaria deusta EU162067, EF155500, EF155501, AJ 390437, AJ 390435 and its synonym Ustulina deusta (Hoffm.) Lind, GenBank number AF 201718. Other higher matches were with members of the Xylariaceae such as Xylaria and Stilbohypoxylon. The sequence alignment of 28 taxa with 635 bp in length placed w12 within the Kretzschmaria clade in the phylogenetic tree, and did not identify the fungus at the species level (tree length 346, CI=0.772, RI=0.833) (Figure 3.8).
37
Figure 3.6 Consensus tree of the Bayesian analysis for w7, w13, w15, w16 and w17 ITS sequences, highlighted within the Hypsizygus clade. Bootstrap values of the parsimony analysis are given.
38
Figure 3.7 Phylogenetic tree generated in UNITE by galaxieBLAST method for ITS sequence of w11 isolate. The numbers on the branches indicate the number of times the partition of the species into the two sets which are separated by that branch occurred among the trees, out of three trees.
Figure 3.8 Phylogenetic tree of the Bayesian analysis of w12 ITS sequence. Bootstrap values are indicated for the parsimony analysis.
39 Lecythophora sp.
Top BLAST hits for isolate w19 matched with 98.2 % similarity with Lecythophora sp. GenBank number HQ660445 isolated from soil, 97.4% similarity with AY219880 isolated from Gaultheria shallon Pursh shrub, 96.6 % similarity with KC007198 isolated from Populus trichocarpa Torr. & A. Gray roots in USA, followed by several unknown ascomycetes likely in the Coniochaetales within the Sordariomycetes class (98.2%), and Phialophora spp. (AY618677) with 96.7 % similarity. Phylogenetic analysis could not clearly assign a species name to w19. However, the isolate can be identified as a species of Lecythophora sp. within the Sordariomycetes (Figure 3.7)
Figure 3.9 Phylogenetic tree generated in UNITE by galaxieBLAST method for ITS sequence of w19 isolate. The numbers on the branches indicate the number of times the partition of the species into the two sets which are separated by that branch occurred among the trees, out of three trees.
Chlorociboria aeruginosa
Top BLAST hits for isolate w18 matched with 99.7 % similarity three Chlorociboria aeruginosa AY755360 from USA, followed by Z81426 from Norway, DQ491501 from USA, and HQ604856 from CAN (BC), with 98.3 % similarity. Phylogenetic analysis carried in UNITE by galaxieBLAST method for ITS sequence also identified unambiguously w18 as C. aeruginosa (Figure 3.8)
40
Figure 3.10 Phylogenetic tree generated in UNITE by galaxieBLAST method for ITS sequence of w18 isolate. The numbers on the branches indicate the number of times the partition of the species into the two sets which are separated by that branch occurred among the trees, out of three trees.
3.3.2 Fungal species distribution in wood samples
Box elder sample (Acer negundo)
This sample displayed pink-pigmented zones (w1) and fine black demarcation lines produced by w2 isolate (Figure 3.11 A). The ITS sequence retrieved from w1 was similar to several Fusarium sp. but could not be identified at the species level. Other sequences such as F. solani, F. ambrosium, and F. oxysporum were similar to w1, with maximum identity higher than 98.9 %.
Pink-pigmented wood tissues in cross section were examined by LM in order to visualize mycelium formation within the wood cells. Pigmented hyphae appeared within the wood cell walls, and were rarely present in the lumina of the wood fibers (Figure 3.12 A), while the pigment diffused within the wood tissue (Figure 3.12 B).
The w2 isolate from the immediate vicinity of the black demarcation (Figure 3.11 A) line was 100% similar to Polyporus squamosus isolated in Germany from Norway maple, and to other five collections identified as P. squamosus from Indiana-USA, Germany and Denmark.
41
10mm 10mm w2
w3 w1
A B
10mm 10mm
w4 w5 w6
w18 C w7 D 10mm w9 w13 w8 w12
w10 w19 E w11 10mm
w16 w17
w15
F
Figure 3.11 – Spalted wood sample investigated for fungal identification: (A) burl of Box elder; (B) burled Buckeye tree harvested from California-USA; (C) Sugar maple from Toronto area; (D) Birch twig from Algonquin Park – Ontario; (E), (F) Sugar maple samples harvested from Toronto area.
42
5µm 5µm
A B
10µm 50µm
C D
50µm 50µm 50µm
E F G
Figure 3.12 Light microscope imaging of spalted wood: (A) (stained with Lacto Fuchsin) and (B) (unstained) cross section of burled boxelder with pink stain, fungal colony identified as Fusarium sp.; (C) cross section of burled buckeye colonized by grey mycelium of Botryosphaeria rhodina within the wood tracheids (arrow); (D) (stained with Toluidine B) cross section in sugar maple with melanized mycelium in vessels and tracheids partially filled (red arrow) or completely filled (double red arrow); (E) Betula sp. colonized by Chlorociboria aeruginosa: radial/tangential section showing ray parenchyma cells colonized by green mycelium-arrow; (F) tangential section of advance colonization by C. aeruginosa, concentrated initially within the ray parenchyma cells (arrow), and gradually dispersed in all neighbouring wood cells; (G) cross section in birch, showing ray parenchyma cells preferentially colonized by green mycelium (arrow).
43
Buckeye sample (Aesculus glabra)
The buckeye sample from California, USA, exhibited deep grey stained zones, (w3, Figure 3.11 B). ITS sequence from w3 matched 100% with several Botryosphaeria rhodina isolated from various wood substrates from Tanzania and Kenya. Microscopic investigations of the burled buckeye sample show no apparent degradation of the wood cells and the fine grey-pigmented hyphae were uniformly distributed within the wood fibers (Figure 3.12 C, arrow).
Sugar maple sample (Acer saccharum)
Three randomly selected sugar maple samples were investigated in this study. The first sample was heavily spalted by black demarcation lines. Four sectors were collected (w4-w7; Figure 3.11 C). The ITS sequence from w4 matched 99.8 % to Oxyporus populinus with one C/T nucleotide substitution of EF011121, an isolate (R-3716) from Forest Products Laboratory, USDA Forest Service, Madison, Wisconsin, isolated from a wood pole (Brochus et al. 2009). The next two highest matches in GenBank, FJ 644282 and FJ644281, were two isolates classified as Oxyporus subpopulinus (B.K.Cui & Y.C.Dai) with four nucleotide substitutions. The w5 sample matched 97.1 % with Atractium crassum (symonym Fusarium merismoides var. crassum Wollenw.), followed with 92% similarity by Neonectia galligena isolated from an apple tree. Isolate w5 was not successfully identified to a species, but all matches were within Hypocreales in the Nectriaceae family. The w6 isolate could not be unidentified at species level, by any available phylogenetic analysis; the first two matches in GenBank with 95 % similarity GU055712 and EU754959 are fungi from Lasiosphaeriaceae family isolated from soil. Isolate w7, correlated to 69 sequences with 99.8 % similarity was undoubtedly identified by phylogenetic analysis as Hypsizygus sp.
The second sugar maple sample also displayed numerous black demarcation lines, and seven sectors were collected (w8-w13, w19; Figure 3.11 E). ITS sequence from w8 and w9 matched 99.8 % to Oxyporus populinus, and their sequences were identical. BLAST in GenBank indicated two nucleotide substitutions of EF011121. The next two highest matches in GenBank, FJ 644282 and FJ644281, were two isolates classified as Oxyporus subpopulinus of unknown origin by four nucleotide substitutions. Isolate w11 was unambiguously identified with Hypocrea rufa Pers., with the first match isolated from the roots of Rhododendron sp. from USA. The sequence of w12 matched 99.2 % with Kretzschmaria sp. and the phylogenetic
44 analysis could not identify the fungus at the species level. Isolate w13 was 99.8 % (one nucleotide substitution) similar with 21 sequences of Hypsizygus marmoreus from China, top match JX046038. Isolate w19 is an unknown ascomycete probably in the Coniochaetales within the Sordariomycetes class, based on top hits in GenBank (Lecythophora sp. 98.2 % and Phialophora sp. 96.7 % similarity).
From the third spalted sample of sugar maple, four sectors were collected (w14-w17; Figure 3.11 F). The ITS sequences of w15, w16 and w17 were matched with Hypsizygus marmoreus. The partial ITS sequence of w15 isolates matched 99.7 % to several strains, first match FJ609280 followed by FJ609257 to FJ609279 and AJ494833 to AJ494835 of unknown origin. The w16 sample was a 100% match with FJ609268 and a 99% match with several strains of the fungus, while the w17 isolate was a 99.8 % match with FJ609279 (differs by a single Y/T polymorphism) followed again by several strains within the same group. However, phylogenetic analysis placed the ITS sequence of the isolates within the polyphyletic clade of Hypsizygus sp. Microscopic examination of the sample revealed black pigment formation deposited in fine demarcation lines, which do not follow a predictable pattern or wood fiber orientation. In addition, fungi did not degrade the wood cells inhabited by sclerotial mycelium (Figure 3.12 D), and the lumens of the cells were sometimes partially filled (Figure 3.12 D, red arrow) or completely filled with melanized mycelium (Figure 3.12 D, double red arrow).
Birch sample (Betula sp.)
This wood sample was a twig of birch with visible bright green pigmentation, deeply diffused into wood tissue (Figure 3.11 D); top BLAST of w18 sequence matched 99.7% similarity with C. aeruginosa AY755360 from USA, and the ITS sequence of the w18 isolate was undoubtedly aligned within the Chlorociboria aeruginosa monophyletic clade. As shown in microscopic imaging, the wood ray parenchyma cells are preferentially colonized by green mycelium (Figure 3.12 E, F and G arrow), and in more advanced stages of colonization, the pigmented hyphae extend more uniformly within the wood cells.
45 3.4 Discussion
By studying spalted samples of six wood species, we identified eight fungal species associated with the black demarcation lines, and three fungal species associated with stained wood. Of these, Polyporus squamosus and Chlorociboria were already known as "spalting fungi" (Campbell and Munson1936, Blanchette et al. 1992). It is notable that the search did not retrieve some of the most commonly reported spalting fungi such as Xylaria polymorpha or Trametes versicolor. This highlights the value of DNA-based identification through DNA barcodes.
From all the fungi identified in this study, of clinical significance is the anamorph of Botryosphaeria rhodina identified in buckeye burl sample from California, which was rarely found as human pathogen, being associated with inflamation of eye cornea (keratitis), and with lesions on nail and subcutaneous tissue (Saha et al. 2010, Summerbel et al. 2004). Two species of Fusarium were also identified in natural spalted wood, one associated with red stain in boxelder, and one from sugar maple with zone line formation. It is known that Fusarium can produce an array of mostly opportunistic mycoses in immunocompromised and immunosuppressed individuals (Sutton and Brandt 2011).
3.4.1. Fungi that produce zone lines
Melanin production did not follow wood fiber orientation, and generally, the wood cells inhabited by melanized mycelium were mildly or not degraded by the fungus. Small melanized hyphae were found exclusively within the lumens of the wood cells; they were concentrated at first along the cell walls as a compact film, followed by formation of a compact matrix of hyphae that sealed the cell lumina (Figure 3.12 D).
Polyporus squamosus
Isolate w2 was unambiguously identified as Polyporus squamosus by phylogenetic analysis, which is a common white rot fungus in living deciduous trees. It is a wound parasite, and sometimes saprotrophic on stumps and dead hardwood trees. The pattern of wood decay often exhibits black demarcation lines that serve as a survival structure separating fungal colonies in wood (Schwarze et al. 2000). A detailed characterization of zone line formation by P. squamosus is given by Campbell and Munson (1936), who observed this phenomenon both in nature and under laboratory conditions.
46 Oxyporus populinus
This fungus was associated with three isolates from different wood samples of sugar maple: w4, w8, w9. The fungus is a common polypore that is parasitic on deciduous trees, especially maple. It is a white rot fungus, and the infection probably occurs through wounds. Its decay strategy has not been extensively evaluated but it is known that the fungus is an aggressive secondary decayer (Tattar et al. 1971, Terho et al. 2007). Although it is not often related to zone line production, Pengler (2000) mentions Oxyporus for enhancing natural spalting appearance of wood in its early growth stages.
Nectriaceae (cf. Fusarium sp.)
Isolate w5 was similar to strain EU860056 Atractium crassum (synonym Fusarium merismoides var. crassum, and anamorph Gibberella sp.), isolated from wooden seawater poles from Germany (Bills et al. 2009). The next higher match was with w5, Neonectria galligena, the primary cause of Nectria canker, also called Target Canker, a disease widespread in North America. Both matches are wood inhabiting fungi from the Nectriaceae family that commonly infect many hardwood trees and cause canker-like damage on the living trees. In this type of infection, zone demarcation lines often appear as a result of wood reaction, but antagonistic reactions with other wood inhabiting fungi are not excluded (Schwarze et al. 2000).
The fungus was not clearly identified by any on-line available search method (UNITE, Fusarium-ID, and Fusarium MLST), nor by phylogenetic analysis in RAxML. Fusarium is a taxonomically complex genus, and those fungi are difficult to identify, due to their high variation and mutation in culture and a unclear morphological differentiation. The available databases might poorly reflect species identification and diversity, and comparisons might lead to unbalanced identifications (O’Donnell 2000, Geiser et al. 2004, O’Donnell et al. 2012). However, it is certain that w5 is a Fusarium sp. within the Nectriaceae.
Hypsizygus marmoreus
Five isolates were identical with Hypsizygus marmoreus: w7, w13, w15, w16 and w17. The last three were removed from different colonies originating on the same wood sample. Their sequences were not identical, suggesting different strains.
47 Hypsizygus marmoreus, synonym Agaricus marmoreus (Peck) and Clitocybe marmorea (Peck) Sacc., is a fairly common parasitic fungus on deciduous trees. Its macroscopic appearance is very similar to Hypsizygus ulmarius (Bull.:Fr) Redhead synonym Lyophyllum ulmarium (Bull.: Fr.) Kiihner. Redhead (1986) and Nagasawa and Arita (1988) established the morphological differences between the two species, and Moncalvo et al. (1993) confirmed the molecular distinction.
The fungus is not an aggressive white rot. In vitro experiments demonstrated limited decay capability, with an average weight loss of 0.70 % after 60 days incubation in beech wood blocks (Ohta 1994). This aspect is probably caused by its weak enzymatic activity; laccase is the only type of enzyme produced by the fungus. In the laboratory, H. marmoreus produced at best 490- 760 Ul-1 of extracellular laccase production compared with 9000–20000 Ul-1 of enzyme activity for Trametes versicolor after 7–14 days of submerged fermentation (Songulashvili et al. 2007). The minimal effect on wood structure makes it a good candidate for commercial spalting.
Phylogenetic analysis of 93 taxa indicate the similarity of w7, w13, w15, w16 and w17 isolates with H. marmoreus sequences mostly form China and Malaysia. One species of H. ulmarius EF421105 included within the assigned clade is a European species. Pleurotus ostreatus HM770897 from Taiwan was also attributed to this clade; however, the absence of morphological data for this sequence could not clarify the correct identity of this isolate (Imtiaj et al. 2011). There are three sequences named H. tessulatus in Genbank (DQ917653, HQ436120, and FJ467372) of European origin, that have higher divergence to our samples than the taxa selected, and that also stand clearly apart in the ITS analyses (data not shown). Based on these results, it can be stated that the investigated isolates belong to H. marmoreus species.
Lecythophora sp.
The w19 isolate is an unknown ascomycete likely in the Coniochaetales within the Sordariomycetes class with highest matches in Lecythophora sp. (synonymy Cadophora, Margarinomyces and Phialophora). The Coniochaetales are saprophytes on wood, dung and soil, with a relatively simple morphology of the ascomata. The anamorphs of this order are classified as Lecythophora sp. (Zhang et al. 2006), but often are confused with other phialophora-like fungi. Their primary morphologic differentiation is comprised of predominant intercalary phialides with smaller incidence of terminal and lateral discrete phialides (Gams
48 2000). Rayner and Todd (1979) also reported the presence of Phialophora sp in the black narrow interaction zones formed by fungi that develop intraspecific antagonism.
Our isolate match AY219880 had a similar DNA sequence to the UAMH 10331 fungus deposited in the University of Alberta Microfungus Collection and Herbarium as salal root endophyte. The fungus was classified as Lecythophora sp. based on morphological aspect on culture and molecular identity of the genus (Lynne Sigler – personal communication).
Kretzschmaria sp.
Many fungi from Xylariaceae are known to produce zone demarcation lines on hardwoods, in particular Kretzschmaria deusta, formerly known as Ustulina deusta. Kretzschmaria deusta is a widespread ascomycetes fungus in northern temperate regions that occurs frequently on living deciduous trees. Spores are dispersed from soil or in mechanical injury to bark (Schwarze et al. 2000). The fungus is often found at the base of the trunk of standing trees and on old stumps, and produces surface black, crusty fruiting bodies. Kretzschmaria deusta is classified as a white-soft rot for its primary degradation of lignin, and as an opportunist fungus invading already significantly damaged trees (Innes et al. 2006).
Previous research on K. deusta has focused on its formation of reaction zones in living deciduous trees (Pearce 1991, Schwarze and Baum 2000, Baum and Schwarze 2002), but Hendry et al. (1993) inferred that Kretzschmaria deusta had similar growth rates and habits in response to host substrate regardless the presence of the living wood cells.
The identification of w12 isolate could not be resolved at the species level by any phylogenetic analysis; however the ITS sequence of the fungus was aligned within the polyphyletic Kretzschmaria sp. clade.
Hypocrea rufa
The w11 isolate was found to be 100% similar with several strains of Trichoderma viride and its teleomorph Hypocrea rufa. Trichoderma species are frequently isolated from forest and agricultural soil, while Hypocrea sp. are commonly found on bark or dead wood and on other fungi (e.g., bracket fungi and agarics). Trichoderma viride is characterized by the formation of pigmented fleshy stromata, from yellow, orange to different shades of brown. According to Jaklitsch et al. (2006), many other species are categorized as “Hypocrea rufa” and
49 “Trichoderma viride”, when in fact the species is relatively scarce, contrary to numerous mentions in the literature (Hagn et al. 2003, Nobe et al. 2004, Osono 2005, Rudresh et al. 2005).
This fungus was not previously associated with spalting formation in wood. However, Trichoderma sp. are known to be antagonistic to many other fungi including white rot fungi (Savoie 2001, Brown et al. 1999), and are often cited as producing antibiotics and acting as a biocontrol agent against soil borne plant-pathogenic fungi (Lieckfeldt et al. 1999). The general antagonistic reaction of wood-decaying fungi is determined by competition in nutrition exploitation. As reported by Popescu et al. (2009), the typical strategy of H. rufa in antagonistic reactions is oxidative stress creating reactive oxygen species (ROS), which assist further in fungal wood decay. If in competition with another fungus with equal virulence and speed reaction, the former can create melanin depositions as stress reaction (Butler et al. 2001). The identity of w11 was unambiguously determined by BLAST and UNITE search.
3.4.2 Staining fungi
Fusarium sp.
Species of the genus Fusarium are difficult to separate, and multilocus phylogenetic methods showed a great deal of species diversity previously under-estimated.
The w1 specimen identified as Fusarium sp. displayed a pink pigment in wood tissue with little indication of decay. Microscopic observations indicate the presence of fine mycelium formation homogenously distributed within the wood cells. Thin hyphae were seldom present in the wood cell lumina, and in sections of 10 µm thickness, the pigment appeared to be diffused throughout the wood structure as well as within the fungal hyphae. In thinner sections of 2 µm the fungal hyphae were observed predominantly in the middle lamellae of the wood structure. Fusarium sp. is commonly known as a plant pathogen, and it was previously identified in pink-pigmented zones characteristic in box elder (Acer negundo) (Demirci and Maden 2006). The pink pigmentation of box elder is a controversial phenomenon, and the role the fungus plays in pigment formation is not clear (Batra et al. 1962, Morse et al. 2002). Fusarium solani (Mart.) Sacc., one of the top matches in GenBank, was previously associated with lignin degradation (Lazovaya et al. 2006), and Fusarium oxysporum, the second top match, characterized by Ouellette et al. (2005) in wood tissue, employs similar strategies of cell wall deterioration and
50 its preference for the middle lamellae. However, in vitro inoculation of sugar maple with Fusarium sp. failed to produce pink pigmentation (Robinson et al. 2011c).
Our molecular analysis confirms the association of this fungus with pink pigment formation in box elder, which might indicate a specificity of fungus-plant interaction, also hypothesized by Demirci and Maden (2006). ITS sequences have been shown to be problematic for distinguishing between closely related Fusarium species. However, based on phylogenetic analysis and search in UNITE, Fusarium-ID and Fusarium MLST database, w1 was identified as a species of Fusarium.
Botryosphaeria sp
The w3 isolate tentatively identified as Botryosphaeria rhodina (Berk, & Curt.), synonym Botryodiplodia theobromae (Patouillard), anamorph Lasiodiplodia theobromae (Pat.) Griffon & Maubl, is an aggressive tropical parasite that damages many plant species, causes dieback and shoot blight in trees and shrubs, and stains in lumber. It was identified as a virulent pathogen in Cocoa trees (Theobroma cacao) (Mbenoun et al. 2007), and in Jew’s Mallow (Nalta Jute, Corchorus olitorius) (Sato et al. 2008) among other species. Beside its ability to infect many plant species, the fungus is also of interest for the fermentation of fragrances (Krasnobajew et al. 1982). It is known that in cultures Lasiodiplodia develops profuse grey mycelium formations. If these formations also occur in wood, this would explain the macroscopic grey color visible on the test sample, that are produced by the dark mycelium formations observed throughout wood cells (Figure 3.12 C).
Chlorociboria aeruginosa
The fungus is saprobic on well-decayed deciduous wood, and is distinguished by its blue-green staining, considered valuable for ornamental purposes (Blanchette et al.1992). Macroscopic differentiation between C. aeruginosa and C. aeruginascens is very difficult, the only reliable distinction being achieved by microscopic observation of the fruiting bodies. The former has larger spores and tomentum hyphae strongly roughened with crystal formation (Ramamurthi et al. 1957, Dixon 1975). Bessette et al. (1997) state that C. aeruginosa unlike C. aeruginascens does not stain wood; however our results indicate that C. aeruginosa is indeed capable of green stain. The first higher match of our isolate (admission number AY755360) in GenBank was classified as C. aeruginosa by Wang et al. (2006). In his studies, the Chlorociboria clade was
51 positioned in the family Helotiaceae by parsimony analysis. The same strain is also mentioned by Johnston and Park (2005), as being similar to C. aeruginosa PDD81292 isolate from rotten wood in White Mountains National Park, Maine, USA. Consistent with these findings, in phylogenetic analysis in UNITE by galaxyBLAST method, the w18 sequence was placed inside the clade established for C. aeruginosa.
Microscopic analysis showed that pigmented hyphae preferentially colonized parenchyma ray cells in incipient stages of invasion. After complete colonization, pigmented granules were deposited along the inner surface of cell walls, sometimes to complete obstructions. In advanced stages of colonization the blue-green pigment appeared to be diffused in cell walls. Chlorociboria species are incapable of degrading cellulose and lignin (Robinson and Laks 2010b), and is more likely that decay fungi previously inhabited the wood. More information on this fungus is presented in Chapter 4.
3.5 Conclusions
In our attempt to gain insight on fungi responsible of wood spalting I identified antagonistic fungi not yet associated with staining or melanin production. I was able to identify three isolates from spalted wood that exhibited stain-like pigment dispersion, and seven specimens associated with melanin formation. Among those, Fusarium sp., Hypocrea rufa, Hypsizygus sp. and Oxyporus populinus were found for the first time associated with black pigmentation in wood, while Botryosphaeria sp. and Fusarium sp. were identified in grey and red stained wood, respectively. All isolates matched with known wood inhabiting fungi, not all of which were previously reported as related to pigment occurrence.
While many basidiomycetes and some ascomycetes fungi are capable of producing black pigment (melanin), other colored pigments can also be produced by certain fungi. Distinct zones colonized in general by aggressive wood decayers, were also adjacent to colonies of the same species delineated by zones of dense melanized mycelium, indicating that intraspecific antagonism and interspecific interaction might be utilized in melanin production. In contrast, staining of wood fiber is caused simply by pigmented hyphae of specific ascomycetes fungi that colonized with minimum damage of the wood cell wall structure.
52
Chapter 4
Morphological and molecular characterization of the two known North American Chlorociboria species and their anamorphs
4.1 Introduction
The blue green staining of wood debris by species of Chlorociboria Seaver ex C.S. Ramamurthi, Korf & L.R. Batra makes it one of the most recognizable genera on the forest ground. This Ascomycota genus is fairly common in temperate forests worldwide, particularly on hardwood debris of sugar maple, poplar, birch, beech, alder, oak, and sometimes also on softwoods pine and cedar (Dixon 1975).
