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Role of non-decay fungi on the weathering of wood Hernandez, Vicente 2012

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ROLE OF NON-DECAY FUNGI ON THE WEATHERING OF WOOD  by  Vicente Hernandez Master in Wood Science and Technology Universidad del Bio-Bio, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Forestry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  September 2012 © Vicente Hernandez, 2012  Abstract In this thesis I hypothesized that the graying of wood exposed outdoors is due to the presence of melanized fungi that are relatively resistant to UV-light. To test this hypothesis I examined the color and chemical changes at wood surfaces exposed to the weather and filtered solar radiation, isolated and identified fungi colonizing wood samples by DNA analysis and microscopy and examined the survival, growth and melanin production of staining fungi under UV, visible or no light. The ability of isolated fungi to decay wood was also tested by evaluating changes in the microstructure, mechanical, viscoelastic and chemical properties of spruce and lime wood incubated with fungi. Finally, I tested a novel non-biocidal approach to reduce the staining of wood by fungi, which employed melanin biosynthesis inhibitors (MBIs). My results support the general hypothesis (above) and reveal that weathered wood surfaces are grayed by the interactive effects of solar radiation and fungal colonization. UVradiation increased the production of melanin by the fungus most frequently isolated from weathered wood (Aureobasidium pullulans), which leads to darker weathered wood surfaces. Decay tests showed that species of Cladosporium, Coniochaeta, Epicoccum, Lewia, Mollisia and Phialocephala, were able to degrade wood tissues. In artificial media, MBIs in combination with UV-radiation blocked the growth of staining fungi, but at wood surfaces MBIs reduced fungal staining irrespective of the type of light that samples were exposed to. I conclude that UV-radiation and melanized fungi interact to influence the color of weathered wood surfaces. Degradation of wood by surface fungi is possible, but the extent of damage probably depends on the presence of conditions that favor microbial decay. Finally, the use of MBIs is a promising approach to control graying of weathered wood surfaces, but further research is required to optimize the treatments and test them outdoors.  ii  Preface Elements of Chapter 5 were presented at the IRG-Americas Regional Meeting; Guanacaste, Costa Rica; 2008, under the title: “The effects of solar radiation on the fungal colonization and color of weathered wood”. I conducted the experimental research, wrote the manuscript and presented the results at the conference. Co-authors and academic supervisors Dr Philip Evans and Dr Colette Breuil, helped with the experimental design, statistical analyses and edited the final manuscript. The citation for the paper is:  Hernandez V., Breuil C., and Evans P.; 2008; “The effects of solar radiation on the fungal colonization and color of weathered wood”; IRG-Americas Regional Meeting; Guanacaste, Costa Rica; IRG/WP 08-10676.  iii  Table of contents Abstract.................................................................................................................................... ii Preface .................................................................................................................................... iii Table of contents ..................................................................................................................... iv List of tables ............................................................................................................................. x List of figures .......................................................................................................................... xiii Acknowledgements ............................................................................................................. xxvii Dedication .......................................................................................................................... xxviii 1. Chapter 1: General introduction ........................................................................................ 1 1.1. Introduction .............................................................................................................. 1  2.  1.2.  General Hypothesis ................................................................................................... 3  1.3.  Scope and importance .............................................................................................. 5  1.4.  Study outline ............................................................................................................. 6  Chapter 2: Literature review .............................................................................................. 8 2.1. Weathering of wood ................................................................................................. 8 2.1.1.  Degradation of wood polymers by solar radiation ....................................................... 10  2.1.2.  Macro and microscopic effect of weathering .............................................................. 12  2.1.3.  Depth of weathering ................................................................................................... 14  2.2.  Biological organisms colonizing weathered wood surfaces ...................................... 15  2.2.1.  Fungi classification...................................................................................................... 17  2.2.2.  Factors affecting fungal survival in wood .................................................................... 21  2.2.3.  Fungi colonizing weathered surfaces .......................................................................... 22  2.2.3.1.  Introduction ............................................................................................................... 22  2.2.3.2.  Organisms colonizing weathered wood....................................................................... 22  2.2.3.3.  Effects of surface fungi on wood ................................................................................. 29  2.2.3.4.  Staining of coated and modified wood ........................................................................ 30  2.3.  Ultraviolet radiation and fungal melanins ............................................................... 32  2.3.1.  Effect of ultraviolet radiation on living cells and fungi ................................................. 32  2.3.2.  Fungal melanins.......................................................................................................... 36  2.3.2.1.  Properties and role of melanins .................................................................................. 36  2.3.2.2.  Synthesis of fungal melanins ....................................................................................... 38  2.4.  Fungal melanin biosynthesis inhibitors .................................................................... 43 iv  2.4.1.  MBIs targeting early stages of DHN melanin biosynthesis ........................................... 44  2.4.2.  MBIs targeting reductase enzymes ............................................................................. 45  2.4.3.  MBIs targeting dehydratase enzymes ......................................................................... 47  2.4.4.  Other inhibitors .......................................................................................................... 49  2.5. 3.  Chapter 3: Fungi colonizing the surface of southern pine exposed to natural weathering 51 3.1. Introduction ............................................................................................................ 51 3.2.  Materials and methods ........................................................................................... 53  3.2.1.  Wood samples and exposure ...................................................................................... 53  3.2.2.  Isolation, purification, identification and storage of fungi ........................................... 54  3.2.3.  Fungal diversity .......................................................................................................... 56  3.2.4.  Growth and color of fungi on solid culture media ....................................................... 57  3.2.5.  Microstructure of wood colonized by fungi ................................................................. 59  3.2.6.  Color of weathered wood and area stained by fungi ................................................... 59  3.2.7.  Chemical changes at weathered wood surfaces .......................................................... 61  3.3.  4.  Summary................................................................................................................. 49  Results .................................................................................................................... 62  3.3.1.  Fungal diversity .......................................................................................................... 62  3.3.2.  Growth and color of isolated fungi .............................................................................. 64  3.3.3.  Fungal colonization under light microscopy ................................................................ 68  3.3.4.  Color of weathered wood and area stained by fungi ................................................... 70  3.3.5.  Moisture content ........................................................................................................ 74  3.3.6.  FTIR spectra of samples exposed outdoors ................................................................. 75  3.4.  Discussion ............................................................................................................... 77  3.5.  Conclusions ............................................................................................................. 84  Chapter 4: Decaying abilities of fungi isolated from weathered wood ............................. 85 4.1. Introduction ............................................................................................................ 85 4.2.  Materials and methods ........................................................................................... 87  4.2.1.  Fungal screening ......................................................................................................... 87  4.2.2.  Decay test................................................................................................................... 90  4.2.2.1.  Experimental design ................................................................................................... 90  4.2.2.2.  Wood samples ............................................................................................................ 91  4.2.2.3.  Fungal inoculation and incubation .............................................................................. 93  v  4.2.2.4.  Mechanical property losses of veneers ....................................................................... 94  4.2.2.5.  Fourier transform infra-red spectroscopy ................................................................... 95  4.2.2.6.  Viscoelastic properties ................................................................................................ 95  4.2.2.7.  Microscopy ................................................................................................................. 96  4.3.  Results .................................................................................................................... 99  4.3.1.  Fungal screening ......................................................................................................... 99  4.3.2.  Decay test................................................................................................................. 101  4.3.2.1.  Mechanical property losses of veneers ..................................................................... 101  4.3.2.1.1. Peak tensile stress ratio ............................................................................................ 102 4.3.2.1.2. Modulus of elasticity (MOE) ratio ............................................................................. 104 4.3.2.1.3. Peak stiffness ratio ................................................................................................... 106 4.3.2.1.4. Peak toughness ratio ................................................................................................ 109  5.  4.3.2.2.  Viscoelastic properties .............................................................................................. 111  4.3.2.3.  Fourier transform infra-red spectroscopy ................................................................. 113  4.3.2.4.  Light microscopy ....................................................................................................... 128  4.3.2.5.  Scanning electron microscopy................................................................................... 137  4.4.  Discussion ............................................................................................................. 140  4.5.  Conclusions ........................................................................................................... 148  Chapter 5: Effects of solar radiation on the colonization of weathered wood by fungi .. 149 5.1 Introduction .......................................................................................................... 149 5.2  Materials and methods ......................................................................................... 150  5.2.1  Experimental design and statistical analyses ............................................................. 150  5.2.2  Wood samples .......................................................................................................... 152  5.2.3  Chemical treatments ................................................................................................ 153  5.2.4  Exposure................................................................................................................... 154  5.2.5  Determination of wood color and area colonized by fungi ........................................ 157  5.2.6  Chemical changes at weathered wood surfaces and isolation and identification of fungi ......................................................................................................................... 157  5.2.7  Fungal ecology and characterization of isolated fungi ............................................... 158  5.3  Results .................................................................................................................. 159  5.3.1  Color of wood after exposure ................................................................................... 159  5.3.2  Area colonized by fungi............................................................................................. 164  vi  6.  5.3.3  Moisture content ...................................................................................................... 170  5.3.4  Chemical changes at weathered wood surfaces ........................................................ 171  5.3.5  Fungal ecology and characterization of isolated fungi ............................................... 173  5.3.5.1  Frequency of isolation .............................................................................................. 179  5.3.5.2  Fungal diversity ........................................................................................................ 181  5.3.5.3  Characterization of fungi on solid culture media ....................................................... 181  5.4  Discussion ............................................................................................................. 186  5.5  Conclusions ........................................................................................................... 191  Chapter 6: Effect of UV radiation on melanization and growth of fungi isolated from weathered wood surfaces ............................................................................................. 192 6.1. Introduction .......................................................................................................... 192 6.2. 6.2.1.  Experimental design ................................................................................................. 194  6.2.2.  Fungi and culturing conditions .................................................................................. 195  6.2.3.  Exposure................................................................................................................... 196  6.2.4.  Determination of radial growth, mycelial color, spores, biomass and melanin production ................................................................................................................ 199  6.3.  7.  Materials and methods ......................................................................................... 194  Results .................................................................................................................. 203  6.3.1.  Melanin concentration ............................................................................................. 203  6.3.2.  Fungal biomass ......................................................................................................... 205  6.3.3.  Spore production ...................................................................................................... 206  6.3.4.  Radial growth of fungal cultures ............................................................................... 208  6.3.5.  Lightness of mycelia.................................................................................................. 209  6.4.  Discussion ............................................................................................................. 211  6.5.  Conclusions ........................................................................................................... 216  Chapter 7: UV light and melanin biosynthesis inhibitors as potential treatments against fungal staining ............................................................................................................... 217 7.1. Introduction .......................................................................................................... 217 7.2.  Materials and methods ......................................................................................... 219  7.2.1.  In-vitro testing of the melanin biosynthesis inhibitors cerulenin, tricyclazole and carpropamid, and the fungicide quinoxyfen .............................................................. 219  7.2.1.1.  Experimental design ................................................................................................. 219  vii  7.2.1.2.  Chemicals and culture media .................................................................................... 220  7.2.1.3.  Inoculation of media with A. pullulans and C. cladosporioides ................................... 221  7.2.1.4.  Exposure to UV and visible light and quantification of number of fungal colonies after exposure .......................................................................................................... 222  7.2.2.  Effect of chemicals and UV radiation on fungal staining of wood .............................. 224  7.2.2.1.  Experimental design ................................................................................................. 224  7.2.2.2.  Wood samples .......................................................................................................... 225  7.2.2.3.  Preparation of solutions and impregnation of wood veneers .................................... 226  7.2.2.4.  Inoculation of media with A. pullulans and exposure of treated wood sections to UV and visible light......................................................................................................... 226  7.2.2.5.  Quantification of staining and color of treated and inoculated veneer sections exposed to UV or visible light.................................................................................... 227  7.2.2.6.  Microscopy ............................................................................................................... 232  7.3.  8.  Results .................................................................................................................. 232  7.3.1.  MBIs tested in malt extract agar ............................................................................... 232  7.3.2.  Effects of MBIs and UV radiation on fungal staining and color of wood ..................... 239  7.3.2.1.  Effect on fungal staining ........................................................................................... 239  7.3.2.2.  Effect on color; comparison of stained wood surfaces .............................................. 243  7.3.2.3.  Effect on color of wood veneers in comparison to unstained wood surfaces ............. 244  7.3.2.4.  Effect of the treatment on the natural color of wood ................................................ 247  7.4.  Discussion ............................................................................................................. 252  7.5.  Conclusions ........................................................................................................... 257  Chapter 8: General discussion, conclusions and suggestions for further research .......... 258 8.1. General discussion ................................................................................................ 258 8.2.  Conclusions ........................................................................................................... 263  8.3.  Suggestion for further research ............................................................................. 265  References ........................................................................................................................... 267 Appendices ........................................................................................................................... 294 Appendix 1: Statistical analysis Chapter 4 ............................................................................. 295 Analysis of variance tensile stress ratio ............................................................................ 295 Analysis of variance modulus of elasticity (MOE) ratio...................................................... 297 Analysis of variance peak stiffness ratio ........................................................................... 299  viii  Analysis of variance peak toughness (work) ratio ............................................................. 301  Appendix 2: Graphic determination of modulus of elasticity, example of calculation ........... 303 Appendix 3: Statistical analysis Chapter 5 ............................................................................. 304 Analysis of variance frequency of isolation of fungi .......................................................... 304 Analysis of variance fungal stains 0 to 40 weeks ............................................................... 306 Analysis of variance color of wood surfaces 0 to 40 weeks ............................................... 370  Appendix 4: Images of fungal colonization evolution in southern pine samples exposed under filter transmitting different wavelengths of solar radiation (Chapter 5) ................................ 550 Appendix 5: Result for reciprocal Simpson index (Chapter 5) ................................................ 555 Appendix 6: Statistical analysis Chapter 6 ............................................................................. 556 Analysis of variance fungal biomass ................................................................................. 556 Analysis of variance lightness fungal mycelia .................................................................... 547 Analysis of variance melanin concentration...................................................................... 559 Analysis of variance radial growth .................................................................................... 560 Analysis of variance spore concentration ......................................................................... 561  Appendix 7: Calibration curves for calculation of fungal melanin concentration (Chapter 6) 563 Appendix 8: Statistical analysis melanin biosynthesis inhibitors tested in artificial media (Chapter 7) ........................................................................................................................... 569 Analysis of variance fungal colonies in plates after exposure artificial media .................... 569  Appendix 9: Statistical analysis melanin biosynthesis inhibitors tested in wood veneers (Chapter 7) ........................................................................................................................... 571 Analysis of variance color differences veneers inoculated ................................................ 571 Analysis of variance color differences veneers inoculated vs not inoculated ..................... 573 Analysis of variance color differences veneers not inoculated .......................................... 575 Analysis of variance fungal stain ratio............................................................................... 577  ix  List of tables Table 2.1: Fungi isolated from wood surface exposed outdoors above the ground. The table also reports the author, substrate and country of isolation. Question mark (?) is featured when information was not available ..................................................................................... 26 Table 2.2: Melanin biosynthesis inhibitors of reductase registered in Japan in 2001 ............. 46 Table 2.3: Melanin biosynthesis inhibitors of dehydratase registered in Japan by 2001 (Kurahashi, 2001) ................................................................................................................. 48 Table 3.1: Density and growth rate of southern pine samples .............................................. 54 Table 3.2: Monthly weather conditions during the exposure period in Vancouver, Canada; reported by Canada’s National Weather Archive .................................................................. 54 Table 3.3: Morphological features of common darks moulds colonizing weathered wood (Barnett and Hunter 1998) ................................................................................................... 56 Table 3.4: Fungi isolated from southern lodgepole pine wood samples after 40 weeks of outdoor exposure in Vancouver, Canada .............................................................................. 63 Table 3.5: Fungal diversity in southern pine wood samples exposed to the weather for 40 weeks in Vancouver, Canada ................................................................................................ 64 Table 3.6: Growth of fungi cultured onto solid malt extract agar (1% Difco) after 7 days of growth.................................................................................................................................. 