"Forestry, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Hernandez, Vicente"@en . "2012-09-28T18:29:59Z"@en . "2012"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "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. UV-radiation 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."@en . "https://circle.library.ubc.ca/rest/handle/2429/43298?expand=metadata"@en . "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 \u00C2\u00A9 Vicente Hernandez, 2012 ii 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. UV- radiation 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. iii Preface Elements of Chapter 5 were presented at the IRG-Americas Regional Meeting; Guanacaste, Costa Rica; 2008, under the title: \u00E2\u0080\u009CThe effects of solar radiation on the fungal colonization and color of weathered wood\u00E2\u0080\u009D. 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; \u00E2\u0080\u009CThe effects of solar radiation on the fungal colonization and color of weathered wood\u00E2\u0080\u009D; IRG-Americas Regional Meeting; Guanacaste, Costa Rica; IRG/WP 08-10676. iv 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 1.2. General Hypothesis ................................................................................................... 3 1.3. Scope and importance .............................................................................................. 5 1.4. Study outline ............................................................................................................. 6 2. 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 v 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. Summary................................................................................................................. 49 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. 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 4. 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 vi 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 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 5. 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 vii 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 6. Chapter 6: Effect of UV radiation on melanization and growth of fungi isolated from weathered wood surfaces ............................................................................................. 192 6.1. Introduction .......................................................................................................... 192 6.2. Materials and methods ......................................................................................... 194 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. 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 7. 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 viii 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. 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 8. 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 ix 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 x 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\u00E2\u0080\u0099s 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 xi 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 xii Table 7.4: Significant effect of, and interaction between exposure to light, chemical treatments and concentration on stained area and color change (\u00CE\u0094E) 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 xiii 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\u00E2\u0080\u0099-bromo-2\u00E2\u0080\u0099,6\u00E2\u0080\u0099- dimethylphenoxy]methyl-4-[(3\u00E2\u0080\u009D-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 xiv 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 xv 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 \u00C2\u00B1SED ........................................................................................................................................... 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 \u00C2\u00B1SED ................................................................................. 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 \u00C2\u00B1SED............................................................................................ 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 \u00C2\u00B1SED ..... 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 xvi ratio close to one indicates that peak stiffness was similar to that of sound wood. Error bars correspond to \u00C2\u00B1SED ............................................................................................................ 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 \u00C2\u00B1SED............................................................................................ 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 \u00C2\u00B1SED ....................................................... 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 \u00C2\u00B1SED............................................................................................ 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 xvii 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 xviii 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 xix 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 xx 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 xxi 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 xxii 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 \u00E2\u0080\u0093 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 1,3,6,8- tetrahydroxynaphthalene (1,3,6,8-THN) to scytalone and 1,3,8-trihydroxynaphthalene (!,3,8- THN) 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 \u00C2\u00B5L of spore solution (1 cell/\u00C2\u00B5L) .............. 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) \u00E2\u0080\u0098curves\u00E2\u0080\u0099 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 xxiii 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 \u00C2\u00B1SED ................................................................................................................................... 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 \u00C2\u00B1SED.................................................................. 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 \u00C2\u00B1SED ..................... 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 xxiv 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 \u00C2\u00B1SED ........................................................................................................................................... 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 (\u00CE\u0094E) 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 \u00C2\u00B1SED ........................................................................................................................................... 244 Figure 7.19: Effects of chemical treatment on color differences (\u00CE\u0094E) 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 \u00C2\u00B1SED .................................................................................................... 245 xxv Figure 7.20: Effects of chemical treatment on color differences (\u00CE\u0094E) 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 \u00C2\u00B1SED ........................................................................ 246 Figure 7.21: Effects of chemical treatments and concentrations on color differences (\u00CE\u0094E) 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 xxvi 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 xxvii 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 \u00E2\u0080\u0098Natural Sciences and Engineering Research Council of Canada\u00E2\u0080\u0099 and the program \u00E2\u0080\u0098Becas Chile\u00E2\u0080\u0099 for their financial support. To technicians, graduate and co-op students in Dr Evans and Dr Breuil\u00E2\u0080\u0099s 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\u00E2\u0080\u0099m sure I would have not been able to reach this point without your unconditional love. To all of you thank you so much! xxviii Dedication To William \u00E2\u0080\u009CBill\u00E2\u0080\u009D New Thanks for sharing your knowledge, wisdom and your sincere friendship with me 1 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\u00E2\u0080\u0099s 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 \u00E2\u0080\u0098weathering\u00E2\u0080\u0099 (Feist, 1983). Weathering can be defined as \u00E2\u0080\u0098surface 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)\u00E2\u0080\u0099 (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 2 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 W\u00C3\u00A4lchli, 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 3 amine light stabilizers are commonly added to finishes, such as varnishes, stains and water- repellents (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 4 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 W\u00C3\u00A4lchli, 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: \u00E2\u0080\u009CThe graying of wood exposed outdoors is due to the presence of melanized fungi with relatively high resistance to UV light\u00E2\u0080\u009D. 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). 5 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 $6.2 billion per annum by 2014 in USA (Freedonia Group, 2011). 6 However, statistics show that one third of the decks installed in the USA are replaced after only a few years of service due to weathering of exposed wood surfaces (Amburgey and Ragon, 2008). The cost of replacing such decks could be in excess of US$ 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 7 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. 8 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 9 Feist (1990) described the changes that occur when wood is weathered as follows: \u00E2\u0080\u009CDuring 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\u00E2\u0080\u009D. 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\u00E2\u0080\u0099s 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, 10 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\u00E2\u0080\u0099s 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 11 it \u00E2\u0080\u0098extends photodegradation into wood beyond the zones affected by UV radiation\u00E2\u0080\u0099 (Kataoka et al. 2007). Lignin is the most sensitive of wood\u00E2\u0080\u0099s 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: \u00E2\u0080\u0098Lignin, which is an amorphous phenolic polymer, is rich in chromophoric groups that strongly absorb UV light\u00E2\u0080\u0099 (Hon 1979). \u00E2\u0080\u0098These groups, including phenolic, double bonds, carbonyls, quinones, quinomethides and biphenyls (Hon 1979), readily interact with UV light to form free radicals\u00E2\u0080\u0099. \u00E2\u0080\u0098These radicals react with molecular oxygen to form new radicals such as peroxides, hydroperoxides, peroxyl and alkoxyl radicals\u00E2\u0080\u0099 (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 \u00E2\u0080\u0098these free radicals are transformed into quinoid structures which accumulate at wood surfaces causing the first color changes during weathering\u00E2\u0080\u0099 (George et al., 2005). Cellulose seems to be more resistant to weathering than lignin as it is only 12 sensitive to wavelengths shorter than 280 nm, and the ozone layer prevents such radiation from reaching the earth\u00E2\u0080\u0099s 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 13 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 14 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 \u00C2\u00B5m 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 \u00CE\u00BCm. 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 \u00CE\u00BCm. 15 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 \u00E2\u0080\u0098biofilm\u00E2\u0080\u0099 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. \u00E2\u0080\u0098A lichen is an association of a fungus and a photosynthetic symbiont resulting in a stable thallus of specific structure\u00E2\u0080\u0099 (Hawksworth and Hill 1984). Around one in five of all known fungi can be \u00E2\u0080\u0098lichenized\u00E2\u0080\u0099, 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 16 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 17 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. 18 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\u00E2\u0080\u0099s 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). 19 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 sub- categories according to the mode of degradation of woody tissues: (1) brown-rot; (2) white- rot; 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. Kingdom Eumycota Phylum Chytridiomycota Phylum Zygomycota Phylum Dykariomycota Sub-Phylum Ascomycotina Sub-Phylum Basidiomycotina Class Ascomycetes Class Saccharomycetes Class Holobasidiomycetes Class Phragmobasidiomycetes Class Teliomycetes 20 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; 21 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). 22 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 M\u00C3\u00B6bius (1924). Both authors described the presence of fungi in wood, but only M\u00C3\u00B6bius attributed the graying of wood surfaces to the presence of fungi. Before M\u00C3\u00B6bius (1924) it was thought that weathered wood became gray due to the accumulation of dirt. Subsequent microscopic studies confirmed M\u00C3\u00B6bius\u00E2\u0080\u0099s 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 carbon- containing 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, 23 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 W\u00C3\u00A4lchli (1969) isolated A. pullulans, Macrosporium sp., Tetracoccosporium sp., Cladosporium sp. and Sclerophoma sp. from weathered wood in the late 1960\u00E2\u0080\u0099s. 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 24 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, 25 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. 26 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 A. pullulans Sell and Walchli (1969); ?; ? Dickinson (1971); Scots pine, WRC; England Lim et al. (2005; 2007); WRC; Vancouver-Canada 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 Lim et al. (2005; 2007); WRC; Vancouver-Canada Acremonium sp. Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Alternaria spp. 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. Doi and Horisawa (2001); sugi; Japan Aspergillus spp. Amburgey (1974); asphalt shingles (wood based); USA Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Brachysporiella sp. Sudayani et al. (2002); Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Candophora sp. Held et al. (2006); ?; Antarctica Cladosporium spp. Sell and Walchli (1969); ?; ? Dickinson (1971); Scots pine, WRC; England Held et al. (2006); ?; Antarctica 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 Curvularia spp. Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Amburgey (1974); asphalt shingles (wood based); USA Doi and Horisawa (2001); sugi; Japan Hansen (2008); ?; Brazil 27 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 Fusarium spp. Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Amburgey (1974); asphalt shingles (wood-base); USA Hansen (2008); ?; Brazil Fusicladium sp. Amburgey (1974); asphalt shingles (wood-base); USA Geotrichum sp. Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Gliomastix sp. 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 Monilia sp. Sudayani et al. (2002); Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Amburgey (1974); asphalt shingles (wood-base); USA 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. Doi and Horisawa (2001); sugi; Japan Sudayani et al. (2002); Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Nigrospora spp. Doi and Horisawa (2001); sugi; Japan Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Hansen (2008); ?; USA Oidiodendron spp. Smith and Swann (1976); WRC; USA, Vancouver Canada Lim et al. (2005; 2007); WRC; Vancouver-Canada Pachnocybe ferruginea Lim et al. (2005; 2007); WRC; Vancouver-Canada Paecilomynes sp. Sudiyani et al. 2002; Albizia, kapur, Mahoni, Nangka, Puspa; Indonesia Penicillium spp. 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 28 Isolate Author; substrate; country Pestalotia sp. Doi and Horisawa (2001); sugi; Japan Phellinus ferreus Lim et al. (2005; 2007); WRC; Vancouver-Canada Phialocephala spp. Lim et al. (2005; 2007); WRC; Vancouver-Canada 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 29 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 3- 4% 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 \u00CE\u00B1-cellulose and 30 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). 31 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 32 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 1,3- dimethylol-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\u00E2\u0080\u0099s surface depends on the output from the sun and the properties of earth\u00E2\u0080\u0099s 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 33 wavelengths, between 260 \u00E2\u0080\u0093 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. 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) \u00E2\u0080\u0098Photoreactivation repair: this mechanism makes possible the repair UV photons absorbed No chemical reaction Photochemical reaction Biologically relevant Potentially biologically irrelevant Obligatory biologically relevant No inactivation due to repair or bypass No biological effect No lethal effect (mutation, growth delay, etc.) Inactivation ? ? Secondary alterations Secondary alterations 34 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\u00E2\u0080\u0099. (2) \u00E2\u0080\u0098Excision 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)\u00E2\u0080\u0099. (3) \u00E2\u0080\u0098Post-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\u00E2\u0080\u0099. (4) \u00E2\u0080\u0098SOS 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\u00E2\u0080\u0099. \u00E2\u0080\u0098This 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\u00E2\u0080\u0099 (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 35 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. 36 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 \u00E2\u0080\u0098secondary metabolism\u00E2\u0080\u0099 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 37 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, \u00CE\u00B3-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, \u00CE\u00B3-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 38 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), \u00CE\u00B3-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). 39 Figure 2.5: Precursors of fungal melanins 40 The biosynthesis of GHB melanins was described for Agaricus bisporus by Fogarty and Tobin (1996): \u00E2\u0080\u009CGHB 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 \u00CE\u00B3-glutaminyl-3,4-benzoquinone (GBQ) and quinoid products of GBQ. The \u00CE\u00B3-glutaminyl moiety of GHB may be removed prior to polymerization by a \u00CE\u00B3-glutaminytransferase present in the fruiting body. The \u00CE\u00B3-glutaminyl residue may thus be transferred to a receptor, liberating 4-aminiphenol (or 4- aminocathechol if the \u00CE\u00B3-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\u00E2\u0080\u009D. 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 41 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; Romero- Martinez 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\u00E2\u0080\u0099s 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). 42 Figure 2.6: DHN melanin biosynthesis 43 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\u00E2\u0080\u0099s 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). 44 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\u00E2\u0080\u0099-bromo-2\u00E2\u0080\u0099,6\u00E2\u0080\u0099-dimethylphenoxy]methyl-4-[(3\u00E2\u0080\u009D-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). 45 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\u00E2\u0080\u0099-bromo-2\u00E2\u0080\u0099,6\u00E2\u0080\u0099-dimethylphenoxy]methyl-4-[(3\u00E2\u0080\u009D- 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). 46 Table 2.2: Melanin biosynthesis inhibitors of reductase registered in Japan in 2001 Chemical group Name Chemical structure Poly chlorinated aromatic compounds PCBA PCMN CPA Fthalide Fused heterocyclic compounds Tricyclazole Pyroquilon Chlobenthiazole PP 389 Phthaladine 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 Cl Cl Cl Cl Cl OH Cl Cl Cl Cl Cl OH CN Cl Cl Cl Cl Cl O CH3 O Cl Cl Cl Cl O O CH3 N S N N N O N S Cl O CH3 N N O CH3 N N N N N Cl CH3 47 (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 \u00E2\u0080\u0098shunt\u00E2\u0080\u0099 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 \u00CE\u00BCg/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). 48 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 Carboxamide derivatives Carpropamid (CAR) Dichlocymet (DCM) Fenoxanil BFS Cyclobutane carboxamid 4-aminoquinazolin dereviates Cl CH3 NH O Cl Cl CH3 CH3 Cl CH3 NH OCl NH2 But Cl O Cl CH3 O NH CH3 CN Prl Cl CH3 NH O OH F Br NH OCH3 F FF CH3 Cl NN NH N NN NH 49 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\u00E2\u0080\u0099s 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 50 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. 51 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 (M\u00C3\u00B6bius, 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 data- bases for the identification to be accurate (Dismukes et al. 2003). Nevertheless, I hypothesize here that the combination of both molecular techniques and microscopy will 52 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\u00C2\u00B0 43\u00E2\u0080\u0099 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. 53 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 \u00C2\u00B1 1 \u00C2\u00B0C and 65 \u00C2\u00B1 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 \u00E2\u0089\u0088 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 RDM\u00C2\u00B3, Delmhorst Instrument Company). Monthly weather conditions for the exposure period are shown in Table 3.2. 54 Table 3.1: Density and growth rate of southern pine samples Board/Block Growth [rings/cm] Basic density [g/cm3] 1 8.28 0.429 2 5.14 0.432 3 4.57 0.560 4 4.50 0.505 5 5.42 0.452 Table 3.2: Monthly weather conditions during the exposure period in Vancouver, Canada; reported by Canada\u00E2\u0080\u0099s National Weather Archive Year Month Mean max. temp. [\u00C2\u00B0C] Mean temp. [\u00C2\u00B0C] Mean min. temp. [\u00C2\u00B0C] Extrem. max. [\u00C2\u00B0C] Extrem. min. [\u00C2\u00B0C] Total rain [mm] Total snow [mm] Total precip. [mm] 2007 Aug 21.9 17.8 13.6 26.7 11.3 8.4 0.0 8.4 2007 Sept 17.6 14.2 10.8 22.4 6.2 73.6 0.0 73.6 2007 Oct 12.4 9.6 6.7 17.3 1.5 155.2 0.0 155.2 2007 Nov 8.9 5.9 2.8 12.8 -3.3 116.2 0.0 116.2 2007 Dec 5.8 3.2 0.6 12.9 -5.3 181.6 19.6 210.6 2008 Jan 5.5 2.8 0.1 10.3 -4.9 122.2 14.2 137.6 2008 Feb 8.6 5.5 2.4 14.1 -2.9 67.4 0.8 68.6 2008 Mar 9.1 5.9 2.7 11.6 -1 72.8 2.4 75.2 2008 Apr 11.3 7.6 3.8 18.8 -2.1 56.8 2.2 62.2 2008 May 16.6 12.8 8.9 29 3.3 43.2 0.0 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. 55 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 \u00E2\u0080\u0093 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 cross- referenced 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 \u00C2\u00B5L nano pure water and 100 \u00C2\u00B5L of glycerol. Stock tubes were stored at -80\u00C2\u00B0C. 56 Table 3.3: Morphological features of common darks moulds colonizing weathered wood (Barnett and Hunter 1998) Genera Features Aureobasidium 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. Alternaria 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. Cladosporium 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 lemon- shaped; often in simple or branched acropetalous chains; parasitic on higher plants or saprophytic. Epicoccum Sporodochia dark, more or less cushion-shaped, variable in size; conidiophores compact or loose, dark, rather short; conidia dark, several- celled (dicyosporous), globose; mostly saprophytic, or weakly parasitic. Phoma 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 / \u00CE\u00A3 (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. 57 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. 58 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 59 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 \u00C2\u00B5m 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 60 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\u00E2\u0080\u0099s 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 61 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 \u00CE\u00BCm (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. 62 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. 63 Table 3.4: Fungi isolated from southern lodgepole pine wood samples after 40 weeks of outdoor exposure in Vancouver, Canada Fungi Phylum Source (Exposure/Rack) Codification Identification Primer sequenced Closest match in Blast (GeneBank) Identity Aureobasidium pullulans (black) Ascomycota Full / Sample 2 2 rDNA / Microscopy ITS4 Aureobasidium pullulans FJ216455 556/561 (99%) Aureobasidium pullulans (black) Ascomycota 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%) Hormonema dematioides Ascomycota Full / Sample 1 6_1S rDNA / Microscopy ITS4 Hormonema dematioides AY253451 566/573 (98%) 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 ITS4 Epicoccum nigrum FJ904918 526/531 (99%) 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 rDNA ITS4 Mollisia minutella DQ008242.1 448/448 (93%) *: Morphological features cross references against fungi identified by DNA analysis 64 Table 3.5: Fungal diversity in southern pine wood samples exposed to the weather for 40 weeks in Vancouver, Canada Sample Fungal richness Simpson index 1 6 3.6 2 5 5 3 7 4.5 4 6 3 5 2 2 Average 5.2 3.6 Total 26 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). 65 Table 3.6: Growth of fungi cultured onto solid malt extract agar (1% Difco) after 7 days of growth Fungi Growth at day 7 Avg (mm) [SD] Truncatella angustata 24.1 [NA] Epicoccum sp. 20.7 [5.2] Phoma sp. 17.9 [1.0] Alternaria sp. 5 15.1 [8.3] Aureobasidium pullulans (white) 13.4 [0.6] Hormonema dematioides 13.4 [2.4] Aureobasidium pullulans (black) 12.9 [2.7] Glonium pusillum 10.2 [NA] Cladosporium sp. 5.7 [NA] Lecythophora sp. 3.6 [NA] Mollisia minutella 2.0 [NA] Table 3.7: Lightness of fungi cultured onto solid media malt extract (agar 1% Difco) after 7 days of growth Fungi Lightness at day 20 Avg (L) [SD] Hormonema dematioides 16.6 [3.6] Cladosporium sp. 24.0 [NA] Alternaria sp. 5 27.5 [0.7] Aureobasidium pullulans (black) 28.0 [NA] Mollisia minutella 29.0 [NA] Glonium pusillum 50.0 [NA] Epicoccum sp. 58.5 [13.3] Truncatella angustata 61.0 [NA] Lecythophora sp. 69.0 [NA] Aureobasidium pullulans (white) 73.0 [NA] Phoma sp. 76.2 [8.5] 66 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 67 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 68 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). 69 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 70 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. Figure 3.6: Area of southern pine wood samples colonized by fungi during 40 weeks of exposure outdoors. Error bars depict standard deviations 0 1 2 3 4 5 6 7 8 0 20 40 60 80 100 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Months of exposure C o lo n iz ed a re a (% ) Weeks of exposure 71 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 72 The color at the surface of southern pine specimens expressed as lightness, redness\u00E2\u0080\u0093 greenness and yellowness\u00E2\u0080\u0093blueness 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). 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\u00E2\u0080\u0093greenness 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 Exposure Control 0 1 2 3 4 5 6 7 8 9 10 40 50 60 70 80 0 4 8 12 16 20 24 28 32 36 40 Months of exposure Li gh tn e ss [L ] Weeks of exposure 73 constant, until week 24, when they became greener ([a] decreased). As with lightness, redness\u00E2\u0080\u0093greenness values showed an inflection point close to week 6 corresponding to pronounced staining of wood by fungi. 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\u00E2\u0080\u0093blueness [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. Exposure Control 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 10 12 0 4 8 12 16 20 24 28 32 36 40 Months of exposure R ed n es s / G re en n es s (a ) Weeks of exposure 74 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 (\u00E2\u0089\u0088 30% moisture content) and appeared to vary depending on the number and severity of rainfall events (Figure 3.11). Exposure Control 0 2 4 6 8 10 0 10 20 30 0 4 8 12 16 20 24 28 32 36 40 Months of exposure Y e e llo w n e ss /B lu e n e ss [b ] Weeks of exposure 75 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. Moisture content Rainfall 1 2 3 4 5 6 7 8 9 10 0 50 100 150 200 12 15 18 21 24 27 30 4 8 12 16 20 24 28 32 36 40 Months of exposure R ai n fa ll [m m ] M o is tu re c o n te n t [ % ] Weeks of exposure 76 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. 77 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, 78 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). 79 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 80 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 81 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 82 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 photo- oxidation 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 83 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 soft- rot 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. 84 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. 85 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 W\u00C3\u00A4lchli, 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). 86 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). 87 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 \u00CE\u00B2-(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 88 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. 89 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 90 Table 4.1: Fungi tested for their ability to synthesize lignolytic and cellulolytic enzymes No Fungi Strain(s) tested 1 Allantophomopsis lycopodina (H\u00C3\u00B6hn.) Carris 1 2 Alternaria sp. 5 3 Aureobasidium pullulans (de Bary) G. Arnaud (black) 6 4 Aureobasidium pullulans (de Bary) G. Arnaud (white) 6 5 Botryosphaeria stevensii Shoemaker 1 6 Botryotinia fuckeliana (de Bary) Whetzel 4 7 Cladosporium cladosporioides (Fresen.) G.A. de Vries 5 8 Cladosporium sp. 1 9 Coniochaeta ligniaria (Grev.) Massee 2 10 Epicoccum nigrum Link 5 11 Epicoccum sp. 1 12 Glonium pusillum H. Zogg 1 13 Hormonema dematioides (Lagerb. & Melin) 7 14 Lecythophora sp. 2 15 Leptosphaerulina chartarum Cec. Roux 1 16 Lewia infectoria (Fuckel) M.E. Barr & E.G. Simmons 2 17 Mollisia minutella (Sacc.) Rehm 1 18 Penicillium expansum Link ex. Thom 1 19 Peniophora aurantiaca (Bresadola) von H\u00C3\u00B6hnel & Litschaue 1 20 Phialocephala sp. 1 21 Phialophora sp. 2 22 Phoma sp. 5 23 Rhizopogon sp. 1 24 Trichoderma viride Pers. 1 25 Truncatella angustata (Pers.) S. Hughes 1 26 Valsa ambiens (Pers.) Fr. 1 27 Trichaptum abietinum (Pers.) Ryvarden (white-rot control) 1 28 Coniophora puteana (Schum. ex Fries) Karst. (brown-rot control) 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 \u00E2\u0080\u0098blocks\u00E2\u0080\u0099 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 91 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\u00E2\u0080\u0099s 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 Wood species Petri dishes 1 17 + control 2 18 . . . . . . . . . . . . . . . . . . . . 12 17 + control 2 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 92 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 \u00CE\u00BCm) 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\u00C2\u00B0C \u00C2\u00B1 1\u00C2\u00B0C at 65% \u00C2\u00B1 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 \u00C2\u00B1 5\u00C2\u00B0C) for 24 hours to a constant weight (as above) and sterilized in autoclave at 121\u00C2\u00B0C and 103.4 kPa for 20 min. Veneers were re- hydrated 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, 93 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) \u00E2\u0080\u0093 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 \u00CE\u00BCL 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 \u00CE\u00BCL 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 94 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\u00C2\u00B0C 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 95 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 96 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\u00C2\u00B0C with a heating rate of 5\u00C2\u00B0C/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 \u00C2\u00B5m 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, 97 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. 98 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) 99 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. 100 Table 4.3: Laccase activity and index for enzymatic activity for carboxymethyl cellulose (CMC) No Fungi Strains tested Laccase activity\u00E2\u0080\u00A0 after 12 days Icmc* after 7 days 1 Mollisia minutella 1 +++ 5.00 2 Rhizopogon sp. 1 - 5.00 3 Coniophora puteana (brown-rot control) 1 - 5.00 4 Phialophora sp. 2 - 2.43-2.59 5 Coniochaeta ligniaria 2 - 2.23-2.55 6 Lecythophora sp. 2 +++ 2.15-2.22 7 Penicillium expansum 1 - 2.04 8 Valsa ambiens 1 - 1.96 9 Botryosphaeria stevensii 1 - 1.82 10 Aureobasidium pullulans (white) 6 - 1.62-2.84 11 Aureobasidium pullulans (black) 6 - 1.56-1.94 12 Cladosporium cladosporioides 5 - 1.51 13 Botryotinia fuckeliana 4 +++ 1.5-1.94 14 Phoma sp. 5 - 1.44-1.99 15 Lewia infectoria 2 - 1.38-1.48 16 Glonium pusillum 1 - 1.33 17 Peniophora aurantiaca 1 - 1.29 18 Cladosporium sp. 1 - 1.31-1.63 19 Epicoccum nigrum 5 - 1.26-1.3 20 Epicoccum sp. 1 - 1.21 21 Leptosphaerulina chartarum 1 - 1.21 22 Alternaria sp. 5 - 1.17-1.25 23 Truncatella angustata 1 - 1.15 24 Trichoderma viride 1 - 1.01 25 Allantophomopsis lycopodina 1 - 0.00 26 Hormonema dematioides 7 - 0.00 27 Phialocephala sp. 1 +++ 0.00 28 Trichaptum abietinum (white-rot control) 1 +++ 0.00 \u00E2\u0080\u00A0Rank of enzymatic activity: negative (-); low (+); medium (++); high (+++) *Icmc: index for range of enzyme activity on carboxymethyl cellulose 101 Table 4.4: Fungi isolated from weathered wood and tested for their ability to breakdown wood Treatment Fungi Code Name Strains tested 1 Alternaria sp. Alt. 3 2 A. pullulans (black) Aur. (black) 3 3 A. pullulans (white) Aur. (white) 3 4 B. fuckeliana Botr. 3 5 Cladosporium sp. Clad. 3 6 C. ligniaria Conio. 2 7 E. nigrum Epic. 3 8 H. dematioides Horm. 3 9 Lecythophora sp. Lecyth. 1 10 L. infectoria Lew. 3 11 M. minutella Moll. 1 12 Phialocephala sp. Phialoc. 1 13 Phialophora sp. Phialop. 2 14 Phoma sp. Phom. 3 15 T. abietinum (white-rot control) Trich. 1 16 C. puteana (brown-rot control) Coniop. 1 17 Chaetomium globosum Kunze ex Fr. (soft-rot control) Chaet. 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 (P- values) of experimental variables (fungi, wood species) and interaction of fungi with wood species on the different response variables. Since in some cases interactions were produced by unusual variations in one or two treatment, the main effects were also included in the results in order to facilitate interpretation of results. 102 Table 4.5: Significant effects of, and interactions between fungal species and wood species, on mechanical properties of veneers exposed to test fungi P-value Source of variation Peak tensile stress MOE Peak stiffness Toughness Fungi <0.001 <0.001 <0.001 <0.001 Wood sp. <0.001 <0.001 <0.001 <0.001 Fungi x Wood sp. <0.001 <0.001 <0.001 <0.001 4.3.2.1.1. Peak tensile stress ratio Fungal species had a significant effect (P-value < 0.001) on the peak tensile stress ratios (maximum tensile stress as a ratio to that of the sound wood control) of wood veneers. Cladosporium sp. and C. ligniaria produced the greatest losses in peak tensile stress, followed by the control fungi, C. globosum and T. abietinum. Less pronounced losses in strengths were produced by Phialocephala sp., M. minutella, L. infectoria and E. nigrum. The rest of the fungi produced tensile stress ratios for treated veneers that were close to one, indicating that peak tensile stress of veneers was similar to that of the sound wood controls, Figure 4.4. 0.2 0.4 0.6 0.8 1.0 1.2 Pe ak t en si le s tr es s ra ti o Fungi 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 \u00C2\u00B1SED 103 Wood species also had a significant effect (P-value < 0.001) on the peak tensile stress ratio of tested veneers. Lime veneers exposed to fungi isolated from weathered wood showed a significantly lower peak tensile ratio than spruce veneers, Figure 4.5. 0.70 0.75 0.80 0.85 0.90 0.95 1.00 Lime Spruce P e ak t e n si le s tr e ss r at io Wood 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 \u00C2\u00B1SED The interaction of fungal species x wood species also had a significant effect (P-value < 0.001) on the peak tensile stress ratio of tested veneers. The interaction was caused by an inconsistent variation in peak tensile stress ratio of wood veneers exposed to Phialophora sp. The peak tensile stress ratio of spruce veneers was generally significantly higher than that of lime wood veneers (Figure 4.5), but in veneers incubated with Phialophora sp., the opposite was the case (circled in Figure 4.6). 104 Lime Spruce l.s.d. -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 P e ak T e n si le s tr e ss r at io Fungi 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. 4.3.2.1.2. Modulus of elasticity (MOE) ratio Fungal species had a significant effect (P-value < 0.001) on the MOE ratio of wood veneers. Cladosporium sp., C. ligniaria and C. globosum produced the highest losses in MOE. Less pronounced losses were caused by Phialocephala sp. and T. abietinum. The rest of the tested fungi produced MOE ratios in veneers that were close to one, indicating that the MOE of the veneers was similar to that of the sound wood controls, Figure 4.7. 105 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 \u00C2\u00B1SED Wood species also had a significant effect (P-value < 0.001) on MOE ratio. Lime veneers exposed to fungi isolated from weathered wood showed significantly lower MOE ratio than spruce veneers, Figure 4.8. 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 \u00C2\u00B1SED 0.2 0.4 0.6 0.8 1.0 1.2 M O E ra ti o Fungi 0.75 0.80 0.85 0.90 0.95 1.00 1.05 Lime Spruce M O E ra ti o Wood 106 The interaction of fungal species x wood species also had a significant effect (P-value < 0.001) on the MOE ratio of tested veneers. Inconsistent variation in MOE ratio of spruce veneers exposed to Cladosporium sp. accounts in part for this interaction. Lime veneers exposed to Cladosporium sp. had the lowest MOE ratio of all veneers (Figure 4.7). However, spruce veneers exposed to Cladosporium sp. showed no losses in MOE, whereas other fungi that caused losses in MOE of lime veneers also caused losses in MOE of spruce veneers (Figure 4.9). Lime Spruce l.s.d. -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 M O E ra ti o Fungi 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 4.3.2.1.3. Peak stiffness ratio Fungal species had a significant effect (P-value < 0.001) on the peak stiffness ratio of wood veneers. Cladosporium sp., C. ligniaria and C. globosum produced the highest losses in peak tensile stress. Less severe losses in stiffness were caused by T. abietinum. All other tested 107 fungi produced peak stiffness ratios in tested veneers that were close to one, indicating that the peak stiffness of veneers was similar to that of the sound wood controls, Figure 4.10. 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 ratio close to one indicates that peak stiffness was similar to that of sound wood. Error bars correspond to \u00C2\u00B1SED Wood species also had a significant effect (P-value < 0.001) on peak stiffness ratio. Lime veneers incubated with fungi isolated from weathered wood showed a significantly lower peak stiffness ratio than spruce veneers, Figure 4.11). 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 P e ak s ti ff n e ss r a ti o Fungi 108 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 \u00C2\u00B1SED The interaction of fungal species x wood species also had a significant effect (P-value < 0.001) on peak stiffness ratio. The interaction occurred for the same reason as the interaction detected for losses in MOE (as expected), Figure 4.12. Lime Spruce l.s.d. -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 P e ak s ti ff n e ss r at io Fungi 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 0.80 0.85 0.90 0.95 1.00 1.05 Lime Spruce P e ak s ti ff n e ss r a ti o Wood 109 4.3.2.1.4. Peak toughness ratio Fungal species had a significant effect (P-value < 0.001) on the peak toughness ratio (maximum amount of energy absorbed by the wood as a ratio to that of the sound wood control) of wood veneers. Cladosporium sp., C. ligniaria and C. globosum produced the greatest losses in peak toughness ratio followed by T. abietinum. Less pronounced losses in toughness were caused by E. nigrum, L. infectoria, M. minutella and Phialocephala sp. A slight increase in peak toughness ratio was caused by C. puteana. All other tested fungi produced peak toughness ratios for tested veneers that were close to one, indicating that the toughness of veneers was similar to that of sound wood, Figure 4.13. 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 \u00C2\u00B1SED 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 To u gh n es s ra ti o Fungi 110 Wood species also had a significant effect (P-value < 0.001) on toughness ratio. Lime veneers incubated with fungi isolated from weathered wood showed a significantly lower toughness ratio than spruce veneers, Figure 4.14. 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 \u00C2\u00B1SED The interaction of fungal species x wood species also had a significant effect (P-value < 0.001) on peak toughness ratio (Figure 4.15). This interaction occurred for the same reason that there was a significant (P-value < 0.001) interaction of fungal species x wood species on peak tensile strength ratio (see Figure 4.6). 0.75 0.80 0.85 0.90 0.95 1.00 Lime Spruce To u gh n e ss ra ti o Wood 111 Lime Spruce l.s.d. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 To u gh n e ss r at io Fungi 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 4.3.2.2. Viscoelastic properties The storage modulus (SM) of lime and spruce samples exposed to different fungi are depicted in Figure 4.16 and Figure 4.17, respectively. SM decreased with temperature for both wood species showing transitions at about 75 \u00C2\u00B0C and 150 \u00C2\u00B0C. The SM of lime wood incubated with Alternaria sp., Cladosporium sp., C. ligniaria, Phialocephala sp., C. globosum and T. abietinum showed lower values than that of untreated wood, and there was no second transition point at 150 \u00C2\u00B0C. In contrast the SM of lime samples incubated with M. minutella, E. nigrum and Lewia sp. was slightly higher than that of the untreated control. Spruce samples did not show an inflection point at about 150 \u00C2\u00B0C and their SM remained lower than that of the sound wood control. The SM of spruce wood incubated with Phialophora sp. had the highest SM of all spruce wood samples exposed to the different fungi. 