Two species of Chlorociboria are recognized in North America. They morphologically correspond to two species originally described from Europe, C. aeruginosa (Oeder) Seaver ex C.S. Ramamurthi, Korf & L.R. Batra and C. aeruginascens (Nyl.) Kanouse ex C.S. Ramamurthi, Korf & L.R. Batra. These two species are distinguished by the size of their ascospores and the smooth versus strongly roughened tomentum hyphae, which are few to numerous in both species, (Dixon 1975, Johnston and Park 2005). However, there has been much confusion between the two taxa especially in the early European literature. Recent studies of the genus identified 17 species of Chlorociboria worldwide (Kirk et al. 2008), most of which are concentrated in the Southern Hemisphere (Johnston and Park 2005). In a study on 15 species of Chlorociboria by Johnston and Park (2005), phylogenetic analyses placed the genus as a monophyletic group within the Helotiales by both parsimony and neighbour-joining analysis, and Wang et al. (2006) placed Chlorociboria species within the Leotiomycetes group, in a clade with the Cyttariales and the Erysiphales.
In the past, Chlorociboria species were placed in Helvella by Oeder (1770), Sowerby (1797) and Persoon (1801), in Peziza by Persoon (1795/96, 1822), Vahl (1797), Fries (1822, 1849) and Greville (1827), in Helotium by Berkley (1860), and in Chlorosplenium by Tulasne & Tulasne (1865). Finally, Seaver (1936) invalidly created the genus Chlorociboria to accommodate these
53 species, and later Seaver (1951) placed them in the family Helotiaceae, tribe Helotieae. The name Chlorociboria was validated by Ramamurthi et al. (1958).
The long-standing confusion between C. aeruginosa and C. aeruginascens was generated by the inconsistent use of the epithets. As discussed by Dixon (1975), the name “aeruginosa” was used first by Oeder (1770) when he proposed the species Helvella aeruginosa, in later agreement with Persoon (1795/96), until Persoon (1801) described again both Peziza aeruginosa and Helvella aeruginosa. Nylander (1868) described Peziza aeruginascens as a species similar to P. aeruginosa Pers. and Chlorosplenium aeruginosum Tul., but with smaller spores. Dixon (1975) examined the original specimen of P. aeruginosa from the Persoon herbarium and identified it as P. aeruginascens Nyl., but he did not select this specimen as lectotype in order to conserve the current usage of the two epithets. In North America, studies by Kanouse (1947) and Ramamurthi et al. (1958) recognized the two distinct species C. aeruginascens Nyl. and C. aeruginosa Oeder.
The anamorph, or asexual reproductive stage for Chlorociboria was first reported by Tulasne and Tulasne (1865) from Europe, as “the spermagonial fungus Sphaeria moriformis (Tod.) var. seated on greenish wood” produced along with Chlorosplenium aeruginosum on the same wood debris, which later Dixon (1975), based on the spore and ascus dimensions, re-identified as Chlorociboria aeruginascens subsp. aeruginascens. Sphaeria moriformis is the perfect state of a pyrenomycete, Bertia moriformis (Tode) De Not., while the anamorph of Chlorociboria was named by Saccardo (1884) as Dothiorella tulasnei, and Höhnel (1911) transferred it as Dothiorina tulasnei. There is no information available on the anamorph of C. aeruginosa in the literature.
The anamorph Dothiorina tulasnei was alternately attributed to either C. aeruginascens or C. aeruginosa (Tulasne and Tulasne 1865, Saccardo 1884 and 1889, Höhnel 1911, Berthet 1964, and Dixon 1975), and more recently to C. argentinensis by Sanchez and Bianchinotti (2007).
The recent report of Dothiorina tulasnei by Sanchez and Bianchinotti (2007) described an anamorph specimen originated from Argentina, although the description seems to differ from those from Australia and the United States; no species differentiation was made, and no molecular investigation was made available. Moreover, due to the differences between the conidiogenesis described by Berthet (1964) and that of the Argentinian specimen, Sanchez and
54 Bianchinotti (2007) concluded that Dothiorina might not be related to Chlorociboria.
The blue-green pigment produced by Chlorociboria fungi has been studied since the 19th century. Greville (1827) suggested that chips of wood are turned green by activity of the fungus, and Gümbel (1858) later named the pigment ioxylin acid (from the Greek ἰός = verdigris and ξύλον = wood). Rommier (1868) extracted a green pigment with alkali solvents, which he named xylindein. Among other properties, the substance was soluble in aqueous solution, and easily dyed silk and wool in a bright blue-green colour. Liebermann (1874) pursued his own investigation, and extracted a crystalline xylindein with low nitrogen content; it was insoluble in most solvents, except in concentrated aqueous solution of sulfuric acid and phenol. Studies on synthesis of xylindein were carried out more recently by Donner et al. (2012). The potential of this pigment is tangible for both craftsmanship and industrial applications, but there has been no significant application to this day, partially because of the lack of readily obtainable natural or synthetic xylindein. It was inferred by Hartig (1882) and Berkely (1860) that craftsmen from Hannover, Germany and Tunbridge Wells, England, might have produced green stained wood for their production of intarsia panels and furniture since the 19th century, by allowing mycelium to grow in wood. Ramsbottom (1963) also mentions artificial inoculation of wood for spalted wood production, and even applied for a patent. More recently, Robinson and Laks (2010b) studied the use of C. aeruginascens for spalted wood production. Other patent applications targeted the ability of xylindein to inhibit plant germination without any hazardous effects on cultivated crops (Shibata et al. 2007), and as an animal cell growth retardant and antioxidant (Shinnosuke et al. 2004). A new compound derived from the green pigment produced by Chlorociboria, and assessed as an anti-osteoporosis, anti-thrombosis, anti-inflammatory, immunosuppressive agent, as well as a diuretic, was the subject of another patent application (Futoshi et al.1996).
This study was derived from the investigation of green stained wood reported in the previous chapter, based on the phenotypic differences between fruiting bodies and stromata-like formations observed on green pigmented wood samples. To clarify whether the differences were due to anamorph and teleomorph expression in the specimens acquired, pure cultures of Chlorociboria species were isolated for the following: (1) to verify con-specificity of the two species recognized in North America with their European counterparts, respectively C. aeruginascens and C. aeruginosa, and infer their origin in a broad biogeographic context; (2) to
55 unambiguously establish the identity of their anamorphs, and the teleomorph-anamorph relationship (sexual versus asexual reproductive stage), and (3) to examine xylindein production of pure cultures of the two species in various artificial media.
4.2 Materials and methods 4.2.1 Specimen examined
Three bright green decaying wood samples were collected on the forest floor from Haliburton forest, Ontario, Canada, and deposited in the Royal Ontario Museum Fungarium (TRTC). The first sample (TRTC167754) is a piece of unidentified hardwood that exhibits cup shaped fruiting bodies that were identified as C. aeruginascens (Figure 4.2 A). The second sample (TRTC167755) is a twig of Acer saccharum (Marshall) covered with minute, black sphere-like fungal structures (Figure 4.2B). The third sample (TRTC167753) is a piece of unidentified hardwood colonized by C. aeruginosa holomorph (the whole fungus, including anamorphs and teleomorph), with characteristic small fruiting bodies, as well as minute black sphere-like fungal structures (Figure 4.1A, B).
Sections of fresh or rehydrated fruiting bodies and conidiomata were hand-made with a razor blade and were mounted in distilled water. Imaging was acquired on a Zeiss Axioplan 1 or an Olympus MVX10 microscope equipped with Olympus DP71 camera.
There was no ITS sequence of European specimens of C. aeruginascens in public databases; two UK strains from the University of Alberta Microfungus Collection and Herbarium (UAMH) were obtained for comparison with North American collections.
4.2.2 Molecular analysis
DNA from the specimens listed in Table 4.1 was extracted for PCR amplification of the ribosomal RNA transcribed spacer region (ITS), which serves as the primary proxy for species identification in fungi using DNA barcodes (Schoch et al. 2012). PCR amplification was carried out in 25 µL reactions containing 10 ng genomic DNA, 10 mM Tris-HCl, 50 mM KCl, 2.5 mM
MgCl2, 0.1% gelatin, 0.8 mg/mL bovine serum albumin, 0.2 mM dNTP, 0.2 µM of ITS1F and ITS4 primers and 1U of Platinum® Taq DNA Polymerase (Life Technologies – Invitrogen).
56 The thermal conditions were: 94°C for 2 minutes followed by five cycles at 94°C for 30 sec., 60°C for 30 sec., 72°C for 1 minute; 30 cycles at 94°C for 30 sec., 55°C for 30 sec., 72°C for 1 minute and a final cycle at 72°C for 5 min. The PCR products were visualized on 1% agarose gel stained with ethidium bromide. The PCR amplicons were purified using QIAquick PCR purification kit (QIAGEN). All purified PCR amplicons were sequenced at both strands using the BigDye Terminator v3.1 Cycle Sequencing Kit (ABI). The sequencing protocol closely followed the ABI protocol. Sequences were edited in Sequencer vs. 4.1 (Gene Corp.) and deposited in GenBank with accession numbers: JX843710-14, JX843721, JX239625, JX948103, and JX948104 (Table 4.1 and Table 4.2).
GenBank was searched to obtain representative ITS sequences for Chlorociboria species (Table 4.2). Sequences were aligned using default settings in MUSCLE v.3.6 software (Edgar, 2004) and afterwards optimized by eye in SE-AL software (Rambaut 2002). Ambiguously aligned characters were excluded for phylogenetic analyses. Phylogenetic analyses were carried out using a Maximum Likelihood (ML) and Bayesian analyses in RAxML v7.0.4 (Stamatakis 2006) and MrBayes v3.2 (Ronquist et al. 2012) respectively. For the ML analysis, 1000 fast bootstrap replicates were estimated using the GTR-CAT (Generalized time-reversible Categorization) model (Stamatakis 2008), and bipartitions were printed into the ML tree with the highest likelihood. For the Bayesian analysis, four simultaneous MCMC (Markov chain Monte Carlo) chains were run with 1,000,000 generations, sampling every 100. A mixed GTR model, which selects the best rate matrix, was implemented. Convergence was evaluated by observing the likelihood of the model reaching a plateau in Tracer v1.5, and the average standard deviation of split frequencies. The sampled trees were summarized in a 50% majority-rule consensus tree with the posterior clade credibility values.
4.2.3 Cultures
Single spores were isolated from apothecia of TRTC167753 (strain DT008) and TRTC167754 (strain DT8315), and from conidiomata TRTC167755 (strain DT007 and DT007a), and cultured on 2% malt extract agar (MEA). Cultures were deposited in University of Alberta Microfungus Collection and Herbarium collection and given UAMH numbers (Table 4.1).
For the growth and pigmentation tests three replicates per strain were grown on ten media, which were as follows: 2% Difco MEA; 2% Difco MEA with 2% sugar maple dust 40 mesh;
57 2% Difco MEA and 0.2% peptone; 2% Difco MEA with 2% sugar maple dust 40 mesh and 0.2% peptone; Oxoid MEA Difco; Oxoid MEA Difco with 2% sugar maple dust 40 mesh; PDA (Potato Dextrose Agar) (Bioshop Canada); PDA with 2% sugar maple dust 40 mesh; 4% agar, and modified Leonian’s agar (Malloch 1981). The cultures were incubated at 21oC in the dark for five weeks, and measurements of the culture diameter and pigmentation were recorded weekly with a digital calliper. To compare the growth and pigmentation of the isolates in various media, data were analysed using Excel dot plot analysis as described by Cleveland and McGill (1984). Fungal cultures on various substrate media were also investigated for sporulation and pigment production ability at the cellular level.
4.3 Results and discussion 4.3.1 Morphological description
4.3.1.1. Chlorociboria aeruginosa (Oeder) Seaver ex C.S. Ramamurthi, Korf & L.R. Batra
≡ Helvella aeruginosa Oeder, Fl. Danic. 3(9): tab. 534:2 (1770)
≡ Peziza aeruginosa (Oeder) Vahl, Fl. Danic. 8: tab. 1200, fig. 1 (1797)
≡ Helotium aeruginosum (Oeder) Gray, Nat. Arr. Brit. Pl. (London) 1: 661 (1821)
≡ Chlorosplenium aeruginosum (Oeder) De Not., Discom.: 22 (1864) [1863]
≡ Chlorociboria aeruginosa (Oeder) Seaver, Mycologia 28(4): 391 (1936)
≡ Peziza aeruginosa Pers. (1795) Pezizaceae
≡ Chlorosplenium aerugineum (Berk.) Sacc. 1889; Dermateaceae
≡ Chlorosplenium discoideum Massee, Brit. Fung. -Fl. (London) 4: 286 (1895)
Teleomorph (TRTC167753): Apothecia develop on stumps or dead fallen wood, heavily stained in bright blue-green color, arising from black basal stroma originating from the ray cells. The fungi inhabit many hardwood species like sugar maple, poplar, elm, beech, and oak as well as on pine, hemlock and cedar. Discs grow up to 5mm in diameter, bright blue-green color, sometimes with white gelatinous substance present on the hymenium (Figure 4.1A). Stipe are 2-
58 3 x 0.5-1 mm, central stipitate, rarely eccentric. Asci cylindric-clavate of 60-90 x 5-7 µm, are bearing eight unicellular ascospores (Figure 4.1C). When released, the spores seem to be surrounded by small-pigmented vesicles up to 0.2µm. The ascospores are fusiform-elliptic, hyaline, and prone to quick germination, 8-12 x 1.5-2 µm (Figure 4.1D)
Anamorph (TRTC167753): Stromatic conidioma globose or cushion-like black structures, spherical or elongated, emerging superficially on decorticated wood, from ray cells of the substrate, 0.5-1 mm (Figure 4.1 B). In section, multilocular chambers irregularly distributed, with numerous hyaline to slightly pigmented phialides 3-7x 0.5-1.5 µm produced on irregular branched conidiophores. Conidia 3-7 x 2-3µm, (Figure 4.1E-F).
Culture DT008 (derived from TRTC167753 teleomorph): Single spore culture initiated growth within the second day of inoculation on 2% MEA, mycelium propagates at a very slow rate, insignificant in the first week, white at first, and beginning pigmentation after approximately one week. At single point inoculation, the culture never reaches full growth on petri dishes (Figure 4.3C, F). Maximum growth is recorded for 2% MEA with 1% 40 mesh wood sawdust (Figure 4.4A). The fungus produces pigment in all media tested, at various rates; the lowest pigmentation is recorded for PDA media (Table 4.2). In 2% MEA, the fungus produces pigmentation within the fungal hyphae of 1-2.5 µm width, also in vesicles on the outer cell wall, either attached to the hyphae, or as a conglomerate of minute vesicles grouped in a single spot (Figure 4.1G, H). There were no conidiospores observed on any of the media tested.
4.3.1.2. Chlorociboria aeruginascens (Nyl.) Kanouse ex C.S. Ramamurthi, Korf & L.R. Batra
≡ Peziza aeruginosa Pers per Pers.:Fr (var.) b. subgrisea Fr. Syst. Mycol 2(1): 130 (1822)
≡ Chlorosplenium aeruginosum sensu Tulasne and Tulasne (1865)
≡ Peziza aeruginascens Nyl.: 42 (1869)
≡ Chlorosplenium aeruginascens (Nyl.) P. Karst., Bidr. Känn. Finl. Nat. Folk 19: 103 (1871)
≡ Dothiorina tulasnei (Sacc.) Höhn., Sber. Akad. Wiss. Wien, Math.-naturw. Kl., Abt. 1 120: 464 [86 repr.] (1911) (anamorph)
59 Teleomorph (TRTC167754): Apothecia develop on stump or dead fallen wood, originating in the ray cells. Common on many hardwood species like sugar maple, poplar, elm, beech, and oak as well as on pine, hemlock and cedar. Disc of maximum 7 mm, bright blue-green color, black when dry. Stipe 3-6 x 1-1.5 mm, eccentric stipitate, rarely central stipitate (Figure 4.2A). Asci cylindric-clavate 30-55 x 3-5 µm, with eight ascospores. The ascospores are unicellular, fusiform to fusiform-elliptic, hyaline, 5-8 x 2-2.5 µm (Figure 4.2C).
Anamorph (TRTC167755): stromatic conidioma are globose moriform black structures, spherical or rarely elongated, emerging superficially on green stained decorticated wood, from wood ray cells, up to 2 mm diameter (Figure 4.2B). In section, distinct multilocular chambers regularly distributed and oriented towards the surface of the stromatic conidioma, with numerous hyaline to slightly pigmented phialides 5-10x 1.5-2 µm produced on irregular branched conidiophores. Conidiospores 3-5 x 2-2.5 µm, are elongated, hyaline or slightly green (Figure 4.2F). The interior of the stromatic conidioma constituted of intricate hyaline gelatinous hyphae, becoming more orientated between the loculi, and green to dark green towards the surface of the stromatic conidioma (Figure 4.2D, E). It was also noted that the wood populated by this fungus only in the anamorph state is less pigmented than the wood occupied entirely by fungus, developing apothecia.
Culture DT8315 (derived from TRTC167754 teleomorph): Single spore cultures initiated growth within the second day of inoculation on 2% MEA, mycelium propagated at a very slow rate, white at first, beginning pigmentation after approximately one week. On the duration of the growth period, 4-8 weeks, a whitish circle surrounds the colony. After eight weeks, the hyphae of 2-5 µm width, were heavily pigmented, and the whole colony became dark green if grown in the dark, with a brown tint if exposed to light. At single point inoculation, the culture never reached full growth on petri dishes (Figure 4.3). As indicated by Robinson et al. (2012b), maximum growth and pigmentation was recorded for 2%MEA with 1% wood sawdust 40 mesh (Figure 4.4). The fungus produced pigment in all media tested, at variable rates (Figure 4.4); the lowest pigmentation was recorded for potato dextrose agar (PDA) (Table 4.3). In 2% MEA, the fungus produced heavy pigmentation within the fungal hyphae, also in vesicles on the outer cell wall, either attached to the hyphae, or as a conglomerate of minute vesicles grouped in a single spot.
60
1.5 mm 0.25 mm
A B B 10 µm 5 µm
C D
20 µm 10 µm
E F
5 µm 5 µm
G H
Figure 4.1 Chlorociboria aeruginosa TRTC 167753; A- young apothecia and stromatic conidioma (arrow) developed on wood surface; B- stromatic conidioma developed on wood surface (arrow), and young apothecia (double arrow); C- ascus from teleomorph; D- ascospore from teleomorph; E- sectioned stromatic conidioma; F- conidiospores from anamorph; G- hyphae from culture with internal pigmentation and external pigmented exudate attached by the cell wall; H - pigmented exudate in culture
61
5 mm 1 mm
d
A B
10 µm 500 µm 100 µm
F
C D E 20µm 20µm
G H
Figure 4.2 Chlorociboria aeruginascens: A- TRTC167754 (teleomorph) apothecia developed on wood surface; B- TRTC167755 (anamorph) stromatic conidioma developed on wood surface; C- ascospores; D- section of stromatic conidioma with loculi (arrow); E- loculus; F- conidiospore; G- pigmented hyphae from culture derived from teleomorph, with external vesicle attached by the cell wall and gelatinous pigmented secretion; H- conidiospores from culture derived from anamorph.
62 Culture DT007 (derived from TRTC167755 anamorph): Same morphological characteristics as described for DT8315, with the addition of conidia produced on 2%MEA, formed directly on hyaline hyphae or on small irregular branched phialides 7-15 x 1.5-2.5 µm. Conidia are unicellular, irregular mostly ovoid, smooth, hyaline to light green color, 3-7 x 2-2.5 µm, which readily germinate (Figure 4.2G, H).
The morphology of Chlorociboria teleomorphs reported here corresponds to that described by Dixon (1975). The morphology of the C. aeruginascens anamorph described in this study corresponds very well with the original description of D. tulasnei given by Tulasne and Tulasne (1865), as also reported in Berthet (1964) and Dixon (1975), and does not concur with the characteristics reported for D. tulasnei by Sanchez and Bianchinotti (2007) from an Argentinian specimen. The lack of detail concerning anamorph morphology from previous studies, creates confusions among Chlorociboria species, and lack of molecular evidence might have led to disagreement regarding the relationship of D. tulasnei with Chlorociboria reported by Sanchez and Bianchinotti (2007).
It was observed that the anamorph states of both fungi are distinct from the “stromata mass” from which the apothecia emerge; the fruiting bodies of the fungus “blooms” from structures smaller than 1mm (Figure 4.2B).
In culture, conidia formation was recorded only for C. aeruginascens strain UAMH11655, which was initially isolated from an anamorph state on a decaying sugar maple twig. These findings are in accordance with the description given by Berthet (1964) for cultures of Chlorosplenium aeruginosum (Oed. Ex S.F. Gray) de Not., and are inconsistent with the conidial description given by Fenwick (1993).
In agreement with the recent revision of the International Code of Nomenclature for Algae, Fungi, and Plants (ICN) (Hawksworth 2011, Norvell 2011), a name for the anamorph of C. aeruginosa was not assign.
63
A B C
D E F
Figure 4.3 Culture of Chlorociboria in 2% MEA (A-C) and 2% MEA with sugar maple saw dust (D-F): C. aeruginascens UAMH 11655 isolated from single ascospore – A, D; C. aeruginascens UAMH11656 isolated from single conidiospore – B, E; C. aeruginosa UAMH11657 isolated from single ascospore – C, F.
50" 50" 50" 45" 45" 45" MEA" 40" 40" 40" 35" 35" 35" MEA+SM" 30" 30" 30" MEA+P" 25" 25" 25" MEA+P+SM" 20" 20" 20" OMEA" 15" 15" 15" OMEA+SM" 10" 10" 10" PDA" mean%diameter%culture%in%mm% 5" 5" 5" PDA+SM" 0" 0" 0" week"1" week2" week3" week4"" week5" week"1" week2" week3" week4"" week5" A B C week"1" week2" week3" week4"" week5" Figure 4.4 Growth assessment in various culture media recorded over a period of five weeks, of C. aeruginosa UAMH11657 (A), C. aeruginascens UAMH11655 (B), and C aeruginascens UAMH11656 (C).
64
4.3.2 Analyses of ITS sequences
A MUSCLE alignment of ITS sequence for 53 specimens representing 15 species, one subspecies, and one unidentified collection of Chlorociboria (Tables 4.1 and 4.2) was 477 bp in length. Sixty-nine positions were ambiguously aligned and removed for phylogenetic analyses. Both Bayesian and ML phylogenetic trees supported monophyly of each species, but relationships between them were generally poorly resolved (data not shown), as in Johnston and Park (2005). The analyses indicates that the unidentified Chlorociboria sp. DQ257350 (from USA) correspond to C. aeruginosa. Results also showed that C. albohymenia, C. campbellensis, C. clavula, C. duriligna, C. halonata, C. procera, and C. spiralis (all from New Zealand) are the most distantly related taxa to C. aeruginascens and C. aeruginosa, in agreement with Johnston and Park (2005). These New Zealand species were subsequently removed from the analyses in order to increase phylogenetic signal for inferring evolutionary relationships between C. aeruginascens, C. aeruginosa, and closely related species. Table 4.1. Material examined in this study and Genbank accession numbers of newly produced ITS sequences. Species Origin of tissue Voucher collection Culture collection GenBank used for ITS number 1 number 1 accession number sequencing C. aeruginosa Single spore culture TRTC167753 DT008 JX843710 derived from = UAMH11657 ascocarp (Ontario, Canada) C. aeruginosa Ascocarp (Ontario, TRTC167753 - JX948103 Canada) (FB1) C. aeruginascens Single spore culture TRTC167754 DT8315 JX843711 derived from = UAMH11655 ascocarp (Ontario, Canada) C. aeruginascens Ascocarp (Ontario, TRTC167754 - JX948104 Canada) (FB2) C. aeruginascens Single spore culture TRTC167755 DT007 JX843712 derived from = UAMH11656 Dothiorina tulasnei anamorph (Ontario, Canada) C. aeruginascens Single spore culture TRTC167755 DT007a JX843721 derived from Dothiorina tulasnei anamorph (Ontario, Canada) C. aeruginascens Culture from single - UAMH7614 JX843714 ascospore (UK) C. aeruginascens Culture from single - UAMH7615 JX843713 ascospore (UK) 1 TRCT: Royal Ontario Museum Fungarium; DT: culture originally produced by the first author (Daniela Tudor); UAMH: cultures deposited at the University of Alberta Microfungus Collection and Herbarium.