65 Table 3.7: Lightness of fungi cultured onto solid media malt extract (agar 1% Difco) after 7 days of growth ..................................................................................................................... 65 Table 4.1: Fungi tested for their ability to synthesize lignolytic and cellulolytic enzymes ...... 90 Table 4.2: Summary of the experimental design used for the decay test .............................. 91 Table 4.3: Laccase activity and index for enzymatic activity for carboxymethyl cellulose (CMC) ........................................................................................................................................... 100 Table 4.4: Fungi isolated from weathered wood and tested for their ability to breakdown wood .................................................................................................................................. 101 Table 4.5: Significant effects of, and interactions between fungal species and wood species, on mechanical properties of veneers exposed to test fungi ................................................ 102 x  Table 5.1: Summary of experimental design used to test the effect of solar radiation on wood surfaces and fungal colonization......................................................................................... 152 Table 5.2: Chemical treatment applied to southern pine wood samples exposed outdoors for 40 weeks in Vancouver (Canada) and exposed to different wavelengths of the solar radiation ........................................................................................................................................... 154 Table 5.3: Filters used to block selected regions of the solar spectrum from reaching samples ........................................................................................................................................... 155 Table 5.4: Fungi isolated from samples exposed to UVA+UVB+Vis.light+IR. Primer sequenced for rDNA identification ITS4 ................................................................................................ 174 Table 5.5: Fungi isolated from samples exposed to UVA+Vis.light+IR. Primer sequenced for rDNA identification ITS4 ..................................................................................................... 175 Table 5.6: Fungi isolated from samples exposed to Vis.light+IR. Primer sequenced for rDNA identification ITS4 ............................................................................................................... 176 Table 5.7: Fungi isolated from samples exposed to IR. Primer sequenced for rDNA identification ITS4 ............................................................................................................... 177 Table 5.8: Fungi isolated from samples exposed to No light. Primer sequenced for rDNA identification ITS4 ............................................................................................................... 178 Table 5.9: Lightness of fungi grown on solid media malt extract agar (1% MEA) ................. 182 Table 5.10: Growth of fungi grown on solid malt extract agar (1% MEA) after 7 days ......... 184 Table 6.1: Summary of experimental design used to test the effect of different light sources on fungal development and melanization ........................................................................... 195 Table 6.2: Significant effects of, and interaction between exposure to light and fungal species on melanin concentration, biomass, radial growth and lightness of fungal cultures ........... 203 Table 7.1: Summary of experimental design used to test the effect of different melanin biosynthesis inhibitors and a fungicide on the survival of fungi .......................................... 220 Table 7.2: Summary of experimental design used to test the effect of a melanin biosynthesis inhibitor and UV radiation on fungal staining of wood ........................................................ 225 Table 7.3: Significant effect of, and interactions between exposure to light, chemical, and fungal species on the number of colonies growing on agar plates ...................................... 232 xi  Table 7.4: Significant effect of, and interaction between exposure to light, chemical treatments and concentration on stained area and color change (ΔE) of fungal and water inoculated spruce veneers surfaces, after 5 days of exposure. Stained area of veneers was analyzed as ratio of stained area of impregnated veneers over control veneers. Natural logarithm (LN) transformation was used to fulfill assumptions of analysis of variance ........ 239 Table A5.1: reciprocal diversity Simpson index for fungi isolated from weathered southern pine samples exposed outdoors under different filters for 40 weeks .................................. 555 Table A7.1: UV-VIS light absorbance and concentration of fungal melanin produced by C. cladosporioides [R2F33] ..................................................................................................... 563 Table A7.2: UV-VIS light absorbance and concentration of fungal melanin produced by A. pullulans [R2F32.2] ............................................................................................................. 564 Table A7.3: UV-VIS light absorbance and concentration of fungal melanin produced by O. piliferum [TAB28] ............................................................................................................... 565 Table A7.4: UV-VIS light absorbance and concentration of fungal melanin produced by A. pullulans [ATCC 42371] ....................................................................................................... 566 Table A7.5: UV-VIS light absorbance and concentration of fungal melanin produced by A. pullulans [R1F22W] ............................................................................................................ 567 Table A7.6: UV-VIS light absorbance and concentration of fungal melanin produced by O. piliferum [Cartapip97] ........................................................................................................ 568  xii  List of figures Figure 2.1: Appearance of weathered Southern pine (Pinus sp.) wood. Note the graying and surface checking of the wood ................................................................................................. 8 Figure 2.2: Phenoxy radicals produced during photodegradation of lignin. (a) Guaiacoxyl radical; (b) Phenacyl radical; and (c) Cetyl radical ................................................................. 11 Figure 2.3: Biological classification of true fungi as described by Kendrick (2000) ................. 19 Figure 2.4: Possible effects of absorption of UV radiation by deoxyribonucleic acid (DNA) (Harm 1980) ......................................................................................................................... 33 Figure 2.5: Precursors of fungal melanins ............................................................................. 39 Figure 2.6: DHN melanin biosynthesis .................................................................................. 42 Figure 2.7: Melanin biosynthesis inhibitors acting on the early stages of the biosynthesis of melanin. (a) Structure of cerulenin (Fleet and Breuil 2002) and (b) [3-[4’-bromo-2’,6’dimethylphenoxy]methyl-4-[(3”-methylphenyl) aminocarbonyl]methyl-1,2,4-oxadiazol-5-one] (KC10017) (Kim et al. 1998) .................................................................................................. 45 Figure 2.8: Compounds that inhibit DHN-melanin biosynthesis in P. oryzae and other brown and black fungi. (a) N-methyl-2-quinolone (MQ), (b) s-triazolo-[4,3-a]quinoline (TQ) and (c) Coumarin (Wheeler and Klich 1995) ..................................................................................... 49 Figure 3.1: Growth rate and fungal mat color measurements. (a) Growth measurement in Photoshop of a fungal colony after 7 days of growth on malt extract agar (MEA) 1%; note the use of the ruler tool to estimate the diametrical growth of the fungal colony; (b) Fungal mat color measurement in Photoshop after 20 days of growth onto MEA 1%; note the original image of the colony, the selection of a relevant area for the measurement, information about the RGB color of the selected pixels (red square right side of the image) and color picker tool for transformation from RGB into CIELab color ..................................................................... 58 Figure 3.2: Measurement using Photoshop of the area of a wood sample stained by fungi. Original image (left) and colored pixels (centre) for quantification of stained area ............... 60 Figure 3.3: Dark fungi isolated from weathered wood, after 20 days of growth on malt extract agar (1% Difco): (a) Hormonema dematioides; (b) Cladosporium sp.; (c) Aureobasidium pullulans; (d) Alternaria sp.; (e) Mollisia minutella; and (f) Glonium pusillum ....................... 66  xiii  Figure 3.4: Light fungi isolated from weathered wood, after 20 days of growth on malt extract agar (1% Difco): (a) Epicoccum nigrum; (b) Phoma sp.; (c) Lecythophora sp.; (d) Aureobasidium pullulans; and (e) Truncatella angustata ...................................................... 67 Figure 3.5: Light microscopy images of sections from southern pine wood samples exposed outdoors for 40 weeks. (a) Tangential longitudinal section showing dark hyphae in degraded rays and tracheids; (b) Radial longitudinal section showing dark hyphae colonizing ray parenchyma cells, but not ray tracheids in rays; (c) Radial section showing dark hyphae colonizing tracheids approximately 200 micrometers beneath the weathered wood surface ............................................................................................................................................. 69 Figure 3.6: Area of southern pine wood samples colonized by fungi during 40 weeks of exposure outdoors. Error bars depict standard deviations.................................................... 70 Figure 3.7: Changes in color and colonized area of southern pine wood samples exposed to weather for 40 weeks in Vancouver, Canada. (a) week 0, (b) week 4, (c) week 8, (d) week 12, (e) week 16, (f) week 20, (g) week 32, (h) week 40 ............................................................... 71 Figure 3.8: Changes in lightness of southern pine wood samples exposed to the weather in Vancouver for 40 weeks. Lightness is expressed using the CIELab system, L [100=white; 0=black]. Error bars depicting standard deviations ............................................................... 72 Figure 3.9: Changes in redness/greenness of southern pine wood samples exposed to the weather in Vancouver for 40 weeks. Redness/greenness is expressed using the CIELab system, a [+60=red; -60=green]. Error bars depict standard deviations ................................ 73 Figure 3.10: Changes in yellowness/blueness of southern pine wood samples exposed to the weather in Vancouver for 40 weeks. Yellowness/blueness is expressed using the CIELab system, b [+60=yellow; -60=blue]. Error bars depict standard deviations ............................. 74 Figure 3.11: Changes in moisture content of southern pine wood samples exposed outdoors for 40 weeks in Vancouver Canada (data available for week 10 to 32). The figure includes the rain that fell (mm) during the exposure trial. Error bars depict standard deviations ............. 75 Figure 3.12: FTIR absorbance spectra of southern pine wood surfaces exposed to the weather for 40 weeks and unexposed control. Exposed sample showing decrease of peaks at 1740, 1655, 1514 and 1462 cm-1 related to lignin and little change in peaks at 1158 and 898 cm-1 related to carbohydrates ...................................................................................................... 76 Figure 4.1: Fungal screening: (a) Trichaptum abietinum after seven days of growth on media containing guiacol (0.2 g/L), the enzymatic activity of the fungus was ranked as high (+++); (b) carboxymethyl cellulose (CMC) assay; measurement of halo diameter using the ruler tool of Photoshop. The fungus in the image is Lecythophora sp. after 14 days of growth in media containing CMC 10 (g/L) stained with Congo red .................................................................. 89 xiv  Figure 4.2: Wood samples after 1 week of exposure to fungi: (a) solid wood samples; (b) wood veneers ....................................................................................................................... 98 Figure 4.3: Equipment for sample preparation and testing; (a) sliding microtome with blade holder and clamping device for wood samples; (b) Instron Universal tensile tester (model 5565) and; (c) Dynamic mechanical analyzer (Perkin Elmer model DMA 7e) ......................... 98 Figure 4.4: Peak tensile stress ratio (peak tensile stress of bioassayed veneer/peak tensile stress sound wood) of wood veneers exposed to fungi isolated from weathered wood. Cladosporium sp. and C. ligniaria produced the highest losses in peak tensile stress followed by the control fungi C. globosum and T. abietinum. Peak tensile stress ratio close to one indicates that tensile stress was similar to that of sound wood. Error bars correspond to ±SED ........................................................................................................................................... 102 Figure 4.5: Peak tensile stress ratio (peak tensile stress of bioassayed veneer/peak tensile stress sound wood) of lime and spruce veneers. Lime veneers treated with fungi isolated from weathered wood showed a significantly lower peak tensile ratio than spruce veneers. Peak tensile stress ratio close to one indicates that tensile stress was similar to that of sound wood. Error bars correspond to ±SED ................................................................................. 103 Figure 4.6: Peak tensile stress ratio (peak tensile stress of bioassayed veneer/peak tensile stress of sound wood) of lime and wood veneers inoculated with fungi isolated from weathered wood. Statistical interaction of fungi x wood (encircled in red) occurred due to the behavior of lime and spruce veneers incubated with Phialophora sp. Peak tensile stress ratio close to one indicates that tensile stress was similar to that of sound wood....................... 104 Figure 4.7: Modulus of elasticity (MOE) ratio (MOE bioassayed veneer/MOE sound wood) of wood veneers exposed to fungi isolated from weathered wood. Cladosporium sp. and C. ligniaria produced the highest losses in MOE followed by C. globosum, Phialocephala sp. and T. abietinum. MOE ratio close to one indicates that MOE was similar to that of sound wood. Error bars correspond to ±SED............................................................................................ 105 Figure 4.8: Modulus of elasticity (MOE) ratio (MOE bioassayed veneer/MOE sound wood) lime and spruce veneers. Lime veneers incubated with fungi isolated from weathered wood showed a significantly lower MOE ratio than spruce veneers. MOE ratio close to one indicated that MOE was similar to that of sound wood. Error bars correspond to ±SED ..... 105 Figure 4.9: Modulus of elasticity (MOE) ratio (MOE bioassayed veneer/MOE sound wood) of lime and wood veneers incubated with fungi isolated from weathered wood. MOE ratio close to one indicates that MOE was similar to that of sound wood ............................................ 106 Figure 4.10: Peak stiffness ratio (peak stiffness bioassayed veneer/peak stiffness sound wood) of wood veneers exposed to fungi isolated from weathered wood. Cladosporium sp., C. ligniaria and C. globosum produced the highest losses in peak tensile stress. Peak stiffness xv  ratio close to one indicates that peak stiffness was similar to that of sound wood. Error bars correspond to ±SED ............................................................................................................ 107 Figure 4.11: Peak stiffness ratio (peak stiffness bioassayed veneer/peak stiffness sound wood) of lime and spruce veneers. Lime veneers incubated with fungi isolated from weathered wood showed a significantly lower peak stiffness ratio than spruce veneers. Peak stiffness ratio close to one indicated that peak stiffness was similar to that of sound wood. Error bars correspond to ±SED............................................................................................ 108 Figure 4.12: Peak stiffness ratio (peak stiffness bioassayed veneer/peak stiffness sound wood) of lime and wood veneers incubated with fungi isolated from weathered wood. Peak stiffness ratio close to one indicates that peak stiffness was similar to that of sound wood control................................................................................................................................ 108 Figure 4.13: Peak toughness ratio (peak toughness bioassayed veneer/peak toughness sound wood) of wood veneers incubated with fungi isolated from weathered wood. Cladosporium sp., C. ligniaria and C. globosum produced the highest losses in peak tensile stress followed by T. abietinum. Peak toughness ratio close to one indicates that peak toughness was similar to that of sound wood. Error bars correspond to ±SED ....................................................... 109 Figure 4.14: Peak toughness ratio (peak toughness treated veneer/peak toughness sound wood) of lime and spruce veneers. Lime veneers treated with fungi isolated from weathered wood showed significantly lower peak stiffness ratio than spruce veneers. Peak toughness ratio close to one indicates that peak toughness was similar to that of sound wood control. Error bars correspond to ±SED............................................................................................ 110 Figure 4.15: Peak toughness ratio (peak toughness bioassayed veneer/peak toughness sound wood) of lime and wood veneers incubated with fungi isolated from weathered wood. Peak toughness ratio close to one indicated that peak toughness was similar to that of sound wood control ...................................................................................................................... 111 Figure 4.16: Storage modulus of lime wood samples after 12 weeks of incubation with fungi isolated from weathered wood, blue arrows indicate zones of viscoelastic transition ........ 112 Figure 4.17:Storage modulus of spruce wood samples after 12 weeks of incubation with fungi isolated from weathered wood, blue arrow indicate a zone of viscoelastic transition ........ 113 Figure 4.18: Normalized FTIR spectra of lime wood exposed to Alternaria sp., A. pullulans (black) and A. pullulans (white). Peaks related to cellulose and hemicelluloses at 1108 and 1737 cm-1 were reduced in size by Alternaria. A. pullulans (black) reduced the sizes of the peaks at 1059, 1108, 1165 and 1737 cm-1 and A. pullulans (white) reduced the sizes of the peaks at 1165 and 1737 cm -1. All fungi increased the peak at 1655 cm-1. The spectrum for the sound wood control is shown for comparison .................................................................... 116 xvi  Figure 4.19: Normalized FTIR spectra of lime wood exposed to B. fuckeliana, Cladosporium sp., and C. puteana. Cladosporium sp. decreased the peak related to cellulose and hemicelluloses 1059 cm-1. C. ligniaria decreased peaks related to cellulose, hemicelluloses and lignin at 1059, 1244, 1376, 1510, 1598 and 1737 cm -1. Both of the latter fungal species increased the peak at 1655 cm-1. No changes in the spectrum of lime wood were produced by B. fuckeliana. The spectrum for the sound wood control is shown for comparison ............. 117 Figure 4.20: Normalized FTIR spectra of lime wood exposed to E. nigrum, H. dematioides and Lecythophora sp. H. dematioides decreased peaks related to cellulose and lignin at 1244, 1376, 1598, 1737 cm-1. Lecythophora sp. increased the peak at 1108 (cellulose and hemicelluloses). All fungi increased the peak at 1655 cm -1, but E. nigrum did not produce any other changes. The spectrum for the sound wood control is shown for comparison .......... 118 Figure 4.21: Normalized FTIR spectra of lime wood exposed to L. infectoria, M. minutella and Phialocephala sp. L. infectoria decreased the peak related to cellulose and hemicelluloses at 1737 cm-1. Phialocephala sp. decreased peaks related to cellulose and hemicelluloses at 1244, 1376 and 1737 cm-1. M. minutella decreased peaks related cellulose, hemicelluloses and lignin at 1244, 1376 1510 and 1737 cm-1. All fungi increased the peak at 1655 cm-1. The spectrum of the sound wood control is shown for comparison .......................................... 119 Figure 4.22: Normalized FTIR spectra of lime wood exposed to Phialophora sp., Phoma sp. and C. globosum. Phialophora sp. decreased the peak related to lignin at 1510 cm -1, Phoma sp. decreased the peak related cellulose and hemicelluloses at 1737 cm -1. C. globosum decreased the peaks at 1244 and 1737 cm-1 related to cellulose and lignin. All fungi increased the peak at 1655 cm-1. The spectrum for the sound wood control is shown for comparison ........................................................................................................................................... 120 Figure 4.23: Normalized FTIR spectra of lime wood exposed to C. puteana and T. abietinum. C. puteana decreased the peaks at 1244 and 1737 cm-1 related to cellulose and lignin. T. abietinum increased the peak at 1165 cm-1 related to cellulose and hemicelluloses and decreased the peaks at 1510 and 1598 cm-1 related to lignin. Both fungal species increased the peak at 1655 cm-1. The spectrum for the sound wood control is shown for comparison ........................................................................................................................................... 121 Figure 4.24: Normalized FTIR spectra of spruce wood exposed to Alternaria sp., A. pullulans (black) and A. pullulans (white). The peak related to cellulose and hemicelluloses 1737 cm-1 was decreased by Alternaria. A. pullulans (black) decreased the peak at 1268 cm-1 (lignin). A. pullulans (white) decreased the peak at 1731 cm-1 (cellulose and hemicelluloses). All fungi increased the peak at 1655 cm-1. The spectrum of the sound wood control is shown for comparison ........................................................................................................................ 122 Figure 4.25: Normalized FTIR spectra of spruce wood exposed to B. fuckeliana, Cladosporium sp., and C. puteana. B. fuckeliana and C. ligniaria decreased the peak at 1505 cm-1 related to xvii  lignin. All fungal species increased the peak at 1655 cm-1. Cladosporium sp. did not produce any further changes. The spectrum of the sound wood control is shown for comparison ... 123 Figure 4.26: Normalized FTIR spectra of spruce wood exposed to E. nigrum, H. dematioides and Lecythophora sp. H. dematioides decreased peaks related to cellulose and lignin at 1268, 1505 and 1737 cm-1. Lecythophora sp. decreased peaks at 1737 (cellulose and hemicelluloses) and 1268, 1462 and 1505 cm-1 (lignin). E. nigrum decreased the peak related to cellulose and hemicelluloses at 1737 cm-1. All fungi increased the peak at 1655 cm-1. The spectrum of the sound wood control is shown for comparison .......................................... 124 Figure 4.27: Normalized FTIR spectra of spruce wood exposed to L. infectoria, M. minutella and Phialocephala sp. L. infectoria decreased the peak related to cellulose and hemicelluloses at 1737 cm-1 and 1462 cm-1 related to lignin. Phialocephala sp. increased the peak at 1268 cm-1 (lignin), M. minutella decreased peaks related to cellulose, hemicelluloses, and lignin at 1268 and 1737 cm-1. All fungi increased the peak at 1655 cm-1. The spectrum of the sound control is shown for comparison ........................................................................ 125 Figure 4.28: Normalized FTIR spectra of spruce wood exposed to Phialophora sp., Phoma sp. and C. globosum. C. globosum decreased the peak at 1737 cm-1 related to cellulose and hemicelluloses. Phialophora and C. globosum increased the peak at 1655 cm-1. No changes were produced in wood exposed to Phoma sp. All fungi increased the peak at 1655 cm-1. The spectrum of the sound wood control is shown for comparison .......................................... 126 Figure 4.29: Normalized FTIR spectra of spruce wood exposed to C. puteana and T. abietinum. C. puteana decreased the peaks at 1268, 1462, 1505 and 1737 cm-1 related to cellulose, hemicelluloses and lignin. T. abietinum decreased the peaks at 1268 cm-1 related to lignin and increased the peak at 1165 cm -1. The spectrum of the sound wood control is shown for comparison ........................................................................................................ 127 Figure 4.30: Light microscopy images of lime wood colonized by (a) Alternaria sp.; (b) A. pullulans (black); (c) A. pullulans (white); (d) B. fuckeliana; (e) C. globosum; (f) Cladosporium sp.; (g) control .................................................................................................................... 130 Figure 4.31: Light microscopy images of lime wood colonized by (a) C. ligniaria; (b) C. puteana; (c) E. nigrum; (d) H. dematioides; (e) Lecythophora sp.; (f) L. infectoria; and (g) control................................................................................................................................ 131 Figure 4.32: Light microscopy images of lime wood colonized by (a) M. minutella; (b) Phialocephala sp.; (c) Phialophora sp.; (d) Phoma sp.; (e) T. abietinum; and (f) sound wood control................................................................................................................................ 132 Figure 4.33: Light microscopy images of spruce wood colonized by (a) Alternaria sp.; (b) A. pullulans (black); (c) A. pullulans (white); (d) B. fuckeliana; (e) C. globosum; (f) Cladosporium sp.; and (g) control ............................................................................................................. 134 xviii  Figure 4.34: Light microscopy images of spruce wood colonized by (a) C. ligniaria; (b) C. puteana; (c) E. nigrum; (d) H. dematioides; (e) Lecythophora sp.; (d) L. infectoria; and (g) Control ............................................................................................................................... 135 Figure 4.35: Light microscopy images of spruce wood colonized by (a) M. minutella; (b) Phialocephala sp.; (c) Phialophora sp.; (d) Phoma sp.; (e) T. abietinum; and (f) sound wood control................................................................................................................................ 136 Figure 4.36: SEM images of lime wood colonized by Cladosporium sp. and A. pullulans (black). a) Cladosporium sp. formed a complex and packed net of hyphae on the surface the veneer; b) and c) Cladosporium sp. eroded the wood and the whole surface was affected; d) higher magnification image of a veneer degraded by Cladosporium sp. revealed that in some cases the wood cells were degraded to more basic sub-units; e) lime wood veneers colonized by A. pullulans showed no sign of decay at the surface despite colonization by hyphae; f) sound wood control ...................................................................................................................... 138 Figure 4.37: SEM images of spruce wood colonized by Cladosporium sp. a) presence of hyphae covering the wood surface; b) higher magnification imagine showing the presence of a complex network of hyphae and spores on the veneer, but no signs of degradation were observed; c) sound wood control ....................................................................................... 