112 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 Alt. Clad. Conio. Epic. Lew. Moll. Phialoc. Chaet. Trich. Control 0 6000 12000 18000 25 35 45 55 65 75 85 95 105 115 125 135 145 155 165 175 185 195 St o ra ge m o d u lu s (M P a ) Temperature (\u00C2\u00B0C) 113 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 4.3.2.3. Fourier transform infra-red spectroscopy FTIR spectra for lime and spruce wood samples exposed to the different fungi are shown in Figure 4.18 to Figure 4.23 and Figure 4.24 to Figure 4.29, respectively. In lime wood incubated with Alternaria sp., A. pullulans (black) and A. pullulans (white), Cladosporium sp., Lewia sp., Phialocephala sp., Phoma sp., C. globosum and C. puteana there were significant decreases, or in some cases elimination of peaks related to cellulose and hemicelluloses. Chaet. Conio. Clad. Trich. Phialoc. Moll. Phialop. Control 0 6000 12000 St o ra ge m o d u lu s (M P a) Temperature (\u00C2\u00B0C) 114 Among the peaks affected were 1059 cm-1, C-O stretching in cellulose (Faix and B\u00C3\u00B6ttcher 1992; Pandey and Theagarajan 1997; Popescu et al. 2010), 1108 cm-1, C-O-H deformation in hemicelluloses and cellulose; (Faix and B\u00C3\u00B6ttcher 1992; Popescu et al. 2010), 1165 cm-1, C-O- C stretching in hemicelluloses and cellulose (Faix and B\u00C3\u00B6ttcher 1992; Pandey and Theagarajan 1997; Popescu et al. 2010), 1244 cm-1, guaiacyl ring and C-O stretching in xylan and lignin (Faix and B\u00C3\u00B6ttcher 1992; Popescu et al. 2010), 1376 cm-1, C-H Deformation, CH3 symmetric deformation in hemicelluloses and cellulose (Faix and B\u00C3\u00B6ttcher 1992; Pandey and Theagarajan 1997; Popescu et al. 2010), 1737 cm-1, C=O stretching of carbonyl and acetyl groups in hemicelluloses (Faix and B\u00C3\u00B6ttcher 1992; Popescu et al. 2010). Conversely, samples inoculated with Lecythophora sp. and T. abietinum showed an increase in the peak at 1108 cm-1. Peaks related to lignin decreased in veneers inoculated with T. abietinum or Phialophora sp. Peaks affected were 1510 cm-1, C=C stretching of substituted aromatic rings in lignin (Harrington et al. 1964; Faix and B\u00C3\u00B6ttcher 1992), 1598 cm-1, C=C stretching of substituted aromatic rings in lignin (Pandey and Theagarajan 1997; Popescu et al. 2010). Decreases in both, cellulose and lignin, peaks were observed in lime wood incubated with C. ligniaria, H. dematioides or Mollisia sp. The peaks most affected were 1059 cm-1, 1244 cm-1, 1376 cm-1, 1510 cm-1, 1598 cm-1 and 1737 cm-1. In addition, all fungi but T. abietinum and B. fuckeliana produced increases in the peak at 1655 cm-1, C=O conjugated stretching of phenolic groups in lignin. Such an increase has been attributed to the increase of carbonyl moieties as decay occurs (Popescu et al. 2010). The spectrum of lime wood incubated with B. fuckeliana was similar to that of the sound wood control. 115 In spruce wood inoculated with Alternaria sp., A. pullulans (white), E. nigrum, Lewia sp. and C. globosum, there was also a significant decrease in peaks related to cellulose and hemicelluloses. Peaks affected were 1462 cm-1, C-H deformation in lignin and carbohydrates (Pandey and Theagarajan 1997) and 1737 cm-1, C=O stretching of carbonyl and acetyl groups in hemicelluloses (Faix and B\u00C3\u00B6ttcher 1992; Pandey and Pitman 2003). A decrease in peaks related to lignin was produced by A. pullulans (black), B. fuckeliana, C. ligniaria, and T. abietinum. Peaks affected were 1268 cm-1, C-O guaiacyl ring breathing, C-O stretching, C-O linkage in guaiacyl aromatic methoxyl groups in lignin (Pandey and Theagarajan 1997) and 1505 cm-1, C=C stretching of substituted aromatic rings in lignin (Harrington et al. 1964). Conversely, samples inoculated with Phialocephala sp. showed an increase in the peak at 1268 cm-1. Decreases of both, cellulose and lignin, peaks was observed in spruce wood inoculated with H. dematioides, Lecythophora sp., Mollisia sp. and C. puteana. The peaks most affected were 1268 cm-1, 1462 cm-1, 1505 cm-1 and 1737 cm-1. All fungi but Phoma sp. and C. puteana increased the peak at 1655 cm-1 (as above). The FTIR spectrum of spruce wood inoculated with Phoma sp. appeared to be unaffected by fungal exposure. 116 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 117 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 118 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 119 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 120 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 121 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 122 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 123 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 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 124 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 125 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 126 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 127 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 128 4.3.2.4. Light microscopy Light microscopy revealed degradation of lime wood by Alternaria sp., C. ligniaria, Cladosporium sp., Phialocephala sp., Phialophora sp. and C. puteana. Wood colonized by C. ligniaria, Phialocephala sp., Phialophora sp. and C. puteana, showed a decay pattern resembling that caused by soft-rot fungi: a mixture of soft-rot decay cavities and erosion of fibers and vessels walls. The decay pattern caused by Cladosporium sp. consisted mainly of erosion of wood cell walls, while Alternaria sp. produced a general disruption of colonized wood tissues, but no clear signs of cell wall degradation. Some damage of wood tissue was found in samples colonized by B. fuckeliana, Lecythophora sp., L. infectoria, M. minutella and T. abietinum. In these samples rupture of vessels cell walls was observed, which may have been due to the enzymatic action of fungi. No degradation was observed in samples colonized by E. nigrum, H. dematioides, A. pullulans (black and white) or Phoma sp. Irrespective of the different patterns of degradation caused by the fungi, most of them colonized parenchyma cells (ray and axial parenchyma) and the lumens of vessels where spores and hyphae accumulated. In samples colonized by Alternaria sp. a general disruption of wood tissues was observed, but no clear pattern of wood cell wall degradation was seen. Samples colonized by A. pullulans (black) and A. pullulans (white) showed no signs of fungal degradation. Samples colonized by B. fuckeliana showed erosion of wood tissues in cells adjacent to vessels. Samples colonized by C. globosum showed clear signs of soft rot decay-presence of soft-rot cavities and erosion of wood cell walls. Samples colonized by Cladosporium sp. showed erosion-type decay of fibers and vessels, but rays appeared to be resistant to degradation 129 (Figure 4.30). Samples colonized by C. ligniaria showed erosion of wood cell walls and soft- rot cavities. Samples colonized by C. puteana showed clear degradation of wood cell walls (erosion) and presence of soft rot-like cavities. Samples colonized by H. dematioides showed the presence of spore aggregations in vessel lumina. In samples colonized by Lecythophora sp. and L. infectoria vessels walls were degraded (Figure 4.31). Samples incubated with M. minutella showed rupture of cell walls. Samples colonized by Phialocephala sp. showed erosion of wood cell walls. Samples colonized by Phialophora sp. showed erosion and rupture of wood cell walls in tissue close to the surface of the sample. Samples incubated with T. abietinum showed degradation of vessels. No signs of degradation were seen in samples incubated with Phoma sp. (Figure 4.32). 130 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 131 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 132 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 Light microscopy showed that spruce wood was much less susceptible to degradation by test fungi than lime. Despite colonization of spruce by fungi little decay was observed. In samples colonized by Alternaria sp., C. globosum and Cladosporium sp. disruption of wood tissues was observed close to the surface of the samples- it seems that cell walls were weakened by the presence of the fungus. Samples colonized by A. pullulans (black) and A. 133 pullulans (white) showed colonization of parenchyma rays and tracheids. Samples colonized by B. fuckeliana showed disruption of wood tissues close to the surface of the sample and detachment of wood cell walls colonized by fungi (Figure 4.33). In samples colonized by C. ligniaria, C. puteana, Lecythophora sp. disruption of wood tissues was observed close to the surface of the samples. In samples colonized by E. nigrum and L. infectoria colonization of parenchyma ray cells and resin canal was observed, but there were no signs of decay. The sample colonized by H. dematioides showed colonization of rays close to the surface and deterioration and staining of the first rows of tracheids (Figure 4.34). Samples incubated with M. minutella and Phialocephala sp. showed disruption of wood tissues close to the surface of the sample and detachment of wood cell walls in cells colonized by fungi. Samples colonized by Phialophora sp. T. abietinum and Phoma sp. showed a general disruption of wood tissues close to the surface of the samples (Figure 4.35). 134 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 135 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 136 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 137 4.3.2.5. Scanning electron microscopy Scanning electron microscopy (SEM) was carried out on lime and spruce wood samples colonized by Cladosporium sp., and lime wood samples colonized by A. pullulans (Figure 4.36 and Figure 4.37). Cladosporium sp. was able to form a compact mycelial mat that evenly covered the surface of lime. The lime wood was very heavily and evenly degraded. Wood fibers appeared to be degraded to more basic sub-units. SEM images suggest that degradation of lime occurred from the direct effects of enzymes diffusing from the hyphal mat towards the wood surface. No cavities or bore holes were observed. In contrast, Cladosporium sp. was not effective at degrading spruce, and the hyphal mat formed by the fungus on spruce was much less compact than that on lime. Similarly, A. pullulans did not produce any changes to the microstructure of spruce or lime wood. 138 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 139 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 140 4.4. Discussion In the introduction to this Chapter, I hypothesized that some of the fungi isolated from weathered wood would be able to degrade wood. Some of my findings support this hypothesis as Cladosporium sp., C. ligniaria, E. nigrum, L. infectoria, M. minutella and Phialocephala sp. were all able to significantly decrease some of the mechanical properties of lime and spruce wood veneers. Peak tensile stress ratio and toughness ratio were the parameters most affected by these fungi. I chose to examine the changes in mechanical properties of lime and spruce veneers caused by fungi, because mechanical properties depend on the integrity of cellulose, hemicelluloses and lignin. Cellulose is responsible for strength in wood fibers mainly due to its high degree of polymerization and linear orientation, while hemicelluloses also contribute to tensile properties as they act as coupling agents between cellulose and lignin (Ifju, 1964; Spiegelberg, 1966). Lignin helps to bind carbohydrates molecules together within the cell wall of wood fibers (Jeffries, 1994). In support of these results, the dynamic stiffness (storage modulus) of samples incubated with these organisms also exhibited lower values than sound wood. Storage modulus (SM) represents the elastic component of a viscoelastic material or the ability of wood to recover from elastic deformation (Menard 1999). The SM of wood is negatively correlated with temperature as the hemicelluloses and lignin become ductile when temperature increases and produce movements in their polymeric side chains (Birkinshaw et al. 1999). A lower dynamic stiffness as was observed for wood samples exposed to some fungi provides evidence of changes to the structure of hemicelluloses and lignin. Changes to the chemical composition of hemicelluloses, lignin and cellulose were confirmed in both spruce and lime 141 by FTIR spectroscopy. FTIR spectroscopy detected changes in functional groups associated with all three of wood\u00E2\u0080\u0099s polymeric constituents in wood exposed to fungi. Early decreases in the mechanical properties of wood as a result of exposure to fungi have been related to degradation of hemicelluloses (Winandy and Morrell 1993). For example, observation of Pinus spp. colonized by brown-rot fungi showed that early losses in bending strength (> 40%) were related to degradation of hemicelluloses. In contrast, later more pronounced strength losses (> 75%) were caused by degradation of cellulose (Curling et al. 2002). Changes in the microstructure of wood colonized by the fungi also provide evidence of the ability of some of the fungi to degrade wood. The decay pattern observed by light microscopy of transverse sections of solid wood samples exposed to fungi consisted of cavities and erosion of cell walls. Such a pattern is typical of soft-rot decay caused by ascomycete fungi (Savory 1954). However, SEM revealed that the surface of highly degraded veneers colonized by Cladosporium sp. was different to that of the surface of solid wood samples. Wood fibers at veneer surfaces appeared to be very heavily degraded by direct enzymatic leakage from fungal hyphae established on the veneer surface. SEM images showed fiber cell walls breaking down to more elemental sub-units. Amongst the different fungi that were tested, results showed that Cladosporium sp. and C. ligniaria caused the greatest losses in peak tensile stress ratio, MOE ratio, peak stiffness ratio and toughness ratio of lime and spruce wood. Changes in FTIR bands for carbohydrates and lignin in spruce and lime provided evidence of the ability of these fungi to modify wood\u00E2\u0080\u0099s polymers. Cladosporium spp. have been reported to possess cellulase, xylanase, mananase, amylase and cellobiose dehydrogenase enzymes (Ghahfarokhi et al. 2004; 142 Nilsson, 1974). C. ligniaria produces cellulase, xylanase, manganese peroxidase and lignin peroxidase (Lopez et al., 2007). The action of these enzymes may account for why both fungi were able to degrade lime and to a lesser extent spruce veneers. Furthermore, Zyani et al. (2009) observed that Cladosporium cladosporioides was able to decay wood in in-vitro tests, although they did not specify the wood species that was tested. Therefore, my findings are consistent with their observations. Cladosporium sp. is a highly melanized fungus which is adapted to the conditions found at wood surfaces exposed outdoors (Park 1982). Moreover, it is frequently isolated from weathered wood (Hansen, 2008). Cladosporium\u00E2\u0080\u0099s cellulolytic abilities may represent another beneficial adaptation for survival at weathered wood surfaces. Likewise, L. infectoria, M. minutella, Phialophora sp and Phialocephala sp. also significantly reduced the tensile properties of wood; and with the exception of M. minutella and L. infectoria in lime wood, all the other fungi I tested were able to modify the storage modulus of wood. The latter observation suggests that wood exposed to fungi was less rigid than the sound wood controls. Fungal species from these genera have been reported to produce soft-rot decay of solid wood and also forest debris (Morrell and Zabel, 1985; Hale and Eaton, 1985; Allmer et al. 2006). Phialophora and Phialocephala fungi also cause soft-rot cavities and erosion-type decay in pine and beech wood (Morrell and Zabel 1985). Light microscopy images here showed that presence of soft- rot cavities in samples of lime wood exposed to some of the fungi, but erosion of cell walls was more frequently observed. Enzymatic erosion is the simplest type of soft-rot decay since it only requires the presence of diffusible enzymes inside cell lumens, whereas soft-rot cavity formation is more complex as it requires fungal hyphae to penetrate into the cell 143 wall, align in the direction of the microfibrils and produce enzymes that dissolve the wall around the hyphae (Nilsson, 1973). Alternaria sp. and E. nigrum reduced the peak tensile stress of lime veneers, but not that of spruce. Both fungi commonly colonize weathered wood (Morrell and Zabel, 1985; Pfeffer et al., 2012), but only Alternaria species have been reported as being able to produce soft-rot decay (Rajderkar 1966; Morrell and Zabel 1985). A. pullulans is probably the most ubiquitous colonizer of weathered wood surfaces (Dickinson 1971). It did not significantly reduce the tensile properties of lime or spruce. However, its effect on the toughness approached statistical significance. A. pullulans together with Alternaria sp. and Cladosporium sp., are well adapted to survive at weathered wood surfaces, as mentioned in Chapter 2. Tests have demonstrated that they can withstand long periods of dryness and high temperatures (Park 1982). Several studies have tried to elucidate whether A. pullulans can degrade wood and model wood compounds. For example, Sharpe and Dickinson (1992) carried out an in-vitro test on the ability of A. pullulans to use cellulose, different hemicellulose monomers and dimers, and lignin model compounds, as sole carbon sources. Their findings suggested that A. pullulans is not able to degrade cellulose, but it can use simple sugars. Accordingly, the authors concluded that wood cell wall carbohydrates need to be broken down possibly to mono or disaccharides before A. pullulans can utilize them (Sharpe and Dickinson 1992). Sharpe and Dickinson (1992), also found that A. pullulans was able to utilize phenolic compounds more readily than oligosaccharides. In support of their findings I observed that the FTIR band at 1268 cm-1 (C-O guaiacyl ring breathing, C-O stretching, C-O linkage in guaiacyl aromatic methoxyl groups lignin; Pandey and Theagarajan 144 1997) appeared to decrease in spruce wood incubated with A. pullulans. According to Bourbonnais and Paice (1987) A. pullulans is able to cleave \u00CE\u00B2-O-4 linkages in model lignin compounds, but it does not have the ability to degrade non-phenolic dimers. This observation indicates that the capacity of A. pullulans to degrade lignin is limited. Nevertheless, fungi with limited ability to degrade wood tissues may increase the overall rate of decay because their enzymes may eventually act on wood that has been degraded by other fungi. For instance, L. hoffmanni can metabolize phenolic compounds, and fungi from the genus Phoma have been isolated from soft-rotted wood (Savory 1954; Bugos et al. 1988). Both of these fungi may contribute to the degradation of wood during the later stages of decay, even though they are unable to degrade sound wood. Lime wood was more susceptible to fungal degradation than spruce. The greater susceptibility of lime to degradation by ascomycetes isolated from weathered wood (here) accords with the observations of Nilsson and Daniel (1989) and Encinas et al. (1998) who reported weight and toughness losses in birch and pine spp. colonized by staining fungi. Faix et al. (1985); Nilsson and Daniel (1989) and Blanchette (1991) all suggested that differences in the lignin content of hardwood (lime) and softwood (spruce) may account for the greater susceptibility of the former to degradation by ascomycetes. Lignin is a significant barrier to hyphae and enzymes since it encrusts cell walls preventing enzymes from hydrolyzing carbohydrates (Winandy and Rowell 2005). Softwoods with a high concentration of guaiacyl lignin units are particularly resistant to fungi that cause soft-rot, whereas wood consisting predominantly of syringyl lignin is more susceptible to soft-rot fungi (Faix et al. 1985; Nilsson and Daniel 1989; Blanchette 1991). 145 The toughness of wood veneers was severely affected by fungi, as mentioned above Toughness is the most sensitive indicator of fungal decay (Wilcox 1978). Some studies have shown that staining fungi cannot alter the toughness of wood (Schirp et al. 2000), whereas other have shown the opposite. For example, Encinas et al. (1998) reported that the blue stain fungus Lasiodiplodia theobromae was able to produce significant losses in toughness in birch and Pine species. Such losses were well correlated with weight losses in the tested samples. Discrepancies on the effect of staining fungi on the mechanical properties of wood, and specifically on toughness properties could be related to the fact that degradation of wood varies with both fungal and wood species (Zabel and Morrell 1992). Nevertheless, here I showed that surface fungi isolated from weathered wood are able to affect the toughness of thin wood veneers. Furthermore, microscopy showed that fungi reducing the toughness of veneers also eroded and produced cavities in wood cell walls. The erosion of wood cell walls during the weathering has been attributed to the action of UV radiation and water (Evans 2008). It is not clear whether fungi also contribute to the erosion of wood cell walls during weathering. As stated in the introduction to this thesis, there is a body of opinion that indicates that fungi colonizing weathered wood are unable to degrade woody tissues (Feist 1983). Thus, references to their effect on wood mainly describe how they discolor and affect the appearance of wood. However, Duncan (1963) described early research suggesting that fungi colonizing weathered wood might degrade wood tissues. My findings for some of the fungi isolated from weathered wood support such suggestions, although the contribution of these organisms to the degradation of wood surfaces in-vivo would depend on conditions at 146 weathered wood surfaces being favorable to decay. Soft-rot decay in weathered western red cedar shingles was documented by Smith and Swann (1976). Their samples were actively colonized by a number of moulds, which produced soft-rot cavities and erosion of wood cell walls. In addition, Seifert (1964) reported that A. pullulans, possibly the most successful colonizer of weathered wood surfaces (Dickinson 1971), was able to produce weight losses and reduce cellulose and pentosans in Scots pine wood. Results here showed that A. pullulans was able to affect the strength properties and chemical composition of wood veneers, but not as much as Cladosporium sp. or C. ligniaria did. Soft-rot can occur slowly at low moisture levels according to Blanchette et al. (1994). Whereas, Worrall et al. (1991) suggested that soft-rot fungi may not differ from basidiomycetes in their preference for moderate moisture conditions. Moisture conditions favoring microbial degradation at wood surfaces are probably met sporadically year round but more commonly during winter and autumn as evaporation rates are slower due to lower temperatures (Denig et al. 2000). Microbial degradation of weathered wood surfaces may be more pronounced in wet tropical climates, which produce conditions at wood surfaces that are more favorable for decay. Furthermore, weathered wood is a modified substrate that is more susceptible to microbial degradation because solar radiation degrades carbohydrates and lignin. Such degradation may facilitate the enzymatic degradation of the remaining wood tissues as suggested by Evans and Banks (1986). The ability of some of the fungi colonizing weathered wood to break down wood tissues in- vitro has been shown in this Chapter. However, further research is needed to elucidate whether the conditions for microbial degradation are met at wood surfaces either 147 sporadically or seasonally in different climates. If such conditions occur it would be pertinent to clarify how much of the erosion at weathered surfaces is due to the action of surface fungi. 148 4.5. Conclusions My observations support the hypothesis that fungi isolated from weathered wood can degrade wood tissues because Cladosporium sp., C. ligniaria, E. nigrum, L. infectoria, M. minutella and Phialocephala sp. were able to significantly reduce the mechanical properties of lime and spruce wood. Tensile stress and toughness were the parameters most affected by fungi. Cladosporium sp., C. ligniaria produced the most dramatic changes in these mechanical properties. As a result veneers colonized by these fungi became very brittle. These fungi caused erosion of cell walls (soft-rot decay type II) and to a lesser extent soft- rot cavities (soft-rot decay type I). A. pullulans, one of the most successful organisms colonizing weathered wood worldwide, produced no significant changes in the tensile properties of incubated wood. However, its effect on the toughness of spruce was nearly significant. The dynamic stiffness of samples exposed to some of the fungi was lower than that of sound wood. Furthermore, some of the fungi were able to degrade wood\u00E2\u0080\u0099s chemical components. Therefore, I conclude that some the fungi colonizing weathered wood surfaces are capable of causing significant degradation of wood particularly hardwood. Further research is necessary to elucidate whether the conditions for microbial degradation are met at wood surfaces either sporadically or seasonally in different climates and how much of the erosion of wood during weathering is caused by the action of surface fungi. 149 5. Chapter 5: Effects of solar radiation on the colonization of weathered wood by fungi 5.1 Introduction Wood exposed outdoors rapidly acquires a rough, gray color, which adversely affects its appearance (Feist 1990). The graying of wood surfaces is caused by the colonization of wood by melanized fungi, which have the ability to metabolize photodegraded wood polymers (Duncan 1963; Dickinson 1971). Melanin in these fungi is apparently synthesized as a protective response against solar UV radiation, but this response may darken the wood (Brisson et al. 1996; Fogarty and Tobin 1996; Butler and Day 1998; Henson et al. 1999). Information in the literature supports the concept that UV radiation increases the fungal staining of wood surfaces, as it has been shown that UV radiation increases melanin production in several fungi (Frederick et al. 1999). In addition, UV radiation may restrict fungal diversity at wood surfaces to those organisms able to survive exposure to energetic radiation, e.g. Aureobasidium pullulans (de Bary) G. Arnaud and Hormonema dematioides Lagerb. & Melin (Ray et al. 2004). In this Chapter I hypothesize that blocking UV radiation from reaching wood surfaces will influence the diversity of fungi colonizing the wood surfaces. In the absence of UV radiation the adaptations of melanized fungi that commonly colonize weathered wood surfaces may not provide a competitive advantage and other fungi might successfully out compete them. In such conditions fungal staining of wood may be less severe than that of wood exposed to the full solar spectrum. This hypothesis was tested by exposing 25 southern pine boards for 40 weeks under polymethylmethacrylate filters which blocked different wavelengths of 150 solar radiation from reaching wood surfaces. Fungi colonizing the samples exposed under different filters were isolated, identified and characterized. Changes in fungal diversity were recorded and the color of wood surfaces exposed under the different filters was measured and related to the ecology of fungi colonizing the samples. The final appearance of exposed wood surfaces was evaluated by measuring the area colonized by fungi and color of wood surfaces. Chemical changes at wood surfaces under different filters were evaluated using FTIR internal reflectance spectroscopy. In summary, in this Chapter I seek to better understand the importance of melanin for fungi colonizing wood surfaces exposed to solar UV radiation and record the frequency of highly melanized fungal hyphae colonizing wood surfaces exposed to the weather. 5.2 Materials and methods 5.2.1 Experimental design and statistical analyses The experiment was initially designed to assess the effect of different chemical treatments and wavelengths of solar radiation on the color of wood surfaces exposed outdoors. However, later on as results became available it was realized that the experiment could also provide important information on the effect of solar radiation on the ecology of fungi colonizing exposed samples. Therefore, initially a split-split-plot design was used to examine the effect of different components of the solar spectrum and four chemical treatments at four different concentrations (chemical loads) on the color of wood surfaces. The design included five decking boards cut from five different trees (blocks), which provided replication at the higher level. Each sample (whole-plot) cut from these decking boards was 151 sub-divided into 4 areas, which were assigned to three treatments plus a control (water) (sub-plots). Such areas were then sub-divided into four strips (sub-sub-plots), which were randomly assigned to the four chemical loads. The samples were exposed in racks under one of five different filters that blocked selected regions of the solar spectrum (Evans et al., 2008). The resulting experimental design accounted for random variation in wood properties of decking samples, that due to exposure of samples under various filters in different testing racks (spatial effects of location of samples between and within racks) and that due to the spatial effect of location of different chemical treatments and chemical loads. Separate analyses of variance (ANOVA) were performed after the first 4 weeks of exposure, every two weeks until week 20 and then at weeks 24, 32 and 40. Data for fungi colonizing and staining the wood were only acquired from untreated areas of the samples. Therefore, such data were analyzed as a factorial experiment with random blocks. ANOVA was performed to examine the effect of filter type (F), fungal species (S) and F x S on the frequency of isolation of fungi and Simpson index for fungal diversity, and filter type on the area colonized by fungi (stained area). Analysis of variance (ANOVA) 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), and as a result of such tests the data for fungal diversity were transformed into natural logarithms and analyzed as logarithms. After ANOVA (p<0.05), significant differences were estimated using Fisher\u00E2\u0080\u0099s least significant test (l.s.d.). Results are presented in graphs as means and either standard error of the differences (s.e.d.) or l.s.d. bars can be used to 152 compare means. The output from the statistical analyses of data in this Chapter is appended to this thesis (Appendix 3). A summary of the experimental design is presented in Table 5.1. Table 5.1: Summary of experimental design used to test the effect of solar radiation on wood surfaces and fungal colonization Blocks Exposure type Wood samples Chemical treatments Chemical loads Strips per sample 1 5 + control 6 3 + water 4 16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 + control 6 3 + water 4 16 5.2.2 Wood samples The same five flat-sawn southern pine boards used for the experiment described in Chapter 3 were used in this experiment. The preparation of samples was the same as that of the samples prepared for the experiment described in Chapter 3. Then, sixteen strips, 20 mm wide, were cut into the upper face of each sample by cutting 15 grooves, 3 to 5 mm deep (transverse to the grain), with a band saw Meber (Model SR-500). Strips were isolated from each other by filling the grooves with a hot melt resin (commercial grade) applied with a heat gun. The end grain of samples was sealed with epoxy resin (Quick cure 5; System three resins, Inc. WA, USA) to restrict rate of drying and the development of end checks. 153 5.2.3 Chemical treatments Three chemicals plus a control were applied to the surface of the wooden samples to test their ability to decrease color changes of wood during exposure to different wavelengths of the solar spectrum. Treatments included: (1) carpropamid, an inhibitor of dihydroxynaphthalene (DHN) fungal melanins (Bayer Crop Science, Germany); (2) acetic acid, a by-product in wood after acetylation (Glacial acetic acid, Fisher Scientific, Nepeam- Ontario, Canada); (3) tinuvin 384, a benzotriazole UV absorber (Ciba Specialty Chemical Corporation, Tarrytown - New York, USA); and (4) distilled water (control). Each chemical treatment was brushed onto one of the four pre-designated areas on the wood surfaces. Each area consisted of 4 strips, which randomly received one of the 4 chemical loads, determined in agreement with the recommendations of the companies that manufacture the chemicals (Figure 5.1). Grooves at the wood surface and the chemical sealant (described at section 5.2.2) prevented the chemicals from diffusing from one strip to another. Details on the chemical treatments can be found in Table 5.2. 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) Testing area 1: water Testing area 2: Tinuvin 384 Testing area 3: Acetic acid Testing area 4: Carpropamid Chem. load 4 3 1 2 Chem. load 3 4 2 1 Chem. load 4 1 3 2 Chem. load 4 3 2 1 154 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 Chemical treatment Solvent Mother solution concentration (ppm) Chemical load Amount applied (mg) Carpropamid Acetone industrial grade 100 1 0.02 2 0.03 3 0.05 4 0.06 Acetic acid Water 100 1 0.01 2 0.03 3 0.04 4 0.06 Tinuvin 384 Mineral spirit 10000 1 1.00 2 2.00 3 3.00 4 5.00 Distilled Water n/a n/a 1 100.00 2 200.00 3 300.00 4 400.00 5.2.4 Exposure Samples were exposed in racks which contained five horizontal openings for different polymethylmethacrylate filters (CRYO Industries, Rockaway, USA). These filters transmitted selected regions of the solar spectrum (Table 5.3). Matching filters on the sides and ends of the openings prevented unfiltered light from reaching the samples. Five samples, cut from the same board, were randomly assigned to the five different areas in each rack. The samples were oriented parallel to the long axis of the racks on 40 mm wide spacer blocks. The construction of the racks is described by Urban (2005); and Evans et al. (2008). Angled aluminum sheet captured rain water and directed it on to the surface of samples (Figure 5.2). This sheeting and the wooden frame were painted dark brown to minimize reflection 155 of light on to the samples. Racks were inspected daily and dust accumulating on the filters was removed when necessary. Samples were exposed outdoors to the weather, \u00E2\u0089\u0088 400 mm above ground for 40 weeks in Vancouver, Canada. The superficial moisture content of the wood samples was measured from week 10 to 32, using a portable resistance-type moisture meter (Delmhorst RDM\u00C2\u00B3, Delmhorst Instrument Company). Meteorological conditions during the exposure trial are shown in Table 3.2 (Chapter 3). During the trial un-weathered southern pine controls samples were kept in the dark in a conditioning room. Table 5.3: Filters used to block selected regions of the solar spectrum from reaching samples Filter No Filter type Light type transmitted Wavelengths blocked [nm] 1 OP-4 UVB+ UVA+Vis.light+IR None 2 GP UVA+Vis.light+IR 260-345 3 OP-2 Vis.light+IR 260-400 4 GP-Black 1146-0 IR 260-760 5 GP-Black 199-0 No light All IR: infra red; Vis: visible light; UVA: ultra violet light type A; UVB: ultra violet light type B 156 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 157 5.2.5 Determination of wood color and area colonized by fungi Every month during the 40 weeks that the wooden samples were exposed under the different filters their color was measured. Samples were removed from the racks every week during the first 4 weeks of exposure, every two weeks until week 20 and then at weeks 24, 32 and 40. The color of the wood was measured using a portable spectrophotometer and expressed using the CIELab color coordinates as described in Chapter 3 (section 3.2.6). Digital images of the wooden samples, scale 1:1; 96 dpi resolution, were obtained using a desktop scanner (Microtek Scan Maker i800) and the area of wood stained by fungi was quantified using Adobe Photoshop CS3 (Extended version 10.0.1, Adobe Systems Incorporated, USA) as described in Chapter 3 (section 3.2.6). Such quantification was performed only in the control areas (treated with water) of the samples. 5.2.6 Chemical changes at weathered wood surfaces and isolation and identification of fungi FTIR spectroscopy was used to examine chemical changes occurring at untreated wood surfaces when samples were exposed under the filters for 40 weeks. Pieces of wood measuring 20 mm (width) x 60 mm (length) x 8 mm (thickness) were sawn from the control area of 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 as described in Chapter 3 (section 3.2.7). The isolation of fungi from the surface of weathered southern pine samples used the method described in Chapter 3 (section 3.2.2). Four wood fragments obtained from the 158 control area of each sample were used for the fungal isolations from wooden samples exposed outdoors for 40 weeks under the different filters. Isolated fungi were identified using both microscopy and DNA analysis. Molecular techniques were used first to identify fungi. Then, their identities were confirmed by examining their morphological features (Table 3.3, Chapter 3). 5.2.7 Fungal ecology and characterization of isolated fungi The frequency of occurrence of fungi (FIF) colonizing each southern pine sample exposed under the different filters was calculated as follows: FIF = number of fungi of the species i in the sample / total number of fungi in the sample FIF results were grouped into seven categories. Six categories for the most frequently occurring fungi, plus one for \u00E2\u0080\u009Cothers\u00E2\u0080\u009D, which comprised a diverse group of fungi isolated one or two times per sample. Results for the five categories isolated from wood exposed under the different filters are presented in graphs. The diversity of fungi colonizing the southern pine samples exposed under the different filters was quantified using the reciprocal Simpson index, as described in Chapter 3 (section 3.2.3). Isolated fungi were grown on 1% malt extract agar. A 5 mm diameter agar plug, from the original fungal culture, was placed on agar in a 150 mm x 15 mm round Petri dish. A digital image of the hyphal mat on each plate (1:1 scale) at standard conditions of illumination, was obtained after 7 days using a desktop scanner (as described in Chapter 3 section 3.2.4). 159 The diameter of the hyphal mat was measured using Photoshop as described in Chapter 3. After 20 days the plates were re-scanned without their lids and the images were used to calculate the lightness of the hyphal mats as described in Chapter 3. 5.3 Results 5.3.1 Color of wood after exposure Independent analyses of variance were performed on color of wood for each exposure period from 1 to 40 weeks. There was no significant effect of the different chemical treatments, chemical loads and their interactions on the color of wood surfaces. However, filter type significantly affected (P-value > 0.001) the color of southern pine wood samples from week 1 to 40 (end of the exposure trial). The color of southern pine specimens expressed using the CIELab color co-ordinates lightness (L), redness\u00E2\u0080\u0093greenness (a) and yellowness\u00E2\u0080\u0093blueness (b) were measured throughout the 40 week exposure trial. The color of samples that were kept in the dark in a conditioning room (20 \u00C2\u00B1 1 \u00CB\u009AC and 65 \u00C2\u00B1 5% r.h.) for the duration of the trial was also measured. During the first 20 weeks of exposure, lightness of samples exposed under filters 1,2, 4 and 5 decreased, although samples exposed to the most energetic radiation (filters 1 and 2) showed lower lightness values. Conversely, the lightness of samples exposed under filter 3 increased during the first 2 weeks of exposure, but thereafter their lightness decreased. After 20 weeks, less pronounced changes in lightness occurred in all samples. Samples exposed under filters that transmitted UV radiation were darker than those exposed under filters that blocked such radiation (Figure 5.3). 160 Significant differences in redness\u00E2\u0080\u0093greenness [a] of samples exposed under the different filters occurred after the first week of exposure (Figure 5.4). After the first week of exposure samples became greener. Samples exposed under filter 1 and 3 reddened until 8 to 10 weeks of exposure, and then they became greener ([a] value decreased). Greening continued until 20 weeks of exposure for samples that were shielded from UV radiation. Samples exposed under the filters that transmitted UV radiation continued to become greener until the end of the exposure trial. It was noticeable that samples exposed to UV radiation initially reddened significantly more than samples shielded from UV radiation, but the latter were generally greener than samples exposed to UV radiation. Significant changes in yellowness\u00E2\u0080\u0093blueness also occurred in all samples except the ones exposed under filter 1, which became more blue (Figure 5.5). After two weeks exposure, samples exposed to UV and visible light (filters 1, 2, and 3) yellowed, but no major changes were observed in samples exposed under filters that blocked UV and visible light. After 4 to 10 weeks of exposure samples became bluer ([b] value decreased). This trend continued for approximately 20 weeks for all samples except the ones exposed under filter 1. Subsequently, the decrease in [b] was less pronounced except for samples exposed under filter 1, which continued to become bluer until the end of the exposure trial. 161 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) filter 1 l.s.d. filter 2 filter 3 filter 4 filter 5 Unexposed 0 2 4 6 8 10 50 60 70 80 0 5 10 15 20 25 30 35 40 45 Months of exposure Li gh tn e ss [ L] Weeks of exposure 1.1 1.3 1.0 1.0 1.0 1.1 1.4 1.6 2.0 1.7 1.7 1.6 1.6 1.7 1.6 162 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) filter 1 l.s.d. filter 2 filter 3 filter 4 filter 5 Unexposed 0 2 4 6 8 10 0 2 4 6 8 10 12 0 5 10 15 20 25 30 35 40 45 Months of exposure R ed n es s - G re en n es s [a ] Weeks of exposure 0.2 0.6 0.4 0.5 0.5 0.6 0.6 0.6 0.6 0.6 0.5 0.6 0.5 0.5 0.4 163 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) filter 1 l.s.d. filter 2 filter 3 filter 4 filter 5 Unexposed 0 2 4 6 8 10 10 15 20 25 30 35 0 5 10 15 20 25 30 35 40 45 Months of exposure Ye llo w n e ss - B lu e n es s [b ] Weeks of exposure 0.7 1.1 0.7 0.7 0.7 0.9 1.0 1.0 1.1 1.1 1.0 1.0 1.0 1.0 0.9 164 5.3.2 Area colonized by fungi The area colonized (stained) by fungi on the control strip of samples (treated with water) was analyzed independently for each exposure period from 1 to 40 weeks. Analysis of variance indicated that after 2 weeks of exposure the area stained by fungi was significantly affected (P-value < 0.001) by filter type. Dark stains started to appear 6 to 8 weeks after the southern pine samples were exposed outdoors under the different filters (Figure 5.6). However, small black fungal colonies appeared as early as the second week of exposure (Figure 5.7). These colonies increased in number over the next four to five weeks. After 12 weeks of exposure, the area colonized by fungi increased noticeably, covering approximately 40% to 90% of the total area of exposed specimens (Figure 5.8). After 20 weeks of exposure greater than 90 percent of the entire surface of all specimens was colonized by microorganisms. Samples exposed under the filter that blocked all solar radiation (filter 5) were colonized faster than the other samples. In contrast, samples exposed under the filter that transmitted the entire solar spectrum were less stained than samples exposed under the other filters (Figure 5.9 and Figure 5.10). The increase in area of samples stained by fungi is shown in Appendix 4. 