65
Table 4.2 Genbank sequences used in this study. Species Origin GenBank Source accession number C. argentinensis Argentina EF520123 Unpublished, Johnston and Park C. argentinensis Argentina EF520124 Unpublished, Johnston and Park C. aeruginosa Canada (British Columbia) HQ604856 Unpublished, Berbee ML et al. C. aeruginosa Canada (Ontario) JX239625 Unpublished, Tudor et al. C. aeruginascens China AY755359 Johnston and Park (2005) C. aeruginosa Europe (Norway) Z81426 Johnston and Park (2005) C. aeruginascens subsp australis New Zealand JN943460 Schoch et al. (2012) C. aeruginascens subsp australis New Zealand JN943459 Schoch et al. (2012) C. aeruginascens subsp australis New Zealand AY947345 Johnston and Park (2005) C. aeruginascens subsp australis New Zealand AY755351 Johnston and Park (2005) C. aeruginascens subsp australis New Zealand AY755350 Johnston and Park (2005) C. aeruginascens subsp australis New Zealand JN943458 Schoch et al. (2012) 1 C. albohymenia New Zealand AY755347 Johnston and Park (2005) C. argentinensis New Zealand JN943457 Schoch et al. (2012) C. argentinensis New Zealand AY755337 Johnston and Park (2005) C. argentinensis New Zealand AY755338 Johnston and Park (2005) C. awakinoana New Zealand JN943461 Schoch et al. (2012) C. awakinoana New Zealand JN943462 Schoch et al. (2012) C. awakinoana New Zealand AY755339 Johnston and Park (2005) C. awakinoana New Zealand AY755340 Johnston and Park (2005) 1 C. campbellensis New Zealand AY755357 Johnston and Park (2005) 1 C. clavula New Zealand JN943465 Schoch et al. (2012) 1 C. clavula New Zealand AY755346 Johnston and Park (2005) 1 C. duriligna New Zealand JN943468 Schoch et al. (2012) 1 C. duriligna New Zealand JN943467 Schoch et al. (2012) 1 C. duriligna New Zealand AY755341 Johnston and Park (2005) 1 C. halonata New Zealand AY755355 Johnston and Park (2005) 1 C. halonata New Zealand AY755356 Johnston and Park (2005) 1 C. halonata New Zealand AY755354 Johnston and Park (2005) 1 C. halonata New Zealand JN943471 Schoch et al. (2012) 1 C. halonata New Zealand JN943469 Schoch et al. (2012) 1 C. halonata New Zealand JN943470 Schoch et al. (2012) C. macrospora New Zealand AY755343 Johnston and Park (2005) C. pardalota New Zealand AY755353 Johnston and Park (2005) C. poutoensis New Zealand AY755352 Johnston and Park (2005) 1 C. procera New Zealand AY755345 Johnston and Park (2005) C. spathulata New Zealand AY755344 Johnston and Park (2005) C. spathulata New Zealand JN943463 Schoch et al. (2012) C. spathulata New Zealand JN943464 Schoch et al. (2012) C. spathulata New Zealand AY755342 Johnston and Park (2005) 1 C. spiralis New Zealand AY755348 Johnston and Park (2005) C. aeruginascens USA AY755358 Johnston and Park (2005) C. aeruginascens USA AY755349 Johnston and Park (2005) C. aeruginosa USA AY755360 Johnston and Park (2005) C. aeruginosa USA DQ491501 Unpublished, Schoch C C. sp. USA DQ257350 Wang et al. (2006) 1 Sequences not used in final analyzes (see text).
66 Sequences alignment of the reduced matrix was 472 bp in length, of which 419 positions were unambiguously aligned and kept for analyses. The resulting phylogenetic tree is depicted in Figure 4.5. The best ML tree had a log likelihood value of -1742.96, while the Bayesian analysis converged at a log likelihood value of -1806.79 (average). The tree was rooted with C. macrospora, as suggested from both earlier analyses and Johnston and Park (2005).
ITS sequence variation within North American, European, and Chinese collections of C. aeruginascens, including sequences derived from single spore cultures of the anamorph Dothiorina tulasnei (DT007 and DT007a; Table 4.1 and Figure 4.5), was in 8 positions (1.69%). This variation corresponds well with the average intraspecific divergence calculated across the Ascomycota (1.96%; Nilsson et al. 2008). Similarly, ITS sequence variation within North American and European collections of C. aeruginosa was in 12 positions (2.54%). Overall, for both C. aeruginascens and C. aeruginosa, the results confirm conspecificity between North American and European collections and unambiguously link their anamorphs to their sexual stage.
The sister group of C. aeruginascens is C. aeruginascens subsp. australis, a taxon so far known only from New Zealand (Johnston and Park 2005). Both geographic distinction and high ITS sequence divergence between the two groups (>7%) suggests that the latter could be recognized at the species level. The sister group of C. aeruginosa consists of two species known only from New Zealand, C. pardalota and C. poutoensis (Johnston and Park 2005). The results suggest a Southern Hemisphere origin for Chlorociboria, in agreement with the phylogeny depicted in Johnston and Park (2005). Non-monophyly of the two holarctic species and their respective sister group relationship with Southern Hemisphere taxa indicate complex migration routes and speciation processes in the genus.
The ITS phylogeny depicted in Figure 4.5 also suggests that specimens identified as C. argentinensis (Dixon 1975, Johnston and Park 2005) may in fact represent two distinct vicariant species, since ITS divergence between the New Zealand and Argentine samples is high. However, similarly to the possible species-level distinction between C. aeruginascens and C. aeruginascens var. australis that was mentioned above, more comprehensive studies in these groups are needed before taxonomic conclusions can be made.
67
Figure 4.5 Bayesian 50% majority-rule consensus tree of ITS sequences. Highlighted clades indicate C. aeruginosa and C. aeruginascens. Support values above branches are shown for clades with Bayesian posterior probabilities > 95% and/or ML bootstrap values higher than 80%%; * - sequences obtained from cultures from anamorphs; ^ - sequences obtained from cultures from teleomorphs; # - sequences obtained from teleomorphs.
68 4.3.3 Laboratory culture and pigmentation
Laboratory cultures on various media indicate differences between the two species studied. In 2% MEA, C. aeruginosa had an initially faster growth rate than C. aeruginascens, and less intense pigmentation; however, the colony sizes are comparable after five weeks of incubation, with little additional growth, while the difference of pigment intensity is maintained (Figure 4.3 A-C). As shown in Figure 4.1 I, J and Figure 4.2 H, I, the mycelium of C. aeruginosa consist of thinner hyphae (1-2.5 µm width) compared to C. aeruginascens that produces hyphae with width up to 5 µm when heavily pigmented. The colour intensity of the culture appears to be also influenced by the amount of extracellular pigment produced in the gelatinous vesicles.
All media tested with the enhancement of sugar maple wood dust stimulated growth and pigmentation of Chlorociboria (Figure 4.4), as already demonstrated by Robinson et al. (2012b). Cultures obtained from apothecia and stroma of C. aeruginascens had similar pigmentation and growth patterns in all media tested.
The growth of both Chlorociboria species was comparable in all media tested, except 4% agar and Leonian’s agar that inhibited considerably hyphal extension for both species (Table 4.3, Figure 4.4).
Table 4.3. Dot plot of mean percentage pigmentation versus growth for cultures of: A – C. aeruginosa UAMH 11657, B - C. aeruginascens UAMH 11655, and C – C. aeruginascens UAMH 11656, after eight weeks of incubation.
In 4% agar, 2% MEA and 2% MEA with wood saw dust, xylindein was produced concomitant with apical growth, while in all the other substrate media, the pigment production appeared to be delayed in both fungal species. Unlike in Fenwich (1993), the in vitro cultures of both Chlorociboria species always produced pigmentation in all media tested, and the colonies produced very few asexual spores, that easily germinated.
69 4.4 Conclusions
Respective conspecificity of North America and European collections of C. aeruginascens and C. aeruginosa were confirmed from ITS sequence data, and it was demonstrated that the two species have distinct Southern Hemisphere origins. Their anamorphs were unambiguously identified: the anamorph of C. aeruginascens is Dothiorina tulasnei, and the anamorph of C. aeruginosa was previously undescribed. In nature, the anamorphs of the two Chlorociboria species are morphologically differentiated based on stromata characteristics and spore size. In vitro cultures of both C. aeruginascens and C. aeruginosa produce xylindein in all the substrate media tested. Asexual spores are rarely produced in culture, but readily germinated.
70
Chapter 5
The influence of moisture content variation on fungal pigment formation in spalted wood
5.1 Introduction
Wood moisture content (MC) is one of the most important conditions that influence fungal behavior and wood colonization patterns (Boddy 1983b). It is established that optimal fungal growth is achieved at 35–50 % MC on a dry weight basis, with a minimum required of 20–30 % necessary for fungal development; the values vary for different fungal species and inhabited wood substrates (Cartwright and Findlay 1958; Rayner and Todd 1979). Cartwright and Findlay (1958) also mention the ability of some fungal mycelium to survive below the fiber saturation point moisture content (26- 27% of the dry weight for most wood species), while spores, as well as mycelium of several fungal species, can survive for many years in dry condition (Schmidt 2006). Very high wood moisture content also inhibits fungal activity in wood substrate by limiting the quantity of the oxygen available in wood, preventing degradation (Cartwright and Findlay 1958; Boddy 1983a,b).
Moisture content of the substrate is not entirely dependent on the environmental conditions. It is known that wood-inhabiting fungi can influence the microclimatic regime in dead wood. Some fungi are able to regulate the moisture content of a substrate to ensure the optimal water availability, or as a strategy in antagonistic reactions, to create an environment stressful for fungal competitors, thereby avoiding combative exclusion (Boddy and Heilmann-Clausen 2008). Miller (1932) demonstrated that wood decomposer fungi like Serpula lacrymans (Wulfen) J. Schröt., Schizopora paradoxa (Schrad.) Donk and Coniophora puteana (Schumach.) P. Karst., could regulate the water content of the colonized habitat. During the initial stages of colonization, the dry conditions are improved for optimal growth by means of cellulose and polysaccharide decomposition, while at higher levels of humidity the water surplus is extracted from wood substrate into aerial mycelium, to ensure the optimal moisture content. By virtue of pigmented zonation lines that they produce, fungi from Xylaria species seem to maintain wood drier than ambient conditions (Boddy et al. 1989; Heilmann-Clausen 2001), while Armillaria species occupy wood wetter than ambient (Lopez-Real and Swift 1975;
71 Chapela and Boddy 1988). As reported by Humar et al. (2001), moisture content of wooden blocks inoculated with Trametes versicolor (L.) Lloyd rose from 10 % to 57 % above the fiber saturation point after 12 weeks of exposure to decay, and opportunistic mold fungi like Penicillium spp. can ensure fungal succession on various wood substrates by improving moisture content in decayed wood (Dix 1985). However, variation in water content levels determines the prevalence of fungal species inhabiting the wood substrate. Based on the moisture content preference, Käärik (1974) indicate three distinctive groups that colonize wood: fungi that occupy wood at 37 – 47 % moisture content, fungi that prefer a drier part of the wood of 17-23% MC, and fungi that had no restriction in terms of water availability.
There are 44 species of basidiomycetes and 10 species of ascomycetes that have been reported to form dark zone lines (Lopez-Real and Swift 1975). Research by Hubert (1924) indicated that fungal melanin deposition in spatial demarcation could be triggered by limited water availability. The reaction to desiccation might cause fungal species like Xylaria polymorpha (Pers.) Grev., Bjerkandera adusta (Willd.) P. Karst., Phellinus igniarius (L.) Quél. and Porodaedalea pini (Brot.) Murrill to develop morphological changes to ensure the survival of the colonies. They produce high resistance, melanin-type pigment that surrounds the fungal community like a barrier, blocking the water exchange within the wood substrate. This may appear as fine delimitation lines in section. Campbell (1933), in his study on zone line formation by X. polymorpha, refers to desiccation as the cause of a particular kind of zone line, as well as for Armillaria mellea (Vahl) P. Kumm (Campbell 1934).
Laboratory studies by Lopez-Real and Swift (1975, 1977) indicate that melanized mycelium formation by A. mellea and Stereum hirsutum (Willd.) Pers., is considered as a normal morphogenetic process, and occurs at any moisture content when growth is possible, except for S. hirsutum at low humidity (< 35% MC), when pigmentation is inhibited at early stages of growth. Studies by Campbell (1933,1934), Lopez-Real (1975), Rayner and Todd (1979), Boddy (1986), and Boddy et al. (1989) describe the formation of melanized mycelium in zone lines, in inter- and intraspecific antagonistic reactions; they offer a perspective on substrate and environmental conditions that influence such formations in natural settings. Lopez-Real and Swift (1975) indicated the existence of a relationship between the moisture content of the substrate during the initial period of colonization and the ability of Armillaria mellea to form melanin. For Stereum hirsutum, high moisture content of the substrate may be a critical factor
72 determining pigment formation. Campbell (1933, 1934) studied in vitro zonation of A. mellea and X. polymorpha, mentioning that optimum moisture is necessary to produce pigmentation; however no specific values were indicated. Rishbeth (1951) studied the behavior of Heterobasidion annosum (Fr.) Bref. and observed that infection is best promoted in freshly cut stumps. Hopp (1938) investigated pigmentation of Ganoderma applanatum (Pers.) Pat. and Phellinus igniarius (L.) Quél. and found that moisture content strongly influenced the antagonistic reactions with pigment production in wood. However, research under laboratory conditions is limited, and a more elaborate and consistent investigation was necessary to determine the optimal conditions for pigmentation for given wood species by fungal species utilized in spalting.
The research was designed for direct applicability in spalting formation and aims to establish a behavior pattern for a group of spalting fungi. This study investigates the direct influence of moisture content of specific wood substrates on fungal pigmentation for spalting production. The ability to manipulate fungal reactions through moisture content changes in wood, offers a chance to enhance the pigmentation intensity and patterns currently available with spalted woods. It also offers an opportunity to add considerable value to underutilized hardwood species.
5.2 Material and methods 5.2.1 Wood and fungal species selection
Two common wood species from southern Ontario, sugar maple (Acer saccharum Marsh.) and beech (Fagus grandifolia L.) were selected for testing, based on their contrasting natural spalting prevalence. The average specific gravity (SG) of the tested wood species was SG=0.68 (0.04 standard deviation) for sugar maple and SG=0.74 (0.03 standard deviation) for beech, calculated based on the measured oven-dry weight and dimensions of five representative 14 mm cubes samples of the wood used for experiments.
Eight fungal species were selected based on their spalting ability (Table 5.1). The ascomycete X. polymorpha is known to produce melanin by a DHN pathway and the basidiomycete T. versicolor probably produces melanin by the catechol pathway (Bell and Wheeler 1986; Taylor
73 et al. 1988). For those fungi, three strains were tested for each species, to determine whether melanin production varied significantly among strains. Xylaria polymorpha strains UAMH 11518, UAMH 11519, UAMH 11520 had been isolated from Acer saccharum in Alberta, MI, USA. Two T. versicolor strains were obtained from the Forest Products Laboratory in Madison, WI, USA: Mad 697 was isolated from a cankered area of Fagus grandifolia in Vermont, USA, and strain R105 was isolated from a dead branch of Malus sp. in New York, USA. The third strain UAMH 11521 was isolated from Acer saccharum in Houghton, MI, USA.
Table 5.1 Wood and fungal species selection Wood species / specific gravity Fungal species Culture (SG) collection number
Sugar maple / SG=0.68 Xylaria polymorpha UAMH 11518 UAMH 11519 UAMH 11520
Beech / SG=0.74 Trametes versicolor Mad 697 FP 72074-R UAMH 11521 Polyporus squamosus UAMH 11653 Polyporus brumalis UAMH 11652 Fomes fomentarius UAMH 11654 Inonotus hispidus F2037 Piptoporus betulinus UAMH 11651 Scytalidium cuboideum UAMH 4802
Other fungal species, one strain per species, were used for additional experiments: Polyporus squamosus (Huds.) Fr. UAMH 11653 isolated from beech in Toronto, ON, Canada, Polyporus brumalis (Pers.) Fr. UAMH 11652 isolated from unidentified dead wood in Toronto area, ON, Canada, Fomes fomentarius (L.) J.J. Kickx UAMH 11654 isolated from birch in Toronto, ON, Canada, Inonotus hispidus (Bull.) P. Karst. F2037 of unknown origin, Piptoporus betulinus UAMH 11651 (Bull.) P. Karst., isolated from sugar maple in Toronto, ON, Canada. One staining fungus investigated for pigment formation was Scytallidium cuboideum (Sacc. & Ellis) Sigler & Kang UAMH 4802 isolated from sodium pentachlorophenate dipped red oak lumber.
Fungal cultures used for inoculation were grown on 95x15 mm Petri dishes with 2% malt extract agar at room temperature for two weeks prior to inoculation.
74 5.2.2 Moisture content test preparation
The moisture content test was performed using a modified decay jar test with vermiculite instead of soil, as outlined in Robinson et al. (2009b, 2012a), to avoid influence of soil substrate on pigment formation. Jars with plastic lids (250 ml) were prepared with 15g vermiculite and variable amounts of distilled water per set.
According to AWPA (2009) Standards, the amount of water added to the jars should be 130% of the water-holding capacity (WHC) of the substrate, in this case vermiculite, for optimum condition of decay. Based on vermiculite WHC, the calculations indicate that 30g of distilled water should be added in each culture jar for standard incubation conditions.
Nine levels of water availability was tested, modifying the amount of water added to each jar, in increments of 5g, including five levels (1-5) under and three levels (7-9) above the standard conditions for decay (level 6) (Table 5.2).
5.2.3 Inoculation and incubation
Culture jars with vermiculite and the specified amounts of distilled water were autoclaved for 30 minutes at 1210C. Five replicates per set of 14 mm wood cubes, preconditioned at 500C overnight to provide an initial dry weight, were surface steam sterilized for 30 minutes under atmospheric pressure at 100°C. After cooling, the wood samples were placed on top of vermiculite in culture jars, and inoculated with a 0.5x2 cm strip of fungal inoculum on top of wood samples. Culture jars were incubated at 270C ± 20C and 80% ± 5% relative humidity in the inoculation chamber, for eight weeks for T. versicolor and F. fomentarius and ten weeks for the other fungi.
At the end of the period of incubation, blocks were removed from jars, gently surface-cleaned to remove mycelium and any traces of vermiculite, and weighed before and after overnight oven drying to determine final moisture content and mass loss. To avoid changes of fungal pigment colors, the drying temperature was modified to 500C instead of 103 ± 2 0C standard for MC testing.
75 5.2.4 Mass loss and pigment assessment
Mass loss was calculated based on oven dried weight of each wood sample measured before and after exposure to fungi.
Dried wood samples were scanned externally on one representative surface of each wood sample with Epson WorkForce 500 scanner at 2400 dpi. Samples were then cut in half along the wood fiber in equal parts, and one internal surface was also scanned. The obtained images (Figure 5.1) were analyzed for pigment evaluation with Scion Image software, following the protocol described in Robinson et al. (2009a), recording the determined percentage of pigment formation on the total scanned surface.
To analyze the importance of moisture content for mass loss and fungal pigment formation, a one-way ANOVA was run, followed by Tukey’s HSD at α = 0.05, using SAS, version 9.2, with fungal inoculation and MC conditions as independent variables, for each wood species.
A B
C D Figure 5.1 Pigment formations in beech by Trametes versicolor (A-extern, C-intern) and Xylaria polymorpha (B-extern, D-intern)
76 5.2.5 Estimation of initial conditions and changes of substrate moisture content
To estimate the wood moisture content in conditions simulated in the experiment, two sets of tests were run for eight weeks, without fungal inoculation, the same as for the main test. Five replicates of wood samples per treatment, initially oven-dried conditioned, were weighed every day for the first week and then weekly for the following period, to monitor changes of moisture content in wood samples.
To minimize mould contamination, wood samples were manipulated and weighed in a sterile laminar flow hood. However, for comparison, wood samples from a second set were kept in sterile conditions and weighed only at the end of the eight-week incubation period. Wood moisture content was calculated based on the final oven-dry weight.
5.3 Results 5.3.1 Initial conditions
Periodical measurement of beech and sugar maple samples kept in culture conditions without fungal inoculation, showed that after one day, wood samples attained 70% of the final moisture content measured at the end of eight weeks; after four days, samples reached approximately 80% of the final moisture content, and after approximately two weeks, wood samples reached equilibrium moisture content (Figure 5.2). There were clear differences among treatments at every stage of testing period.
Comparisons between the final results of wood samples exposed to the same moisture content settings, but kept in sterile and unsterile conditions, indicated a slightly positive difference in water uptake of the sterile wood samples, that might be explained by water loss due to periodic handling for weight measurements (Figure 5.3). Based on the measured MC after eight weeks of conditioning in sterile conditions, sugar maple’s water absorption capacity was higher than that of beech, for the same amount of water availability (Table 5.3). The same general trend was observed when wood was exposed to fungal activity. However, in this case, the final moisture content was significantly influenced by the fungal species that colonized the wood (Table 5.3). By comparison, S. cuboideum maintained MC levels lower than the normal uptake of both wood
77 species, while F. fomentarium maintained MC levels similar with the normal uptake in beech, and lower in sugar maple. The final moisture content was calculated based on the oven dry weight at 500C, and it might not reflect the absolute moisture content value, since drying of the wood samples at this temperature might still retain moisture.
110" 110" 100" 100" 90" 90" 80" 80" 1" 1" 70" 2" 70" 2" 60" 3" 60" 3" 4" 50" 50" 4" 5" 5" 40" 40" 6" 6" 30" 7" 30" 7" 8" 20" 20" 8"
mean"moisture"content"%" 9"
mean"moisture"content"%" 9" 10" 10" 0" 0" B
A day2" day3" day4" day5" day6" day1"" day2" day3" day4" day5" day6" day1"" week1"week2"week3"week4"week5"week6"week7"week8" week1"week2"week3"week4"week5"week6"week7"week8" Figure 5.2 Moisture content at various water level additions in beech (A) and sugar maple (B) incubated in culture condition without fungal inoculation, measured over eight weeks period.
100" set1"(sterile)" 100" set1"(sterile)" set2"(unsterile)" set2"(unsterile)" 80" 80"
60" 60"
40" 40"
20" 20" mean"moisture"content"%" mean"moisture"content"%" 0" 0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 1" 2" 3" 4" 5" 6" 7" 8" 9" treatment" A B treatment"
Figure 5.3 Initial moisture content at various water level additions in beech (A) and sugar maple (B) incubated in sterile and unsterile condition without fungal inoculation, measured over eight weeks period. The error bars represent the standard deviation of five replicates.
78 Table 5.2 Estimated moisture content (%) values for each treatment of the wood species tested, based on the final moisture content of wood conditioned for eight weeks in sterile conditions.
Treatment level Grams of water added per Beech Sugar maple jar MC/ st.dev. MC/ st.dev. 1 5 g 21/3 19/4 2 10 g 25/3 22/2 3 15 g 29/3 31/2 4 20 g 37/4 36/2 5 25 g 40/4 44/2 6 30 g 50/3 59/3 7 35 g 56/4 70/1 8 40 g 59/3 82/6 9 45 g 59/3 88/9
Table 5.3 Final moisture content in wood samples (five replicates per set) incubated with various fungal species: tv = T. versicolor (average of three strains); xp = X. polymorpha (average of three strains); ih = I. hispidus; pbr = P. brumalis; pbt = P. betulinus; ff = F. fomentarius; psq = P. squamosus; sc = S. cuboideum; - = no fungal inoculation.
Fungal species tv xp ih pbr pbt ff psq sc - Treatment g Mean % moisture content /standard deviation water/jar beech 5 27 /5 21 /2 23 /5 27 /3 31 /2 27 /3 25 /5 13 /4 21 /3 10 34 /4 33 /4 32 /4 33 /3 31 /2 31 /2 30 /2 23 /10 25 /3 15 37 /6 35 /3 34 /5 37 /4 38 /4 34 /8 33 /5 33 /3 29 /3 20 39 /3 40 /6 44 /5 41 /2 40 /3 43 /4 40 /5 43 /4 37 /4 25 45 /8 43 /6 50 /6 44 /4 53 /3 53 /7 51 /7 46 /5 40 /4 30 46 /7 49 /5 54 /7 44 /2 52 /3 56 /4 58 /5 47 /3 50 /3 35 50 /10 50 /5 55 /5 45 /8 55 /9 52 /7 58 /14 47 /3 56 /4 40 58 /11 54 /5 58 /2 55 /6 53 /5 52 /11 62 /13 47 /5 59 /4 45 63 /14 60 /6 79 /8 59 /9 62 /2 77 /16 88 /10 49 /5 59 /3 sugar 5 24 /5 23 /3 19 /4 29 /3 18 /7 8 /3 24 /1 9 /4 19 /4 maple 10 33 /5 31 /3 33 /3 33 /5 35 /3 10 /2 29 /1 15 /1 22 /2 15 37 /6 39 /8 36 /6 36 /3 46 /2 13 /2 31 /4 20 /3 31 /2 20 41 /4 46 /3 66 /11 52 /6 66 /3 23 /4 34 /3 25 /2 36 /2 25 47 /7 51 /3 71 /9 59 /6 80 /8 24 /6 43 /2 25 /4 44 /2 30 47 /6 56 /5 77 /5 72 /4 91 /5 37 /6 44 /4 31 /4 59 /13 35 55 /6 57 /5 81 /9 82 /3 95 /6 47 /5 45 /4 37 /5 70 /11 40 76 /8 71 /7 89 /7 85 /2 96 /7 53 /3 48 /2 37 /3 82 /6 45 89 /8 78 /6 90 /4 87 /4 93 /3 54 /3 58 /3 37 /2 88 /8
79 5.3.2 Mass loss
An important factor in spalting formation is the retaining of minimal loss after wood exposure to fungal activity correlated with maximum pigmentation. Comparing the results of on both wood species, there was no significant treatment that ensured minimal loss in both wood species inoculated with tested fungi. However, lower levels of moisture content (averaging 21 – 38%) attained by treatments level 1, 2 and 3 of water resulted in stimulation of mass loss in beech inoculated with X. polymorpha, P. brumalis and P. betulinus (Figure 5.3A). Mass loss induced by F. fomentarius was greater in beech samples, while the rest of the fungi degraded sugar maple samples more (Figure 5.3 A and B).