139 Figure 5.1: Distribution of chemical treatments, testing areas and chemical loads. The figure shows the treatments applied to sample 3 (block 1) exposed under a filter transmitting all wavelengths of solar spectrum (Filter 1) ............................................................................. 153 Figure 5.2: Rack used for exposure of wood to different wavelengths of the solar radiation. (a) and (b) engineering drawings of the rack featuring angled aluminum sheets; (c) actual view of the rack and the five different polymethylmethacrylate filters ............................... 156 Figure 5.3: Lightness (L) of southern pine wood samples during 40 weeks of exposure under polymethylmethacrylate filters. Lightness is expressed using the CIELab parameter, L [100=white; 0=black]. Filter 1 transmitted UVB+UVA+Vis.light+IR; Filter 2 transmitted UVA+Vis.light+IR; Filter 3 transmitted Vis.light+IR; Filter 4 transmitted IR; and Filter 5 transmitted no radiation (L.s.d. bars for comparison of means only apply for the specific week in which they are located) ......................................................................................... 161 Figure 5.4: Redness-greenness (a) of southern pine wood samples during 40 weeks of exposure under polymethylmethacrylate filters. Redness/greenness is expressed using the CIELab parameter, a [+60=red; -60=green]. Filter 1 transmitted UVB+UVA+Vis.light+IR; Filter 2 transmitted UVA+Vis.light+IR; Filter 3 transmitted Vis.light+IR; Filter 4 transmitted IR; and Filter 5 transmitted no radiation (L.s.d. bars for comparison of means only apply for the specific week in which they are located)............................................................................. 162 xix  Figure 5.5: Yellowness-blueness (b) of southern pine wood samples during 40 weeks of exposure under polymethylmethacrylate filters. Yellowness/blueness is expressed using the CIELab parameter, b [+60=yellow; -60=blue]. Filter 1 transmitted UVB+UVA+Vis.light+IR; Filter 2 transmitted UVA+Vis.light+IR; Filter 3 transmitted Vis.light+IR; Filter 4 transmitted IR; and Filter 5 transmitted no radiation (L.s.d. bars for comparison of means only apply for the specific week in which they are located)............................................................................. 163 Figure 5.6: Area of southern pine wood samples colonized by fungi during 40 weeks of exposure under different polymethylmethacrylate filters. Filter 1 transmitted UVB+UVA+Vis.light+IR; Filter 2 transmitted UVA+Vis.light+IR; Filter 3 transmitted Vis.light+IR; Filter 4 transmitted IR; and Filter 5 transmitted no radiation. After 12 weeks of exposure the total area of specimens stained by fungi ranged from 40 % to 90 %. After 20 weeks exposure, greater than 90 percent of the area of specimens was stained. L.s.d. bars for comparison of means apply only for the specific week in which they are located ...................................... 165 Figure 5.7: Appearance of southern pine wood samples exposed to the weather for 2 weeks in Vancouver, Canada, under a polymethylmethacrylate filter transmitting all wavelengths of solar radiation (Filter 1). Blue arrows show black dots attributable to early stages of fungal colonization ........................................................................................................................ 166 Figure 5.8: Appearance of southern pine wood samples exposed to the weather for 12 weeks in Vancouver, Canada, under filters 1 (a), 2 (b), 3 (c), 4 (d), 5 (e) and control sample stored in a conditioning room (f) ....................................................................................................... 167 Figure 5.9: Appearance of southern pine wood samples exposed to the weather for 16 weeks in Vancouver, Canada, under filters 1 (a), 2 (b), 3 (c), 4 (d), 5 (e) and control sample stored in a conditioning room (f) ....................................................................................................... 168 Figure 5.10: Appearance of southern pine wood samples exposed to the weather for 40 weeks in Vancouver, Canada, under filters 1 (a), 2 (b), 3 (c), 4 (d), 5 (e) and control sample stored in a conditioning room (f) ........................................................................................ 169 Figure 5.11: Changes in moisture content of southern pine wood samples during 40 weeks of exposure under polymethylmethacrylate filters in Vancouver, Canada (data available from week 10 to 32). The figure includes the monthly rainfall total during the exposure trial. Analysis of variance revealed no significant differences in the weekly moisture contents of samples exposed under the different filters ....................................................................... 170 Figure 5.12: Normalized FTIR absorbance spectra of southern pine wood surfaces exposed to the weather for 40 weeks under polymethylmethacrylate filters and unexposed control. Filter 1 transmitted UVB+UVA+Vis.light+IR; Filter 2 transmitted UVA+Vis.light+IR; Filter 3 transmitted Vis.light+IR; Filter 4 transmitted IR; and Filter 5 transmitted no radiation. Exposed samples showed decreases in peaks at 1740, 1514 and 1462 cm -1 related to lignin xx  and 1158 cm-1 related to carbohydrates. The spectrum of the unexposed control is included for comparison ................................................................................................................... 172 Figure 5.13: Frequency of isolation of fungi from southern pine samples exposed to different wavelengths of solar radiation under polymethylmethacrylate filters (results averaged across filter type and expressed as ratio of occurrence) ................................................................ 180 Figure 5.14: Frequency of isolation of fungi from southern pine samples exposed to different wavelengths of solar radiation under polymethylmethacrylate filters. Factor responsible for the interaction of filter type x fungal species (encircled in red). Results expressed as ratio of frequency of occurrence ..................................................................................................... 180 Figure 5.15: Fungi isolated from weathered wood after 20 days of growth on 1% malt extract agar arranged from the darkest to the lightest: (a) A. pullulans (black); (b) H. dematioides; (c) Cladosporium sp.; (d) A. lycopodina; (e) Alternaria sp.; (f) Lewia sp.; (g) B. stevensii; (h) E. nigrum; (i) Leptosphaerulina sp.; (j) Phialocephala sp.; (k) A. pullulans (white); (l) Phoma sp.; (m) Penicillium sp.; (n) V. ambiens; (o) C. ligniaria; (p) Lecythophora sp.; (q) B. fuckeliana; (r) Peniophora sp.; (s) T. viride; and (t) Rhizopogon sp. ............................................................ 183 Figure 5.16: Fungi isolated from weathered wood after 20 days of growth on 1% malt extract agar arranged from the fastest to the slowest growing species: (a) T. viride; (b) V. ambiens; (c) A. lycopodina; (d) B. stevensii; (e) B. fuckeliana; (f) Lewia sp.; (g) Peniophora sp.; (h) Alternaria sp.; (i) E. nigrum; (j) Leptosphaerulina sp.; (k) Phoma sp.; (l) Penicillium sp.; (m) H. dematioides; (n) A. pullulans (black); (o) A. pullulans (white); (p) Phialocephala sp.; (q) Cladosporium sp.; (r) Lecythophora sp.; (s) C. ligniaria; and (t) Rhizopogon sp. ................... 185 Figure 6.1: Transmittance of a quartz glass lid to UV (340 nm) and visible light (450 nm approx.), Petri dish glass is shown. Transmittance was measured using a UV-VIS spectrophotometer (Varian Model Cary 50 Bio) ................................................................. 196 Figure 6.2: UV and visible light exposure units and irradiance charts. (a) UV exposure unit, the unit included 2 UV bulbs 340 nm, 40 W (Q-Lab Corp.); (b) visible light exposure unit, the unit included 2 fluorescent bulbs 450 nm approx. F40L/AQ/ECO wide spectrum, 40W (General electric); (c) irradiance chart for UV tubes; and (d) irradiance chart for visible light tubes. Irradiance charts were kindly provided by the manufacturers ............................................ 198 Figure 6.3: Determination of spore concentration by hemocytometer counting ................. 200 Figure 6.4: Melanin production of fungi isolated from weathered wood (including controls) after 7 days of growth under UV or visible light, or when grown in the dark. L.s.d. (least significant difference bar) ................................................................................................... 205  xxi  Figure 6.5: Production of biomass by fungi isolated from weathered wood (including controls) after 7 days of growth under UV or visible light, or when grown in the dark. L.s.d. (least significant difference bar) ................................................................................................... 206 Figure 6.6: Production of spores by fungi isolated from weathered wood (including controls) after 7 days of growth under UV or visible light or when grown in the dark. L.s.d. (least significant difference bar) ................................................................................................... 207 Figure 6.7: Radial growth (LN [1 + radial growth]) of fungi isolated from weathered wood (including controls) after 7 days growth under UV or visible light or when grown in the dark. L.s.d. (least significant difference bar) ................................................................................ 209 Figure 6.8: Lightness of mycelia from fungi isolated from weathered wood (including control) after 7 days of growth under UV or visible light or when grown in the dark. No measurements were performed for Ophiostoma fungi exposed under UV radiation. L.s.d. (least significant difference bar). Lightness is expressed using the CIE parameter L, 0: black – 100: white .... 210 Figure 7.1: Chemical structures of three melanin biosynthesis inhibitors (MBIs) and a fungicide used to inhibit growth of A. pullulans and C. cladosporioides. (a) cerulenin, inhibitor of melanin biosynthesis at the polyketide synthase step; (b) tricyclazole, inhibitor of polyhydroxynaphthalene reductase in the enzymatic reduction of 1,3,6,8tetrahydroxynaphthalene (1,3,6,8-THN) to scytalone and 1,3,8-trihydroxynaphthalene (!,3,8THN) to vermelone; (c) carpropamid, inhibitor of the dehydratase enzyme in the enzymatic dehydration of scytalone into 1,3,8-THN and dehydration for the conversion of vermelone into 1,8-dihydroxynaphthalene; and (d) quinoxyfen, disruptor of early cell signaling events in fungal cells ......................................................................................................................... 221 Figure 7.2: Screen-shot of the software used to count the number of fungal colonies in each plate ................................................................................................................................... 223 Figure 7.3: Inoculation of spruce veneers with 50 µL of spore solution (1 cell/µL) .............. 227 Figure 7.4: Color measurement of stained area on spruce veneer sections inoculated with A. pullulans and exposed for 5 days under UV or visible light: (a) adjustment of tonal range; (b) stained pixels selected using threshold adjustment; (c) ‘curves’ function of the software used to adjust the tonal range; and (d) threshold adjustment..................................................... 230 Figure 7.5: Color measurement of stained spruce veneers inoculated with A. pullulans and exposed for 5 days under UV or visible light: (a) Use of histogram in Photoshop to acquire information about the RGB color of the image; and (b) color picker tool for transformation of RGB into CIELab color ......................................................................................................... 231  xxii  Figure 7.6: Average number of fungal colonies growing on malt extract agar in Petri dishes exposed to either UV or visible light. Results averaged across plates containing different MBIs (plus control) and inoculated with A. pullulans or C. cladosporioides. Error bars correspond to ±SED ................................................................................................................................... 234 Figure 7.7: Average number of fungal colonies growing on malt extract agar in Petri dishes containing different MBIs, the fungicide quinoxyfen, or acetone (as control). Results averaged across plates exposed to UV and visible light and inoculated with A. pullulans or C. cladosporioides. Error bars correspond to ±SED.................................................................. 234 Figure 7.8: Average number of colonies of A. pullulans and C. cladosporioides growing on malt extract agar in Petri dishes. Results averaged across plates containing different chemicals and exposed to UV or visible light. Error bars correspond to ±SED ..................... 235 Figure 7.9: Average number of fungal colonies growing on malt extract agar in Petri dishes containing the MBIs carpropamid, cerulenin and tricyclazole, the fungicide quinoxyfen, and acetone (control plates); and exposed to UV or visible light. Results averaged across plates inoculated with A. pullulans or C. cladosporioides. L.s.d. bar for comparison of means ...... 235 Figure 7.10: Average number of fungal colonies growing on malt extract agar in Petri dishes exposed to UV or visible light, and inoculated with either A. pullulans or C. cladosporioides. Results averaged across plates containing melanin biosynthesis inhibitors, quinoxyfen or acetone. L.s.d. bar for comparison of means ...................................................................... 236 Figure 7.11: Effects of chemical types (MBIs, fungicide [quinoxyfen] or acetone [control]) and exposure to UV radiation or visible light on growth of A. pullulans on artificial media. Concentration of MBIs and quinoxyfen = 10 ppm; acetone in control plates was added at a level that was the same as that used to dissolve the MBIs .................................................. 237 Figure 7.12: Effects of chemical types (MBIs, fungicide [quinoxyfen] or acetone [control]) and exposure to UV radiation or visible light on growth of C. cladosporioides on artificial media. Concentration of MBIs and quinoxyfen = 10 ppm; acetone in control plates was added at a level that was the same as that used to dissolve the MBIs .................................................. 238 Figure 7.13: Appearance of spruce veneer sections impregnated with carpropamid or quinoxyfen, inoculated with spores of A. pullulans and exposed for 5 days to UV radiation: (a) carpropamid control veneer (impregnated with acetone); (b) veneer impregnated with carpropamid at 3000 ppm; (c) veneer impregnated with carpropamid at 6000 ppm; (d) quinoxyfen control veneer (impregnated with acetone); (e) veneer impregnated with quinoxyfen at 3000 ppm; and (f) veneer impregnated with quinoxyfen at 6000 ppm. Veneers impregnated with carpropamid stained significantly less than the control. In contrast, impregnation with quinoxyfen appeared to encourage fungal colonization ........................ 240  xxiii  Figure 7.14: Appearance of spruce veneer sections impregnated with carpropamid or quinoxyfen, inoculated with spores of A. pullulans and exposed for 5 days to visible light: (a) carpropamid control veneer (impregnated with acetone); (b) veneer impregnated with carpropamid at 3000 ppm; (c) veneer impregnated with carpropamid at 6000 ppm; (d) quinoxyfen control veneer (impregnated with acetone); (e) veneer impregnated with quinoxyfen at 3000 ppm; and (f) veneer impregnated with quinoxyfen at 6000 ppm. Veneers impregnated with carpropamid stained less than the control. The presence of quinoxyfen appeared to encourage melanization of A. pullulans .......................................................... 241 Figure 7.15: Effect of chemical treatment on staining (evaluated as LN (1 + Stained area ratio)) of spruce veneers. Results averaged across veneer sections treated with different concentrations of chemicals and exposed to UV or visible light. Error bars correspond to ±SED ........................................................................................................................................... 242 Figure 7.16: Magnified appearance of spruce veneer sections impregnated with carpropamid or quinoxyfen, inoculated with spores of A. pullulans and exposed for 5 days to UV radiation: (a) carpropamid control veneer (impregnated with acetone); (b) veneer impregnated with carpropamid at 3000 ppm; (c) veneer impregnated with carpropamid at 6000 ppm; (d) quinoxyfen control veneer (impregnated with acetone); (e) veneer impregnated with quinoxyfen at 3000 ppm; and (f) veneer impregnated with quinoxyfen at 6000 ppm. Greater staining of sections treated with quinoxyfen was observed ................................................ 242 Figure 7.17: Magnified appearance of spruce veneer sections impregnated with carpropamid or quinoxyfen, inoculated with spores of A. pullulans and exposed for 5 days to visible light: (a) carpropamid control veneer (impregnated with acetone); (b) veneer impregnated with carpropamid at 3000 ppm; (c) veneer impregnated with carpropamid at 6000 ppm; (d) quinoxyfen control veneer (impregnated with acetone); (e) veneer impregnated with quinoxyfen at 3000 ppm; and (f) veneer impregnated with quinoxyfen at 6000 ppm. Less staining of wood samples was observed compared to sections exposed to UV radiation .... 243 Figure 7.18: Effects of chemical treatment on color differences (ΔE) of spruce veneers treated with carpropamid or quinoxyfen; inoculated with spores of A. pullulans v. spruce control veneers (impregnated with acetone) inoculated with A. pullulans, after 5 days of exposure to UV and visible light. Results averaged across veneer sections treated with different concentrations of chemical and exposed to UV or visible light. Error bars correspond to ±SED ........................................................................................................................................... 244 Figure 7.19: Effects of chemical treatment on color differences (ΔE) of spruce veneers impregnated with carpropamid or quinoxyfen inoculated with spores of A. pullulans v. spruce veneers impregnated with carpropamid or quinoxyfen and not inoculated with the fungus, after 5 days of exposure to either UV or visible light. Results averaged across veneer sections treated with different concentrations of chemicals and exposed to UV or visible light. Error bars correspond to ±SED .................................................................................................... 245 xxiv  Figure 7.20: Effects of chemical treatment on color differences (ΔE) of spruce veneers impregnated with either carpropamid or quinoxyfen and inoculated with spores of A. pullulans v. spruce veneers sections impregnated with either carpropamid or quinoxyfen and not inoculated with the fungus, after 5 days of exposure to either UV or visible light. Results averaged across veneer sections treated with different chemicals and exposed to UV or visible light. Error bars correspond to ±SED ........................................................................ 246 Figure 7.21: Effects of chemical treatments and concentrations on color differences (ΔE) of spruce veneers impregnated with carpropamid or quinoxyfen and inoculated with spores of A. pullulans v. spruce veneers impregnated with carpropamid or quinoxyfen and not inoculated with the fungus, after 5 days of exposure to either UV or visible light. Results averaged across veneer sections exposed to UV or visible light. L.s.d. bar is shown for comparison of means ......................................................................................................... 246 Figure 7.22: Appearance of spruce control (not inoculated) veneer sections impregnated with carpropamid or quinoxyfen and exposed to UV radiation for 5 days: (a) carpropamid control veneer (impregnated with acetone); (b) veneer impregnated with carpropamid at 3000 ppm; (c) veneer impregnated with carpropamid at 6000 ppm; (d) quinoxyfen control veneer (impregnated with acetone); (e) veneer impregnated with quinoxyfen at 3000 ppm; and (f) veneer impregnated with quinoxyfen at 6000 ppm ............................................................ 248 Figure 7.23: Appearance of spruce veneer control (not inoculated) sections not inoculated and impregnated with carpropamid or quinoxyfen and exposed to visible light for 5 days: (a) carpropamid control veneer (impregnated with acetone); (b) veneer impregnated with carpropamid at 3000 ppm; (c) veneer impregnated with carpropamid at 6000 ppm; (d) quinoxyfen control veneer (impregnated with acetone); (e) veneer impregnated with quinoxyfen at 3000 ppm; and (f) veneer impregnated with quinoxyfen at 6000 ppm ......... 249  Figure 7.24: Magnified appearance of spruce veneer control (not inoculated) sections impregnated with carpropamid or quinoxyfen and exposed to UV radiation for 5 days: (a) carpropamid control veneer (impregnated with acetone); (b) veneer impregnated with carpropamid at 3000 ppm; (c) veneer impregnated with carpropamid at 6000 ppm; (d) quinoxyfen control veneer (impregnated with acetone); (e) veneer impregnated with quinoxyfen at 3000 ppm; and (f) veneer impregnated with quinoxyfen at 6000 ppm. Veneers were not stained by A. pullulans, as expected .................................................................... 250 Figure 7.25: Magnified appearance of spruce veneer control (not inoculated) sections impregnated with carpropamid or quinoxyfen and exposed to visible light for 5 days: (a) carpropamid control veneer (impregnated with acetone); (b) veneer impregnated with carpropamid at 3000 ppm; (c) veneer impregnated with carpropamid at 6000 ppm; (d) quinoxyfen control veneer (impregnated with acetone); (e) veneer impregnated with quinoxyfen at 3000 ppm; and (c) veneer impregnated with quinoxyfen at 6000 ppm. Veneers were not stained by A. pullulans, as expected .................................................................... 251 xxv  Figure A2.1: Tensile stress vs strain of lime wood veneer (block 1) incubated with Mollisia sp. red triangle used to calculate the modulus of elasticity directly from the figure ................. 303 Figure A4.1: Appearance of southern pine wood samples exposed to weather for 40 in Vancouver, Canada, under a polymethylmethacrylate filter transmitting UVB+UVA+Vis.light+IR (Filter 1). (a) week 0, (b) week 4, (c) week 8, (d) week 12, (e) week 16, (f) week 20, (g) week 32, (h) week 40 ................................................................................. 550 Figure A4.2: Appearance of southern pine wood samples exposed to weather for 40 in Vancouver, Canada, under a polymethylmethacrylate filter transmitting UVA+Vis.light+IR (Filter 2). (a) week 0, (b) week 4, (c) week 8, (d) week 12, (e) week 16, (f) week 20, (g) week 32, (h) week 40 ................................................................................................................... 551 Figure A4.3: Appearance of southern pine wood samples exposed to weather for 40 in Vancouver, Canada, under a polymethylmethacrylate filter transmitting Vis.light+IR (Filter 3). (a) week 0, (b) week 4, (c) week 8, (d) week 12, (e) week 16, (f) week 20, (g) week 32, (h) week 40 .............................................................................................................................. 552 Figure A4.4: Appearance of southern pine wood samples exposed to weather for 40 in Vancouver, Canada, under a polymethylmethacrylate filter transmitting IR (Filter 4). (a) week 0, (b) week 4, (c) week 8, (d) week 12, (e) week 16, (f) week 20, (g) week 32, (h) week 40 . 553 Figure A4.5: Appearance of southern pine wood samples exposed to weather for 40 in Vancouver, Canada, under a polymethylmethacrylate filter blocking all wavelengths of solar radiation (Filter 5). (a) week 0, (b) week 4, (c) week 8, (d) week 12, (e) week 16, (f) week 20, (g) week 32, (h) week 40..................................................................................................... 554 Figure A7.1: Calibration curve absorbance vs concentration C. cladosporioides ................. 563 Figure A7.2: Calibration curve absorbance vs concentration A. pullulans [R2F32.2] ............ 564 Figure A7.3: Calibration curve absorbance vs concentration O. piliferum [TAB28] .............. 565 Figure A7.4: Calibration curve absorbance vs concentration A. pullulans [ATCC 42371] ...... 566 Figure A7.5: Calibration curve absorbance vs concentration A. pullulans [R1F22W] ........... 567 Figure A7.6: Calibration curve absorbance vs concentration O. piliferum [Cartapip97] ....... 