165 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 filter 1 l.s.d. filter 2 filter 3 filter 4 filter 5 0 2 4 6 8 10 0 10 20 30 40 50 60 70 80 90 100 110 120 0 5 10 15 20 25 30 35 40 45 Months of exposure St ai n e d a re a (% ) Weeks of exposure 5.3 7.0 6.9 5.6 4.9 3.6 2.7 2.7 2.3 2.3 166 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 167 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) 168 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) 169 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) 170 5.3.3 Moisture content The superficial moisture content of the southern pine wood samples was measured every week from weeks 10 to 32 of the exposure trial. The moisture content of samples was always below the fiber saturation point and appeared to vary depending on the number and severity of rainfall events. Analysis of variance revealed no significant differences (P-value > 0.05) in the weekly moisture contents of samples exposed under the different filters (Figure 5.11). Filter 1 Filter 2 Filter 3 Filter 4 Filter 5 Rainfall 1 2 3 4 5 6 7 8 9 10 -40 10 60 110 160 210 10 13 16 19 22 25 28 4 8 12 16 20 24 28 32 36 40 Months of exposure R ai n fa ll [m m ] M o is tu re c o n te n t [% ] Weeks of exposure 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 171 5.3.4 Chemical changes at weathered wood surfaces FTIR spectra of samples exposed to the weather under the polymethylmethacrylate filters for 40 weeks and unexposed controls are shown in Figure 5.12. After exposure, wavenumbers of peaks at 1514 and 1462 cm-1 were smaller than those in the control, although the decreases in peak heights were more pronounced for samples exposed to the most energetic radiation (filter 1, 2 and 3). 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). The peaks at a wavenumber of 1740 cm-1 decreased for all samples, and the peak at 1655 cm-1 decreased only for samples exposed under filter 1 and increased for samples exposed under filters 4 and 5. These peaks correspond to conjugated C-O absorptions which typically increase during the early stages of weathering, and then decrease with extended exposure to solar radiation (Anderson et al., 1991; Pandey and Pitman, 2003; Williams, 2005). On the other hand, the peak at a wavenumber of 1158 cm-1 (C-O-C stretching in pyranose rings in cellulose and hemicelluloses, Huang et al. 2008), decreased in comparison to the peak in the unexposed control. Again decreases in peak height were more pronounced for samples exposed under filters that transmitted more energetic radiation. 172 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 and 1158 cm-1 related to carbohydrates. The spectrum of the unexposed control is included for comparison 173 5.3.5 Fungal ecology and characterization of isolated fungi A total of 126 fungi from 21 different genera were isolated from the southern pine samples exposed under the different polymethylmethacrylate filters for 40 weeks. All of the fungi except for one belonged to the phylum ascomycota. The exception was a basidiomycete fungus from the genera Rhizopogon, which was isolated from a sample shielded from light (filter 5). Several very well known colonizers of weathered wood were isolated including Aureobasidium pullulans, Hormonema dematioides, Cladosporium sp., Alternaria sp., Phoma sp, and Epicoccum nigrum Link. Other fungi isolated were Allantophomopsis lycopodina (H\u00C3\u00B6hn.) Carris, Botryosphaeria stevensii Shoemaker, Botryotinia fuckeliana (de Bary) Whetzel, Coniochaeta ligniaria (Grev.) Massee, Lecythophora sp., Leptosphaerulina chartarum Cec. Roux, Lewia infectoria (Fuckel) M.E. Barr & E.G. Simmons, Penicillium expansum Link ex. Thom, Peniophora aurantiaca (Bresadola) von H\u00C3\u00B6hnel & Litschaue, Phialocephala sp., Phialophora sp., Rhizopogon sp., Trichoderma viride Pers., Valsa ambiens (Pers.) Fr., and Exophiala sp. A list of fungi isolated from samples exposed under the different filters and the methods used to identify them are given in Table 5.4 to Table 5.8. In addition, as in Chapter 3, further characterization of isolated A. pullulans in solid media revealed that two types were present: a dark-type and a white-type. The latter white fungus melanized approximately one week after being inoculated onto 1% MEA. 174 Table 5.4: Fungi isolated from samples exposed to UVA+UVB+Vis.light+IR. Primer sequenced for rDNA identification ITS4 Fungi Rack Codification Identification Closest match in Blast GenBank Acc No. Identity Aureobasidium pullulans 3 1_3 Microscopy Aureobasidium pullulans (white) 3 4 Microscopy Aureobasidium pullulans (white) 4 3 Microscopy Aureobasidium pullulans (black) 1 4W rDNA Aureobasidium pullulans AY225167.1 550/554 (99%) Aureobasidium pullulans (black) 2 5_3 Microscopy Aureobasidium pullulans (black) 5 3_1 Microscopy Aureobasidium sp. (black) 4 5 rDNA Aureobasidium sp. AM901687.1 555/557 (99%) Epicoccum nigrum 1 3W rDNA Epicoccum nigrum FJ904918.1 524/528 (99%) Epicoccum nigrum 2 2 rDNA Epicoccum nigrum FJ424264.1 518/521 (99%) Epicoccum sp. 3 2 Microscopy Epicoccum sp. 4 1 Microscopy Epicoccum sp. 4 2 Microscopy Epicoccum sp. 5 1 Microscopy Epicoccum sp. 5 2 Microscopy Hormonema dematioides 1 5W rDNA Hormonema dematioides AY253451.1 557/565 (98%) Hormonema dematioides 3 8 Microscopy Hormonema dematioides 4 4_1 Microscopy Hormonema dematioides 5 3_2 Microscopy Botryosphaeria stevensii 2 3_2 rDNA Botryosphaeria stevensii EU856766.1 532/535 (99%) Botryotinia fuckeliana 3 7 rDNA Botryotinia fuckeliana EF207415.1 507/513 (98%) Alternaria sp. 2 4 Microscopy Cladosporium sp. 1 2W rDNA Cladosporium sp. GU214631.1 528/530 (99%) Phoma sp. 3 6 rDNA Phoma herbarum AY337712.1 469/475 (98%) Coniochaeta ligniaria 3 5_1 rDNA Coniochaeta ligniaria AY198390.1 521/525 (99%) Penicillium sp. 3 1_1 rDNA Penicillium expansium FJ008997.1 554/556 (99%) Phialophora sp. 3 3 rDNA Phialophora sp. AY618679.1 503/505 (99%) 175 Table 5.5: Fungi isolated from samples exposed to UVA+Vis.light+IR. Primer sequenced for rDNA identification ITS4 Fungi Rack Codification Identification Closest match in Blast GenBank Acc No. Identity Aureobasidium pullulans (white) 1 2W rDNA Aureobasidium pullulans GQ376094.1 551/556 (99%) Aureobasidium pullulans (white) 2 6 Microscopy Aureobasidium pullulans (white) 3 2 Microscopy Aureobasidium pullulans (white) 4 3 Microscopy Aureobasidium pullulans (white) 5 3 Microscopy Aureobasidium pullulans (black) 2 5 rDNA Aureobasidium pullulans AM901687.1 543/547 (99%) Aureobasidium pullulans (black) 3 5_1 Microscopy Aureobasidium pullulans (black) 4 5 Microscopy Hormonema dematioides 1 6_1W rDNA Hormonema dematioides AY253451.1 557/567 (98%) Hormonema dematioides 2 2 rDNA Hormonema dematioides AY253451.1 558/563 (99%) Hormonema dematioides 3 3_1 rDNA Hormonema dematioides AY253451.1 545/551 (98%) Hormonema dematioides 4 4_1 Microscopy Hormonema dematioides 5 1 Microscopy Cladosporium sp. 1 3 Microscopy Cladosporium sp. 4 1_2 Microscopy Cladosporium sp. 5 2 Microscopy Epicoccum nigrum 1 5W rDNA Epicoccum nigrum AF455403.1 513/520 (98%) Epicoccum sp. 4 2 Microscopy Botryotinia fuckeliana 5 4 rDNA Botryotinia fuckeliana EU128648.1 501/504 (98%) Botryotinia sp. 2 4 Microscopy Alternaria sp. 1 1 Microscopy Phoma sp. 2 3 rDNA Phoma sp. FJ903335.1 507/509 (99%) Leptosphaerulina sp. 3 4 rDNA Leptosphaerulina sp. AM901681.1 562/564 (99%) 176 Table 5.6: Fungi isolated from samples exposed to Vis.light+IR. Primer sequenced for rDNA identification ITS4 Fungi Rack Codification Identification Closest match in Blast GenBank Acc No. Identity Aureobasidium pullulans (white) 2 1_1 Microscopy Aureobasidium pullulans (white) 3 5_3 Microscopy Aureobasidium pullulans (white) 4 1 Microscopy Aureobasidium pullulans (white) 5 1 Microscopy Aureobasidium pullulans (white) 1 5S rDNA Aureobasidium pullulans AF121283.1 516/516 (100%) Aureobasidium pullulans (black) 1 1_2S rDNA Aureobasidium sp. AM901687.1 548/554 (98%) Aureobasidium pullulans (black) 2 4 rDNA Aureobasidium pullulans DQ787427.1 527/531 (99%) Aureobasidium pullulans (black) 3 3_2 rDNA Aureobasidium sp. AM901687.1 559/565 (98%) Aureobasidium pullulans (black) 4 2 rDNA Aureobasidium sp. AM901687.1 556/556 (100%) Aureobasidium pullulans (black) 5 3 rDNA Aureobasidium sp. AM901687.1 553/555 (99%) Hormonema dematioides 2 2 rDNA Hormonema dematioides AY253451.1 565/569 (99%) Hormonema dematioides 4 3 Microscopy Hormonema dematioides 5 9 Microscopy Phoma sp. 1 5_2 rDNA Phoma sp. AM901684.1 521/537 (97%) Phoma sp. 4 5 rDNA Phoma herbarum AY337712.1 509/516 (98%) Phoma sp. 5 8 rDNA Phoma herbarum AY337712.1 504/512 (98%) Alternaria sp. 1 4 Microscopy Alternaria sp. 3 2_1 Microscopy Cladosporium sp. 2 3 rDNA Cladosporium cladosporioides GQ241276.1 501/505 (99%) Cladosporium sp. 5 2 Microscopy Epicoccum sp. 2 5 Microscopy Epicoccum sp. 5 4 Microscopy Botryotinia sp. 5 5 rDNA Botryotinia fuckeliana EU128648.1 505/511 (98%) Coniochaeta ligniaria 3 5_4 Microscopy Lewia sp. 5 11 rDNA Lewia infectoria GQ376103.1 561/568 (98%) Phialophora sp. 3 4A rDNA Phialophora sp. AY618679.1 503/505 (99%) 177 Table 5.7: Fungi isolated from samples exposed to IR. Primer sequenced for rDNA identification ITS4 Fungi Rack Codification Identification Closest match in Blast GenBank Acc No. Identity Aureobasidium pullulans (white) 3 2_1 rDNA Aureobasidium pullulans GQ376094.1 551/554 (99%) Aureobasidium pullulans (white) 2 7 Microscopy Aureobasidium pullulans (white) 3 5 Microscopy Aureobasidium pullulans (white) 4 4 Microscopy Aureobasidium pullulans (black) 1 7_1W rDNA Aureobasidium pullulans DQ787427.1 528/531 (99%) Aureobasidium pullulans (black) 5 6 Microscopy Cladosporium sp. 1 4 Microscopy Cladosporium sp. 2 5 Microscopy Cladosporium sp. 3 1 Microscopy Cladosporium sp. 4 3 Microscopy Epicoccum nigrum 5 8 rDNA Epicoccum nigrum FJ424264.1 511/514 (99%) Epicoccum nigrum 2 1_1 rDNA Epicoccum nigrum AF455403.1 508/511 (99%) Epicoccum nigrum 3 3 Microscopy Epicoccum nigrum 4 7 Microscopy Hormonema dematioides 1 1_2W rDNA Hormonema dematioides AY253451.1 552/564 (97%) Hormonema dematioides 4 2_1 rDNA Hormonema dematioides AY253451.1 541/552 (98%) Phoma sp. 2 2 rDNA Phoma herbarum AY337712.1 498/507 (98%) Phoma sp. 5 1 rDNA Phoma herbarum AY337712.1 501/508 (98%) Lewia sp. 4 1_1 rDNA Lewia infectoria AF4555012.1 520/539 (96%) Lewia sp. 5 2 rDNA Lewia infectoria EF104194.1 528/531 (99%) Allantophomopsis lycopodina 4 5 rDNA Allantophomopsis lycopodina AB041243.1 498/498 (100%) Botryotinia sp. 3 4 rDNA Botryotinia fuckeliana GU062311.1 470/471 (99%) Exophiala sp. 1 2S rDNA Exophiala xenobiotica DQ182589.1 531/534 (99%) Lecythophora sp. 1 1 rDNA Lecythophora sp. AY219880.1 521/543 (95%) Phialocephala sp. 5 4_1 rDNA Phialocephala sp. AY524844.1 778/836 (93%) Trichoderma viride 4 6 rDNA Trichoderma viride FJ872073.1 548/548 (100%) 178 Table 5.8: Fungi isolated from samples exposed to No light. Primer sequenced for rDNA identification ITS4 Fungi Rack Codification Identification Closest match in Blast GenBank Acc No. Identity Cladosporium sp. 1 1 Microscopy Cladosporium sp. 3 6 Microscopy Cladosporium sp. 4 3_1 Microscopy Cladosporium sp. 4 3_2 Microscopy Cladosporium sp. 5 3 Microscopy Cladosporium sp. 5 4 Microscopy Aureobasidium pullulans (black) 3 2_2 rDNA Aureobasidium pullulans GQ376094.1 556/561 (99%) Aureobasidium pullulans (black) 2 5 Microscopy Aureobasidium pullulans (black) 5 1_1 Microscopy Aureobasidium pullulans (white) 3 2_1_1 rDNA Aureobasidium pullulans GQ376094.1 533/537 (99%) Aureobasidium pullulans (white) 5 2 Microscopy Epicoccum nigrum 1 3 Microscopy Epicoccum nigrum 2 3 Microscopy Epicoccum nigrum 3 3 Microscopy Epicoccum nigrum 4 7 Microscopy Epicoccum nigrum 5 6 Microscopy Hormonema dematioides 2 1 Microscopy Hormonema dematioides 3 1_1 Microscopy Alternaria alternata 4 1_1 rDNA Alternaria alternata FN179367.1 499/500 (99%) Alternaria tenuissima 4 2 rDNA Alternaria tenuissima FJ827038.1 499/501 (99%) Alternaria sp. 1 2 Microscopy Leptosphaerulina sp. 1 4W rDNA Leptosphaerulina chartarum DQ384571.1 465/470 (98%) Peniophora sp. 4 4 rDNA Peniophora aurantiaca AF210825.1 586/607 (96%) Rhizopogon sp. 4 3_3 rDNA Rhizopogon sp. AF377159.1 394/466 (84%) Valsa ambiens 2 6 rDNA Valsa ambiens EF447369.2 530/531 (99%) 179 5.3.5.1 Frequency of isolation Analysis of variance showed a significant effect of species (P-value < 0.001) and a significant interaction of filter type x fungal species (P-value = 0.018) on the parameter FIF (frequency of occurrence of fungi). There was no significant effect of filter type (P-value > 0.05) on FIF. A. pullulans was more frequently isolated from the southern pine samples than any other fungal species. In contrast, the frequency of isolation of Alternaria sp. and Phoma sp. was significantly (P-value < 0.05) lower than that of other fungi (Figure 5.13). The interaction of filter type x fungal species occurred because the frequency of occurrence of A. pullulans was significantly higher than that of all other fungi on samples exposed under filters 1, 2 and 3, whereas under filters 4 and 5 some other fungi were more frequently isolated. For example, under IR and in the absence of light (filter 4 and 5, respectively) Cladosporium sp., Epicoccum sp. and \u00E2\u0080\u009Cothers\u00E2\u0080\u009D were more frequently isolated. The occurrence of H. dematioides was considerably lower after UV radiation was blocked from reaching samples, Figure 5.14. 180 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) 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 0 0.1 0.2 0.3 0.4 Fr e q u e n cy o f Is o la ti o n ( ra ti o ) Fungi isolated from southern pine samples A. pull. H. dem. Clad. sp. Alt. sp. E. nigr. Pho. sp. Others l.s.d. 0 0.1 0.2 0.3 0.4 0.5 1 2 3 4 5 Fr eq u en cy o f Is o la ti o n ( ra ti o ) Filter type 181 5.3.5.2 Fungal diversity Analysis of variance showed that there was no significant effect of filter type on the diversity of fungi isolated from the southern pine wood samples (P-value = 0.839), but the average diversity was lower in samples exposed under filter 1. Results for reciprocal Simpson index are appended to this thesis (Appendix 5). 5.3.5.3 Characterization of fungi on solid culture media The lightness of fungal mycelia after 20 days of growth expressed using the CIELab coordinate (L) is shown in Table 5.9. A. pullulans, H. dematioides, Cladosporium sp., A. lycopodina and Alternaria sp. possessed the darkest mycelia whereas Lecythophora sp., B. fuckeliana, Peniophora sp., Trichoderma viride and Rhizopogon sp. were the lightest. Scanned images of fungi growing on malt extract agar arranged from the darkest to the lightest fungi are shown in Figure 5.15. 182 Table 5.9: Lightness of fungi grown on solid media malt extract agar (1% MEA) Fungi Lightness (L) Ave SD Rhizopogon sp. 83.95 [NA] T. viride 83.19 [NA] Peniophora sp. 81.08 [NA] B. fuckeliana 80.55 [4.19] Lecythophora sp. 80.5 [NA] C. ligniaria 76.92 [0.35] V. ambiens 76.44 [NA] Penicillium sp. 74.98 [NA] Phoma sp. 74.32 [5.88] A. pullulans (white) 70.49 [9.10] Phialocephala sp. 54.73 [1.81] Leptosphaerulina sp. 52.99 [4.60] E. nigrum 52.19 [18.56] B. stevensii 47.88 [NA] Lewia sp. 41.04 [18.42] Alternaria sp. 34.14 [10.01] A. lycopodina 21.62 [NA] Cladosporium sp. 18.81 [4.54] H. dematioides 14.63 [6.75] A. pullulans (black) 14.62 [9.73] The radial growth of isolated fungi after 7 days is expressed as mm growth per week (Table 5.10.). The less melanized fungi, which were isolated more frequently from samples under the filter that shielded wood from UV radiation, grew faster than black fungi. For example, A. lycopodina, B. stevensii, B. fuckeliana, T. viride and V. ambiens, grew the fastest, at 30 to 35 mm per week. More pigmented fungi like Alternaria sp., E. nigrum, Lewia sp. and Peniophora sp. grew at a rate of 20 to 25 mm per week. The remaining fungi, including very dark fungi such as A. pullulans, H. dematioides and Cladosporium sp. grew even more slowly (6 to 17 mm per week). Scanned images of fungi growing on malt extract agar arranged from the fastest to the slowest growing fungi are shown in Figure 5.16. 183 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. 184 Table 5.10: Growth of fungi grown on solid malt extract agar (1% MEA) after 7 days Fungi Radial growth Ave SD T. Viride 35.45 [NA] V. ambiens 34.28 [NA] A. lycopodina 32.32 [NA] B. stevensii 32.24 [NA] B. fuckeliana 30.41 [11.34] Lewia sp. 24.38 [5.10] Peniophora sp. 23.72 [NA] Alternaria sp. 23.59 [3.53] E. nigrum 23.36 [6.06] Leptosphaerulina sp. 17.45 [0.49] Phoma sp. 16.63 [3.75] Penicillium sp. 16.33 [NA] H. dematioides 13.9 [4.87] A. pullulans (black) 12.53 [3.09] A. pullulans (white) 12.47 [2.59] Phialocephala sp. 11.01 [3.47] Cladosporium sp. 10.36 [2.25] Lecythophora sp. 9.01 [NA] C. ligniaria 7.61 [0.12] Rhizopogon sp. 6.88 [NA] 185 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. 186 5.4 Discussion Filter type did not have a statistically significant effect on the diversity of fungi isolated from southern pine samples exposed to the weather, but A. pullulans was more common in samples exposed to more energetic wavelengths (UVB, UVA and visible light). Furthermore, it was isolated less frequently from samples exposed under filters that blocked UV and visible light. Similarly, H. dematioides was also more prevalent in samples exposed to UV light. The fungi that were most frequently isolated from samples exposed to UV and visible light were often highly melanized. This finding accords with results for the color of wood samples exposed under the different filters. Samples exposed to UV and visible light (Filters 1 \u00E2\u0080\u0093 3) were darker than samples exposed under filters that blocked such radiation (Filters 4 \u00E2\u0080\u0093 5). The literature mentions that melanized fungi are better able to survive exposure to more energetic radiation (Wang and Casadevall 1994; Kawamura et al. 1999). In addition, A. pullulans has been reported to be able to metabolize simple sugars and phenolic compounds, which are generated at wood surfaces due to photodegradation of cellulose hemicelluloses and lignin (Bourbonnais and Paice, 1987; Sharpe and Dickinson, 1992). Furthermore, A. pullulans synthesizes \u00E2\u0080\u0098Pullulan\u00E2\u0080\u0099, a polysaccharide that enables its blastospores to adhere to weathered wood surfaces (Bardage and Bjurman 1998). These characteristics undoubtedly provide A. pullulans with competitive advantages when colonizing wood surfaces exposed outdoors. It is possible that other black fungi may share some of these features. For example, A. pullulans and H. dematioides are morphologically similar (Ray et al. 2004) and both colonize weathered wood surfaces. Like A. pullulans, H. 187 dematioides was frequently isolated from samples exposed to UV radiation, but was less frequently isolated from samples exposed under filters that blocked UV radiation. In the absence of UV and visible light the diversity of fungi colonizing wood was slightly greater than that of samples exposed to more energetic radiation. Evidence in the literature supports the finding that the growth of less melanized fungi may be favored by the absence of UV radiation (Singaravelan et al. 2008). FTIR spectroscopy of weathered samples revealed a reduction of bands at 1514 and 1462 cm-1 (stretching vibration of carbonyl groups in lignin benzene rings and C-H deformations in lignin, respectively; Anderson et al. 1991; Pandey and Pitman 2003). Such changes were less pronounced in samples shielded from UV radiation indicating less photodegradation of lignin. For such samples the concentration of simple phenolic compounds that support fungi adapted to metabolize these substances may be limited (Bourbonnais and Paice, 1987; Feist, 1990; Schoeman and Dickinson, 1997). The use of different amounts of carpropamid, acetic acid and tinuvin 384 did not have a significant effect on the color of wood surfaces exposed under the different filters. Tinuvin 384, a liquid UV absorber developed for coatings (Ciba, 1998), was applied directly onto the wood surface without the addition of a binder to prevent it from leaching it is likely that acetic acid and carpropamid were also leached from samples by rain. Color changes in samples exposed under some of the filters resembled those of samples that were fully exposed to the weather (Chapter 3). Color changes during the first 8 weeks of exposure were probably due to photodegradation of wood. Thereafter, the wood\u00E2\u0080\u0099s color was strongly influenced by the colonization of wood surfaces by fungi. The initial color changes in wood exposed outdoors are mainly due to photodegradation of lignin. Feist and 188 Hon (1984) mentioned that photodegradation of lignin causes wood to become red and yellow, which accords with my findings. However, after 8 weeks exposure, fungal staining began to influence the wood color, but samples were not fully stained by fungi until approximately 20 weeks of exposure. Wood surfaces exposed to more energetic radiation were darker than those shielded from such wavelengths. Such darkening appeared to be associated with the presence of a greater proportion of dark, melanized, fungi. Conversely, samples shielded from UV/visible radiation were colonized more frequently by less highly melanized fungi and tended to be greener. Melanin in hyphae and spores of fungi colonizing wood surfaces is responsible for the staining of wood (Brisson et al. 1996) and its biosynthesis can be increased by the presence of UV radiation (Frederick et al. 1999). Therefore, the presence of UV and visible light seems to increase the severity of fungal staining at weathered wood surfaces. My results suggest that UV radiation influences fungal ecology and the color of weathered wood surfaces exposed outdoors. Accordingly, in the absence of UV radiation the adaptations of certain fungi may not provide a competitive advantage and other fungi become more prevalent. A total of 126 fungi from 21 different genera were isolated in this experiment. A number of these organisms are associated with the staining of wood, but the role played by many of the others species is not completely clear. My results suggest that the fungi most likely to be responsible for staining of southern pine samples were A. pullulans, H. dematioides, Cladosporium sp. and Alternaria sp. Other fungi like Epicoccum nigrum and Phoma sp. were also frequently isolated, but they do not possess highly melanized hyphae. Their contribution to staining may come from their spores and propagules (Barnett and Hunter 189 1998). In addition to staining, the literature and results in Chapter 4 suggests that some of the \u00E2\u0080\u0098other\u00E2\u0080\u0099 fungi isolated here might produce soft-rot decay of wood surfaces. For example Cladosporium cladosporioides, Lewia infectoria, Phialophora sp., Phialocephala sp., Alternaria sp. and C. ligniaria have all been found to produce soft-rot (Rajderkar, 1966; Hale and Eaton, 1985; Morrell and Zabel, 1985; Allmer et al. 2006; Zyani et al., 2009). In addition, L. hoffmanni can metabolize phenolic compounds, and Phoma spp. have been isolated from soft-rotted wood (Savory 1954; Bugos et al. 1988). The presence of these fungi at the surface of weathered wood suggests that soft-rot could occur if the conditions were favorable for fungal growth. Soft-rot fungi were poorly melanized when grown in solid media. Therefore, a valid question is how these fungi can withstand the unfavorable conditions present at wood surfaces exposed to weathering? Two mechanisms are proposed to account for this. The first one is the use of sporulative strategies, for example, propagules such as sporodochia and spore aggregations, which are resistant to UV radiation (Barnett and Hunter, 1998; Rotem and Aust, 1991); and the second is colonization and growth of soft-rot fungi under the surface of wood that is heavily colonized by staining fungi. The hyphae of staining fungi are rich in melanin, which strongly absorbs UV radiation between 250 to 700 nm (Suryanarayanan et al. 2004). The melanin concentrated in the fungi colonizing the weathered surface layer may absorb part of the UV radiation that is incident upon the surface, thereby reducing the amount that reaches sub-surface layers. As a result less highly melanized fungi might be able to grow in this sub-surface layer. Differences in melanin production by the different fungi isolated here were not examined. Therefore, future work should focus on gaining a better understanding of the relationship 190 between exposure to UV light and the production of melanin by staining fungi. Understanding the colonization of weathered wood by fungi is also a key step in developing new protective treatments to maintain the color and appearance of wood exposed outdoors. 191 5.5 Conclusions The experimental results in this Chapter show that changes in fungal ecology of wood surfaces occurred when UV and visible light were blocked from reaching the surface of wood. Under UV and visible light A. pullulans was the dominant fungus colonizing southern pine wood samples, but when such radiation was blocked other fungi became more common. Results also indicate that color changes at exposed wood surfaces during the first 8 weeks of exposure seem to be related to photodegradation of wood. Thereafter, changes appeared to be influenced to a greater extent by the staining of wood by fungi. Therefore, I conclude that solar radiation is an important factor affecting the fungal flora at wood surfaces, and also the color of weathered wood surfaces. Due to their frequency of isolation and the fact that they possess dark mycelia, A. pullulans, H. dematioides, Cladosporium sp. and Alternaria sp. seem to be the fungi most responsible for the grey colorization of weathered wood surfaces. However, the role played by a number of other isolated fungi is unclear. It is possible they could cause soft-rot decay immediately below the weathered wood surface. The results in this chapter enlarge our understanding of aesthetic disfiguration of wood surfaces exposed outdoors. However, the results may also be relevant to situations where wood surfaces are shielded from UV radiation for example by building components or beneath semi-transparent finishes. 192 6. Chapter 6: Effect of UV radiation on melanization and growth of fungi isolated from weathered wood surfaces 6.1. Introduction A large number of black moulds colonize and stain weathered wood surfaces, but the stain only extends few millimeters into the wood (Duncan 1963; Dickinson 1971; Savory 1973). The black-blue stain caused by these fungi occurs because the fungal hyphae growing within wood\u00E2\u0080\u0099s cell lumens, parenchyma cells and resin canals are heavily pigmented. These pigments, which absorb visible radiation and hence are dark brown, are referred to as melanins (Brisson et al., 1996; Butler and Day, 1998). Fungi synthesize melanins via enzymatic or auto-oxidative reactions of phenols, amino acid derivates or amino sugars (Paim et al. 1990; Butler and Day 1998). Melanin in fungal hyphae enhances the survival of fungi under environmental stresses (Henson et al. 1999; Butler and Day 2001). For example, melanin present in fungal conidia reduces damage caused by UV light, solar radiation, \u00CE\u00B3- radiation, and X-rays (Fogarty and Tobin 1996; Butler and Day 1998; Henson et al. 1999). Melanins may also play a role in fungal resistance to desiccation and extreme temperatures (Fogarty and Tobin 1996; Butler and Day 1998). The degree of protection provided by melanin against UV light is proportional to the concentration of melanin in fungal cell walls (Butler and Day, 1998; Durrell, 1964; Fogarty and Tobin, 1996). Accordingly, non-melanized hyphae are more susceptible to UV radiation than melanized ones when they are exposed to different doses of UV light at 254 nm (Wang and Casadevall, 1994). Similarly, Kawamura et al. (1999) found that melanin conferred UV tolerance to Alternaria alternata, and Frederick et al. (1999) found that hyaline hyphae of the fungus G. graminis var. graminis 193 melanized upon irradiation with UV light. As a result the melanized hyphae were more tolerant to UV radiation compared to non-melanized (mutant) hyphae of the same fungal species. Consequently, pigmented (melanized) fungi may have adaptive advantages in environments where they are exposed to UV radiation. It is well documented that the most successful organisms colonizing weathered wood surfaces outdoors are black/dark moulds (Duncan, 1963; Sell, 1968; Dickinson, 1971; Sharpe and Dickinson, 1993). It has been suggested that these dark moulds occupy this niche because they are capable of withstanding long periods of dry conditions, relatively high temperatures and high levels of UV radiation at exposed wood surfaces (Duncan, 1963). It seems likely that these adaptations are due in part to their ability to synthesize melanin. In this chapter I hypothesize that fungi isolated from weathered wood will respond to elevated levels of UV radiation by increasing their production of melanin and as a result will be able to survive such exposure better than fungi that lack the ability to respond in the same way. To test this hypothesis the melanin, biomass and spore production, radial growth and mycelial color of Aureobasidium pullulans (de Bary) G. Arnaud [strains R2F32.2 and R1F22W] and Cladosporium cladosporioides (Fresen.) de Vries [strain R2F33], isolated from weathered wood, were evaluated under three conditions, (1) exposure to artificial UV (340 nm); (2) exposure to visible light (450 nm); (3) and complete darkness. Two albino fungi: A. pullulans [strain ATCC 42371] and Ophiostoma piliferum (Fries) Syd. & P. Syd [strain Cartapip97]; and one pigmented O. piliferum [strain TAB28] were used as controls. 194 6.2. Materials and methods 6.2.1. Experimental design A factorial experiment was designed to examine the effect of different light conditions on melanin, biomass and spore production, radial growth and mycelial color of wood surface fungi grown on artificial media. The design included 6 blocks, which provided replication at the higher level, three light conditions: UV light, visible light and darkness, and 6 different fungi. Analysis of variance (ANOVA) was used to examine the effect of fixed factors (light conditions and fungal species) on factors of interest. The analysis of data was performed using the Software Genstat v. 12 (VSN International 2009). The assumptions of ANOVA (ANOVA) were tested prior to the analysis (normality of residuals and homogeneity of variances), and as a result the spore concentration and radial growth data were transformed into natural logarithm (LN) and analyzed as logarithms. Significant differences between means were estimated using Fisher\u00E2\u0080\u0099s least significant test (l.s.d.). Results are presented in graphs as means and these means can be compared using the relevant standard error of the differences (s.e.d.) or l.s.d. bars. Detailed statistic outputs of the analyses in this chapter are appended to this thesis (Appendix 6). A summary of the experimental design is presented in Table 6.1. 195 Table 6.1: Summary of experimental design used to test the effect of different light sources on fungal development and melanization Blocks Exposure (light sources) Fungal species Petri dishes 1 3 6 18 . . . . . . . . . . . . . . . . . . . . 6 3 6 18 6.2.2. Fungi and culturing conditions Six ascomycete fungi were selected including three isolates from weathered wood: Aureobasidium pullulans (de Bary) G. Arnaud [strains R2F32.2 and R1F22W] and Cladosporium cladosporioides (Fresen.) de Vries [strain R2F33], which were selected because they were frequently isolated from weathered wood and were deeply pigmented. Three control fungi were used: albino species of A. pullulans [strain ATCC 42371] and non pigmented Ophiostoma piliferum (Fries) Syd. & P. Syd [strain Cartapip97] and a pigmented Ophiostoma strain, O. piliferum [strain TAB28]. The albino A. pullulans was donated by Viance LLC. Albino and pigmented controls were included in the experiment to compare their melanin production under dark and light conditions with those of test fungi. All fungi were cultured in 100 mm x 15 mm Petri dishes with 1% MEA Difco media at room temperature, and sub-cultured in identical plates every two weeks to ensure the cultures were fresh. For the experiment 60 mm x 15 mm Petri dishes with 1% MEA Difco and a cellophane layer were used. These plates were inoculated with 7 mm (diameter) agar plugs containing fresh fungal mycelia. The plate\u00E2\u0080\u0099s lids were replaced by UV transparent quartz 196 glass disks 63.5 (diameter) x 1.6 (thickness) mm (Technical Glass Products, Inc. Painesville, OH, USA) which transmit radiation between 245 to 780 nm (Figure 6.1). After inoculation the plates were sealed with parafilm, and the fungi were allowed to grow in the dark at room temperature for two days before they were exposed to light. Digital (TIFF) images of the plates were taken using a Microtek Scan Maker i800 scanner, as described above (Chapter 3, section 3.2.4). 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) 6.2.3. Exposure Plates containing test fungi and controls were exposed to different light conditions in separate exposure units. These units were rectangular boxes made of oriented strand boards. The boxes were painted on the inside with matt paint to reduce reflection, and a sheet of black cloth at the front of the box prevented external light from shining into the Quartz glass disk Petri dish glass 0 10 20 30 40 50 60 70 80 90 100 245 260 320 365 450 505 610 625 635 645 670 770 780 Tr an sm it ta n ce [% ] Wavelength [nm] 197 box, but there was still circulation of fresh air into the interior of the box. The unit that exposed fungi to UV light contained 2 UV tubes 340 nm, 40 W (Q-Lab Corp.) (Figure 6.2a). The other two units contained 2 fluorescent tubes (F40L/AQ/ECO wide spectrum 40W, General electric) (Figure 6.2b), or no light source. Irradiance charts for both tubes types were kindly provided by the manufacturers (Figure 6.2 c and d). Fungi were exposed in these boxes to 1700 (\u00C2\u00B5W x m-2) of UV radiation or 114 (\u00C2\u00B5mol x s-1 x m-2) of visible light for 7 days. The experiment was performed in a conditioning room at 20\u00C2\u00B0C \u00C2\u00B1 1\u00C2\u00B0C at 65% \u00C2\u00B1 5% r.h. 198 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 199 6.2.4. Determination of radial growth, mycelial color, spores, biomass and melanin production 6.2.4.1. Measurement of radial growth of fungal colonies Digital images of the plates were obtained at the start and the end of the experiment using a desktop scanner (as described in section 6.2.2). Images were analyzed using Adobe Photoshop CS3 Extended, version 10.0.1 (Adobe System Incorporated, USA). The ruler tool of the software was used to quantify the radial growth of the fungi during the 7 day exposure period as described in Chapter 3 (Figure 3.1a). For comparative purposes radial growth (mm/week) was expressed as a function of fungal biomass [(mm/week)/mg biomass]. 6.2.4.2. Lightness of mycelia Images of the plates without their quartz glass lid after exposure were acquired using a desktop scanner (as described in section 6.2.2). The lightness of the mycelial mats was estimated using these images as described in Chapter 3 (Figure 3.1b) (Papadakis et al. 2000). Images were loaded into Photoshop and the lightness of mycelia was measured and expressed using the CIE coordinate, L (lightness on scale of 0, [black] to 100 [white]) (International Commission on Illumination 2007). 200 6.2.4.3. Spore production Three mililiters of nano pure water were placed on the surface of each exposed plate. Fungal spores in the moistened mycelial mat were loosened from the mat using a sterile glass rod. The supernatant from each plate was collected in separate 15 mL falcon tubes. The concentration of spores in each tube was determined with a hemocytometer (La Fontaine, Germany) in accord with manufacturer\u00E2\u0080\u0099s guidelines (Figure 6.3) (Smith et al., 1988). The concentration of spores was expressed as colony forming units per mL per week, but it was also expressed as a function of the fungal biomass [((CFU/mL)/week)/fungal biomass] to be consistant with the expression of radial growth. Figure 6.3: Determination of spore concentration by hemocytometer counting 6.2.4.4. Mycelial biomass Mycelial mats growing on top of cellophane layers were scraped from the plates using a sterile scalpel and transferred onto pre-weighed and labeled glass plates. The plates were 201 placed in an oven and dried (100 \u00C2\u00B1 5 \u00C2\u00B0C) for 24 h and then allowed to cool for 2 h in a glass dessicator over silica gel. The plates were re-weighed using an analytical balance (A & D; Model GR-200 from B.C. Scale Co. Ltd; 210 g x 0.0001 g). Biomass produced by fungi in each plate was expressed as mg/week. 6.2.4.5. Isolation of melanin Dry fungal biomass was rehydrated with 2 mL of nano pure water and placed in separate 5 mL collection tubes containing ceramic beads. One mL of water was added to each tube. Tubes were vortex-mixed until fungal mycelia in the tubes disintegrated. Extraction of melanin from hyphae used hot NaOH according to the method of Gadd (1982). This method involved autoclaving the fungal suspensions in 50 mL glass vials containing 10 mL of 1M NaOH for 1.5 hours at 121\u00C2\u00B0C. The supernatants were then collected and the vials were re- filled with 10 mL of 1M NaOH and the extraction procedure was repeated. Twenty mL of supernatant was collected from each tube and filtered using 40 \u00C2\u00B5m sterile cell strainers Fisherbrand (Fisher Scientific, AB; Canada). Melanin in the solutions was precipitated by adding 5 mL of 7M HCl (final pH approx. 1) to each tube and leaving the tubes overnight at room temperature. Tubes were centrifuged (3700 rpm, 15 min.) and the raw melanin (pellet) residues were purified by acid hydrolysis using the method of Bell and Wheeler (1986). Purification used sealed glass vials containing 5 mL 7M HCl and stored at 100\u00C2\u00B0C for 12 hours. The tubes were allowed to cool and centrifuged (as above). Pigments were dissolved in 5 mL 1M NaOH overnight, purified from solid by centrifugation and transferred into new 15 mL falcon tubes. 202 6.2.4.6. Melanin concentration The concentration of melanin in material extracted from fungal mycelium (above) was calculated using absorbance of UV/Vis. light at 420 nm measured using a UV-VIS spectrophotometer (Varian Model Cary 50 Bio) (Singaravelan et al., 2008). Melanin in the solutions was precipitated with HCl (as above), washed with nano pure water, dried and weighed. Then, known amounts of dried melanin, were dissolved in 1M NaOH and the absorbance of light at 420 nm was measured for different solution concentrations (50, 20, 10, 5, 2, 1.42, 1 and 0.5 percent). Absorbance data were used to plot a calibration curve, for absorbance versus concentration of melanin (data available in Appendix 7). These curves were used to calculate the concentrations of the purified melanin in the original parent solutions. Finally the amount of melanin produced by the cultured fungi after seven days of growth in each plate was calculated as: CM plate = CM extracted solution x SW solution / biomass Where: CM plate: concentration of melanin in each plate (mg melanin / mg biomass)/week CM extracted solution: concentration of melanin in each extracted solution (calculated from calibration curve and expressed as mg melanin/g solution) SW solution: standard weight of each extracted melanin solution (5.20 g) biomass: fungal biomass per plate (mg) 203 6.3. Results There were significant effects of exposure to light (E), fungal species (F) and interactions of these two factors (ExF) on melanin concentration, biomass, spore concentration, radial growth and lightness of fungal cultures. 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 P-value Source of variation melanin concentration biomass LN (1+spore concentration) LN (1+ radial growth) lightness Exposure 0.032 <.001 <.001 <.001 <.001 Fungi <.001 <.001 <.001 <.001 <.001 Exposure x Fungi 0.007 <.001 <.001 <.001 <.001 6.3.1. Melanin concentration Melanin production of fungi isolated from weathered wood and controls are depicted in Figure 6.4. The melanin concentration of A. pullulans increased with exposure to both visible and UV light. However, variation occurred between strains. A. pullulans [R2F32.2] showed no significant difference in melanin production when grown under UV or visible light. Melanin concentration of this strain when grown in the absence of light was, however, significantly lower than those of cultures grown under UV or visible light. A. pullulans [R1F22W] behaved differently. This strain produced significantly more melanin when grown under UV light than when it was grown under visible light or in the absence of light. Furthermore, there was no significant difference in the melanin production of cultures grown under visible light or in the dark. The production of melanin by A. pullulans [ATCC 42371], an albino control, showed the same trends as that of A. pullulans [R2F32.2]; although it produced higher concentrations of melanin than the other strains when grown 204 under UV or visible light. It should be noted that A. pullulans [ATCC 42371] is classified as an albino in the American Type Culture Collection, however, my results indicate that it can synthesize melanin. Therefore it should not be classified as an albino strain. Cladosporium cladosporioides [R2F33], showed no significant differences in melanin production when grown under UV or visible light or in the dark (although the difference in melanin production of cultures grown under UV light or in absence of light was almost significant). This fungus produces high amount of melanin in the dark unlike A. pullulans which showed decreased production of melanin in the absence of light. A. pullulans produced more melanin than C. cladosporioides, although the difference was not statistically significant. The control fungi O. piliferum [TAB28] and [Cartapip97] were unable to synthesize melanin under UV radiation. Under visible light both fungi produced small amounts of pigmentation. However, in the absence of light O. piliferum [TAB28] produced larger amount of pigments. O. piliferum [Cartapip97] produced only small amounts of melanin when grown under visible light or in the dark. 205 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) 6.3.2. Fungal biomass The biomass of fungi grown under different light conditions is shown in Figure 6.5. The two strains of A. pullulans [R2F32.2] and [R1F22W], again behaved differently. A. pullulans [R2F32.2] produced the highest amount of biomass when grown under visible light, but there was no difference in biomass production when it was grown in the dark or under UV light. The production of biomass by A. pullulans [R1F22W] increased significantly when it was exposed to less energetic radiation (visible or no light). The A. pullulans control behaved in the same way as A. pullulans [R2F32.2], although the amount of biomass produced by this fungus was significantly lower than that of the fungi isolated from weathered wood. C. cladosporioides [R2F33] behaved in the same way as A. pullulans [R1F22W] and [ATCC 42371]. Significant differences occurred in cultures grown under UV light or in the dark, although the amount of biomass produced by cultures grown under UV UV light Vis. light No light l.s.d. 0.00 0.05 0.10 0.15 0.20 0.25 A. pull. [R2F32.2 ] A. pull. (albino) [ATCC 42371] A. pull. (white) [R1F22W] Clad. [R2F33] O. pilif. [TAB28] O. pilif. (albino) [Cartapip97] [m g m el an in / m g b io m as s] /w ee k Fungi 206 light was not different from that of cultures of A. pullulans [R2F32.2]. The O. piliferum controls [TAB28] and [Cartapip97], did not grow under UV radiation and their production of biomass when grown under visible light or in the dark was not significantly different from that of C. cladosporioides and A. pullulans [R1F22W]. The biomass of O. piliferum [Cartapip97] cultures was significantly higher when they were grown in the dark than when they were grown under visible light (biomass was almost double that of cultures grown under visible light). 