5.3.3 Influence of induced condition on pigment formation
External black to dark brown pigmentation by T. versicolor (average of three strains) was higher with the low water availabilities with maximum pigmentation at treatment level 2 (with 10 g of water) for sugar maple (average MC 33%) and treatment level 3 (15 g water) for beech (average MC 37%). Internal pigmentation in sugar maple was also stimulated by low water content. The large standard deviations for the degree of pigmentation (Figure 5.5 A an B) reflect the high variability of fungi used for this experiment.
External zone lines and black pigmentation produced by X. polymorpha (average of three strains) in beech and sugar maple was significant at treatments levels 2 and 3 (with 10 and 15 g of water respectively, corresponding to average MCs of 32% and 37% respectively) for both wood species (P<0.0001). Internal pigmentation was highest in sugar maple at low moisture content (Figure 5.6). Unlike T. versicolor, this fungus demonstrated smaller deviations among the data and a greater consistency in pigment formation among the three strains.
80
30"
A" A" 25" " A"" A" BA"" " " A" A" A" A" " A" 5"g" 20" A" " """"""" 1 " " A" " 10"g"2 BA" " 15"g" A" " 3 BA" A" 20"g" 15" B" BA" " B" " " B" 4 " " " 25"g" " 5 BC" 30"g" BA" 6 10" B" " A" " C" 35"g" mean%%%mass%loss% A" BC" 7 " B" A" A"A"A" " A" A"" A" " " 40"g" " " " " " 8 " " " A" 45"g" C" A"" A"A" C" DC" 5" A" C" C" DC" A" A"A"A" 9 " " " " C"C" C" " " B" " " " D" DC"" DC" A"" A" " " A" " " B" " " B" B" A" " " D"" " B" " " B" A" A"A"A"A" " " " " " " " " " " " " " 0" tv" xp" ih" pbr" pbt" ff" psq" sc" A%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%fungal%species%in%beech%%%%%%%%%%%
30"
25"
BA" 5"g"1 " A" BA" BA" 10"g"2 20" " A"" BA"BA"" BA" BA" " 15"g"3 " " " BA" " C" A" " 20"g"4 15" " B" " B" BA" " B" 25"g"5 A" " " B" " BA" " A" 30"g"6 B" " A" " B" BA" A" BA" 10" " BA" BA" BA" " BA" BA" 35"g"7 " " BA" " " A" A" A"A" A" " BA"" " " BA" A" A"A" A" mean%%%mass%loss% " BA"B" 40"g" C" " BA" B" A" BA" 8 C" B" B" " B" " 45"g" " B"B"B"B" C" BA" BA" 9 5" " " BA" C" BA" BA" BA" BA" D"DC" BC" C" 0" tv" xp" ih" pbr" pbt" ff" psq" sc" B%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%fungal%species%in%sugar%maple% Figure 5.4 Mass loss of beech (A) and sugar maple (B) wood samples incubated with various fungal species: tv = T. versicolor (average of three strains); xp = X. polymorpha (average of three strains); ih = I. hispidus; pbr = P. brumalis; pbt = P. betulinus; ff = F. fomentarius; psq = P. squamosus; sc = S. cuboideum. Error bars represent standard deviation of five replicates per set. Different letters represent significant differences at α = 0.05 within each category of fungus/wood species combination.
14" A" " 12" 5" 10" 10" 15" 8" 20" A" " 25" 6" A" B" A" " 30" 4" " B" " B" 35" B" " B" B" " " B"" " B" B" A" 40" mean%%%pigment%forma.on% 2" B" B" " B" A" B" A" A" " B" " A" BA"" B" B" A" " " " " A" A" B"B"" B" A" A" " A" " A" " " " " " " " " " " " 45" 0" " " " " ezl_b" ezl_sm" ep_b" ep_sm" izl_b" izl_sm" ip_b" ip_sm" 81
14" #A#
1" 12" 2" ###A# 10" 3" 4" 8" #A# 5" 6" 6" A# 7" B# 4" ###B###B# 8" mean%%%pigment%forma.on% ###B# ##B# B# ##B# #B####B#B## 9" ####B# ####B# #A# 2" ##A# B# ##B# A# A# A# A# ####B# #B# #A#A# A# ###A# #A# A# #BA# #B##B##B#
0" ezl_b" ezl_sm" ep_b" ep_sm" izl_b" izl_sm" ip_b" ip_sm"
TV# Figure 5.5 Pigment production in beech (b) and sugar maple (sm) by Trametes versicolor (average of three strains) at various moisture content values in eight weeks of incubation: ezl – external zone lines; ep – external pigmentation; izl – internal zone lines; ip – internal pigmentation. Error bars represent one standard deviation of five replicates per set. Different letters represent significant differences at α = 0.05 within each fungus/wood species combination.
Figure 5.6 Pigment production in beech (b) and sugar maple (sm) by Xylaria polymorpha (average of three strains) at various moisture content values in ten weeks of incubation: ezl – external zone lines; ep – external pigmentation; izl – internal zone lines; ip – internal pigmentation. Error bars represent one standard deviation of five replicates per set. Different letters represent significant differences at α = 0.05 within each fungus/wood species combination.
82
In the case of I. hispidus and P. squamosus, there was no significant moisture content treatment condition that enhanced pigmentation; however, there was a trend of enhanced dark brown pigmentation observed at higher levels of moisture content (Figure 5.7 and Figure 5.8).
Polyporus brumalis had significant black external pigmentation in sugar maple at treatments level 9 (45g of water) (P<0.0001), while in beech the pigmentation was stimulated at lower levels of moisture content. The same tendency was observed for F. fomentarius (Figure 5.9 and Figure 5.10). Piptoporus betulinus, the only brown rot fungus investigated, produced no pigmentation in either wood species tested.
The red stain fungus S. cuboideum had significant external pigmentation at treatments level 4 for both wood species tested (P<0.0001) at eight weeks of incubation, and there was also significant internal pigmentation in sugar maple (P<0.0001) at the same treatment (Figure 5.11).
Figure 5.7 Pigment production in beech (b) and sugar maple (sm) by Inonotus hispidus at various moisture content values in eight weeks of incubation: ezl – external zone lines; ep – external pigmentation; izl – internal zone lines; ip – internal pigmentation. Error bars represent one standard deviation of five replicates per set. Different letters represent significant differences at α = 0.05 within each fungus/wood species combination.
83
Figure 5.8 Pigment production in beech (b) and sugar maple (sm) by Polyporus squamosus at various moisture content values in eight weeks of incubation: ezl – external zone lines; ep – external pigmentation; izl – internal zone lines; ip – internal pigmentation. Error bars represent one standard deviation of five replicates per set. Different letters represent significant differences at α = 0.05 within each fungus/wood species combination.
Figure 5.9 Pigment production in beech (b) and sugar maple (sm) by Fomes fomentarius at various moisture content values in eight weeks of incubation: ezl – external zone lines; ep – external pigmentation; izl – internal zone lines; ip – internal pigmentation. Error bars represent one standard deviation of five replicates per set. Different letters represent significant differences at α = 0.05 within each fungus/wood species combination.
84
Figure 5.10 Pigment production in beech (b) and sugar maple (sm) by Polyporus brumalis at various moisture content values in eight weeks of incubation: ezl – external zone lines; ep – external pigmentation; izl – internal zone lines; ip – internal pigmentation. Error bars represent one standard deviation of five replicates per set. Different letters represent significant differences at α = 0.05 within each fungus/wood species combination.
Figure 5.11 Pigment production in beech (b) and sugar maple (sm) by Scytallidium cuboideum at various moisture content values in eight weeks of incubation: ezl – external zone lines; ep – external pigmentation; izl – internal zone lines; ip – internal pigmentation. Error bars represent one standard deviation of five replicates per set. Different letters represent significant differences at α = 0.05 within each fungus/wood species combination.
85 As expected, MC levels that produced maximum pigmentation were highly variable among white rot fungi and substrates. Trametes versicolor and X. polymorpha produced pigmentation at lower levels of MC in both wood species, while the rest of the spalting fungi tested produced pigmentation at higher levels in sugar maple (Table 5.4)
Table 5.4 Summary of most important treatment levels that produced maximum external pigmentation for each fungus/wood species combination.
Fungal specie Treatment level Sugar maple Beech Trametes versicolor 2 3 Xylaria polymorpha 3 3 Inonotus hispidus 4 2 Polyporus squamosus 7 3 Fomes fomentarius 8 - Polyporus brumalis 9 1 Scytalidium cuboideum 4 4
5.4 Discussion
For commercial and aesthetic purposes, spalting should result in a high intensity of wood pigmentation, with minimal loss in strength and integrity of the wood after exposure to fungal activity. The natural wood resistance to decay is influenced by many other factors such as wood extractives and ambient temperature. In this test, wood integrity was reflected by mass loss measurements at the end of period of incubation at various levels of moisture content. Although none of the treatments significantly influenced wood degradation, it was observed that sugar maple samples proved to have a general higher degradability than beech, with few exceptions. Polyporus brumalis and S. cuboideum degraded both wood species at the same rate, and F. fomentarius was more effective for beech degradation, within the same treatments of water availability. The observations reported by Bell and Wheeler (1986) based on microscopic analysis, in regard to the lack of wood degradation in spalted wood, would refer strictly to the wood cells that contain zone line formation, and in natural spalting formation in wood substrate exposed to a variety of other stress factors beside moisture content variation.
The theory that solely low water availability can induce desiccation stress on fungi, and inhibit wood degradation as a result of pigment formation has not been proven in this research. If the
86 expression of melanin production limits the secretion of lytic enzymes necessary for host tissue degradation by genetic regulation (Henson et al. 1999), the wood degradation would be minimized only partially in a small portion of the wood substrate. In this case, the wood integrity could be partially retained, by minimizing the wood exposure to fungal activity and therefore achieving fungal pigmentation characteristic to spalting in a shorter period of time.
The moisture content in wood substrates can fluctuate considerably, and is influenced by the relative humidity of the microclimate, the fungal decomposition activity and substrate hygroscopicity and uniformity (Chapela and Boddy 1988; Boddy et al. 1989; Heilmann-Clausen 2001; Boddy and Heilmann-Clausen 2008). From the analysis of moisture content of wood samples exposed to the same testing condition, without fungal inoculation, and based on the predicted values of the initial moisture content that the wood substrate was able to achieve for each treatment, it was determined that sugar maple absorbed more water than beech. This can be explained, by its lower density (the same amount of water in the wood results in a higher % MC) and higher permeability that allows more water from the vermiculite to enter the wood (Samuel and Samuel 2010).
Due to periodic removal of wood samples for weighing, the measured MC of the wood samples varied over the experiment period. This variation could be explained either by loss of moisture through evaporation, or by changes to the position of the wood samples in the vermiculite. However, comparison of final results of this experiment with another set of sterile wood samples, kept in the same condition and measured only for final moisture content after eight weeks, indicates that the final MC of the two sets of wood samples were comparable (Figure 5.2). According to Zabel and Morrell (1992) the most favorable condition for the growth of fungi in wood lies between 40-80 % MC, when free diffusion of enzymes can take place within the film of liquid water that coat the cell lumen walls, but where some air spaces remain for gas diffusion. The formation of black pigmented delimitation zones requires a similar situation, where free water still exists in the lumina of the wood cells, and the levels of concentration of
CO2 are above atmospheric conditions, with the presence of atmospheric levels of oxygen at least for the initial exposure (Lopez-Real and Swift 1975). The present study shows that the fungal pigment formation is a more complex process, as the pigmentation produced by fungal species investigated varies from one species to another, based on water availability and wood species to be spalted.
87 Trametes versicolor produced pigmentation at a lower moisture content, at estimated MC of 22 - 31 % for sugar maple, obtained by the treatment levels 2 and 3, and at estimated MC of 29 % for beech, obtained by the treatment level 3. The treatments with most significant pigment production did not significantly affect mass loss. Xylaria polymorpha produced maximum pigmentation at an estimated 22-31 % MC in sugar maple, obtained by the treatment levels 2 and 3, and estimated 21-29 % MC in beech, obtained by the same treatment levels, which indicates that pigmentation of X. polymorpha is influenced by MC treatment levels and not by wood species investigated. However, the same moisture content that stimulated pigmentation in beech also resulted in the highest mass loss. In the case of I. hispidus no treatment was significant for pigmentation in any of the wood species studied. There was no internal pigmentation recorded for beech, while in sugar maple no pigmentation occurred under 59% estimated MC. Polyporus squamosus developed maximum pigmentation in sugar maple at 70 % estimated MC, obtained by the treatment 7, while F. fomentarius and P. brumalis had significant pigmentation at 82-88 % estimated MC for sugar maple (treatment level 8 and 9). These moisture contents are higher than the optimum moisture contents for growth of most fungi and this supports the earlier conclusion that low MC does not necessarily promote melanin production. The pink staining fungus S. cuboideum developed more uniform pigmentation, significant at 36 - 37 % estimated MC, obtained by the treatment level 4 for both wood species. Although all tested fungi developed a certain degree of external pigmentation, the recorded internal pigmentation was considerably limited. This could be explained by the lack of competition with other fungi to colonize the small wood sample or by the variation of MC distribution, since the wood samples could retain more water at the surface. However, the preponderant formations of external pigmentation indicate a good potential for spalting.
The differences in the reaction among fungal species to various moisture contents might be the outcome of fungal specificity to certain conditions and substrate constraints, as part of successional colonization that ensure wood decomposition (Boddy 1983a; Boddy and Rayner 1983ab; Cooke and Rayner 1984; Boddy 1986; Rayner 1986; Boddy et al. 1989). It is also known that many of the studied fungi produce different types of black pigmentation, using various phenolic compounds from wood substrates as precursors for melanin biosynthesis (Bell and Wheeler 1986; Butler et al. 2001). Those cumulative observations emphasize once more the intricate interactions of fungal species in wood decay.
88
5.5 Conclusions
The reaction of fungi to various levels of moisture content demonstrated that spalting is a complex process that cannot be summarized in a general trend for all the spalting fungi tested. This research indicated that spalting could be stimulated by controlling moisture content of the substrate, and optimal conditions for pigmentation are specific to wood and fungal species involved in the process. Pigmentation was stimulated at low water concentrations in the case of T. versicolor and X. polymorpha for both wood species tested, while I. hispidus, P. squamosus, P. brumalis and S. cuboideum showed a tendency of enhanced pigmentation at higher moisture content levels. Moisture content levels tested did not significantly affect wood mass loss.
For direct applicability in industrial spalting production, pairing of fungal strains should be considered based on their similarity of pigment production at similar MC values. A special consideration should be given to fungi like X. polymorpha that produce maximum pigmentation at lower values of MC, and proved to be more consistent in strain variation.
89
Chapter 6
The influence of pH on pigment formation by lignicolous fungi
6.1 Introduction
Fungal pigmentation is affected by the availability of nutrients, and by pH and environmental circumstances (Lichter and Mills 1998). However, the conditions responsible for this pigment formation are unclear. It is not known what triggers melanin formation in the absence of antagonistic reactions and extreme environmental conditions. Previous research has shown that fungi such as the ascomycete Xylaria polymorpha (Pers.) Grev. and the basidiomycete Trametes versicolor (L.) Lloyd are capable of producing black pigment in monoculture (Rayner and Todd 1977, 1979; Williams et al. 1981; Robinson et al. 2007), by different biosynthesis pathways (Bell and Wheeler 1986). Many ascomycete fungi, including X. polymorpha, synthesize pentaketide melanin from the polyketide monomer 1,8- dihydroxynaphthalene (DHN). Basidiomycete fungi may produce melanin from phenols and catechol precursors, characterized by a dark brown colour, in contrast to the true black appearance of DHN melanin (Wheeler 1983, Butler et al. 2001). Taylor et al. (1988) argue that T. versicolor produces melanin by the catechol pathway, and Wheeler and Bell (1988) indicate that the presence of phenols produces dark pigmentation of the wood substrate, especially at pH levels above 7.
The fungal specificity to pH tolerance is reflected by the evolution of each fungus within its ecological niche and the role it plays in the overall degradation process. The hydrogen ion concentration in the wood at any given time, orchestrates the inhibition or stimulation of spore germination or mycelium propagation of the various fungi present in wood at any time. Pearce (1991) and Schmidt (2006) argue that the pH of the wood substrate varies within the tree, and this factor can influence enzymatic activity by genetic regulation, with direct impact on wood degradation and metabolism. The pH of wood also changes in various stages of decay, due to fungal metabolic regulation activity (Humar et al. 2001), assuring the advance of degradation by sequential colonization by various fungal species. All of these aspects of pH variation play an important role in fungal biodiversity distribution and species succession as wood decays.
90 Although there are many studies on the effects of wood pH on fungi, the main focus is on the wood degradation, and there is no information available related to fungal pigmentation. The goal of this research was to determine to what extent the pH conditions influence fungal pigment formation, and whether the substrate pH variation is a determinant stress factor that could trigger pigmentation by spalting fungi. Two common North American wood species with contrasting natural spalting prevalence, sugar maple (Acer saccharum Marsh), which readily spalts (Robinson et al. 2012c), and American beech (Fagus grandifolia Ehrh.), which rarely spalts, were tested against X. polymorpha and T. versicolor. To extend the comparison of fungal melanin production behavior by different biosynthesis pathways, along with production of other types of pigments, we tested additional ascomycetes and basidiomycetes fungi in malt extract agar at different pH values. The ability to alter fungal pigments through pH changes in wood offers a chance to broaden the colour palette currently available with spalted woods. It also offers an avenue to spalt wood that is not easily spalted, a process that could add considerable value to underutilized hardwood species.
6.2 Materials and methods 6.2.1 Wood and fungi species selection
Two wood species harvested in southern Ontario were selected for testing: sugar maple (Acer saccharum Marshall) and beech (Fagus grandifolia Ehrh.) The average oven-dry specific gravity of the wood species used for testing was SG=0.68 for sugar maple and SG=0.74 for beech.
To test the variation of pigment formation among fungal species, nine fungi were selected based on their spalting ability, from which two fungi were also tested for variation of pigment formation among different strains. Wood testing involved three strains of the ascomycete Xylaria polymorpha (Pers.) Grev.: strains UAMH 11518, UAMH 11519, and UAMH 11520; and three strains of the basidiomycete Trametes versicolor (L.) Lloyd: strain MAD 697 from the Forest Products Laboratory in Madison, WI, USA, isolated from a cankered area of Fagus grandifolia in Vermont, USA; strain R105 from the Forest Products Laboratory in Madison, WI, USA, isolated from a dead branch of Malus sp. in New York, USA, and strain UAMH 11521, isolated from Acer saccharum in Houghton, MI, USA.
91 In vitro tests on agar media utilized the same strains of X. polymorpha and T. versicolor, along with an additional seven fungal species, one strain per species, as follows: Polyporus squamosus (Huds.) Fr. UAMH 11653, isolated from beech in Toronto, ON, Canada; Polyporus brumalis (Pers.) Fr UAMH 11652, isolated from sugar maple in the Toronto area, ON, Canada; Fomes fomentarius (L.) J.J. Kickx UAMH 11654, isolated from birch in Haliburton Forest, ON, Canada; and Inonotus hispidus (Bull.) P. Karst. F2037, of unknown origin. Staining fungi investigated for pigment formation were Monascus ruber Tiegh. UAMH416, isolated from silage in West Virginia, USA; Chlorociboria aeruginascens (Nyl.) Kanouse ex C.S. Ramamurthi, Korf & L.R. Batra UAMH 11655, isolated from sugar maple in Haliburton Forest, ON, Canada; and Scytalidium cuboideum (Sacc. & Ellis) Sigler & Kang UAMH 4802, isolated from Na-pentachlorophenate-dipped red oak lumber.
All fungi used for inoculation were grown on 95 x 15 mm petri dishes with 2% malt extract agar at 21°C for 2 weeks prior to inoculation. These conditions are considered the standard optimal growing conditions for most North American fungi (AWPA, 2009, E10-06).
6.2.2. Test procedure
6.2.2.1. pH test preparation
To avoid the influence of soil substrates on fungal pigment formation, the experiments were performed using a modified decay jar test with vermiculite instead of soil (Robinson et al. 2009b). Sugar maple and beech 14-mm cubes, nine replicates per set, were weighed and treated under vacuum for 1 h with potassium phosphate buffer adjusted to pH 4.5, 5, 5.5, 6, and 6.5. The pH range was selected within the normal values characteristic for wood. After treatment, wood blocks were weighed to determine retention, and kept overnight at 4°C for conditioning. One additional set was added as controls.
To expand on the fungal behavior in regard to pH variation of the substrate, petri dishes of 2% malt extract agar, five replicates per set, were prepared with distilled water adjusted to a pH range from 2 to 8 in increments of 0.5. One additional set per treatment was used without fungus inoculation to measure the final pH of the media, and one additional set without any treatment of the distilled water was added as a control.
92 6.2.2.2. Inoculation and incubation
Culture jars with vermiculite and distilled water were sterilized in an autoclave for 30 min and cooled overnight in a laminar flow hood. Wood blocks cut into 14-mm cubes were first conditioned at 50°C to provide an initial dry weight and then surface steam sterilized for 30 min under conditioned pressure at 100°C, and placed in vermiculite after cooling, with three blocks placed per jar. A strip of inoculum of roughly 0.5 x 2 cm was placed on top of the wood blocks. Inoculated jars were incubated at 27°C ± 2°C and 80% ± 5% relative humidity for 8 weeks for T. versicolor strains, and 10 weeks for X. polymorpha strains. At the end of the incubation period, blocks were removed from jars, gently brushed to remove mycelium and any traces of vermiculite, and weighed before and after overnight oven drying at 50°C to determine moisture content and mass loss.
Petri dishes with 2% MEA (malt extract agar) adjusted to various pH values were inoculated and incubated at 21°C for 4 weeks.
6.2.2.3. Pigment assessment
Dried wood samples were scanned with an Epson WorkForce 500 scanner at 2400 dpi, first on one external side with the most pigment occurrence, and then on an internal face, after the blocks were cut in half to expose a radial section. Zone demarcation lines that define the fungal colony in wood (zl), and pigment deposition in clusters or general pigmentation (p), were considered for analysis. External and internal pigment evaluation was performed with Scion Image software, following the protocol described in Robinson et al. (2009a). Data were analyzed with a one-way ANOVA followed by Tukey HSD using SAS, version 9.2.
Fungal cultures that produce pigments other than melanin were evaluated in petri dishes for red, blue, yellow, and green pigment formation using a CIE L*a*b* Konica Minolta CM-2002 spectrophotometer, and only values for a* (green/red) and b* (blue/yellow) space color parameters were utilized. For melanin formation in pure fungal culture on agar, petri dishes were scanned with an Epson WorkForce 500 scanner at 2400 dpi, using the same pigment evaluation method in Scion Image software.
93 6.2.2.4. Estimation of initial conditions and changes of substrate pH
To estimate changes in wood pH after treatment with the phosphate buffer with adjusted pH, three sets of readings and five representative replicates per treatment were measured. Wood samples were treated, conditioned, and steam-sterilized, and pH measurements were recorded before and after complete exposure to decay fungi. The pH value was determined by an extraction method; wood samples dried at 50oC overnight were ground on a Wiley mill and passed through a 40-mesh screen, and 1 g of wood dust from each sample was placed in a glass vial. Ten milliliters of previously boiled and cooled distilled de-ionized water was added to each vial. The mixture was stirred for 5 min. and kept in a closed vial for 25 min. The extract was filtered and the pH of the solution was measured with a glass electrode on a digital Orion SA520 pH meter.
To avoid precipitation of media in acid solution during sterilization, 16.8g of MEA powder from Difco Laboratories was initially diluted in 150 g distilled water and 350 g of phosphate buffer adjusted to the required pH with hydrochloric acid (HCl), and the two solutions were mixed after sterilization and then poured into petri dishes. The adjusted pH of MEA media was measured from the liquefied substrate in five replicates per treatment and in the control.
6.3 Results 6.3.1 Initial conditions
The methodology used for pH determination provides a close estimate of the true value of wood pH (Campbell and Bryant 1941), and should be adequate for comparison purposes. The purpose of the study on initial conditions was to determine pH change of treated wood with the phosphate buffer and the pH change after fungal decay. The results show that after treatment, the pH values of the two wood species were similar within the treatments, and control beech samples had an average pH of 4.29, slightly more acidic than sugar maple samples, which measured on average pH of 4.72. As shown in Table 6.1, the buffer capacity of the wood influenced the final pH values of phosphate buffer treated samples. For the treatment with phosphate buffer adjusted to pH 5 it was observed that wood samples of both wood species recorded on average a minor drop of pH value, for unknown reasons.