568  xxvi  Acknowledgements Dr Phil Evans, I want to express in these few lines my gratitude and admiration for your ability as a scientist and supervisor. I have met only a few people with the self-discipline and determination that you have. I greatly appreciate the opportunity that you gave to learn how to conduct scientific research, your mentoring and support during the last five years. I also express my gratitude to Dr Colette Breuil, who was the first person to encourage me to come to UBC and pursue a Ph.D degree, and later on supervised my research on microbiology of organisms colonizing weathered wood. Many thanks also to Dr Alan Preston, who showed great interest in my work and progress, and supported my research by giving me the albino strain of Aureobasidium pullulans that was tested in Chapter 6. Special thanks to Alice Obermajer from Canfor Pulp Research and Development in Vancouver BC, Canada, who allowed me t use their Instron tensile testing machine (Chapter 4). Thanks also to the ‘Natural Sciences and Engineering Research Council of Canada’ and the program ‘Becas Chile’ for their financial support. To technicians, graduate and co-op students in Dr Evans and Dr Breuil’s labs, who supported my research and were always willing to help with my research and made my experience as Ph.D student really enjoyable. Special thanks to Dr Arash Jamali, Ian Cullis, Siti Hazneza Abdul Hamid, Dr Jahangir Chowdhury, Dr (c) Sepideh Alamouti and Vincent Wang, for your help and encouragement. To my friends German, Bill, Faride, Stephanie and Jane, thanks for your friendship and support during the difficult times. And finally to my beloved girls Marcela and Josefina, and my parents, I’m sure I would have not been able to reach this point without your unconditional love. To all of you thank you so much!  xxvii  Dedication  To William “Bill” New Thanks for sharing your knowledge, wisdom and your sincere friendship with me  xxviii  1. Chapter 1: General introduction 1.1. Introduction Wood has historically been an important material for construction. Since ancient times it has been favored over other construction materials due to its widespread availability and low cost (Duncan 1963). Even today, with remarkable technological advances in material sciences, wood’s aesthetic properties confer advantages which add extra value to its other well known structural and environmentally-friendly credentials. Unfortunately, the aesthetic properties of wood are rapidly lost when it is exposed outdoors. Wood exposed outdoors rapidly interacts with the environment and it is particularly susceptible to surface degradation called ‘weathering’ (Feist, 1983). Weathering can be defined as ‘surface degradation resulting from environmental factors that can permanently change the natural appearance of wood surfaces, decreasing their aesthetic value by producing discoloration, checks and cracks, which are often accompanied by various forms of distortion (cup, twist, etc)’ (Feist, 1990; Evans, 2008). The environmental factors responsible for the weathering of wood are: (1) solar radiation, (2) moisture (water in its different states), (3) molecular oxygen, (4) heat, (5) pollutants and (6) microorganisms and insects (Evans 2008). Of the above mentioned factors, solar radiation is the most important factor responsible for chemical changes at weathered wood surfaces. Elevated levels of solar radiation occur at wood surfaces exposed outdoors. For example, on a clear day the amount of solar radiation reaching the earth is approximately 1000 W/m2. This is composed of 5% (UV radiation), 45% (visible light) and 50% (Infra-red light) (Evans et al. 2005). UV radiation and visible light from  1  solar radiation are responsible for the depolymerization of lignin, which causes the color of wood to change (yellowing and browning), because unsaturated lignin breakdown products accumulate at the surface of wood (Gellerstendt and Gierer, 1975; Feist and Hon, 1984). Also, photo-depolymerization of lignin affects the integrity of the middle lamella which results in the separation of wood cells and causes micro-checking. Over time micro-checks can develop into macro-checks (Miniutti, 1974; Evans, 2008). Furthermore, UV radiation also depolymerizes cellulose and hemicelluloses creating low molecular weight carbohydrates at wood surfaces (Bourbonnais and Paice 1987; Schoeman and Dickinson 1997; Evans 2008). Hence, UV radiation creates a nutrient rich surface layer in wood exposed outdoors. Such layer is an important food source for a number of microorganisms, especially fungi. Many fungi have been found colonizing weathered wood and metabolizing simple sugars and phenolic photodegradation products (Seifert, 1964; Sell and Wälchli, 1969; Bourbonnais and Paice, 1987; Schoeman and Dickinson, 1997). An important proportion of the fungi colonizing weathered wood are black ascomycetes, which cause the staining of wood surfaces due to the dark pigment (melanin) in their hyphae and spores (Brisson et al. 1996; Chedgy, 2006). The graying of wood by these fungi is one of the defining features of weathered wood (Feist 1990; Evans 2008). Fungi responsible for the staining of weathered wood are often accompanied by other fungi which do not seem to contribute to staining. The role played by these organisms is not clear, but there is some evidence that they may be involved in the decay of wood (Schmidt and French 1976). The effects of such microorganisms and those of other factors involved in the weathering of wood can be blocked by various treatments. For example, UV absorbers and hindered 2  amine light stabilizers are commonly added to finishes, such as varnishes, stains and waterrepellents (Evans 2008). Fungicides and wood preservatives have long been used to protect wood from fungi and other microorganisms. However, some fungi can grow underneath finishes, and others have shown tolerance to wood preservatives (Savory 1973; Kim et al. 2007). The number of biocides that can be used as wood preservatives has been restricted, and there is a need to develop new ways of controlling the decay and discoloration of wood by fungi (Evans, 2003). Blocking the production of pigments (melanin) inside fungal hyphae might be one way of controlling fungal stains in weathered wood. In addition, blocking of melanin biosynthesis would make fungi more susceptible to the damaging effects of UV radiation, which might eventually kill them. This research examines the colonization of wood surfaces exposed outdoors by fungi. I seek to understand the effects of fungi on the wood and examine the complex interaction between solar radiation and fungal colonization. I also aim to generate new approaches to eliminate or decrease fungal stains based on the combined effects of UV radiation and inhibition of fungal melanin biosynthetic pathways.  1.2. General Hypothesis UV radiation is very energetic and harmful to most living organisms (Diffey, 1991; Ranby and Rabek, 1975). Living organisms, including fungi, synthesize melanin to protect themselves from solar radiation and other stressing factors, such as high temperatures and desiccation (Fogarty and Tobin 1996; Butler and Day 1998; Henson et al. 1999). These factors can all be  3  found at wood surfaces exposed outdoors. Studies have indicated that, at wood surfaces exposed outdoors, the predominant fungal flora is dominated by black moulds (Duncan, 1963; Seifert, 1964; Sell and Wälchli, 1969). These fungi apparently can use the melanin in their hyphae to provide a competitive advantage and prevail at wood surfaces. However, as a result the same fungi cause the staining of wood surfaces exposed outdoors as the melanin in their hyphae and spores stains the first few layer of cells at exposed wood surfaces (Brisson et al. 1996). Such staining decreases the aesthetic and economic value of wood and wood product exposed outdoors, as mentioned above.  Base on this information, the general hypothesis for this Thesis is: “The graying of wood exposed outdoors is due to the presence of melanized fungi with relatively high resistance to UV light”.  The treatments used to block the staining of wood generally focus on killing the staining fungi using biocides, but there has been no research that examines the possibility of reducing staining by blocking the biosynthetic pathway of fungal melanins. Melanin biosynthesis inhibitors (MBIs) are chemical substances produced to interrupt the enzymatic pathway involved in the biosynthesis of fungal melanins (Kurahashi 2001). They are commonly used in agriculture as a foliar treatment to prevent blast rice disease produced by the fungus Magnoporthe grisea (Kurahashi 2001). This ascomycete synthesizes dehydroxynaphtalene (DHN) melanin similar to many of the fungi colonizing weathered wood (Bell and Wheeler 1986).  4  If the general hypothesis of this thesis is correct we should be able to use melanin synthesis blockers as a preservative treatment since blocking melanin production may decrease fungal resistance against UV light, possibly leading to the destruction of staining fungi. One problem with this approach is that the biosynthesis of melanin is complex and can vary from one fungal species to another, and some of the different MBIs have different modes of action (Butler and Day, 1998; Kurahashi, 2001). Hence, individual MBIs may not be effective in blocking melanin biosynthesis in all species.  1.3. Scope and importance The scope of this thesis is to study the relationship between fungi colonizing and staining weathered wood, and UV radiation within the solar spectrum. I seek to obtain fundamental information on the fungi involved in the weathering of wood and their interactive response to exposure to UV radiation under controlled conditions. Also, I seek to generate a new approach to control the graying of weathered wood based on the use of fungal melanin biosynthesis inhibitors and the sterilizing effects of UV radiation. I also perform fundamental research to isolate and characterize fungi colonizing weathered wood and examine their ability to degrade wood. The aesthetic disfiguration of wood exposed outdoors significantly decreases the value of wood and wood products. This problem is economically important as illustrated by the problem that the weathering of wood causes for the use of wood for decking. This market is forecasted to reach $6.2 billion per annum by 2014 in USA (Freedonia Group, 2011).  5  However, statistics show that one third of the decks installed in the USA are replaced after only a few years of service due to weathering of exposed wood surfaces (Amburgey and Ragon, 2008). The cost of replacing such decks could be in excess of US$ 1.5 billion. This generates a negative impression of wood as a building material for outdoors uses, which has led to its substitution by other materials such as wood plastic composites. My research focuses on fungi colonizing wood surfaces in Vancouver BC, Canada, but the results might be reasonably extrapolated to different regions of earth with similar climate and flora. It is important to note that the research does not encompass other organisms which colonize weathered wood, such as, algae, bacteria and mosses because these organisms do not appear to be involved in the graying of weathered wood.  1.4. Study outline In this chapter (Chapter 1) the general introduction and rationale for the thesis are presented. Chapter 2 reviews the literature on: (1) the weathering of wood; (2) deleterious effects of UV radiation on wood; (3), biological organisms colonizing weathered wood; (4), fungi colonizing weathered surfaces and their possible effects on wood; (5) effect of UV radiation on living cells; (6), fungal melanins and MBIs. In Chapter 3, the fungi colonizing weathered wood exposed outdoors in Vancouver, Canada, are isolated, identified and characterized. Emphasis is given to the use of molecular techniques (DNA analysis) to efficiently identify fungi. In the following chapter (Chapter 4), the ability of fungi isolated from weathered wood to degrade wood surfaces is studied using several techniques  6  including examination of the effects of fungi on the mechanical and viscoelastic properties of wood (peak tensile stress, modulus of elasticity, peak stiffness, peak toughness and storage modulus). Chapter 5 examines the effect of UV radiation within the solar spectrum on the staining of weathered wood. Insights into the effect of UV radiation on the color of weathered wood are provided by the results presented in this chapter. Chapter 6 complements the previous chapter because it examines the effect of UV radiation on staining fungi growing on artificial culture media. This chapter also examines how UV radiation influences the production of melanin by staining fungi. The last experimental chapter (Chapter 7) seeks to demonstrate the potential use of melanin inhibitors and UV light as a novel treatment to block the fungal staining of wood surfaces. Promising results of in-vitro tests are presented in this chapter. In the final chapter (Chapter 8), I discuss the results of all of the experimental chapters and relate them to the general hypothesis and aims of the thesis. I make conclusions and suggest future research that should be performed to strengthen my findings and conclusions.  7  2. Chapter 2: Literature review This chapter describes the literature on the weathering of wood and the colonization of wood surfaces by fungi that cause the graying of weathered wood. The review focuses on the key literature that is relevant to my thesis.  2.1. Weathering of wood Weathering of wood is caused by damaging reactions that occur at wood surfaces when they are exposed outdoors. These reactions, which are caused by various environmental factors (mentioned in Chapter 1), permanently change the appearance of wood and decrease its appeal (Figure 2.1).  Figure 2.1: Appearance of weathered Southern pine (Pinus sp.) wood. Note the graying and surface checking of the wood  8  Feist (1990) described the changes that occur when wood is weathered as follows: “During weathering the original surfaces become rough as the grain raises, the wood checks, and the checks grow into large cracks. Boards cup, warp, and pull away from fasteners. Surface color changes, the wood gathers dirt and mildew and becomes unsightly”. The environmental factors responsible for the weathering of wood are solar radiation, water, molecular oxygen, heat, pollutants, microorganisms and insects (Evans 2008). Solar radiation is the most important factor responsible for the weathering of wood. Solar radiation can be absorbed by all of wood’s main structural polymers (cellulose, hemicelluloses and lignin), depending on the wavelength of the incident light (Kalnins, 1966). Wood exposed outdoors also gains and loses moisture, which causes dimensional changes that generate surface and internal stresses, leading to checking and warping (Feist 1990). The swelling of wood by water may also open up inaccessible regions of the cell wall making them accessible to other environmental factors that may increase the depth of weathering, according to Feist and Hon (1984). Water in the form of rain can also wash and leach photodegraded wood products from wood surfaces (Derbyshire and Miller 1981). Molecular oxygen contributes to the weathering of wood as most of the processes related to wood photodegradation are oxidative. Molecular oxygen plays a fundamental role in the formation of peroxy radicals, which is a key step in the photodegradation of lignin and holocellulose (Feist and Hon, 1984). Photochemical reactions are accelerated by heat from solar radiation. Many chemical reactions involved in weathering are increased as temperature increases (Maddock, 1920). Wood surfaces exposed outdoors are also contaminated by dust, smoke particles and volatile pollutants, for example, sulfur compounds (Spedding, 1970; Williams, 9  1987). Atmospheric sulfur dioxide, in the form of acid rain, may reduce the mechanical properties of wood surfaces exposed in polluted environments (Raczkowski 1980). A diverse range of fungi, algae, lichens and insects are able to colonize and attack weathered wood surfaces. In most cases the damage is superficial. Nevertheless, most modern studies on the weathering of wood point out that these microorganisms are responsible for the graying and staining of weathered wood (Duncan, 1963; Feist, 1990). However, the precise nature of the damage caused by micro-organisms colonizing weathered wood surfaces has not been fully elucidated. This topic will be examined in greater depth in this literature review. The damage to wood surfaces caused by insects is not described in the literature except for the superficial erosion caused by wasps and hornets that use fragments of weathered wood to make their paper-like nests (Schmolz et al. 2000).  2.1.1. Degradation of wood polymers by solar radiation Solar radiation degrades wood because it is absorbed by wood’s molecular components. The extent of degradation depends on the wavelength of the incident radiation. The critical wavelengths to dissociate the most important bonds in wood are 346, 334 and 289 nm, corresponding to carbon-carbon, carbon-oxygen, and carbon-hydrogen bonds, respectively (Evans 2008). These wavelengths are found in the UV components of solar radiation (Diffey 1991). Thus, UV radiation is the most damaging portion of the solar spectrum. In addition, the violet light component of visible light has sufficient energy to photodegrade lignin, and  10  it ‘extends photodegradation into wood beyond the zones affected by UV radiation’ (Kataoka et al. 2007). Lignin is the most sensitive of wood’s polymers to photodegradation (Derbyshire and Miller 1981), but the complex mechanisms involved in the photodegradation of lignin have not been completely clarified. However, the process can be summarized as follows: ‘Lignin, which is an amorphous phenolic polymer, is rich in chromophoric groups that strongly absorb UV light’ (Hon 1979). ‘These groups, including phenolic, double bonds, carbonyls, quinones, quinomethides and biphenyls (Hon 1979), readily interact with UV light to form free radicals’. ‘These radicals react with molecular oxygen to form new radicals such as peroxides, hydroperoxides, peroxyl and alkoxyl radicals’ (Kalnins, 1966). George et al. (2005) noted that the main free radicals resulting from the photodegradation of lignin are phenoxy radicals (Figure 2.2).  Figure 2.2: Phenoxy radicals produced during photodegradation of lignin. (a) Guaiacoxyl radical; (b) Phenacyl radical; and (c) Cetyl radical  According to their review ‘these free radicals are transformed into quinoid structures which accumulate at wood surfaces causing the first color changes during weathering’ (George et al., 2005). Cellulose seems to be more resistant to weathering than lignin as it is only  11  sensitive to wavelengths shorter than 280 nm, and the ozone layer prevents such radiation from reaching the earth’s surface. However, cellulose is rapidly depolymerized during natural weathering (Derbyshire and Miller 1981; Evans et al. 1996). The depolymerization of cellulose is linked to the formation of aromatic radicals and/or presence of metal ions. In the presence of promoters, such as metal ions and certain dyes, free radicals can be formed even when cellulose is exposed to wavelengths longer than 340 nm (Hon 1975; Feist and Hon 1984). When cellulose in wood is subjected to sunlight, its glycosidic linkages are cleaved causing a loss of strength and degree of polymerization (Derbyshire and Miller, 1981). Hon and Chang (1984) suggested that UV light absorbed by lignin can help to degrade cellulose by energy transfer mechanisms. Nevertheless, cellulose rich surfaces are produced by the photodegradation of lignin at wood surfaces exposed to natural weathering (Feist, 1990) Hemicelluloses seem to be affected by solar radiation in the much same way as cellulose (Feist and Hon 1984). Hemicelluloses, particularly those containing xylose and arabinose, are depolymerized during weathering and leached from wood surfaces (Evans et al. 1992). Leachates from weathered wood surfaces contain a high proportion of mannose and xylose, suggesting the degradation of galactoglucomannan and arabinoglucoronoxylan, respectively (Evans et al. 1992).  2.1.2. Macro and microscopic effect of weathering The first visible effects of weathering at wood surfaces are color changes (Feist, 1990; George et al., 2005; Evans, 2008). Color changes at weathered wood surfaces are initially 12  due to the accumulation of photodegraded lignin fragments in the wood which turns the wood yellow or brown (Gellerstendt and Gierer, 1975; Feist and Hon, 1984). Later, wood starts to turn gray; becoming darker after a few months of outdoor exposure. As mentioned above, the graying and darkening of weathered wood surfaces is attributed to colonization of the wood by staining fungi (Duncan 1963). However, the accumulation of dust and pollutants at wood surfaces also contributes to the graying of weathered wood. Other obvious physical effects of weathering at wood surfaces are the formation of macro-checks and cracks. Checks and cracks are caused by the separation of fibers due to surface and internal stresses resulting from moisture gradients and shrinkage and swelling of inner and outer wood layers (Panshin and De Zeeuw 1980). The photodegradation of lignin also increases the susceptibility of surface layers of wood to check because lignin plays an important role in bonding wood cells together (Evans et al. 2008). Cells at exposed wood surfaces are eroded, but the erosion of weathered wood surfaces is highly dependent on the density of wood (Evans et al. 2005). Thus, the rate of erosion of lower density earlywood is higher than that of latewood. Feist (1983) noted that wood erodes at a rate of 6 to 3 mm per century, for softwoods and hardwoods, respectively. At the microscopic level the effects of weathering are most noticeable in the middle lamella. The high concentration of lignin in this layer makes it very susceptible to UV radiation (Feist 1990). Damage to the middle lamella can be seen in both transverse and longitudinal sections (Feist 1990). Bordered and half bordered pits are also very susceptible to weathering; and small checks originating from pit apertures have been observed in many weathered softwoods (Miniutti, 1974; Chang et al. 1982; Evans, 1989). Checks in tracheid 13  walls follow the microfibril angle of the S2 layer of the secondary wall (Feist 1990). Separation of tracheids and fibers occurs due to erosion of the middle lamella and this, plus the presence of microchecks, causes small sections of cell wall to detach, which produces a progressive loss of integrity of exposed surfaces (Evans, 2008). Thinning and delamination of different cell wall layers can be observed in weathered wood. Thin walled cells, for example epithelial cells in resin canals are more susceptible to weathering than thicker walled cells (Evans, 1989).  2.1.3. Depth of weathering The depth to which weathering extends into wood is related to how deep light penetrates wood. The depth of color changes in wood exposed to weathering acts as a guide to the depth of penetration of wood by light. Browne and Simonson (1957) described two layers in weathered wood: (1) a gray layer, 125 µm in thickness; and (2) a brown layer ranging from 0.51 mm to 2.54 mm in thickness. UV and visible light are not able to penetrate wood to a depth of 2.54 mm. Hence, Browne and Simonson (1957) explained their observation that weathered wood contained a brown layer up to 2.54 mm deep by stating that free radicals formed in outer layers may migrate deeper into the wood and react with the wood producing color changes. Kataoka et al. (2004) found photo-induced changes in Japanese cedar earlywood exposed to artificial solar radiation to a depth of up to 75 μm. They also found an exponential decrease in light penetration with wood depth, but sufficient photochemically active light was present which could degrade wood at a depth of 700 μm.  14  2.2. Biological organisms colonizing weathered wood surfaces A wide range of organisms are able to colonize wood surfaces exposed outdoors. These organisms create a ‘biofilm’ at wood surfaces which can include, fungi, bacteria and algae (Gaylarde and Morton 1999; Sailer et al. 2010). Algae are a very diverse photosynthetic group of plants lacking roots, leafy shoots and vascular tissues (Hoek et al. 1995). They often disfigure the surface of buildings located in shaded areas with high humidity. Algae growing on surfaces require little nutrients, because they can photosynthesize (Gaylarde and Morton 1999). Coccoid green algae that reproduce by autosporulation are suited to environments found at wood surfaces. For example, the coccoid green alga Hylodesmus singaporensis gen. et sp. nov. grows at decayed wood surfaces (Elias et al. 2010). Other algal species found on wood in shaded areas are Protococcus viridis, Chlorococcum sp., Hormidium sp. and Cyanophyceae sp. (Ohba et al. 2001). Algae such as Chlorococcum sp. and Amphora sp. are even able to grow beneath a coat of varnish (de Souza and Gaylarde, 2002). The moisture content at weathered wood surfaces is not always suitable for algae, but they can survive dry periods by developing a symbiotic relation with fungi to form lichens. ‘A lichen is an association of a fungus and a photosynthetic symbiont resulting in a stable thallus of specific structure’ (Hawksworth and Hill 1984). Around one in five of all known fungi can be ‘lichenized’, and across the spectrum of lichenizable fungi about 46% of them belong to the phylum ascomycota (Hawksworth and Hill 1984). Little information is available on the colonization of weathered wood surfaces by lichens, but Schmidt and French (1976) described the colonization of weathered shingles exposed in Portland, Oregon, by the lichen Lecidea granulose (Hoffm.) Ach. They also discuss whether the 15  lichenization of Aureobasidium pullulans (de Bary) G. Arnaud, one of the most common fungi isolated from weathered wood, might be involved in colonization of wood shingles by lichens. Bacteria can also colonize wood surfaces exposed outdoors. Bacteria are unicellular prokaryotes, but some forms such as those found in the Actinomycetes can form chains of cells and have filamentous forms. Many bacteria are adapted for growth on surfaces and they can rapidly exploit a wide range of energy sources. Some of them are very resistant to environmental extremes (Zabel and Morrell, 1992). Bacteria can be present in sufficient numbers to exert adverse effects on apparently clean surfaces. They are notable for their ability to grow at low concentrations of oxygen. Hence, they can be very active in anoxic wet environments and beneath biofilms formed on surfaces exposed outdoors (Gaylarde and Morton 1999). Several bacterial species can damage wood. For example, Clostridium xylanolyticum is able to cause tunneling decay (Zabel and Morrell, 1992). This bacterium produces a xylanase enzyme, which seems to be very active even under anaerobic conditions (Rogers and Baecker 1991). Other members of the genera Clostridium can produce cellulase enzymes, which are even more effective at degrading wood (Boutelje and Bravery 1968; Greaves 1971). Bacillus polymixa can breakdown pectin in pits and consequently increase the permeability of wood (Knuth and McCoy 1961). Bacteria can attack wood even when it has been treated with preservatives (Singh et al. 1992; Eaton 1994). Insects can also affect wood exposed outdoors. Insects live in wood or use it as a food source, but in both cases the wood is chewed into small fragments (Zabel and Morrell, 1992). Insects can benefit from the modified characteristic of weathered wood. For 16  example; termites and wasps frequently excavate weathered wood surfaces. Termites excavate wood by chewing on it, but the digestion of wood is due to the action of enzymes from symbiotic protozoa and bacteria that live in their gut (Breznak and Brune 1994). Termite colonization of wood depends mainly on its moisture content and natural durability (Zabel and Morrell, 1992). Paper wasps, genera Polistinae, and other social wasps, such as yellow jackets and hornets (Vespinae), construct paper covers for their nests using weathered or decayed wood. The covers are made by removing and intensively chewing the weathered wood and using saliva as an adhesive (Schmolz et al., 2000). Weathered or rotten wood is preferred by the insects over sound wood. Other insects that attack weathered wood surfaces are carpenter bees and carpenter ants. Carpenter bees excavate galleries in wood to construct their nests. The galleries are used as a depot for eggs, nectar and pollen (Keasar, 2010). Carperter bees generally attack uncoated softwood, but Zabel and Morrell (1992) reported that after weathering almost all wood species were susceptible to attack by carpenter bees. Carpenter ants behave in similar way, excavating galleries in the wood (Hansen and Klotz 2005). In both cases wood is not used as a food source.  2.2.1. Fungi classification Fungi are very successful at colonizing wood surfaces exposed outdoors, as mentioned above. Fungi are eukaryotic heterothophs belonging to the monophyletic group eumycota (Kendrick, 2000). The fungal body, known as thallus, is formed by multicellular filamentous structures called hyphae. Some fungi form a complex net from their hyphae called mycelia. 17  Other fungi may form yeast (yeast-like fungi) or may grow using both stages (dimorphic fungi) (Kendrick 2000). The hyphal system is adapted to penetrate, externally digest, absorb and metabolize a wide range of organic materials (Zabel and Morrell 1992). A wide range of fungi can colonize wood in trees or when it is used for timber products. Some fungi utilize simple products accumulated in cell lumens, resin canals and parenchyma cells of trees. Other fungi can directly attack the wood’s structural polymers producing decay. The extension and type of damage depends on the type of fungi colonizing the wood. Not all fungi are part of the eumycota kingdom. Certain slime moulds (Phyla: myxosteida, dictyostelida, labyrinthulida, plasmodiophorida) as well as certain chromistan organisms (Phyla: hyphochytriomycota, oomycota) do not belong to the eumycota, but they are still classified as fungi. The main streams of fungi in the eumycota kingdom are part of the phyla: Chytridiomycota, Zygomycota and Dikariomycota. The last phylum includes most of the wood-colonizing fungi in the subphyla ascomycotina and basidiomycotina (Kendrick 2000) (Figure 2.3).  