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) 6.3.3. Spore production The production of spores by the fungi when they were grown under different conditions expressed as LN [1+ (unit forming colonies/mL)/mg biomass] is depicted in Figure 6.6. The production of spores by all of the tested fungi appeared to be affected by the presence of UV Vis. light No light l.s.d. 0 2 4 6 8 10 A. pull. [R2F32.2 ] A. pull. (albino) [ATCC 42371] A. pull. (white) [R1F22W] Clad. [R2F33] O. pilif. [TAB28] O. pilif. (albino) [Cartapip97] m g b io m as s/ w ee k Fungi 207 radiation. In the case of A. pullulans [R2F32.2] the presence of UV and visible radiation decreased the production of spores in comparison to that of cultures grown in the dark. The production of spores by A. pullulans control [ATCC 42371] increased when the energy of the incident light decreased (visible light) or when the fungus was grown in the dark. On the other hand, spore production by isolates of A. pullulans [R1F22W] peaked when they were exposed to visible light. The behavior of C. cladosporioides was very similar to that of the A. pullulans control, as spore production of cultures grown under visible light and in the dark was similar. O. piliferum [TAB28] and [Cartapip97] behaved differently. These fungi were unable to produce spores in presence of UV radiation. Nevertheless, under visible light they produced significantly higher amounts of spores than when they were grown in the dark. 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) UV visible light No light l.s.d. 0 1 2 3 4 5 6 A. pull. [R2F32.2 ] A. pull. (albino) [ATCC 42371] A. pull. (white) [R1F22W] Clad. [R2F33] O. pilif. [TAB28] O. pilif. (albino) [Cartapip97] LN [1 +( u n it fo rm in g co lo n ie s/ m L) /m g b io m as s] Fungi 208 6.3.4. Radial growth of fungal cultures The growth of fungal cultures (measured as described in section 3.2.4) when they were exposed to different light conditions is depicted in Figure 6.7 (analyzed as LN [1+ (mm/week)/mg biomass]. The growth of all of the fungi isolated from weathered wood was affected by UV radiation. All fungi grew well in the dark and under visible light. A. pullulans [R2F32.2] grew significantly faster under visible light than when grown in the dark. However, the differences in growth of A. pullulans [R2F32.2] and [ATCC 42371] when grown under visible light or in the dark were not statistically significant. C. cladosporioides behaved in a similar way to A. pullulans [ATCC 42371]. The growth of O. piliferum controls [TAB28] and [Cartapip97] again was seriously affected by UV radiation, as no growth occurred when the fungi were exposed to UV radiation. However, both strains of O. piliferum grew well under visible light and in the absence of light. 209 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) 6.3.5. Lightness of mycelia The lightness of mycelium from fungi isolated from weathered wood and the controls are depicted in Figure 6.8. The results indicate that all strains of A. pullulans were dramatically affected by exposure to UV radiation. The lightness of A. pullulans [R2F32.2] under UV radiation and visible light was similar, but in the absence of light fungal mycelia was significantly lighter. A. pull [R1F22W] and [ATCC42371] behaved in the same way. They became lighter when grown under less energetic light. Similarly, mycelia of C. cladosporioides was lighter when it was grown under less energetic light, but significant differences in lightness were only found in cultures grown under UV radiation or in the absence of light. The control O. piliferum [TAB28] was lighter when grown under visible light UV Vis. light No light l.s.d. 0.0 0.5 1.0 1.5 2.0 2.5 A. pull. [R2F32.2 ] A. pull. (albino) [ATCC 42371] A. pull. (white) [R1F22W] Clad. [R2F33] O. pilif. [TAB28] O. pilif. (albino) [Cartapip97] LN [1 +( m m /w e e k) /m g b io m as s] Fungi 210 (significantly), but [Cartapip97] showed no significant variation in lightness, irrespective of the light it was exposed to. 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 \u00E2\u0080\u0093 100: white UV Vis. No light l.s.d. 0 5 10 15 20 25 30 35 40 A. pull. [R2F32.2 ] A. pull. (albino) [ATCC 42371] A. pull. (white) [R1F22W] Clad. [R2F33] O. pilif. [TAB28] O. pilif. (albino) [Cartapip97] Li gh tn e ss Fungi 211 6.4. Discussion The radial growth, biomass and spore production of fungi isolated from weathered wood and grown on artificial media was affected by exposure to UV light. Under UV light only A. pullulans and C. cladosporioides, and A. pullulans strain [ATCC42371] grew, generated biomass and spores and synthesized melanin. A. pullulans strain [ATCC42371] was expected to act as an albino control, but contrary to expectations it produced more melanin when exposed to UV and visible light than the other microorganisms. Therefore, for the remainder of this discussion results for A. pullulans [ATCC42371] will be grouped along with those of the other strains of A. pullulans, but the stability of its albino condition will be discussed later. The amount of melanin synthesized by A. pullulans and C. cladosporioides increased when they were exposed to UV radiation. The tendency of fungi to increase melanin production under UV radiation has been reported for the fungus Gaeumannomyces graminis (Frederick et al., 1999), but it has not been reported before for fungi isolated from weathered wood. Thus, my experimental data support the hypothesis that A. pullulans and C. cladosporioides are able to increase melanin production when exposed to UV radiation, an adaptation that would be beneficial to their survival at weathered wood surfaces. The ability of melanized moulds to tolerate elevated levels of UV radiation, high temperatures, water deficiency and chemical and radioactive pollution has been documented before (Fogarty and Tobin 1996; Henson et al. 1999; Butler and Day 1998; 2001; Dadachova et al. 2007). My findings also accord with those of Singaravelan et al. (2008), who described the ability of Aspergillus niger to increase melanin production as an adaptive response to elevated levels of solar 212 radiation. In this experiment two strains of A. pullulans, isolated from weathered wood, were tested. These strains were selected because they produced different patterns of melanization when grown on artificial media. A. pullulans [RF2F32.2] produced melanized mycelia almost simultaneously as it grew. In contrast, A. pullulans [R1F22W] produced colorless mycelia during the first two weeks of growth and then there was mild melanization of its mycelial mat. When exposed to UV light both strains were able to produce melanin. This finding suggest that melanin biosynthesis in both of these fungi is an adaptation that can be enhanced by exposure to UV radiation. Accordingly, both strains synthesized less melanin when they were grown under visible light or in the dark. Exposure to visible light or the absence of light, favored the growth and spore production of tested fungi. According to Griffin (1996) \u00E2\u0080\u009CThe effect of light on sporulation and growth can vary from inductive to inhibitive even at different stages of the same sporulative process\u00E2\u0080\u009D. Accordingly, results here indicate that exposure to UV radiation influenced sporulation. Nevertheless, differences in the production of biomass by the different A. pullulans strains were observed. These differences might be the result of biological variations within species. C. cladosporioides behaved differently, for example it showed no significant differences in the production of melanin when exposed to UV or visible light or when grown in the dark. Apparently this fungus produces melanin not only when exposed to UV radiation. Studies on Cladosporium sp. have pointed out that its spores are widely distributed all over the world and are an important component of the biological particles that are suspended in the atmosphere (Iannone et al. 2011). Melanization of spores may be advantageous when they are subjected to low temperatures and high levels of UV radiation in the upper atmosphere. 213 Similarly, O. pilif. [TAB28] produced an equal amount of melanin when grown under visible light or in the absence of light. This fungus is a wild-type strain able to synthesize melanin. Its natural habitat is in insect galleries under the bark of conifer trees where it is not exposed to UV radiation (Perry 1991). UV radiation has been reported to penetrate wood surfaces to depths of approximately 200 micrometers approximately, but it would be unable to penetrate the bark of conifer trees (Kataoka et al. 2004). The albino control O. piliferum [Cartapip97] used here was originally developed as a bio-control agent for removal of extractives from wood chips prior to pulping. According to Behrendt et al. (1995) it is not able to synthesize melanin. However, I found that it produced small amounts of spectroscopically active substances (absorbance at 420 nm), suggesting that it may produce melanin. However, it is important to point out that the method for melanin extraction used here has not been optimized to obtain pure melanins (Gadd 1982; Rosas et al. 2000). Therefore, the presence of small amounts of chromophores in extracts does not conclusively prove that melanin was present. My finding that common fungi isolated from weathered wood produce more melanin when they are exposed to UV and visible radiation changes our understanding of the discoloration of wood surfaces exposed outdoors. For example, it becomes clear that UV radiation and fungi may interact to produce darker surfaces because in presence of UV radiation A. pullulans became darker and more heavily melanized. On the other hand, the presence of a layer extending to a depth of 100 micrometers at wood surfaces, which contains heavily melanized fungi may shield the underlying wood from UV radiation. Fungal melanin absorbs wavelengths between from 250 to 700 nm, but peak absorbance is at 250 nm 214 (Suryanarayanan et al. 2004). Hence, melanized fungi colonizing weathered wood surfaces may protect lignin in sub-surface wood tissues from photodegradation because it strongly absorbs UV radiation (Kalnins, 1966). The \u00E2\u0080\u0098shielding effect\u00E2\u0080\u0099 of melanized fungi might explain why the rate of erosion of wood surfaces exposed to natural weathering decreases over time whereas the rate of erosion of wood exposed to accelerated weathering in the absence of fungal colonization stays constant (Liu 2011). In accord with this suggestion Sailer et al. (2010) proposed a treatment to encourage the complete colonization of wood surfaces by A. pullulans. Sailer et al. reasoned that such a highly melanized biofilm could protect wood surfaces from photodegradation. A. pullulans strain [ATCC42371] is reported to be an albino mutant by Gadd and De Rome (1988); and Gadd et al. (1990). Its definition as an albino implies total inability to synthesize melanin, and, at the genetic level, inhibition of the expression of the polyketide synthase gene (Fleet and Breuil 2002; Starr et al. 2010). My results show that A. pullulans strain [ATCC42371] was able to produce pigmentation when exposed to UV and visible light, although, under normal culturing condition in the laboratory (1% malt extract agar), the isolate was colorless. My results suggest that A. pullulans [ATCC42371] should be re- classified as a white strain similar to those that I isolated from weathered wood surfaces in Chapters 3 and 5, and others reported by previous studies (Schoeman and Dickinson, 1997). The main hypothesis proposed at the start of this chapter can be accepted in part. Further research to better understand the complex interaction between exposure to UV radiation, fungal colonization and the weathering of wood would be desirable. In particular research should examine the occurrence of fungi at wood surfaces and their influence on the depth 215 and extent of photodegradation of underlying woody tissues. It would also be interesting to compare the diversity and ecology of organisms at wood surfaces with those found beneath the surface. 216 6.5. Conclusions My results show that A. pullulans, one of the most successful fungi at colonizing weathered wood, has the ability to increase its production of melanin when exposed to UV radiation. This could be an adaptive response to the high levels of UV radiation found at wood surfaces exposed outdoors. Conversely, C. cladosporioides did not produce more melanin when it was exposed to UV radiation. Therefore, I conclude that not all fungi need such an adaptive response to survive exposure to UV radiation at wood surfaces outdoors, although it seems that fungi lacking this response need to be highly melanized to grow at weathered wood surfaces. Further research on other fungi isolated from weathered wood is needed to strengthen these conclusions. My results also indicate that our current understanding of the discoloration of weathered wood surfaces needs to be revised, as it is clear that darker staining is produced by the interaction of UV radiation and colonization of wood surfaces by fungi. More research on the influence of staining fungi at wood surfaces on the depth and extent of photodegradation of sub-surface woody tissues is needed to better understand the role that fungi play in the weathering of wood. Results in this chapter have provided new insights into the adaptive response of surface fungi to the high levels of UV radiation that they are exposed to at weathered wood surfaces. The next chapter will study the response of two surface fungi to UV radiation when they are prevented from synthesizing the melanin biopolymer that protects them from UV radiation. 217 7. Chapter 7: UV light and melanin biosynthesis inhibitors as potential treatments against fungal staining 7.1. Introduction Discoloration of wood surfaces exposed to weathering has been attributed to the colonization of wood by melanized fungi (Duncan, 1963; Dickinson, 1971). Melanin synthesized by these fungi protects their cells against the deleterious effects of solar UV radiation, extreme temperatures and desiccation (Fogarty and Tobin 1996; Butler and Day 1998; Henson et al. 1999). However, melanin does not re-radiate absorbed radiation as visible wavelengths (Butler and Day, 1998). Therefore, wood colonized by melanized fungi is a blue/dark color due to the photochemical properties of the melanin contained in the fungal cells (Brisson et al., 1996). Melanin synthesis by fungi can be blocked by specific chemicals. These chemicals have been extensively used in agriculture as fungicides, for example to prevent blast rice disease (Kurahashi 2001). The chemicals target specific enzymes involved in the synthesis of dehydroxynaphthalene (DHN) melanins by ascomycetes. DHN melanins are synthesized by some of the fungi that colonize weathered wood surfaces (Kawamura et al., 1997; Kogej et al., 2004). Therefore in principle melanin biosynthesis inhibitors (MBIs) should be able to reduce the level of fungal melanization and staining of weathered wood surfaces. Furthermore, if surface staining fungi lack sufficient melanin, when exposed outdoors, their death might be hastened because many of them seem to tolerate solar UV radiation primary because of their ability to synthesize melanin (Durrell 1964; Wang and Casadevall 1994; Kawamura et al. 1999). 218 In this chapter I hypothesize that the interruption of melanin biosynthesis in fungi colonizing weathered wood can be achieved by using MBIs. If this occurs fungal staining at wood surfaces would decrease because of high mortality of melanized fungi following exposure to UV radiation. Two in-vitro experiments were carried out to test this hypothesis. In the first one, three different MBIs, cerulenin, tricyclazole and carpropamid, were added to artificial media (1% malt extract agar). Spores of two melanized fungi that were frequently isolated from weathered wood, Aureobasidium pullulans (de Bary) G. Arnaud and Cladosporium cladosporioides (Fresen.) de Vries, were inoculated onto the plates. The plates were exposed to artificial UV or visible light and the growth of the fungi on the plates was examined. For the second experiment spruce veneers were impregnated with carpropamid, which were then inoculated with spores of A. pullulans. Veneers were exposed to artificial UV or visible light. The effectiveness of the treatment was evaluated by measuring the staining and color of the treated veneers and untreated controls. 219 7.2. Materials and methods 7.2.1. In-vitro testing of the melanin biosynthesis inhibitors cerulenin, tricyclazole and carpropamid, and the fungicide quinoxyfen 7.2.1.1. Experimental design A factorial experiment was designed to test the effects of different melanin biosynthesis inhibitors (MBIs) on the survival of fungal colonies grown on artificial media and exposed to either UV or visible light. The experiment included three MBIs, two fungal species, and two exposure conditions. Controls consisting of plates supplemented with a fungicide and the solvent used to prepare the MBIs (acetone) were also included. The design accounted for random variation in media preparation, fungal inoculation, exposure and spatial distribution of the plates under the different light sources. Analysis of variance (ANOVA) was used to examine the effect of fixed factors and their interactions on the number of fungal colonies that grew on plates. Analysis of data was performed using the software Genstat v. 12 (VSN International 2009). The assumptions of ANOVA were tested prior to the analysis (normality of residuals and homogeneity of variances). However, no transformation of data was required. Significant differences (p < 0.05) between means were tested using Fisher\u00E2\u0080\u0099s least significant test (l.s.d.). Results are presented in graphs as means and these means can be compared using either standard error of the differences (s.e.d.) or l.s.d. bars. The detailed statistic output for the analysis of data is appended to this thesis (Appendix 8). A summary of the experimental design is presented in Table 7.1. 220 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 Blocks Exposure (light sources) Chemicals supplemented Concentrations tested Fungal species Petri dishes 1 2 4 + control 1 2 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 4 + control 1 2 20 7.2.1.2. Chemicals and culture media Three MBIs were selected based on their ability to interrupt the biosynthetic pathway for DHN melanins. MBIs were: (1) Cerulenin [(2R,3S)-3-[(4E,7E)-nona-4,7-dienoyl]oxirane-2- carboxamide], (2) tricyclazole [5-methyl-1,2,4-triazolo[3,4-b]benzothiazole], and (3) carpropamid [(1R*,3S*)-2,2-dichloro-N-[1-(4-chlorophenyl)+ethyl]-1-ethyl-3- methylcyclopropanecarboxamide] (Figure 7.1 a, b and c, respectively). In addition the fungicide quinoxyfen [5,7-dichloro-4-(4-fluorophenoxy)] quinoline was tested because of its efficacy against powdery mildews (Coghlan et al. 1991) (Figure 7.1d). Chemicals were purchased as powders from Sigma-Aldrich Co, St Louis, MO, USA. Stock solutions of these chemicals at 100 ppm were prepared in acetone (industrial grade) and added to autoclaved malt extract agar (MEA) (1% Difco), when still liquid (45\u00C2\u00B0C approx.) under magnetic stirring, until the final desired concentration of 10 ppm was reached. Control plates were supplemented with acetone at a level similar to that used to dissolve the MBIs. Media supplemented with these chemicals and acetone was poured into 60 x 15 mm Petri dishes. Plates were stored at 4\u00C2\u00B0C until they were inoculated with test fungi. 221 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 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN) to scytalone and 1,3,8-trihydroxynaphthalene (!,3,8-THN) 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 7.2.1.3. Inoculation of media with A. pullulans and C. cladosporioides Aureobasidium pullulans (de Bary) G. Arnaud [strain R2F32.2] and Cladosporium cladosporioides (Fresen.) de Vries [strain R2F33] were used as the test fungi. These fungi were selected based on their frequency of isolation from weathered wood (Chapters 3 and 5) and their ability to synthesize melanin (Chapter 6). Fungi were cultured in 100 x 15 mm Petri dishes with 1% MEA Difco media at room temperature, and sub-cultured in similar plates every two weeks to ensure that fresh material was available for the duration of the experiment. Spores in the plates were harvested under sterile conditions after 1 week by flooding the plates with 3 mL of nano pure water. Spores produced by the fungi were 222 loosened from fungal mycelia using a sterile glass rod. The supernatants were collected in 15 mL falcon tubes and the concentration of spores in these solutions was determined using a hemocytometer (La Fontaine, Germany) in accord with manufacturer\u00E2\u0080\u0099s guidelines. Then the supernatants were adjusted to one spore per \u00C2\u00B5L (Figure 6.3). Plates containing MBIs were inoculated with 50 \u00C2\u00B5L of spore solutions, which were evenly spread onto the media using a glass rod. The lids of the plates were replaced by quartz glass disks 63.5 (diameter) x 1.6 (thickness) mm (Technical Glass Products, Inc. Painesville, OH, USA) which transmitted radiation between 245 to 780 nm (Figure 6.1). The plates were sealed with parafilm and fungi were allowed to grow in a dark room at ambient temperature for 24 hours before they were exposed to UV radiation or visible light. 7.2.1.4. Exposure to UV and visible light and quantification of number of fungal colonies after exposure Plates containing the tested fungi and control were exposed to UV or visible light in separate wooden boxes, painted inside with matt paint to reduce reflection of light, as described in Chapter 6 (section 6.2.3). A box with no light source acted as a control. Fungi were exposed in these boxes to 1700 (\u00C2\u00B5W x m-2) of UV radiation or 114 (\u00C2\u00B5mol x s-1 x m-2) of visible light for 7 days. The experiment was performed in a conditioning room at 20\u00C2\u00B0C \u00C2\u00B1 1\u00C2\u00B0C and 65% \u00C2\u00B1 5% r.h. After 7 days of exposure to UV or visible light, digital images of the plates without their lids, at a resolution of 300 dpi, were obtained using a Microtek Scan Maker i800 desktop scanner. Digital images of the mycelial mats were loaded into Adobe Photoshop CS3 223 Extended, version 10.0.1 (Adobe System Incorporated, USA) and observed at a magnification of 150%. Individual fungal colonies within each plate were manually counted and labeled using the software\u00E2\u0080\u0099s counting tool (Figure 7.2). Figure 7.2: Screen-shot of the software used to count the number of fungal colonies in each plate 224 7.2.2. Effect of chemicals and UV radiation on fungal staining of wood 7.2.2.1. Experimental design A factorial experiment was used to test the effect of the MBI carpropamid and the fungicide quinoxyfen on the staining and color of wood veneers inoculated with an aqueous solution of fungal spores or water and exposed to UV or visible light. The experimental used two exposure conditions (UV and visible light), two chemicals at two concentrations, and one fungal species. The control was veneers inoculated with sterile water. Five blocks provided replication at the higher level. The resulting design accounted for random variations in the media preparation, impregnation of chemical into the wood, inoculation with fungi, exposure and spatial distribution of plates in each light box. Analysis of variance (ANOVA) was used to determine the effects of the fixed factors (chemicals and light conditions) and random effects on the area of wood stained by fungi and color differences of samples. Analysis of data used Genstat v. 12 (VSN International 2009). The assumptions of ANOVA were tested prior to the analysis (as metioned in section 7.2.1.1) and as a result fungal staining (ratio of stained area of treated veneers divided by stained area of untreated veneers) was transformed into natural logarithms. Significant differences (p<0.05) were tested using Fisher\u00E2\u0080\u0099s least significant test (l.s.d.). Results are presented in graphs as means, and either standard error of the differences (s.e.d.) or l.s.d. bars can be used to compare means. Statistical output for this section is appended to this thesis (Appendix 9). A summary of the experimental design is presented in Table 7.2. 225 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 Blocks Exposure (light sources) Chemicals supplemented Concentrations tested Fungal species Wood species Petri dishes 1 2 2 + control 2 1 1 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 2 + control 2 1 1 16 7.2.2.2. Wood samples White spruce (Picea glauca, Moench (Voss)) was selected for this experiment because it is susceptible to staining and its homogeneous properties make easy to cut thin veneers from it (Forest Products Laboratory, 1999; Harrington, 1988). Wood veneers measuring 18 mm (radial) x 25 mm (tangential) x 85 mm (longitudinal) were cut from blocks of white spruce as described in Chapter 4. Veneers were placed on glass plates and clamped at their ends as described in Chapter 4, and dried at room temperature for seven days. Each veneer was cut with a scissor to produce approximately 4 sections, each 20 mm in length. These sections were mixed together and randomly allocated to the different treatments. Pieces of veneer were oven dried (100 \u00C2\u00B1 5\u00C2\u00B0C) for 24 hours and sterilized in an autoclave at 121\u00C2\u00B0C and 103.4 kPa for 20 min. They were then placed in sterile Petri dishes and sealed until they were needed. 226 7.2.2.3. Preparation of solutions and impregnation of wood veneers The melanin biosynthesis inhibitor (MBI) carpropamid and the fungicide quinoxyfen were used to treat wood sections. Fifty mL solutions of these chemicals in acetone (industrial grade) at concentrations of 3000 or 6000 ppm were prepared in 100 mL glass beakers. Five batches of 4 veneers sections were immersed in these solutions for 24 hours. Each batch represented a \u00E2\u0080\u0098block\u00E2\u0080\u0099. Similarly, control veneers were impregnated with pure acetone (as above). Treated veneer sections were dried at room temperature for 3 days inside a laminar flux chamber that provided a clean environment and favored the evaporation of solvent. Sections were transferred onto 60 mm x 15 mm Petri dishes containing water-agar media (1 %). The water-agar medium was used to maintain moisture content and support fungal growth in the veneers when they were exposed to different light sources in-vitro. Veneer sections were placed on the center of the plates and allowed to re-hydrate for 3 hours before they were inoculated with fungi. 7.2.2.4. Inoculation of media with A. pullulans and exposure of treated wood sections to UV and visible light A. pullulans [strain R2F32.2] was grown in 100 mm x 15 mm Petri dishes with 1% MEA (Difco) at room temperature, and sub-cultured in similar plates every two weeks to ensure that fresh material was available for the experiment. After a week of growth, spores in the plates were harvested, under sterile conditions, by flooding the plates with 3 mL of nano pure water. The spores produced by the fungi were loosened from mycelia with a sterile glass rod. The supernatants were collected in 15 mL falcon tubes and the concentration of 227 spores in these solutions was measured with a hemocytometer (as described in section 7.2.1.3) and adjusted to a concentration of 1 spore per \u00C2\u00B5L. Veneer sections on the water- agar media were inoculated with 50 \u00C2\u00B5L of spore solution (Figure 7.3). Solutions were evenly spread onto the sections with a glass rod. The plates were sealed using quartz glass disks as lids and parafilm (as described in section 7.2.1.3), and kept in a dark room for 24 hours. Veneer sections were exposed to UV, visible or no light at 20\u00C2\u00B0C \u00C2\u00B1 1\u00C2\u00B0C at 65% \u00C2\u00B1 5% r.h. as described in section 7.2.1.4. Figure 7.3: Inoculation of spruce veneers with 50 \u00C2\u00B5L of spore solution (1 cell/\u00C2\u00B5L) 7.2.2.5. Quantification of staining and color of treated and inoculated veneer sections exposed to UV or visible light After 5 days of exposure, digital images of the plates without their lids, at a resolution of 300 dpi, were obtained using a desktop scanner (as described in section 7.2.1.4). Measurements of the staining of veneer sections (stained area) involved adjusting the tonal 228 range of images with Photoshop to help visualize fungal staining (Figure 7.4 a and c). Then, the pixels corresponding to fungal stain were visualized in black by adjusting the threshold level of images (Figure 7.4 b and d). Finally, the number of black pixels in the image\u00E2\u0080\u0099s histogram was used to calculate the stained area of the veneer section as a percentage of the total number of pixels in the image. The color of veneer sections was evaluated using the CIELab color system. Digital images (TIFF format) of the exposed samples (inoculated and without fungi) were loaded into Photoshop. Color measurements used the entire exposed surface contained in each image. Color was initially expressed using the RGB color system obtained using a color histogram (Figure 7.5a). The average RGB color was obtained and converted to the equivalent colors in the CIELab system in Photoshop (Figure 7.5b). Photoshop provides CIELab color using the standard scale of 0 to 100, for lightness, but redness-greenness and blueness-yellowness are expressed at 255 levels in scales ranging from -127 to 128. Therefore, redness- greenness and blueness-yellowness were transformed into the normal CIELab scale (-60 to +60) using the following equation (Papadakis et al., 2000): a* = [120 x (a + 128) / 255] - 60 Where: a* = CIELab color from -60 to + 60 a = CIELab color provided by Photoshop 229 The CIElab parameters were used to calculate the color difference between treated veneers and controls. Color differences were calculated using following equation (ASTM, 1993): \u00CE\u0094E = [(L2 \u00E2\u0080\u0093L1) 2 + (a2 \u00E2\u0080\u0093 a1) 2 + (b2 \u00E2\u0080\u0093 b1) 2]1/2 Where: \u00CE\u0094E = color difference L1, a1 and b1 = CIElab color components of control veneer L2, a2 and b2 = CIElab color components of treated veneer 230 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) \u00E2\u0080\u0098curves\u00E2\u0080\u0099 function of the software used to adjust the tonal range; and (d) threshold adjustment 231 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 232 7.2.2.6. Microscopy Growth of fungi on the surface of wood veneer sections was confirmed by observing the sections under a stereomicroscope (Wild Makroskop M420; Wild Leitz Canada, Willowdale, Ontario). Images of the wood surfaces were acquired using a digital camera (Nikon Coolpix S8100. Nikon Corp. Japan). 7.3. Results 7.3.1. MBIs tested in malt extract agar The results of analysis of variance of the effect of different melanin biosynthesis inhibitors on number of fungal colonies that grew on agar plates are shown in Table 7.3. There were significant effects (p-value < 0.001) of exposure (E), chemicals (Ch) and fungi (F) on the number of colonies in plates. The interactions of E x Ch and E x F were also significant. Main effects were included in the results to facilitate the interpretation of the results. 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 Colonies after exposure Source of variation p-value Exposure (E) <.001 Chemical (Ch) <.001 Fungi (F) <.001 ExCh <.001 ExF <.001 ChxF 0.092 ExChxF 0.195 The number of fungal colonies growing in the plates was significantly lower when the plates were exposed to UV radiation compared to plates exposed to visible light (Figure 7.6). 233 Cerulenin, carpropamid and quinoxyfen also reduced the number of colonies growing on the media compared to the control, whereas tricyclazole had no such effect. However, there were no significant differences in colony numbers on plates containing quinoxyfen and carpropamid, or between plates containing carpropamid and cerulenin (Figure 7.7). The number of colonies of C. cladosporioides growing on plates was significantly higher than that of A. pullulans (Figure 7.8). A significant interaction between exposure and chemical occurred because the number of fungal colonies growing on plates containing MBIs was significantly lower when the plates were exposed under UV light compared to those on plates exposed to visible light, except for plates containing tricyclazole (Figure 7.9). The interaction between exposure and fungi occurred because the number of colonies of A. pullulans on plates exposed to either UV or visible light was similar whereas the number of colonies of C. cladosporioides was significantly lower on plates exposed to UV light compared to those exposed to visible light (Figure 7.10). Plates exposed to UV and visible light and inoculated with A. pullulans and C. cladosporioides are shown in Figure 7.11 and Figure 7.12, respectively. 234 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 \u00C2\u00B1SED 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 \u00C2\u00B1SED 0 40 80 120 160 UV Vis. Light A ve ra ge n u m b e r o f fu n ga l c o lo n ie s o n M EA Exposure 0 40 80 120 160 Quinoxyfen Carpropamid Cerulenin Tricyclazole Control A ve ra ge n u m b er o f fu n ga l c o lo n ie s o n M EA Chemicals 235 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 \u00C2\u00B1SED 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 0 40 80 120 160 A. pullulans C. Cladiosporioides A ve ra ge n u m b e r o f fu n ga l c o lo n ie s o n M EA Fungi UV visible light l.s.d. 0 40 80 120 160 Quinoxyfen Carpropamid Cerulenin Tricyclazole Control A ve ra ge n u m b er o f fu n ga l c o lo n ie s o n M EA Chemicals 236 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 A. pull. Clad. l.s.d. 50 100 150 200 UV Vis. Light A ve ra ge n u m b e r o f fu n ga l c o lo n ie s o n M EA Exposure 237 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 238 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 239 7.3.2. Effects of MBIs and UV radiation on fungal staining and color of wood Table 7.4 summarizes the results of the analyses of variance of the effect of fixed factors (exposure to light, chemical treatment and concentration and their interactions) on fungal staining and color of veneer sections (both inoculated with fungi and not inoculated controls). Table 7.4: Significant effect of, and interaction between exposure to light, chemical treatments and concentration on stained area and color change (\u00CE\u0094E) 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 P-value Source of variation LN [1 + stained area ratio] \u00CE\u0094E inoculated veneers \u00CE\u0094E no fungi veneers \u00CE\u0094E inoculated veneers v. no fungi veneers Exposure (E) 0.398 0.16 0.583 0.259 Chemical (Ch) <.001 <0.001 0.892 <0.001 Concentration (C) 0.492 0.268 0.598 <0.001 E x Ch 0.106 0.506 0.865 0.46 E x C 0.56 0.35 0.735 0.12 Ch x C 0.17 0.691 0.808 <0.001 E x Ch x C 0.666 0.913 0.92 0.57 7.3.2.1. Effect on fungal staining Fungal stains in veneer sections exposed to either UV or visible light are shown in Figure 7.13 and Figure 7.14, respectively. Staining of veneer sections was significantly affected (p < 0.001) by the type of chemical impregnated into the sections, but not by the other factors. However, the interaction of chemical and exposure approached statistical significance (p = 0.106) (Table 7.4), although reduction of staining due to such an interaction was not apparent visually (Figure 7.13 and Figure 7.14). Veneer sections impregnated with carpropamid showed significantly less staining than those impregnated with quinoxyfen 240 (Figure 7.15). The fungi in the veneer sections had melanized hyphae, but in sections exposed to UV radiation hyphae were darker than those exposed to visible light. Light microscopy confirmed presence of A. pullulans hyphae in veneer sections and this observation discounts the presence of contamination due to other microorganisms (Figure 7.16 and Figure 7.17). The light contrast used to obtain the micrographs helped to see the presence of highly melanized hyphae in veneer sections exposed to UV radiation. 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 241 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 242 0 0.2 0.4 0.6 0.8 1 1.2 Carpropamid Quinoxyfen LN [1 + (r at io s ta in e d a re a) ] Chemicals 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 \u00C2\u00B1SED 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 243 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 7.3.2.2. Effect on color; comparison of stained wood surfaces Determination of color difference (\u00CE\u0094E) between sections inoculated with fungi and untreated stained sections made it possible to compare how effective carpropamid and quinoxyfen were against fungal staining. The color difference of veneer sections impregnated with carpropamid and quinoxyfen (inoculated with spores of A. pullulans) against control veneers (impregnated with acetone and inoculated with spores of A. pullulans) was significantly affected (p < 0.001) by chemical treatment (Table 7.4). \u00CE\u0094E was significantly higher in veneers sections impregnated with carpropamid, indicating that the 244 color of such sections was different to that of the heavily stained control veneer sections (Figure 7.18). 0 1 2 3 Carpropamid Quinoxyfen C o lo r d if fe re n ce ( \u00CE\u0094 E) Chemicals Figure 7.18: Effects of chemical treatment on color differences (\u00CE\u0094E) 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 \u00C2\u00B1SED 7.3.2.3. Effect on color of wood veneers in comparison to unstained wood surfaces Determination of color differences (\u00CE\u0094E) between veneer sections inoculated with A. pullulans and sections that were not inoculated with fungus provides another measure of the effectiveness of carpropamid and quinoxyfen at reducing fungal staining. \u00CE\u0094E of inoculated veneer sections v. not inoculated sections was significantly affected (p < 0.001) by chemical treatment (Table 7.4). Sections treated with carpropamid had significantly lower \u00CE\u0094E than veneers impregnated with quinoxyfen (Figure 7.19), indicating that color of veneer sections treated with carpropamid was similar to that of sound veneers. \u00CE\u0094E was also affected by the concentration of chemicals (p < 0.001), as veneer sections treated with 245 carpropamid and quinoxyfen at 3000 and 6000 ppm had a significantly lower \u00CE\u0094E than control veneers impregnated with acetone (Figure 7.20). However, the interaction between chemicals and concentrations was also significant (p < 0.001). This interaction occurred because as the concentration of carpropamid increased the color differences of veneer sections decreased, whereas no such effect of concentration was seen in sections treated with quinoxyfen (Figure 7.21). 0 2 4 Carpropamid Quinoxyfen C o lo r d if fe re n ce ( \u00CE\u0094 E) Chemicals Figure 7.19: Effects of chemical treatment on color differences (\u00CE\u0094E) 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 \u00C2\u00B1SED 246 0 2 4 0 3000 6000 C o lo r d if fe re n ce ( \u00CE\u0094 E) Concentration of chemicals Figure 7.20: Effects of chemical treatment on color differences (\u00CE\u0094E) 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 \u00C2\u00B1SED Carpropamid Quinoxyfen l.s.d. 0 2 4 0 3000 6000 C o lo r d if fe re n ce ( \u00CE\u0094 E) Concentration of chemicals Figure 7.21: Effects of chemical treatments and concentrations on color differences (\u00CE\u0094E) 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 247 7.3.2.4. Effect of the treatment on the natural color of wood There was no significant effect of experimental factors on the staining and color of uninoculated veneer sections. Veneers sections that were not inoculated with spores of A. pullulans but treated with carpropamid and quinoxyfen were \u00E2\u0080\u0098cleaner\u00E2\u0080\u0099 (less discolored) after exposure to UV radiation or visible light, and showed no color differences in comparison with control veneers impregnated with acetone (Figure 7.22 and Figure 7.23). As expected veneer surfaces \u00E2\u0080\u0098yellowed\u00E2\u0080\u0099 due to exposure to UV radiation. Determination of these color differences was relevant to verify whether the chemical treatments had an effect on the natural color of wood surfaces. In addition, light microscopy confirmed that these veneer sections were not colonized by A. pullulans (Figure 7.24 and Figure 7.25). 