94
Table 6.1. pH variation in beech and sugar maple samples after treatment with phosphate buffer at different pH-values
Wood sp./Treatment 4.50 5.00 5.50 6.00 6.50 Control samples
Beech (average) 4.85 4.65 4.93 5.26 5.46 4.29 Standard deviation 0.22 0.16 0.14 0.20 0.24 0.23
Sugar maple (average) 4.88 4.87 5.00 5.32 5.53 4.72 Standard deviation 0.07 0.13 0.05 0.03 0.34 0.05
Measurements of the MEA after setting in petri dishes indicate that the pH adjustment was consistent and the treatment was reliable. Untreated media had pH = 5.5 on average, while treated media had an average pH close to the target value (Figure 6.1).
Figure 6.1 pH value of MEA measured after setting of media treated with phosphate buffer for pH adjustment.
95 6.3.2 Influence of induced condition on decayed wood
6.3.2.1 pH changes
At the end of the incubation period, wood samples were measured for pH values. The three Trametes versicolor and Xylaria polymorpha strains had similar responses to the pH treatments so their results are combined in Figure 6.2 and 6.3. Trametes versicolor demonstrated high variability of final pH for the different pH treatments treatment at pH 4.5 to 5.5, but with little differentiation on the final pH values for treatments of pH 6 and higher. Beech samples generally had higher final pH values for the acidic treatments compared with maple. Control samples registered a drop of the pH for both wood species after decay to approximately pH 4 (Figure 6.2).
Figure 6.2 Final pH value in beech (b) and sugar maple (sm) sample treated with phosphate buffer, decayed by T. versicolor, strains: tv1 - Mad 697, tv2 - R105, tv3 - UAMH 11521. Error bars represent one standard deviation.
Figure 6.3 Final pH value in beech (b) and sugar maple (sm) sample treated with phosphate buffer, decayed by X. polymorpha, strains: x1 – UAMH 11518, x2 – UAMH 11519, x3 – UAMH 11520. Error bars represent one standard deviation.
96
Xylaria polymorpha demonstrated more consistency regarding the final pH value measured. The pH of control samples with this fungus increased from pH 4.3 up to 5.7 for beech and from approximatelly pH 4.7 up to 5.6 for sugar maple, on average (Figure 6.3).
In agreement with the known tendency of fungi to modifying the pH of the substrate to more suitable values, to more acidic values in the case of basidiomycete fungi, and more alkaline values in the case of ascomycetes fungi (Humar et al. 2001), both wood species decayed by T. versicolor had a pH between 4 and 5, lower than that for wood decayed by X. polymorpha, with a pH range from 5 to 5.8.
6.3.2.2. Moisture content and mass loss
There was no significant corelation between mass loss and pH levels. It was found that beech samples inoculated with T. versicolor and X. polymorpha had the lowest mass loss rates (14% and 6%, respectively) for pH 5 treatment, with moisture contents at 50% and 48%, respectively (Figure 6.4).
Sugar maple samples treated with pH 5.5 buffer inoculated with T. versicolor had the lowest mass loss (15%) of samples inoculated with this species, and also the lowest moisture content of wood samples (about 55%), comparable to the moisture content acquired by wood without fungal activity. This suggests that the fungus activity could have been partially inhibited by the treatment. Sugar maple inoculated with X. polymorpha had a mass loss under 7% for all treatments except for the pH 6.5 treatment, which showed no inhibition, and had a mass loss comparable to that of the control samples (28%) (Figure 6.4).
97
A B
C D
Figure 6.4 Mass loss (A, C) and moisture content (B, D) of decayed sugar maple (sm) and beech (b) samples by T. versicolor (tv) and X. polymorpha (xp). Error bars represent one standard deviation.
6.3.2.3. Pigment formation
To test the importance of wood pH for fungal pigment formation, a one-way ANOVA was run for each wood species, followed by Tukey’s HSD at α = 0.05, with fungal inoculation and pH treatments as independent variables. Pigment formation was significantly higher for beech treated with pH 4.5 and inoculated with T. versicolor for external pigmentation (P<0.0001) and for external zone lines (P<0.0001), and for pH 5 and inoculated with X. polymorpha for external pigmentation (P=0.0004) (Figure 6.5 A). In sugar maple samples, T. versicolor and X. polymorpha reacted at treatment with pH 4.5 buffer with significant external zone lines and pigment formation (P<0.0001), and T. versicolor produced significantly more internal zone lines in samples treated with pH 5.5 (P<0.0001) (Figure 6.5 B).
98
A
B
Figure 6.5 Pigment formation in beech (A) and sugar maple (B) treated with phosphate buffer at various pH values, inoculated with T. versicolor (tv) and X. polymorpha (xp). ezl – external zone lines; ep – external pigmentation; izl – internal zone lines; ip – internal pigmentation. The horizontal axis represents the buffer pH used for treatment vs final pH. Error bars represent one standard deviation. Different letters represent significant differences at α = 0.05 within each category.
99 6.3.3 Influence of pH variation in pigment formation on agar
Statistical analysis of data obtained from scanned petridiches indicates that significant fungal pigmentation occurred between pH 4 and 5.5 for most fungi tested, values that also comprised the range of the maximum fungal growth rate (Table 6.2). Trametes versicolor and X. polymorpha, tested as multiple strains, exhibited growth variation within strains. In agar, maximum pigmentation was registered at pH 4 and control samples for two strains of T. versicolor, while one strain developed no pigmentation. Two of the X. polymorpha strains also exhibited maximum pigmentation at pH 4, while one strain expanded the pH range for highest pigmentation from 4 to 5.
CIE L*a*b* color space paramenters obtained from measurements with spectrophotometer, average of three readings, were statistically analised in the case of staining fungi. The green fungus C. aeruginascens displayed a change in colour spectra with the pH variation, within the a* color space parameter (red/green). In acidic media the fungus produced a yellow-green pigment that became dark green in the neutral pH, and slowly faded into a light green-brown pigment, with significant maximum pigmentation at pH 2.5 and 5.
The pigment produced by M. ruber evolved from light yellow in the acidic range to a bright red in the basic range, with maximum pigment intensity and growth at pH 5.
For the staining fungus S. cuboideum, two types of pigmentation were noted: a red pigment measured in a* color space parameter, was constantly produced at all pH values of the agar, with maximum intensity registerd at pH 6 as well as in control samples,, and a dark-blue pigmentation measured in b* color space parameter, which was absent in acidic conditions and reached significant maximum intensity at pH 8.
The results of the experiments set up on malt extract agar were consistent with the tests on wood substrate in regard to the range of pH in which fungal pigmentation occurs, and is important for industrial application. Most of fungi tested showed pigmentation of the mycelia between pH 4 and 5.5, and both T. versicolor and X. polymorpha showed distinctive dissimilarity among strains.
100 Table 6.2. Fungal pigment formation in MEA at various pH values. Fungal growth in culture: intermediate = - , maximum = ¢; minimum values were marked by blank space. Melanin producing fungi were analized based on SCION image analysis, and stainig fungi were statistically analized based on CIE-L*a*b* measurements. Means with the same letter are not significantly different.
treatment control pH pH pH pH pH pH pH pH pH pH pH pH pH fungal specie 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8
T. versicolor !! ! " " ! ! ! ! ! " " !" !" Mad697 B B B B A B B B B B B B B B
T. versicolor ! " " " " ! ! !! " R105 A C C CB B A C C C C C C C C
T. versicolor ! " ! ! ! ! ! ! ! ! ! " UAMH 11521 A C C CB B A C C C C C C C C
P. squamosus ! " " " " ! " " " " UAMH 11653 BA B B B B BA BA A BA BA BA BA B B
P. brumalis ! " " ! ! ! ! ! ! ! " " " UAMH 11652 DE F F FE BC BC BA BA A A F F F F
F. fomentarius ! " " " " ! " " " " !!" UAMH 11654 DEC E E BDAC BA A BAC BDAC BADC DEC DE DE E E
I. hispidus ! " " " " " ! " " " " " F2037 EGDFC G G BAC EGDFC A BAC BDAC BA EBDACEBDFC EGDF GF EGF
X. polymorpha ! - - - - - ! ! - - - - UAMH 11518 B D D CB B A CBD CBD CD D D D D D
X. polymorpha ! - - - - - ! ! ! - - - UAMH 11519 A D D D D A B CB CD CD D CD D D
X. polymorpha ! - - - - ! ------UAMH 11520 BA E CD BC BCD BA BA A ECD E E E E E
C. aeruginascens ------! - - - - UAMH 11655 B D A D D B B A D B C D D D
M. ruber ------! ------UAMH416 BA B B B BA BA BA A BA BA BA BA BA BA (a*-red pigment)
S. cuboideum ! - - ! ! ! ! ! ! ! - - - - (a*-red pigment) A BC BA BAC BAC BC BC BA BC A DC ED EF F (b*-blue pigment) EDF ED F EF EDF ED D EDF ED EDF C BC BA A
UAMH4802
101 6.4 Discussion
Wood pH changes differently based on the wood and fungal species interaction. Wood samples used in these experiments had an initial average pH value of 4.29 for beech and 4.72 for sugar maple, as measured in control samples, and after treatment with buffer solution at different pH values, beech samples kept their acidity compared with those of sugar maple samples. The specific wood chemistry and physical properties of each species trigger specific fungal gene expressions according to the circumstances. The wood pH affects fungal cell capacity for nutrient uptake by influencing the net charge of the outer membrane surface and cytoplasm. The degree of dissociation of nutrients by ionization of organic acids or bases in the wood substrate is also pH dependent. In response, fungi alter the pH or the substrate to create favorable condition for wood decomposition (Griffin 1994, Deacon 1997) by genetic regulation of selective uptake and exchange of ions. In agreement with those observations, after eight weeks of incubation, T. versicolor modified the pH of both beech and sugar maple towards acidic values (4.07 to 4.75 for beech and 3.99 to 4.70 for sugar maple), compared with X. polymorpha, which modified the pH to slightly higher values (5.17 to 5.7 for beech, and 5.32 to 5.60 for sugar maple).
The modification of the pH of wood substrates through decomposition, require variable time for wood structure collapse and fungal decay strategies, based on fungal species involved in the process (Lambert et al. 1980, Holmer et al. 1997). The average mass loss of samples inoculated with the ascomycete X. polymorpha was 8% over the range of pH 4.5 to 6.5 treatment in beech, and 6 % average in sugar maple after 10 weeks of incubation. The average mass loss by Trametes versicolor was 23 % for beech and 20 % for sugar maple over the range of pH 4.5 to 6.5 treatment, after 8 weeks of incubation. Although the treatments with adjusted pH buffer were not statistically significant for decrease of mass loss, there are indications that treatment with buffer at pH 5 was the most efficient in minimizing wood degradation (mass loss) in beech for both fungi, while sugar maple decomposition was lowest at pH 4.5 for X. polymorpha and at pH 5.5 for T. versicolor. As expected, the degradation by T. versicolor at the above indicated treatments with low level of decomposing activity correspond with the lowest final moisture contents, while the final moisture content values produced by X. polymorpha were comparable in all samples, probably due to overall slow degradation ability characteristic to this fungus. The direct proportionality between mass loss and moisture content, as a result of wood enzymatic
102 degradation that results in release of water molecules, was also demonstrated by Eriksson et al. (1990). Specific fungal enzymes use molecular oxygen to oxidize phenols and polyphenols from the wood substrate for melanin biosynthesis. It was inferred by Duckworth and Coleman (1970) and Nagai et al. (2003) that those types of enzymes are stable over the pH range 4 to 7. Similarly, most fungi produced optimum growth in the same pH range (4 to 7), all well within the normal range of the wood (Griffin 1994). The results from this experiment are in agreement with these findings. Significant pigmentation produced by T. versicolor was recorded in beech treated with pH 4.5, and in sugar maple at pH 4.5 for external pigmentation, and pH 5.5 for internal zone lines. Those treatments provided a similar initial pH measured value of about 5 after the treatment, a condition that provides intermediate fungal activity. Xylaria polymorpha reacts mainly to low pH. Significant pigmentation was observed for beech treated with pH 5 (after treatment pH 4.65 on average), and for sugar maple treated with pH 4.5 buffer (after treatment pH 4.88 on average). Wood treatments that stimulate pigmentation did not significantly inhibit fungal decomposition activity. This could be explain by the necessity of a certain fraction of lignin enzymatic decomposition into phenolic products that will serve as melanin precursors.
The results from the agar experiment reveal an interesting trend of pigmentation for staining fungi. All three species tested were capable of producing pigmentation for all pH values tested, with variation of colors and intensities, indicating their effectiveness in limiting the acces of other fungi within the colonized space. Monascus ruber produced pigmentation from yellow at pH 2 to red at pH 8. Chlorociboria aeruginascens had a limited range of pH favourable for pigmentation from pH 2.5 to 7.5, with a color range from yellow green to brown green, respectively. Scytalidium cuboideum developed pigmentation from red at pH 2 to dark blue at pH 8. The red pigment was present at all pH values of the agar, with maximum intensity registered at pH 6. Dark-blue pigmentation was absent in acidic conditions, reaching maximum intensity at pH 8. These findings confirmed previous research by Chidester (1940) that indicated the production of tyrian blue pigment in addition to the red pigmentation in culture media of S. cuboideum, and research of Golinski et al. (1995) that observed a change in colour of the red pigment to blue, when dissolved in solvents of higher polarity (methanol for example) or in basic solutions. The functional role of this pigment might have an important role in fungal virulence. The fungus does not cause significant wood cell wall decay (Schmidt and Dietz 1985); its tolerance and adaptability to a wide range of substrate pH values might be reflected in
103 its ability to colonize many types of wood species and to compete with other fungi (Robinson et al. 2011ac).
6.5 Conclusions
This research indicates that pH of the substrate potentially plays an important role in fungal melanin formation, which occurs within the pH range from 4.5 to 5.5. Wood-inhabiting fungi demonstrated the capacity to produce melanin at pH values within the normal range of the wood substrate pH, even when stresses such as temperature, moisture content variation, and antagonistic reactions were absent. Fungi T. versicolor and X. polymorpha tested on wood substrates were mainly pigment-active with the pH 4.5 treatment. Xylaria polymorpha can also produce pigmentation in beech treated with pH 5. All stainig fungi tested produced pigmentation for all pH values tested, with variation of colors and intensities, specific to each fungal species.
While it is apparent that the pH of the wood substrate is directly related to melanin formation, pH is not the sole determining factor for melanin formation. A multitude of other factors, both environmental and chemical, could also be responsible and interact within a complex relationship. Future research should investigate gene expression and regulation to identify the process by which information encoded in fungal DNA directs the synthesis of melanin, to further elucidate other highly probable pathways of melanin creation and stimulation.
104
Chapter 7
Fungal melanin formation from catechol and L- Dopa precursors
7.1 Introduction and background
Fungi are known to produce various types of melanins by different biosynthesis pathways in wood substrate (Wheeler 1983, Bell and Wheeler 1986, Fogarty and Tobin 1996, Butler and Day 1998, Henson et al. 1999, Jacobson 2000, Butler et al. 2001). The most common melanin precursors are by-products derived from wood degradation, and is generally assumed that ascomycetes fungi like Xylaria polymorpha predominantly synthesize melanin from 1,8 dihydroxynaphthalene (DHN), while basidiomycetes fungi like Trametes versicolor usually produce catechol melanin from γ – glutaminyl – 3, 4 – dihydroxybenzene precursor, also known as GDHB melanin, and more rarely from L – dihydroxyphenylalanine (L- Dopa) (Turner 1971, Wheeler and Bell 1985).
Catechol melanins are considered the most common black pigments in nature; in addition to fungi, they are also produced by plants and animals (Nicolaus et al. 1964). In the process of fungal melanin biosynthesis, it was demonstrated that catechol is an activator of tyrosinase, an enzyme involved in the last step of melanin production (Duckworth and Coleman 1970). According to Piattelli et al. (1965), and based on the similarity between the natural pigment and synthetic pigment produced from enzymatic oxidation of catechol, the spores of Ustilago maydis (Persoon) Roussel are protected by a pigment similar to catechol melanin. The presence of catechol was reported to restore pigment formation in fungal albino mutants of Verticillium dahliae Kleb., (Wheeler et al. 1976, Wheeler et al. 1978). However, it appears that the pigmentation of this fungus does not rely solely on catechol precursors, since L-Dopa or indolic derivatives of L-Dopa have also been proposed as substrates for melanin biosynthesis by V. dahliae and several other fungi (Ellis and Griffiths 1974, 1975).
A few fungi synthesize melanin via L-Dopa, in a pathway similar to mammalian melanin biosynthesis, with two possible originating molecules, L-Dopa or tyrosine. The L-Dopa precursor molecule is oxidized to dopaquinone by copper-dependent laccase, while the alternative tyrosine precursor is first converted to L-Dopa and then to dopaquinone. The same
105 enzyme, tyrosinase, carries out both steps (Land et al. 2004, Langfelder et al. 2003, Riley 1997, Williamson 1997). Both melanin pathways were found to be expressed by the ascomycete Sporothrix schenckii Hektoen and C.F.Perkins, a common soil fungus that decomposes plant material and is also a human pathogen causing subcutaneous infection (Almeida-Paes et al. 2009). The basidiomycete Cryptococcus neoformans (San Felice) Vuill. has a complex ability to assemble melanin pigments from various phenolic substrates like L-Dopa and other catecholamines (Eisenman et al. 2007). Research by Zhong et al. (2008) also described the complex process of melanin formation in C. neoformans; it was demonstrated that melanin polymers contain chemical components derived from sources other than L-Dopa polymerization, that covalently link L-Dopa derived products and polysaccharide components and may attach this pigment to cell wall structures.
Melanin polymers are difficult to study and analyze, in part due to their complex structures that vary with the nature of the host substrate and with physical condition of the environment. Also it is difficult to isolate melanin molecules from the fungal tissue within the host substrate without alteration of its own functional structure. The main types of melanin biosynthesis in fungi can be tested using the property of specific chemical substances to inhibit fungal melanin formation. Tricyclazole, along with pyroquilon and phthalide are known fungicides commercially use for control of rice blast disease caused by Pyricularia oryzae Cavara (Kubo and Furusawa, 1991; Sisler, 1986). The presence of these compounds inhibits the enzymatic reduction of hydroxynapthalene compounds to scytalone and vermelone precursors of DHN melanin (Wheeler and Greenblatt 1988). The fungicide tricyclazole can cause red/brown pigmentation instead of the normal dark/black appearance of melanin even at low concentrations (2-5 ppm) in the substrate. However, it was reported that some fungal species that produce DHN melanin do not promptly react to such low concentrations (Butler et al. 2009). The DHN melanin inhibiting compounds have no effect on Dopa melanins, which react only to specific inhibitors like tropolone, kojic acid, 2-mercaptobenzimidazole and diethyldithiocarbamate (Kahn and Andrawis 1985, Yoshimoto et al. I985).
Although several spalting fungi were studied for the melanin type produced, there is no research available that investigates to what extent the presence of various melanin precursors in wood substrate influence zone line formation or pigmentation. Also, it is not known if fungi are able to assemble melanin from multiple precursors. This study was designed to investigate melanins
106 produced by several lignolytic fungi involved in the spalting process, and their reaction to the addition of catechol and L-Dopa in the substrate.
7.2 Material and Methods 7.2.1 Wood and fungal species selection
Wood species tested were harvested within southern Ontario, based on their ability to spalt: sugar maple (Acer saccharum Marshall) which develops extensive fungal pigmentation and zone lines, and beech (Fagus grandifolia Ehrh.) which more rarely spalts. The average oven-dry specific gravities of the samples tested were SG=0.68 for sugar maple and SG=0.74 for beech.
Three fungi were selected based on their pigment production ability: the ascomycete Xylaria polymorpha (Pers.) Grev., and two basidiomycetes fungi, Trametes versicolor (L.) Lloyd and Inonotus hispidus (Bull.) P. Karst., (Table 7.1). Fungi used in the experiment were grown on 95x15 mm Petri dishes with 2% malt extract agar at room temperature for two weeks prior to inoculation.
Table 7.1 Substrate and fungal species selection for catechol and L-Dopa test. Substrate Fungal species Culture collection number Malt Extract Agar (MEA) Trametes versicolor Mad 697 Sugar maple (SG=0.68) Xylaria polymorpha UAMH 11520 Beech (SG=0.74) Inonotus hispidus F2037
7.2.2 Test procedure
7.2.2.1 Test preparation, inoculation and incubation
To test the influence of synthetic precursors on fungal pigment production in wood substrate, wood blocks were inoculated with fungi following a modified protocol for a decay jar test; soil was replaced with vermiculite, as outlined in Robinson et al. (2009b), to avoid influence of soil substrates on pigment formation. Sugar maple and beech 14 mm cubes, nine replicas per set, were weighed and then treated under vacuum for one hour with chemical solutions of catechol (Sigma-Aldrich) and L-Dopa (Sigma-Aldrich) adjusted for 1ppm, 7ppm, 10ppm, 70ppm, 100ppm, and 700ppm retention. The levels were chosen based on the results of preliminary tests. One additional set was added in each test as control samples. After treatment, wood blocks
107 were kept overnight at 40 C for conditioning, followed by steam sterilization and inoculation in sterile jars containing vermiculite. Based on their virulence, jars inoculated with T. versicolor were incubated for eight weeks, X. polymorpha jars were incubated for ten weeks, and I. hispidus jars were incubated for 12 weeks. At the end of the incubation period, blocks were evaluated for decay (weight loss of samples oven dried before and after exposure) and external/internal spalting amounts.
To test the influence of catechol and L-Dopa on fungal growth in culture media, petri dishes with 1ppm, 7ppm, 10ppm, 70ppm, 100ppm, and 700ppm concentrations in 1% agar were prepared. Fungi were inoculated in petri dishes and incubated at 210C. The mean culture growth and pigmentation diameter was evaluated by measurements of the diameter in petridishes, recorded using a digital caliper every two days in the first week and weekly for up to one month.
7.2.2.2 Pigment assessment
For pigment assessment, wood samples were scanned with Epson WorkForce 500 scanner at 2400 dpi on one external surface face with the highest pigment occurrence, and on an internal face, after the blocks were cut in half to expose a radial section. External and internal pigment evaluation was performed with Scion Image software, following the protocol described in Robinson et al. (2009a), and data recorded represents the percentage of pigmentation from the total image of scanned external or internal surface. To determine the most significant treatment for each fungus, data were analyzed with a one-way ANOVA followed by Tukey HSD using SAS, version 9.2.
7.2.2.3 Tricyclazole test
To test the inhibition of DHN melanin formation, 14 fungi were investigated. In addition to T. versicolor, X. polymorpha, and I. hispidus tested for pigmentation, the following fungal species were added to the tricyclazole test: Polyporus squamosus (Huds.) Fr., Kretzschmaria deusta (Hoffm.) P.M.D. Martin, Phellinus igniarius (L.) Quél., Phoma exigua Sacc., Fomes fomentarius (L.) Fr., Fusarium sp., Chlorociboria aeruginascens Kanouse ex C.S. Ramamurthi, Korf & L.R. Batra, Polyporus brumalis (Pers.) Fr., Scytalidium cuboideum (Sacc. & Ellis) Sigler & Kang, Armillaria novae-zelandiae (G. Stev.) Boesew., and Ophiostoma sp.. They were cultured on in MEA substrate at 10 ppm concentration tricyclazole (5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole, 75% technical grade BEAMTM, Eli Lilly & Co., Indiana) kindly
108 donated by Dr. Michael J. Butler of the University of Western Ontario, London, Canada. The agar test investigated three replicas per fungus and per treatment for the 14 fungal species, and one additional control set.
The same tricyclazole concentration (10ppm) was tested in beech and sugar maple wood substrates, on 14 mm cubic wooden blocks with nine replicates per set of three fungal species (T. versicolor, X. polymorpha, I. hispidus), and one set of control samples. Test preparation, inoculation and incubation followed the same procedure as described above. The influence of tricyclazole on melanin inhibition was recorded as positive or negative (i.e. no effect), based on visual assessment of pigment color modification (Butler et al. 2009).
7.3 Results 7.3.1 Fungal reaction to catechol and L-Dopa in agar substrate
Trametes versicolor incubated in MEA with 10 ppm catechol concentration reached a maximum level of pigmentation after one week of incubation, followed by melanin degradation, while colonies exposed to 100 ppm catechol maintained a linear extension of pigmented area. Control samples and 1 ppm concentration of catechol produced no pigmentation (Figure 7.1A and Figure 7.2). The growth of the colonies was inhibited by catechol up to two weeks at 10 and 100 ppm concentrations, until the fungus managed to degrade and overcome the toxicity of the catechol additive to the substrate (Figure 7.1B).