18  Kingdom Eumycota  Phylum Chytridiomycota  Phylum Zygomycota  Sub-Phylum Ascomycotina  Class Ascomycetes  Class Phragmobasidiomycetes  Phylum Dykariomycota  Sub-Phylum Basidiomycotina  Class Saccharomycetes  Class Holobasidiomycetes  Class Teliomycetes  Figure 2.3: Biological classification of true fungi as described by Kendrick (2000)  Fungi can also be classified as decaying or staining fungi. Decay fungi fall into three subcategories according to the mode of degradation of woody tissues: (1) brown-rot; (2) whiterot; and (3) soft-rot (Zabel and Morrell, 1992). Brown-rot breaks down cellulose and hemicelluloses, but decomposition of lignin is limited (Cartwright and Findlay, 1958; Green and Highley, 1997). Brown-rot rapidly degrades cellulose and the S2 layer of the wood cell wall, but highly lignified wall layers such as the middle lamella appear to be resistant to degradation (Eriksson et al. 1990). Brown-rotted wood is brittle, heavily cracked and powdery (Schwarze 2007). White-rot fungi can degrade lignin as well as cellulose and hemicelluloses. 19  White-rot fungi are classified into two types that cause simultaneous rot and selective delignification, respectively. In the former, lignin and carbohydrates are degraded simultaneously whereas selective delignification involves removal of lignin from cell walls before the holcellulose is degraded (Zabel and Morrell, 1992). Soft-rot is different from white and brown rot mainly due to the different way it degrades cell walls layers. Soft-rot is chemically more similar to brown-rot than white-rot, as carbohydrates are decomposed while lignin is only slightly modified (Savory, 1954; Greaves and Levy, 1965; Schwarze, 2007). Soft-rot decay is sub-classified into Type 1 and 2. In Type 1 decay cavities are formed inside the S2 layer of the secondary wall, while in Type 2 discrete notches are eroded in the cell wall layer adjacent to lumens (Zabel and Morrell 1992; Schwarze 2007). Soft-rot fungi require less moisture than basidiomycete fungi (Duncan 1963). Staining fungi belong predominantly to the sub-phylum ascomycotina, but they include a wide variety of pathogenic and non-pathogenic fungi, plus an important number of moulds. Two groups of staining fungi can be distinguished; (1) sap-staining fungi and; (2) surface staining fungi. Sap-staining fungi can be further classified into pathogenic or non-pathogenic fungi. In both cases fungi develop by metabolizing substances accumulated in the parenchyma cells of trees, logs or unseasoned wood. Fungal staining can extend throughout the sapwood (Zabel and Morrell, 1992; Krokene and Solheim, 1998). Surface staining fungi include a great number of moulds, which colonize wood surfaces creating black and dark stains that only extend few millimeters underneath the wood surface (Duncan 1963;  20  Dickinson 1971; Savory 1973). These fungi play a predominant role in changing the color of weathered wood to grey (as mentioned above).  2.2.2. Factors affecting fungal survival in wood Fungal development in wood requires the presence of water, oxygen, moderate temperatures, nutrients, appropriate pH, nitrogen, vitamins and minerals. The moisture content of wood needs to be slightly greater than the fiber saturation point. Free water in cell lumens is a reactant in hydrolysis and a diffusion medium for enzymes. It also solubilizes substrate molecules, and acts as a solvent or wood-capillary swelling agent (Zabel and Morrell 1992). Most fungi are obligate aerobes or in other words they require free oxygen for metabolic reactions (Scheffer 1986). The metabolic activities of fungi, such as digestion, assimilation, respiration and translocation are affected by temperature (Cochrane 1958). Metabolites within the wood in trees are used by fungi to create a wide range of compounds needed for their growth and development, including chitin, glucan, nucleotides, enzymes, proteins and lipids (Zabel and Morrell, 1992). The pH of wood primarily affects substrate availability, rate of exoenzymatic reactions, exoenzyme stability, cell permeability, extracellular components and solubility of minerals and vitamins (Zabel and Morrell, 1992). Nitrogen is required by fungi to synthesize proteins and other cell constituents or products such as nucleoproteins, lipoproteins, enzymes and chitin in hyphal cell walls. Many fungi also require thiamine, as well as phosphorous, potassium, magnesium and sulfur, trace amounts of iron, zinc, copper manganese and molybdenum (Cochrane, 1958; Griffin, 1981; Zabel and Morrell, 1992).  21  2.2.3. Fungi colonizing weathered surfaces 2.2.3.1. Introduction  The presence of fungi in weathered wood was first noticed by Schacht (1863) and later by Möbius (1924). Both authors described the presence of fungi in wood, but only Möbius attributed the graying of wood surfaces to the presence of fungi. Before Möbius (1924) it was thought that weathered wood became gray due to the accumulation of dirt. Subsequent microscopic studies confirmed Möbius’s observations that the graying of weathered wood is almost exclusively the result of growth of dark colored fungi at the wood surface (Duncan 1963; Dickinson 1971).  2.2.3.2. Organisms colonizing weathered wood The fungi colonizing weathered wood surfaces are moulds, which can grow on most carboncontaining materials including wood, leather, plastic, food and paints. Wood-staining moulds have dark hyphae and spores, but their growth on weathered wood seems to be limited to periods of high humidity or intermittent rain (Kuhne et al. 1970; Hansen 2008). Nevertheless, the surface moulds that colonize weathered wood are capable of withstanding dry conditions and the relatively high temperatures at wood surfaces (Duncan 1963). The growth of moulds occurs after their spores alight and germinate on wood surfaces. After germination, hyphae, ramify through the wood cells, by penetrating cell lumina,  22  bordered pits and rays. Hyphae of fungi colonizing softwoods are most prominent in rays and resin ducts. Here the fungi metabolize sugars, starches, resin acids and hemicelluloses for growth. The walls of the ray parenchyma and epithelial cells surrounding resin ducts are often destroyed, leaving elongated open channels that increase the permeability of the affected wood. This effect may contribute to pronounced fluctuations in the surface moisture content of wood (Duncan 1963). Fungi colonizing weathered wood have been isolated and identified by several researchers. Sell and Wälchli (1969) isolated A. pullulans, Macrosporium sp., Tetracoccosporium sp., Cladosporium sp. and Sclerophoma sp. from weathered wood in the late 1960’s. However, A. pullulans was isolated from weathered wood before this by Seifert (1964). Subsequently, Dickinson (1971) isolated a range of mould fungi from Scots pine (Pinus sylvestris L.) and Western red cedar (Thuja plicata Donn ex D.Don), in England and Sweden. The main species he isolated were A. pullulans, Cladosporium sp., Alternaria sp., Stemphylium sp. and Torula sp. Later, and based on more isolations, he pointed out that A. pullulans was the main fungus responsible for the graying of weathered wood. More recent studies have observed that A. pullulans also frequently colonizes painted wood surfaces (Amburgey, 1974; Schmidt and French, 1976; Bardage and Bjurman, 1998). The frequent isolation of A. pullulans from weathered and painted wood surfaces seems to be related to its ability to metabolize photodegraded lignin product from weathered wood surfaces and also its capacity to withstand desiccation and high temperatures (Park 1982; Schoeman and Dickinson 1996; 1997). These characteristics may give it an advantage over many other moulds that colonize wood surfaces. The ubiquitous colonization of wood by moulds is also clearly related to 23  their successful modes of propagation. According to Hansen (2008) airborne conidia are easily carried by the wind for long distances, even from one continent to another. Thus, spores are abundant everywhere in the world. Therefore the successful colonization of a newly exposed wood surface will largely depend on the substrate and its surface microclimate. Mould fungi are able to colonize wood surfaces even in an extreme climate like that in Antarctica. For example, four species of soft rot fungi, Candophora sp., Cladosporium sp., Hormonena dematioides, sp., Lecythophora hoffmannii and Penicillium sp. were isolated from a 40+ years old wood structure at New Harbor, Antarctica by Held et al. (2006). More recently fungal diversity on weathered western red cedar fences and decks exposed in Vancouver, Canada, was examined by Lim et al. (2005; 2007). They isolated a wide range of basidiomycetes and ascomycetes. The ascomycetes they isolated were Oidiodendron griseum, Rhinocladiella atrovirens, 2 species of Sporothrix, several species of Phialophora, Acanthophysium lividocaeruleum, Coniophora puteana, Dacrymyces stillatus, Hyphoderma praetermissum, Pachnocybe ferruginea, Phellinus ferreus, A. pullulans, Exophiala heteromorpha, Phialocephala dimorphospora, Rhinocladiella atrovirens, and Umbelopsis autotrophica. An earlier study isolated A. pullulans, Cladosporium spp., Oidiodendron spp., Penicillium spp., Phialocephala spp., Raffaelea sp., Rhinocladiella spp., Sepsonema sp., Sporothrix spp., Trichoderma spp., from weathered Western red cedar shingles and shakes (Smith and Swann, 1976). A comprehensive review of fungi isolated from wood surfaces exposed outdoors (above the ground) around the world shows that the most frequent fungus isolated from weathered wood is A. pullulans (Table 2.1). This fungus is followed, in decreasing order of importance, 24  by species of Cladosporium, Penicillium, Phialocephala, Alternaria, Curvularia, Fusarium, Nigrospora, Rhinocladiella, Sporothrix, and Trichoderma. All these organisms have been isolated from virtually all continents (excepting Africa for which data are not available) from durable and non durable wood species and in some cases from preservative treated wood. However, the review also indicates that other fungi are able to colonize weathered wood. Such fungi have only been isolated once or twice but they are a highly diverse group of microorganisms distributed across at least 46 genera.  25  Table 2.1: Fungi isolated from wood surface exposed outdoors above the ground. The table also reports the author, substrate and country of isolation. Question mark (?) is featured when information was not available Isolate  Author; substrate; country Sell and Walchli (1969); ?; ? Dickinson (1971); Scots pine, WRC; England Lim et al. (2005; 2007); WRC; Vancouver-Canada  A. pullulans  Kim et al. (2007); treated radiata pine; Korea Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Amburgey (1974); asphalt shingles (wood based); USA Schmidt and French (1976); lauan, cedar and redwood; USA Hansen (2008); ?; USA, Thailand, Brazil Smith and Swann (1976); WRC; USA, Vancouver Canada Doi and Horisawa (2001); sugi; Japan  Acanthophysium lividocaeruleum Acremonium sp.  Alternaria spp.  Lim et al. (2005; 2007); WRC; Vancouver-Canada Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Dickinson (1971); Scots pine, WRC; England Amburgey (1974); asphalt shingles (wood based); USA Hansen (2008); ?; Germany, Malaysia, USA, Thailand, Brazil Doi and Horisawa (2001); sugi; Japan  Arthrinium sp. Aspergillus spp. Brachysporiella sp. Candophora sp.  Doi and Horisawa (2001); sugi; Japan Amburgey (1974); asphalt shingles (wood based); USA Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Sudayani et al. (2002); Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Held et al. (2006); ?; Antarctica Sell and Walchli (1969); ?; ? Dickinson (1971); Scots pine, WRC; England Held et al. (2006); ?; Antarctica  Cladosporium spp.  Kim et al. (2007); treated radiata pine; Korea Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Hansen (2008); ?; Germany, Malaysia, USA, Thailand, Brazil Smith and Swann (1976); WRC; USA, Vancouver Canada  Coniophora puteana  Lim et al. (2005; 2007); WRC; Vancouver-Canada Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Amburgey (1974); asphalt shingles (wood based); USA  Curvularia spp.  Doi and Horisawa (2001); sugi; Japan Hansen (2008); ?; Brazil  26  Isolate  Author; substrate; country  Dacrymyces stillatus  Lim et al. (2005; 2007); WRC; Vancouver-Canada  Epicoccum sp.  Doi and Horisawa (2001); sugi; Japan  Exophiala heteromorpha  Lim et al. (2005; 2007); WRC; Vancouver-Canada  Fumago sp.  Amburgey (1974); asphalt shingles (wood based); USA Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Amburgey (1974); asphalt shingles (wood-base); USA  Fusarium spp.  Hansen (2008); ?; Brazil Fusicladium sp.  Gliomastix sp.  Amburgey (1974); asphalt shingles (wood-base); USA Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Doi and Horisawa (2001); sugi; Japan  Hormonema dematioides  Held et al. (2006); ?; Antarctica  Hyalodendron sp.  Kim et al. (2007); treated radiata pine; Korea  Hyphoderma praetermissum  Lim et al. (2005; 2007); WRC; Vancouver-Canada  Lecythophora hoffmannii  Held et al. (2006); ?; Antarctica  Macrosporium sp.  Sell and Walchli (1969); ?; ?  Melasmia sp.  Amburgey (1974); asphalt shingles (wood-base); USA Sudayani et al. (2002); Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Amburgey (1974); asphalt shingles (wood-base); USA  Geotrichum sp.  Monilia sp. Monochaetia sp.  Amburgey (1974); asphalt shingles (wood-base); USA  Mucor sp.  Amburgey (1974); asphalt shingles (wood-base); USA  Nectria sp.  Doi and Horisawa (2001); sugi; Japan  Neurospora spp.  Nigrospora spp.  Oidiodendron spp. Pachnocybe ferruginea Paecilomynes sp.  Penicillium spp.  Doi and Horisawa (2001); sugi; Japan Sudayani et al. (2002); Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Doi and Horisawa (2001); sugi; Japan Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Hansen (2008); ?; USA Smith and Swann (1976); WRC; USA, Vancouver Canada Lim et al. (2005; 2007); WRC; Vancouver-Canada Lim et al. (2005; 2007); WRC; Vancouver-Canada Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Held et al. (2006); ?; Antarctica Kim et al. (2007); treated radiata pine; Korea Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Amburgey (1974); asphalt shingles (wood-base); USA Hansen (2008); ?; Germany Smith and Swann (1976); WRC; USA, Vancouver Canada  27  Isolate  Author; substrate; country  Pestalotia sp.  Doi and Horisawa (2001); sugi; Japan  Phellinus ferreus  Lim et al. (2005; 2007); WRC; Vancouver-Canada Lim et al. (2005; 2007); WRC; Vancouver-Canada  Phialocephala spp.  Smith and Swann (1976); WRC; USA, Vancouver Canada Kim et al. (2007); treated radiata pine; Korea Lim et al. (2005; 2007); WRC; Vancouver-Canada  Phoma spp.  Hansen (2008); ?; Malaysia, Thailand, Brazil Kim et al. (2007); treated radiata pine; Korea  Pithomyces spp.  Amburgey (1974); asphalt shingles (wood-base); USA  Pithomyces spp.  Doi and Horisawa (2001); sugi; Japan  Raffaelea sp.  Smith and Swann (1976); WRC; USA, Vancouver Canada  Rhinocladiella spp.  Lim et al. (2005; 2007); WRC; Vancouver-Canada Smith and Swann (1976); WRC; USA, Vancouver Canada  Sclerophoma sp.  Sell and Walchli (1969); ?; ?  Scolecobasidium sp.  Amburgey (1974); asphalt shingles (wood-base); USA  Sepsonema sp.  Smith and Swann (1976); WRC; USA, Vancouver Canada  Sordaria sp.  Doi and Horisawa (2001); sugi; Japan  Sphaeropsis sp.  Amburgey (1974); asphalt shingles (wood-base); USA  Sporothrix spp.  Lim et al. (2005; 2007); WRC; Vancouver-Canada Smith and Swann (1976); WRC; USA, Vancouver Canada  Stemphylium sp.  Dickinson (1971); Scots pine, WRC; England Hansen (2008); ?; Germany  Tetracoccosporium sp.  Sell and Walchli (1969); ?; ?  Thielaviopsis sp.  Amburgey (1974); asphalt shingles (wood-base); USA  Torula sp.  Dickinson (1971); Scots pine, WRC; England Amburgey (1974); asphalt shingles (wood-base); USA  Trematisphaeria sp.  Amburgey (1974); asphalt shingles (wood-base); USA  Trichocladium sp.  Amburgey (1974); asphalt shingles (wood-base); USA  Trichoderma spp.  Amburgey (1974); asphalt shingles (wood-base); USA Smith and Swann (1976); WRC; USA, Vancouver Canada Kim et al. (2007); treated radiata pine; Korea  Umbelopsis autotrophica  Lim et al. (2005; 2007); WRC; Vancouver-Canada  28  2.2.3.3. Effects of surface fungi on wood The growth of moulds at wood surfaces can produce a range of colors, including black, gray, green, purple and red. Heavy colonization of wood surfaces by mould can also produce characteristic mould-like odors, and their spores represent a potential cause of allergies (Zabel and Morrell, 1992). A number of moulds have the ability to attack pit membranes, and this effect of moulds on the structure of wood has been used to develop biological treatments to increase the permeability of difficult-to-treat wood species (Schulz, 1956). Others moulds are antagonist to decay fungi (Hulme and Shields, 1972) and others can detoxify wood preservatives (Brown, 1953). Some moulds isolated from weathered wood can also cause soft-rot decay. Such fungi include Alternaria sp., Phialophora sp., Lecythophora hoffmannii, Coniochaeta ligniaria, Phoma sp., Aspergillus sp., Penicillium sp., Trichoderma sp. (Savory 1954; Rajderkar 1966; Bugos et al. 1988; Zabel and Morrell 1992; Lim et al. 2007). In fact this phenomenon was observed in weathered western red cedar shingles over 30 years ago by Smith and Swann (1976). A. pullulans is able to depolymerize carbohydrates and previous studies have shown that it can cause weight losses of 7% and 34% when grown on cellulose and hemicelluloses, respectively (Seifert, 1964). In addition, A. pullulans exhibits cellulase, polygalacturonase, pectinesterase and laccase activity suggesting that it is capable of attacking carbohydrates directly in lignified cell walls (Dickinson 1971). Indirect evidence of the ability of moulds to degrade wood is available from a study carried out by Merrill et al. (1965). They examined the effects of common moulds on fiberboards, and found that most of the moulds caused strength and weight losses. Chemical analyses showed that they were able to reduce the α-cellulose and 29  hemicellulose content of the fiberboards. In addition, Alternaria sp. and Penicillium sp. were able to reduce the lignin content of the fiberboards (Merrill et al. 1965). Today, it is known that hemicelluloses influence the mechanical properties of wood (Curling et al. 2002), and their degradation may account for the strength losses of fiberboards that Merrill observed (Merrill et al. 1965).  2.2.3.4. Staining of coated and modified wood Wood is still susceptible to fungal attack by moulds even when it is covered by coatings. Alternaria sp., Phoma sp., Cladosporium sp., Stemphylum sp. and A. pullulans have all been isolated from coated wood (Duncan 1963; Savory 1973). These fungi can sometimes grow within the finish without colonizing the wood, by using some of the chemical components of the coating as a food source, for example oil-based binders (Duncan 1963; Savory 1973). Evidence for this is that A. pullulans grows on paints applied to metals (Savory 1973). A number of theories have been proposed to explain the colonization of coated wood by mould fungi. The first postulates that spores land on wood prior to the application of coatings and germinate later using moisture from within the wood (Duncan 1963; Savory 1973). A second theory suggests that fungi grow directly on finishes and penetrate into the wood using imperfections in the coating, raised fibers, or via enzymatic mechanisms (Duncan 1963; Savory 1973). Once fungi colonize the wood surface under the coating, the growth of hyphae can generate mechanical stresses which cause the coating to blister, fracture and finally fail (Duncan 1963).  30  According to Dickinson (1971) the most effective treatment at preventing fungal colonization of finished wood is a pre-treatment containing a water repellent and fungicide. However, good control of fungi has also been obtained using a primer containing a mix of fungicides (propiconazole + 3-Iodo-2-propynyl butylcarbamate (IPBC), 0.5+0.2 %, respectively) (Hannu and Ahola 1998). Fungi colonizing weathered wood, however, exhibit some tolerance to preservative treatments. This behavior includes tolerance to preservatives such as chromated copper arsenate (CCA). For example, Kim et al. (2007) isolated 16 species from the genera Phoma, Cladosporium, Penicillium, Aureobasidium, Phialophora, and Trichoderma from CCA-treated radiata pine (Pinus radiata D.Don). They concluded that staining fungi are more tolerant to CCA salts than basidiomycete fungi (Kim et al. 2007). Cladosporium sp. and Aspergillus sp., are also tolerant of the fungicides found in some preservative formulations. According to Shirikawa et al. (2002) paint containing a mix of preservatives was able to prevent the growth of large numbers of microorganisms on wood. However, it could not inhibit the growth of Cladosporium sp. and Aspergillus sp. The use of photocatalytic substances such as TiO2 has been shown to be effective against microorganisms growing on concrete and other materials surfaces (Gumy et al., 2006), but this approach has not been tested on weathered wood. Fungi also seem to be able to colonize modified wood surfaces. Wood surface fungi have been reported colonizing thermally and chemically modified wood. Raberg et al. (2006) reported colonization of thermally modified Norway spruce (Picea abies (L) H.Karst.) by Mucor sp. and Hormonema dematioides; and colonization of acetylated Scots pine by Cladosporium sp. and Phoma sp. Recently, a wide range of fungi were found colonizing 31  specimens of Scots pine (Pinus sylvestris L.) and European beech (Fagus sylvatica L.) modified with an amino-alkyl-functional oligomeric siloxane, sodium water glass or 1,3dimethylol-4,5-dihydroxyethylene urea (DMDHEU) (Pfeffer et al. 2012). In such work Trichoderma sp. and Epicoccum sp. were the predominant fungi isolated from the modified woods, but DMDHEU modified wood was only colonized by A. pullulans.  2.3. Ultraviolet radiation and fungal melanins 2.3.1. Effect of ultraviolet radiation on living cells and fungi The ultraviolet (UV) region of the electromagnetic spectrum has been subdivided into three regions: UVA (400-320 nm); UVB (320-290 nm); and UVC (290-200 nm). The division between UVB and UVC at 290 nm is chosen because ultraviolet radiation at wavelengths shorter than 290 nm is unlikely to be present in terrestrial sunlight, except at high altitudes (Henderson 1977). The quantity and quality of UV light reaching the earth’s surface depends on the output from the sun and the properties of earth’s atmosphere, but UVB is the most important part of the terrestrial UV spectrum in terms of its damaging effects on biological organisms and materials (Diffey 1991). The biological effects of UV light start with its photochemical absorption by biological molecules. The biological molecules that are most susceptible to UV radiation are nucleic acids and proteins, and their nucleotides which act as chromophores (absorbers of light) (Harm 1980). In nucleic acids like deoxyribonucleic acid (DNA) the nucleotides are adenine, guanine, thymine and cytosine. DNA nucleotides absorb UV radiation at slightly different  32  wavelengths, between 260 – 265 nm. In contrast, proteins absorb less UV radiation than DNA, and at wavelengths closer to 280 nm (Diffey 1991). The products of UV absorption are mainly derivates of pyrimidine (pyridime dimers). In addition, DNA and proteins in cells cross-link when they are exposed to UV radiation (Patrick and Rahn 1976). Cells exposed to UV radiation can reach a state of inactivation, losing their ability to reproduce (Diffey 1991). The range of responses of DNA in biological organisms to UV radiation is summarized in Figure 2.4. UV photons absorbed  No chemical reaction  Biologically relevant  No inactivation due to repair or bypass  No biological effect  Photochemical reaction  Potentially biologically irrelevant  Secondary alterations  ? Secondary alterations  Obligatory biologically relevant  ?  No lethal effect (mutation, growth delay, etc.)  Inactivation  Figure 2.4: Possible effects of absorption of UV radiation by deoxyribonucleic acid (DNA) (Harm 1980)  Living cells have the ability to repair their DNA despite the damage caused by UV exposure. Repairing mechanisms have been identified and are described here according to Freifelder's (1987) terminology. (1) ‘Photoreactivation repair: this mechanism makes possible the repair 33  of DNA by the separation of a photoreactivating enzyme attached to the resultant pyrimidine dimers in the presence of radiation between 330 and 600 nm. The separation leaves a repaired section of DNA’. (2) ‘Excision repair: this repair process takes places in the dark. The defective zone of DNA is excised by enzymes and then replaced with normal nucleotides utilizing the complementary base pairing information in the interactive strand (in case the complementary strand is intact)’. (3) ‘Post-replication repair: UV damaged DNA can replicate in such a way that gaps are left in the daughter strand opposite the damaged sites. Subsequently the gaps are filled by DNA synthesis’. (4) ‘SOS repair: this mechanism is not fully understood, but it is thought to include a bypass system that allows the growth of the DNA chain across the damaged site’. ‘This is achieved at the cost of fidelity of replication, and a great deal of evidence now indicates that SOS repair is the major cause of ultraviolet induced mutagenesis’ (Freifelder 1987). Living cells in fungal hyphae and spores are susceptible to solar radiation and especially to UV light. Exposure to solar radiation has been shown to be one of the most important factors affecting the survival of fungi (Rotem and Aust, 1991). The inactivation of microorganisms by light depends on the wavelength of the incident light, its intensity, and other physical and chemical parameters such as temperature, and substrate conditions (roughness and nutrients). The concentration of microorganisms at the exposed surface also plays an important role (Ozcelik, 2007; Schoenen and Kolch, 1992). The germicidal effect of UV light is well known and it is routinely used in air handling units (Levetin et al. 2001). Such units contain UV lamps that are able to reduce spore concentrations in air ducts. The effectiveness of such systems has been demonstrated against Cladosporium sp., and 34  Alternaria sp. spores (Levetin et al. 2001). Shorter wavelengths closer to 254 nm have greater fungicidal effects than longer ones such as 354 nm which, according to Ozcelik (2007) are unable to inactivate moulds even after 75 minutes of exposure. Nevertheless such exposure may decrease growth rates of fungi. Accordingly, Cagan and Svercel (2001) found that the radial growth of the fungus Beauveria bassiana decreased with an increase in time of exposure to UV light with an average wavelength of 253.7 nm. In contrast, other fungi exhibited different behavior to solar radiation or UV radiation (Rotem and Aust 1991). In some fungi their survival when exposed to UV radiation was proportional to the melanin content of their spores walls (Durrell 1964). For example, Wang and Casadevall (1994) found that non-melanized hyphae were more susceptible to UV radiation than melanized ones when exposed to different doses of UV light with a wavelength peak at a 254 nm. Kawamura et al. (1999) found that melanin conferred UV tolerance to Alternaria alternata. Frederick et al. (1999) found that exposure to UV light resulted in the melanization of hyaline hyphae of the fungus G. graminis var. graminis. As a result the hyphae became more tolerant to UV radiation compared to the hyphae of a non-melanized mutant. Melanin also confers UV tolerance to most spores and propagules (Henson et al. 1999). Another mechanism used by fungi to tolerate exposure to UV radiation involves the aggregation of spores and propagueles. For example, Rotem and Aust (1991) found a higher survival ratio for spores exposed to UV radiation when they formed aggregates.  