248 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 249 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 250 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 251 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 252 7.4. Discussion My results support the hypothesis that melanin biosynthesis inhibitors (MBIs) can inhibit the germination of spores, and the filamentous growth of two highly melanized fungi (A. pullulans and C. cladosporioides). Inhibition of growth and pigmentation of different blue- sapstaining fungi was reported by Fleet and Breuil (2002). They showed that both the growth and melanin biosynthesis pathway of such fungi were affected by the melanin biosynthesis inhibitors (MBIs) cerulenin, carpropamid and tricyclazole. My hypothesis that melanin-inhibited-fungi would be more susceptible to UV radiation was confirmed by my first experiment with A. pullulans and C. cladosporioides grown on MEA supplemented with the different MBIs and exposed to UV radiation. Melanin confers protection against different environmental factors, including UV radiation (Fogarty and Tobin 1996; Butler and Day 1998; Henson et al. 1999). Inhibition of enzymes in the dihydroxynaphthalene (DHN) melanin biosynthetic pathway by MBIs evidently decreased the tolerance of A. pullulans and C. cladosporioides to artificial UV radiation (340 nm). Differences in the effectiveness of the MBIs at inhibiting growth of fungi on plates exposed to UV light may be related to the different modes of action of cerulenin, tricyclazole and carpropamid, and the amount and nature of the intermediate products generated from the inhibition of steps in the DHN melanin biosynthetic pathway. For example, carpropamid is an inhibitor of scytalone dehydratase, an enzyme responsible for converting scytalone to 1,3,8 \u00E2\u0080\u0093 trihydroxynaphthalene by water elimination. The compound also inhibits a second dehydration step in the conversion of vermelone to 1,8-DHN. Cultures supplemented with carpropamid show accumulation of scytalone, as reported by Tsuji et al. (1997) for the 253 fungus Colletotrichum lagenarium. Tricyclazole inhibits two reductase enzymes (1,3,8- trihydroxynpahthalene reductase and 1,3,6,8-tetrahydroxynaphthalene reductase) which control the transformation of 1,3,6,8- tetrahydroxynaphthalene into scytalone and 1,3,8- trihydroxynpahthalene into vermelone (Wheeler and Greenblatt 1988). There is evidence indicating that flaviolin and 2-hydroxyjuglone are the intermediate melanin products produced by the action of tricyclazole in DHN melanin-producing fungi. Accumulation of flaviolin and 2-hydroxyjuglone has been reported by several authors for cultures of W. dermatitidis, H. werneckii, P. triangularis, and T. salinum supplemented with tricyclazole (Wheeler and Stipanovic 1985; Kogej et al. 2004). The intermediate melanin products generated by the action of carpropamid and tricyclazole have different photo-chromatic properties that might explain the differences in their effectiveness at inhibiting germination of spores of A. pullulans and C. cladosporioides exposed to UV radiation. For example, intermediate melanin products absorb limited amounts of radiation around 340 nm, but they have a peak absorption at 280 nm (Romero-Martinez et al., 2000). On the other hand, cerulenin affects the early stages of melanin production by inhibiting the enzyme polyketide synthase (Kubo et al. 1986), but it can also inhibit fatty acid synthase which is critical for physiological processes in many fungi (Kubo et al., 1986). Since the inhibition of DHN melanin pathway by cerulenin occurs at an early stage there is no accumulation of intermediate melanin product, which could explain the differences in its effectiveness compared to other MBIs (Fleet and Breuil, 2002). However, unlike carpropamid and tricyclazole, cerulenin only interferes with one enzymatic reaction in the DHN melanin pathway. This might limit its inhibitory properties at low concentrations, such as the 10 ppm 254 concentration used in my experiment. Amongst the three MBIs inhibitors tested, carpropamid was the most effective at decreasing the survival of fungal spores during exposure to UV radiation. Hence, the inhibition of scytalone dehydratase may be the most effective target in the melanin biosynthesis pathway of A. pullulans and C. cladosporioides, which may increase the susceptibility of these fungi to UV radiation and consequently inhibit their growth under UV-rich environments such as wood surfaces exposed outdoors. Carpropamid was able to reduce the staining of wood veneers by A. pullulans, but there was no synergistic effect of carpropamid and UV exposure (unlike the results from in-vitro tests). This discrepancy may be related to the higher concentrations of carpropamid used to treat veneers. The concentration of carpropamid was deliberately increased when treating spruce veneers to ensure that sufficient chemical was available that could restrict the germination of A. pullulans spores. The amount of chemical applied to veneers was not optimized to find a lower dose that would act in combination with UV radiation to restrict staining of veneers by A. pullulans. In addition to the melanin biosynthesis inhibitors (MBIs) tested here, the fungicide quinoxyfen was also tested in artificial media and on wood veneers. Quinoxyfen was developed to act against powdery mildew fungi in different crops (Coghlan et al., 1991). The active compound in this fungicide appears to be different from those of other biocides. However, tests performed in Blumeria graminis showed that, in the same way as MBIs, quinoxyfen can affect the appressorium development, which requires the presence of a high concentration of melanin (Wheeler et al. 2003). Hence, my interest in testing quinoxyfen here. Quinoxyfen was slightly more effective than carpropamid at inhibiting the 255 growth of A. pullulans and C. cladosporioides in agar plates exposed to UV or visible light. Quinoxyfen interferes with fungal growth by promoting the production of tubular cells instead of appressoria (Wheeler et al. 2003). These tubular cells might be susceptible to UV radiation? In contrast, quinoxyfen was less effective when used to treat wood veneers, and A. pullulans responded to its presence by becoming darker, possibly because the concentration of quinoxyfen was too low for it to have a fungicidal effect. Today there is increasing concern about the toxicity of chemicals used as wood preservatives and great interest in new more environmentally friendly preservatives with lower mammalian toxicity (Evans, 2003). The use of MBIs in combination with UV radiation to restrict fungal staining of wood surfaces is a new approach to \u00E2\u0080\u0098preserving wood\u00E2\u0080\u0099. However, further research needs to be done to find the concentration of MBIs that can work synergistically with UV radiation to control fungal staining. Other areas that require attention are, for example, testing of MBIs together or using them in combination with biocides that are currently used to control staining fungi. The mode of action of the three MBIs tested here differs from each other. Therefore, it would be interesting to examine any possible additive effects among them. In addition, other fungi need to be tested to confirm that MBIs can restrict fungal staining at wood surfaces exposed outdoors. MBIs are organic molecules, which are photo-sensitive and they might be photo-degraded at exposed wood surfaces. Therefore, consideration needs to be given to protecting them against this effect. It is also possible that they could be leached from wood surfaces as suggested by results in Chapter 5. Therefore ways of grafting or binding them to wood surfaces may need to be 256 developed. MBIs may be better suited under a coating or used in combination with hydrophobic additives such as oils and waxes. 257 7.5. Conclusions My experimental results show that melanin biosynthesis inhibitors (MBIs) are able to inhibit the growth of two of the most common fungi isolated from weathered wood surfaces (A. pullulans and C. cladosporioides). There was evidence that the fungi\u00E2\u0080\u0099s ability to withstand the deleterious effects of UV radiation was reduced when they were grown on media containing MBIs. In addition, carpropamid (one of the MBIs tested) reduced the staining of wood surfaces inoculated with spores of A. pullulans exposed to artificial UV radiation in- vitro. This outcome is very interesting because, if reproduced at wood surfaces exposed outdoors, it may reduce the amount of chemical needed to prevent fungal staining, which might decrease the environmental impacts and cost of the preservative treatment. To my knowledge this is the first attempt to use this approach to control staining by the black moulds that colonize weathered wood surfaces. Therefore, I conclude that the use of MBIs as anti-stain agents appears sufficiently promising to do further full-scale tests and outdoor trials. Such trials should seek to optimize the concentration of MBIs to produce a synergistic effect with solar UV radiation, and possibly use MBIs as mixed formulations and with additives to prevent their photodegradation and leaching from exposed wood surfaces. 258 8. Chapter 8: General discussion, conclusions and suggestions for further research 8.1. General discussion In this thesis I hypothesized that the graying of wood surfaces exposed outdoors was due to the presence of melanized fungi with resistance to UV radiation. The experimental results in Chapters 3, 5 and 6 support this hypothesis. The results in Chapter 4 also provide new information on the role that such fungi have on the micro-structural properties of wood. Chapter 7 suggests that the staining of wood surfaces exposed outdoors by melanized fungi can be decreased or eliminated by inhibiting melanin production, thereby increasing the susceptibility of the fungi to the deleterious effects of solar radiation. The interactive effects of UV radiation and colonization of wood surfaces by fungi has received little attention with the exception of studies that have shown that photodegradation of lignin provides a source of carbon for some of the fungi that colonize weathered wood. It is clear from my findings that the interactive effects of UV radiation and fungi play a more significant role in the surface degradation of wood exposed outdoors. Examination of the colonization of wood exposed outdoors by fungi revealed that changes in the appearance of wood surfaces were clearly driven by the interactive effect of solar radiation and fungal colonization. During the first four to eight weeks of outdoor exposure, yellowing of wood surfaces occurred due to photodegradation of lignin. These findings accord with those of other researchers (Gellerstendt and Gierer, 1975; Feist and Hon, 1984; Feist, 1990). Thereafter surfaces became darker, bluer and greener and finally acquired a grey color, again as others have observed (Duncan, 1963; Feist, 1990). The fungi isolated 259 from exposed surfaces were mainly from the ascomycota phylum. A. pullulans and H. dematioides were frequently isolated. These two species are recognized wood stainers, have dark mycelia and spores and have been widely documented as colonizing wood surfaces exposed outdoors (Seifert, 1964; Dickinson, 1971; Amburgey, 1974; Schmidt and French, 1976; Bardage and Bjurman, 1998; Held et al. 2006). Other fungi that were frequently isolated were E. nigrum and species of Phoma, which do not possess black hyphae, but produce aggregations of black spores and dark protective structures (sporodochia and pycnidia, respectively) (Barnett and Hunter, 1998; Rotem and Aust, 1991). Other fungi (Cladosporium spp. and Alternaria spp.) were isolated less frequently. All the aforementioned observations confirm the results of previous studies of the colonization of weathered wood by fungi. New information was generated by my experiment that examined the colonization of wood exposed under polymethylmethacrylate filters. This experiment showed that when wood samples were exposed to the full solar spectrum under a filter they were colonized by the same organisms that colonize fully exposed surfaces. However, when energetic radiation (UV and visible light) was blocked from reaching the surface, the black fungi (A. pullulans and H. dematioides) were isolated less frequently and less melanized fungi became more common. This finding hinted at the comparative advantage given by black pigmentation to fungi colonizing wood surfaces exposed to UV and visible light. A subsequent experiment showed that A. pullulans increased its melanin production when exposed to UV radiation, in accord with the findings of previous studies that have shown that melanized fungi are better able to survive exposure to UV radiation than hyaline (white) fungi (Wang and Casadevall, 1994). However, 260 not all black fungi increased their melanin production when exposed to UV radiation. For example, a fungus from the genera Cladosporium was naturally melanized irrespectively of the radiation it was exposed to. The interactive effects of UV radiation and fungal colonization had an effect on the color of weathered wood. For example, wood samples exposed under filters that transmitted UV and visible radiation tended to be darker, in accords with their frequent colonization by black fungi and the ability of such fungi to increase their melanin production in the presence of UV and visible light. On the other hand, samples exposed to less energetic radiation (IR and no light) showed less pronounced darkening and they tended to be greener. As stated above, these results point to a complex relationship between the surface photodegradation of wood, UV radiation and colonization of wood surfaces by fungi. Based on my experimental results and information available in literature the relationship between photodegradation of wood and fungal colonization can be summarized as follows: wood surfaces exposed outdoors are rapidly photodegraded by solar radiation which produces the first color changes seen at weathered wood surfaces (Gellerstendt and Gierer, 1975; Feist and Hon, 1984). Wood photodegradation products that accumulate at wood surfaces provide a carbon source for fungal spores that alight on wood surfaces (Schoeman and Dickinson, 1997). Days after exposure and subject to the availability of water, colonization of wood surfaces by fungi begins as small black colonies (spots) at the wood surface. Subsequently these colonies spread and produce darkening (graying) of the wood surface due to the presence of melanized fungi (Chedgy, 2006). Black fungi are common colonizers of wood surfaces exposed to the full solar spectrum. These fungi use the melanin contained in their cells to protect themselves against the damaging 261 effects of UV radiation and visible light. However, UV radiation can also promote increased melanin production in some of these black fungi, which may lead to darker wood surfaces. The number of hyaline (white) fungi discovered colonizing wood surfaces exposed to weathering was surprising in view of my findings and those of other researchers that melanin protects fungi exposed to solar radiation. These fungi do not produce high amounts of melanin. Therefore, it is valid to question how they survive the conditions found at wood surfaces exposed to the weather? Their survival may be explained in part by their reproductive strategies, such as those described above for Phoma and Epicoccum species. A second explanation is that they are protected by association with fungi that possess melanin. Melanin can confer protection against UV radiation, extreme temperatures and desiccation, conditions all found at wood surfaces exposed outdoors (Fogarty and Tobin 1996; Henson et al. 1999; Butler and Day 1998; 2001; Dadachova et al. 2007). Thus, black fungi growing at the outer wood surface may confer protection to hyaline fungi growing immediately below the exposed surface layers. If this occurs then it is possible that a symbiotic relationship could exist between both types of fungi. This suggestion is based on results obtained in Chapter 4, which showed that some hyaline fungi could decay woody tissues. After the photodegradation of wood tissues has reached an advanced stage, carbon sources from such degradation may become limited. Decay of the remaining tissue by hyaline fungi may provide carbon sources for black fungi, which protect the hyaline fungi growing in the sub-surface layers? The ability of certain fungi isolated from weathered wood to breakdown wood tissues (Chapter 4 of this thesis) has a number of important implications that are worthy of 262 discussion. Firstly, my finding that species such as Cladosporium sp., C. ligniaria, E. nigrum, L. infectoria, M. minutella and Phialocephala sp. can significantly reduce the mechanical properties of thin wood veneers helps to change the idea that fungi at weathered wood surfaces simply alter the appearance of wood. Secondly, my findings raise a number of questions about the damage that these fungi produce in-vivo and how much they contribute to the erosion of wood surfaces exposed outdoors. Erosion of wood surfaces during weathering is thought to be caused only by the combined action of UV radiation and water. However, my results suggest that microbial degradation could contribute to surface erosion. The occurrence of conditions that favor decay are possibly a limiting factor, but it certainly seems possible that fungi accelerate the erosion of weathered wood in situations where the moisture content at wood surfaces favor microbial colonization. Fungal staining during weathering affects the appearance of wood, which decreases its value as a construction material. Furthermore, the maintenance and replacement of weathered wooden cladding is costly (Amburgey and Ragon, 2008). Therefore, protection of wood against this type of damage is commercially important. Wood can be protected against photodegradation by using coatings and various additives, for example UV absorbers and hindered amine stabilizers. Protection of wood against fungal colonization relies on the use of biocides, which are indiscriminate and do not specifically target the organisms that cause weathered wood to become grey. My experimental results (Chapters 5, 6 and 7) suggest that in principle it is possible to use an alternative approach to decrease and possibly eliminate fungal stains at wood surfaces exposed outdoors. This approach involves preventing staining fungi from synthesizing melanin using chemicals applied at low 263 concentrations. Fungi prevented from synthesizing melanin were lighter and also appeared to be more susceptible to UV radiation. An experiment showed that two of the common fungi isolated from weathered wood A. pullulans and C. cladosporioides, could be prevented from growing by the combination of the melanin biosynthesis inhibitor (MBI) carpropamid and UV radiation. The approach when tested with wood veneers did not produce a statistically significant interaction between carpropamid and UV radiation on fungal growth, but positive effects were achieved with the MBI on its own. It is possible that lower doses of MBIs in wood might achieve the desired synergistic effect with UV radiation, similar to that seen in artificial media. This might decrease the toxicity of treated samples, with obvious environmental benefits. A possible drawback of the treatment is the relatively high cost of the MBIs tested. However, in the near future the demand for more environmentally friendly and less toxic preservative treatments may justify their use. 8.2. Conclusions My results demonstrated that melanized fungi are responsible for the graying of wood surfaces exposed outdoors. However, initial color changes at wood surfaces exposed to weathering were due to photodegradation of lignin. As anticipated solar radiation affected the colonization of wood surfaces by fungi. Solar radiation interfered with the ecology of wood surfaces by encouraging colonization of the wood by melanized fungi (A. pullulans and H. dematioides). Furthermore, in the presence of UV radiation A. pullulans increased its production of melanin, apparently as an adaptive measure. Such an adaptation probably 264 gives the fungus a competitive advantage when colonizing wood surfaces exposed to the weather. However, as a result the wood surface became darker. Therefore, I conclude that UV radiation and staining fungi interact to influence the color of wood surfaces exposed outdoors. Fungi colonizing weathered wood surfaces consist of a diverse group of ascomycetes. Among them, black ascomycetes with relatively high resistance to UV radiation, were found colonizing weathered wood surfaces in association with a number of less melanized fungi. Some of these fungi were able to reduce the strength properties of thin wood veneers (in- vitro). The type of degradation produced by one of these fungi appeared to be different from that of soft-rot decay (Type 1 or 2). Therefore I conclude that some fungi colonizing weathered wood surfaces can degrade woody tissues, but the extent of degradation probably depends on wood species and presence of conditions at wood surfaces that favor microbial decay. The use of melanin biosynthesis inhibitors (MBIs) and UV radiation to decrease or eliminate fungal stains in wood surfaces subjected to artificial weathering was explored. MBIs were able to block melanin production in fungi isolated from weathered wood making the fungi more susceptible to UV radiation. At low doses, MBIs tested against fungi in artificial media, acted synergistically with artificial UV radiation (at wavelengths within the solar spectrum) to inhibit the development of fungi from fungal spores. However, such a synergistic effect was not reproduced with treated wood veneers, but the MBI carpropamid was able to decrease fungal staining irrespective of the presence of UV or visible light. I conclude that the use of MBIs on their own or in combination with UV radiation is a promising approach 265 to controlling the fungi responsible for the graying of weathered wood surfaces, but further research is required to optimize the system and test it against a much greater range of fungi. The research in this thesis provides some new insights into the role played by the fungi that colonize weathered wood surfaces. However, as normally occurs in science, new knowledge is also accompanied by new questions. Therefore, I suggest further research that is needed to more fully explore some of my findings and to develop new treatments to reduce fungal staining of weathered wood surfaces. 8.3. Suggestion for further research This thesis described a number of experiments that were performed to better understand the role of non-decay fungi on the weathering of wood. My findings produced a number of new research questions, which could not be answered here. One important question concerns the ecological relationship between the different fungi colonizing weathered wood surfaces. For example, melanized and hyaline (white) fungi were found growing together at weathered wood surfaces. I speculated on a possible synergistic relationship between the two types of fungi. However, a better understanding of their ecological interaction is needed and could be achieved by isolating fungi in different layers from the surface to the sub-surface of weathered wood samples. Using this approach it should be possible to confirm or reject the hypothesis that black fungi protect hyaline fungi in weathered wood from exposure to UV radiation. A second area that would benefit from 266 further research is the extent to which fungi degrade weathered wood in-vivo. It is important to find out whether conditions conductive for such decay exist at wood surfaces exposed outdoors and if so whether they occur sporadically or seasonally. Also, it is important to establish whether fungi colonizing weathered wood increase the erosion of wood surfaces outdoors or reduce erosion by shielding the wood from UV radiation. A number of experiments could be performed to answer these questions. These experiments could include the use of chemicals (biocides) to restrict fungal colonization of wood surfaces and measurements of erosion of treated and untreated wood surfaces exposed to the weather. Future research should also focus on finding the optimum concentration of the MBI carpropamid to treat wood samples and achieve synergy with UV radiation in controlling fungal growth in wood. My research did not use mixes of different MBIs to control fungal staining. The MBIs tested here possess different modes of action that could have additive effects against a broader spectrum of fungi. Future large scale outdoor trials to test whether MBIs can restrict the fungal staining of wood should be carried out. However, this brings other problems to consider such as, protecting MBIs which are organic compounds, from photodegradation. Another issue that should be addressed is the development of methods to prevent the leaching of MBIs from wood. This might be achieved by grafting the MBIs to wood or incorporating them in or under a hydrophobic polymer matrix. 267 References Adams, J. (2009). Species richness: patterns in the diversity of life. Berlin; New York: Springer; Chichester, UK: Springer. Allmer, J., Vasiliasuskas, R., Ihrmark, K., Stenlid, J., & Dahlberg, A. (2006). Wood-inhabiting fungal communities in woody debris of Norway spruce (Picea abies (L.) Karst.), as reflected by sporocarps, mycelial isolation and T-RFLP identification. FEMS Microbiology ecology, 55(1), 57\u00E2\u0080\u009367. Amburgey, T. (1974). Organisms causing discoloration and deterioration of asphalt roofing shingles. Forest Prod. J., 24(6), 52\u00E2\u0080\u009354. Amburgey, T., & Ragon, K. (2008). \u00E2\u0080\u009CTreating\u00E2\u0080\u009D treated wood-Decks (SCH report No. 8). Mississippi State University. Anderson, E., Pawlak, Z., Owen, N., & Feist, W. (1991). Infrared studies of wood weathering. Part I: Softwoods. Applied Spectroscopy, 45(4), 641\u00E2\u0080\u0093647. ASTM. (1993). Standard test for calculation of color differences from instrumentally measured color coordinates. ASTM D2244. Philadelphia: American Society of Testing Materials. Balajee, S., Sigler, L., & Brandt, M. (2007). DNA and the classical way: identification of medically important molds in the 21st century. Medical Mycology: Official Publication of the International Society for Human and Animal Mycology, 45(6), 475\u00E2\u0080\u0093 490. 268 Bardage, S. L., & Bjurman, J. (1998). Isolation of an Aureobasidium pullulans polysaccharide that promotes adhesion of blastospores to water-borne paints. Can. J. Microbiol., 44(10), 954\u00E2\u0080\u0093958. Barnett, H. L., & Hunter, B. B. (1998). Illustrated genera of imperfect fungi. St. Paul, Minn.: APS Press. Behrendt, C., Blanchette, R., & Farrell, R. (1995). Biological control of blue-stain fungi in wood. Phytopathology, 85(1), 92\u00E2\u0080\u009397. Bell, A., & Wheeler, M. (1986). Biosynthesis and function of fungal melanins. Annual Review of Phytopathology, 24(1), 411\u00E2\u0080\u0093451. Birkinshaw, M., McCarthy, C., Regan, C., Hale, N., Cahill, D., & McCourt, M. (1999). The thermomechanical behavior of wood subject to fungal decay. Holzforschung, 53(5), 459\u00E2\u0080\u0093464. Blanchette, R. (1991). Delignification by wood-decay fungi. Annual Review of Phytopathology, 29(1), 381\u00E2\u0080\u0093403. Blanchette, R., Haight, J., Koestler, R., Hatchfield, P., & Arnold, D. (1994). Assessment of deterioration in archaeological wood from ancient Egypt. Journal of the American Institute for Conservation, 33(1), 55\u00E2\u0080\u009370. Bodig, J. (1982). Mechanics of Wood and Wood Composites. New York: Van Nostrand Reinhold. Bourbonnais, R., & Paice, M. (1987). Oxidation and reduction of lignin-related aromatic compounds by Aureobasidium pullulans. Applied Microbiology and Biotechnology, 26(2), 164\u00E2\u0080\u0093169. 269 Boutelje, J., & Bravery, A. (1968). Observations on bacterial attack of piles supporting a Stockholm building. Journal of the Institute of Wood Science, (20), 47\u00E2\u0080\u009347. Breznak, J., & Brune, A. (1994). Role of microorganisms in the digestion of lignocellulose by termites. Annual Review of Entomology, 39(1), 453\u00E2\u0080\u0093487. Brisson, A., Gharibian, S., Eagen, R., Leclerc, D., & Breuil, C. (1996). Localization and characterization of melanin granules produced by the sap-staining fungus Ophiostoma piceae. Material und Organismen, 30(1), 23\u00E2\u0080\u009332. Brown, F. (1953). Mercury-tolerant penicillia causing discoloration in northern white pine lumber. Journa of the Forest Products Research Society, 3, 67\u00E2\u0080\u009369. Browne, F., & Simonson, H. (1957). The penetration of light into wood. Forest Prod. J., 7(10), 303\u00E2\u0080\u0093314. Bugos, R., Sutherland, J., & Adler, J. (1988). Phenolic compound utilization by the soft rot fungus Lecythophora hoffmannii. Applied and Environmental Microbiology, 54(7), 1882\u00E2\u0080\u00931885. Butler, M., & Day, A. (1998). Fungal melanins: a review. Can. J. Microbiol., 44(12), 1115\u00E2\u0080\u0093 1136. Butler, M., & Day, A. (2001). Pathogenic properties of fungal melanins. Mycologia, 93(1), 1\u00E2\u0080\u0093 8. Caesar-TonThat, T., Van Ommen, F., Geesey, G., & Henson, J. (1995). Melanin production by a filamentous soil fungus in response to copper and localization of copper sulfide by sulfide-silver staining. Applied and Environmental Microbiology, 61(5), 1968\u00E2\u0080\u00931975. 270 Cagan, L., & Svercel, M. (2001). The influence of ultraviolet light on pathogenicity of entomopathogenic fungus Beauveria bassiana (balsamo) vuillemin to the European corn borer, Ostrinia nubilalis HBN. (Lepidoptera: crambidae). Journal of Central European Agriculture, 2(3-4), 228\u00E2\u0080\u0093233. Cartwright, K., & Findlay W. (1958). Decay of timber and its prevention (2nd ed.). London. Chang, S., Hon, D., & Feist, W. (1982). Photodegradation and photoprotection of wood surfaces. Wood Fiber and Science, 14(2), 104\u00E2\u0080\u0093117. Chedgy, R. (2006). The role of extractive depletion in the fungal colonization of western redcedar (Master Degree Thesis). University of British Columbia. Choi, Y.-W., Hyde, K., & Ho, W. H. (1999). Single spore isolation of fungi. Fungal Diversity, (3), 29\u00E2\u0080\u009338. Ciba. (1998). Tinuvin 384-2, light stabilizer. Ciba Specialty Chemicals, Coating effects segment, Edition 2 4 98. Cochrane, V. (1958). Physiology of fungi. New York: Wiley. Coghlan, M., Krumkalns, E., Caley, B., Hall, H., & Arnold, W. (1991). Novel agents for the control of cereal and grape powdery mildew. Synthesis and chemistry of agrochemicals II, ACS symposium Series (Baker D., Feyes J. and Moberg W., Vol. 443, pp. 538\u00E2\u0080\u0093552). Washington DC: American Chemical Society. Cooper, L. A., & Gadd, G. M. (1984). Differentiation and melanin production in hyaline and pigmented strains of Microdochium bolleyi. Antonie van Leeuwenhoek, 50(1), 53\u00E2\u0080\u009362. 271 Crawford, R., Carpenter, S., Mayfield, J., & Martin, R. (1987, July). Fungi from foliage of Arctostaphylos patula, Castanopsis chrysophylla, and Ceanothus velutinus. U.S. Forest Service. Cronin, L., Tiffney Jr, W., & Eveleigh, D. (2000). The graying of cedar shingles in a maritime climate--a fungal basis? Journal of Industrial Microbiology and Biotechnology, 24(5), 319\u00E2\u0080\u0093322. Curling, S., Clausen, C., & Winandy, J. (2002). Relation between mechanical properties, weight loss, and chemical composition of wood during incipient brown-rot decay. Forest Prod. J., 52(7-8), 34\u00E2\u0080\u009339. Dadachova, E., Bryan, R., Huang, X., Moadel, T., Schweitzer, D., Aisen, P., Nosanchuk, J., et al. (2007). Ionizing radiation changes the electronic properties of melanin and enhances the growth of melanized fungi. PloS ONE, 2(5). doi:10.1371/journal.pone.0000457 de Souza, A., & Gaylarde, C. (2002). Biodeterioration of varnished wood with and without biocide: implications for standard test methods. International Biodeterioration & Biodegradation, 49(1), 21\u00E2\u0080\u009325. Denig, J., Wengert, E., & Simpson, W. (2000). Drying hardwood lumber (General technical report FPL No. GTR-FPL-118) (p. 138). Forest Products Laboratory. Derbyshire, H., & Miller, E. (1981). The photodegradation of wood during solar irradiation. Holz als Roh- und Werkstoff, 39(8), 341\u00E2\u0080\u0093350. Dickinson, D. (1971). Disfigurement of decorative timbers by blue stain fungi. B.W.P.A. Annual convention (pp. 151\u00E2\u0080\u0093169). Presented at the B.W.P.A. Annual convention. 272 Diffey, B. (1991). Solar ultraviolet radiation effects on biological systems. Phys. Med. Biol., 36(3), 299\u00E2\u0080\u0093328. Dismukes, W., Pappas, P., & Sobel, J. (2003). Clinical mycology / edited by William E. Dismukes, Peter G. Pappas, Jack D. Sobel. Oxford; New York: Oxford University Press. Doi, S., & Horisawa, S. (2001). Fungi isolated from the surfaces of Sugi (Cryptomeria japonica) heartwood lumbers exposed at six test sites of Japanese islands. High- performance utilization of wood for outdoor uses (Y. Imamura.). Japan. Duncan, C. (1963). Role of microorganisms in the weathering of wood and degradation of exterior finishes. Official Digest Federation Societies Paints Technology, 35(465), 1003\u00E2\u0080\u00931012. Durrell, L. (1964). The composition and structure of walls of dark fungus spores. Mycopathologia, 23(4), 339\u00E2\u0080\u0093345. Eaton, R. (1994). Bacterial decay of ACQ-treated wood in a water cooling tower. International Biodeterioration & Biodegradation, 33(3), 197\u00E2\u0080\u0093207. Elias, M., Nemcova, Y., Skaloud, P., Neustupa, J., Kaufnerova, V., & Sejnohova, L. (2010). Hylodesmus singaporensis gen. et sp. nov., a new autosporic subaerial green alga (Scenedesmaceae, Chlorophyta) from Singapore. Int. J. Syst. Evol. Microbiol., 60(5), 1224\u00E2\u0080\u00931235. Encinas, O., Bjorn, H., & Geoffrey, D. (1998). Changes in toughness and fracture characteristics of wood attacked by the blue stain fungus Lasiodiplodia theobromae. Holzforschung, 52(1), 82\u00E2\u0080\u009388. 273 Eriksson, K., Blanchette, R., & Ander, P. (1990). Microbial and enzymatic degradation of wood and wood components. Berlin: Springer-Verlag. Evans, P. (1988). A note on assessing the deterioration of thin wood veneer during weathering. Wood and Fiber Science, 20(4), 487\u00E2\u0080\u0093492. Evans, P. (1989). Structural changes in Pinus radiata during weathering. Journal of the Institute of Wood Science, 11(5), 172\u00E2\u0080\u0093181. Evans, P. (2003). Emerging technologies in wood protection. Forest Prod. J., 53(1), 14\u00E2\u0080\u009322. Evans, P. (2008). Weathering and photoprotection of wood. Development of Wood Preservative Systems, Symposium Series (Schulz, T., Nicholas, D.). American Chemical Society. Evans, P., & Banks, W. (1986). Physicochemical factors affecting the degradation of wood surfaces by Diplodia-natalensis. Material Und Organismen, 21(3), 203\u00E2\u0080\u0093212. Evans, P., Chowdhury, M., Mathews, B., & Schmalzl, K. (2005). Weathering and surface protection of wood. Handbook of Environmental Degradation of Materials (pp. 277\u00E2\u0080\u0093 297). William Andrew. Evans, P., Michell, A., & Schmalzl, K. (1992). Studies of the degradation and protection of wood surfaces. Wood Science and Technology, 26(2), 151\u00E2\u0080\u0093163. Evans, P., Thay, P., & Schmalzl, K. (1996). Degradation of wood surfaces during natural weathering. Effects on lignin and cellulose and on the adhesion of acrylic latex primers. Wood Science and Technology, 30(6), 411\u00E2\u0080\u0093422. 274 Evans, P., Urban, K., & Chowdhury, M. (2008). Surface checking of wood is increased by photodegradation caused by ultraviolet and visible light. Wood Science and Technology, 42(3), 251\u00E2\u0080\u0093265. Faix, O., & B\u00C3\u00B6ttcher, J. H. (1992). The influence of particle size and concentration in transmission and diffuse reflectance spectroscopy of wood. Holz als Roh- und Werkstoff, 50(6), 221\u00E2\u0080\u0093226. Faix, O., Mozuch, M., & Kirk, T. (1985). Degradation of Gymnosperm (Guaiacyl) vs. Angiosperm (Syringyl/Guaiacyl) lignins by Phanerochaete chrysosporium. Holzforschung, 39(4), 203\u00E2\u0080\u0093208. Feist, W. (1983). Weathering and protection of wood. 19th annual meeting of the American wood Preservers Association (Vol. 79, pp. 195\u00E2\u0080\u0093205). Presented at the 19th Annual meeting of the American wood preservers association. Feist, W. (1990). Outdoor wood weathering and protection. Archaeological Wood (pp. 263\u00E2\u0080\u0093 298). American Chemical Society. Feist, W., & Hon, D. (1984). Chemistry of weathering and protection. The chemistry of solid wood (pp. 401\u00E2\u0080\u0093451). American chemical society. Fleet, C., & Breuil, C. (2002). Inhibitors and genetic analysis of scytalone dehydratase confirm the presence of DHN-melanin pathway in sapstain fungi. Mycol. Res., 106(11), 1331\u00E2\u0080\u00931339. Fogarty, R., & Tobin, J. (1996). Fungal melanins and their interactions with metals. Enzyme and Microbial Technology, 19(4), 311\u00E2\u0080\u0093317. 275 Forest Products Laboratory. (1999). Wood handbook - Wood as an engineering material. Madison WI, USA: Forest Products Laboratory. Department of Agriculture, Forest Service. Frederick, B., Caesar-TonThat, T., Wheeler, M., Sheehan, K., Edens, W., & Henson, J. (1999). Isolation and characterization of Gaeumannomyces graminis var. graminis melanin mutants. Mycol. Res., 103(1), 99\u00E2\u0080\u0093110. Freedonia Group. (2011). Wood & competitive decking (Market research No. 2718). Cleveland, OH, USA: The Freedonia Group. Freifelder, D. (1987). Microbial genetics. Boston, Ma.: Jones and Bartlett. Gadd, G. (1982). Effects of media composition and light on colony differentiation and melanin synthesis in Microdochium bolleyi. Trans. Br. mycol. Soc., 78(1), 115\u00E2\u0080\u0093122. Gadd, G. M., & De Rome, L. (1988). Biosorption of copper by fungal melanin. Applied Microbiology and Biotechnology, 29(6), 610\u00E2\u0080\u0093617. Gadd, G. M., Gray, D. J., & Newby, P. J. (1990). Role of melanin in fungal biosorption of tributyltin chloride. Applied Microbiology and Biotechnology, 34(1), 116\u00E2\u0080\u0093121. Gaylarde, C., & Morton, L. (1999). Deteriogenic biofilms on buildings and their control: A review. Biofouling: The Journal of Bioadhesion and Biofilm Research, 14(1), 59. Gellerstendt, G., & Gierer, J. (1975). The reactions of lignin during neutral sulfite pulping. Part V. The reactions of alpha-(4-Hydroxy-3-methoxyphenyl)-glycerol-beta-guaiacyl ether with sulfite and their dependence on pH. Acta Chemica Scandinavica, 29b, 561\u00E2\u0080\u0093570. 276 George, B., Suttie, E., Merlin, A., & Deglise, X. (2005). Photodegradation and photostabilisation of wood - the state of the art. Polymer Degradation and Stability, 88(2), 268\u00E2\u0080\u0093274. Ghahfarokhi, M., Fazli, A., Lotfi, A., & Abyaneh, M. (2004). Cellobiose dehydrogenase production by the genus Cladosporium. Iranian Biomedical Journal, 8(2), 107\u00E2\u0080\u0093111. Greaves, H. (1971). The bacterial factor in wood decay. Wood Science and Technology, 5(1), 6\u00E2\u0080\u009316. Greaves, H., & Levy, J. (1965). Comparative degradation of the sapwood of scots pine, beech, and birch by Lenzitestrabea, Polystictus versicolor, Chaetomium globosum and Bacillus polymyxa. J. Inst. Wood Sc., 15, 55\u00E2\u0080\u009363. Green, F., & Highley, T. (1997). Mechanism of brown-rot decay: paradigm or paradox. International Biodeterioration & Biodegradation, 39(2-3), 113\u00E2\u0080\u0093124. Griffin, D. (1981). Fungal Physiology. New York: Wiley. Griffin, D. (1996). Fungal physiology. John Wiley and Sons. Gumy, D., Morais, C., Bowen, P., Pulgarin, C., Giraldo, S., Hajdu, R., & Kiwi, J. (2006). Catalytic activity of commercial of TiO2 powders for the abatement of the bacteria (E. coli) under solar simulated light: Influence of the isoelectric point. Applied Catalysis B: Environmental, 63(1-2), 76\u00E2\u0080\u009384. Gutzmer, R., Mommert, S., Kuttler, U., Werfel, T., & Kapp, A. (2004). Rapid identification and differentiation of fungal DNA in dermatological specimens by LightCycler PCR. J. Med Microbiol, 53(12), 1207\u00E2\u0080\u00931214. 277 Hale, M., & Eaton, R. (1985). The ultrastructure of soft rot fungi. I. Fine hyphae in wood cell walls. Mycologia, 77(3), 447\u00E2\u0080\u0093463. Hannu, V., & Ahola, P. (1998). Resistance of painted wood to mould fungi. Part 3: The effect of weathering, wood and fungicides on mold growth. IRG annual meeting. Presented at the In IRG annual meeting, Netherlands: The international research group on wood preservation. Hansen, K. (2008). Molds and moldicide formulations for exterior paints and coatings. ACS Symposium series 982, Development of commercial wood preservaties (pp. 198\u00E2\u0080\u0093 213). Hansen, L., & Klotz, J. (2005). Carpenter ants of the United States and Canada. Cornell University Press. Harm, W. (1980). Biological effects of ultraviolet radiation. Cambridge, Eng., New York: Cambridge University Press. Harrington, K., Higgins, H., & Michell, A. (1964). Infrared spectra of Eucalyptus regnans F. Muell and Pinus radiata D.Don. Holzforschung, 18(4), 108\u00E2\u0080\u0093113. Harrington, T. (1988). Leptographium root disease on conifers. (F. Cobb, Ed.). American Phytopathological Society. Hawksworth, D., & Hill, D. (1984). The lichen-forming fungi. Glasgow: Blackie. Held, B. W., Jurgens, J. A., Duncan, S. M., Farrell, R. L., & Blanchette, R. A. (2006). Assessment of fungal diversity and deterioration in a wooden structure at New Harbor, Antarctica. Polar Biology, 26(6), 526\u00E2\u0080\u0093531. Henderson, S. (1977). Daylight and its spectrum. New York, American Elsevier Pub. Co. 278 Henson, J., Butler, M., & Day, A. (1999). The dark side of the mycelium: Melanin of phytopathogenic fungi. Annu. Rev. Phytopathol., 37, 447\u00E2\u0080\u0093471. Hewitt, G. (2000). New modes of action of fungicides. Pesticide outlook, 28\u00E2\u0080\u009332. Himelick, E. (1982). Pine blue-stain associated with the pine wilt syndrome. Journal of Arboriculture, 8(8), 212\u00E2\u0080\u0093216. Hoek, C., Mann, D., & Jahns, H. (1995). Algae: an introduction to phycology. Cambridge: Cambridge University Press. Hon, D. (1979). On possible chromophoric structures in wood pulps- A survey of the present state of knowledge. Polym. Plast. Technol. Eng., 12(2), 159\u00E2\u0080\u0093179. Hon, D., & Chang, S. (1984). Surface degradation of wood by ultraviolet light. Journal of Polymer Science: Polymer Chemistry Edition, 22(9), 2227\u00E2\u0080\u00932241. Hon, N. (1975). Formation of free radicals in photoirradiated cellulose. III. Effect of photosensitizers. Journal of Polymer Science: Polymer Chemistry Edition, 13(8), 1933\u00E2\u0080\u00931941. Huang, A., Zhou, Z., Liu, J., Fei, B., & Sun, S. (2008). Distinction of three wood species by Fourier infrared spectroscopy and two-dimensional correlation IR spectroscopy. Journal of Molecular Structure, 883-884, 160\u00E2\u0080\u0093166. Hulme, M., & Shields, J. (1972). Effect of primary fungal infection upon secondary colonization of birch bolts. Material und Organismen, 7, 177\u00E2\u0080\u0093188. Iannone, R., Chernoff, D., Pringle, A., Martin, T., & Bertram, A. (2011). The ice nucleation ability of one the most abundant types of fungal spores found in the atmosphere. Atmos. Chem. Phys., 11, 1191\u00E2\u0080\u00931201. 279 Ifju, G. (1964). Tensile strength behavior as a function of cellulose in wood. Forest Prod. J., 14(8), 366\u00E2\u0080\u0093372. International Commission on Illumination. (2007). Colorimetry: Understanding the CIE System. Vienna, Austria: CIE/Commission internationale de l\u00E2\u0080\u0099eclairage; Wiley- Interscience. Jeffries, T. (1994). Biodegradation of lignin and hemicelluloses. Biochemistry of Microbial Degradation (C. Ratledge., pp. 233\u00E2\u0080\u0093277). Dordrecht, Netherlands: Kluwer Academic Publishers. Kalnins, M. (1966). Surface characteristics of wood as they affect durability of finishes. Part II. Photochemical degradation of wood. U.S. Forest service research paper, FPL 57. Kataoka, Y., Kiguchi, M., & Evans, P. (2004). Photodegradation depth profile and penetration of light in Japanese cedar earlywood (Cryptomeria japonica D.Don) exposed to artificial solar radiation. Surface Coating International Part B: Coatings Transactions, 87(B3), 149\u00E2\u0080\u0093234. Kataoka, Y., Kiguchi, M., Williams, R., & Evans, P. (2007). Violet light causes photodegradation of wood beyond the zone affected by ultraviolet radiation. Holzforschung, 61(1), 23\u00E2\u0080\u009327. Kawamura, C., Moriwaki, J., Kimura, N., Fujita, Y., Fuji, S., Hirano, T., Koizumi, S., et al. (1997). The melanin biosynthesis genes of Alternaria alternata can restore pathogenicity of the melanin-deficient mutants of Magnaporthe grisea. Molecular Plant-Microbe Interactions: MPMI, 10(4), 446\u00E2\u0080\u0093453. 280 Kawamura, C., Tsujimoto, T., & Tsuge, T. (1999). Targeted disruption of a melanin biosynthesis gene affects conidial development and UV tolerance in the Japanese pear pathotype of Alternaria alternata. Mol. Plant-Microbe Interact., 12(1), 59\u00E2\u0080\u009363. Keasar, T. (2010). Large carpenter bees as agricultural pollinators. Psyche, 2010(i), 1\u00E2\u0080\u00937. Kendrick, B. (2000). The fifth kingdom (3rd ed.). Waterloo, Ont.: Mycology Publications. Kiiskinen, L.