When agar substrate was treated with L-Dopa, the process of pigmentation was initiated more rapidly at 100 ppm, reaching maximum intensity after one week from inoculation (Figure 7.3 A and B), after which the process of bleaching was initiated (Figure 7.4 1D and 2D).
The fungus was able to process both phenolic additives and the accumulation of pigmentation within the fungal mycelium was obvious in both cases. The main difference in pigmentation patterns was the more rapid decomposition of L-Dopa melanin than that of catechol melanin.
109
A B Figure 7.1 The effect of catechol on pigmentation (A) and growth (B) of Trametes versicolor culture in 1% agar. Data shown are the means of five replicates.
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Figure 7.2 Trametes versicolor colonies in 1% agar: 1A,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm catechol, C- 10ppm catechol, D-100ppm catechol on agar.
A B
Figure 7.3 The effect of L-Dopa on pigmentation (A) and growth (B) of Trametes versicolor culture in 1% agar. Data shown are the means of five replicates.
110
1A!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1B!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1C!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1D!
2A!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!2B!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!2C!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!2D! !
Figure 7.4 Trametes versicolor colonies in 1% agar: 1A,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm L-Dopa, C-10ppm L-Dopa, D- 100ppm L-Dopa on agar.
Xylaria polymorpha treated with 100 ppm catechol reached maximum pigmentation also at one week after inoculation followed shortly by complete degradation of melanin (Figure 7.5A and Figure 7.6). Although the colonies started to develop mycelium before control samples, the highest level of catechol concentration resulted in growth inhibition of fungal colonies (Figure 7.5B). The treatment with L-Dopa had no effect on either pigmentation or growth of X. polymorpha colonies (Figures 7.7 and 7.8). At high concentration of catechol, the fungus produced nonspecific pigmentation of mycelium indicating a possible similarity or tolerance for this type of phenolic compound. However, the L-Dopa compound showed no affinity with this fungus, which was able to degrade it without any melanin formation.
B A Figure 7.5 The effect of catechol on pigmentation (A) and growth (B) of Xylaria polymorpha culture in 1% agar. Data shown are the means of five replicates.
111
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Figure 7.6 Xylaria polymorpha colonies in 1% agar: 1 A,B,C,D, after one week from inoculation; 2 A,B,C,D, after one month from inoculation; A-control, B-1ppm catechol, C- 10ppm catechol, D-100ppm catechol on agar.
A B Figure 7.7 The effect of L-Dopa on pigmentation (A) and growth (B) of Xylaria polymorpha culture in 1% agar. Data shown are the means of five replicates.
1A!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1B!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1C!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1D!
2A!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!2B!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!2C!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!2D! ! Figure 7.8 Xylaria polymorpha colonies in 1% agar: 1A,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm L-Dopa, C-10ppm L-Dopa, D-100ppm L-Dopa on agar.
112 At higher concentrations of phenolic substance in agar, a light coloration of media was observed, due to weak oxidation in the presence of atmospheric oxygen. The degradation of L- Dopa was visible as bleaching of the substrate with the increase of mycelium expansion (Figure 7.9). Area of pigmentation by Inonotus hispidus was not affected by catechol, but the intensity of pigmentation was the highest at 100 ppm concentration, accompanied by the highest inhibition of fungal growth (Figure 7.9A,B and Figure 7.10). However, the pigment produced under catechol influence appeared to be much darker than the yellow-brown of the natural pigmentation. The pigmentation produced under L-Dopa treatment was much closer in color to the natural occurrence, and also this substance did not inhibit growth (Figure 7.11). The only notable change in pattern of pigmentation was the more pronounced zonation that marked the colony boundaries, which was accompanied as usual by the diffusion of the pigment within the agar substrate (Figure 7.12 2D).
A B Figure 7.9 The effect of catechol on pigmentation (A) and growth (B) of Inonotus hispidus culture in 1% agar. Data shown are the means of five replicates.
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Figure 7.10 Inonotus hispidus colonies in 1% agar: 1A,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm catechol, C-10ppm catechol, D-100ppm catechol on agar.
113
A B Figure 7.11 The effect of L-Dopa on pigmentation (A) and growth (B) of Inonotus hispidus culture in 1% agar. Data shown are the means of five replicates.
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Figure 7.12 Inonotus hispidus colonies in 1% agar: 1A,B,C,D, after one week from inoculation; 2A,B,C,D, after one month from inoculation; A-control, B-1ppm L-Dopa, C-10ppm L-Dopa, D- 100ppm L-Dopa on agar.
7.3.2 Pigment formation in wood substrate
To test the influence of catechol and L-Dopa on fungal pigment formation in wood samples, a one-way ANOVA was run, followed by Tukey’s HSD at α = 0.05, with fungal inoculation and induced compounds retentions as independent variables, for each wood species.
Internal pigment production (ip), external zone lines formation (ezl) and external pigmentation were significantly higher for sugar maple samples inoculated with T. versicolor and treated to 700 ppm catechol retention, while internal zone line formation was significant for treatment with 100 ppm catechol (P<0.0001) (Figure 7.13A).
114 There was no significant effect in beech samples; however there was a similar tendency for highest internal pigmentation and zone line formation for 100 ppm catechol retention (Figure 7.13A).
Figure 7.13 Pigmentation and zone line formation by T. versicolor in wood substrate. Data shown are the means of nine replicates. The graph shows means of pigmentation surface in percentages. Bars of a given pigmentation type with different letters represent significantly different means; ezl = external zone lines; ep = external pigmentation; izl = internal zone lines; ip = internal pigmentation. A- T. versicolor in beech treated with catechol; B- T. versicolor in sugar maple treated with catechol; C- T. versicolor in beech treated with L-Dopa; D- T. versicolor in sugar maple treated with L-Dopa.
Treatment with L-Dopa did not produce statistically significant differences for any of the retention values in both sugar maple and beech samples. However, there was a noticeable increase in pigmentation compared with control samples, which was more evident for sugar maple (Figure 7.14). Based on mass loss of tested wood samples, fungal decomposition activity under the influence of catechol treatment was inhibited in sugar maple, while in beech there was
115 no change in mass loss compared with control samples. L-Dopa treatments had no impact on mass loss at any of the retention values tested (Table 7.2).
Table 7.2 Summary of data for untransformed mass loss for tested fungi in sugar maple and beech. Data shown are the means of nine replicates. Mean % mass loss /standard deviation T. versicolor X. polymorpha I. hispidus Catechol Sugar maple control 14.3/2.2 7.9/2.3 7.3/1.3 1ppm 10.9/1.9 7.1/0.7 5.8/1.4 7ppm 5.2/2 4.5/2.2 6.2/1.9 10ppm 7.2/3.5 4.4/2.1 5.8/2.9 70ppm 8.0/3.4 6.0/2.7 5.3/1.5 100ppm 5.7/3.9 6.1/1.1 6.3/2.1 700ppm 3.4/5.2 5.5/4.0 5.8/1.9 Beech control 13.1 /8.1 6.4/2.0 6.9/1.0 1ppm 8.0/7.9 7.1/1.9 5.5/1.0 7ppm 11.3/9.1 4.3/3.2 4.9/1.6 10ppm 11.6/6 5.7/1.9 5.9/1.6 70ppm 13.3/6.6 5.7/3.2 4.8/1.1 100ppm 13.2/6.8 6.9/2 5.7/1.2 700ppm 9.7/6.3 6.6/5.2 6.3/1.2 L- Dopa Sugar maple control 14.4 /1.5 8.4/ 2.1 8.6/ 1.3 1ppm 13.1/2.5 8.8/1.4 11.1/1.5 7ppm 14.1/3.2 10.8/1.0 9.3/1.4 10ppm 14.6/2.6 8.1/1.2 10.6/1.5 70ppm 14.7/3.1 9.1/1.9 10.9/1.0 100ppm 14.4/3.2 9.7/2.4 11.3/2.9 700ppm 13.4/2.6 11.5/1.6 9.9/2.5 Beech control 13.9 /4.4 8.3/2.3 7.0/2.3 1ppm 8.9/2.9 6.1/1.7 6.6/2.1 7ppm 16.4/5.6 7.0/2.0 4.0/2.3 10ppm 15.2/5.8 7.7/1.3 3.7/2.1 70ppm 13.7/2.5 7.5/1.3 5.7/2.4 100ppm 13.0/3.4 7.2/2.7 4.6/1.4 700ppm 9.1/1.7 8.6/2.4 4.6/2.8
Pigmentation of X. polymorpha and I. hispidus were not significantly influenced by any of the two tested phenolic compounds introduced to sugar maple and beech substrate, except for internal pigmentation of X. polymorpha in beech at 100 ppm retention of L-Dopa (Figure 7.14 A-D, and Figure 7.15 A-D). Fungal decomposition activity of X. polymorpha was stimulated by L-Dopa in sugar maple, while catechol had a weak inhibitory effect on both wood species inoculated with I. hispidus (Table 7.2).
116
Figure 7.14 Pigmentation and zone line formation by X. polymorpha in wood substrate. Data shown are the means of nine replicates. The graph shows means of pigmentation surface in percentages. Bars of a given pigmentation type with different letters represent significantly different means at the 0.05 level of confidence; ezl = external zone lines; ep = external pigmentation; izl = internal zone lines; ip = internal pigmentation. A- X. polymorpha in beech treated with catechol; B- X. polymorpha in sugar maple treated with catechol; C- X. polymorpha in beech treated with L-Dopa; D- X. polymorpha in sugar maple treated with L-Dopa.
117
Figure 7.15 Pigmentation and zone line formation by I. hispidus in wood substrate. Data shown are the means of nine replicates. The graph shows means of pigmentation surface in percentages. Bars of a given pigmentation type with different letters represent significantly different means at the 0.05 level of confidence; ezl = external zone lines; ep = external pigmentation; izl = internal zone lines; ip = internal pigmentation. A- I. hispidus in beech treated with catechol; B- I. hispidus in sugar maple treated with catechol; C- I. hispidus in beech treated with L-Dopa; D- I. hispidus in sugar maple treated with L-Dopa.
7.3.3 Fungal reaction to tricyclazole, an inhibitor to DHN melanin
DHN melanin produced by fungi grown in the presence of tricyclazole fungicide changed from dark brown or black to a reddish-brown pigment. The clear distinction in melanin coloration between the treated and untreated cultures observed in X. polymorpha, K. deusta, P. exigua and Ophiostoma sp. indicates that the pigment produced by those fungi is DHN melanin. In T. versicolor, F. fomentarius and P. brumalis, there was no indication of pigment inhibition, as well as for non-melanin producing fungi Fusarium sp. and S. cuboideum. The pigment produced
118 by C. aeruginascens, known as xylindein was modified in color by the tricyclazole treatment. The pigment changed from bright green to slightly brown-green color (Table 7.3). The results of the tricyclazole test in wood substrate could not be clearly evidenced, due to the similarity of wood color with melanin color modification under tricyclazole treatment.
Table 7.3 Wood and fungal species selection for tricyclazole test. Negative (no effect) (-) or positive (changes in color) (+) reaction to tricyclazole indicates the DHN type of melanin; (na) color change of melanin could not be clearly differentiated from wood color. Substrate Fungal species Type of fungi Culture collection Reaction to number tricyclazole Malt Extract Trametes versicolor Basidiomycota Mad 697 - Agar (MEA) R105 - UAMH 11521 - Xylaria polymorpha Ascomycota UAMH 11649 + UAMH 11518 + UAMH 11519 + UAMH 11520 + Polyporus squamosus Basidiomycota UAMH 11653 - Inonotus hispidus Basidiomycota F2037 - Kretzschmaria deusta Ascomycota DT28510 + Phellinus igniarius Basidiomycota UAMH8178 - Phoma exigua Ascomycota UAMH6956 + Fomes fomentarius Basidiomycota UAMH 11654 - Fusarium sp. Ascomycota DAOM213213 - UAMH7066 - Chlorociboria Ascomycota UAMH 7615 + aeruginascens Polyporus brumalis Basidiomycota UAMH 11652 - Scytalidium cuboideum Ascomycota UAMH4802 - Armillaria novae- Basidiomycota UAMH7962 - zelandiae Ophiostoma sp. Asco mycota FTK387T + Wood species / specific gravity Trametes versicolor Basidiomycota Mad 697 na Xylaria polymorpha Ascomycota UAMH 11649 na Sugar maple / Inonotus hispidus Basidiomycota F2037 na SG=0.68 Fomes fomentarius Basidiomycota UAMH 11654 na Beech/SG=0.74
119 7.4 Discussion
The results from the in vitro test using T. versicolor, X. polymorpha and I. hispidus on agar and wood blocks indicates that addition of catechol and L-Dopa have various effects on fungal pigmentation.
The most significant pigmentation produced by T. versicolor was recorded for internal pigmentation and zone line formations in beech treated with 100 ppm catechol, while pigmentation in sugar maple was significant for external pigmentation and zone line formation when treated with catechol at 700 ppm. Comparison between wood species at certain treatment reveal that T. versicolor produced more pigmentation in beech when incubated in wood samples treated with catechol, while external pigmentation in sugar maple was higher in samples treated with L-Dopa. Comparison between treatments of the same wood species indicates significant higher overall pigmentation (ep, ezl, ip and izl) in beech with catechol, and higher external pigmentation on sugar maple with L-Dopa. The lowest mass loss was registered for sugar maple treated with catechol at 700 ppm, and coincides with the most significant pigmentation produced by T. versicolor. These findings are very significant for commercial spalting, where the goal is to obtain melanin production at low wood degradation.
The agar test of T. versicolor registered maximum pigmentation at week 1 and 2 in catechol at 10 ppm, followed by the degradation of melanin initially formed, and at week 4 in 100 ppm catechol. Maximum pigmentation with L-Dopa in the agar test was recorded in week 1 and 2 for L-Dopa treatment for 100 ppm retention, followed by melanin degradation. In agar, the culture growth was not significantly inhibited by any treatment.
Melanin produced by T. versicolor under tricyclazole treatment in agar test showed no color modification, indicating that this fungus do not produce DHN melanin type. Although the fungus was able to absorb and transform both catechol and L-Dopa into melanin, at high levels of concentration, the intensity of the pigment produced in agar, and the occurrence of internal pigmentation in wood blocks indicated that catechol is the most favourable phenolic precursor for melanin production by T. versicolor. Pigmentation and growth inhibition of T. versicolor cultures when catechol was introduced at early stages of colonization in agar, followed by degradation of catechol and stimulation of mycelium growth after 13 days, was also reported by Hart and Hillis (1974) and by Taylor et al. (1988). The catechol melanin is more susceptible to
120 degradation or bleaching if ligninase/melaninase enzymes produced by white rot fungi are well established (Butler et al. 1998a). This aspect might be an effect of pH modification by fungus to create a favourable environment for wood degradation and fungal growth, as demonstrated in Chapter 6.
The bimodal mechanism of melanin formation, as well as the differences in reaction to various wood species could be related to the genetic regulation in response to plant reaction to fungal infection. The toxic effect of phenolic compounds produced by trees in early stages of fungal infection is counteracted by the ability of fungi to absorb and transform those substances for individual protection (Eslyn and Aho 1982). The survival of T. versicolor is ensured by its ability to tolerate and utilize a larger array of phenolic compounds (Smith et al. 1981, Freer and Detroy 1982), until the fungus is well established and the enzymatic activity is effective to decompose accumulated phenols (Taylor et al. 1988).
The results of this research indicate that X. polymorpha was significantly more responsive in pigmentation to wood treatment with catechol, and the reaction to wood substrate variation was not significant. The same consistent activity of X. polymorpha was observed in the case of mass loss, which was also not significantly influenced by any treatment. The agar tests provide more information on the pigmentation reaction of X. polymorpha to substances tested. The reaction to catechol at 100ppm recorded significant growth inhibition and maximum pigmentation in week 1 followed shortly by melanin degradation. The incubation of X. polymorpha in L-Dopa had no significant influence on pigmentation and fungal growth, and the tricyclazole results clearly indicate that the fungus naturally produces DHN melanin type. The study indicates that X. polymorpha has also a bimodal mechanism for melanin assembly, although not all recognized phenolic precursors produce a reaction for pigmentation from this fungus.
Xylariaceous ascomycetes like Xylaria have often been regarded as white-rot fungi (Kirk 1971, Rogers 1979, Pointing et al. 2003); however, these fungi were also classified as soft-rot due to the distinctive longitudinal cavities in wood cell walls (Worrall 1997). Despite the large amount of information available on these fungi, intensely studied for the importance of secondary metabolites to the pharmaceutical industry (Abate et al. 1997, Espada et al. 1997, Stadler and Hellwig 2005), the biogenetic pathways that have developed for decomposition of wood and other organic substrates remain unclear (Stadler 2011). Little is known about the chemical changes in decomposition process of the Xylariaceae, in part due to lack of analytical work
121 dedicated to elucidation of the natural function of secondary metabolites between the endophytic and saprotrophic behaviour of Xylariaceae. The consistency of pigmentation and degradation abilities could be a consequence of the slow growth and degradation activity of X. polymorpha, and might just reflect the equilibrium in the fungus-host relationship in regard to the fungus reaction to phenolic compounds produced by trees in response to fungal degradation (Whalley 1996, Edwards et al. 2003). The fungus showed pigmentation reaction to most common phenolic melanin precursors derived from lignin degradation, catechol and DHN, and was able to readily degrade L-Dopa.
Inonotus hispidus is known to produce two phenolic pigments, the brown pigment hispidin [6- (3',4'-dihydroxystyryl)-4-hydroxy-2-pyrone] (Edward et al. 1961, Klaar and Steglich 1977) and a yellow metabolite hispolon [6-(9,10-dihydroxyphenyl)-3,5(E)-hexadien-4-ol-2-one] (Ali et al. 1996). The former is probably produced by phenylalanine oxidation (Perrin and Towers 1973a). When exposed to catechol there was no significant increase of pigment formation in either wood species (data not shown). However, in both agar and wood substrate tests, at 100 and 700ppm respectively of catechol concentration, the fungus synthesized significantly more pigmentation. Based on weight loss percentage in wood samples, neither catechol nor L-Dopa inhibited fungal activity, and the fungus was able to uptake and transform both phenols, without degradation of the pigment. The biosynthesis pathways by which those pigments are produced are still unresolved, although many precursors and protein bound compounds were reported as associated with hispidin production (Perrin and Towers 1973a, Perrin and Towers 1973b, Fiasson 1982, Gonindard et al. 1997). It was hypothesized that hispidin may polymerize during maturation of fungi and that the UV light initiates a sequential increase in the activity of enzymes involved in production of hispidin (Nambudiri et al. 1973, Vance et al. 1974).
The change in color of melanin pigments produced by K. deusta, P. exigua and Ophiostoma sp. in the presence of 10 ppm tricyclazole in MEA indicate the DHN nature of melanin produced by those fungi. An exception is the green-pigmented C. aeruginascens, which is known to produce the pigment xylindein (Blackburn et al. 1962) and it was never associated with melanin production. The change in color of xylindein in agar treated with tricyclazole might be related to the change in pH of MEA when treated with tricyclazole (see Chapter 6). The changes in color from black or brown to red-brown, specific to tricyclazole inhibition of DHM melanin were not
122 clearly distinguished in wood substrate due to interference of the initial wood color in the red spectrum.
7.5 Conclusions
This research indicates distinct reactions of tested fungi to catechol and L- Dopa. The bioassay has provided evidence that T. versicolor and I. hispidus were able to absorb and transform both substances into pigmentation in both agar and wood substrate, which indicates multiple biosynthesis pathways for melanin assembly. Xylaria polymorpha was able to utilize only catechol for pigmentation, and not L- Dopa. However, the tricyclazole test indicates that X. polymorpha as well as K. deusta, P. exigua and Ophiostoma sp. produce DHN melanin.
These results are particularly of interest for spalting wood production, indicating that pretreatment of wood substrate with catechol at 700 ppm may accelerate and enhance pigmentation when inoculated with certain basidiomycetes or ascomycetes fungi, while reducing the amount of wood degradation. Investigations on the pigmentation and melanin decomposition threshold should be taken into consideration to establish the optimal time of incubation for maximum pigmentation in spalting production.
123
Chapter 8
Microscopic investigation of fungal pigment formation and its morphology in wood substrates
8.1 Introduction and background
Microscopy is often used to characterize fungal morphology and to study pigmentation formation and ultrastructure, in particular, to analyze the effect of growing conditions, gene modification and inhibitory effects of antifungal treatments. The zone lines formed by certain fungi in the invaded wood tissues have presented an intriguing problem to scientists. In spite of the tremendous variety of structure, formation, and significance, fungal pigmentation is little understood, while the accounts of its study in the literature are numerous (Anagnost et al. 1994, Bell and Wheeler 1986, Butler et al. 2005, Robinson et al. 2010b). However, comparative studies of pigment formation in various fungi in the same wood substrate, or of one fungus in various wood substrates are scarce. This type of research could help elucidate some important factors that might influence and specify pigmentation patterns.
8.1.1 Melanins
Fungal melanins have been described in many developmental stages, such as sclerotial formation, aging of hyphae, sporogenesis, and hyphal pigmentation due to wounding or as result of light exposure and extreme environmental conditions.
It was demonstrated by light microscopy that the peripheral ovoid bodies formed by certain wood inhabiting fungi like Xylaria polymorpha (Pers.) Grev, Armillaria mellea (Vahl) P. Kumm and Polyporus squamosus (Huds.) Fr. consist of compacted black bladder-like hyphae found in the lumina of wood cells that are practically unaltered. The matrix of bladder hyphae forms an amorphous black matter (Campbell 1933, Campbell 1934, Campbell and Munson 1936).
The ultrastructure of melanin formation within the fungal cells could be visualized by electron microscopy. To preserve cell integrity and to enhance image contrast, the general protocol of wood sample preparation for Transmission Electron Microscope (TEM) includes pre-staining
124 with 2% osmium tetroxide, fixating in 2% glutaraldehyde, dehydrating and embedding in Spurr’s resin. Staining ultrathin sections with lead citrate is usually performed before examination with TEM (Chaffey 2002). However, it was often inferred that the high density of melanin pigments provides sufficient contrast of melanin in electron microscopy of even unstained biological samples (Thathachari 1973, 1976, Rosas et al. 2000).
Detailed research on fungal melanin was done by Ellis and Griffiths (1974), who studied melanized mycelium of Verticillium dahliae Kleb., Humicola grisea Traaen, Epicoccum nigrum Link, Colletotrichum coccodes (Wallr.) S. Hughes and Amorphotheca resinae Parbery. The study revealed that fungal melanin is confined to the fungus cell wall region, either externally or within the hyphal or spores cell walls. In some cases the accumulation of electron-opaque granules of melanin was associated with verrucose excrescences with size variations from 30 to 200 nm. Further imaging investigations with TEM and Scanning Electron Microscopy (SEM) on melanin formation of Phomopsis spp., exposed to short periods of light, revealed similar structural characteristics (Ellis and Griffiths 1975).
Based on the similarities of the ultrastructure of melanin formation of a wild type isolate and the scytalone treated albino mutant of V. dahliae, Wheeler et al. (1976) concluded that scytalone is a natural precursor of melanin, as previously reported by Bell et al. (1976a). Imaging analysis by light, SEM and TEM revealed that granular melanin occurred in sclerotial cell walls, forming a matrix that encased the fungal walls of the wild type isolate as well as of the albino mutant when treated with scytalone. However, the melanin produced by the same albino mutant treated with catechol, dihydroxyphenylalanine (Dopa), and other phenols, had different properties than the wild type or scytalone melanins; while the surfaces of sclerotial hyphae of the wild-type isolate appeared rough due to dense melanin granule formations, the surface of albino mutant and mutants treated with melanin precursors were relatively smooth. The sample preparation procedure for TEM imaging described by Wheeler et al. (1976), involved pre-staining with either osmium tetroxide or with potassium permanganate, fixation, dehydration and embedding in Spurr’s resin, and further staining of thin sections with lead citrate. The authors concluded that sections pre-stained with potassium permanganate were generally more contrasting than those pre-stained with osmium tetroxide. The sections prepared for SEM were also pretreated with osmium tetroxide, dehydrated and finally coated with Au:Pd alloy (60:40).