35  2.3.2. Fungal melanins 2.3.2.1. Properties and role of melanins Fungal melanins are high molecular weight, dark brown or black pigments formed by enzymatic or auto-oxidative polymerization of phenols and amino acid derivates or amino sugars, which are synthesized from carbohydrates by fungi during biosynthetic processes (Butler and Day, 1998; Paim et al. 1990). Melanin pigments are not essential for fungal growth. In fact, their synthesis is sometimes classified as ‘secondary metabolism’ and both pigmented and albino strains of the same fungi may exist (Henson et al. 1999). However, pigmented fungi may have comparative advantages when growing in certain environments (Butler and Day, 1998; Fogarty and Tobin, 1996). Hence, melanin can account for approximately 30 percent of the dry weight of a fungal cell. This quantity underscores its importance to fungi (Butler and Day 1998). Melanins can be found within or outside cell walls. The latter occurs via secretion of phenol compounds, which are subsequently oxidized, or through secretion of phenol oxidases enzymes to oxidize phenolics compounds in the medium external to the fungus. An example of this process occurs in A. pullulans, which releases extracellular granules of melanin (Butler and Day, 1998; Fogarty and Tobin, 1996). In general, melanins from different organisms share some common characteristics. They are often sparingly soluble in alkali and generally insoluble in water, aqueous acids, and common organic solvents, and they can interact with metals (Butler and Day, 1998; Caesar-TonThat et al. 1995; Fogarty and Tobin, 1996). For example, supernatant culture fluids from Cladosporium resinae and A. pullulans, containing extracellular melanin, can  36  bind Cu. Melanin from A. pullulans is also produced in response to Cu, Co, Pb, Hg, Cd, Fe, Mn, Ag, Al, and Ni, but not Mg, or Zn (Caesar-TonThat et al. 1995). The dark color of melanins occurs because they do not re-radiate absorbed radiation as visible light (Butler and Day 1998). An impressive characteristic of fungal melanins is that they may exist as free radicals which are easily formed under various conditions such as incubation at increased temperature, irradiation with UV, γ-rays, or reaction with chemical reductants (Fogarty and Tobin 1996). In this sense, melanins are unique biopolymers because they contain stable free radicals that can act as proton receivers or donors; although they can be reduced by silver ions and oxidized by H2O2 (Fogarty and Tobin 1996; Henson et al. 1999). Several studies have shown that the presence of melanin enhances the survival of fungi exposed to environmental stress. The melanin present in fungal conidia reduces damage caused by UV light, solar radiation, γ-radiation, and X rays. The degree of protection against UV radiation is proportional to the concentration of melanin in conidial walls (Fogarty and Tobin 1996; Henson et al. 1999; Butler and Day 1998; 2001). Melanins may also provide fungi with increased resistance to desiccation and extreme temperatures. Melanins are synthesized in fungal pathogenesis by fungi to develop turgor in appressoria, and to increase virulence (Fogarty and Tobin 1996; Henson et al. 1999; Butler and Day 1998; 2001). Melanins provide protection against lysis in natural soils and protection against oxidizing agents (Butler and Day 1998). They also act as a physical boundary between the cell and its often hostile surroundings. Thus, melanin isolates the fungus from physical and biological stresses including poisons (Butler and Day 2001). Some melanins can bind drugs such as chlorpromazine and chloroquine. It is possible that some fungicides can be bound to 37  and inactivated by fungal melanins in a similar fashion (Butler and Day 1998; 2001). Melanins can also limit the leakage of useful compounds from fungal cells (Butler and Day 2001).  2.3.2.2. Synthesis of fungal melanins Tyrosine, 3,4-dihydroxyphenylalanine (DOPA), γ-glutaminyl-4-hydroxybenzene (GHB), catechol, catecholamines, and 1,8-dihydroxynaphthalene (DHN) are the known precursors of fungal melanins (Fogarty and Tobin 1996). These precursors generate 4 different types of fungal melanins: DOPA, GHB, Chatechol and DHN (Figure 2.5). DOPA melanins are heteropolymers made from a number of different compounds derived from tyrosine and DOPA (Butler and Day, 1998; Fogarty and Tobin, 1996). The biosynthetic pathway of DOPA melanins starts when tyrosine is hydroxylated to form DOPA followed by formation of DOPA-quinone by dehydrogenation of DOPA (Fogarty and Tobin 1996). DOPA melanins are able to switch incident visible, UV, and infrared energy into heat by converting the electronic energy of the radiation into vibrational and rotational activity in the molecular structure of the melanin. DOPA melanin is synthesized by basidiomycete fungi (Butler and Day 1998).  38  Figure 2.5: Precursors of fungal melanins  39  The biosynthesis of GHB melanins was described for Agaricus bisporus by Fogarty and Tobin (1996): “GHB melanin is generated from the precursor glutaminyl-4-hydroxybenzene, synthesized via the shikimic acid pathway. The shikimic acid is o-hydroxylated, followed by dehydrogenation of diphenol and polymerization of γ-glutaminyl-3,4-benzoquinone (GBQ) and quinoid products of GBQ. The γ-glutaminyl moiety of GHB may be removed prior to polymerization by a γ-glutaminytransferase present in the fruiting body. The γ-glutaminyl residue may thus be transferred to a receptor, liberating 4-aminiphenol (or 4aminocathechol if the γ-glutaminylmoiety from GDHB is removed), which can be converted to very reactive oxidized intermediates, such as 2-hydroxy-4-iminoquinone. The intermediates can then polymerize to yield melanin”. As for DOPA melanin, it is well accepted that GHB melanin is produced by fruiting bodies of basidiomycetes. Cathecol melanin contains percentages of carbon, hydrogen, nitrogen and carboxyl groups, but its biosynthesis is still unclear (Fogarty and Tobin 1996). The starting molecule for the DHN melanin pathway is 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), which is formed by the head-to-tail joining and cyclization of acetate molecules. After that an alternating pair of reduction and dehydration reactions results in the formation of an immediate precursor (the monomer) to the melanin polymer, which is DHN. In brief, 1,3,6,8-THN is reducted to scytalone, and a dehydration reaction then forms 1,3,8-trihydroxynaphthalene (THN). A second reduction reaction forms vermelone from 1,3,8-THN, which is converted to DHN by a second dehydration reaction, and DHN is finally polymerized in a final step to form DHN melanin (Figure 2.6) (Fogarty and Tobin 1996). DHN melanins are synthesized by a number of ascomycetous and imperfect fungi, mainly 40  filamentous fungi. Among them are: Sporothrix shenckii, Alternaria alternata, A. pullulans, Cladosporium carrionii, Cladosporium bantianum and Cladosporium cladosporioides, G. graminis, Magnaporthe grisea, C. lagenarium, Cochliobolus heterostrophus, and Aspergillus sp. (Caesar-TonThat et al. 1995; Kawamura et al. 1997; Henson et al. 1999; RomeroMartinez et al. 2000; Kogej et al. 2004). However, the complex factors involved in the biosynthesis of DHN melanin can generate slightly different polymers in different fungi. As a result, color differences can be found between melanins from different fungi. These differences are related to the amounts and wavelengths of light that melanins absorb, and with the polymer’s structure, size, crosslinking, oxidation state, cellular location, and complexation with other cellular components (Henson et al. 1999). Comparison of melanins derived from DOPA, DHN, GHB, and catechol shows that they have similar (but not identical) chemical and physical properties. One explanation for this similarity, which is supported by Fourier transform infrared spectroscopy, is that they all contain identical functional groups (Fogarty and Tobin 1996).  41  Figure 2.6: DHN melanin biosynthesis  42  2.4. Fungal melanin biosynthesis inhibitors Fungal melanin biosynthesis inhibitors (MBIs) are chemical substances initially developed as systemic and multi-systemic fungicides against rice blast disease (Kurahashi 2001). Their mode of action is based on impeding the penetration of fungal hyphae inside plant’s tissues by affecting the thickening mechanism of fungal appressoria (Kurahashi 2001). Appressoria need to attain a specific turgor to penetrate plant tissue, and this is achieved by the accumulation of melanin. Production of melanin can be blocked by MBIs which impedes the thickening of appressoria and consequently prevents the penetration of plant tissues by the rice blast fungus (Kubo 2005). The fungus responsible for blast rice disease (M. grisea) and fungi responsible for other infections in crops are normally filamentous ascomycetes which synthesize melanin via 1,8-DHN. The synthesis of DHN melanin can be interrupted by MBIs which target the different enzymes involved in the biosynthetic pathways of DHN-melanin (Kim et al. 1998). The target site where MBIs act vary according to the enzyme they target. In general, MBIs are able to block three different enzymatic pathways: (1) at the earliest stages of melanin biosynthesis (possibly before and on pentaketide formation or cyclization); (2) at the reductive stage (reductase enzyme inhibited); and (3) at the dehydrate stage (dehydratase enzyme inhibited) (Figure 2.6). Melanin biosynthesis inhibitors are also useful for providing insights into the different pathways involved in the synthesis of melanin. The inhibition of specific enzymatic activity hints at the biosynthetic process involved in melanin synthesis. This research involves analyzing the chemicals that accumulate due to the action of MBIs (Butler and Day, 1998).  43  2.4.1. MBIs targeting early stages of DHN melanin biosynthesis The  compound  cerulenin  [(2R,3S)-3-[(4E,7E)-nona-4,7-dienoyl]oxirane-2-carboxamide]  (Figure 2.7a) is a strong inhibitor of melanin biosynthesis at the polyketide synthase step of DHN synthesis. Cerulenin also inhibits the enzyme fatty acid synthase, a physiologically critical enzyme. Therefore at low concentrations cerulenin is able to inhibit fungal growth in-vitro (Fleet and Breuil 2002). The fungicide KC10017 [3-[4’-bromo-2’,6’-dimethylphenoxy]methyl-4-[(3”-methylphenyl) aminocarbonyl]methyl-1,2,4-oxadiazol-5-one] (Figure 2.7b) also blocks DHN-melanin biosynthesis at the earliest stage of melanin biosynthesis. The target sites for this chemical are the reaction steps prior to 1,3,6,8-THN formation, namely pentaketide synthesis and /or pentaketide cyclization (Kim et al. 1998). According to Kim et al. (1998) the fungicide is very effective at blocking the biosynthesis of melanin by M. grisea, but when it was tested against other microorganisms like A. alternata and C. lagenarium it did not cause color changes in mycelia suggesting that it did not act as a melanin biosynthesis inhibitor. Kim et al. (1998) accounted for this discrepancy by suggesting that the biosynthetic pathway prior to 1,3,6,8-THN formation for M. grisea and A. alternata and C. lagenarium might be different, or alternatively that the structure of the enzyme blocked by KC10017 in M. grisea might be different from that in A. alternata and C. lagenarium (Kim et al. 1998).  44  Figure 2.7: Melanin biosynthesis inhibitors acting on the early stages of the biosynthesis of melanin. (a) Structure of cerulenin (Fleet and Breuil 2002) and (b) [3-[4’-bromo-2’,6’-dimethylphenoxy]methyl-4-[(3”methylphenyl) aminocarbonyl]methyl-1,2,4-oxadiazol-5-one] (KC10017) (Kim et al. 1998)  2.4.2. MBIs targeting reductase enzymes A second target site for MBIs is the enzymatic reduction of 1,3,6,8-THN to scytalone and 1,3,8-THN  to  vermelone.  This  can  be  achieved  by  blocking  the  enzyme  polyhydroxynaphthaline reductase (Kurahashi and Pontzen 1998; Kim et al. 1998; Kubo et al. 1996; 2005). The list of MBIs that block the reductase enzyme system and are registered as fungicides in Japan are listed in Table 2.2 (Kurahashi, 2001).  45  Table 2.2: Melanin biosynthesis inhibitors of reductase registered in Japan in 2001 Chemical group  Name  Chemical structure OH Cl  Cl  PCBA Cl  Cl Cl  CN OH Cl  Cl  PCMN  Cl  Poly chlorinated aromatic compounds  Cl Cl  O O Cl  CPA  CH3 Cl  Cl  Cl Cl O  Cl O  Fthalide Cl  Cl Cl  CH3 N N  Tricyclazole  N S O  N  Pyroquilon Cl  Fused heterocyclic compounds  H3C N  Chlobenthiazole  O S  N  N N  PP 389  N N CH3  O  Cl N  Phthaladine  N CH3  Tricyclazole is the reductase inhibitor that has been most widely studied. Tricyclazole was first developed as a fungicide, but it has been widely used in studies of melanin biosynthesis 46  (Cooper and Gadd, 1984; Fleet and Breuil, 2002; Kogej et al., 2004; Romero-Martinez et al., 2000). The effect of tricyclazole on pigmented fungal strains in-vitro is to induce hyphae to become pink initially. The hyphae then darken to red and brown as the fungal colony ages (Cooper and Gadd, 1984). These color changes are due to the accumulation of ‘shunt’ products from the blocked pathway. Flaviolin and 2-hydroxyjuglone (2-HJ) (Figure 2.6) are auto-oxidative products of 1,3,6,8-THN and 1,3,8-THN, respectively, and they have been isolated from cultures treated with tricyclazole (Butler and Day, 1998; Kogej et al., 2004). Wheeler and Klich (1995) evaluated the inhibition of pigmentation in Penicillium and Aspergillus species using several MBIs. They showed that tricyclazole, chlobenthiazone and pyroquilon were the most successful treatments, followed by phthalide, PCBA, and others. They also noticed that the fungicide chlobenthiazone did not inhibit mycelial growth at a concentration of 8 μg/mL. According to Cooper and Gadd (1984), tricyclazole might affect other types of melanins because it was able to inhibit induced colorization by DOPA and indole, which are precursors of the tyrosine type melanin.  2.4.3. MBIs targeting dehydratase enzymes A third target for fungal MBIs is the enzymatic dehydration of scytalone into 1,3,8-THN by elimination of water, and also a second dehydration reaction for the conversion of vermelone into 1,8-DHN (Kurahashi and Pontzen 1998; Kubo et al. 1996; 2005). The fungicides that target these reactions were developed later than reductase inhibitors; Kurahashi (2001) published a list of MBIs inhibitors of dehydratase that were registered in Japan in 2001 (Table 2.3). 47  The fungicide from this list that has been most commonly tested is carpropamid [(1R*,3S*)2,2-dichloro-N-[1-(4-chlorophenyl)+ethyl]-1-ethyl-3-methylcyclopropanecarboxamide]. Carpropamid is used as a foliar fungicide (Kurahashi and Pontzen 1998; Kurahashi et al. 1999; 2001; Hewitt 2000; Rohilla et al. 2001). It has also been used in laboratory studies to confirm the presence of the DHN-melanin pathway in fungi (Fleet and Breuil, 2002).  Table 2.3: Melanin biosynthesis inhibitors of dehydratase registered in Japan by 2001 (Kurahashi, 2001) Chemical group  Name  Chemical structure CH3  Carpropamid (CAR)  Cl  O  Cl  NH CH3 Cl  CH3  Cl  CH3  Dichlocymet (DCM)  O But  NH  NH2  Cl Cl  Cl  Carboxamide derivatives  CH3  Fenoxanil  CH3  NH O  Prl CN  O  CH3  O  OH  NH  BFS Cl  F CH3  O Cl  Cyclobutane carboxamid  NH F F  Br N  N NH  4-aminoquinazolin dereviates  N  N NH  N  48  CH3 F  2.4.4. Other inhibitors Other MBIs are also mentioned in the literature. For example, Wheeler and Klich (1995) mention the ability of MQ (N-methyl-2-quinolone), TQ (s-triazolo-[4,3-a]quinoline) and coumarin (Figure 2.8 (a), (b) and (c), respectively) to inhibit the melanization of P. oryzae. However, there is no information on the metabolic targets for these molecules.  Figure 2.8: Compounds that inhibit DHN-melanin biosynthesis in P. oryzae and other brown and black fungi. (a) N-methyl-2-quinolone (MQ), (b) s-triazolo-[4,3-a]quinoline (TQ) and (c) Coumarin (Wheeler and Klich 1995)  2.5. Summary This literature review provides background information on the weathering of wood, biological agents colonizing wood surfaces with emphasis on moulds colonizing wood surfaces, effect of UV radiation on microorganisms, fungal melanins and chemical inhibition of melanin biosynthesis. This information enables the reader of this thesis to understand the experimental chapters that follow. This review shows that only a few studies have examined the effect of moulds on the structural properties of wood and its polymeric constituents. Such studies do not conclusively establish whether moulds can degrade wood’s structural tissues. Similarly, the effect of UV radiation on the growth, survival and melanization of fungi have been studied in general, but the effect of UV radiation on the growth and melanization of moulds 49  colonizing weathered wood has not been examined. In addition, the control of surface fungi to prevent the graying of wood has been restricted to the use of fungicides. The possibility of using melanin biosynthesis inhibitors to reduce the staining and graying of weathered wood has not been examined. This thesis intends to fill these gaps and provide new information to enhance our understanding of the role of non-decay fungi on the weathering of wood, with emphasis on ability of wood surface moulds to decay wood, effects of UV radiation on melanization and growth of surface moulds and prevention of graying of weathered wood surfaces using chemicals that inhibit the biosynthetic pathways of fungal melanins.  50  3. Chapter 3: Fungi colonizing the surface of southern pine exposed to natural weathering 3.1. Introduction Early observations of fungi causing the graying of wood exposed outdoors date back to the 19th and early 20th century, as mentioned in Chapter 2 (Möbius, 1924; Schacht, 1863), but the fungi colonizing weathered wood surfaces were not identified until the mid 20th century (Duncan 1963). A comprehensive list of fungi isolated from weathered wood surfaces around the world was tabulated in Chapter 2. Many of the organisms colonizing weathered wood have remarkable ability to grow in adverse environments (Duncan 1963), but their diversity normally depends on wood species (substrate), exposure conditions and climate (Hansen 2008). Most of the species isolated from weathered wood were identified using their morphological features (observed under the light microscope). This method of identification requires great skill and experience to produce accurate results (Gutzmer et al. 2004), because many fungal species share similar morphological features. On the other hand, identification of fungi using DNA analysis, can be more accurate (Ray et al. 2004; Balajee et al. 2007). In such analysis ribosomal genes are the most common targeted genes used for differentiating fungi at the genus and species levels. Genes are multiple copied, sequenced and blasted against genes from known (identified) organisms. The drawback of this technique is that the gene sequences of the target organisms must be available in databases for the identification to be accurate (Dismukes et al. 2003). Nevertheless, I hypothesize here that the combination of both molecular techniques and microscopy will  51  be highly effective at identifying the different fungi colonizing wood surfaces exposed outdoors. The aim of the research in this chapter was to isolate, identify and characterize the fungi colonizing untreated wood surfaces exposed outdoors. Southern pine wood was the test substrate because it is a commercially important wood species and it is prone to fungal staining (Himelick, 1982). Southern pine is a generic name given to most pine species whose major range is in the United States south of the Mason-Dixon line (lat. 39° 43’ N.). Southern pine comprises at least 10 species, all hard pines-diploxylon members of the genus Pinus, family Pinaceae, and order Coniferales, e.g. P. palustris, P. elliottii, P. tadea, P. echinata, P. glabra, and others (Koch, 1972). Fungi growing on wood samples exposed outdoors for 40 weeks were isolated and identified using both molecular techniques and microscopy. The growth rate and mycelia color of the fungi were then measured in solid culture media. The morphology, color and area of exposed wood surfaces affected by stain were also quantified. Chemical changes occurring at weathered wood surfaces were assessed using Fourier transform infra red spectroscopy (FTIR). Fungi isolated from weathered southern pine surfaces were used for subsequent experimentation in Chapters 4 and 5.  52  3.2. Materials and methods 3.2.1. Wood samples and exposure Five flat-sawn southern pine boards measuring 381 mm x 1397 mm x 24000 mm, supplied by CSI (now Viance) in North Carolina, USA, were used in this experiment. The growth rate and wood density of sample boards is shown in Table 3.1 below. The boards were stored in a conditioning room at 20 ± 1 °C and 65 ± 5% relative humidity (r.h.) for 24 weeks (12% equilibrium moisture content), and were cross-cut to produce 5 samples (one per board), each 320 mm long. These samples were planed on their tangential faces with growth rings oriented convex to the face (bark-side up) using a Martin T54 thickness planer. Then, sixteen strips, 20 mm wide, were made on the exposed face of each sample by cutting transversally to the grain 15 grooves, 3 to 5 mm in depth, with a band saw (Meber, Model SR-500). The strips, intended to facilitate measurement of stained area, were isolated from each other by filling the grooves with a hot melt resin (commercial grade) applied with a heating gun. The end grain on samples was sealed with epoxy resin (Quick cure 5; System three resins, Inc. WA, USA), to minimize further drying and development of checks. Samples were exposed outdoors to the weather, at ≈ 400 mm above the ground for 40 weeks in Vancouver, Canada. The 40 weeks (August to May) included many sunny days and periods when samples were exposed to heavy rainfall. The superficial moisture content of the samples was measured during the most rainy months of the exposure trial (week 10 to 32) using a portable resistance-type moisture meter (Delmhorst RDM³, Delmhorst Instrument Company). Monthly weather conditions for the exposure period are shown in Table 3.2.  53  Table 3.1: Density and growth rate of southern pine samples  Board/Block 1 2 3 4 5  Growth [rings/cm] 8.28 5.14 4.57 4.50 5.42  Basic density [g/cm3] 0.429 0.432 0.560 0.505 0.452  Table 3.2: Monthly weather conditions during the exposure period in Vancouver, Canada; reported by Canada’s National Weather Archive  Year 2007 2007 2007 2007 2007 2008 2008 2008 2008 2008  Month Aug Sept Oct Nov Dec Jan Feb Mar Apr May  Mean max. temp. [°C] 21.9 17.6 12.4 8.9 5.8 5.5 8.6 9.1 11.3 16.6  Mean temp. [°C] 17.8 14.2 9.6 5.9 3.2 2.8 5.5 5.9 7.6 12.8  Mean min. temp. [°C] 13.6 10.8 6.7 2.8 0.6 0.1 2.4 2.7 3.8 8.9  Extrem. max. [°C]  Extrem. min. [°C]  26.7 22.4 17.3 12.8 12.9 10.3 14.1 11.6 18.8 29  11.3 6.2 1.5 -3.3 -5.3 -4.9 -2.9 -1 -2.1 3.3  Total rain [mm] 8.4 73.6 155.2 116.2 181.6 122.2 67.4 72.8 56.8 43.2  Total snow [mm] 0.0 0.0 0.0 0.0 19.6 14.2 0.8 2.4 2.2 0.0  Total precip. [mm] 8.4 73.6 155.2 116.2 210.6 137.6 68.6 75.2 62.2 43.2  3.2.2. Isolation, purification, identification and storage of fungi The isolation of fungi from the surface of weathered southern pine samples used the method of Lim et al. (2005). A small fragment of wood, was excised from under the wood surfaces using a sharp scalpel and seeded directly onto 1% malt extract agar (MEA) Difco Different fungi growing on agar were separated by simple replication on to new plates, or by single spore isolation as described by Choi et al. (1999). Fungal isolations were performed on all samples after they were exposed to 40 weeks of natural weathering. 54  Isolated fungi were identified using both molecular techniques and microscopy, as mentioned above. Molecular techniques were used first to identify fungi and their identities were confirmed by examining their morphological features (Table 3.3). Some fungi had specific morphological characteristics that made it easier to identify them using light microscopy (Barnett and Hunter 1998). Identification using molecular techniques involved the extraction, amplification, purification and sequencing of fungal ribosomal DNA (rDNA). rDNA extraction was carried out using a modified version of the method developed by Lim et al. (2005). Modifications included the use of TES buffer as an extraction buffer and mechanical breakage of fungal cells by stirring the solution for 3 minutes at 600 rpm using a sterile stainless steel rod. The internal transcribed spacer (ITS) region of the rDNA was amplified using the universal primers ITS4 – ITS5 (Schmidt and Moreth 2002). Purification used the QIAquick PCR purification kit for enzymatic reactions (Quiagen Sciences Maryland, USA), and sequencing was performed at the DNA Synthesis and Sequencing Facility, at Macrogen (Seoul, Korea). The information obtained from the sequences was crossreferenced  in  the  GeneBank  data-base  website  (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). This data-base identifies similarities of the unknown fungus with those of known fungi in the data-base. Fungi were identified to the level of the genus or species depending on the information available. Stocks of isolated and purified fungi were prepared by placing 4 to 6 agar plugs (5 mm in diameter) of isolated fungi into 2 mL screw-cap collection tubes filled with 900 µL nano pure water and 100 µL of glycerol. Stock tubes were stored at -80°C.  55  Table 3.3: Morphological features of common darks moulds colonizing weathered wood (Barnett and Hunter 1998)  Genera Aureobasidium  Alternaria  Cladosporium  Epicoccum  Phoma  Features Mycelium not extensive, hyaline when young, becoming dark with age, black and shiny in old cultures, bearing abundant conidia laterally; conidia (blastospores) subhyaline to dark, 1-celled, ovoid, producing other conidia by budding; saprophytic or weakly parasitic; common in soil. Conidiophores dark, mostly simple; determinate or sympodial, rather short or elongate; conidia (porospores) dark, typically with both cross and longitudinal septa; various shapes, obclavate to elliptical or ovoid, frequently borne acropetally in apical or branched appendages; parasitic or saprophytic on plant material. Conidiophores tall, dark, upright, branched variously near the apex, clustered or single; conidia (blastospores) dark, 1 or 2 celled, variable in shape and size, ovoid to cylindrical and irregular, some typically lemonshaped; often in simple or branched acropetalous chains; parasitic on higher plants or saprophytic. Sporodochia dark, more or less cushion-shaped, variable in size; conidiophores compact or loose, dark, rather short; conidia dark, severalcelled (dicyosporous), globose; mostly saprophytic, or weakly parasitic. Pycnidia dark, ostiolate, lenticular to globose, immersed in host tissue, erumpent or with a short beak piercing the epidermis; conidiophores short; conidia small, 1 celled, hyaline, ovoid to elongate; parasitic, producing spots, principally on leaves.  