-L., Ratto, M., & Kruus, K. (2004). Screening for novel laccase-producing microbes. Journal of Applied Microbiology, 97(3), 640\u00E2\u0080\u0093646. Kim, J. J., Kang, S. M., Choi, Y.-S., & Kim, G.-H. (2007). Microfungi potentially disfiguring CCA- treated wood. International Biodeterioration & Biodegradation, 60(3), 197\u00E2\u0080\u0093201. Kim, J.-C., Min, J.-Y., Kim, H. T., Kim, B. S., Kim, Y. S., Kim, B. T., Yu, S. H., et al. (1998). Target site of a new antifungal compound KC10017 in the melanin biosynthesis of Magnaporthe grisea. Pesticide Biochemistry and Physiology, 62(2), 102\u00E2\u0080\u0093112. Knuth, D., & McCoy, E. (1961). Bacterial deterioration of pine logs in pond storage. Forest Prod. J., 12(9), 437\u00E2\u0080\u0093442. Koch, P. (1972). Utilization of the Southern Pines (Vol. 1). U.S. Department of Agriculture, Forest Service. Kogej, T., Wheeler, M., Lanisnik, T., & Gunde-Cimerman, N. (2004). Evidence for 1,8- dihydroxynaphthalene melanin in three halophilic black yeast grown under saline and non-saline conditions. FEMS Microbiology Letters, 232(2), 203\u00E2\u0080\u0093209. Krokene, P., & Solheim, H. (1998). Pathogenicity of four blue-stain fungi associated with aggressive and nonagressive bark beetles. Ecology and Population Biology, 88(1), 39\u00E2\u0080\u009344. 281 Kubo, Y. (2005). Studies on mechanisms of appressorial penetration by Colletotrichum lagenarium. J. Gen. Plant. Pathol., 71(6), 451\u00E2\u0080\u0093453. Kubo, Y., Katoh, M., Furusawa, I., & Shishiyama, J. (1986). Inhibition of melanin biosynthesis by cerulenin in appressoria of Colletotrichum lagenarium. Experimental Mycology, 10(4), 301\u00E2\u0080\u0093306. Kubo, Y., Takano, Y., Endo, N., Yasuda, N., Tajima, S., & Furusawa, I. (1996). Cloning and structural analysis of the melanin biosynthesis gene SCD1 encoding scytalone dehydratase in Colletotrichum lagenarium. Applied and Environmental Microbiology, 62(12), 4340\u00E2\u0080\u00934344. Kuhne, H., Leukens, U., Sell, J., & W\u00C3\u00A4lchli, O. (1970). Untersuchungen an bewitterten Holzoberfl\u00C3\u00A4chen\u00E2\u0080\u0094erste Mitteilung: Raster-elektronenmikroskopische Beobachtungen an Vergrauungspilzen. Holz als Roh- und Werkstoff, 28(6), 223\u00E2\u0080\u0093229. Kurahashi, Y. (2001). Melanin biosynthesis inhibitors (MBIs) for control of rice blast. Pesticide outlook, 12(1), 32\u00E2\u0080\u009335. Kurahashi, Y., & Pontzen, R. (1998). Carpropamid: a new melanin biosynthesis inhibitor. Pflanzenschutz-Nachrichten Bayer, 51(3), 245\u00E2\u0080\u0093256. Kurahashi, Y., Sakawa, S., Sakuma, H., Tanaka, K., Haenssler, G., & Yamaguchi, I. (1999). Effect of carpropamid on secondary infection by rice blast fungus. Pesticide Science, 55(1), 31\u00E2\u0080\u009337. Leightley, L. (1980). A rapid screening method for determining soft-rot decay ability. Mycology, 72(3), 632\u00E2\u0080\u0093637. 282 Levetin, E., Shaughnessy, R., Rogers, C., & Scheir, R. (2001). Effectiveness of germicidal UV radiation for reducing fungal contamination within air handling units. Applied and Environmental Microbiology, 67(8), 3712\u00E2\u0080\u00933715. Lim, Y.-W., Chedgy, R., Amirthalingam, S., & Breuil, C. (2007). Screening fungi tolerant to Western red Cedar (Thuja plicata Donn) extractives. Part 2. Development of a feeder strip assay. Holzforschung, 61(2), 195\u00E2\u0080\u0093200. Lim, Y.-W., Kim, J.-J., Chedgy, R., Morris, P., & Breuil, C. (2005). Fungal diversity from western redcedar fences and their resistance to \u00CE\u00B2-thujaplicin. Antonie van Leeuwenhoek, 87(2), 109\u00E2\u0080\u0093117. Liu, C. (2011). Use of confocal profilometry to quantify erosion of wood and screen chemicals for their ability to photostabilize wood (Master of Science Thesis). The University of British Columbia, Vancouver, Canada. Lopez, M. J., Vargas-Garcia, M., Suarez-Estrella, F., Nichols, N., Dien, B. S., & Moreno, J. (2007). Lignocellulose-degrading enzymes produced by the ascomycete Coniochaeta lignaria and related species: Application for a lignocellulosic substrate treatment. Enzyme and Microbial Technology, 40(4), 794\u00E2\u0080\u0093800. Maddock, W. (1920). Principles of general physiology. Longmans, Green. Maria, G., & Sridhar, K. (2002). Richness and diversity of filamentous fungi on woody litter of mangroves along the west coast of India. Current science, 83(12), 1573\u00E2\u0080\u00931580. Menard, K. (1999). Dynamic mechanical analysis. CRC Press. 283 Merrill, W., French, D., & Hossfeld, R. (1965). Effect of common molds on physical and chemical properties of wood fiberboard. Part II of a series of wood fiberboard studies. Tappi, 48(8), 470\u00E2\u0080\u0093474. Miniutti, V. (1974). Preliminary observations. Microscale changes in cell structure at softwood surfaces during weathering. Forest Prod. J., 14(12), 571\u00E2\u0080\u0093576. M\u00C3\u00B6bius, M. (1924). \u00C3\u009Cber graues und schwarzes Holz. Bot. Ges., 42, 341\u00E2\u0080\u0093344. Morrell, J., & Zabel, R. (1985). Wood strength and weight losses caused by soft rot fungi isolated from treated southern pine utility poles. Wood and Fiber Science, 17(1), 132\u00E2\u0080\u0093143. Nicholas, D., & Jin, Z. (1996). Use of compression strength loss for measuring decay in the soil block test (No. IRG/WP/96-20083) (pp. 1\u00E2\u0080\u009311). The international research group on wood preservation. Nilsson, T, & Daniel, G. (1989). Chemistry and microscopy of wood decay by some higher ascomycetes. Holzforschung, 43(1), 11\u00E2\u0080\u009318. Nilsson, Thomas. (1973). Studies on wood degradation and cellulolytic activity of microfungi (Report No. 104). Studia forestalia Suecica. Stockholm: Royal College of Forestry. Nilsson, Thomas. (1974). The degradation of cellulose and the production of cellulase, xylanase, mannanase and amylase by wood attacking microfungi (Report No. 114). Studia forestalia Suecica. Stockholm: Royal College of Forestry. Ohba, N., Tsujimoto, Y., & Imamura, Y. (2001). Development of accelerated outdoor- exposure test method of soiling and evaluation of algal growth on exterior materials. High-performance utilization of wood for outdoor uses (Y. Imamura.). Japan. 284 Ozcelik, B. (2007). Fungi/bactericidal and static effects of ultraviolet light in 254 and 354 nm wavelengths. Research Journal of Microbiology, 2(1), 42\u00E2\u0080\u009349. Paajanen, L. (1994). Structural changes in primed Scots pine and Norway spruce during weathering. Materials and structures, 27(168), 237\u00E2\u0080\u0093244. Paim, S., Linhares, L., Mangrich, A., & Martin, J. (1990). Characterization of fungal melanins and soil humic acids by chemical analysis and infrared spectroscopy. Biol Fertil Soils, 10(1), 72\u00E2\u0080\u009376. Pandey, K. K., & Theagarajan, K. S. (1997). Analysis of wood surfaces and ground wood by diffuse reflectance (DRIFT) and photoacoustic (PAS) Fourier transform infrared spectroscopic techniques. Holz als Roh- und Werkstoff, 55(6), 383\u00E2\u0080\u0093390. Pandey, K., & Pitman, A. (2003). FTIR studies of the changes in wood chemistry following decay by brown-rot and white-rot fungi. International biodeterioration & biodegradation, 52(3), 151\u00E2\u0080\u0093160. Panshin, A., & De Zeeuw, C. (1980). Textbook of wood technology: structure, identification, properties, and uses of the commercial woods of the United States and Canada (4th ed.). New York: McGraw-Hill. Papadakis, S., Abdul-Malek, S., Kandem, R., & Yam, K. (2000). A versatile and inexpensive technique for measuring color of foods. Food Technology, 54(12), 48\u00E2\u0080\u009351. Park, D. (1982). Phylloplane fungi: Tolerance of hyphal tips to drying. Trans. Br. mycol. Soc., 79(1), 174\u00E2\u0080\u0093178. 285 Patrick, M., & Rahn, R. (1976). Photochemistry of DNA and polynucleotides: photoproducts. Photochemistry and Photobiology of Nucleic Acids, Biology (Wang S. Y., Vol. II, pp. 35\u00E2\u0080\u009391). New York: Academic Press. Peciulyte, D. (2007). Isolation of cellulolytic fungi from waste paper gradual recycling materials. Ekologija, 53(4), 11\u00E2\u0080\u009318. Peet, R. (1974). The measurement of species diversity. Annual Review of Ecology and Systematics, 5(1), 285\u00E2\u0080\u0093307. Perry, T. (1991). A synopsis of the taxonomic revisions in the genus Ceratocystis including a review of bluestaining species associated with Dendroctonus bark beetles ( No. SO- 86). USDA Forest Serv. Gen. Tech. Pfeffer, A., Hoegger, P., K\u00C3\u00BCes, U., & Militz, H. (2012). Fungal colonisation of outside weathered modified wood. Wood Science and Technology, 46(1-3), 63\u00E2\u0080\u009372. Popescu, C.-M., Popescu, M.-C., & Vasile, C. (2010). Characterization of fungal degraded lime wood by FT-IR and 2D IR correlation spectroscopy. Microchemical Journal, 95(2), 377\u00E2\u0080\u0093387. Raberg, U., Bijelovic, J., Land, C., Bardage, S., & Terziev, N. (2006). Identification of fungi colonising coated and modified wood exposed outdoors using sequencing and T- RFLP profiling. International Research Group on Wood Protection, Document No: IRG/WP 06-20326. Raczkowski, J. (1980). Seasonal effects on the atmospheric corrosion of spruce micro- sections. Holz als Roh- und Werkstoff, 38(6), 231\u00E2\u0080\u0093234. 286 Rajderkar, N. (1966). Decay of wood by Alternaria and Penicillium and chief methods of control. Mycopathology, 30(2), 149\u00E2\u0080\u0093151. Ranby, B., & Rabek, J. (1975). Photodegradation, photo-oxidation and photostabilization of polymers. Journal of Polymer Science: Polymer Letters Edition, 13(10), 621\u00E2\u0080\u0093622. Ray, M., Dickinson, D., & Buck, M. (2004). Aureobasidium or Hormonema? A genetic Approach. IRG 35th Annual Meeting. Ljubljana, Slovenia: The international research group on wood preservation. Rogers, G., & Baecker, A. (1991). Clostridium xylanolyticum sp. nov., an anaerobic Xylanolytic bacterium from decayed Pinus patula wood chips. Int. J. Syst. Bacteriol., 41(1), 140\u00E2\u0080\u0093143. Rohilla, R., Singh, U., & Singh, R. L. (2001). Uptake and translocation of carpropamid in rice (Oryza sativa L). Pest Management Science, 57(3), 239\u00E2\u0080\u0093247. Romero-Martinez, R., Wheeler, M., Guerrero-Plata, A., Rico, G., & Torres-Guerrero, H. (2000). Biosynthesis and functions of melanin in Sporothrix schenckii. Infection and Immunity, 68(6), 3696\u00E2\u0080\u00933703. Rosas, A., Nosanchuk, J., Gomez, B., Edens, W., Henson, J., & Casadevall, A. (2000). Isolation and serological analyses of fungal melanins. Journal of Immunological Methods, 244(1-2), 69\u00E2\u0080\u009380. Rotem, J., & Aust, H. (1991). The effect of ultraviolet and solar radiation and temperature on survival of fungal propagules. Journal of Phytopathology, 133(1), 76\u00E2\u0080\u009384. Sailer, M., van Nieuwenhuijzen, E., & Knol, W. (2010). Forming of a functional biofilm on wood surfaces. Ecological Engineering, 36(2), 163\u00E2\u0080\u0093167. 287 Savory, J. (1954). Breakdown of timber by ascomycetes and fungi imperfecti. Ann. Appl. Biol., 41(2), 336\u00E2\u0080\u0093347. Savory, J. (1973). Effects of timber micro-organisms on paint performance. J. Oil Col. Chem., 56, 247\u00E2\u0080\u0093250. Sazci, A., Radford, A., & Erenler, K. (1986). Detection of cellulolytic fungi by using Congo red as an indicator: a comparative study with the dinitrosalicyclic acid reagent method. Journal of Applied Bacteriology, 61(6), 559\u00E2\u0080\u0093562. Schacht, H. (1863). Ueber die Veranderungen durch Pilze in abgestorbenen Pflanzensellen. Jahrbucher fur wissenschaftliche Botanik. Berlin: Verlag von august Hirschawald. Scheffer, T. (1986). O2 requirements for growth and survival of wood-decaying and sapwood-staining fungi. Can. J. Bot., 64(9), 1957\u00E2\u0080\u00931963. Schirp, A., Farrell, R., & Kreber, B. (2000). Capability of staining fungi to cause structural changes in New Zealand radiata pine: Toughness testing and enzyme production. Maderas Ciencia y Tecnologia, 2(2), 119\u00E2\u0080\u0093129. Schmidt, E., & French, D. (1976). Aureobasidium pullulans on wood shingles. Forest Prod. J., 26(7), 34\u00E2\u0080\u009337. Schmidt, O., & Moreth, U. (2002). Data bank of rDNA-ITS sequences from building-rot fungi for their identification. Wood Science and Technology, 36(55), 429\u00E2\u0080\u0093433. Schmolz, E., Bruders, N., Daum, R., & Lamprecht, I. (2000). Thermoanalytical investigations on paper covers of social wasps. Thermochimica Acta, 361(1-2), 121\u00E2\u0080\u0093129. 288 Schoeman, M., & Dickinson, D. (1996). Aureobasidium pullulans can utilize simple aromatic compunds as a sole source of carbon in liquid culture. Letters in Applied Microbiology, 22(2), 129\u00E2\u0080\u0093131. Schoeman, M., & Dickinson, D. (1997). Growth of Aureobasidium pullulans on lignin breakdown products at weathered wood surfaces. Mycologist, 11(4), 168\u00E2\u0080\u0093172. Schoenen, D., & Kolch, A. (1992). Photoreactivation of E. coli depending on light intensity after UV irradiation. Zentralblatt F\u00C3\u00BCr Hygiene Und Umweltmedizin = International Journal of Hygiene and Environmental Medicine, 192(6), 565\u00E2\u0080\u009370. Schulz, G. (1956). Exploratory tests to increase preservative penetration in spruce and aspen by mold infection. Forest Prod. J., 6(2), 77\u00E2\u0080\u009380. Schwarze, F. (2007). Wood decay under the microscope. Fungal Biology Reviews, 21(4), 133\u00E2\u0080\u0093170. Seifert, K. (1964). Changes of the chemical wood components by blue rot Pullularia pullulans (de Bary) Berkout (= Aureobasidium pullulans (de Bary) Arnaud). Holz als Roh- und Werkstoff, 22(11), 445\u00E2\u0080\u0093449. Sell, J. (1968). Untersuchungen \u00C3\u00BCber die Besiedelung von unbehandeltem und angestrichenem Holz durch Bl\u00C3\u00A4uepilze. European Journal of Wood and Wood Products, 26(6), 215\u00E2\u0080\u0093222. Sell, J., & W\u00C3\u00A4lchli, O. (1969). Changes in the surface texture of weathered-exposed wood. Material und Organismen, 4(2), 81\u00E2\u0080\u009387. Sexton, C., Corden, M., & Morrell, J. (1993). Assessing fungal decay of wood by small-scale toughness tests. Wood and Fiber Science, 25(4), 375\u00E2\u0080\u0093383. 289 Sharpe, P., & Dickinson, D. (1992). Blue stain in service on wood surface coatings. Part 1: The nutritional requirements of Aureobasidium pullulans. In IRG Annual Meeting (Vol. IRG/WP 1556\u00E2\u0080\u009392). Harrogate, U.K.: The international research group on wood preservation. Sharpe, P., & Dickinson, D. (1993). Blue stain in service on wood surface coatings. Part 3: Nutritional capability of Aureobasidium pullulans compared to other fungi commonly isolated from wood surface coatings (Vol. IRG/WP/93\u00E2\u0080\u009310035). Presented at the In IRG Annual Meeting, Orlando, USA: The international research group on wood preservation. Sherwood, M. (1973). Microfungi of the H. J. Andrews experimental forest a preliminar checklist ( No. 58). Plant patology department. Corvallis Oregon: Oregon State University. Shirikawa, M., Gaylarde, C., Gaylarde, P., John, V., & Gambale, W. (2002). Fungal colonization and succession on newly painted buildings and the effect of biocide. FEMS Microbiology ecology, 39(2), 165\u00E2\u0080\u0093173. Singaravelan, N., Grishkan, I., Beharav, A., Wakamatsu, K., Ito, S., & Nevo, E. (2008). Adaptive melanin response of the soil fungus Aspergillus niger to UV radiation stress at \u00E2\u0080\u009CEvolution Canyon\u00E2\u0080\u009D, Mount Carmel, Israel. PloS ONE, e2993, 3(8). doi:10.1371/journal.pone.0002993 Singh, A., Hedley, M., Page, D., Han, C., & Atisongkroh, K. (1992). Microbial degradation of CCA-treated cooling tower timbers. IAWA bulletin, 13(2), 215\u00E2\u0080\u0093231. 290 Smith, C., Slade, S., Nordheim, E., Cascino, J., Harris, R., & Andrews, J. (1988). Sources of variability in the measurement of fungal spore yields. Applied Microbiology and Biotechnology, 54(6), 1430\u00E2\u0080\u00931435. Smith, R., & Swann, G. (1976). Colonization and degradation of western red cedar shingles and shakes by fungi. Material und Organismen, 3, 253\u00E2\u0080\u0093262. Spedding, D. (1970). Sorption of sulphur dioxide by indoor surfaces. 2. Wood. Journal of Applied Chemistry of the USSR, 20(7), 226\u00E2\u0080\u0093228. Spiegelberg, H. (1966). The effect of hemicelluloses on the mechanical properties of individual fibers (Doctor of Philosophy Thesis). Lawrence University, Appleton, Wisconsin, USA. Starr, C., Evers, C., & Starr, L. (2010). Biology: Concepts and Applications. Cengage Learning. Sudiyani, Y., Horisawa, S., Chen, K., Doi, S., & Imamura, Y. (2002). Changes in surface properties of tropical wood species exposed to the Indonesian climate in relation to mold colonies. Journal of wood science, 48(6), 542\u00E2\u0080\u0093547. Suryanarayanan, T., Ravishankar, J., Venkatesan, G., & Murali, T. (2004). Characterization of the melanin pigment of a cosmopolitan fungal endophyte. Mycological Research, 108(8), 974\u00E2\u0080\u0093978. Tsui, C., Wang, B., Khadempour, L., Alamouti, S., Bohlmann, J., Murray, B., & Hamelin, R. (2010). Rapid identification and detection of pine pathogenic fungi associated with mountain pine beetles by padlock probes. Journal of Microbiological Methods, 83(1), 26\u00E2\u0080\u009333. 291 Tsuji, G., Takeda, T., Furusawa, I., Horino, O., & Kubo, Y. (1997). Carpropamid, an anti-Rice blast fungicide, inhibits scytalone dehydratase activity and appressorial penetration in Colletotrichum lagenarium. Pesticide Biochemistry and Physiology, 57(3), 211\u00E2\u0080\u0093 219. Urban, K. (2005). The effect of solar radiation on the surface checking of lodgepole pine (Master of Science Thesis). The University of British Columbia, Vancouver, Canada. Viswanath, B., Chandra, M., Pallavi, H., & Reddy, B. (2008). Screening and assessment of laccase producing fungi isolated from different environmental samples. African Journal of Biotechnology, 7(8), 1129\u00E2\u0080\u00931133. Wang, Y., & Casadevall, A. (1994). Decreased susceptibility of melanized Cryptococcus neoformans to UV light. Applied and Environmental Microbiology, 60(10), 3864\u00E2\u0080\u0093 3866. Wang, Z., Chen, T., Gao, Y., Breuil, C., & Hiratsuka, Y. (1995). Biological degradation of resin acids in wood chips by wood-inhabiting fungi. Applied and Environmental Microbiology, 61(1), 222\u00E2\u0080\u0093225. Wethern, J. (1959). Pulp and chemical potential for western red cedar utilization. Forest Prod. J., 9(Sept), 308\u00E2\u0080\u0093313. Wheeler, I., Hollomon, D., Gustafson, G., Mitchell, J., Longhurst, C., Zhang, Z., & Gurr, S. (2003). Quinoxyfen perturbs signal transduction in barley powdery mildew (Blumeria graminis f.sp. hordei). Molecular Plant Pathology, 4(3), 177\u00E2\u0080\u0093186. doi:10.1046/j.1364-3703.2003.00165.x 292 Wheeler, M., & Greenblatt, G. (1988). The inhibition of melanin biosynthetic reactions in Pyricularia oryzae by compounds that prevent rice blast disease. Experimental Mycology, 12(2), 151\u00E2\u0080\u0093160. Wheeler, M., & Klich, M. (1995). The effects of tricyclazole, pyroquilon, phthalide, and related fungicides on the production of conidial wall pigments by Penicillium and Aspergillus species. Pesticide Biochemistry and Physiology, 52(2), 125\u00E2\u0080\u0093136. Wheeler, M., & Stipanovic, R. (1985). Melanin biosynthesis and the metabolism of flaviolin and 2-hydroxyjuglone in Wangiella dermatitidis. Archive of Microbiology, 142(3), 234\u00E2\u0080\u0093241. Wilcox, W. (1978). Review of literature on the effects of early stages of decay on wood strength. Wood and Fiber Science, 9(4), 252\u00E2\u0080\u0093257. Williams, R. (1987). Acid effects on accelerated wood weathering. Forest Prod. J., 37(2), 37\u00E2\u0080\u0093 38. Williams, R. (2005). Weathering of wood. Handbook of chemistry and wood composites (Boca Raton., pp. 139\u00E2\u0080\u0093185). CRC Press. Winandy, J., & Morrell, J. (1993). Relationship between incipient decay, strength, and chemical composition of Douglas-fir heartwood. Wood and Fiber Science, 25(3), 278\u00E2\u0080\u0093288. Winandy, J., & Rowell, R. (2005). Chemistry of wood strength. Handbook of wood chemistry and wood composites (pp. 303\u00E2\u0080\u0093347). Boca Raton, Fla: CRC Press. Worrall, J., Anagnost, S., & Wang, C. (1991). Conditions for soft rot of wood. Can. J. Microbiol., 37(11), 869\u00E2\u0080\u0093847. 293 Zabel, R., & Morrell, J. (1992). Wood microbiology\u00E2\u0080\u00AF: decay and its prevention. San Diego: Academic Press. Zyani, M., Mortabit, D., Mostakim, M., Iraqui, M., Haggoud, A., Ettayebi, M., & Koraichi, S. (2009). Cellulolytic potential of fungi in wood degradation from an old house at the Medina of Fez. Annals of Microbiology, 59(4), 699\u00E2\u0080\u0093704. 294 Appendices Appendixes can be found in the DVD attached to this thesis. List of appendices Appendix 1: Statistical analysis Chapter 4 Appendix 2: Graphic determination of modulus of elasticity, example of calculation Appendix 3: Statistical analysis Chapter 5 Appendix 4: Images of fungal colonization evolution in southern pine samples exposed under filter transmitting different wavelengths of solar radiation (Chapter 5) Appendix 5: Result for reciprocal Simpson index (Chapter 5) Appendix 6: Statistical analysis Chapter 6 Appendix 7: Calibration curves for calculation of fungal melanin concentration (Chapter 6) Appendix 8: Statistical analysis melanin biosynthesis inhibitors tested in artificial media (Chapter 7) Appendix 9: Statistical analysis melanin biosynthesis inhibitors tested in wood veneers (Chapter 7) 295 Appendix 1: Statistical analysis Chapter 4 Analysis of variance tensile stress ratio Variate: Tensile_stress_ratio Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Block stratum 10 1.30944 0.13094 3.43 Block.Dish stratum Fungi 17 20.63514 1.21383 31.76 <.001 Residual 148 (22) 5.65690 0.03822 0.82 Block.Dish.Area stratum W_specie 1 2.30182 2.30182 49.17 <.001 Fungi.W_specie 17 7.38402 0.43435 9.28 <.001 Residual 158 (22) 7.39683 0.04682 Total 351 (44) 40.76775 Message: the following units have large residuals. Block 5 0.120 s.e. 0.058 Block 6 -0.118 s.e. 0.058 Block 1 Dish 4 0.378 s.e. 0.120 Block 1 Dish 18 0.479 s.e. 0.120 Block 8 Dish 5 0.358 s.e. 0.120 Block 9 Dish 18 -0.429 s.e. 0.120 Block 1 Dish 18 Area 1 -0.402 s.e. 0.137 Block 1 Dish 18 Area 2 0.402 s.e. 0.137 Block 2 Dish 2 Area 1 0.376 s.e. 0.137 Block 2 Dish 2 Area 2 -0.376 s.e. 0.137 Block 9 Dish 3 Area 1 -0.401 s.e. 0.137 Block 9 Dish 3 Area 2 0.401 s.e. 0.137 Block 9 Dish 13 Area 1 -0.390 s.e. 0.137 Block 9 Dish 13 Area 2 0.390 s.e. 0.137 296 Tables of means Variate: Tensile_stress_ratio Grand mean 0.849 Fungi A. pull (B) A. pull (W) Alt Botry Chaet glob 0.920 0.983 0.907 1.034 0.431 Fungi Clad Con put Conioch Control Epicoc 0.339 1.115 0.391 1.000 0.903 Fungi Hormonema Lecyth Lewia Mollisia Phialocephala 1.051 0.983 0.908 0.850 0.808 Fungi Phialophora Phoma Trichaptum 1.005 0.979 0.674 W_specie Lime Spruce 0.773 0.925 Fungi W_specie Lime Spruce A. pull (B) 0.999 0.841 A. pull (W) 0.985 0.981 Alt 0.847 0.967 Botry 1.098 0.970 Chaet glob 0.101 0.760 Clad -0.004 0.682 Con put 0.979 1.251 Conioch 0.137 0.646 Control 1.000 1.000 Epicoc 0.846 0.959 Hormonema 1.044 1.058 Lecyth 1.073 0.894 Lewia 0.874 0.941 Mollisia 0.788 0.913 Phialocephala 0.537 1.080 Phialophora 1.097 0.913 Phoma 0.970 0.988 Trichaptum 0.540 0.808 Standard errors of differences of means Table Fungi W_specie Fungi W_specie rep. 22 198 11 s.e.d. 0.0589 0.0217 0.0879 d.f. 148 158 304.57 Except when comparing means with the same level(s) of Fungi 0.0923 d.f. 158 (Not adjusted for missing values) 297 Analysis of variance modulus of elasticity (MOE) ratio Variate: MOE_ratio Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Block stratum 10 3.70210 0.37021 13.93 Block.Dish stratum Fungi 17 11.02075 0.64828 24.39 <.001 Residual 148 (22) 3.93356 0.02658 0.66 Block.Dish.Area stratum W_specie 1 3.13308 3.13308 77.64 <.001 Fungi.W_specie 17 8.93710 0.52571 13.03 <.001 Residual 158 (22) 6.37573 0.04035 Total 351 (44) 33.76693 Message: the following units have large residuals. Block 7 0.203 s.e. 0.097 Block 1 Dish 18 0.293 s.e. 0.100 Block 8 Dish 5 0.278 s.e. 0.100 Block 9 Dish 18 -0.336 s.e. 0.100 Block 1 Dish 3 Area 1 0.368 s.e. 0.127 Block 1 Dish 3 Area 2 -0.368 s.e. 0.127 Block 1 Dish 13 Area 1 0.449 s.e. 0.127 Block 1 Dish 13 Area 2 -0.449 s.e. 0.127 Block 7 Dish 4 Area 1 0.384 s.e. 0.127 Block 7 Dish 4 Area 2 -0.384 s.e. 0.127 Block 8 Dish 5 Area 1 -0.519 s.e. 0.127 Block 8 Dish 5 Area 2 0.519 s.e. 0.127 298 Tables of means Variate: MOE_ratio Grand mean 0.894 Fungi A. pull (B) A. pull (W) Alt Botry Chaet glob 0.926 0.953 0.959 1.023 0.567 Fungi Clad Con put Conioch Control Epicoc 0.511 1.047 0.615 1.000 0.987 Fungi Hormonema Lecyth Lewia Mollisia Phialocephala 1.006 0.988 1.004 1.020 0.742 Fungi Phialophora Phoma Trichaptum 1.020 0.933 0.791 W_specie Lime Spruce 0.805 0.983 Fungi W_specie Lime Spruce A. pull (B) 0.933 0.920 A. pull (W) 0.911 0.995 Alt 1.009 0.910 Botry 1.009 1.036 Chaet glob 0.164 0.970 Clad -0.013 1.035 Con put 0.999 1.096 Conioch 0.404 0.825 Control 1.000 1.000 Epicoc 0.939 1.035 Hormonema 0.963 1.049 Lecyth 1.042 0.934 Lewia 0.945 1.063 Mollisia 0.942 1.098 Phialocephala 0.539 0.944 Phialophora 1.002 1.038 Phoma 0.957 0.909 Trichaptum 0.746 0.835 Standard errors of differences of means Table Fungi W_specie Fungi W_specie rep. 22 198 11 s.e.d. 0.0492 0.0202 0.0780 d.f. 148 158 297.09 Except when comparing means with the same level(s) of Fungi 0.0857 d.f. 158 (Not adjusted for missing values) 299 Analysis of variance peak stiffness ratio Variate: Peak_stiffness_ratio Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Block stratum 10 3.85027 0.38503 10.83 Block.Dish stratum Fungi 17 13.67544 0.80444 22.62 <.001 Residual 148 (22) 5.26328 0.03556 0.71 Block.Dish.Area stratum W_specie 1 3.59532 3.59532 72.09 <.001 Fungi.W_specie 17 6.97775 0.41046 8.23 <.001 Residual 158 (22) 7.87991 0.04987 Total 351 (44) 38.62168 Message: the following units have large residuals. Block 1 Dish 5 0.334 s.e. 0.115 Block 7 Dish 5 -0.362 s.e. 0.115 Block 8 Dish 5 0.313 s.e. 0.115 Block 8 Dish 7 -0.322 s.e. 0.115 Block 9 Dish 18 -0.325 s.e. 0.115 Block 1 Dish 3 Area 1 0.448 s.e. 0.141 Block 1 Dish 3 Area 2 -0.448 s.e. 0.141 Block 1 Dish 13 Area 1 0.536 s.e. 0.141 Block 1 Dish 13 Area 2 -0.536 s.e. 0.141 Block 8 Dish 5 Area 1 -0.421 s.e. 0.141 Block 8 Dish 5 Area 2 0.421 s.e. 0.141 300 Tables of means Variate: Peak_stiffness_ratio Grand mean 0.921 Fungi A. pull (B) A. pull (W) Alt Botry Chaet glob 0.891 1.026 1.008 1.007 0.536 Fungi Clad Con put Conioch Control Epicoc 0.474 1.044 0.581 1.000 1.035 Fungi Hormonema Lecyth Lewia Mollisia Phialocephala 1.045 1.029 1.052 1.049 0.952 Fungi Phialophora Phoma Trichaptum 1.066 0.969 0.812 W_specie Lime Spruce 0.826 1.016 Fungi W_specie Lime Spruce A. pull (B) 0.928 0.854 A. pull (W) 0.950 1.102 Alt 1.027 0.989 Botry 1.028 0.985 Chaet glob 0.166 0.905 Clad -0.012 0.961 Con put 0.948 1.140 Conioch 0.372 0.790 Control 1.000 1.000 Epicoc 0.956 1.113 Hormonema 0.991 1.100 Lecyth 0.994 1.064 Lewia 0.960 1.143 Mollisia 0.977 1.120 Phialocephala 0.814 1.090 Phialophora 1.017 1.115 Phoma 0.970 0.968 Trichaptum 0.773 0.850 Standard errors of differences of means Table Fungi W_specie Fungi W_specie rep. 22 198 11 s.e.d. 0.0569 0.0224 0.0881 d.f. 148 158 300.53 Except when comparing means with the same level(s) of Fungi 0.0952 d.f. 158 (Not adjusted for missing values) 301 Analysis of variance peak toughness (work) ratio Variate: Peak_work_ratio Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Block stratum 10 5.2188 0.5219 4.51 Block.Dish stratum Fungi 17 33.0956 1.9468 16.82 <.001 Residual 148 (22) 17.1253 0.1157 0.61 Block.Dish.Area stratum W_specie 1 2.3098 2.3098 12.12 <.001 Fungi.W_specie 17 9.8291 0.5782 3.03 <.001 Residual 158 (22) 30.1147 0.1906 Total 351 (44) 90.4632 Message: the following units have large residuals. Block 1 Dish 4 0.750 s.e. 0.208 Block 1 Dish 18 0.636 s.e. 0.208 Block 1 Dish 18 Area 1 -0.756 s.e. 0.276 Block 1 Dish 18 Area 2 0.756 s.e. 0.276 Block 2 Dish 2 Area 1 1.058 s.e. 0.276 Block 2 Dish 2 Area 2 -1.058 s.e. 0.276 302 Tables of means Variate: Peak_work_ratio Grand mean 0.874 Fungi A. pull (B) A. pull (W) Alt Botry Chaet glob 0.991 1.076 0.911 1.123 0.370 Fungi Clad Con put Conioch Control Epicoc 0.261 1.270 0.294 1.000 0.859 Fungi Hormonema Lecyth Lewia Mollisia Phialocephala 1.155 1.049 0.859 0.795 0.898 Fungi Phialophora Phoma Trichaptum 1.047 1.111 0.663 W_specie Lime Spruce 0.798 0.950 Fungi W_specie Lime Spruce A. pull (B) 1.111 0.870 A. pull (W) 1.107 1.045 Alt 0.745 1.078 Botry 1.248 0.998 Chaet glob 0.069 0.671 Clad 0.001 0.521 Con put 1.047 1.493 Conioch 0.050 0.538 Control 1.000 1.000 Epicoc 0.782 0.936 Hormonema 1.168 1.141 Lecyth 1.155 0.943 Lewia 0.847 0.871 Mollisia 0.729 0.861 Phialocephala 0.554 1.242 Phialophora 1.244 0.850 Phoma 1.041 1.181 Trichaptum 0.459 0.867 Standard errors of differences of means Table Fungi W_specie Fungi W_specie rep. 22 198 11 s.e.d. 0.1026 0.0439 0.1669 d.f. 148 158 292.85 Except when comparing means with the same level(s) of Fungi 0.1862 d.f. 158 (Not adjusted for missing values) 303 Appendix 2: Graphic determination of modulus of elasticity, example of calculation 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 Modulus of elasticity (MOE) = (\u00CE\u0094 stress / \u00CE\u0094 strain) MOE = (25285173.1 - 15456205.1) / (0.011158 - 0.008097) MOE = 3210593954 N/m2 Peak tensile stress = 23941131.7 N/m2 0 5000000 10000000 15000000 20000000 25000000 30000000 0 0 .0 0 0 6 1 0 4 9 9 0 .0 0 1 2 2 3 8 8 5 0 .0 0 1 8 4 0 6 8 2 0 .0 0 2 4 4 7 7 6 9 0 .0 0 3 0 6 4 5 6 7 0 .0 0 3 6 7 4 8 0 3 0 .0 0 4 2 8 5 3 0 2 0 .0 0 4 8 9 8 9 5 0 .0 0 5 5 0 9 1 8 6 0 .0 0 6 1 2 2 8 3 5 0 .0 0 6 7 3 9 3 7 0 .0 0 6 8 4 4 8 8 2 St re ss Strain Tensile Stress - Strain Lime - Mollisia sp. \u00CE\u0094Stress \u00CE\u0094Strain Peas tensile stress 304 Appendix 3: Statistical analysis Chapter 5 Analysis of variance frequency of isolation of fungi Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 0.00000 0.00000 0.00 block.*Units* stratum filter 4 0.00000 0.00000 0.00 1.000 fungi 6 1.06571 0.17762 12.19 <.001 filter.fungi 24 0.63406 0.02642 1.81 0.018 Residual 136 1.98119 0.01457 Total 174 3.68096 Message: the following units have large residuals. block 2 *units* 28 -0.2857 s.e. 0.1064 block 3 *units* 7 0.3056 s.e. 0.1064 305 Tables of means Variate: freq Grand mean 0.1429 filter 1 2 3 4 5 0.1429 0.1429 0.1429 0.1429 0.1429 fungi Alternaria sp. Aureobasidium pullulans 0.0574 0.3054 fungi Cladosporium sp. Epicoccum sp. 0.1211 0.1509 fungi Hormonema dematioides Others 0.1372 0.1718 fungi Phoma sp. 0.0562 filter fungi Alternaria sp. Aureobasidium pullulans 1 0.0500 0.3111 2 0.0400 0.3500 3 0.0900 0.4171 4 0.0000 0.2386 5 0.1071 0.2100 filter fungi Cladosporium sp. Epicoccum sp. 1 0.0500 0.2389 2 0.1300 0.0800 3 0.0400 0.0686 4 0.1586 0.1586 5 0.2271 0.2086 filter fungi Hormonema dematioides Others 1 0.1889 0.1389 2 0.2200 0.1400 3 0.1186 0.1371 4 0.0686 0.2857 5 0.0900 0.1571 filter fungi Phoma sp. 1 0.0222 2 0.0400 3 0.1286 4 0.0900 5 0.0000 306 Standard errors of differences of means Table filter fungi filter fungi rep. 35 25 5 d.f. 136 136 136 s.e.d. 0.02885 0.03414 0.07633 Least significant differences of means (5% level) Table filter fungi filter fungi rep. 35 25 5 d.f. 136 136 136 l.s.d. 0.05706 0.06751 0.15096 Analysis of variance fungal stains 0 to 40 weeks Variate: W1 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 0.00045807 0.00011452 1.97 rack.sample stratum Exposure 6 0.00020982 0.00003497 0.60 0.727 Residual 24 0.00139711 0.00005821 0.88 rack.sample.area stratum treatment 3 0.00013568 0.00004523 0.68 0.565 Exposure.treatment 18 0.00125571 0.00006976 1.05 0.413 Residual 84 0.00556556 0.00006626 1.00 rack.sample.area.strip stratum chem_charg 3 0.00012939 0.00004313 0.65 0.583 Exposure.chem_charg 18 0.00126200 0.00007011 1.06 0.394 treatment.chem_charg 9 0.00054106 0.00006012 0.91 0.519 Exposure.treatment.chem_charg 54 0.00363311 0.00006728 1.02 0.451 Residual 336 0.02226224 0.00006626 Total 559 0.03684976 307 Message: the following units have large residuals. rack 5 sample 2 0.00572 s.e. 0.00158 rack 3 sample 1 area 3 0.01431 s.e. 0.00315 rack 3 sample 3 area 3 0.01371 s.e. 0.00315 rack 3 sample 4 area 3 0.01011 s.e. 0.00315 rack 5 sample 2 area 1 0.01843 s.e. 0.00315 rack 3 sample 1 area 3 strip 1 -0.01909 s.e. 0.00631 rack 3 sample 1 area 3 strip 2 -0.01909 s.e. 0.00631 rack 3 sample 1 area 3 strip 3 -0.01909 s.e. 0.00631 rack 3 sample 1 area 3 strip 4 0.05726 s.e. 0.00631 rack 3 sample 3 area 3 strip 4 0.05486 s.e. 0.00631 rack 3 sample 4 area 3 strip 4 0.04046 s.e. 0.00631 rack 5 sample 2 area 1 strip 1 -0.02457 s.e. 0.00631 rack 5 sample 2 area 1 strip 2 0.07371 s.e. 0.00631 rack 5 sample 2 area 1 strip 3 -0.02457 s.e. 0.00631 rack 5 sample 2 area 1 strip 4 -0.02457 s.e. 0.00631 Tables of means Variate: W1 Grand mean 0.00067 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 0.00114 0.00119 0.00084 0.00000 0.00000 0.00154 0.00000 treatment acetic acid carpropamid tinuvin water 0.00048 0.00088 0.00133 0.00000 chem_charg 1 2 3 4 0.00068 0.00000 0.00065 0.00136 Exposure treatment acetic acid carpropamid tinuvin water filter 1 0.00000 0.00000 0.00457 0.00000 filter 2 0.00000 0.00000 0.00477 0.00000 filter 3 0.00337 0.00000 0.00000 0.00000 filter 4 0.00000 0.00000 0.00000 0.00000 filter 5 0.00000 0.00000 0.00000 0.00000 full 0.00000 0.00614 0.00000 0.00000 None 0.00000 0.00000 0.00000 0.00000 308 Exposure chem_charg 1 2 3 4 filter 1 0.00000 0.00000 0.00457 0.00000 filter 2 0.00477 0.00000 0.00000 0.00000 filter 3 0.00000 0.00000 0.00000 0.00337 filter 4 0.00000 0.00000 0.00000 0.00000 filter 5 0.00000 0.00000 0.00000 0.00000 full 0.00000 0.00000 0.00000 0.00614 None 0.00000 0.00000 0.00000 0.00000 treatment chem_charg 1 2 3 4 acetic acid 0.00000 0.00000 0.00000 0.00193 carpropamid 0.00000 0.00000 0.00000 0.00351 tinuvin 0.00273 0.00000 0.00261 0.00000 water 0.00000 0.00000 0.00000 0.00000 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 0.00000 0.00000 0.00000 0.00000 carpropamid 0.00000 0.00000 0.00000 0.00000 tinuvin 0.00000 0.00000 0.01829 0.00000 water 0.00000 0.00000 0.00000 0.00000 filter 2 acetic acid 0.00000 0.00000 0.00000 0.00000 carpropamid 0.00000 0.00000 0.00000 0.00000 tinuvin 0.01909 0.00000 0.00000 0.00000 water 0.00000 0.00000 0.00000 0.00000 filter 3 acetic acid 0.00000 0.00000 0.00000 0.01349 carpropamid 0.00000 0.00000 0.00000 0.00000 tinuvin 0.00000 0.00000 0.00000 0.00000 water 0.00000 0.00000 0.00000 0.00000 filter 4 acetic acid 0.00000 0.00000 0.00000 0.00000 carpropamid 0.00000 0.00000 0.00000 0.00000 tinuvin 0.00000 0.00000 0.00000 0.00000 water 0.00000 0.00000 0.00000 0.00000 filter 5 acetic acid 0.00000 0.00000 0.00000 0.00000 carpropamid 0.00000 0.00000 0.00000 0.00000 tinuvin 0.00000 0.00000 0.00000 0.00000 water 0.00000 0.00000 0.00000 0.00000 full acetic acid 0.00000 0.00000 0.00000 0.00000 carpropamid 0.00000 0.00000 0.00000 0.02457 tinuvin 0.00000 0.00000 0.00000 0.00000 water 0.00000 0.00000 0.00000 0.00000 None acetic acid 0.00000 0.00000 0.00000 0.00000 carpropamid 0.00000 0.00000 0.00000 0.00000 tinuvin 0.00000 0.00000 0.00000 0.00000 water 0.00000 0.00000 0.00000 0.00000 309 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 0.001206 0.000973 0.000973 0.002535 d.f. 24 84 336 107.99 Except when comparing means with the same level(s) of Exposure 0.002574 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 0.002535 0.001946 0.005129 d.f. 255.19 413.54 443.99 Except when comparing means with the same level(s) of Exposure 0.002574 0.005148 d.f. 336 413.54 treatment 0.001946 d.f. 336 Exposure.treatment 0.005148 d.f. 336 Exposure.chem_charg 0.005148 d.f. 413.54 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 0.002490 0.001935 0.001914 0.005024 d.f. 24 84 336 107.99 Except when comparing means with the same level(s) of Exposure 0.005119 d.f. 84 310 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 0.004992 0.003825 0.010079 d.f. 255.19 413.54 443.99 Except when comparing means with the same level(s) of Exposure 0.005063 0.010120 d.f. 336 413.54 treatment 0.003827 d.f. 336 Exposure.treatment 0.010127 d.f. 336 Exposure.chem_charg 0.010120 d.f. 413.54 Analysis of variance Variate: W2 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 0.032655 0.008164 0.21 rack.sample stratum Exposure 6 12.509154 2.084859 52.38 <.001 Residual 24 0.955231 0.039801 5.04 rack.sample.area stratum treatment 3 0.012641 0.004214 0.53 0.661 Exposure.treatment 18 0.054858 0.003048 0.39 0.987 Residual 84 0.663816 0.007903 0.85 rack.sample.area.strip stratum chem_charg 3 0.027087 0.009029 0.98 0.405 Exposure.chem_charg 18 0.107357 0.005964 0.64 0.864 treatment.chem_charg 9 0.110122 0.012236 1.32 0.224 Exposure.treatment.chem_charg 54 0.437373 0.008100 0.87 0.720 Residual 336 3.110674 0.009258 Total 559 18.020967 311 Message: the following units have large residuals. rack 1 sample 1 0.1363 s.e. 0.0413 rack 2 sample 3 -0.1296 s.e. 0.0413 rack 2 sample 6 0.0932 s.e. 0.0413 rack 1 sample 1 area 1 -0.1216 s.e. 0.0344 rack 1 sample 1 area 3 0.0972 s.e. 0.0344 rack 2 sample 3 area 2 -0.1111 s.e. 0.0344 rack 2 sample 3 area 3 0.1444 s.e. 0.0344 rack 3 sample 5 area 3 0.0989 s.e. 0.0344 rack 3 sample 5 area 4 -0.1166 s.e. 0.0344 rack 4 sample 5 area 2 0.1290 s.e. 0.0344 rack 4 sample 5 area 4 -0.1047 s.e. 0.0344 rack 5 sample 2 area 1 0.1010 s.e. 0.0344 rack 5 sample 2 area 4 -0.0938 s.e. 0.0344 rack 1 sample 1 area 1 strip 1 -0.2741 s.e. 0.0745 rack 1 sample 1 area 1 strip 3 0.5086 s.e. 0.0745 rack 1 sample 1 area 2 strip 2 0.4340 s.e. 0.0745 rack 1 sample 1 area 2 strip 3 -0.2479 s.e. 0.0745 rack 1 sample 1 area 2 strip 4 -0.3020 s.e. 0.0745 rack 1 sample 1 area 3 strip 1 -0.5247 s.e. 0.0745 rack 1 sample 1 area 3 strip 3 0.5299 s.e. 0.0745 rack 2 sample 3 area 1 strip 2 -0.2462 s.e. 0.0745 rack 2 sample 3 area 1 strip 3 0.2564 s.e. 0.0745 rack 2 sample 3 area 3 strip 3 0.2231 s.e. 0.0745 rack 2 sample 3 area 4 strip 1 -0.3201 s.e. 0.0745 rack 2 sample 3 area 4 strip 3 0.3710 s.e. 0.0745 rack 3 sample 5 area 3 strip 3 0.2738 s.e. 0.0745 rack 3 sample 5 area 4 strip 1 -0.2451 s.e. 0.0745 rack 4 sample 5 area 1 strip 3 0.3098 s.e. 0.0745 rack 4 sample 5 area 1 strip 4 -0.3125 s.e. 0.0745 rack 5 sample 2 area 1 strip 2 0.3834 s.e. 0.0745 rack 5 sample 2 area 1 strip 3 -0.2537 s.e. 0.0745 Tables of means Variate: W2 Grand mean 0.0703 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 0.0406 0.0077 0.0065 0.0012 0.0012 0.4350 0.0000 treatment acetic acid carpropamid tinuvin water 0.0774 0.0710 0.0685 0.0643 chem_charg 1 2 3 4 0.0759 0.0584 0.0739 0.0730 312 Exposure treatment acetic acid carpropamid tinuvin water filter 1 0.0391 0.0409 0.0403 0.0420 filter 2 0.0024 0.0095 0.0095 0.0095 filter 3 0.0167 0.0047 0.0024 0.0024 filter 4 0.0048 0.0000 0.0000 0.0000 filter 5 0.0047 0.0000 0.0000 0.0000 full 0.4741 0.4422 0.4277 0.3960 None 0.0000 0.0000 0.0000 0.0000 Exposure chem_charg 1 2 3 4 filter 1 0.0405 0.0238 0.0540 0.0440 filter 2 0.0048 0.0048 0.0167 0.0047 filter 3 0.0000 0.0024 0.0047 0.0190 filter 4 0.0000 0.0000 0.0000 0.0048 filter 5 0.0000 0.0000 0.0024 0.0024 full 0.4862 0.3779 0.4398 0.4360 None 0.0000 0.0000 0.0000 0.0000 treatment chem_charg 1 2 3 4 acetic acid 0.0756 0.0572 0.0924 0.0844 carpropamid 0.0890 0.0671 0.0483 0.0798 tinuvin 0.0832 0.0636 0.0543 0.0731 water 0.0559 0.0458 0.1008 0.0547 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 0.0191 0.0191 0.0477 0.0706 carpropamid 0.0667 0.0190 0.0294 0.0485 tinuvin 0.0571 0.0381 0.0469 0.0190 water 0.0190 0.0191 0.0920 0.0381 filter 2 acetic acid 0.0000 0.0000 0.0095 0.0000 carpropamid 0.0000 0.0191 0.0095 0.0095 tinuvin 0.0191 0.0000 0.0095 0.0095 water 0.0000 0.0000 0.0382 0.0000 filter 3 acetic acid 0.0000 0.0000 0.0000 0.0666 carpropamid 0.0000 0.0095 0.0000 0.0095 tinuvin 0.0000 0.0000 0.0095 0.0000 water 0.0000 0.0000 0.0095 0.0000 filter 4 acetic acid 0.0000 0.0000 0.0000 0.0191 carpropamid 0.0000 0.0000 0.0000 0.0000 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0000 0.0000 0.0000 filter 5 acetic acid 0.0000 0.0000 0.0095 0.0095 carpropamid 0.0000 0.0000 0.0000 0.0000 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0000 0.0000 0.0000 full acetic acid 0.5102 0.3810 0.5801 0.4252 carpropamid 0.5563 0.4220 0.2992 0.4912 tinuvin 0.5063 0.4071 0.3143 0.4832 water 0.3720 0.3016 0.5658 0.3446 None acetic acid 0.0000 0.0000 0.0000 0.0000 carpropamid 0.0000 0.0000 0.0000 0.0000 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0000 0.0000 0.0000 313 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 0.03154 0.01063 0.01150 0.03985 d.f. 24 84 336 55.48 Except when comparing means with the same level(s) of Exposure 0.02811 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 0.04110 0.02258 0.06607 d.f. 66.86 418.79 278.60 Except when comparing means with the same level(s) of Exposure 0.03043 0.05973 d.f. 336 418.79 treatment 0.02300 d.f. 336 Exposure.treatment 0.06085 d.f. 336 Exposure.chem_charg 0.05973 d.f. 418.79 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 0.06510 0.02113 0.02262 0.07984 d.f. 24 84 336 55.48 Except when comparing means with the same level(s) of Exposure 0.05590 d.f. 84 314 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 0.08204 0.04438 0.13006 d.f. 66.86 418.79 278.60 Except when comparing means with the same level(s) of Exposure 0.05985 0.11741 d.f. 336 418.79 treatment 0.04524 d.f. 336 Exposure.treatment 0.11970 d.f. 336 Exposure.chem_charg 0.11741 d.f. 418.79 Analysis of variance Variate: W3 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 0.053170 0.013293 0.34 rack.sample stratum Exposure 6 13.159952 2.193325 55.80 <.001 Residual 24 0.943390 0.039308 4.74 rack.sample.area stratum treatment 3 0.012742 0.004247 0.51 0.675 Exposure.treatment 18 0.046322 0.002573 0.31 0.997 Residual 84 0.697054 0.008298 0.87 rack.sample.area.strip stratum chem_charg 3 0.026898 0.008966 0.94 0.422 Exposure.chem_charg 18 0.158559 0.008809 0.92 0.551 treatment.chem_charg 9 0.102381 0.011376 1.19 0.299 Exposure.treatment.chem_charg 54 0.414593 0.007678 0.80 0.834 Residual 336 3.207419 0.009546 Total 559 18.822481 315 Message: the following units have large residuals. rack 1 sample 1 0.1326 s.e. 0.0410 rack 2 sample 3 -0.1211 s.e. 0.0410 rack 2 sample 6 0.0929 s.e. 0.0410 rack 1 sample 1 area 1 -0.1150 s.e. 0.0353 rack 1 sample 1 area 3 0.0918 s.e. 0.0353 rack 2 sample 3 area 2 -0.1039 s.e. 0.0353 rack 2 sample 3 area 3 0.1657 s.e. 0.0353 rack 3 sample 5 area 3 0.0971 s.e. 0.0353 rack 3 sample 5 area 4 -0.1159 s.e. 0.0353 rack 4 sample 5 area 2 0.1236 s.e. 0.0353 rack 4 sample 5 area 4 -0.0982 s.e. 0.0353 rack 5 sample 2 area 4 -0.1169 s.e. 0.0353 rack 1 sample 1 area 1 strip 1 -0.2718 s.e. 0.0757 rack 1 sample 1 area 1 strip 2 -0.2282 s.e. 0.0757 rack 1 sample 1 area 1 strip 3 0.5015 s.e. 0.0757 rack 1 sample 1 area 2 strip 2 0.4292 s.e. 0.0757 rack 1 sample 1 area 2 strip 3 -0.2527 s.e. 0.0757 rack 1 sample 1 area 2 strip 4 -0.2591 s.e. 0.0757 rack 1 sample 1 area 3 strip 1 -0.5200 s.e. 0.0757 rack 1 sample 1 area 3 strip 3 0.5251 s.e. 0.0757 rack 2 sample 3 area 1 strip 2 -0.2674 s.e. 0.0757 rack 2 sample 3 area 4 strip 1 -0.3367 s.e. 0.0757 rack 2 sample 3 area 4 strip 3 0.3638 s.e. 0.0757 rack 3 sample 5 area 3 strip 3 0.2810 s.e. 0.0757 rack 3 sample 5 area 4 strip 1 -0.2499 s.e. 0.0757 rack 3 sample 5 area 4 strip 3 0.2274 s.e. 0.0757 rack 4 sample 5 area 1 strip 3 0.2885 s.e. 0.0757 rack 4 sample 5 area 1 strip 4 -0.2959 s.e. 0.0757 rack 5 sample 2 area 1 strip 2 0.3906 s.e. 0.0757 rack 5 sample 2 area 1 strip 3 -0.2466 s.e. 0.0757 Tables of means Variate: W3 Grand mean 0.0757 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 0.0489 