Melanin biofilm formations involved in sporogenesis of Agaricus bisporus (J.E. Lange) Imbach
125 were studied by TEM microscopy by Hegnauer et al. (1985). For comparison of native and synthetic melanin obtained by γ-glutaminyl-4-hydroxybenzene (GHB) and tyrosinase, the spores covered with melanin were subjected to melanin extraction. The precipitated melanin was fixed in 2.5 % glutaraldehyde, post-fixed with osmium tetroxide, dehydrated and embedded in Spurr’s epoxy resin for visualization. Thin sections of the samples were further stained with 2% aqueous uranyl acetate and Reynold’s lead citrate before examination with TEM. The results indicated the presence of two types of melanin structure: partly amorphous and partly granular plate-like particles 50-100 nm in diameter, and electron-opaque round particles of 30 to 200 nm. However, the evidence of this study remained inconclusive, due to harsh chemical treatments applied for melanin extraction. Wheeler and Bell (1988) further identified, by electron microscopy, three types of melanin formation: wall-bound, extracellular and cytoplasmic melanins; however the latter, identified in Aspergillus niger Tiegh., was less common, while wall-bound and extracellular melanins were formed by many fungi regardless of the nature of the phenolic precursors used for melanin biosynthesis. Evidence that the electron-dense materials in fungal cell walls were melanin pigments was also reported by Bell and Wheeler (1986). Electron-dense granules were observed only in pigmented cells and not in hyaline fungal cells. Also, normal patterns of electron-dense granule formations appeared when melanin precursors were available to albino mutants. Moreover, the introduction of the melanin synthesis inhibitor, tricyclazole, affected electron-dense granule formation in several fungal species tested that produced 1,8 dihydroxynaphthalene (DHN) melanin, mostly characteristic of ascomycetes fungi (Bell and Wheeler 1986).
A quantitative assay of cell wall melanin was developed by Butler and Lachance (1987), based on the high affinity of dye Azure A for DHN and DOPA melanin, as previously described by Bull (1970) and Nicolaus et al. (1964). Although the procedure was sensitive, nondestructive and rapid (30 min), the range of linearity of the azure A method was narrower when assessing whole cells of the black yeast Phaeococcomyces nigricans (M.A. Rich & A.M. Stern) de Hoog compared to the extracted and digested melanin of the same fungus.
Another technique of melanin localization was developed using the enhanced accumulation of DHN melanin in cell walls of the rice plant pathogen Gaeumannomyces graminis var. graminis
(Sacc.) Arx & D.L. Olivier, exposed to CuSO4 (Caesar-Tonthat et al. 1995). Since considerable amounts of CuS was absorbed in the melanin layer, a sulfide-silver staining technique proved
126 successful in visualization and localization of CuS associated with the melanin layer of cell walls. Silver also precipitates in hyphal septa, which suggests that septa also melanize. However, silver precipitation did not occur near hyphal tips nor in tricyclazole treated hyphae or their septa. Those findings are in agreement with previous research (Henson et al. 1999, Butler and Lachance 1987) that observed the binding of the Azure A dye to the septa of melanized hyphae of G. graminis. A copper sulfide-silver staining technique for fungal melanin detection in electron microscopy was further developed by Butler et al. (2005), based on Dancher’s method (1981), and it proved to be more efficient in labeling melanin produced in the black yeast Phaeococcomyces, in the G. graminis var. graminis, and in the sporidial cells of Microbotryum violaceum (Pers.) G. Deml & Oberw.
Innovative research that tested the antibody response to melanin precursors and phaeomelanin was reported by Kammeyer et al. (1992) and Liu and Jimbow (1993), respectively. The immune response of fungal melanin was first investigated in the human pathogen Cryptoccocus neoformans (San Felice) Vuill. by Casadevall and Scharff (1991). When grown on phenolic media, the basidiomycetes C. neoformans produced black pigmentation that formed a protective capsule for the cells (Polacheck and Kwon-Chung 1988). Research on melanin contribution to virulence of this fungus, led to investigation of binding capacity of anti-melanin monoclonal antibody to glucuronoxylomannan (GXM) (Nosanchuck and Casadevall 1997). Glucuronoxylomannan was determined as the major polysaccharide component of capsular C. neoformans (Cherniak and Sundstrom 1994). Further research on the antibody response to fungal melanin in mice demonstrated that melanin can be immunogenic, and the antibody response was evaluated by enzyme-linked immunosorbent assay (ELISA), immunofluorescence and agglutination analysis (Nosanchuck et al. 1998). Beside serological methods used to study the antibody response to melanin, phage display techniques were also used to identify melanin- binding peptides for C. neoformans (Nosanchuck et al. 1999) and for Alternaria alternata (Fr.) Keissl. (Carzaniga et al. 2002).
To investigate the effectiveness of several melanin antibodies generated in response to polymerized fungal melanin MAb 11B11, 6D2 and 5C11, extensive studies were carried out by Rosas et al. (2000). They were tested against L-Dopa melanin from C. neoformans, synthetic melanin, and melanin from Sepia officinalis L. (ink melanin), indicating MAb 6D2 as the most reliable antibody. Immunogold electron microscopy indicated that MAb 6D2 and 11B11 have
127 more than one binding site to melanin, and immunofluorescence microscopy demonstrated that melanin antibodies binding properties were reduced by high salt concentration of the antibody solution (Rosas et al. 2000, Casadeval et al. 2000, Rosas et al. 2002, Dadakova and Casadevall 2005, Dadakova et al. 2008).
8.1.2 Staining fungi
The morphology of wood staining fungal pigments is difficult to visualize in wood substrate. Most of the pigments produced by staining fungi are soluble in water, acetone and alcohol, and this might impede sample preparation for imaging with electron microscopy such that the pigmentation would be preserved in its natural state. However, many characteristics of pigment formation of staining fungi were studied from fungal cultures grown on various agar media in petri dishes (Fenwich 1993, Kang et al. 2011, Robinson et al. 2010b).
It is well known that pigments produced by Scytalidium cuboideum (Sacc. & Ellis) Sigler & Kang species diffuse into wood or agar substrate to precede hyphae invasion. As described by Chidester (1940), the fungus grows relative rapidly in culture, producing numerous clusters of spores, at first of ivory – yellow colour, to become pink and later, dark spots of tyrian blue mycelium appear in the underside of aged cultures. Research of Golinski et al. (1995) described the change in colour of the red pigment to blue when dissolved in basic solutions.
It was observed that the fungus stained pine wood samples within three weeks after inoculation, and pigmentation was more concentrated around resin accumulations, and at higher moisture contents of the substrate. In general, these pigments have antifungal properties, are soluble in water and other solvents and easily diffuse in wood tissue. They help to ensure the integrity of fungal colonies, food resources, and space sequestration (Brian 1957).
Although S. cuboideum was described as strongly cellulolytic (Sigler and Carmichael 1976, Sigler and Carmichael 1983), decay tests indicate that S. cuboideum shows little significant decay potential in either soft- or hardwood species (Schmidt and Diez 1985, Robinson et al. 2011c). Light and electron microscopy studies of S. cuboideum inoculated on pine indicated that the fungus could be categorized as a soft rot type 1, as the S2 cell wall contains diffuse cavities or is sometimes completely degraded, with the S3 remaining intact (Anagnost et al. 1994).
The pigment produced by green staining fungus Chlorociboria aeruginascens Kanouse ex C.S.
128 Ramamurthi, Korf & L.R. Batra has been intensively studied, and it was determined that the fungus has no apparent degradation capacity of cellulose and lignin (Robinson and Laks 2010b). The wood color is altered due to the presence of pigmented mycelium within the wood cells, and only in advanced stages of colonization does the blue-green pigment seem to be diffused into wood fibers. Detailed characterization of the fruiting bodies formation of North American species was given by Dixon (1975) from analysis with light microscopy, and the patterns of wood colonization of the pigmented mycelium of Chlorociboria were studied by Blanchette et al. (1992). The research identified the fungus by light and electron microscopy analysis, in one of the intarsia panels of the Gubbio studiolo, produced in Italy in the 15th century. Blanchette et al. (1992) described deposits of dark-green and yellowish pigments, located in some fibers and vessels, and more predominantly in ray parenchyma cells. TEM imaging of the fresh material studied for comparison with the 500-year-old green stained veneer, revealed that hyphae within the wood cells displayed accumulation of pigmented substances around the outer cell walls. The pigmented hyphae were present in unaltered wood cells, as well as in cells; a type 2 erosion of the secondary wall, typical of white rot degradation, was evident.
This study investigates the fungal pigment deposition within the wood cells by various spalting fungi, to determine whether the various types of melanin can be morphologically differentiated, and to acquire imaging of staining fungi in wood. It also investigates whether various types of fungal melanins in wood substrate can be immunolabeled by an available primary monoclonal melanin antibody. The binding properties of melanin with various chemical elements are not completely understood; therefore, I sought to use methods that minimize morphological changes in the research material. The particular focus of this investigation is on wood spalting fungi, as I was interested to explore the pigmentation patterns that could impact spalting production.
8.2 Materials and methods 8.2.1 Wood and fungal species
Wood samples of four species, colonized by one or more of seven species of spalting fungi, were studied (Table 8.1). The specimens were acquired from natural or in vitro spalted wood, from a particular zone that displayed zone line formation or concentrated stain pigmentation.
129 Table 8.1 Substrate and fungal species selection for microscopy analysis. LM – light microscopy, TEM – transmission electron microscopy, SEM– scanning electron microscopy, FL – fluorescence microscopy, CM – confocal microscopy. Substrate Fungal species/ treatment Condition Investigation method Sugar maple Oxyporus populinus in vivo LM, TEM, SEM, FL, CM Acer saccharum (Schumach.) Donk Marshall Trametes versicolor (L.) Lloyd in vitro TEM, FL Trametes versicolor in catechol in vitro TEM, FL Xylaria polymorpha (Pers.) Grev. in vitro TEM, FL Inonotus hispidus (Bull.) P. Karst. in vitro TEM Scytalidium cuboideum in vitro TEM (Sacc. & Ellis) Sigler & Kang TEM (red and blue) Chlorociboria aeruginascens in vitro (Nyl.) Kanouse ex C.S. Ramamurthi, Korf & L.R. Batra pretreated with T. versicolor Beech Trametes versicolor in vitro TEM, FL Fagus grandifolia Trametes versicolor in catechol in vitro TEM, FL Ehrh. Xylaria polymorpha in vitro TEM, FL Inonotus hispidus in vitro TEM Scytalidium cuboideum in vitro TEM
Aspen Scytalidium cuboideum in vitro TEM Populus sp. Chlorociboria aeruginascens in vitro TEM
Yellow Birch Fomes fomentarium in vivo TEM, FL Betula alleghaniensis (L.) J.J. Kickx Britt.
8.2.2 Chemical fixation for copper sulfide-silver staining technique
Silver-staining procedures were investigated for their effectiveness in identifying fungal melanins formation at the cell wall level. Wood sections of 1-2 µm were treated with a 10mM copper sulphate solution in distilled water for 1-10h, and after washing were treated with 1% sodium sulfide solution in distilled water for 1h in the dark. After another series of washing with distilled water, the samples were dried and developed in a 20 mL solution of 22 mg silver lactate and 170 mg hydroquinone in a citrate buffer for 1 to 10 min at 26 0C, followed by fixation in 2% glutaraldehyde in phosphate buffer, and examined with Axioplan light microscope with DP71 camera.
8.2.3 Chemical fixation for SEM
To maintain the original form of the specimen and to preserve the cellular ultrastructure so it could withstand the effects of succeeding steps in the preparative protocol, the 1-5 µm sections acquired from spalted wood were suspended overnight in primary fixative of 2.5%
130 glutaraldehyde, post-fixed in 1% osmium tetroxide, followed by ethanol dehydration and HMDS (hexamethyldisiloxane) infiltration. The samples were dried overnight in a fume hood. The specimens were mounted on aluminum stubs with double-sided carbon tape and sputter coated with gold-palladium using Cressington Sputter Coater 108. The examination was performed on Hitachi S2500 SEM at 20 kV.
Alternative specimen preparation omitted fixation and post-fixation with osmium, and included coating with gold-palladium of critical point dried samples.
8.2.4 Chemical fixation for TEM and LM
To ensure structural integrity at the cell level and to provide electron contrast, samples of spalted wood were fixed overnight in 2.5% (alternative 2%) glutaraldehyde and post-fixed in 1% osmium tetroxide in the dark. After dehydration with ethanol, the samples were embedded in Spurr’s or LR White resin and polymerized.
For LM imaging, the embedded samples were sectioned with a glass knife in semi-thin sections and stained with toluidine blue and methylene blue for 1 min, or alternatively with Fuchsine for staining fungal tissue. Semi-thin sections were examined with Olympus MVX10 microscope equipped with Olympus DP71 camera.
Ultrathin sections were obtained by sectioning with a diamond knife. Selected sections were stained with 3% uranyl acetate, post-stained with Reynold’s lead citrate. Stained and unstained samples were examined with Philips PW6006 transmission electron microscope.
8.2.5 Immunolocalization procedure for TEM
To localize the antibody binding on fungal melanins in vivo and in vitro, immunogold labeling was performed on spalted wood tissue. Ultrathin tissue sections of samples embedded in LR white resin were placed on nickel grids with Formvar film, treated with 10% H2O2 for 10 min., and then washed with phosphate buffered saline (PBS) solution. Grids were blocked in SuperBlock Blocking Buffer (Thermo Scientific) in PBS for 4 h at room temperature. Grids were then incubated overnight at 4°C in 5 µg/ml of MAb 6D2 (provided by Dr. Arturo Casadevall, Albert Einstein College of Medicine, Bronx, NY). After being washed with PBS, the grids were incubated in secondary antibody at 1:1,000 dilution of immunogold conjugate for electron microscopy (EM) Goat antibody mouse immunoglobulin M (IgM): 5nm (BBI
131 International) for 2 h at room temperature. Grids were then washed with PBS, fixed in 2% glutaraldehyde, and examined using a 100 CX transmission electron microscope Philips PW6006, and images were acquired with AMT V600 software. The significant binding of the melanin antibody was determined based on particle counting in the target region compared with background area. Positive and negative control samples were used to test the efficacy of the antibody binding to melanin. Plates blocked with SuperBlock Blocking Buffer without melanin were used as negative controls.
8.2.6 Immunolocalization procedure for SEM
To identify cell wall-binded fungal melanin particle, 5 µm thick samples of wood with positive and negative melanin formation, fixed in 1.25% glutaraldehyde overnight at 40C were blocked for nonspecific bindings with PBS + 4% (1%) BSA (alternately added 4-1% milk) for 1h, incubated for 30 min to 3h in 20µg/ml MAb 6D2 in PBS, treated after washing series with 10nm protein A-gold (1:100) for 1h, and (alternatively) exposed to silver enhancement reagent (SEKL15 from BBI) for 5-20 min. After washing and drying, samples were coated with gold- palladium and examined with Hitachi S2500 SEM. Control samples were used to test each step and alternatives of the procedure.
Wood samples 5 µm thick, fixed in 1.25% glutaraldehyde overnight at 40C were blocked for nonspecific bindings with PBS + 4% (1%) BSA for 1h, incubated for 3h in 20µg/ml MAb 6D2 in PBS, treated after washing series with immunogold conjugate EM Goat antibody mouse IgM:5nm (BBI International) 1:100 for 1h, and (alternatively) exposed to silver enhancement reagent (SEKL15 from BBI) for 5-20 min. After washing and drying, samples were coated with gold-palladium and examined with Hitachi S2500 SEM.
8.2.7 Immunofluorescence analyses of spalted wood.
Due to the nature of the specific binding of primary antibody to melanin molecules, the images of spalted wood samples labelled with the fluorescent dye obtained by immunofluorescence analysis are seen in high contrast against a dark background, allowing for very precise and sensitive observations.
Thin sections 0.5-1µm of spalted wood samples and sound wood used as controls, embedded in LR White resin were first immersed in SuperBlock Blocking Buffer in PBS for 4 h at room
132 temperature to block nonspecific bindings, and incubated with 20 µg/ml of MAb 6D2 overnight at 4°C, followed by conjugation to the Alexa Fluor 488 Goat anti mouse IgM (µ chain) (Invitrogen) 1:200 after washing. Tissue samples were again washed with PBS. A mounting solution of 50% glycerol, 50% PBS and 0.1MN propylgallate (P3130 Sigma –Aldrich) and coverslip were applied, and the samples were examined using an Axioplan microscope with fluorescein isothiocyanate filter, and Olympus DP71 camera, and a Quorum WaveFX / FRAP spinning disk confocal microscope.
The efficacy of the antibody binding to melanin was tested by positive and negative labelling of control samples. Positive control was tested for melanin suspension 1:4, obtained using the protocol described by Harki et al. (1997), and sepia melanin (Sigma Aldrich) adsorbed onto a nitrocellulose membrane (BIO-RAD) in SuperBlock Blocking Buffer in PBS, based on enzyme- linked immunosorbent assay method, as described by Rosas et al. (2000). Plates blocked with SuperBlock Blocking Buffer without melanin were used as negative controls. The omitting of the primary antibody from the labeling protocol was used to identify non-specific binding of secondary antibody (the conjugated Alexa Fluor dye 488) on melanin positive samples.
8.3 Results 8.3.1 Melanin detection with the copper sulfide - silver staining techniques.
Imaging analyses with LM and SEM of wood samples treated as described by Butler et al. (2005) indicated that the procedure could not clearly detect fungal melanin in wood substrate. The samples of melanin produced by X. polymorpha in sugar maple were more densely stained than melanin of T. versicolor in sugar maple or beech following the same protocol. The results indicate that the sulfide-silver amplification method resulted in heavy unspecific precipitation even in nonmelanized wood samples. Silver particles were bound to wood cell walls, as well as to the middle lamellae, regardless of the time exposure of samples to either copper sulphate or silver development (images not shown).
133 8.3.2 Immunofluorescence and immunogold analysis of melanin
Pigment of melanin produced by Oxyporus populinus, Trametes versicolor, Xylaria polymorpha, Fomes fomentarius, and Inonotus hispidus in wood substrate were successfully immunolabeled by MAb 6D2 melanin antibody. Absence of labelling of control samples confirmed the overall efficacy of the immunofluorescence procedure, and the efficacy of the antibody binding to melanin was also confirmed by positive labelling (results not shown).
Oxyporus populinus
Sugar maple samples with zone line formations by O. populinus were analyzed by LM, SEM, TEM, Fluorescence and confocal microscopy. The apparent black line formation is composed of dense mycelium that fills the lumina of the wood cells (Figure 8.1A, E). Two types of melanin deposits were distinguished. The first type is a dense layer of oval melanin granules, usually of 50 - 100 nm, bonded to the wood cell wall; it is deposited in cavities characteristic of white rot degradation of the cell wall (Figure 8.1B and C, single white arrow). With this type, round and bigger granules of melanin, up to 1.5 µm in diameter, with rugose surfaces, are also often observed in well established zone line formations (Figure 8.1B and C, double white arrows). The second type of melanin deposit forms conglomerates of hollow, melanized fungal hyphae (Figure 8.1 B, E and F, red double arrows). Hyphae are from 2 to 5 µm in diameter with 500 to 600 nm melanized wall thickness; they tend to completely fill wood cell lumina. The surface of the melanized outer wall is also of rugose nature (Figure 8.1D red arrows).
134
1.5mm 1 μm
A B 1.5 μm 5 μm
C D
0.25 mm 30 μm μm
F E F
Figure 8.1 Acer saccharum samples with zone lines formations by Oxyporus populinus; (A) LM of wood cross section with melanized mycelium in wood cells; (B) TEM of parenchima ray in longitudinal section, melanized mycelium organized in clusters (red double arrows); (C) SEM of wood tracheid with melanin deposits; small oval melanin granule deposits (white single arrows); round and bigger granules of melanin, (double white arrows); (D) SEM of melanized mycelium with rugose surface of the outer wall (red single arrows); (E and F) SEM of tangential section in wood sample; melanized mycelium organized in clusters in vessels (red double arrows).
135 As observed from immunofluorescence imaging, the melanin antibody MAb 6D2 successfully binds to melanin deposits from wood cells (Figure 8.2 A and B - arrow), while negative control samples are not successfully labeled (Figure 8.2 C and D). Confocal microscopy provided a 3D imaging of selective binding of the tested melanin antibody (Figure 8.3).
10 µm 10 µm
A B
10 µm 10 µm
C D
Figure 8.2 Immunofluorescence of fungal melanin in cross section of parenchyma rays of Acer saccharum with melanized mycelium of Oxyporus populinus in fluorescence light (A) and in bright field (B); arrow indicate labeling of melanin with Alexa Fluor dye conjugated with secondary antibody; control samples of sugar maple in cross section of parenchyma rays, with no fungal inoculation, in fluorescence light (C) and in bright field (D).
136
A 1010 µmµm B 1010 µmµm C 10 µm
Figure 8.3 Imaging of Acer saccharum ray parenchyma with melanized mycelium of Oxyporus populinus in cross section, obtained by confocal microscopy. (A) fluorescence image; (B) phase contrast image; (C) merged confocal image.
Trametes versicolor
The investigated melanin pigments formed by T. versicolor in sugar maple and beech substrate, as well as in the same wood substrate treated with catechol before fungal inoculation, showed no morphological differences between catechol treated and untreated wood (Figure 8.4 A and B, and Figure 8.5 A and B) substrates; however, there were differences between the wood species (Figure 8.4 A versus Figure 8.5 A). In sugar maple, small melanin granules 100 - 250 nm released by active hyphae tend to assemble without much initial binding to the cell wall, whereas beech cell walls and hyphal surroundings are heavily coated with a dense layer of melanin deposits (Figure 8.4 and Figure 8.5). Potential ongoing melanin production activity is indicated by the presence of melanized hyphae within the wood cells.
137
2 µm 2 µm
A B 7.5 µm 7.5 µm
C D 7.5 µm 7.5 µm
E F
Figure 8.4 Imaging by TEM (A and B) of non-osmicated sections, and immuno-FL of Acer saccharum with melanin produced by T. versicolor (A), (C) and (E) in monoculture and (B), (D) and (F) pretreated with catechol before fungal inoculation; (A) - TEM image of cross section of wood tracheids; (B) - TEM image of cross section in wood tracheids; (C) and (D) - fluorescence image of cross section in wood tracheids with melanin; (E) and (F) - LM image of same C and D respectively cross section in wood tracheids.
138
2 µm 2 µm
A B 7.5 µm 7.5 µm
C D
7.5 µm 7.5 µm
E F
Figure 8.5 Imaging by TEM (A and B) of non-osmicated sections, and immuno-FL of Fagus grandifolia with melanin produced by T. versicolor (A), (C), (E) in monoculture, and (B), (D), (F) pretreated with catechol before fungal inoculation; (A), (B) - TEM images of cross section of tracheids; (C) and (D) - immunofluorescence images of cross section in wood tracheids with melanin; (E) and (F) - LM image of same C and D cross section respectively.
139
Xylaria polymorpha
Similar type and size of oval granules, 50-150 nm in sugar maple, and 70-150 nm in beech, were observed in the case of X. polymorpha, that assemble from layer to conglomerate from the inner wood cell walls of both tested species. SEM of sclerotial fungal hyphae appeared hollow in section, and the remaining “host” was comprised of melanized fungal cell walls with rugose surface (Figure 8.6, arrow).
Figure 8.6 Imaging by TEM (A and B), and FL of melanin produced by X. polymorpha in sugar maple (A, C and E) and in beech (B, D and F), and SEM of sugar maple in cross section with melanin layer deposition on the cell wall of the wood vessel (G), longitudinal section on wood tracheid filled with melanized mycelium (H).
Fomes fomentarius
The studied samples of birch with black zone lines, formed by white rot basidiomycete F. fomentarius, had no layer of melanin deposits along the wood cell wall. Instead the hyphae within the lumen seemed heavily pigmented in the fungal cell; however melanin immunolabeling was registered also at the cell wall level. (Figure 8.7 C - arrow). Immunofluorescence imaging also indicated melanin production activity (Figure 8.7 A and B).
Inonotus hispidus
Pigmentation produced by I. hispidus was observed as deposition of very small 100 - 200 nm granules along the sugar maple cell walls (Figure 8.8 A - arrow). Dense bigger granules (300 - 850 nm) were also observed along the wood cell walls (Figure 8.8 A - double arrows). The melanin granule depositions are not present in wood cells subject to active fungal decomposition (Figure 8.8 B). Melanin production activity was also immunolabeled within the fungal hyphae as well as in their cell walls (Figure 8.8 C red arrow).
140
2 µm 2 µm
A B
7.5 µm 7.5 μm
C D
7.5 µm 7.5 µm
E F
2.5 µm 3 µm
G H Figure 8.6 Imaging of Acer saccharum (A, C and E) and in Fagus grandifolia (B, D and F with melanin produced by X. polymorpha; (A) and (B) TEM imaging of vessels in cross section; (C) and (D) immunofluorescence imaging of vessels in cross section; (E) and (F) LM imaging of vessels in cross section; (G) - SEM of Acer saccharum cross section with melanin layer deposition on the cell wall of the wood vessel; (H) - longitudinal section on Acer saccharum tracheids filled with hollow melanized mycelium (arrow).
141
7.5 µm 7.5 µm 1 µm
A B C
Figure 8.7 Imaging of natural melanin produced by Fomes fomentarius in Betula alleghaniensis. (A) - imaging by light and (B) -immunofluorescence microscopy of cross section in ray cells with melanin; (C) immunolabelling of melanin in fungal cell wall (arrow) in TEM imaging of natural melanin produced by F. fomentarius.
2 µm 2 µm 50 nm
A B C
Figure 8.8 Immunolabeling of melanin produced by I. hispidus in Acer saccharum. (A) - TEM imaging of cross section of Acer saccharum tracheid cells with melanin granule deposition; (B) - TEM imaging of cross section of Acer saccharum tracheid cells with melanized hyphae; (C) - TEM imaging tangential section of fungal hyphae with immunolabeled melanin formations in hyphal cell wall (arrow).