3.2.3. Fungal diversity The diversity of fungi colonizing weathered southern pine samples was assessed using two measures: (1) fungal richness and (2) reciprocal Simpson index. Fungal richness is simply the total number of species isolated per sample (Adams 2009) and the reciprocal Simpson index corresponds to the number of fungal species that in theory must be colonizing the wood after exposure (Peet 1974). The reciprocal Simpson index is calculated using the following formula (Maria and Sridhar 2002): Reciprocal Simpson index = [1 / Σ (pi)2] Where, pi = proportion of individuals that species i contributes to the total per sample. Simpson index was calculated separately for each weathered southern pine sample. 56  3.2.4. Growth and color of fungi on solid culture media Isolated fungi were grown on 1% MEA Difco. A 5 mm diameter agar plug, from the original fungal culture, was placed on agar in a 150 mm x 15 mm Petri dish. Under standard conditions of illumination a digital image of the hyphal mat from each plate (1:1 scale) was obtained after 7 days using a desktop scanner (Microtek Scan Maker i800). The diameter of the hyphal mat was digitally measured with the ruler tool of the software Adobe Photoshop CS3 Extended, version 10.0.1 (Adobe Systems Incorporated, USA), Figure 3.1a. The plates were re-scanned without their lids after 20 days and the images were used to digitally measure the color of the hyphal mats (using Photoshop, as above). Color measurement of hyphal mats involved the selection of a relevant portion of mycelia in the image and the evaluation of red-green-blue (RGB) color of the selection using the color histogram provided by the software. The average RGB color was registered and then entered in the picker color tool of the software, which provides equivalent colors in different color systems, including the CIELab system, Figure 3.1b. Color of fungal mats was recorded using the CIELab color coordinates, L (lightness on scale of 0, [black] to 100 [white]), a* (+60 [red] to -60 [green]) and b* (+60 [yellow] to -60 [blue]) (International Commission on Illumination 2007). Only lightness results are presented and discussed here.  57  Figure 3.1: Growth rate and fungal mat color measurements. (a) Growth measurement in Photoshop of a fungal colony after 7 days of growth on malt extract agar (MEA) 1%; note the use of the ruler tool to estimate the diametrical growth of the fungal colony; (b) Fungal mat color measurement in Photoshop after 20 days of growth onto MEA 1%; note the original image of the colony, the selection of a relevant area for the measurement, information about the RGB color of the selected pixels (red square right side of the image) and color picker tool for transformation from RGB into CIELab color  58  3.2.5. Microstructure of wood colonized by fungi The microstructure of wood colonized by fungi was examined using light microscopy. Pieces of wood measuring 10 mm x 10 mm were cut from the surface of exposed southern pine specimens and soaked in distilled water for 2 days. Each water-saturated block was clamped in a microtome and 20 µm sections were cut from the block using a disposable blade (Type S35, Feather Safety Razor Co., Japan) bolted to a microtome blade-holder. Sections were  dehydrated in ethanol (industrial grade) for 2 days and then transferred to a saturated solution of safranin (BDH Chemical Ltd, England) in ethanol for 2 days. Each stained section was placed on a droplet of DPX (dibutyl phthalate xylene) mountant (Fluka Analytical, Germany) on a glass slides measuring 76 mm x 26 mm x 1 mm (Matsunami Glass Ind. Ltd. Japan), covered with a glass cover slip measuring 22 mm x 40 mm x 0.20 mm (Fisher Finest Premium Cover Glass, Fisher Scientific, Pittsburgh, USA), and dried at room temperature for 48 hours. The sections were examined using a light microscope (Carl Zeiss, Germany) at various magnifications. An Olympus DP71 digital camera attached to the microscope was used to take photographs of fungi colonizing the wood sections.  3.2.6. Color of weathered wood and area stained by fungi The color of wood samples exposed to the weather was measured periodically. Samples were removed from the weathering racks and their color was measured: weekly during the first 4 weeks of exposure, every two weeks until week 20 and then at weeks 24, 32 and 40. Color expressed in CIELab color coordinates (as shown in section 3.2.4) was measured using  59  a portable spectrophotometer (Minolta CM-2600d). After color measurements, digital images of wood samples, scale 1:1; 96 dpi resolution, were taken with a desktop scanner (as above) to assess the area of wood stained by fungi. Digital images were examined using Photoshop (as above) at increased magnification (150 %) for fungal stains, and an additional transparent layer (same pixels size and resolution) was added to each picture. In this layer the area colonized by fungi was manually colored with Photoshop’s brush tool. The number of dark colored pixels, measured with the automatic counting tool of the software, divided by the total number of pixels in the layer, multiplied by 100 was recorded as the stained area (Figure 3.2).  Figure 3.2: Measurement using Photoshop of the area of a wood sample stained by fungi. Original image (left) and colored pixels (centre) for quantification of stained area  60  3.2.7. Chemical changes at weathered wood surfaces FTIR spectroscopy was used to examine chemical changes occurring at wood surfaces exposed outdoors. Pieces of wood measuring 20 mm (width) x 60 mm (length) x 8 mm (thickness) were sawn from each sample and stored for 5 days in a vacuum desiccator over silica gel. Direct reflectance (ATR-IR) FTIR spectra of weathered (gray) surfaces were obtained using a single bounce attenuated total reflectance accessory (PikeMiracle, PIKE technologies, WI, USA) attached to a spectrometer (Perkin Elmer Spectrum one, Waltham MA, USA). The penetration of infrared radiation into the wood sample was expected to be approximately 1.2 μm (Evans et al. 2008). Spectra of the fingerprint region 1800 to 800 (cm 1  ) represented 16 accumulations at 8 cm-1 of resolution. Relevant peaks in the spectra were  highlighted in the Spectrum software (v 5.3.1) on a PC attached to the spectrometer.  61  3.3. Results 3.3.1. Fungal diversity A total of 26 isolates from 10 different genera, all in the phylum ascomycota, were isolated from the five replicate (boards) weathered southern pine samples. Of the 10 genera 4 were identified exclusively by DNA analysis, representing 15 % of the total isolates; 2 genera were identified exclusively by light microscopy, representing only 12 % of the total isolates; and 4 genera were identified using both techniques, representing 73 % of the total isolates (Table 3.4). The fungal richness on samples varied from 2 to 7, and the Simpson index from 2 to 5 (Table 3.5). Among the isolated fungi several were very well known colonizers of weathered wood including Aureobasidium pullulans, Hormonema dematioides, Cladosporium sp., Alternaria sp., and Phoma sp. Other fungi isolated were Truncatella angustata (Pers.) S. Hughes, Glonium pusillum Zogg Zogg H., Mollisia minutella (Sacc.) Rehm and a fungus from the genus Lecythophora. In addition, further characterization of isolated A. pullulans on solid media revealed that two varieties were present: a dark-type and a white-type. The latter white fungus melanized approximately one week after being seeded onto 1% MEA.  62  Table 3.4: Fungi isolated from southern lodgepole pine wood samples after 40 weeks of outdoor exposure in Vancouver, Canada Phylum  Source (Exposure/Rack)  Codification  Identification  Primer sequenced  Aureobasidium pullulans (black)  Ascomycota  Aureobasidium pullulans (black)  Ascomycota  Full / Sample 2  2  rDNA / Microscopy  ITS4  Aureobasidium pullulans  FJ216455  556/561 (99%)  Full / Sample 3  1_1  Microscopy*  Aureobasidium pullulans (white)  Ascomycota  Full / Sample 3  3  rDNA / Microscopy  ITS4  Aureobasidium pullulans  AF455533  549/564 (97%)  Aureobasidium pullulans (black)  Ascomycota  Full / Sample 4  4  Microscopy*  Hormonema dematioides  Ascomycota  Full / Sample 3  5_2  Microscopy*  Hormonema dematioides  Ascomycota  Full / Sample 4  1  Microscopy*  Hormonema dematioides  Ascomycota  Full / Sample 5  4  Microscopy*  Hormonema dematioides  Ascomycota  Full / Sample 1  1S  rDNA / Microscopy  ITS4  Hormonema dematioides  AY253451  561/571 (98%)  ITS4  Hormonema dematioides  AY253451  566/573 (98%)  ITS4  Epicoccum nigrum  FJ904918  526/531 (99%)  Fungi  Closest match in Blast (GeneBank)  Identity  Hormonema dematioides  Ascomycota  Full / Sample 1  6_1S  rDNA / Microscopy  Alternaria sp.  Ascomycota  Full / Sample 1  3.2  Microscopy  Alternaria sp.  Ascomycota  Full / Sample 1  1  Microscopy  Cladosporium sp.  Ascomycota  Full / Sample 3  4_1  Microscopy  Epicoccum nigrum  Ascomycota  Full / Sample 1  7W  rDNA / Microscopy  Epicoccum sp.  Ascomycota  Full / Sample 3  2  Microscopy*  Epicoccum sp.  Ascomycota  Full / Sample 3  6  Microscopy*  Epicoccum sp.  Ascomycota  Full / Sample 4  5  Microscopy*  Epicoccum sp.  Ascomycota  Full / Sample 5  2  Microscopy*  Phoma herbarum  Ascomycota  Full / Sample 4  6  Microscopy*  Phoma sp.  Ascomycota  Full / Sample 1  4S  rDNA / Microscopy  ITS4  Phoma sp.  AM901684  532/535 (99%)  Phoma sp.  Ascomycota  Full / Sample 2  1  rDNA / Microscopy  ITS4  Phoma herbarum  DQ132841  510/519 (98%)  Phoma sp.  Ascomycota  Full / Sample 4  2  rDNA / Microscopy  ITS4  Phoma herbarum  DQ132841  514/526 (97%)  Phoma sp.  Ascomycota  Full / Sample 4  3  rDNA / Microscopy  ITS4  Phoma herbarum  AY337712  463/471 (98%)  Truncatella angustata  Ascomycota  Full / Sample 2  4  rDNA  ITS4  Truncatella angustata  AF405306  557/558 (99%)  Glonium pusillum  Ascomycota  Full / Sample 2  1_1  rDNA  ITS4  Glonium pusillum  EU552134.1  507/509 (99%)  Lecythophora sp.  Ascomycota  Full / Sample 2  5_1  rDNA  ITS4  Lecythophora sp.  AY219880.1  528/539 (97%)  Mollisia minutella Ascomycota Full / Sample 3 4_2 *: Morphological features cross references against fungi identified by DNA analysis  rDNA  ITS4  Mollisia minutella  DQ008242.1  448/448 (93%)  63  Table 3.5: Fungal diversity in southern pine wood samples exposed to the weather for 40 weeks in Vancouver, Canada  Sample 1 2 3 4 5 Average Total  Fungal richness 6 5 7 6 2 5.2 26  Simpson index 3.6 5 4.5 3 2 3.6 6.76  3.3.2. Growth and color of isolated fungi The radial growth of fungi after 7 days is expressed as mm of growth per week (Table 3.6). Epicoccum sp., T. angustata and Phoma sp. grew the fastest, 17 and 24 mm per week, respectively. A. pullulans and H. dematioides grew at similar rates, of around 13 mm per week. Other fungi grew more slowly particularly Mollisia minutella (2 mm), Lecythophora sp. (3.5 mm) and Cladosporium sp. (5.6 mm). Lightness of fungi after 20 days of growth expressed as the CIE L coordinate is shown in Table 3.7. A. pullulans (black), H. dematioides, Cladosporium sp. and Mollisia sp. produced the darkest mycelia whereas A. pullulans (white variety), Alternata sp., Epicoccum sp., T. angustata and G. pusillum were lighter. Hyaline (white) growth was shown by Phoma sp. and Lecythophora sp. (Table 3.7). Scanned images of fungi growing on MEA show the variation in color of the different fungi that were isolated from weathered wood and these images accord with color measurements (Figure 3.3 and Figure 3.4).  64  Table 3.6: Growth of fungi cultured onto solid malt extract agar (1% Difco) after 7 days of growth Growth at day 7  Fungi Truncatella angustata Epicoccum sp. Phoma sp. Alternaria sp. 5 Aureobasidium pullulans (white) Hormonema dematioides Aureobasidium pullulans (black) Glonium pusillum Cladosporium sp. Lecythophora sp. Mollisia minutella  Avg (mm) 24.1 20.7 17.9 15.1 13.4 13.4 12.9 10.2 5.7 3.6 2.0  [SD] [NA] [5.2] [1.0] [8.3] [0.6] [2.4] [2.7] [NA] [NA] [NA] [NA]  Table 3.7: Lightness of fungi cultured onto solid media malt extract (agar 1% Difco) after 7 days of growth Lightness at day 20  Fungi Hormonema dematioides Cladosporium sp. Alternaria sp. 5 Aureobasidium pullulans (black) Mollisia minutella Glonium pusillum Epicoccum sp. Truncatella angustata Lecythophora sp. Aureobasidium pullulans (white) Phoma sp.  65  Avg (L) 16.6 24.0 27.5 28.0 29.0 50.0 58.5 61.0 69.0 73.0 76.2  [SD] [3.6] [NA] [0.7] [NA] [NA] [NA] [13.3] [NA] [NA] [NA] [8.5]  Figure 3.3: Dark fungi isolated from weathered wood, after 20 days of growth on malt extract agar (1% Difco): (a) Hormonema dematioides; (b) Cladosporium sp.; (c) Aureobasidium pullulans; (d) Alternaria sp.; (e) Mollisia minutella; and (f) Glonium pusillum  66  Figure 3.4: Light fungi isolated from weathered wood, after 20 days of growth on malt extract agar (1% Difco): (a) Epicoccum nigrum; (b) Phoma sp.; (c) Lecythophora sp.; (d) Aureobasidium pullulans; and (e) Truncatella angustata  67  3.3.3. Fungal colonization under light microscopy Visual examination of end-grain of samples exposed to the weather for 40 weeks revealed that some of the samples were stained all the way through. Light microscopy revealed that fungi colonized and degraded parenchyma cells in the rays. Also, they were present in adjacent longitudinal tracheids. Hyphae penetrated the wood via ray parenchyma cells rather than via tracheids or ray tracheids. Hyphae grew longitudinally using the lumens of tracheids as a pathway (Figure 3.5).  68  Figure 3.5: Light microscopy images of sections from southern pine wood samples exposed outdoors for 40 weeks. (a) Tangential longitudinal section showing dark hyphae in degraded rays and tracheids; (b) Radial longitudinal section showing dark hyphae colonizing ray parenchyma cells, but not ray tracheids in rays; (c) Radial section showing dark hyphae colonizing tracheids approximately 200 micrometers beneath the weathered wood surface  69  3.3.4. Color of weathered wood and area stained by fungi Dark stains appeared on the surface of the southern pine samples 6 to 8 weeks after they were exposed outdoors. The increase in the percentage of the area of samples stained by fungi is shown in Figure 3.6. There was some evidence of fungal growth on wood surfaces as early as the second week of exposure. At this stage, small black fungal colonies were present, which increased in number over the next four weeks (week 6). After 8 weeks of exposure, the area colonized by fungi increased noticeably, covering approximately 50 % of the total area of samples. This increase coincided with an increase in the number of rainfall episodes. After 10 weeks exposure, the entire surface of the specimens was colonized by microorganisms. Subsequently there were only small changes in the color of the exposed surfaces. Evolution of wood graying is depicted in Figure 3.7.  0  1  2  3  Months of exposure 4 5  6  7  8  Colonized area (%)  100 80 60 40 20 0 0  2  4  6  8  10  12  14  16  18  20  22  24  26  28  30  32  Weeks of exposure  Figure 3.6: Area of southern pine wood samples colonized by fungi during 40 weeks of exposure outdoors. Error bars depict standard deviations  70  Figure 3.7: Changes in color and colonized area of southern pine wood samples exposed to weather for 40 weeks in Vancouver, Canada. (a) week 0, (b) week 4, (c) week 8, (d) week 12, (e) week 16, (f) week 20, (g) week 32, (h) week 40  71  The color at the surface of southern pine specimens expressed as lightness, redness– greenness and yellowness–blueness using the CIELab color space system, was measured throughout the 40 week exposure trial. Color measurements were also made on samples that were kept in a dark conditioning room for the duration of the trial. Samples became darker even after one week of exposure, but then their color remained the same until week 8. Afterwards, there was further darkening which coincided with the increase in colonization of samples by fungi. Lightness plateaued after 14 weeks of exposure (Figure 3.8).  0  1  2  3  Months of exposure 4 5 6  7  8  9  10  36  40  80 Control Lightness [L]  70  60  50 Exposure 40 0  4  8  12  16  20  24  28  32  Weeks of exposure Figure 3.8: Changes in lightness of southern pine wood samples exposed to the weather in Vancouver for 40 weeks. Lightness is expressed using the CIELab system, L [100=white; 0=black]. Error bars depicting standard deviations  Redness–greenness of exposed samples is shown in Figure 3.9. Samples became redder over the first 6 weeks of the trial, but thereafter their redness decreased as they became greener. From week 14 to week 24 the redness/greenness of samples remained relatively 72  constant, until week 24, when they became greener ([a] decreased). As with lightness, redness–greenness values showed an inflection point close to week 6 corresponding to pronounced staining of wood by fungi.  0  1  2  3  Months of exposure 4 5 6  7  8  9  10  36  40  Redness / Greenness (a)  12 10 8 6 Control  4 2  Exposure 0 0  4  8  12  16  20  24  28  32  Weeks of exposure Figure 3.9: Changes in redness/greenness of southern pine wood samples exposed to the weather in Vancouver for 40 weeks. Redness/greenness is expressed using the CIELab system, a [+60=red; -60=green]. Error bars depict standard deviations  Yellowness–blueness [b] values of samples during the exposure trial are depicted in Figure 3.10. Changes in [b] are similar to those of redness. Yellowness increased initially reaching a maximum at the end of the first week and then stayed approximately constant until week 4. Thereafter, yellowness of samples decreased until week 14, when it stayed approximately the same for the remainder of the exposure trial. As with the previous color components, [b] showed an inflection point after 6 weeks corresponding to extensive colonization of samples by fungi.  73  Yeellowness/Blueness [b]  0  Months of exposure 4 6  2  8  10  30 Control 20  10 Exposure 0 0  4  8  12  16  20  24  28  32  36  40  Weeks of exposure Figure 3.10: Changes in yellowness/blueness of southern pine wood samples exposed to the weather in Vancouver for 40 weeks. Yellowness/blueness is expressed using the CIELab system, b [+60=yellow; 60=blue]. Error bars depict standard deviations  3.3.5. Moisture content The superficial moisture content of the southern pine wood samples was measured from weeks 10 to 32 of the exposure trial. The moisture content of samples was always below the fiber saturation point (≈ 30% moisture content) and appeared to vary depending on the number and severity of rainfall events (Figure 3.11).  74  1  2  3  4  Months of exposure 5 6 7  8  9  10  30  200  Moisture content 150  24 21  100 Rainfall  18  Rainfall [mm]  Moisture content [%]  27  50 15 12  0 4  8  12  16  20  24  28  32  36  40  Weeks of exposure Figure 3.11: Changes in moisture content of southern pine wood samples exposed outdoors for 40 weeks in Vancouver Canada (data available for week 10 to 32). The figure includes the rain that fell (mm) during the exposure trial. Error bars depict standard deviations  3.3.6. FTIR spectra of samples exposed outdoors FTIR spectra of samples exposed to the weather for 40 weeks and unexposed controls are shown in Figure 3.12.  75  Figure 3.12: FTIR absorbance spectra of southern pine wood surfaces exposed to the weather for 40 weeks and unexposed control. Exposed sample showing decrease of peaks at 1740, 1655, 1514 and 1462 cm-1 related to lignin and little change in peaks at 1158 and 898 cm-1 related to carbohydrates  After exposure, peaks at 1514 and 1462 cm-1 decreased in size in comparison to those in the spectrum of the control. These peaks correspond to stretching vibration of carbonyl groups in lignin benzene rings and C-H deformations in lignin, respectively (Anderson et al. 1991; Pandey and Pitman, 2003). Peaks at 1740 and 1655 cm-1 also decreased during weathering. These peaks correspond to conjugated C-O absorptions which typically increase at early stages of weathering and then decrease after extended exposure (Anderson et al., 1991; Pandey and Pitman, 2003; Williams, 2005). These changes indicate a decrease in the lignin content of samples. Conversely, peaks at 898 and 1158 cm-1, corresponding to C-H stretching and C-O-C stretching in pyranose rings in cellulose and hemicelluloses (Huang et al. 2008), showed little change.  76  3.4. Discussion The isolation and identification of fungi conducted in this Chapter revealed that only fungi belonging to the ascomycota phylum were able to colonize southern pine wood surfaces exposed outdoors and above ground for 40 weeks in Vancouver, Canada. An average of 5 fungal isolates was recovered per sample (fungal richness), but the number of fungal species expected to be found in each sample was estimated at 4 (average reciprocal Simpson index per sample). Neither of these two parameters (fungal richness and reciprocal Simpson index) have been used before to quantify fungal diversity in wood surfaces exposed outdoors. A. pullulans, H. dematioides, Epicoccum nigrum and Phoma sp. were the most frequently isolated fungi and they represented more than 70% of the fungal flora. Therefore, they were the main colonizers of weathered southern pine here and they are probably also responsible for the changes in color of wood during weathering. A. pullulans has been frequently isolated from weathered wood and coatings, as mentioned in Chapter 2 (Seifert 1964; Dickinson 1971; Amburgey 1974; Schmidt and French 1976; Bardage and Bjurman 1998). Physiological studies on A. pullulans have shown that it can metabolize simple sugars and phenolic compounds, which are chemically similar to the photodegradation products of hemicelluloses and lignin, respectively (Bourbonnais and Paice 1987; Schoeman and Dickinson 1996; 1997). Furthermore, A. pullulans is able to synthesize a polysaccharide (pullulan) that allows its blastospores to adhere to wood and enhances its asexual reproduction (Bardage and Bjurman 1998). Also, A. pullulans produces highly melanized mycelia which is a desirable attribute for a microorganism exposed to UV radiation, 77  fluctuating temperatures and high intermitant availability of water at weathered wood surfaces (Fogarty and Tobin 1996; Butler and Day 1998; 2001; Henson, Butler, and Day 1999). Microorganisms with these characteristics would be well adapted to weathered wood surfaces. I isolated two varieties of A. pullulans. One was darkly pigmented while the other was much lighter. Physiological differences between strains of A. pullulans have been reported by Schoeman and Dickinson (1997). They attributed these differences to biological adaptations related to the environments that the strains inhabited. Another fungus, H. dematioides, which is similar morphologically to A. pullulans, has also been isolated from weathered wood surfaces (Held et al., 2006). It is possible that the two species are physiologically similar, which would explain my observation of the frequent isolation of H. dematioides from weathered southern pine samples. Epicoccum nigrum and Phoma sp. have also been found colonizing weathered wood surfaces (Doi and Horisawa, 2001; Hansen, 2008). These fungi have colorless rather than melanized hyphae. Therefore, they must use a different mechanism to that used by A. pullulans and H. dematioides to survive at the surface of weathered wood. According to the literature, E. nigrum is able to produce black sporodochia (spore aggregations). This structure increases the survival of spores exposed to UV radiation (Barnett and Hunter, 1998; Rotem and Aust, 1991). On the other hand, Phoma sp. produces dark structures known as pycnidium. Inside the pycnidium spores are kept safe until released (Barnett and Hunter 1998). Another survival strategy that hyaline fungi might use when colonizing wood exposed outdoors is to grow underneath darker fungi. In weathered wood the dark layer extends to a depth of a few millimeters (Duncan 1963). This dark color is due to the presence of melanized fungi (Dickinson 1971). 