0.0172 0.0119 0.0018 0.0012 0.4493 0.0000 treatment acetic acid carpropamid tinuvin water 0.0828 0.0755 0.0753 0.0694 chem_charg 1 2 3 4 0.0790 0.0638 0.0790 0.0811 316 Exposure treatment acetic acid carpropamid tinuvin water filter 1 0.0533 0.0433 0.0545 0.0444 filter 2 0.0095 0.0262 0.0143 0.0190 filter 3 0.0285 0.0095 0.0024 0.0071 filter 4 0.0048 0.0000 0.0000 0.0024 filter 5 0.0047 0.0000 0.0000 0.0000 full 0.4789 0.4493 0.4562 0.4126 None 0.0000 0.0000 0.0000 0.0000 Exposure chem_charg 1 2 3 4 filter 1 0.0452 0.0309 0.0611 0.0583 filter 2 0.0048 0.0262 0.0262 0.0119 filter 3 0.0000 0.0047 0.0047 0.0381 filter 4 0.0000 0.0024 0.0000 0.0048 filter 5 0.0000 0.0000 0.0024 0.0024 full 0.5028 0.3827 0.4589 0.4526 None 0.0000 0.0000 0.0000 0.0000 treatment chem_charg 1 2 3 4 acetic acid 0.0756 0.0626 0.0965 0.0966 carpropamid 0.0917 0.0766 0.0496 0.0839 tinuvin 0.0873 0.0649 0.0679 0.0812 water 0.0613 0.0512 0.1021 0.0628 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 0.0191 0.0286 0.0571 0.1086 carpropamid 0.0667 0.0285 0.0294 0.0485 tinuvin 0.0761 0.0381 0.0658 0.0381 water 0.0190 0.0286 0.0920 0.0381 filter 2 acetic acid 0.0000 0.0190 0.0190 0.0000 carpropamid 0.0000 0.0762 0.0095 0.0190 tinuvin 0.0191 0.0000 0.0286 0.0095 water 0.0000 0.0095 0.0477 0.0190 filter 3 acetic acid 0.0000 0.0095 0.0000 0.1046 carpropamid 0.0000 0.0095 0.0000 0.0286 tinuvin 0.0000 0.0000 0.0095 0.0000 water 0.0000 0.0000 0.0095 0.0191 filter 4 acetic acid 0.0000 0.0000 0.0000 0.0191 carpropamid 0.0000 0.0000 0.0000 0.0000 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0095 0.0000 0.0000 filter 5 acetic acid 0.0000 0.0000 0.0095 0.0095 carpropamid 0.0000 0.0000 0.0000 0.0000 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0000 0.0000 0.0000 full acetic acid 0.5102 0.3810 0.5896 0.4347 carpropamid 0.5754 0.4220 0.3087 0.4912 tinuvin 0.5158 0.4166 0.3715 0.5211 water 0.4099 0.3111 0.5658 0.3635 None acetic acid 0.0000 0.0000 0.0000 0.0000 carpropamid 0.0000 0.0000 0.0000 0.0000 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0000 0.0000 0.0000 317 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 0.03135 0.01089 0.01168 0.04006 d.f. 24 84 336 57.44 Except when comparing means with the same level(s) of Exposure 0.02881 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 0.04121 0.02297 0.06685 d.f. 69.09 418.41 288.35 Except when comparing means with the same level(s) of Exposure 0.03090 0.06077 d.f. 336 418.41 treatment 0.02336 d.f. 336 Exposure.treatment 0.06179 d.f. 336 Exposure.chem_charg 0.06077 d.f. 418.41 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 0.06470 0.02165 0.02297 0.08021 d.f. 24 84 336 57.44 Except when comparing means with the same level(s) of Exposure 0.05729 d.f. 84 318 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 0.08222 0.04515 0.13157 d.f. 69.09 418.41 288.35 Except when comparing means with the same level(s) of Exposure 0.06077 0.11946 d.f. 336 418.41 treatment 0.04594 d.f. 336 Exposure.treatment 0.12155 d.f. 336 Exposure.chem_charg 0.11946 d.f. 418.41 Analysis of variance Variate: W4 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 0.06681 0.01670 0.35 rack.sample stratum Exposure 6 15.26489 2.54415 53.04 <.001 Residual 24 1.15120 0.04797 5.51 rack.sample.area stratum treatment 3 0.00785 0.00262 0.30 0.825 Exposure.treatment 18 0.05030 0.00279 0.32 0.996 Residual 84 0.73090 0.00870 0.84 rack.sample.area.strip stratum chem_charg 3 0.03362 0.01121 1.08 0.357 Exposure.chem_charg 18 0.20165 0.01120 1.08 0.370 treatment.chem_charg 9 0.08335 0.00926 0.89 0.531 Exposure.treatment.chem_charg 54 0.41719 0.00773 0.75 0.906 Residual 336 3.48143 0.01036 Total 559 21.48919 319 Message: the following units have large residuals. rack 1 sample 1 0.1489 s.e. 0.0453 rack 2 sample 3 -0.1052 s.e. 0.0453 rack 2 sample 6 0.1200 s.e. 0.0453 rack 1 sample 1 area 1 -0.1125 s.e. 0.0361 rack 1 sample 1 area 3 0.1464 s.e. 0.0361 rack 2 sample 3 area 1 -0.1187 s.e. 0.0361 rack 2 sample 3 area 3 0.1591 s.e. 0.0361 rack 4 sample 5 area 2 0.1254 s.e. 0.0361 rack 4 sample 5 area 4 -0.0940 s.e. 0.0361 rack 5 sample 2 area 4 -0.1028 s.e. 0.0361 rack 1 sample 1 area 1 strip 1 -0.3100 s.e. 0.0788 rack 1 sample 1 area 1 strip 3 0.4823 s.e. 0.0788 rack 1 sample 1 area 2 strip 2 0.4007 s.e. 0.0788 rack 1 sample 1 area 2 strip 3 -0.2906 s.e. 0.0788 rack 1 sample 1 area 3 strip 1 -0.5244 s.e. 0.0788 rack 1 sample 1 area 3 strip 2 -0.2630 s.e. 0.0788 rack 1 sample 1 area 3 strip 3 0.4824 s.e. 0.0788 rack 1 sample 1 area 3 strip 4 0.3050 s.e. 0.0788 rack 2 sample 3 area 2 strip 3 0.2585 s.e. 0.0788 rack 2 sample 3 area 4 strip 1 -0.3082 s.e. 0.0788 rack 2 sample 3 area 4 strip 3 0.3828 s.e. 0.0788 rack 3 sample 5 area 3 strip 3 0.2929 s.e. 0.0788 rack 3 sample 5 area 4 strip 1 -0.3140 s.e. 0.0788 rack 4 sample 5 area 1 strip 3 0.2505 s.e. 0.0788 rack 4 sample 5 area 1 strip 4 -0.2674 s.e. 0.0788 rack 5 sample 2 area 1 strip 2 0.4572 s.e. 0.0788 rack 5 sample 2 area 1 strip 3 -0.2466 s.e. 0.0788 rack 5 sample 2 area 4 strip 1 0.2383 s.e. 0.0788 Tables of means Variate: W4 Grand mean 0.0860 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 0.0744 0.0238 0.0148 0.0018 0.0012 0.4861 0.0000 treatment acetic acid carpropamid tinuvin water 0.0899 0.0877 0.0865 0.0799 chem_charg 1 2 3 4 0.0908 0.0727 0.0892 0.0913 320 Exposure treatment acetic acid carpropamid tinuvin water filter 1 0.0628 0.0789 0.0854 0.0705 filter 2 0.0142 0.0381 0.0214 0.0214 filter 3 0.0285 0.0143 0.0047 0.0119 filter 4 0.0048 0.0000 0.0000 0.0024 filter 5 0.0047 0.0000 0.0000 0.0000 full 0.5145 0.4826 0.4942 0.4530 None 0.0000 0.0000 0.0000 0.0000 Exposure chem_charg 1 2 3 4 filter 1 0.0761 0.0499 0.0943 0.0773 filter 2 0.0095 0.0333 0.0381 0.0142 filter 3 0.0024 0.0095 0.0095 0.0381 filter 4 0.0000 0.0024 0.0000 0.0048 filter 5 0.0000 0.0000 0.0024 0.0024 full 0.5480 0.4136 0.4802 0.5026 None 0.0000 0.0000 0.0000 0.0000 treatment chem_charg 1 2 3 4 acetic acid 0.0824 0.0694 0.1005 0.1075 carpropamid 0.1066 0.0847 0.0659 0.0934 tinuvin 0.0981 0.0744 0.0815 0.0921 water 0.0762 0.0621 0.1089 0.0723 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 0.0286 0.0381 0.0571 0.1275 carpropamid 0.1142 0.0474 0.0863 0.0675 tinuvin 0.1235 0.0666 0.1038 0.0475 water 0.0379 0.0475 0.1301 0.0666 filter 2 acetic acid 0.0000 0.0285 0.0285 0.0000 carpropamid 0.0000 0.0952 0.0286 0.0285 tinuvin 0.0286 0.0000 0.0477 0.0095 water 0.0095 0.0095 0.0477 0.0190 filter 3 acetic acid 0.0000 0.0095 0.0000 0.1046 carpropamid 0.0000 0.0190 0.0095 0.0286 tinuvin 0.0000 0.0095 0.0095 0.0000 water 0.0095 0.0000 0.0190 0.0191 filter 4 acetic acid 0.0000 0.0000 0.0000 0.0191 carpropamid 0.0000 0.0000 0.0000 0.0000 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0095 0.0000 0.0000 filter 5 acetic acid 0.0000 0.0000 0.0095 0.0095 carpropamid 0.0000 0.0000 0.0000 0.0000 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0000 0.0000 0.0000 full acetic acid 0.5482 0.4096 0.6086 0.4916 carpropamid 0.6323 0.4315 0.3371 0.5294 tinuvin 0.5348 0.4450 0.4095 0.5876 water 0.4764 0.3681 0.5658 0.4016 None acetic acid 0.0000 0.0000 0.0000 0.0000 carpropamid 0.0000 0.0000 0.0000 0.0000 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0000 0.0000 0.0000 321 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 0.03463 0.01115 0.01217 0.04303 d.f. 24 84 336 52.76 Except when comparing means with the same level(s) of Exposure 0.02950 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 0.04446 0.02384 0.07043 d.f. 63.29 419.08 262.45 Except when comparing means with the same level(s) of Exposure 0.03219 0.06308 d.f. 336 419.08 treatment 0.02433 d.f. 336 Exposure.treatment 0.06438 d.f. 336 Exposure.chem_charg 0.06308 d.f. 419.08 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 0.07147 0.02217 0.02393 0.08632 d.f. 24 84 336 52.76 Except when comparing means with the same level(s) of Exposure 0.05866 d.f. 84 322 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 0.08883 0.04686 0.13868 d.f. 63.29 419.08 262.45 Except when comparing means with the same level(s) of Exposure 0.06332 0.12398 d.f. 336 419.08 treatment 0.04786 d.f. 336 Exposure.treatment 0.12664 d.f. 336 Exposure.chem_charg 0.12398 d.f. 419.08 Analysis of variance Variate: W6 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 0.03918 0.00980 0.21 rack.sample stratum Exposure 6 16.88594 2.81432 59.67 <.001 Residual 24 1.13197 0.04717 4.61 rack.sample.area stratum treatment 3 0.00807 0.00269 0.26 0.852 Exposure.treatment 18 0.14473 0.00804 0.79 0.711 Residual 84 0.85984 0.01024 0.82 rack.sample.area.strip stratum chem_charg 3 0.03842 0.01281 1.02 0.382 Exposure.chem_charg 18 0.28069 0.01559 1.25 0.221 treatment.chem_charg 9 0.10613 0.01179 0.94 0.487 Exposure.treatment.chem_charg 54 0.52708 0.00976 0.78 0.865 Residual 336 4.19905 0.01250 Total 559 24.22110 323 Message: the following units have large residuals. rack 1 sample 1 0.1396 s.e. 0.0450 rack 2 sample 3 -0.1043 s.e. 0.0450 rack 2 sample 6 0.1304 s.e. 0.0450 rack 1 sample 1 area 1 -0.1203 s.e. 0.0392 rack 1 sample 1 area 3 0.1647 s.e. 0.0392 rack 2 sample 3 area 1 -0.1248 s.e. 0.0392 rack 2 sample 3 area 3 0.1627 s.e. 0.0392 rack 4 sample 5 area 2 0.1431 s.e. 0.0392 rack 1 sample 1 area 1 strip 1 -0.3029 s.e. 0.0866 rack 1 sample 1 area 1 strip 3 0.4894 s.e. 0.0866 rack 1 sample 1 area 2 strip 2 0.3720 s.e. 0.0866 rack 1 sample 1 area 2 strip 3 -0.2716 s.e. 0.0866 rack 1 sample 1 area 3 strip 1 -0.5530 s.e. 0.0866 rack 1 sample 1 area 3 strip 2 -0.2820 s.e. 0.0866 rack 1 sample 1 area 3 strip 3 0.4918 s.e. 0.0866 rack 1 sample 1 area 3 strip 4 0.3431 s.e. 0.0866 rack 2 sample 3 area 4 strip 1 -0.2749 s.e. 0.0866 rack 2 sample 3 area 4 strip 3 0.3780 s.e. 0.0866 rack 3 sample 3 area 2 strip 2 0.3353 s.e. 0.0866 rack 3 sample 3 area 3 strip 4 0.2824 s.e. 0.0866 rack 3 sample 5 area 3 strip 3 0.2709 s.e. 0.0866 rack 3 sample 5 area 4 strip 1 -0.3306 s.e. 0.0866 rack 4 sample 5 area 1 strip 3 0.2577 s.e. 0.0866 rack 4 sample 5 area 1 strip 4 -0.2602 s.e. 0.0866 rack 5 sample 2 area 1 strip 2 0.4211 s.e. 0.0866 Tables of means Variate: W6 Grand mean 0.1106 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 0.1243 0.0654 0.0398 0.0113 0.0089 0.5246 0.0000 treatment acetic acid carpropamid tinuvin water 0.1113 0.1124 0.1144 0.1043 chem_charg 1 2 3 4 0.1156 0.0964 0.1136 0.1168 324 Exposure treatment acetic acid carpropamid tinuvin water filter 1 0.0961 0.1240 0.1566 0.1204 filter 2 0.0499 0.0903 0.0500 0.0713 filter 3 0.0547 0.0333 0.0214 0.0499 filter 4 0.0119 0.0119 0.0190 0.0024 filter 5 0.0167 0.0119 0.0000 0.0071 full 0.5502 0.5154 0.5537 0.4791 None 0.0000 0.0000 0.0000 0.0000 Exposure chem_charg 1 2 3 4 filter 1 0.1307 0.1164 0.1466 0.1035 filter 2 0.0571 0.0784 0.0714 0.0546 filter 3 0.0190 0.0308 0.0309 0.0785 filter 4 0.0143 0.0071 0.0190 0.0048 filter 5 0.0071 0.0047 0.0095 0.0143 full 0.5813 0.4374 0.5178 0.5620 None 0.0000 0.0000 0.0000 0.0000 treatment chem_charg 1 2 3 4 acetic acid 0.1082 0.0802 0.1236 0.1333 carpropamid 0.1297 0.1064 0.0874 0.1260 tinuvin 0.1321 0.0948 0.1182 0.1125 water 0.0925 0.1041 0.1252 0.0954 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 0.0665 0.0666 0.0762 0.1751 carpropamid 0.1902 0.1139 0.1053 0.0866 tinuvin 0.1995 0.1235 0.2273 0.0761 water 0.0665 0.1614 0.1775 0.0761 filter 2 acetic acid 0.0760 0.0379 0.0570 0.0286 carpropamid 0.0381 0.1426 0.0665 0.1138 tinuvin 0.0571 0.0285 0.0858 0.0285 water 0.0570 0.1045 0.0762 0.0475 filter 3 acetic acid 0.0191 0.0190 0.0286 0.1521 carpropamid 0.0000 0.0285 0.0381 0.0666 tinuvin 0.0286 0.0190 0.0190 0.0190 water 0.0285 0.0569 0.0379 0.0761 filter 4 acetic acid 0.0095 0.0000 0.0190 0.0191 carpropamid 0.0191 0.0000 0.0286 0.0000 tinuvin 0.0286 0.0190 0.0285 0.0000 water 0.0000 0.0095 0.0000 0.0000 filter 5 acetic acid 0.0191 0.0000 0.0381 0.0095 carpropamid 0.0095 0.0095 0.0000 0.0285 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0095 0.0000 0.0191 full acetic acid 0.5672 0.4382 0.6465 0.5489 carpropamid 0.6513 0.4506 0.3732 0.5864 tinuvin 0.6112 0.4736 0.4665 0.6636 water 0.4954 0.3871 0.5848 0.4491 None acetic acid 0.0000 0.0000 0.0000 0.0000 carpropamid 0.0000 0.0000 0.0000 0.0000 tinuvin 0.0000 0.0000 0.0000 0.0000 water 0.0000 0.0000 0.0000 0.0000 325 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 0.03434 0.01209 0.01336 0.04412 d.f. 24 84 336 58.36 Except when comparing means with the same level(s) of Exposure 0.03199 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 0.04600 0.02611 0.07547 d.f. 73.98 419.45 303.84 Except when comparing means with the same level(s) of Exposure 0.03535 0.06909 d.f. 336 419.45 treatment 0.02672 d.f. 336 Exposure.treatment 0.07070 d.f. 336 Exposure.chem_charg 0.06909 d.f. 419.45 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 0.07087 0.02405 0.02628 0.08831 d.f. 24 84 336 58.36 Except when comparing means with the same level(s) of Exposure 0.06362 d.f. 84 326 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 0.09167 0.05133 0.14851 d.f. 73.98 419.45 303.84 Except when comparing means with the same level(s) of Exposure 0.06954 0.13580 d.f. 336 419.45 treatment 0.05257 d.f. 336 Exposure.treatment 0.13908 d.f. 336 Exposure.chem_charg 0.13580 d.f. 419.45 Analysis of variance Variate: W8 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 18999.31 4749.83 0.92 rack.sample stratum Exposure 6 168632.35 28105.39 5.47 0.001 Residual 24 123263.41 5135.98 48.06 rack.sample.area stratum treatment 3 433.69 144.56 1.35 0.263 Exposure.treatment 18 4261.70 236.76 2.22 0.008 Residual 84 8976.56 106.86 1.79 rack.sample.area.strip stratum chem_charg 3 52.56 17.52 0.29 0.830 Exposure.chem_charg 18 603.47 33.53 0.56 0.925 treatment.chem_charg 9 394.28 43.81 0.73 0.678 Exposure.treatment.chem_charg 54 2869.02 53.13 0.89 0.693 Residual 336 20061.61 59.71 Total 559 348547.96 327 Message: the following units have large residuals. rack 1 sample 1 43.31 s.e. 14.84 rack 2 sample 3 -32.18 s.e. 14.84 rack 3 sample 5 -35.55 s.e. 14.84 rack 5 sample 2 43.30 s.e. 14.84 rack 1 sample 1 area 2 -11.56 s.e. 4.00 rack 4 sample 1 area 2 14.73 s.e. 4.00 rack 4 sample 1 area 4 -11.25 s.e. 4.00 rack 4 sample 5 area 2 22.81 s.e. 4.00 rack 4 sample 5 area 3 -20.32 s.e. 4.00 rack 5 sample 2 area 2 -11.56 s.e. 4.00 rack 2 sample 3 area 1 strip 3 20.83 s.e. 5.99 rack 2 sample 3 area 4 strip 2 -17.86 s.e. 5.99 rack 3 sample 5 area 4 strip 3 26.68 s.e. 5.99 rack 4 sample 1 area 2 strip 4 -18.93 s.e. 5.99 rack 4 sample 4 area 4 strip 1 18.33 s.e. 5.99 rack 4 sample 5 area 1 strip 2 47.80 s.e. 5.99 rack 4 sample 5 area 1 strip 3 -19.82 s.e. 5.99 rack 4 sample 5 area 1 strip 4 -21.38 s.e. 5.99 rack 4 sample 5 area 2 strip 1 -60.86 s.e. 5.99 rack 4 sample 5 area 2 strip 2 28.41 s.e. 5.99 rack 4 sample 5 area 2 strip 3 21.21 s.e. 5.99 rack 4 sample 5 area 4 strip 2 56.43 s.e. 5.99 rack 4 sample 5 area 4 strip 3 -18.71 s.e. 5.99 rack 4 sample 5 area 4 strip 4 -20.78 s.e. 5.99 Tables of means Variate: W8 Grand mean 8.35 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 3.42 0.05 0.04 1.39 2.82 50.74 0.00 treatment acetic acid carpropamid tinuvin water 7.70 7.42 9.67 8.61 chem_charg 1 2 3 4 8.88 8.13 8.24 8.16 328 Exposure treatment acetic acid carpropamid tinuvin water filter 1 3.64 4.37 4.00 1.68 filter 2 0.03 0.06 0.05 0.07 filter 3 0.06 0.02 0.02 0.05 filter 4 1.31 0.85 0.16 3.24 filter 5 0.01 3.60 1.16 6.50 full 48.86 43.04 62.30 48.76 None 0.00 0.00 0.00 0.00 Exposure chem_charg 1 2 3 4 filter 1 4.70 2.96 3.90 2.13 filter 2 0.03 0.06 0.06 0.07 filter 3 0.04 0.03 0.04 0.04 filter 4 1.40 1.54 1.07 1.55 filter 5 3.20 1.85 5.28 0.96 full 52.78 50.46 47.36 52.36 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 10.12 6.88 7.25 6.56 carpropamid 7.34 7.79 7.83 6.72 tinuvin 9.85 10.31 9.12 9.41 water 8.20 7.54 8.77 9.95 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 8.24 2.39 3.84 0.07 carpropamid 3.95 5.21 4.17 4.13 tinuvin 3.80 3.95 4.02 4.23 water 2.81 0.29 3.56 0.07 filter 2 acetic acid 0.01 0.04 0.01 0.07 carpropamid 0.06 0.08 0.09 0.03 tinuvin 0.03 0.07 0.03 0.06 water 0.02 0.05 0.10 0.12 filter 3 acetic acid 0.05 0.07 0.07 0.05 carpropamid 0.02 0.02 0.03 0.02 tinuvin 0.03 0.00 0.02 0.04 water 0.05 0.04 0.05 0.06 filter 4 acetic acid 0.00 1.43 0.54 3.26 carpropamid 2.93 0.03 0.42 0.02 tinuvin 0.64 0.00 0.00 0.00 water 2.03 4.69 3.31 2.94 filter 5 acetic acid 0.03 0.00 0.02 0.01 carpropamid 2.46 5.56 6.39 0.00 tinuvin 0.00 0.07 4.13 0.45 water 10.29 1.77 10.57 3.36 full acetic acid 62.48 44.22 46.29 42.45 carpropamid 41.96 43.65 43.72 42.81 tinuvin 64.43 68.06 55.63 61.09 water 42.24 45.93 43.80 63.09 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 329 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 11.331 1.236 0.924 11.680 d.f. 24 84 336 27.06 Except when comparing means with the same level(s) of Exposure 3.269 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 11.527 2.021 12.423 d.f. 25.70 353.39 34.58 Except when comparing means with the same level(s) of Exposure 2.444 5.348 d.f. 336 353.39 treatment 1.847 d.f. 336 Exposure.treatment 4.887 d.f. 336 Exposure.chem_charg 5.348 d.f. 353.39 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 23.387 2.457 1.817 23.962 d.f. 24 84 336 27.06 Except when comparing means with the same level(s) of Exposure 6.501 d.f. 84 330 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 23.708 3.975 25.230 d.f. 25.70 353.39 34.58 Except when comparing means with the same level(s) of Exposure 4.806 10.517 d.f. 336 353.39 treatment 3.633 d.f. 336 Exposure.treatment 9.613 d.f. 336 Exposure.chem_charg 10.517 d.f. 353.39 Analysis of variance Variate: W10 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 42703.7 10675.9 1.81 rack.sample stratum Exposure 6 473741.2 78956.9 13.36 <.001 Residual 24 141872.3 5911.3 9.00 rack.sample.area stratum treatment 3 2054.6 684.9 1.04 0.378 Exposure.treatment 18 8288.4 460.5 0.70 0.800 Residual 84 55144.8 656.5 3.37 rack.sample.area.strip stratum chem_charg 3 76.0 25.3 0.13 0.942 Exposure.chem_charg 18 2688.1 149.3 0.77 0.738 treatment.chem_charg 9 2950.9 327.9 1.68 0.091 Exposure.treatment.chem_charg 54 8347.3 154.6 0.79 0.848 Residual 336 65393.0 194.6 Total 559 803260.4 331 Message: the following units have large residuals. rack 3 sample 2 -36.60 s.e. 15.92 rack 3 sample 6 33.72 s.e. 15.92 rack 4 sample 7 37.96 s.e. 15.92 rack 5 sample 3 36.66 s.e. 15.92 rack 1 sample 4 area 3 -28.82 s.e. 9.92 rack 1 sample 7 area 1 -28.22 s.e. 9.92 rack 1 sample 7 area 3 27.44 s.e. 9.92 rack 2 sample 4 area 4 -27.37 s.e. 9.92 rack 3 sample 6 area 4 -34.26 s.e. 9.92 rack 4 sample 2 area 1 -29.47 s.e. 9.92 rack 5 sample 3 area 1 -27.08 s.e. 9.92 rack 1 sample 4 area 1 strip 3 54.24 s.e. 10.81 rack 1 sample 4 area 1 strip 4 -34.97 s.e. 10.81 rack 1 sample 4 area 2 strip 1 -32.45 s.e. 10.81 rack 1 sample 4 area 2 strip 4 55.85 s.e. 10.81 rack 1 sample 4 area 4 strip 2 34.79 s.e. 10.81 rack 1 sample 7 area 2 strip 1 -33.50 s.e. 10.81 rack 1 sample 7 area 2 strip 4 44.80 s.e. 10.81 rack 2 sample 4 area 1 strip 1 -34.20 s.e. 10.81 rack 2 sample 4 area 1 strip 3 35.98 s.e. 10.81 rack 4 sample 2 area 4 strip 4 -45.80 s.e. 10.81 rack 4 sample 4 area 1 strip 4 37.61 s.e. 10.81 rack 4 sample 7 area 1 strip 1 -51.91 s.e. 10.81 rack 5 sample 3 area 1 strip 1 -44.33 s.e. 10.81 rack 5 sample 3 area 1 strip 3 35.33 s.e. 10.81 rack 5 sample 3 area 1 strip 4 39.16 s.e. 10.81 rack 5 sample 6 area 3 strip 3 32.81 s.e. 10.81 rack 5 sample 6 area 3 strip 4 -44.20 s.e. 10.81 Tables of means Variate: W10 Grand mean 36.43 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 21.58 22.16 28.01 43.14 40.11 100.00 0.00 treatment acetic acid carpropamid tinuvin water 36.34 38.33 33.35 37.70 chem_charg 1 2 3 4 35.87 36.58 36.88 36.39 332 Exposure treatment acetic acid carpropamid tinuvin water filter 1 20.57 25.50 21.12 19.15 filter 2 32.44 21.99 15.01 19.21 filter 3 28.84 31.83 23.34 28.03 filter 4 40.99 48.39 37.38 45.82 filter 5 31.52 40.60 36.64 51.66 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 Exposure chem_charg 1 2 3 4 filter 1 24.08 18.60 25.24 18.42 filter 2 23.57 22.29 19.86 22.92 filter 3 27.70 30.22 30.19 23.93 filter 4 37.82 44.42 41.95 48.38 filter 5 37.90 40.55 40.90 41.08 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 39.20 37.38 35.56 33.20 carpropamid 32.74 40.59 40.56 39.43 tinuvin 33.79 32.93 30.99 35.71 water 37.74 35.43 40.41 37.21 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 25.68 20.23 22.20 14.18 carpropamid 24.59 22.84 26.02 28.53 tinuvin 25.04 19.10 22.72 17.60 water 21.01 12.22 30.01 13.36 filter 2 acetic acid 46.27 25.37 25.83 32.29 carpropamid 11.00 32.60 21.00 23.38 tinuvin 16.67 17.45 12.82 13.08 water 20.36 13.73 19.79 22.94 filter 3 acetic acid 32.54 35.42 29.20 18.22 carpropamid 30.26 33.26 35.07 28.72 tinuvin 13.04 28.79 28.46 23.07 water 34.95 23.42 28.04 25.73 filter 4 acetic acid 40.86 40.69 42.28 40.11 carpropamid 34.34 56.72 50.00 52.49 tinuvin 41.30 28.98 27.82 51.40 water 34.79 51.29 47.71 49.50 filter 5 acetic acid 29.05 39.97 29.42 27.62 carpropamid 29.02 38.70 51.80 42.90 tinuvin 40.44 36.18 25.10 44.85 water 53.08 47.33 57.29 48.93 full acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 333 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 12.157 3.062 1.667 14.036 d.f. 24 84 336 41.34 Except when comparing means with the same level(s) of Exposure 8.102 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 12.743 4.209 15.981 d.f. 28.95 250.35 68.74 Except when comparing means with the same level(s) of Exposure 4.412 11.137 d.f. 336 250.35 treatment 3.335 d.f. 336 Exposure.treatment 8.823 d.f. 336 Exposure.chem_charg 11.137 d.f. 250.35 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 25.090 6.090 3.280 28.340 d.f. 24 84 336 41.34 Except when comparing means with the same level(s) of Exposure 16.112 d.f. 84 334 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 26.064 8.290 31.884 d.f. 28.95 250.35 68.74 Except when comparing means with the same level(s) of Exposure 8.678 21.934 d.f. 336 250.35 treatment 6.560 d.f. 336 Exposure.treatment 17.356 d.f. 336 Exposure.chem_charg 21.934 d.f. 250.35 Analysis of variance Variate: W12 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 54957.3 13739.3 2.36 rack.sample stratum Exposure 6 526440.6 87740.1 15.09 <.001 Residual 24 139584.1 5816.0 10.15 rack.sample.area stratum treatment 3 3824.6 1274.9 2.22 0.091 Exposure.treatment 18 6752.2 375.1 0.65 0.845 Residual 84 48155.1 573.3 3.49 rack.sample.area.strip stratum chem_charg 3 1063.1 354.4 2.16 0.093 Exposure.chem_charg 18 2641.8 146.8 0.89 0.587 treatment.chem_charg 9 1070.5 118.9 0.72 0.687 Exposure.treatment.chem_charg 54 8491.1 157.2 0.96 0.563 Residual 336 55197.4 164.3 Total 559 848177.9 335 Message: the following units have large residuals. rack 2 sample 5 -35.60 s.e. 15.79 rack 1 sample 7 area 1 -38.39 s.e. 9.27 rack 1 sample 7 area 4 28.27 s.e. 9.27 rack 3 sample 3 area 2 23.79 s.e. 9.27 rack 4 sample 3 area 1 -39.22 s.e. 9.27 rack 4 sample 3 area 3 33.42 s.e. 9.27 rack 1 sample 2 area 1 strip 1 -50.32 s.e. 9.93 rack 1 sample 2 area 1 strip 3 42.08 s.e. 9.93 rack 1 sample 2 area 1 strip 4 29.57 s.e. 9.93 rack 1 sample 2 area 2 strip 4 -33.30 s.e. 9.93 rack 1 sample 2 area 4 strip 4 -39.50 s.e. 9.93 rack 1 sample 5 area 3 strip 1 40.69 s.e. 9.93 rack 1 sample 5 area 3 strip 3 -31.20 s.e. 9.93 rack 1 sample 7 area 2 strip 1 -33.57 s.e. 9.93 rack 2 sample 4 area 4 strip 4 -30.10 s.e. 9.93 rack 3 sample 2 area 4 strip 1 -32.14 s.e. 9.93 rack 3 sample 4 area 1 strip 1 -35.58 s.e. 9.93 rack 4 sample 4 area 1 strip 1 -41.94 s.e. 9.93 rack 5 sample 4 area 1 strip 1 -46.74 s.e. 9.93 rack 5 sample 4 area 4 strip 4 -37.21 s.e. 9.93 rack 5 sample 7 area 1 strip 1 -35.09 s.e. 9.93 rack 5 sample 7 area 1 strip 3 33.32 s.e. 9.93 rack 5 sample 7 area 4 strip 4 -30.89 s.e. 9.93 Tables of means Variate: W12 Grand mean 69.97 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 70.38 71.16 69.36 86.24 92.69 100.00 0.00 treatment acetic acid carpropamid tinuvin water 73.62 69.85 66.24 70.20 chem_charg 1 2 3 4 69.24 68.13 71.66 70.88 336 Exposure treatment acetic acid carpropamid tinuvin water filter 1 73.58 71.26 67.13 69.53 filter 2 86.14 70.60 61.14 66.75 filter 3 76.92 64.44 64.08 71.99 filter 4 85.66 87.72 82.68 88.91 filter 5 93.04 94.89 88.62 94.20 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 Exposure chem_charg 1 2 3 4 filter 1 71.27 71.27 70.26 68.70 filter 2 71.26 68.72 72.61 72.04 filter 3 67.56 65.59 75.66 68.63 filter 4 81.25 84.66 86.98 92.08 filter 5 93.32 86.66 96.08 94.69 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 74.41 71.41 74.23 74.42 carpropamid 66.95 67.21 74.21 71.02 tinuvin 67.29 64.91 65.19 67.55 water 68.30 68.98 73.00 70.52 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 74.85 76.93 69.21 73.32 carpropamid 74.96 64.30 71.45 74.34 tinuvin 67.21 71.10 66.19 64.03 water 68.08 72.74 74.19 63.12 filter 2 acetic acid 86.52 87.12 85.92 84.98 carpropamid 59.05 76.12 75.96 71.28 tinuvin 67.65 57.36 57.89 61.67 water 71.83 54.27 70.66 70.24 filter 3 acetic acid 71.39 76.24 83.21 76.82 carpropamid 55.42 57.28 82.32 62.75 tinuvin 67.13 63.58 64.07 61.56 water 76.31 65.24 73.06 73.36 filter 4 acetic acid 89.57 73.46 83.04 96.57 carpropamid 84.66 87.73 89.72 88.78 tinuvin 81.57 82.30 81.26 85.59 water 69.21 95.14 93.91 97.38 filter 5 acetic acid 98.57 86.13 98.24 89.22 carpropamid 94.55 85.02 100.00 100.00 tinuvin 87.50 80.03 86.94 100.00 water 92.66 95.48 99.15 89.52 full acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 337 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 12.058 2.862 1.532 13.726 d.f. 24 84 336 39.31 Except when comparing means with the same level(s) of Exposure 7.571 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 12.559 3.903 15.417 d.f. 28.23 245.20 62.07 Except when comparing means with the same level(s) of Exposure 4.053 10.325 d.f. 336 245.20 treatment 3.064 d.f. 336 Exposure.treatment 8.106 d.f. 336 Exposure.chem_charg 10.325 d.f. 245.20 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 24.887 5.691 3.013 27.756 d.f. 24 84 336 39.31 Except when comparing means with the same level(s) of Exposure 15.057 d.f. 84 338 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 25.716 7.687 30.817 d.f. 28.23 245.20 62.07 Except when comparing means with the same level(s) of Exposure 7.973 20.338 d.f. 336 245.20 treatment 6.027 d.f. 336 Exposure.treatment 15.945 d.f. 336 Exposure.chem_charg 20.338 d.f. 245.20 Analysis of variance Variate: W14 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 22266.7 5566.7 1.67 rack.sample stratum Exposure 6 568970.9 94828.5 28.48 <.001 Residual 24 79903.4 3329.3 9.68 rack.sample.area stratum treatment 3 3072.6 1024.2 2.98 0.036 Exposure.treatment 18 5659.0 314.4 0.91 0.564 Residual 84 28885.8 343.9 2.38 rack.sample.area.strip stratum chem_charg 3 606.0 202.0 1.40 0.243 Exposure.chem_charg 18 3045.2 169.2 1.17 0.282 treatment.chem_charg 9 1165.3 129.5 0.90 0.528 Exposure.treatment.chem_charg 54 7863.6 145.6 1.01 0.464 Residual 336 48508.7 144.4 Total 559 769947.1 339 Message: the following units have large residuals. rack 1 sample 3 -32.52 s.e. 11.95 rack 2 sample 5 -37.58 s.e. 11.95 rack 1 sample 5 area 1 -21.40 s.e. 7.18 rack 1 sample 7 area 4 19.56 s.e. 7.18 rack 3 sample 1 area 1 -19.18 s.e. 7.18 rack 3 sample 1 area 4 23.14 s.e. 7.18 rack 4 sample 3 area 1 -34.83 s.e. 7.18 rack 1 sample 2 area 1 strip 1 -53.01 s.e. 9.31 rack 1 sample 2 area 1 strip 3 37.78 s.e. 9.31 rack 1 sample 2 area 2 strip 4 -31.18 s.e. 9.31 rack 1 sample 2 area 4 strip 4 -39.15 s.e. 9.31 rack 1 sample 5 area 1 strip 1 -34.96 s.e. 9.31 rack 1 sample 5 area 1 strip 4 32.48 s.e. 9.31 rack 1 sample 5 area 3 strip 1 27.96 s.e. 9.31 rack 1 sample 5 area 4 strip 4 -37.16 s.e. 9.31 rack 1 sample 7 area 1 strip 1 -35.61 s.e. 9.31 rack 1 sample 7 area 2 strip 1 -39.32 s.e. 9.31 rack 3 sample 3 area 1 strip 1 -28.75 s.e. 9.31 rack 4 sample 4 area 1 strip 1 -36.47 s.e. 9.31 rack 5 sample 4 area 4 strip 4 -30.79 s.e. 9.31 rack 5 sample 5 area 1 strip 1 -28.12 s.e. 9.31 Tables of means Variate: W14 Grand mean 75.26 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 75.92 84.73 77.16 95.15 93.88 100.00 0.00 treatment acetic acid carpropamid tinuvin water 77.25 75.36 71.41 77.03 chem_charg 1 2 3 4 75.29 74.04 76.89 74.84 Exposure treatment acetic acid carpropamid tinuvin water filter 1 79.02 78.09 73.83 72.74 filter 2 89.87 86.93 70.53 91.58 filter 3 82.18 71.82 73.45 81.21 filter 4 94.29 95.39 92.16 98.75 filter 5 95.39 95.32 89.88 94.94 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 340 Exposure chem_charg 1 2 3 4 filter 1 76.59 79.99 76.11 70.99 filter 2 88.04 82.45 85.80 82.62 filter 3 73.25 76.16 81.86 77.39 filter 4 94.12 92.82 97.40 96.25 filter 5 95.02 86.85 97.06 96.60 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 77.78 74.98 78.34 77.89 carpropamid 72.54 75.01 77.61 76.30 tinuvin 73.27 69.54 71.32 71.51 water 77.57 76.62 80.29 73.64 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 76.46 82.76 76.76 80.09 carpropamid 84.34 78.77 70.55 78.69 tinuvin 72.51 78.12 76.80 67.90 water 73.03 80.31 80.34 57.28 filter 2 acetic acid 92.10 89.90 89.20 88.26 carpropamid 76.04 90.90 90.57 90.23 tinuvin 88.59 60.77 66.77 66.00 water 95.42 88.23 96.66 86.00 filter 3 acetic acid 79.10 83.45 85.63 80.53 carpropamid 58.11 73.87 85.66 69.63 tinuvin 71.47 75.15 70.28 76.89 water 84.31 72.15 85.88 82.51 filter 4 acetic acid 96.79 82.60 98.09 99.68 carpropamid 93.52 95.97 96.51 95.58 tinuvin 91.17 92.73 94.99 89.76 water 94.99 100.00 100.00 100.00 filter 5 acetic acid 100.00 86.17 98.70 96.70 carpropamid 95.74 85.53 100.00 100.00 tinuvin 89.11 80.03 90.36 100.00 water 95.23 95.66 99.16 89.71 full acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 341 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 9.123 2.216 1.436 10.441 d.f. 24 84 336 40.08 Except when comparing means with the same level(s) of Exposure 5.864 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 9.698 3.332 12.342 d.f. 30.61 307.07 76.80 Except when comparing means with the same level(s) of Exposure 3.800 8.815 d.f. 336 307.07 treatment 2.872 d.f. 336 Exposure.treatment 7.599 d.f. 336 Exposure.chem_charg 8.815 d.f. 307.07 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 18.829 4.408 2.825 21.102 d.f. 24 84 336 40.08 Except when comparing means with the same level(s) of Exposure 11.661 d.f. 84 342 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 19.790 6.556 24.578 d.f. 30.61 307.07 76.80 Except when comparing means with the same level(s) of Exposure 7.474 17.345 d.f. 336 307.07 treatment 5.650 d.f. 336 Exposure.treatment 14.948 d.f. 336 Exposure.chem_charg 17.345 d.f. 307.07 Analysis of variance Variate: W16 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 21168.8 5292.2 2.14 rack.sample stratum Exposure 6 589554.1 98259.0 39.68 <.001 Residual 24 59435.4 2476.5 9.75 rack.sample.area stratum treatment 3 1564.6 521.5 2.05 0.113 Exposure.treatment 18 2341.0 130.1 0.51 0.945 Residual 84 21337.6 254.0 2.10 rack.sample.area.strip stratum chem_charg 3 625.1 208.4 1.72 0.162 Exposure.chem_charg 18 2615.9 145.3 1.20 0.258 treatment.chem_charg 9 638.1 70.9 0.59 0.809 Exposure.treatment.chem_charg 54 6234.3 115.5 0.95 0.571 Residual 336 40679.7 121.1 Total 559 746194.6 343 Message: the following units have large residuals. rack 1 sample 3 -34.84 s.e. 10.30 rack 2 sample 5 -25.98 s.e. 10.30 rack 1 sample 5 area 1 -22.33 s.e. 6.17 rack 3 sample 1 area 1 -20.96 s.e. 6.17 rack 3 sample 1 area 4 19.35 s.e. 6.17 rack 1 sample 2 area 1 strip 1 -51.76 s.e. 8.52 rack 1 sample 2 area 1 strip 3 34.13 s.e. 8.52 rack 1 sample 2 area 1 strip 4 27.92 s.e. 8.52 rack 1 sample 2 area 4 strip 4 -36.88 s.e. 8.52 rack 1 sample 5 area 1 strip 1 -30.08 s.e. 8.52 rack 1 sample 5 area 1 strip 2 -28.61 s.e. 8.52 rack 1 sample 5 area 1 strip 4 33.41 s.e. 8.52 rack 1 sample 5 area 3 strip 1 27.14 s.e. 8.52 rack 1 sample 5 area 4 strip 4 -31.70 s.e. 8.52 rack 1 sample 7 area 1 strip 1 -36.05 s.e. 8.52 rack 1 sample 7 area 2 strip 1 -37.36 s.e. 8.52 rack 2 sample 1 area 4 strip 1 25.84 s.e. 8.52 rack 2 sample 1 area 4 strip 4 -28.20 s.e. 8.52 rack 2 sample 4 area 4 strip 4 -28.57 s.e. 8.52 rack 3 sample 3 area 1 strip 1 -26.05 s.e. 8.52 rack 4 sample 3 area 1 strip 1 -45.58 s.e. 8.52 rack 5 sample 4 area 4 strip 4 -37.10 s.e. 8.52 Tables of means Variate: W16 Grand mean 77.80 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 80.34 89.89 82.81 96.32 95.21 100.00 0.00 treatment acetic acid carpropamid tinuvin water 79.70 76.90 75.53 79.05 chem_charg 1 2 3 4 77.93 77.11 79.45 76.69 Exposure treatment acetic acid carpropamid tinuvin water filter 1 82.26 79.72 80.06 79.33 filter 2 90.31 89.23 84.78 95.22 filter 3 90.49 78.24 79.76 82.76 filter 4 97.52 95.54 93.01 99.22 filter 5 97.35 95.56 91.08 96.83 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 344 Exposure chem_charg 1 2 3 4 filter 1 80.42 83.55 80.54 76.86 filter 2 91.00 91.42 92.58 84.54 filter 3 81.17 81.71 87.50 80.88 filter 4 95.77 94.83 98.15 96.55 filter 5 97.17 88.27 97.41 97.97 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 80.30 78.47 80.16 79.89 carpropamid 74.55 76.67 79.61 76.78 tinuvin 76.85 74.95 76.57 73.74 water 80.03 78.36 81.47 76.34 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 80.12 85.84 80.80 82.28 carpropamid 85.02 80.90 73.01 79.96 tinuvin 75.57 86.23 84.08 74.34 water 80.97 81.26 84.25 70.85 filter 2 acetic acid 92.10 90.66 89.77 88.72 carpropamid 77.27 93.99 93.64 92.04 tinuvin 98.38 83.95 86.91 69.88 water 96.26 97.10 100.00 87.52 filter 3 acetic acid 89.87 91.54 90.55 90.01 carpropamid 69.82 79.25 94.10 69.80 tinuvin 77.85 81.66 78.48 81.04 water 87.11 74.38 86.88 82.67 filter 4 acetic acid 100.00 90.37 100.00 99.69 carpropamid 93.83 96.20 96.51 95.63 tinuvin 92.35 92.73 96.07 90.88 water 96.89 100.00 100.00 100.00 filter 5 acetic acid 100.00 90.89 100.00 98.53 carpropamid 95.89 86.34 100.00 100.00 tinuvin 93.79 80.08 90.45 100.00 water 99.01 95.77 99.19 93.36 full acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 345 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 7.868 1.905 1.315 8.998 d.f. 24 84 336 39.96 Except when comparing means with the same level(s) of Exposure 5.040 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 8.426 2.969 10.830 d.f. 31.51 328.20 81.90 Except when comparing means with the same level(s) of Exposure 3.480 7.856 d.f. 336 328.20 treatment 2.630 d.f. 336 Exposure.treatment 6.959 d.f. 336 Exposure.chem_charg 7.856 d.f. 328.20 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 16.240 3.788 2.587 18.186 d.f. 24 84 336 39.96 Except when comparing means with the same level(s) of Exposure 10.023 d.f. 84 346 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 17.173 5.842 21.544 d.f. 31.51 328.20 81.90 Except when comparing means with the same level(s) of Exposure 6.844 15.455 d.f. 336 328.20 treatment 5.174 d.f. 336 Exposure.treatment 13.689 d.f. 336 Exposure.chem_charg 15.455 d.f. 328.20 Analysis of variance Variate: W16 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 21168.8 5292.2 2.14 rack.sample stratum Exposure 6 589554.1 98259.0 39.68 <.001 Residual 24 59435.4 2476.5 9.75 rack.sample.area stratum treatment 3 1564.6 521.5 2.05 0.113 Exposure.treatment 18 2341.0 130.1 0.51 0.945 Residual 84 21337.6 254.0 2.10 rack.sample.area.strip stratum chem_charg 3 625.1 208.4 1.72 0.162 Exposure.chem_charg 18 2615.9 145.3 1.20 0.258 treatment.chem_charg 9 638.1 70.9 0.59 0.809 Exposure.treatment.chem_charg 54 6234.3 115.5 0.95 0.571 Residual 336 40679.7 121.1 Total 559 746194.6 347 Message: the following units have large residuals. rack 1 sample 3 -34.84 s.e. 10.30 rack 2 sample 5 -25.98 s.e. 10.30 rack 1 sample 5 area 1 -22.33 s.e. 6.17 rack 3 sample 1 area 1 -20.96 s.e. 6.17 rack 3 sample 1 area 4 19.35 s.e. 6.17 rack 1 sample 2 area 1 strip 1 -51.76 s.e. 8.52 rack 1 sample 2 area 1 strip 3 34.13 s.e. 8.52 rack 1 sample 2 area 1 strip 4 27.92 s.e. 8.52 rack 1 sample 2 area 4 strip 4 -36.88 s.e. 8.52 rack 1 sample 5 area 1 strip 1 -30.08 s.e. 8.52 rack 1 sample 5 area 1 strip 2 -28.61 s.e. 8.52 rack 1 sample 5 area 1 strip 4 33.41 s.e. 8.52 rack 1 sample 5 area 3 strip 1 27.14 s.e. 8.52 rack 1 sample 5 area 4 strip 4 -31.70 s.e. 8.52 rack 1 sample 7 area 1 strip 1 -36.05 s.e. 8.52 rack 1 sample 7 area 2 strip 1 -37.36 s.e. 8.52 rack 2 sample 1 area 4 strip 1 25.84 s.e. 8.52 rack 2 sample 1 area 4 strip 4 -28.20 s.e. 8.52 rack 2 sample 4 area 4 strip 4 -28.57 s.e. 8.52 rack 3 sample 3 area 1 strip 1 -26.05 s.e. 8.52 rack 4 sample 3 area 1 strip 1 -45.58 s.e. 8.52 rack 5 sample 4 area 4 strip 4 -37.10 s.e. 8.52 Tables of means Variate: W16 Grand mean 77.80 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 80.34 89.89 82.81 96.32 95.21 100.00 0.00 treatment acetic acid carpropamid tinuvin water 79.70 76.90 75.53 79.05 chem_charg 1 2 3 4 77.93 77.11 79.45 76.69 Exposure treatment acetic acid carpropamid tinuvin water filter 1 82.26 79.72 80.06 79.33 filter 2 90.31 89.23 84.78 95.22 filter 3 90.49 78.24 79.76 82.76 filter 4 97.52 95.54 93.01 99.22 filter 5 97.35 95.56 91.08 96.83 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 348 Exposure chem_charg 1 2 3 4 filter 1 80.42 83.55 80.54 76.86 filter 2 91.00 91.42 92.58 84.54 filter 3 81.17 81.71 87.50 80.88 filter 4 95.77 94.83 98.15 96.55 filter 5 97.17 88.27 97.41 97.97 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 80.30 78.47 80.16 79.89 carpropamid 74.55 76.67 79.61 76.78 tinuvin 76.85 74.95 76.57 73.74 water 80.03 78.36 81.47 76.34 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 80.12 85.84 80.80 82.28 carpropamid 85.02 80.90 73.01 79.96 tinuvin 75.57 86.23 84.08 74.34 water 80.97 81.26 84.25 70.85 filter 2 acetic acid 92.10 90.66 89.77 88.72 carpropamid 77.27 93.99 93.64 92.04 tinuvin 98.38 83.95 86.91 69.88 water 96.26 97.10 100.00 87.52 filter 3 acetic acid 89.87 91.54 90.55 90.01 carpropamid 69.82 79.25 94.10 69.80 tinuvin 77.85 81.66 78.48 81.04 water 87.11 74.38 86.88 82.67 filter 4 acetic acid 100.00 90.37 100.00 99.69 carpropamid 93.83 96.20 96.51 95.63 tinuvin 92.35 92.73 96.07 90.88 water 96.89 100.00 100.00 100.00 filter 5 acetic acid 100.00 90.89 100.00 98.53 carpropamid 95.89 86.34 100.00 100.00 tinuvin 93.79 80.08 90.45 100.00 water 99.01 95.77 99.19 93.36 full acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 349 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 7.868 1.905 1.315 8.998 d.f. 24 84 336 39.96 Except when comparing means with the same level(s) of Exposure 5.040 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 8.426 2.969 10.830 d.f. 31.51 328.20 81.90 Except when comparing means with the same level(s) of Exposure 3.480 7.856 d.f. 336 328.20 treatment 2.630 d.f. 336 Exposure.treatment 6.959 d.f. 336 Exposure.chem_charg 7.856 d.f. 328.20 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 16.240 3.788 2.587 18.186 d.f. 24 84 336 39.96 Except when comparing means with the same level(s) of Exposure 10.023 d.f. 84 350 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 17.173 5.842 21.544 d.f. 31.51 328.20 81.90 Except when comparing means with the same level(s) of Exposure 6.844 15.455 d.f. 336 328.20 treatment 5.174 d.f. 336 Exposure.treatment 13.689 d.f. 336 Exposure.chem_charg 15.455 d.f. 328.20 Analysis of variance Variate: W18 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 11058.43 2764.61 2.41 rack.sample stratum Exposure 6 628804.85 104800.81 91.41 <.001 Residual 24 27516.86 1146.54 8.44 rack.sample.area stratum treatment 3 586.96 195.65 1.44 0.237 Exposure.treatment 18 3074.65 170.81 1.26 0.238 Residual 84 11414.88 135.89 1.74 rack.sample.area.strip stratum chem_charg 3 474.53 158.18 2.03 0.110 Exposure.chem_charg 18 1868.60 103.81 1.33 0.165 treatment.chem_charg 9 411.25 45.69 0.59 0.809 Exposure.treatment.chem_charg 54 3750.95 69.46 0.89 0.691 Residual 336 26204.62 77.99 Total 559 715166.62 351 Message: the following units have large residuals. rack 1 sample 3 -24.30 s.e. 7.01 rack 1 sample 5 -15.27 s.e. 7.01 rack 1 sample 3 area 1 -12.81 s.e. 4.51 rack 1 sample 5 area 1 -17.97 s.e. 4.51 rack 1 sample 5 area 2 11.79 s.e. 4.51 rack 3 sample 1 area 1 -24.22 s.e. 4.51 rack 3 sample 1 area 4 12.90 s.e. 4.51 rack 1 sample 2 area 1 strip 1 -49.28 s.e. 6.84 rack 1 sample 2 area 4 strip 4 -29.17 s.e. 6.84 rack 1 sample 3 area 2 strip 4 -21.48 s.e. 6.84 rack 1 sample 5 area 1 strip 1 -26.46 s.e. 6.84 rack 1 sample 5 area 1 strip 2 -30.73 s.e. 6.84 rack 1 sample 5 area 1 strip 3 23.55 s.e. 6.84 rack 1 sample 5 area 1 strip 4 33.65 s.e. 6.84 rack 1 sample 5 area 3 strip 1 23.91 s.e. 6.84 rack 1 sample 5 area 4 strip 4 -32.23 s.e. 6.84 rack 2 sample 5 area 4 strip 1 26.14 s.e. 6.84 rack 2 sample 6 area 1 strip 1 -23.41 s.e. 6.84 rack 3 sample 1 area 2 strip 1 -24.04 s.e. 6.84 rack 3 sample 3 area 1 strip 1 -26.40 s.e. 6.84 rack 4 sample 3 area 1 strip 1 -26.16 s.e. 6.84 rack 5 sample 4 area 4 strip 4 -30.19 s.e. 6.84 Tables of means Variate: W18 Grand mean 81.51 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 88.80 94.01 90.78 99.37 97.64 100.00 0.00 treatment acetic acid carpropamid tinuvin water 81.99 80.59 80.50 82.96 chem_charg 1 2 3 4 81.72 80.77 82.93 80.63 Exposure treatment acetic acid carpropamid tinuvin water filter 1 89.33 88.78 88.19 88.89 filter 2 90.41 96.59 90.95 98.08 filter 3 96.52 81.88 90.54 94.18 filter 4 99.74 99.34 98.83 99.57 filter 5 97.96 97.57 95.02 100.00 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 352 Exposure chem_charg 1 2 3 4 filter 1 89.12 91.20 87.76 87.10 filter 2 95.05 93.38 96.96 90.64 filter 3 89.95 89.87 95.86 87.44 filter 4 98.81 99.19 100.00 99.47 filter 5 99.12 91.76 99.93 99.75 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 81.80 81.69 82.09 82.39 carpropamid 79.72 80.91 83.13 78.61 tinuvin 81.99 78.58 81.90 79.54 water 83.38 81.90 84.61 81.96 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 86.03 91.60 86.91 92.79 carpropamid 92.46 93.08 84.66 84.92 tinuvin 89.47 90.47 86.61 86.19 water 88.54 89.66 92.87 84.51 filter 2 acetic acid 92.28 90.75 89.83 88.78 carpropamid 90.59 98.48 99.82 97.49 tinuvin 98.65 87.01 98.19 79.94 water 98.70 97.27 100.00 96.35 filter 3 acetic acid 94.30 97.66 97.90 96.21 carpropamid 75.52 84.56 97.44 69.99 tinuvin 91.85 90.88 88.74 90.67 water 98.13 86.36 99.36 92.87 filter 4 acetic acid 100.00 98.94 100.00 100.00 carpropamid 99.47 100.00 100.00 97.89 tinuvin 97.50 97.82 100.00 100.00 water 98.29 100.00 100.00 100.00 filter 5 acetic acid 100.00 92.87 100.00 98.99 carpropamid 100.00 90.28 100.00 100.00 tinuvin 96.48 83.89 99.72 100.00 water 100.00 100.00 100.00 100.00 full acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 353 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 5.354 1.393 1.056 6.233 d.f. 24 84 336 42.56 Except when comparing means with the same level(s) of Exposure 3.686 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 5.875 2.299 7.890 d.f. 34.69 357.40 104.46 Except when comparing means with the same level(s) of Exposure 2.793 6.082 d.f. 336 357.40 treatment 2.111 d.f. 336 Exposure.treatment 5.585 d.f. 336 Exposure.chem_charg 6.082 d.f. 357.40 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 11.050 2.771 2.076 12.575 d.f. 24 84 336 42.56 Except when comparing means with the same level(s) of Exposure 7.331 d.f. 84 354 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 11.930 4.521 15.645 d.f. 34.69 357.40 104.46 Except when comparing means with the same level(s) of Exposure 5.493 11.960 d.f. 336 357.40 treatment 4.153 d.f. 336 Exposure.treatment 10.987 d.f. 336 Exposure.chem_charg 11.960 d.f. 357.40 Analysis of variance Variate: W20 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 4129.59 1032.40 1.97 rack.sample stratum Exposure 6 652197.44 108699.57 207.06 <.001 Residual 24 12599.11 524.96 5.17 rack.sample.area stratum treatment 3 