142 8.3.3 Staining pigments
Scytalidium cuboideum
Pigmented wood samples inoculated with S. cuboideum in monoculture were studied from sugar maple in red and blue pigmentation areas, and beech and aspen wood from red pigmentation areas. In sugar maple, precipitation of grouped 50 - 200 nm pigment granules, without apparent binding to the wood cell walls accompanied by round compact homogenous vesicle-like, up to 1µm formations (Figure 8.9 A) were observed by TEM imaging. Much denser accumulation of pigments was observed in the sugar maple samples dyed blue by S. cuboideum (100 - 150 nm). The large vesicles were rare and amorphous, and the decomposition of wood cell walls was more intense (Figure 8.9 D). In red pigmented beech samples, the pigmented granules were very small (50-100 nm) and less frequent, with no vesicle formation (Figure 8.9 C). In red stained aspen, amorphous vesicles with rare small pigment granule precipitations (up to 80 nm) were observed along the wood cell walls that displayed type 1 and 2 soft rot decay cavities in S2 and S3 layers of the wood cell walls (Fig. 8.9 D).
1.5 µm 1 µm
A B
2 µm 2 µm
C D
Figure 8.9 TEM imaging of pigments produced by S. cuboideum in cross section of (A) Acer saccharum vessel, (B) - Acer saccharum tracheid, (C) – Fagus grandifolia tracheids and vessel, and (D) - Populus sp. tracheids. All wood samples displayed red pigmentation except sugar maple (B), which developed blue pigmentation.
143 Chlorociboria aeruginascens
The xylindein formation in sugar maple pretreated with T. versicolor and sterilized before inoculation with C. aeruginascens (Figure 8.10 B), and aspen samples inoculated with the same fungus in monoculture (Figure 8.10 A), followed the same patterns of pigment deposition as melanins. The deposits were assembled into a 125-250 nm layer on the inner wall of the wood cell lumen, along with clusters of pigmented mycelium within the lumen. They were accompanied occasionally by homogenous pigmented vesicle-like formations up to 4 µm, and were more pronounced in sugar maple. The sterile aspen sample inoculated with monoculture of C. aeruginascens, in vitro, also displayed cell wall erosion. However, the results were inconclusive due to the lack of investigation of the initial state of the wood substrate tested, before inoculation with monoculture of C. aeruginascens.
2 µm 2.5 µm
A B
Figure 8.10 TEM imaging of tracheids in longitudinal section, with green pigment produced by C. aeruginascens (A) - in monoculture inoculated in Populus sp., and (B) – pretreated with T. versicolor in Acer saccharum.
144 8.4 Discussion
Melanin formations cannot be easily differentiated among fungal species and their wood substrates; however there are some noteworthy distinctive characteristics. The two types of pigment assembly, the granule-dense layer attached to the wood cell lumen walls, and the conglomerate of sclerotial hyphae that tend to obstruct wood cells, are present in almost all pigmented wood inhabiting fungi; however, granule dimensions and assembly patterns can vary (Table 8.2). The coating of the wood lumen might help ensure a secure territory and resist fungal competitors. The complete obstruction of successive wood cells at the boundaries of each fungal colony forms the melanin outer layer that ensures its protection. The hypothesis of initial formation of a pigmented layer is supported by the observation that it can be present without hyphal conglomerates, while assembled conglomerates within the wood cells are always contained within this type of granular layer. Bell and Wheeler (1986) argued that extracellular melanins are derived either from secretion of phenol oxidases into the external environment to oxidize available phenolic compounds, or by secretion of phenols into the external environment where they are oxidized by autolysis. These extracellular melanins might contribute to the deposits along the wood cell walls. These authors also investigated the so-called wall-bound melanin, found in most melanized fungi in hyphae, conidia and sclerotial cell walls. Wall- bound melanin forms various patterns of electron-dense granules, that may occur in the outer or inner part of the fungal cell walls (Pirt and Rowley1969, Jones 1970, Ellis and Griffiths1974, Ellis and Griffiths1975, Benitez et al. 1976, Jackson and Gay1976 Wheeler et al. 1976). Bell and Wheeler (1986) also note the lack of degradation of the wood cells inhabited by melanized hyphae; it is more advantageous for fungi to preserve undamaged wood cell walls, to ensure structural integrity of the melanized layer. It could be speculated that the expression of melanin production and enzymatic degradation exclude each other by genetic regulation.
The present research characterized fungal pigmentation in the wood substrate, and the described findings might serve as explanation for various results reported for the chemical structure of melanin (Tian et al. 2003, Zhong et al. 2008, Dadakova et al. 2008, Casadevall et al. 2012) as well as for the multiple genes identified in melanin biogenesis (Henson et al. 1999, Williamson 1997, Kimura and Tsuge 1993, Woo et al. 2010, Wang and Breuil 2002).
145 Table 8.2 Summary of the microscopy methods and results obtained from the study of Xp = Xylaria polymorpha, Tv = Trametes versicolor, Ff = Fomes fomentariu; Ih = Inonotus hispidus, Sc = Scytalidium cuboideum, Ca = Chlorociboria aeriginascens in SM = sugar maple, B = beech, Bi = birch, Ap = aspen.
Sample type Staining Immuno labeling Microscopy Results Fungus/wood/treatment method Xp/Sm, Xp/B, Tv/SM, Silver - LM Unspecific binding to wood tissue Tv/B staining Op/SM Alexa Fl Mab6D2 LM, SEM, Two types of melanin deposits, rugose surface 488 (primary antibody) TEM of melanin Gold conjugate Immunolabelling of melanin, selective binding secondary antibody FL, CM of melanin antibody Tv/SM, Alexa Fl Mab6D2 LM, TEM Two types of melanin deposits, rugose surface Tv/SM/catechol 488 (primary antibody) of melanin FL Immunolabelling of melanin, selective binding of melanin antibody Tv/B, Tv/B/catechol Alexa Fl Mab6D2 LM, TEM Morphologic differences of melanin deposition 488 (primary antibody) in SM and B; same type of melanin deposits in Gold conjugate treated-untreated wood secondary antibody FL Immunolabelling of melanin Xp/SM, Xp/B Alexa Fl Mab6D2 LM, TEM, Two types of melanin deposits, rugose surface 488 (primary antibody) SEM of melanin Gold conjugate Immunolabelling of melanin secondary antibody FL Ff/Bi Alexa Mab6D2, LM, TEM One type of gregarious melanin deposits, no FL 488 Gold conjugate layer along the wood cells walls secondary antibody, FL Immunolabelling of melanin granule and at the fungal cell walls level Ih/SM - Mab6D2 TEM Two types of melanin deposits, Gold conjugate Immunolabelling of melanin granule and at the secondary antibody fungal cell walls level Sc/SM, Sc/B, Sc/Ap - - TEM Two types of pigment precipitation
Ca/Ap, Ca/SM - - TEM Two types of granular pigment deposits
Melanin deposits produced in vitro by T. versicolor in sugar maple are attached to the wood cell walls, while in the same substrate pretreated with catechol, those bindings are obviously absent (Figure 8.4). That was not the case in beech, where T. versicolor was not influenced by catechol treatment in regard to pigmentation pattern and melanin assembly (Figure 8.5). The pigmentation patterns by this fungus not only differ in the same wood species, if catechol was introduced into the substrate, but also differ between sugar maple to beech samples inoculated under the same conditions. There is strong evidence that the structure of melanin produced by one fungus varies with the nature of the wood substrate, which can directly influence the existing chemical bonds with the wood polysaccharides along the wood cell walls, as demonstrated by Zhong et al. (2008). There is yet no indication how and if the structural function of those chemical bonds have any influence on the melanin properties; however any attempts to analyze extracted melanin particles, fail to demonstrate the chemical bonds between
146 melanin and wood cell walls, due to treatment with harsh chemicals during extraction (Butler et al. 2001). Simon and Ito (2004) also argued that in the natural environment, melanins are associated with proteins and metal ions bound to the functional groups of the biopolymers, which influence the overall structure of these melanin formations.
If taken into consideration that melanin appearance might reflect variation in the chemical structure, it can be hypothesized that, three types of melanin molecules could be expressed with various organizational structures in fully developed zonations: small granules, big granules and melanized external hyphal layers, as observed in this study on O. populinus zone line formation in sugar maple in vivo (Figure 8.1 to 8.3).
The morphology of melanin formation produced by X. polymorpha demonstrated more consistency within the wood substrate, and resembles the morphology reported by Campbell (1933). As expected, the fungus developed decay cavities specific to white rot fungi, especially in beech. Wood cells within the zone line formations contain small melanin granules that form a firmly bound layer to the wood cell walls, and melanized hyphae (Figure 8.6).
There are no melanin granule depositions along the wood lumen, in the case of zone lines formation in birch colonized by F. fometarius. The immunolabeling with melanin antibody of fungal mycelium in birch wood, indicates melanin activity within the fungal cell walls (Figure 8.7 A and B). However, the immunolocalization with melanin antibody could not be clearly differentiated within the dense dark organelles formed in the hyphal cells, (Figure 8.7 C). The mature zone lines formed by F. fometarius are assembled from conglomerates of melanized hyphae within the wood cells, and the precise pattern of hyphal melanization is not clear from this investigation. The lack of melanin deposits along the wood cell lumen is also supported by the similar description and LM imaging of decay and melanin production patterns of F. fometarius given by Schwarze et al. (2000).
Other distinct patterns of pigmentation are observed in the case of I. hispidus. There are two known phenolic pigments produced by I. hispidus identified by extraction analysis: the brown pigment hispidin characterized by Edwards et al. (1961), Klaar and Steglich (1977) and a much lighter brown-yellow metabolite known as hispolon (Ali et al. 1996). It was hypothesized that hispidin may polymerize during maturation of fungi and its development is increased by exposure to light (Nambudiri et al. 1973, Vance et al. 1974). However, their morphology was
147 never described from native infected wood substrates or from agar media. Two distinctive types of layered pigment deposition along the wood cell walls were observed, each assembled from granules up to 200 nm and 850 nm respectively, as well as immonogold labelled melanization within the hyphal cell wall (Figure 8.8 A and B). The specific pigmented layers might be the result of the bi-modal nature of pigment production by I. hispidus.
The distinctive melanin structures and assemblies produced by the wood inhabiting fungi that were studied, indicate that an accurate description of chemical structure of fungal melanin is not possible by extraction methods. The fact that each fungus produces multiple melanin-type pigment structures at any given time should be accounted for.
The selective binding of the melanin antibody, might be related with the enzymatic activity of the fungal hyphae in regard to melanin production, since freshly exposed melanin particles are not reactive with the antibody. This observed effect may reflect the specificity of the antibody for which the product was generated (Rosas et al. 2000). Immunolabeling of fungal melanin in wood substrate for SEM visualization using silver enhancement technique was not successful, due to nonspecific binding to wood substrates of silver particles used in this protocol.
The mycelium of S. cuboideum is also found in the lumina of wood cells. Red and blue pigment stains were observed in the sample of sugar maple, while beech and aspen samples investigated developed only red stain. The red pigments produced by this fungus appear as fine deposits along the wood cell walls, which could be a result of extracellular secretions or as cell-bound pigmentation. Also big vesicle-like formations, round and up to 1 µm in sugar maple, and amorphous in aspen, could be observed. The blue pigment in sugar maple appears as a more defined layer than the red deposits, consisting of up to 10x20 nm ovoid, dense granules. In the present study, decay cavities were observed in sugar maple and aspen, characteristic of early white-rot erosion caused by some basidiomycetes, or type 2 soft-rot degradation caused by various ascomycetes (Blanchette et al. 1990, Eriksson et al. 1990, Nilsson1985). Although it was demonstrated by Sigler and Carmichael (1976, 1983) that S. cuboideum is strongly celluloytic, the fungus does not seem to induce significant decay in red oak cell walls (less than 1% mass loss) (Schmidt and Dietz 1985). In this study, no decay cavities were observed in wood cells of beech, and only pigment deposits along the lumen were observed (Figure 8.9).
148 Aspen colonized in vitro with Chlorociboria aeruginascens in monoculture showed no decomposition activity of the wood cell walls. The inability of C. aeruginascens to decompose wood was also reported by Robinson and Laks (2010b). The investigated samples of sugar maple pretreated with white-rot fungus displayed similar features of Chlorociboria infected wood samples from a 500 year old intarsia panel, reported by Blanchette et al. (1992). The pigmented electron-dense deposits, more pronounced in samples from sugar maple, were observed within the nondegraded cells as well as within the cell that shows white-rot erosion of the cell walls.
Pigmentation could be readily visualized even without pre- and post-staining of analyzed sections for TEM with osmium tetroxide and uranyl acetate respectively, due to the electron- dense properties of the examined pigments. However, the acquired image quality might have been improved by those techniques.
8.5 Conclusions
The results of this research indicate a bi- or multi-modal activity of melanin production and assembly by wood inhabiting fungi, and identify possible variations of melanin formation mechanisms, influenced by fungal and wood species involved in the process. Immunolabeling with an available melanin antibody confirmed the melanin nature of the pigments produced by O. populinus, T. versicolor, X. polymorpha, F. fomentarius, and I. hispidus. Future research of fungal melanin chemical structure should focus on nondestructive analysis methods combined with advanced imaging techniques.
149
Chapter 9
Synthesis and Conclusions
This thesis makes several contributions to our understanding of the fungal pigmentation process in wood substrate. Specifically the research contributes to the understanding of fungal diversity involved in spalted wood, the influence of environmental conditions, and the influence of wood substrate composition, on fungal pigmentation. The research findings, taken separately or combined, deliver a significant contribution to the current knowledge in the field of fungal degradation patterns in wood, with direct applicability to spalting production in creating value added wood products.
9.1 Major findings and relevance 9.1.1 Fungal communities involved in spalted wood
In our attempt to gain insight on fungal communities involved in wood pigmentation, three fungal isolates from natural spalted wood that exhibited stain-like pigment dispersion, and seven specimens associated with melanin formation were identified from ITS sequence data and parsimony analysis. All isolates matched with known wood inhabiting fungi, not all of which were previously associated with pigment occurrence in wood. Lasiodiplodia theobromae was identified in grey stained burl from buckeye (Aesculus glabra), Fusarium sp. was identified in red stained Manitoba maple (Acer negundo), and Fusarium merismoides and Trichoderma viride colonized sugar maple with zone lines formations. Multiple colonies of the white rot fungi Hypsizygus marmoreus and Oxyporus populinus were also confirmed in spalted sugar maple (Acer saccharum).
150 Chlorociboria aeruginascens and Chlorociboria aeruginosa were confirmed in green stained wood from ITS sequence data, and it was demonstrated that the two species have distinct Southern Hemisphere origins. Their anamorphs were unambiguously identified, and the anamorph of C. aeruginosa was characterized in this study for the first time.
9.1.2 The influence of environmental conditions on fungal pigmentation activity
The evaluation of the moisture content and pH of wood substrate supports the hypothesis that those conditions are important factors that determine fungal pigment intensity and stimulation. The research indicates that optimal moisture content conditions for pigmentations are specific to each wood and fungal species combination. It was determined that pigmentation was stimulated at low water concentrations in the case of T. versicolor and X. polymorpha inoculated in both beech and sugar maple, while I. hispidus, P. squamosus, P. brumalis and S. cuboideum showed a tendency of enhanced pigmentation at higher levels of moisture content (35%-55%). Moisture contents tested did not significantly affect wood mass loss.
The experiments conducted in agar and wood substrates indicate that the pH value directly influences melanin formation and pigment colour range and intensity in the case of staining fungi. Fungi associated with zone line formation were able to produce melanin within the pH range from 4.5 to 5.5, representing the normal pH range of the wood substrates. Trametes versicolor strains were mainly pigment-active when inoculated on beech and sugar maple substrates adjusted to pH 4.5, and Xylaria polymorpha strains produced pigmentation in beech treated with pH 4.5 and 5, and sugar maple treated with pH 4.5 buffers. Pigment colour produced by Scytalidium cuboideum in agar adjusted to pH range from 2 to 8 varied from bright red in the acidic media, to dark blue in agar adjusted to alkaline pH values. Chlorociboria species produced pigmentation within the green range, from bright yellow-green in acidic media (pH 2-3.5), blue green within the normal range of wood pH (4.5-6) to darker olive-green at higher pH values (6.5-8).
Although it was demonstrated that moisture content and pH values affect fungal pigment formation, other environmental and chemical characteristics could also significantly alter and interact within a complex relationship
151 9.1.3 The influence of wood substrate composition
The experimental research of fungal response to catechol and L-Dopa melanin precursors introduced in agar, beech and sugar maple substrate, indicates distinct reactions of all three tested fungi. The study provided evidence that T. versicolor and I. hispidus were able to absorb and transform both substances into pigmentation in both agar and wood substrate, which indicates multiple biosynthesis pathways for melanin assembly. Xylaria polymorpha utilized only catechol, and degraded L-Dopa before any pigmentation occurred within the fungal colony. The standard test with tricyclazole for determination of DHN nature of the melanin indicated that X. polymorpha as well as Kretzschmaria deusta, Phoma exigua and Ophiostoma sp. produce DHN melanin.
Imaging analysis of natural melanin and melanin produced by stimulation with catechol and L- Dopa precursors showed a bi- or multi-modal activity of melanin production and assembly by wood inhabiting fungi, and identified possible variations of melanin formation mechanisms, influenced by fungal and wood species involved in the process. Immuno-labeling with an available melanin antibody confirmed the melanin nature of the pigments produced by Oxyporus populinus, Trametes versicolor, Xylaria polymorpha, Fomes fomentarius, and Inonotus hispidus.
9.2 Significance of original contribution 9.2.1 Original scientific contribution
The research presented in this thesis makes original contributions to the overall knowledge and understanding of fungal pigment formation, demonstrated by analytical investigations and experimental research.
• This research identifies several fungal species like Oxyporus populinus, Hypsizygus sp., Hypocrea rufa, and Botryosphaeria sp., previously not associated with spalting
formation.
• It solves the anamorph-teleomorph relationship for the two known North American Chlorociboria species.
• The anamorph of Chlorociboria aeruginosa was characterized for the first time.
152
• It was demonstrated that white rot fungi are capable to produce melanin from various precursors.
• The morphology of pigment assembly varies based on fungal species and wood substrate involved in spalting process.
9.2.2 Contribution to original applications in developing methodology for spalting production
As a value added process, spalting can considerably enhance timber value, especially of underutilized wood species. The results of this experimental research contribute to the development and improvement of the methodologies used for industrial production of spalted wood. For spalting logs or timber of hardwood species, we demonstrated the potential of inoculation with specific fungal species, the incubation in certain conditions, and/or certain pre- treatments of the wood substrate to shorten and improve the appearance of pigmentation patterns. We recommend the following guidelines:
• Fungal strains used as inoculum on timber or logs should be pre-tested to assess the maximum pigmentation ability in regard with the moisture content and the wood species to be spalted.
• Pairing of fungal strains should be considered based on their similarity of pigment production at proximate moisture content values.
• Fungal species like Oxyporus populinus and Hypsizygus sp. should be included in spalting process due to their enhanced ability to produce zone line formations.
• The adjustment of wood substrate to pH 4.5 can enhance melanin production in wood treated with Trametes versicolor and Xylaria polymorpha.
• The variation of wood pH can be used to control the color range of pigments produces by Chlorociboria aeruginascens., Monascus ruber and Scytalidium cuboideum staining fungi.
153
• The pre-treatment of wood substrate with catechol can be used for most spalting fungi to enhance melanin production.
9.3 Future directions and concluding remarks
Fungal pigment production is a complex process, and is influenced by fungal species, wood substrate composition and environmental conditions. Future research of melanin function, biosynthesis, chemical structure and interactions among environmental factors that cause melanin formation should focus on nondestructive analysis methods combined with advanced imaging techniques. I also recommend the investigation of gene expression and regulation to identify the process by which information encoded in fungal DNA directs the synthesis of melanin, to further elucidate other highly probable pathways of melanin creation and stimulation.
To further contribute to the development of industrial production of spalted wood, I recommend the study of the temperature effect on stimulation fungal pigmentation, and also the interactions between temperature, moisture content, wood pH versus fungal species combinations.
154
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Appendix
Data and results presented in Chapters 3 to 9 of this thesis include seven co-authored manuscripts at various stages of preparation or publication, and three contributions to scientific conferences. As primary author I was responsible for initiating and designing the research, carrying out the laboratory and fieldwork, conducting statistical analyses, writing the manuscripts, and delivering presentations. The co-authors of these manuscripts and presentations made significant contributions to these studies in regards to experimental design, manuscript editing, and/or financial support.
To this date, the research in this thesis have been presented as follow:
Chapter 3:
Tudor, D., Moncalvo, J.M., Cooper, P.A., (2012) Identification of fungi in spalted wood with ITS barcode markers. In preparation.
Chapter 4:
Tudor, D., Margaritescu, S., Santiago Sánchez-Ramírez, S., Robinson, S.C., Cooper, P.A., Moncalvo, J.M. (2012) Morphological and molecular characterization of the two known North American Chlorociboria species and their anamorphs. In preparation.
Tudor, D. (2012) The characterization and use of Chlorociboria spp. Haliburton Forest and Wildlife Reserve. – oral presentation.
Chapter 5:
Tudor, D., Robinson, S.C., Cooper, P.A. (2012) The influence of moisture content variation on fungal pigment formation in spalted wood. Applied Microbiology and Biotechnology Express 2:69.
Tudor, D., Robinson, S.C., Cooper, P.A. (2011) The influence of moisture content and wood pH variation on fungal melanin formation in wood substrates. International Research Group on
188 Wood Protection IRG 11-10759. Queenstown, New Zealand. – oral presentation and paper submission.
Chapter 6:
Tudor, D., Robinson, S.C., Cooper, P.A. (2013) The influence of pH on pigment formation by lignicolous fungi. International Biodeterioration and Biodegradation 80:22-28
Tudor, D. (2011) The influence of moisture content and wood pH variation on fungal melanin formation in wood substrates. Forest Biomaterials Science Seminar, University of Toronto, – oral presentation.
Chapter 7:
Tudor, D., Robinson, S.C., Krigstin, S., Cooper, P.A. Fungal melanin formation from catechol and L-Dopa precursors by Trametes versicolor, Xylaria Polymorpha and Inonotus hispidus. In preparation.
Tudor, D., Robinson, S.C., Cooper, P.A. (2011) Effects of chemical treatment on fungal melanin stimulation for spalting, Annual meeting of Canadian Wood Preservation Association, Halifax, Canada. – oral presentation and paper submission.
Chapter 8:
Tudor, D., Robinson, S.C., Sage, T., Krigstin, S., Cooper, P.A. Microscopic investigation of fungal pigment formation and its morphology in wood substrates. In preparation.
Also, the adjacent experimental research of the results applicability were the subject of an art exposition entitled “Spalting” (September 2012), at the University of Toronto Art Center, comprised of 24 pieces of wood panels “painted” with fungal pigments as abstract art, and the artistic potential of spalted wood was also presented as follow:
Tudor, D., Krigstin, S., Cooper, P.A. (2012) Trees and Fungi -The Legacy of Richness and Darkness. Annual Art History Symposium, York University, Toronto, Canada. – oral presentation.
Other scientific contributions:
189 Robinson, S.C., Tudor, D., Hipson, S., Snider, H., Ng, S., Korshikov, E., Cooper, P.A. (2013) Methods of inoculating Acer spp., Populus spp., and Fagus grandifolia logs for commercial spalting applications. Journal of Wood Science in press. doi:10.1007/s10086-013-1335-5
Robinson, S.C., Tudor, D., Mansourian, Y., Cooper, P.A. (2012) The effects of several commercial wood coatings on the deterioration of biological pigments in wood exposed to UV light. Wood Science and Technology 47(3): 457-466
Robinson, S.C., Tudor, D., Snider, H., Cooper, P.A. (2012) Stimulating growth and xylindein production of Chlorociboria aeruginascens in agar-based systems. AMB Express 2:15.
Robinson, S.C., Tudor, D., Cooper, P.A. (2011) Feasibility of using red pigment producing fungi to stain wood for decorative applications. Canadian Journal of Forest Research 41:1722- 1728.
Robinson, S.C., Tudor, D., Cooper, P.A. (2011) Utilizing pigment-producing fungi to add commercial value to American beech (Fagus grandifolia). Applied Microbiology and Biotechnology 93(3):1041-1048.
Robinson, S.C., Tudor, D., Cooper, P.A. (2011) Promoting fungal pigment formation in wood by utilizing a modified decay jar method. Wood Science and Technology 46:841-849.
Robinson, S.C., Tudor, D., Cooper, P.A. (2011) Wood preference of spalting fungi in urban hardwood species. Int. Biodeterioration and Biodegradation 65:1145-1149.
Copyright permission letters were obtained for the images presented in Figure 2.2.