78  The melanin concentrated in this layer may absorb part of the harmful UV radiation that reaches the wood surface. Organisms and also the wood itself, below this layer, may be shielded from UV light and hence sub-surface fungi may not need to be highly melanized to survive. Other fungi isolated during this trial were Lecythophora sp., Truncatella angustata, Glonium pusillum and Mollisia minutella. Each of these fungal species was isolated only once. Most of them are recognized pathogens of trees, plants and fruits, and are normally found on wood debris and soil (Sherwood 1973; Crawford et al. 1987; Allmer et al. 2006; Held et al. 2006). Lecythophora sp. has also been reported degrading resin acids from lodgepole pine chips, which may help it to colonize wood surfaces (Wang et al. 1995). Identification of fungi using DNA analysis was particularly valuable because there was little information on some of the fungal species growing in exposed wood surfaces, and also for the identification and separation of H. dematioides sp. and A. pullulans sp. These two species are very difficult to identify and separate using their morphological features (Ray et al. 2004). Some other fungi had distinctive morphological characteristics and were easily recognized under the microscope. Hence, it was not necessary to use DNA analysis to identify them. Identification of fungal species isolated only once was difficult. In such cases, DNA sequencing was essential. Identification of organisms using more complex molecular techniques, for example, sequencing of specific genes, can be very accurate (Tsui et al. 2010), and makes the use of microscopy redundant. Nevertheless, such techniques are costly and consequently they are normally limited to very specific situations. In contrast, in this chapter, DNA identifications were achieved by sequencing only the DNA strand amplified by the primer ITS4. This approach did not decrease the efficiency of the 79  technique, but made it less expensive, since overall costs for sequencing were reduced by fifty percent. Therefore, the use of basic DNA identifications complemented those achieved using microscopy and this combined approach proved to be a suitable and affordable way to identify microorganisms colonizing weathered southern pine wood. Previous research on fungal flora colonizing wood surfaces exposed outdoors in Vancouver has focused on fungi that colonize western red cedar. This wood is widely used for outdoor applications due to his natural durability (Wethern, 1959). Comparison of fungal species isolated here in southern pine with those isolated from western red cedar revealed that certain fungi colonize both wood species. For example, Smith and Swann (1976) isolated and identified fungi colonizing western red cedar shingles exposed for 5 to 28 years outdoors. From a total of 708 isolates approximately 14 different genera were isolated. Philophora and Rhinocladiella were the most frequently isolated genera, but also A. pullulans, and Cladosporium spp., as well as species of basidiomycetes, actinomycetes and bacteria were also frequently isolated. In two other studies Lim et al. (2005; 2007) found a wide range of basidiomycetes and ascomycetes growing on western red cedar decks and fences. They frequently isolated A. pullulans from western red cedar. Studies performed outside Vancouver support the ability of the fungi I isolated to colonize weathered wood and also tolerate diverse climatic conditions. For example, Cronin et al. (2000) isolated and identified fungi responsible for the graying of white cedar (Thuja occidentalis L.) shingles in maritime climates. They isolated fungi directly from wood pieces and identified by microscopy A. pullulans, Alternaria sp., Penicillium sp. and Cladosporium sp. Sudiyani et al. (2002) exposed several tropical wood species outdoors in Indonesia and 80  isolated the moulds colonizing the woods. Identification of organisms was performed by light microscopy. Fourteen different fungal genera were identified in the subphylum ascomycotina, 3 were basidiomycetes and 1 was an actinomycete, but also several organisms were unidentified. Like here, Aureobasidium and Cladosporium were frequently isolated. In the extreme conditions of Antarctica, Held et al. (2006) was able to isolate fungi from 5 different genera. Through microscopy and DNA analysis they were able to identify Cadophora, Cladosporium, Hormonema, Lecythophora and Penicillium species (Held et al., 2006). Three species from these 5 genera were isolated here in southern pine, indicating the ability of these species to withstand adverse climatic conditions. Hence, my results are in partial agreement with those of other studies because the fungi isolated from southern pine wood samples here have been found colonizing a variety of wood substrates exposed outdoors, not only in Vancouver, but also in diverse locations and climates around the world. Differences between my results and those of other studies, e.g. number of genera and species isolated and absence of basidiomycetes, may be attributed to differences in substrates, climate, size and methods for sampling and length of time that samples were exposed to the weather. Sampling method in particular may have influenced the results obtained by several authors in the past. The method of sampling used here was selected according to the target organisms that I was seeking to isolate. For example, in my samples, fungi with the ability to grow in the thin layer of weathered wood were of interest. Therefore an appropriate method of sampling this layer was chosen. Other sampling methods, for example scratching or swabbing the surface may have inflated the number of fungi isolated by other studies because these techniques can isolate fungi (via mycelia and 81  spores) that are opportunistically present at the wood surface, but do not colonize weathered wood. Changes in the color of southern pine wood exposed outdoors appear to be due initially to photodegradation of wood and thereafter to colonization of the surface by fungi. Photodegraded wood surfaces turned red and yellow initially probably because of photooxidation of lignin and the accumulation of unsaturated aromatic compounds in the wood (Feist and Hon, 1984; Gellerstendt and Gierer, 1975). Accordingly, FTIR spectroscopy showed a decrease in the functional groups assigned to lignin (1514 cm -1 stretching vibration of carbonyl groups in benzene rings and 1462 cm-1 C-H deformations in lignin, Anderson et al. 1991; Pandey and Pitman 2003) and a relative increase in the groups assigned to cellulose (898 cm-1 C-H stretching and 1158 cm-1 C-O-C stretching in pyranose rings in cellulose and hemicelluloses, Huang et al. 2008). After 8 weeks of exposure wood surfaces became darker (L decreased). This color change coincided with significant colonization of the weathered surface by fungi. Later, after 14 weeks of exposure, the darkening of the wood surface tended to stabilize, coinciding with the complete staining of the wood surface by fungi. The two main fungi isolated from weathered wood were black, supporting previous suggestions in the literature that the graying of wood exposed outdoors is due to colonization of weathered wood surfaces by fungi. The diversity and types of fungi colonizing wood exposed outdoors must be taken into account when developing treatments to prevent the unwanted graying of wood exposed outdoors. The organisms isolated most frequently here (and by other related studies) should be used in bioassays to test the effectiveness of biocides at preventing the fungal 82  staining of weathered wood. Complementary experiments should be performed to increase our understanding of the effects that ascomycetes fungi have on the properties of wood surfaces. For example, some ascomycetes isolated from wood surfaces are regarded as softrot fungi (Savory 1954; Rajderkar 1966; Bugos et al. 1988; Zabel and Morrell 1992; Lim et al. 2005; Lopez et al. 2007). There have been no studies that have examined in detail whether fungi colonizing weathered wood can cause significant degradation of the wood. Hence, the next chapter (Chapter 4) examines whether the fungi isolated from weathered southern pine wood here are able to cause significant degradation of wood.  83  3.5. Conclusions The combination of molecular techniques and microcopy can complement each other making identification of fungi isolated from weathered wood surfaces faster, more affordable and accurate. Furthermore, identification of fungi (to the level of genus) is possible using these methods without the need for highly trained personnel. Ascomycete fungi dominated the fungal flora isolated from southern pine wood exposed outdoors for 40 weeks in Vancouver, Canada. A. pullulans, H. dematioides, Epicoccum nigrum and Phoma sp. were the fungi most frequently isolated from weathered southern pine wood. It is likely that these microorganisms posses adaptations that enable them to survive at weathered wood surfaces. These adaptations may include high level of melanization, abilities to metabolize wood extractives, sugars and photodegradation product, and appropriate reproductive strategies. Ascomycete fungi colonizing wood surfaces exposed outdoors are responsible for the graying of weathered wood (as other have noted), but color changes at wood surfaces, during the first weeks of outdoor exposure (0 to 8 weeks) involve yellowing and reddening, which is probably due to photodegradation of lignin. Color changes related to fungal colonization became more pronounced after approximately 8 weeks of outdoors exposure outdoor, and complete graying of the surface occurred after 14 weeks exposure. The fungi responsible for such graying are the black fungi that were frequently isolated here, A. pullulans and H. dematioides.  84  4. Chapter 4: Decaying abilities of fungi isolated from weathered wood 4.1. Introduction Fungi colonizing weathered wood surfaces include a broad spectrum of micro-organisms, but wood decaying basidiomycetes do not seem to predominate (Duncan, 1963; Seifert, 1964; Sell and Wälchli, 1969; Dickinson, 1971; Feist, 1990). The fungi colonizing weathered wood disfigure the wood to a depth of a few millimeters (Duncan 1963; Dickinson 1971; Savory 1973), but there is a body of opinion that suggests that they are unable to degrade the wood (Feist 1983). This opinion is underpinned by studies which have failed to detect soft-rot cavities in the walls of tracheids at weathered wood surfaces (Evans, 1989; Paajanen, 1994) and the fact that environmental conditions at weathered wood surfaces are generally unfavorable for microbial degradation (Evans 2008). However, Smith and Swann (1976) have a different opinion. Their histological studies on weathered western red cedar shingles found evidence of soft-rot cavities and enzymatic erosion of wood cell walls. Furthermore, cellulolytic and lignolytic fungi that have the ability to produce soft-rot decay are frequent colonizers of weathered wood (Savory 1954; Rajderkar 1966; Bugos et al. 1988; Zabel and Morrell 1992; Lim et al. 2005). Therefore, it seems reasonable to assume that under certain circumstances, fungal degradation of wood surfaces (particularly the occurrence of soft-rot decay) may occur when wood weathers. In addition, such degradation might be enhanced by the photo-induced delignification of wood surfaces as suggested by Evans and Banks (1986).  85  The techniques used to assess soft-rot decay such as microscopy and measurement of weight loss are not very good at detecting the early stages of soft-rot. In contrast, measurement of wood strength losses is far more sensitive to early decay (Wilcox, 1978; Morrell and Zabel, 1985; Sexton et al., 1993; Nicholas and Jin, 1996). In this chapter, I hypothesize that some of the fungi isolated from weathered wood will be able to degrade wood tissues and such degradation will lead to losses in the mechanical properties of wood. To test this hypothesis a range of fungi isolated from weathered wood surfaces (in Chapter 3) were screened for their ability to produce cellulolytic and lignolytic enzymes. Then, they were used in a bioassay, which measured changes in mechanical properties of wood exposed to the different fungi. In addition other techniques including dynamic mechanical analysis, FTIR spectroscopy and light and scanning electron microscopy were used to examine whether fungi were able to break down the wood, and identify the type of degradation caused by the fungi (if any).  86  4.2. Materials and methods 4.2.1. Fungal screening Fungi isolated from weathered wood in Chapter 3 were screened for their ability to synthesize lignolytic and cellulolytic enzymes in-vitro (Table 4.1). Laccase producing organisms were indentified by their ability to breakdown the aromatic compound guaiacol, a widely used lignin model (Kiiskinen et al. 2004). When fungi are inoculated on solid media containing guaiacol, fungi able to produce laccase form reddish-brown halos around their mycelia, as their lignollytic enzymes breakdown the guiacol (Figure 4.1a) (Kiiskinen et al. 2004). Five mm (diameter) agar plugs from different cultures of surface fungi were transferred onto 150 mm x 15 mm Petri dishes with solid media containing: peptone (3 g/l), glucose (10 g/l), KH2PO4 (0.6 g/l), ZnSO4 (0.001 g/l), K2HPO4 (0.4 g/l), FeSO4 (0.0005 g/l), MnSO4 (0.05 g/l), MgSO4 (0.5 g/l), agar (20 g/l) and guaicol (0.2 g/l) (Viswanath et al. 2008). The enzymatic activity after one week of growth was ranked visually according to the intensity and extension of the reddish-brown halos as follows: (1) negative (-); (2) low (+); (3) medium (++); and (4) high (+++). On the other hand, the ability of surface fungi to produce cellulolytic enzymes was tested using a carboxymethyl cellulose (CMC) assay (Peciulyte 2007). In this CMC assay, fungi are grown on solid media containing CMC as a sole source of carbon. During this assay cellulolytic enzymes break down the CMC. The enzymatic reaction can be visualized by adding Congo red dye to the growth medium. Congo red strongly bonds to contiguous β-(1-4)-bound-D-glucopyranosyl units (Sazci et al. 1986). At the end of the bioassay Congo red is removed from the medium using a solution  87  of 1M NaCl, but yellower halos remain in areas where cellulolytic enzymes were active. Enzymatic activity is quantified using an index for enzyme activity for CMC (Icmc), as follows: Icmc = Clear or yellower halo diameter/Fungi colony diameter (Peciulyte 2007). Specifically in my experiment 5 mm (diameter) agar plugs from the original cultures were transferred onto 150 mm x 15 mm Petri dish with solid media containing: NH4NO3 (1.6g/L), Na2HPO4 (0.5g/L), K2HPO4 (0.65 g/L), MgSO4.7H2O (3 g/L), CaCl2.2H2O (0.4 g/L), yeast extract (0.3 g/L), Triton X100 (0.1 g/L), agar (15 g/L) and CMC (10 g/L). After a period of incubation for 14 days, cultures were flooded with Congo red dye (1% aqueous solution) and 1M NaCl for 15 and 20 minutes, respectively. Diameter of fungi colonies and clear halos were calculated using image analysis of digital pictures. Digital images of the fungal colonies on each plate, 1:1 scale; under standard conditions of illumination were obtained using a desktop scanner (Microtek Scan Maker i800). The diameter of each hyphal mat and clear halos were digitally measured with the ruler tool of the software Adobe Photoshop CS3 Extended, version 10.0.1 (Adobe System Incorporated, USA), Figure 4.1b. Fungi showing strong enzymatic activity and those most frequently isolated from weathered wood were selected for subsequent experimentation. White-rot and brown-rot decay fungi, and a known soft-rot fungus were used as controls.  88  Figure 4.1: Fungal screening: (a) Trichaptum abietinum after seven days of growth on media containing guiacol (0.2 g/L), the enzymatic activity of the fungus was ranked as high (+++); (b) carboxymethyl cellulose (CMC) assay; measurement of halo diameter using the ruler tool of Photoshop. The fungus in the image is Lecythophora sp. after 14 days of growth in media containing CMC 10 (g/L) stained with Congo red  89  Table 4.1: Fungi tested for their ability to synthesize lignolytic and cellulolytic enzymes No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28  Fungi Allantophomopsis lycopodina (Höhn.) Carris Alternaria sp. Aureobasidium pullulans (de Bary) G. Arnaud (black) Aureobasidium pullulans (de Bary) G. Arnaud (white) Botryosphaeria stevensii Shoemaker Botryotinia fuckeliana (de Bary) Whetzel Cladosporium cladosporioides (Fresen.) G.A. de Vries Cladosporium sp. Coniochaeta ligniaria (Grev.) Massee Epicoccum nigrum Link Epicoccum sp. Glonium pusillum H. Zogg Hormonema dematioides (Lagerb. & Melin) Lecythophora sp. Leptosphaerulina chartarum Cec. Roux Lewia infectoria (Fuckel) M.E. Barr & E.G. Simmons Mollisia minutella (Sacc.) Rehm Penicillium expansum Link ex. Thom Peniophora aurantiaca (Bresadola) von Höhnel & Litschaue Phialocephala sp. Phialophora sp. Phoma sp. Rhizopogon sp. Trichoderma viride Pers. Truncatella angustata (Pers.) S. Hughes Valsa ambiens (Pers.) Fr. Trichaptum abietinum (Pers.) Ryvarden (white-rot control) Coniophora puteana (Schum. ex Fries) Karst. (brown-rot control)  Strain(s) tested 1 5 6 6 1 4 5 1 2 5 1 1 7 2 1 2 1 1 1 1 2 5 1 1 1 1 1 1  4.2.2. Decay test 4.2.2.1. Experimental design An experiment was designed to test the effect of fungi isolated from weathered wood on the tensile properties of two wood species. Twelve ‘blocks’ provided replication at the higher level. Each block included 18 treatments (17 fungi plus a control), which were randomly assigned to 18 Petri dishes. The internal area of each Petri dish was subdivided into two; a hardwood (lime, Tilia vulgaris Hayne) and a softwood (White spruce, Picea 90  glauca, Moench (Voss)) were randomly assigned to the two sectors within each dish. The resulting split-plot design accounted for random variation in fungal growth and wood properties. Analysis of variance (ANOVA) was used to examine the effect of fungal species and wood species and the interactions of these factors on the mechanical properties of thin wood veneers (see below). The analysis of data was performed using the software Genstat v. 12 (VSN International 2009). The assumptions of ANOVA were tested prior to the final analysis (normality of residuals and homogeneity of variances). After ANOVA (p<0.05), significant differences were estimated using Fisher’s least significant test (l.s.d.). Results are presented in graphs featuring means and either standard error of the differences (s.e.d.) or l.s.d bars for the different tested parameters. The detailed output of the statistical analyses in this chapter is appended to this thesis (Appendix 1). A summary of the experimental design is presented in Table 4.2. Table 4.2: Summary of the experimental design used for the decay test  Blocks Fungal species 1 17 + control . . . . . . . . . . 12 17 + control  Wood species 2 . . . . . 2  Petri dishes 18 . . . . . 18  4.2.2.2. Wood samples Two non-durable wood species were used as test substrates for the bioassay. White spruce was selected because of its susceptibility to fungal degradation and homogeneous properties (Forest Products Laboratory 1999). Lime wood was selected because previous 91  work demonstrated that thin wood veneers from this wood species can be successfully used to detect degradation of fungi when tested in tension (Evans and Banks 1986). Wood veneers were cut from white spruce and lime using the method described by Evans (1988). Blocks measuring 18 mm (radial) x 25 mm (tangential) x 85 mm (longitudinal) were cut from five different lime and white spruce boards. These blocks were soaked in distilled water for 5 days. Individual blocks were firmly clamped in a custom-made sample holder attached to a sliding microtome (Spencer Lens Co. Buffalo, USA; Figure 4.3a) with the radial face uppermost. Eighty micrometers (80 μm) veneers were cut from each block using a disposable stainless steel blade (Type S35, Feather Safety Razor Co., Japan) mounted in a blade holder. Veneers were placed on glass plates and clamped at their ends using strips of  Perspex and butterfly clips. Restrained veneers were air dried in a conditioning room at 20°C ± 1°C at 65% ± 5% r.h. for seven days. Each veneer was labeled using a pencil and their thickness and weights were measured with a digital micrometer (Lorentz & Wettre HWS 5781) and an analytical balance A & D (Model GR-200 from B.C. Scale Co. Ltd; 210 g x 0.0001 g), respectively. Veneers were then oven dried (100 ± 5°C) for 24 hours to a constant weight (as above) and sterilized in autoclave at 121°C and 103.4 kPa for 20 min. Veneers were rehydrated by soaking them in nano-pure sterile water under sterile conditions. The effect of fungi on the microstructure of wood used small lime and white spruce samples. These samples measured 35 mm (longitudinal) x 12 mm (radial) x 2.5 mm (tangential), and were cut and planed from parent boards and then labeled with pencil. They were then conditioned for 14 days, oven dried until they reached constant weight,  92  sterilized in an autoclave, and re-hydrated with nano-pure water under sterile conditions (as above).  4.2.2.3. Fungal inoculation and incubation Black colored and control fungi were tested for their ability to breakdown wood veneers and solid wood samples. Three or two or sometimes one isolate were used per treatment. Not all of the test fungi were able to produce spores on solid media. Therefore, fungi were inoculated from aqueous solutions containing a known and standard concentration of fungal mycelia. To obtain such solutions 1% w/v malt extract agar (MEA) – Difco Petri dishes, overlaid with a layer of cellophane were inoculated with five agar plugs (5 mm in diameter) from original fungal cultures. After two weeks when fungi had completely covered the cellophane layer the fungal mycelia was collected in 1.5 mL screw-cap tubes using a sterile scalpel. Then, 500 μL of nano-pure water was added to the tube and mycelia were crushed using a sterile stainless steel rod and the solution was stirred for 3 minutes at 100 rpm. Crushed mycelia was then transferred to 50 mL falcon tubes and diluted with nano-pure water until a total volume of 40 mL was obtained. The dry weight of fungal mycelia in 3 mL of solution was used to estimate fungal biomass per mL. Later, fungal biomass concentrations were adjusted to 2.13 x 10-4 g/mL. Petri dishes (150 mm x 15 mm) with 1% MEA and cellophane were then inoculated with 1000 μL of fungal solutions. The inoculum was evenly spread over the cellophane using a glass rod. Inoculated cultures were left for approximately 48 hours until clear signs of new mycelial growth was noted. Then the cellophane sheets were transferred onto new plates containing the following mineral media 93  designed to encourage soft-rot fungal decay: NH4NH3 (6 g/L), K2HPO4 (4 g/L), KH2PO4 (5 g/L), MgSO4.7H2O (4 g/L), thiamine HCl (0.02 g/L) and agar (15 g/L) (Leightley 1980). Wood veneers and solid wood samples were allocated to segments inside the Petri dishes, as mentioned above. The dishes were then sealed using plastic foil (The Glad Company, USA) and incubated for 12 weeks under sterile conditions at 20°C in dark room, Figure 4.2.  4.2.2.4. Mechanical property losses of veneers All veneers exposed to fungi for 12 weeks were conditioned (as above) for 14 days. Tensile strength (ability to resist an applied stress in tension) tests were carried out using an Instron Universal Tension Tester (model 5565, Figure 4.3b) using 20 mm/min cross-head speed and 38.1 mm span-length. Data collected from each test were used to plot stress-strains curves for each veneer (see Appendix 2). Stress (amount of force for a given area unit) and strain (deformation per unit of the original length) were calculated as follows (Bodig, 1982):  Stress = force applied / Area tested Strain = displacement / original length  Stress-strains curves on graphs were used to determine: (1) peak tensile stress (PTS, maximum tensile stress value) and (2) modulus of elasticity (MOE, slope of the curve). PTS and MOE were used to calculate the peak work done, which is equivalent to the maximum toughness (ability of the material to absorb and distribute energy within itself) of the  94  samples (PWD, peak toughness), and peak stiffness (PS, maximum stiffness), as follows (Bodig, 1982):  PWD = PTS2 / (2 x MOE) PS = Peak force applied / peak displacement  Mechanical property losses results of veneers are expressed as the ratio of matched controls.  4.2.2.5. Fourier transform infra-red spectroscopy Fourier transform infra-red spectroscopy was used to examine chemical changes at the surface of wood veneers exposed to fungi. A small piece of veneer measuring 10 mm (tangential) x 10 mm (longitudinal) was cut from the parent veneer using scissors. Pieces of veneers were stored for 5 days in a vacuum desiccator over silica gel. Direct reflectance (ATR-IR) FTIR spectra of veneers surfaces were obtained using a single bounce attenuated total reflectance accessory, as described in Chapter 3 (section 3.2.7).  4.2.2.6. Viscoelastic properties The viscoelastic properties of solid wood samples were quantified because of their sensitivity to small polymeric changes such as those produced by enzymatic fungal degradation. The dynamic elastic response or storage modulus (SM) of solid wood samples 95  exposed to fungi that caused the greatest losses in tensile strength was measured. Solid wood samples measuring 35 mm (longitudinal) x 12 mm (radial) x 2.5 mm (tangential) were reduced in size to 1 (tangential) x 3 (radial) x 25 (longitudinal) mm and tested in a dynamic mechanical analyser (DMA, Perkin Elmer model DMA 7e, Figure 4.3c). The test was performed as follows: (1) double cantilever bending geometry; (2) 20 mm span-length; (3) temperature range of 25 to 200°C with a heating rate of 5°C/min; (4) frequency 1Hz; and (5) ratio static/dynamic charge 550/500 mN.  4.2.2.7. Microscopy The microstructure of solid wood samples exposed to fungi was examined using light microscopy. Pieces of wood measuring 10 mm (radial) x 2.5 mm (tangential) x 10 mm (longitudinal) mm were cut from the surface of exposed lime and spruce specimens and soaked in distilled water for 2 days. Each water-saturated block was clamped in a microtome (as above) and 20 µm sections were cut from the block using a disposable stainless steel blade (Type S35, Feather Safety Razor Co., Japan) bolted to a microtome blade-  holder. Sections were dehydrated in ethanol (industrial grade) for 2 days and then soaked in a saturated solution of safranin (BDH Chemical Ltd, England) in ethanol for 2 days. Each stained section was placed on a droplet of DPX (dibutyl phthalate xylene) mountant (Fluka Analytical, Germany) on a glass slide measuring 76 mm x 26 mm x 1 mm (Matsunami Glass Ind. Ltd. Japan), covered with a glass cover slip, 22 mm x 40 mm x 0.20 mm in size (Fisher Finest Premium Cover Glass, Fisher Scientific, Pittsburgh, USA). Slides were dried at room temperature for 48 hours. The sections were examined using a light microscope (Carl Zeiss, 96  Germany) at various magnifications. An Olympus DP71 digital camera attached to the microscope was used to take photographs of fungi colonizing wood. Scanning electron microscopy (SEM) was used to examine structural changes in veneers exposed to fungi. A small piece of veneer measuring 5 (radial) mm x 5 (longitudinal) mm, was cut from the parent veneer using scissors and glued to aluminum stubs using nylon nail polish as an adhesive. The stubs containing the veneers were stored for 5 days in a vacuum desiccator over silica gel. The stubs were coated with a 10 nm layer of gold using a sputter coater (Nanotech SEMPrep II) and then examined using a Zeiss Ultraplus field emission scanning electron microscope at an accelerating voltage of 5 kV. Secondary electron images of veneers were obtained and saved as TIFF files.  97  Figure 4.2: Wood samples after 1 week of exposure to fungi: (a) solid wood samples; (b) wood veneers  Figure 4.3: Equipment for sample preparation and testing; (a) sliding microtome with blade holder and clamping device for wood samples; (b) Instron Universal tensile tester (model 5565) and; (c) Dynamic mechanical analyzer (Perkin Elmer model DMA 7e)  98  4.3. Results 4.3.1. Fungal screening The results for lacasse activity and index for enzyme activity of fungi on CMC are shown in Table 4.3. Five fungi showed lignolytic activity, while 24 out of 28 exhibited cellulolytic activity on CMC. The enzymatic activity of the fungi and their frequency of isolation on weathered wood (Chapter 3) and in other studies were used as criteria to select fungi for the decay test described below. Selected organisms are shown in Table 4.4.  99  Table 4.3: Laccase activity and index for enzymatic activity for carboxymethyl cellulose (CMC) No  Fungi  Strains tested  Laccase activity† after 12 days  Icmc* after 7 days  1 2  Mollisia minutella Rhizopogon sp.  1 1  +++ -  5.00 5.00  3 4  Coniophora puteana (brown-rot control) Phialophora sp.  1 2  -  5.00 2.43-2.59  5 6  Coniochaeta ligniaria Lecythophora sp.  2 2  +++  2.23-2.55 2.15-2.22  7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22  Penicillium expansum Valsa ambiens Botryosphaeria stevensii Aureobasidium pullulans (white) Aureobasidium pullulans (black) Cladosporium cladosporioides Botryotinia fuckeliana Phoma sp. Lewia infectoria Glonium pusillum Peniophora aurantiaca Cladosporium sp. Epicoccum nigrum Epicoccum sp. Leptosphaerulina chartarum Alternaria sp.  1 1 1 6 6 5 4 5 2 1 1 1 5 1 1 5  +++ -  2.04 1.96 1.82 1.62-2.84 1.56-1.94 1.51 1.5-1.94 1.44-1.99 1.38-1.48 1.33 1.29 1.31-1.63 1.26-1.3 1.21 1.21 1.17-1.25  23 24 25 26 27  Truncatella angustata Trichoderma viride Allantophomopsis lycopodina Hormonema dematioides Phialocephala sp.  1 1 1 7 1  +++  1.15 1.01 0.00 0.00 0.00  28  Trichaptum abietinum (white-rot control)  1  +++  0.00  †Rank of enzymatic activity: negative (-); low (+); medium (++); high (+++) *Icmc: index for range of enzyme activity on carboxymethyl cellulose  100  Table 4.4: Fungi isolated from weathered wood and tested for their ability to breakdown wood Treatment  Fungi  Code Name  Strains tested  1 2 3 4 5 6 7  Alternaria sp. A. pullulans (black) A. pullulans (white) B. fuckeliana Cladosporium sp. C. ligniaria E. nigrum  Alt. Aur. (black) Aur. (white) Botr. Clad. Conio. Epic.  3 3 3 3 3 2 3  8 9  H. dematioides Lecythophora sp.  Horm. Lecyth.  3 1  10 11 12 13 14 15 16 17  L. infectoria M. minutella Phialocephala sp. Phialophora sp. Phoma sp. T. abietinum (white-rot control) C. puteana (brown-rot control) Chaetomium globosum Kunze ex Fr. (soft-rot control)  Lew. Moll. Phialoc. Phialop. Phom. Trich. Coniop. Chaet.  3 1 1 2 3 1 1 1  4.3.2. Decay test 4.3.2.1. Mechanical property losses of veneers Analysis of variance showed a significant effect (P-value < 0.001) of fungal species (F), wood species (W) and interaction of FxW, on the different mechanical properties of spruce and lime veneers tested in tension (peak tensile stress ratio, modulus of elasticity (MOE) ratio, peak stiffness ratio, and toughness ratio). Table 4.5 shows the statistical significance (Pvalues) of experimental variables (fungi, wood species) and