345.59 115.20 1.13 0.340 Exposure.treatment 18 1887.64 104.87 1.03 0.433 Residual 84 8527.32 101.52 2.37 rack.sample.area.strip stratum chem_charg 3 265.12 88.37 2.06 0.105 Exposure.chem_charg 18 1001.33 55.63 1.30 0.187 treatment.chem_charg 9 415.67 46.19 1.08 0.380 Exposure.treatment.chem_charg 54 2884.64 53.42 1.24 0.129 Residual 336 14421.69 42.92 Total 559 698675.15 355 Message: the following units have large residuals. rack 1 sample 3 -19.75 s.e. 4.74 rack 3 sample 1 -10.39 s.e. 4.74 rack 1 sample 3 area 2 10.47 s.e. 3.90 rack 1 sample 5 area 1 -16.37 s.e. 3.90 rack 3 sample 1 area 1 -21.15 s.e. 3.90 rack 3 sample 1 area 4 12.06 s.e. 3.90 rack 3 sample 4 area 1 9.84 s.e. 3.90 rack 3 sample 4 area 4 -11.59 s.e. 3.90 rack 1 sample 2 area 1 strip 1 -42.72 s.e. 5.07 rack 1 sample 2 area 4 strip 4 -28.56 s.e. 5.07 rack 1 sample 3 area 1 strip 1 -19.27 s.e. 5.07 rack 1 sample 3 area 4 strip 4 -18.65 s.e. 5.07 rack 1 sample 5 area 1 strip 1 -42.32 s.e. 5.07 rack 1 sample 5 area 1 strip 3 22.73 s.e. 5.07 rack 1 sample 5 area 1 strip 4 23.52 s.e. 5.07 rack 1 sample 5 area 4 strip 4 -22.52 s.e. 5.07 rack 2 sample 5 area 4 strip 1 15.15 s.e. 5.07 rack 2 sample 6 area 1 strip 1 -16.75 s.e. 5.07 rack 3 sample 1 area 2 strip 1 -21.34 s.e. 5.07 rack 3 sample 4 area 1 strip 4 18.76 s.e. 5.07 rack 4 sample 7 area 2 strip 1 17.92 s.e. 5.07 rack 5 sample 7 area 1 strip 2 18.74 s.e. 5.07 Tables of means Variate: W20 Grand mean 83.40 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 92.78 96.58 96.07 99.88 98.48 100.00 0.00 treatment acetic acid carpropamid tinuvin water 83.56 82.27 83.29 84.48 chem_charg 1 2 3 4 83.63 82.82 84.42 82.73 Exposure treatment acetic acid carpropamid tinuvin water filter 1 93.08 91.87 91.77 94.38 filter 2 91.95 97.23 98.17 98.99 filter 3 100.00 89.18 97.14 97.96 filter 4 100.00 100.00 99.53 100.00 filter 5 99.88 97.62 96.44 100.00 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 356 Exposure chem_charg 1 2 3 4 filter 1 92.57 93.02 94.03 91.48 filter 2 96.62 96.28 97.52 95.91 filter 3 96.70 96.47 99.41 91.70 filter 4 99.53 100.00 100.00 100.00 filter 5 100.00 93.94 100.00 100.00 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 83.35 83.38 83.53 83.97 carpropamid 82.49 82.42 84.78 79.40 tinuvin 83.89 81.94 84.27 83.07 water 84.79 83.52 85.11 84.48 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 88.87 93.14 94.12 96.19 carpropamid 93.19 93.71 93.84 86.75 tinuvin 94.66 92.69 92.35 87.38 water 93.56 92.56 95.80 95.60 filter 2 acetic acid 94.59 91.04 90.60 91.57 carpropamid 91.89 99.43 99.82 97.77 tinuvin 100.00 95.16 99.65 97.84 water 100.00 99.51 100.00 96.45 filter 3 acetic acid 100.00 100.00 100.00 100.00 carpropamid 92.35 93.32 99.78 71.26 tinuvin 94.43 100.00 97.86 96.27 water 100.00 92.55 100.00 99.28 filter 4 acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 98.11 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 filter 5 acetic acid 100.00 99.50 100.00 100.00 carpropamid 100.00 90.48 100.00 100.00 tinuvin 100.00 85.76 100.00 100.00 water 100.00 100.00 100.00 100.00 full acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 357 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 3.623 1.204 0.783 4.554 d.f. 24 84 336 54.67 Except when comparing means with the same level(s) of Exposure 3.186 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 4.043 1.814 5.798 d.f. 37.06 308.26 135.15 Except when comparing means with the same level(s) of Exposure 2.072 4.799 d.f. 336 308.26 treatment 1.566 d.f. 336 Exposure.treatment 4.144 d.f. 336 Exposure.chem_charg 4.799 d.f. 308.26 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 7.477 2.395 1.540 9.127 d.f. 24 84 336 54.67 Except when comparing means with the same level(s) of Exposure 6.336 d.f. 84 358 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 8.191 3.569 11.466 d.f. 37.06 308.26 135.15 Except when comparing means with the same level(s) of Exposure 4.075 9.442 d.f. 336 308.26 treatment 3.081 d.f. 336 Exposure.treatment 8.150 d.f. 336 Exposure.chem_charg 9.442 d.f. 308.26 Analysis of variance Variate: W24 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 4063.56 1015.89 1.95 rack.sample stratum Exposure 6 652508.29 108751.38 208.78 <.001 Residual 24 12501.48 520.89 5.17 rack.sample.area stratum treatment 3 338.92 112.97 1.12 0.345 Exposure.treatment 18 1857.59 103.20 1.02 0.443 Residual 84 8467.51 100.80 2.37 rack.sample.area.strip stratum chem_charg 3 254.30 84.77 1.99 0.115 Exposure.chem_charg 18 995.70 55.32 1.30 0.186 treatment.chem_charg 9 401.65 44.63 1.05 0.402 Exposure.treatment.chem_charg 54 2843.21 52.65 1.24 0.136 Residual 336 14312.43 42.60 Total 559 698544.64 359 Message: the following units have large residuals. rack 1 sample 3 -19.67 s.e. 4.72 rack 3 sample 1 -10.35 s.e. 4.72 rack 1 sample 3 area 2 10.50 s.e. 3.89 rack 1 sample 5 area 1 -16.41 s.e. 3.89 rack 1 sample 5 area 3 9.78 s.e. 3.89 rack 3 sample 1 area 1 -21.10 s.e. 3.89 rack 3 sample 1 area 4 11.91 s.e. 3.89 rack 3 sample 4 area 1 9.80 s.e. 3.89 rack 3 sample 4 area 4 -11.57 s.e. 3.89 rack 1 sample 2 area 1 strip 1 -42.72 s.e. 5.06 rack 1 sample 2 area 4 strip 4 -28.56 s.e. 5.06 rack 1 sample 3 area 1 strip 1 -19.06 s.e. 5.06 rack 1 sample 3 area 4 strip 4 -18.82 s.e. 5.06 rack 1 sample 5 area 1 strip 1 -42.33 s.e. 5.06 rack 1 sample 5 area 1 strip 3 22.66 s.e. 5.06 rack 1 sample 5 area 1 strip 4 23.56 s.e. 5.06 rack 1 sample 5 area 4 strip 4 -22.38 s.e. 5.06 rack 2 sample 6 area 1 strip 1 -16.34 s.e. 5.06 rack 3 sample 1 area 2 strip 1 -21.11 s.e. 5.06 rack 3 sample 4 area 1 strip 4 18.76 s.e. 5.06 rack 4 sample 7 area 2 strip 1 17.92 s.e. 5.06 rack 5 sample 7 area 1 strip 2 18.72 s.e. 5.06 Tables of means Variate: W24 Grand mean 83.42 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 92.88 96.55 96.08 99.95 98.48 100.00 0.00 treatment acetic acid carpropamid tinuvin water 83.57 82.31 83.31 84.50 chem_charg 1 2 3 4 83.64 82.84 84.43 82.78 Exposure treatment acetic acid carpropamid tinuvin water filter 1 93.12 92.08 91.84 94.48 filter 2 91.99 97.25 97.94 99.01 filter 3 100.00 89.23 97.14 97.97 filter 4 100.00 100.00 99.81 100.00 filter 5 99.88 97.62 96.44 100.00 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 360 Exposure chem_charg 1 2 3 4 filter 1 92.59 93.12 94.07 91.75 filter 2 96.40 96.31 97.52 95.97 filter 3 96.70 96.52 99.41 91.71 filter 4 99.81 100.00 100.00 100.00 filter 5 100.00 93.94 100.00 100.00 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 83.35 83.39 83.54 84.00 carpropamid 82.50 82.45 84.78 79.51 tinuvin 83.92 81.97 84.27 83.08 water 84.79 83.55 85.13 84.51 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 88.87 93.16 94.19 96.27 carpropamid 93.20 93.76 93.85 87.51 tinuvin 94.73 92.82 92.35 87.48 water 93.56 92.73 95.89 95.76 filter 2 acetic acid 94.59 91.07 90.60 91.71 carpropamid 91.97 99.43 99.82 97.79 tinuvin 99.05 95.21 99.66 97.85 water 100.00 99.52 100.00 96.54 filter 3 acetic acid 100.00 100.00 100.00 100.00 carpropamid 92.35 93.47 99.78 71.30 tinuvin 94.43 100.00 97.87 96.27 water 100.00 92.60 100.00 99.28 filter 4 acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 99.22 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 filter 5 acetic acid 100.00 99.51 100.00 100.00 carpropamid 100.00 90.48 100.00 100.00 tinuvin 100.00 85.76 100.00 100.00 water 100.00 100.00 100.00 100.00 full acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 361 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 3.609 1.200 0.780 4.537 d.f. 24 84 336 54.69 Except when comparing means with the same level(s) of Exposure 3.175 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 4.027 1.807 5.776 d.f. 37.06 308.16 135.20 Except when comparing means with the same level(s) of Exposure 2.064 4.781 d.f. 336 308.16 treatment 1.560 d.f. 336 Exposure.treatment 4.128 d.f. 336 Exposure.chem_charg 4.781 d.f. 308.16 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 7.448 2.386 1.534 9.093 d.f. 24 84 336 54.69 Except when comparing means with the same level(s) of Exposure 6.314 d.f. 84 362 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 8.159 3.556 11.423 d.f. 37.06 308.16 135.20 Except when comparing means with the same level(s) of Exposure 4.060 9.408 d.f. 336 308.16 treatment 3.069 d.f. 336 Exposure.treatment 8.120 d.f. 336 Exposure.chem_charg 9.408 d.f. 308.16 Analysis of variance Variate: W32 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 3073.36 768.34 1.43 rack.sample stratum Exposure 6 659370.22 109895.04 203.97 <.001 Residual 24 12930.70 538.78 8.35 rack.sample.area stratum treatment 3 220.62 73.54 1.14 0.338 Exposure.treatment 18 1104.01 61.33 0.95 0.522 Residual 84 5418.19 64.50 2.63 rack.sample.area.strip stratum chem_charg 3 97.70 32.57 1.33 0.265 Exposure.chem_charg 18 585.87 32.55 1.33 0.168 treatment.chem_charg 9 226.74 25.19 1.03 0.418 Exposure.treatment.chem_charg 54 1861.58 34.47 1.41 0.039 Residual 336 8240.80 24.53 Total 559 693129.80 363 Message: the following units have large residuals. rack 1 sample 3 -20.93 s.e. 4.81 rack 3 sample 1 -10.53 s.e. 4.81 rack 1 sample 3 area 2 9.92 s.e. 3.11 rack 3 sample 1 area 1 -20.36 s.e. 3.11 rack 3 sample 1 area 3 8.41 s.e. 3.11 rack 3 sample 1 area 4 11.41 s.e. 3.11 rack 3 sample 4 area 4 -9.99 s.e. 3.11 rack 1 sample 2 area 1 strip 1 -24.75 s.e. 3.84 rack 1 sample 2 area 4 strip 4 -24.43 s.e. 3.84 rack 1 sample 3 area 1 strip 1 -18.92 s.e. 3.84 rack 1 sample 3 area 3 strip 1 -15.26 s.e. 3.84 rack 1 sample 3 area 4 strip 4 -18.37 s.e. 3.84 rack 1 sample 5 area 1 strip 1 -33.39 s.e. 3.84 rack 1 sample 5 area 1 strip 3 12.04 s.e. 3.84 rack 1 sample 5 area 4 strip 4 -17.26 s.e. 3.84 rack 3 sample 1 area 2 strip 1 -20.83 s.e. 3.84 rack 3 sample 4 area 1 strip 4 14.89 s.e. 3.84 rack 4 sample 7 area 2 strip 1 14.11 s.e. 3.84 rack 5 sample 7 area 1 strip 2 14.11 s.e. 3.84 Tables of means Variate: W32 Grand mean 83.91 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 93.68 97.12 97.57 100.00 98.98 100.00 0.00 treatment acetic acid carpropamid tinuvin water 83.66 83.24 83.80 84.93 chem_charg 1 2 3 4 83.96 83.75 84.54 83.38 Exposure treatment acetic acid carpropamid tinuvin water filter 1 93.50 92.86 92.26 96.11 filter 2 92.09 97.32 99.19 99.87 filter 3 100.00 94.54 97.18 98.56 filter 4 100.00 100.00 100.00 100.00 filter 5 100.00 97.96 97.94 100.00 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 364 Exposure chem_charg 1 2 3 4 filter 1 92.63 94.81 94.79 92.49 filter 2 96.68 97.22 97.54 97.03 filter 3 98.38 98.30 99.43 94.18 filter 4 100.00 100.00 100.00 100.00 filter 5 100.00 95.90 100.00 100.00 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 83.36 83.47 83.76 84.03 carpropamid 83.47 83.48 84.95 81.06 tinuvin 84.19 83.51 84.29 83.19 water 84.80 84.53 85.14 85.26 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 88.90 93.16 95.65 96.27 carpropamid 93.20 94.12 95.07 89.04 tinuvin 94.81 94.25 92.44 87.53 water 93.62 97.69 96.01 97.10 filter 2 acetic acid 94.61 91.14 90.66 91.95 carpropamid 92.11 99.43 99.82 97.91 tinuvin 100.00 98.56 99.67 98.54 water 100.00 99.77 100.00 99.72 filter 3 acetic acid 100.00 100.00 100.00 100.00 carpropamid 99.00 98.95 99.78 80.44 tinuvin 94.50 100.00 97.95 96.27 water 100.00 94.25 100.00 100.00 filter 4 acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 filter 5 acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 91.86 100.00 100.00 tinuvin 100.00 91.75 100.00 100.00 water 100.00 100.00 100.00 100.00 full acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 365 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 3.670 0.960 0.592 4.279 d.f. 24 84 336 42.76 Except when comparing means with the same level(s) of Exposure 2.540 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 3.913 1.404 5.066 d.f. 30.96 290.46 82.34 Except when comparing means with the same level(s) of Exposure 1.566 3.716 d.f. 336 290.46 treatment 1.184 d.f. 336 Exposure.treatment 3.132 d.f. 336 Exposure.chem_charg 3.716 d.f. 290.46 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 7.575 1.909 1.164 8.630 d.f. 24 84 336 42.76 Except when comparing means with the same level(s) of Exposure 5.051 d.f. 84 366 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 7.980 2.764 10.077 d.f. 30.96 290.46 82.34 Except when comparing means with the same level(s) of Exposure 3.081 7.314 d.f. 336 290.46 treatment 2.329 d.f. 336 Exposure.treatment 6.161 d.f. 336 Exposure.chem_charg 7.314 d.f. 290.46 Analysis of variance Variate: W40 Source of variation d.f. s.s. m.s. v.r. F pr. rack stratum 4 3073.36 768.34 1.43 rack.sample stratum Exposure 6 659370.22 109895.04 203.97 <.001 Residual 24 12930.70 538.78 8.35 rack.sample.area stratum treatment 3 220.62 73.54 1.14 0.338 Exposure.treatment 18 1104.01 61.33 0.95 0.522 Residual 84 5418.19 64.50 2.63 rack.sample.area.strip stratum chem_charg 3 97.70 32.57 1.33 0.265 Exposure.chem_charg 18 585.87 32.55 1.33 0.168 treatment.chem_charg 9 226.74 25.19 1.03 0.418 Exposure.treatment.chem_charg 54 1861.58 34.47 1.41 0.039 Residual 336 8240.80 24.53 Total 559 693129.80 367 Message: the following units have large residuals. rack 1 sample 3 -20.93 s.e. 4.81 rack 3 sample 1 -10.53 s.e. 4.81 rack 1 sample 3 area 2 9.92 s.e. 3.11 rack 3 sample 1 area 1 -20.36 s.e. 3.11 rack 3 sample 1 area 3 8.41 s.e. 3.11 rack 3 sample 1 area 4 11.41 s.e. 3.11 rack 3 sample 4 area 4 -9.99 s.e. 3.11 rack 1 sample 2 area 1 strip 1 -24.75 s.e. 3.84 rack 1 sample 2 area 4 strip 4 -24.43 s.e. 3.84 rack 1 sample 3 area 1 strip 1 -18.92 s.e. 3.84 rack 1 sample 3 area 3 strip 1 -15.26 s.e. 3.84 rack 1 sample 3 area 4 strip 4 -18.37 s.e. 3.84 rack 1 sample 5 area 1 strip 1 -33.39 s.e. 3.84 rack 1 sample 5 area 1 strip 3 12.04 s.e. 3.84 rack 1 sample 5 area 4 strip 4 -17.26 s.e. 3.84 rack 3 sample 1 area 2 strip 1 -20.83 s.e. 3.84 rack 3 sample 4 area 1 strip 4 14.89 s.e. 3.84 rack 4 sample 7 area 2 strip 1 14.11 s.e. 3.84 rack 5 sample 7 area 1 strip 2 14.11 s.e. 3.84 Tables of means Variate: W40 Grand mean 83.91 Exposure filter 1 filter 2 filter 3 filter 4 filter 5 full None 93.68 97.12 97.57 100.00 98.98 100.00 0.00 treatment acetic acid carpropamid tinuvin water 83.66 83.24 83.80 84.93 chem_charg 1 2 3 4 83.96 83.75 84.54 83.38 Exposure treatment acetic acid carpropamid tinuvin water filter 1 93.50 92.86 92.26 96.11 filter 2 92.09 97.32 99.19 99.87 filter 3 100.00 94.54 97.18 98.56 filter 4 100.00 100.00 100.00 100.00 filter 5 100.00 97.96 97.94 100.00 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 368 Exposure chem_charg 1 2 3 4 filter 1 92.63 94.81 94.79 92.49 filter 2 96.68 97.22 97.54 97.03 filter 3 98.38 98.30 99.43 94.18 filter 4 100.00 100.00 100.00 100.00 filter 5 100.00 95.90 100.00 100.00 full 100.00 100.00 100.00 100.00 None 0.00 0.00 0.00 0.00 treatment chem_charg 1 2 3 4 acetic acid 83.36 83.47 83.76 84.03 carpropamid 83.47 83.48 84.95 81.06 tinuvin 84.19 83.51 84.29 83.19 water 84.80 84.53 85.14 85.26 Exposure treatment chem_charg 1 2 3 4 filter 1 acetic acid 88.90 93.16 95.65 96.27 carpropamid 93.20 94.12 95.07 89.04 tinuvin 94.81 94.25 92.44 87.53 water 93.62 97.69 96.01 97.10 filter 2 acetic acid 94.61 91.14 90.66 91.95 carpropamid 92.11 99.43 99.82 97.91 tinuvin 100.00 98.56 99.67 98.54 water 100.00 99.77 100.00 99.72 filter 3 acetic acid 100.00 100.00 100.00 100.00 carpropamid 99.00 98.95 99.78 80.44 tinuvin 94.50 100.00 97.95 96.27 water 100.00 94.25 100.00 100.00 filter 4 acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 filter 5 acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 91.86 100.00 100.00 tinuvin 100.00 91.75 100.00 100.00 water 100.00 100.00 100.00 100.00 full acetic acid 100.00 100.00 100.00 100.00 carpropamid 100.00 100.00 100.00 100.00 tinuvin 100.00 100.00 100.00 100.00 water 100.00 100.00 100.00 100.00 None acetic acid 0.00 0.00 0.00 0.00 carpropamid 0.00 0.00 0.00 0.00 tinuvin 0.00 0.00 0.00 0.00 water 0.00 0.00 0.00 0.00 369 Standard errors of differences of means Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 s.e.d. 3.670 0.960 0.592 4.279 d.f. 24 84 336 42.76 Except when comparing means with the same level(s) of Exposure 2.540 d.f. 84 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 s.e.d. 3.913 1.404 5.066 d.f. 30.96 290.46 82.34 Except when comparing means with the same level(s) of Exposure 1.566 3.716 d.f. 336 290.46 treatment 1.184 d.f. 336 Exposure.treatment 3.132 d.f. 336 Exposure.chem_charg 3.716 d.f. 290.46 Least significant differences of means (5% level) Table Exposure treatment chem_charg Exposure treatment rep. 80 140 140 20 l.s.d. 7.575 1.909 1.164 8.630 d.f. 24 84 336 42.76 Except when comparing means with the same level(s) of Exposure 5.051 d.f. 84 370 Table Exposure treatment Exposure chem_charg chem_charg treatment chem_charg rep. 20 35 5 l.s.d. 7.980 2.764 10.077 d.f. 30.96 290.46 82.34 Except when comparing means with the same level(s) of Exposure 3.081 7.314 d.f. 336 290.46 treatment 2.329 d.f. 336 Exposure.treatment 6.161 d.f. 336 Exposure.chem_charg 7.314 d.f. 290.46 Analysis of variance color of wood surfaces 0 to 40 weeks Analysis of variance week 1 Variate: L Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 203.640 50.910 1.62 block.board stratum exposure 5 3247.592 649.518 20.70 <.001 Residual 20 627.563 31.378 1.13 block.board.area stratum treatment 3 114.951 38.317 1.38 0.255 exposure.treatment 15 167.494 11.166 0.40 0.974 Residual 72 1995.746 27.719 3.61 block.board.area.Strip stratum Chem_Charge 3 51.084 17.028 2.22 0.086 exposure.Chem_Charge 15 245.000 16.333 2.13 0.009 treatment.Chem_Charge 9 85.084 9.454 1.23 0.275 exposure.treatment.Chem_Charge 45 353.529 7.856 1.02 0.436 Residual 286 (2) 2193.402 7.669 Total 477 (2) 9188.195 371 Message: the following units have large residuals. block 3 board 4 2.34 s.e. 1.14 block 3 board 5 area 3 -5.55 s.e. 2.04 block 5 board 3 area 1 Strip 2 6.26 s.e. 2.14 Tables of means Variate: L Grand mean 74.64 exposure full I.R. none UVA UVB Vis light 69.90 74.48 76.79 75.86 73.09 77.73 treatment acetic acid carpropamid tinuvin water 73.87 74.88 75.20 74.61 Chem_Charge high low medium Very high 75.18 74.40 74.36 74.63 exposure treatment acetic acid carpropamid tinuvin water full 68.72 70.01 71.08 69.77 I.R. 73.28 74.83 75.80 73.99 none 75.31 77.21 77.22 77.41 UVA 75.19 75.63 76.98 75.66 UVB 73.70 72.59 72.81 73.25 Vis light 77.05 79.00 77.30 77.56 exposure Chem_Charge high low medium Very high full 70.23 70.43 68.86 70.06 I.R. 75.01 75.37 74.40 73.11 none 76.69 75.17 77.62 77.67 UVA 76.77 75.80 75.38 75.51 UVB 73.91 73.35 71.89 73.20 Vis light 78.45 76.29 77.98 78.20 treatment Chem_Charge high low medium Very high acetic acid 74.36 73.79 73.99 73.36 carpropamid 75.48 73.77 74.88 75.39 tinuvin 75.96 75.33 74.06 75.45 water 74.90 74.72 74.49 74.31 372 exposure treatment Chem_Charge high low medium Very high full acetic acid 70.13 67.63 67.97 69.13 carpropamid 70.18 70.39 69.38 70.11 tinuvin 70.18 72.87 69.28 72.01 water 70.45 70.83 68.83 68.98 I.R. acetic acid 73.48 75.91 73.63 70.11 carpropamid 76.60 73.56 75.00 74.15 tinuvin 76.11 76.88 74.49 75.70 water 73.85 75.14 74.49 72.49 none acetic acid 75.58 73.46 77.05 75.13 carpropamid 77.54 76.36 76.31 78.64 tinuvin 76.93 76.12 77.85 77.97 water 76.69 74.74 79.26 78.95 UVA acetic acid 75.49 74.22 75.72 75.32 carpropamid 75.28 74.22 76.64 76.39 tinuvin 79.46 78.01 74.73 75.73 water 76.86 76.74 74.43 74.60 UVB acetic acid 73.87 75.67 71.71 73.56 carpropamid 74.48 70.00 72.72 73.16 tinuvin 74.73 73.41 69.77 73.34 water 72.56 74.34 73.37 72.74 Vis light acetic acid 77.60 75.84 77.89 76.88 carpropamid 78.80 78.11 79.20 79.89 tinuvin 78.37 74.67 78.23 77.94 water 79.01 76.54 76.60 78.11 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 0.626 0.481 0.253 1.197 d.f. 20 72 286 90.30 Except when comparing means with the same level(s) of exposure 1.177 d.f. 72 373 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 0.825 0.650 1.607 d.f. 57.91 205.49 243.98 Except when comparing means with the same level(s) of exposure 0.619 1.593 d.f. 286 205.49 treatment 0.506 d.f. 286 exposure.treatment 1.238 d.f. 286 exposure.Chem_Charge 1.593 d.f. 205.49 (Not adjusted for missing values) Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.886 0.680 0.358 1.692 d.f. 20 72 286 90.30 Except when comparing means with the same level(s) of exposure 1.665 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.166 0.919 2.272 d.f. 57.91 205.49 243.98 Except when comparing means with the same level(s) of exposure 0.876 2.252 d.f. 286 205.49 treatment 0.715 d.f. 286 exposure.treatment 1.751 d.f. 286 exposure.Chem_Charge 2.252 d.f. 205.49 (Not adjusted for missing values) Least significant differences of means (5% level) 374 Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.848 1.355 0.704 3.362 d.f. 20 72 286 90.30 Except when comparing means with the same level(s) of exposure 3.319 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 2.334 1.813 4.476 d.f. 57.91 205.49 243.98 Except when comparing means with the same level(s) of exposure 1.724 4.440 d.f. 286 205.49 treatment 1.407 d.f. 286 exposure.treatment 3.447 d.f. 286 exposure.Chem_Charge 4.440 d.f. 205.49 (Not adjusted for missing values) Analysis of variance Variate: a Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 52.758 13.190 2.22 block.board stratum exposure 5 661.387 132.277 22.26 <.001 Residual 20 118.855 5.943 1.54 block.board.area stratum treatment 3 14.674 4.891 1.26 0.293 exposure.treatment 15 32.193 2.146 0.55 0.899 Residual 72 278.444 3.867 2.81 375 block.board.area.Strip stratum Chem_Charge 3 6.770 2.257 1.64 0.180 exposure.Chem_Charge 15 23.106 1.540 1.12 0.339 treatment.Chem_Charge 9 16.868 1.874 1.36 0.205 exposure.treatment.Chem_Charge 45 78.789 1.751 1.27 0.126 Residual 287 (1) 395.129 1.377 Total 478 (1) 1666.813 Message: the following units have large residuals. block 3 board 1 1.076 s.e. 0.498 block 1 board 3 area 1 2.173 s.e. 0.762 block 2 board 5 area 2 2.072 s.e. 0.762 block 1 board 5 area 3 Strip 3 -2.720 s.e. 0.907 block 1 board 5 area 3 Strip 4 3.519 s.e. 0.907 block 2 board 5 area 1 Strip 3 3.005 s.e. 0.907 block 3 board 5 area 3 Strip 4 -3.165 s.e. 0.907 block 5 board 6 area 2 Strip 3 2.693 s.e. 0.907 Tables of means Variate: a Grand mean 5.984 exposure full I.R. none UVA UVB Vis light 7.844 6.131 5.552 5.127 6.970 4.284 treatment acetic acid carpropamid tinuvin water 6.223 5.833 5.802 6.080 Chem_Charge high low medium Very high 5.903 6.143 6.051 5.842 exposure treatment acetic acid carpropamid tinuvin water full 7.935 7.997 7.207 8.237 I.R. 6.609 5.605 6.150 6.159 none 6.161 5.442 5.223 5.380 UVA 5.322 5.252 4.678 5.254 UVB 6.825 6.940 6.969 7.144 Vis light 4.483 3.763 4.582 4.306 376 exposure Chem_Charge high low medium Very high full 7.812 7.737 8.148 7.680 I.R. 6.165 6.110 6.104 6.144 none 5.729 6.125 5.175 5.177 UVA 4.743 5.153 5.449 5.160 UVB 6.934 6.935 7.222 6.787 Vis light 4.032 4.795 4.204 4.102 treatment Chem_Charge high low medium Very high acetic acid 6.280 6.226 6.136 6.248 carpropamid 5.736 6.298 5.738 5.561 tinuvin 5.498 5.963 6.284 5.462 water 6.096 6.085 6.044 6.097 exposure treatment Chem_Charge high low medium Very high full acetic acid 7.660 7.972 8.556 7.554 carpropamid 7.984 7.904 8.226 7.874 tinuvin 7.490 6.790 7.658 6.892 water 8.114 8.282 8.152 8.402 I.R. acetic acid 7.210 5.630 7.048 6.550 carpropamid 5.314 6.824 4.498 5.784 tinuvin 5.962 6.232 6.846 5.560 water 6.174 5.756 6.022 6.684 none acetic acid 5.840 7.296 5.180 6.328 carpropamid 5.298 5.568 5.858 5.044 tinuvin 5.430 5.852 4.884 4.726 water 6.348 5.786 4.778 4.610 UVA acetic acid 5.260 5.602 5.094 5.332 carpropamid 5.316 5.822 5.150 4.720 tinuvin 4.006 4.060 5.820 4.826 water 4.390 5.130 5.734 5.764 UVB acetic acid 7.190 5.934 7.020 7.156 carpropamid 6.804 7.618 6.766 6.572 tinuvin 6.066 6.986 8.300 6.524 water 7.676 7.202 6.804 6.896 Vis light acetic acid 4.522 4.920 3.920 4.570 carpropamid 3.702 4.050 3.930 3.370 tinuvin 4.034 5.858 4.194 4.242 water 3.872 4.352 4.774 4.228 377 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 0.2726 0.1795 0.1071 0.4683 d.f. 20 72 287 84.67 Except when comparing means with the same level(s) of exposure 0.4397 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 0.3548 0.2582 0.6525 d.f. 55.59 239.41 253.03 Except when comparing means with the same level(s) of exposure 0.2624 0.6324 d.f. 287 239.41 treatment 0.2142 d.f. 287 exposure.treatment 0.5247 d.f. 287 exposure.Chem_Charge 0.6324 d.f. 239.41 (Not adjusted for missing values) Standard errors of differences of means 378 Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.3854 0.2539 0.1515 0.6623 d.f. 20 72 287 84.67 Except when comparing means with the same level(s) of exposure 0.6219 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.5018 0.3651 0.9228 d.f. 55.59 239.41 253.03 Except when comparing means with the same level(s) of exposure 0.3710 0.8943 d.f. 287 239.41 treatment 0.3030 d.f. 287 exposure.treatment 0.7421 d.f. 287 exposure.Chem_Charge 0.8943 d.f. 239.41 (Not adjusted for missing values) 379 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 0.8040 0.5061 0.2982 1.3169 d.f. 20 72 287 84.67 Except when comparing means with the same level(s) of exposure 1.2397 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.0054 0.7192 1.8174 d.f. 55.59 239.41 253.03 Except when comparing means with the same level(s) of exposure 0.7303 1.7617 d.f. 287 239.41 treatment 0.5963 d.f. 287 exposure.treatment 1.4606 d.f. 287 exposure.Chem_Charge 1.7617 d.f. 239.41 (Not adjusted for missing values) Analysis of variance Variate: b Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 193.012 48.253 3.90 block.board stratum exposure 5 7183.577 1436.715 116.25 <.001 Residual 20 247.177 12.359 1.34 block.board.area stratum treatment 3 18.869 6.290 0.68 0.565 exposure.treatment 15 144.988 9.666 1.05 0.417 Residual 72 662.691 9.204 3.12 380 block.board.area.Strip stratum Chem_Charge 3 27.156 9.052 3.07 0.028 exposure.Chem_Charge 15 42.079 2.805 0.95 0.507 treatment.Chem_Charge 9 42.809 4.757 1.61 0.111 exposure.treatment.Chem_Charge 45 216.364 4.808 1.63 0.010 Residual 287 (1) 846.301 2.949 Total 478 (1) 9593.434 Message: the following units have large residuals. block 4 board 6 -2.102 s.e. 0.718 block 1 board 3 area 1 3.414 s.e. 1.175 block 5 board 6 area 4 -3.117 s.e. 1.175 block 1 board 4 area 2 Strip 1 4.481 s.e. 1.328 block 1 board 4 area 2 Strip 4 -4.505 s.e. 1.328 block 1 board 5 area 2 Strip 4 -4.611 s.e. 1.328 block 1 board 5 area 3 Strip 3 -4.167 s.e. 1.328 block 5 board 6 area 2 Strip 3 4.623 s.e. 1.328 block 5 board 6 area 2 Strip 4 -4.292 s.e. 1.328 Tables of means Variate: b Grand mean 26.625 exposure full I.R. none UVA UVB Vis light 32.156 25.095 24.795 24.102 31.654 21.950 treatment acetic acid carpropamid tinuvin water 26.910 26.366 26.548 26.677 Chem_Charge high low medium Very high 26.715 26.897 26.645 26.246 exposure treatment acetic acid carpropamid tinuvin water full 31.857 32.529 31.422 32.816 I.R. 25.409 24.159 26.179 24.634 none 25.292 24.812 24.254 24.820 UVA 24.663 24.451 23.062 24.230 UVB 31.950 31.502 31.502 31.661 Vis light 22.291 20.742 22.866 21.901 381 exposure Chem_Charge high low medium Very high full 32.207 32.408 32.247 31.763 I.R. 25.466 25.176 24.906 24.833 none 25.066 25.420 24.429 24.264 UVA 23.565 24.094 24.658 24.090 UVB 32.189 31.567 31.755 31.104 Vis light 21.793 22.716 21.872 21.419 treatment Chem_Charge high low medium Very high acetic acid 27.092 27.076 26.616 26.859 carpropamid 26.486 26.972 26.351 25.656 tinuvin 26.118 27.037 27.043 25.993 water 27.163 26.503 26.568 26.474 exposure treatment Chem_Charge high low medium Very high full acetic acid 32.418 32.090 32.166 30.754 carpropamid 32.220 32.708 32.880 32.310 tinuvin 31.214 31.776 31.594 31.106 water 32.976 33.058 32.348 32.882 I.R. acetic acid 25.824 23.904 26.224 25.684 carpropamid 24.202 26.752 21.822 23.860 tinuvin 26.456 26.216 27.248 24.798 water 25.384 23.832 24.328 24.992 none acetic acid 24.694 26.960 23.934 25.582 carpropamid 24.516 25.050 25.470 24.214 tinuvin 24.352 25.128 23.982 23.556 water 26.704 24.542 24.332 23.704 UVA acetic acid 24.408 25.074 24.936 24.236 carpropamid 24.792 25.002 24.762 23.250 tinuvin 22.056 21.868 24.822 23.502 water 23.004 24.434 24.112 25.372 UVB acetic acid 32.608 31.682 30.956 32.554 carpropamid 32.498 31.274 31.836 30.400 tinuvin 30.716 32.126 32.514 30.652 water 32.936 31.186 31.714 30.810 Vis light acetic acid 22.598 22.744 21.478 22.344 carpropamid 20.688 21.044 21.334 19.902 tinuvin 21.912 25.108 22.100 22.344 water 21.974 21.968 22.576 21.086 382 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 0.3930 0.2769 0.1568 0.7069 d.f. 20 72 287 87.66 Except when comparing means with the same level(s) of exposure 0.6784 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 0.5148 0.3878 0.9705 d.f. 56.85 224.82 251.39 Except when comparing means with the same level(s) of exposure 0.3840 0.9500 d.f. 287 224.82 treatment 0.3135 d.f. 287 exposure.treatment 0.7680 d.f. 287 exposure.Chem_Charge 0.9500 d.f. 224.82 (Not adjusted for missing values) 383 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.5559 0.3917 0.2217 0.9996 d.f. 20 72 287 87.66 Except when comparing means with the same level(s) of exposure 0.9594 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.7281 0.5485 1.3726 d.f. 56.85 224.82 251.39 Except when comparing means with the same level(s) of exposure 0.5430 1.3435 d.f. 287 224.82 treatment 0.4434 d.f. 287 exposure.treatment 1.0861 d.f. 287 exposure.Chem_Charge 1.3435 d.f. 224.82 (Not adjusted for missing values) 384 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.1595 0.7808 0.4363 1.9867 d.f. 20 72 287 87.66 Except when comparing means with the same level(s) of exposure 1.9125 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.4581 1.0808 2.7032 d.f. 56.85 224.82 251.39 Except when comparing means with the same level(s) of exposure 1.0688 2.6475 d.f. 287 224.82 treatment 0.8727 d.f. 287 exposure.treatment 2.1376 d.f. 287 exposure.Chem_Charge 2.6475 d.f. 224.82 (Not adjusted for missing values) Analysis of variance week 2 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 183.691 45.923 0.33 block.board stratum exposure 5 2917.945 583.589 4.23 0.009 Residual 20 2761.097 138.055 5.33 block.board.area stratum treatment 3 164.561 54.854 2.12 0.106 exposure.treatment 15 251.601 16.773 0.65 0.826 Residual 72 1866.594 25.925 2.95 385 block.board.area.Strip stratum Chem_Charge 3 19.789 6.596 0.75 0.523 exposure.Chem_Charge 15 96.547 6.436 0.73 0.751 treatment.Chem_Charge 9 118.107 13.123 1.49 0.150 exposure.treatment.Chem_Charge 45 238.213 5.294 0.60 0.979 Residual 288 2530.019 8.785 Total 479 11148.164 Message: the following units have large residuals. block 4 board 1 5.24 s.e. 2.40 block 5 board 4 -5.29 s.e. 2.40 block 4 board 3 area 1 Strip 3 -7.61 s.e. 2.30 block 5 board 2 area 3 Strip 1 -7.04 s.e. 2.30 Tables of means Variate: L Grand mean 74.13 exposure full I.R. none UVA UVB Vis light 70.84 74.77 76.10 73.38 71.70 77.98 treatment acetic acid carpropamid tinuvin water 73.64 74.24 75.04 73.59 Chem_Charge high low medium Very high 74.38 74.27 73.88 73.99 exposure treatment acetic acid carpropamid tinuvin water full 70.34 69.93 71.89 71.19 I.R. 72.41 75.94 76.48 74.25 none 75.64 76.64 77.19 74.94 UVA 73.59 73.45 73.43 73.04 UVB 72.22 70.77 72.24 71.57 Vis light 77.66 78.72 79.01 76.55 exposure Chem_Charge high low medium Very high full 70.74 70.79 69.89 71.92 I.R. 74.92 74.77 74.52 74.87 none 76.05 76.43 76.58 75.34 UVA 73.57 73.53 73.16 73.25 UVB 72.63 71.63 71.03 71.50 Vis light 78.33 78.48 78.07 77.05 386 treatment Chem_Charge high low medium Very high acetic acid 74.08 73.34 73.84 73.31 carpropamid 74.49 73.97 74.37 74.12 tinuvin 75.05 75.13 74.07 75.91 water 73.87 74.65 73.22 72.61 exposure treatment Chem_Charge high low medium Very high full acetic acid 70.66 69.55 69.99 71.16 carpropamid 70.21 69.50 68.89 71.10 tinuvin 70.20 71.98 70.40 74.99 water 71.87 72.15 70.29 70.44 I.R. acetic acid 73.17 71.93 71.95 72.60 carpropamid 75.94 75.74 75.50 76.56 tinuvin 77.46 76.03 75.77 76.65 water 73.12 75.35 74.87 73.66 none acetic acid 75.10 76.41 76.23 74.82 carpropamid 77.66 75.35 77.68 75.85 tinuvin 76.63 78.41 76.35 77.38 water 74.83 75.56 76.05 73.32 UVA acetic acid 74.21 73.37 74.60 72.16 carpropamid 72.94 72.48 74.02 74.34 tinuvin 73.22 73.87 73.06 73.57 water 73.90 74.38 70.95 72.94 UVB acetic acid 72.56 72.12 72.21 71.99 carpropamid 71.71 71.14 70.44 69.81 tinuvin 74.90 70.68 69.72 73.66 water 71.38 72.59 71.77 70.53 Vis light acetic acid 78.80 76.65 78.06 77.13 carpropamid 78.50 79.61 79.71 77.06 tinuvin 77.91 79.81 79.11 79.22 water 78.12 77.85 75.41 74.80 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 1.314 0.465 0.271 1.643 d.f. 20 72 288 44.92 Except when comparing means with the same level(s) of exposure 1.139 d.f. 72 387 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 1.434 0.660 2.004 d.f. 28.29 232.68 95.95 Except when comparing means with the same level(s) of exposure 0.663 1.617 d.f. 288 232.68 treatment 0.541 d.f. 288 exposure.treatment 1.326 d.f. 288 exposure.Chem_Charge 1.617 d.f. 232.68 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.858 0.657 0.383 2.323 d.f. 20 72 288 44.92 Except when comparing means with the same level(s) of exposure 1.610 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.027 0.933 2.834 d.f. 28.29 232.68 95.95 Except when comparing means with the same level(s) of exposure 0.937 2.286 d.f. 288 232.68 treatment 0.765 d.f. 288 exposure.treatment 1.875 d.f. 288 exposure.Chem_Charge 2.286 d.f. 232.68 388 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 3.875 1.310 0.753 4.679 d.f. 20 72 288 44.92 Except when comparing means with the same level(s) of exposure 3.210 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 4.151 1.839 5.625 d.f. 28.29 232.68 95.95 Except when comparing means with the same level(s) of exposure 1.845 4.505 d.f. 288 232.68 treatment 1.506 d.f. 288 exposure.treatment 3.690 d.f. 288 exposure.Chem_Charge 4.505 d.f. 232.68 Analysis of variance Variate: a Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 109.664 27.416 1.09 block.board stratum exposure 5 906.451 181.290 7.24 <.001 Residual 20 501.068 25.053 5.36 block.board.area stratum treatment 3 13.666 4.555 0.97 0.410 exposure.treatment 15 33.972 2.265 0.48 0.941 Residual 72 336.610 4.675 2.71 block.board.area.Strip stratum Chem_Charge 3 1.390 0.463 0.27 0.848 exposure.Chem_Charge 15 28.593 1.906 1.11 0.350 treatment.Chem_Charge 9 18.186 2.021 1.17 0.312 exposure.treatment.Chem_Charge 45 75.967 1.688 0.98 0.514 Residual 288 496.247 1.723 Total 479 2521.813 389 Message: the following units have large residuals. block 4 board 1 -2.442 s.e. 1.022 block 3 board 5 area 1 2.311 s.e. 0.837 block 1 board 5 area 3 Strip 2 3.023 s.e. 1.017 block 1 board 5 area 4 Strip 2 3.619 s.e. 1.017 block 4 board 4 area 4 Strip 3 3.567 s.e. 1.017 block 5 board 2 area 3 Strip 1 3.208 s.e. 1.017 Tables of means Variate: a Grand mean 6.975 exposure full I.R. none UVA UVB Vis light 8.578 6.288 5.905 7.289 8.770 5.019 treatment acetic acid carpropamid tinuvin water 7.038 7.014 6.696 7.150 Chem_Charge high low medium Very high 6.911 6.944 7.055 6.989 exposure treatment acetic acid carpropamid tinuvin water full 8.559 8.748 8.430 8.574 I.R. 7.177 5.812 5.927 6.237 none 5.994 5.770 5.531 6.325 UVA 7.069 7.579 7.135 7.373 UVB 8.417 9.176 8.420 9.066 Vis light 5.014 4.999 4.736 5.329 exposure Chem_Charge high low medium Very high full 8.799 8.526 8.888 8.099 I.R. 6.180 6.374 6.654 5.945 none 5.966 6.010 5.571 6.074 UVA 6.979 7.308 7.480 7.389 UVB 8.503 8.816 8.881 8.880 Vis light 5.043 4.630 4.858 5.546 treatment Chem_Charge high low medium Very high acetic acid 6.932 6.960 7.011 7.251 carpropamid 6.996 7.171 6.945 6.943 tinuvin 6.621 6.782 7.105 6.276 water 7.097 6.862 7.158 7.484 390 exposure treatment Chem_Charge high low medium Very high full acetic acid 8.072 8.486 9.272 8.408 carpropamid 8.744 8.960 9.276 8.010 tinuvin 9.610 7.738 8.878 7.494 water 8.770 8.918 8.124 8.482 I.R. acetic acid 6.964 7.260 7.754 6.728 carpropamid 5.528 6.030 6.488 5.200 tinuvin 5.460 6.306 6.236 5.704 water 6.766 5.898 6.136 6.146 none acetic acid 6.222 6.004 5.332 6.418 carpropamid 5.384 6.818 4.986 5.890 tinuvin 5.740 5.276 5.816 5.292 water 6.516 5.940 6.148 6.694 UVA acetic acid 6.724 6.980 7.094 7.478 carpropamid 7.672 8.076 7.422 7.144 tinuvin 6.788 6.940 7.752 7.058 water 6.730 7.234 7.650 7.876 UVB acetic acid 8.572 8.006 8.142 8.948 carpropamid 8.904 8.788 9.068 9.944 tinuvin 7.172 9.726 9.346 7.436 water 9.364 8.742 8.966 9.190 Vis light acetic acid 5.036 5.022 4.470 5.526 carpropamid 5.742 4.352 4.430 5.472 tinuvin 4.958 4.708 4.604 4.672 water 4.436 4.438 5.926 6.514 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 0.5596 0.1974 0.1198 0.6989 d.f. 20 72 288 44.76 Except when comparing means with the same level(s) of exposure 0.4835 d.f. 72 391 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 0.6146 0.2864 0.8643 d.f. 29.02 244.51 100.30 Except when comparing means with the same level(s) of exposure 0.2935 0.7016 d.f. 288 244.51 treatment 0.2397 d.f. 288 exposure.treatment 0.5870 d.f. 288 exposure.Chem_Charge 0.7016 d.f. 244.51 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.7914 0.2791 0.1695 0.9884 d.f. 20 72 288 44.76 Except when comparing means with the same level(s) of exposure 0.6837 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.8692 0.4051 1.2222 d.f. 29.02 244.51 100.30 Except when comparing means with the same level(s) of exposure 0.4151 0.9922 d.f. 288 244.51 treatment 0.3389 d.f. 288 exposure.treatment 0.8302 d.f. 288 exposure.Chem_Charge 0.9922 d.f. 244.51 392 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.6509 0.5565 0.3335 1.9911 d.f. 20 72 288 44.76 Except when comparing means with the same level(s) of exposure 1.3630 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.7777 0.7979 2.4248 d.f. 29.02 244.51 100.30 Except when comparing means with the same level(s) of exposure 0.8170 1.9543 d.f. 288 244.51 treatment 0.6671 d.f. 288 exposure.treatment 1.6340 d.f. 288 exposure.Chem_Charge 1.9543 d.f. 244.51 Analysis of variance Variate: b Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 201.926 50.481 0.28 block.board stratum exposure 5 4442.499 888.500 4.89 0.004 Residual 20 3631.580 181.579 21.58 block.board.area stratum treatment 3 16.419 5.473 0.65 0.585 exposure.treatment 15 36.286 2.419 0.29 0.995 Residual 72 605.803 8.414 2.49 block.board.area.Strip stratum Chem_Charge 3 3.699 1.233 0.36 0.779 exposure.Chem_Charge 15 51.625 3.442 1.02 0.436 treatment.Chem_Charge 9 15.720 1.747 0.52 0.862 exposure.treatment.Chem_Charge 45 180.694 4.015 1.19 0.204 Residual 286 (2) 967.006 3.381 Total 477 (2) 9973.390 393 Message: the following units have large residuals. block 4 board 1 -5.605 s.e. 2.751 block 5 board 2 -6.176 s.e. 2.751 block 5 board 3 6.592 s.e. 2.751 block 4 board 4 area 4 3.008 s.e. 1.123 block 1 board 5 area 3 Strip 1 4.589 s.e. 1.419 block 1 board 5 area 3 Strip 4 -4.431 s.e. 1.419 block 4 board 4 area 4 Strip 3 5.017 s.e. 1.419 block 5 board 2 area 2 Strip 1 4.340 s.e. 1.419 Tables of means Variate: b Grand mean 28.377 exposure full I.R. none UVA UVB Vis light 30.119 26.524 25.598 30.865 32.764 24.389 treatment acetic acid carpropamid tinuvin water 28.144 28.479 28.262 28.620 Chem_Charge high low medium Very high 28.419 28.463 28.393 28.231 exposure treatment acetic acid carpropamid tinuvin water full 29.534 30.146 30.460 30.336 I.R. 26.925 26.123 26.319 26.729 none 24.995 25.891 25.497 26.009 UVA 30.559 31.165 30.471 31.263 UVB 32.339 32.973 32.524 33.219 Vis light 24.512 24.576 24.301 24.164 exposure Chem_Charge high low medium Very high full 30.339 30.276 30.345 29.517 I.R. 26.389 26.830 26.827 26.051 none 25.843 25.853 25.417 25.280 UVA 30.158 30.987 31.040 31.273 UVB 33.062 32.979 32.547 32.467 Vis light 24.724 23.851 24.182 24.796 treatment Chem_Charge high low medium Very high acetic acid 28.247 28.058 28.099 28.173 carpropamid 28.575 28.635 28.637 28.071 tinuvin 28.082 28.555 28.489 27.924 water 28.774 28.604 28.347 28.756 394 exposure treatment Chem_Charge high low medium Very high full acetic acid 29.192 29.412 30.042 29.492 carpropamid 29.824 30.730 31.158 28.874 tinuvin 31.622 29.614 30.690 29.916 water 30.720 31.348 29.492 29.786 I.R. acetic acid 26.604 27.424 27.570 26.104 carpropamid 25.396 26.536 27.340 25.220 tinuvin 25.670 26.914 26.074 26.620 water 27.886 26.446 26.324 26.260 none acetic acid 25.684 25.210 24.222 24.866 carpropamid 25.072 27.426 25.738 25.330 tinuvin 25.818 25.302 25.924 24.946 water 26.798 25.474 25.786 25.978 UVA acetic acid 30.189 30.208 31.324 30.514 carpropamid 31.132 31.484 31.372 30.674 tinuvin 28.888 30.568 31.494 30.934 water 30.422 31.688 29.972 32.971 UVB acetic acid 32.888 32.196 31.718 32.556 carpropamid 33.682 32.208 32.548 33.454 tinuvin 32.094 34.094 32.590 31.320 water 33.586 33.420 33.332 32.538 Vis light acetic acid 24.926 23.898 23.720 25.506 carpropamid 26.342 23.424 23.668 24.872 tinuvin 24.398 24.838 24.164 23.806 water 23.232 23.246 25.178 25.000 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 1.5066 0.2648 0.1679 1.6079 d.f. 20 72 286 25.81 Except when comparing means with the same level(s) of exposure 0.6486 d.f. 72 395 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 1.5481 0.3932 1.7585 d.f. 22.29 256.42 36.80 Except when comparing means with the same level(s) of exposure 0.4112 0.9633 d.f. 286 256.42 treatment 0.3357 d.f. 286 exposure.treatment 0.8223 d.f. 286 exposure.Chem_Charge 0.9633 d.f. 256.42 (Not adjusted for missing values) Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 2.1306 0.3745 0.2374 2.2739 d.f. 20 72 286 25.81 Except when comparing means with the same level(s) of exposure 0.9173 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.1893 0.5561 2.4869 d.f. 22.29 256.42 36.80 Except when comparing means with the same level(s) of exposure 0.5815 1.3623 d.f. 286 256.42 treatment 0.4748 d.f. 286 exposure.treatment 1.1630 d.f. 286 exposure.Chem_Charge 1.3623 d.f. 256.42 (Not adjusted for missing values) 396 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 4.4444 0.7465 0.4672 4.6757 d.f. 20 72 286 25.81 Except when comparing means with the same level(s) of exposure 1.8286 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 4.5369 1.0952 5.0399 d.f. 22.29 256.42 36.80 Except when comparing means with the same level(s) of exposure 1.1445 2.6826 d.f. 286 256.42 treatment 0.9345 d.f. 286 exposure.treatment 2.2890 d.f. 286 exposure.Chem_Charge 2.6826 d.f. 256.42 (Not adjusted for missing values) Analysis of variance week 3 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 223.759 55.940 1.96 block.board stratum exposure 5 4873.958 974.792 34.17 <.001 Residual 20 570.569 28.528 1.39 block.board.area stratum treatment 3 69.557 23.186 1.13 0.342 exposure.treatment 15 135.898 9.060 0.44 0.960 Residual 72 1474.141 20.474 2.90 397 block.board.area.Strip stratum Chem_Charge 3 25.185 8.395 1.19 0.314 exposure.Chem_Charge 15 113.108 7.541 1.07 0.385 treatment.Chem_Charge 9 100.597 11.177 1.58 0.119 exposure.treatment.Chem_Charge 45 361.153 8.026 1.14 0.263 Residual 288 2031.166 7.053 Total 479 9979.092 Message: the following units have large residuals. block 2 board 4 2.90 s.e. 1.09 block 5 board 1 area 4 4.79 s.e. 1.75 block 1 board 4 area 2 Strip 3 -7.13 s.e. 2.06 block 1 board 5 area 4 Strip 2 -6.55 s.e. 2.06 block 4 board 5 area 3 Strip 3 7.02 s.e. 2.06 Tables of means Variate: L Grand mean 73.07 exposure full I.R. none UVA UVB Vis light 67.47 74.70 76.79 73.45 70.45 75.54 treatment acetic acid carpropamid tinuvin water 72.49 73.09 73.56 73.13 Chem_Charge high low medium Very high 73.42 73.10 72.93 72.81 exposure treatment acetic acid carpropamid tinuvin water full 66.99 66.67 68.50 67.71 I.R. 73.79 74.69 75.94 74.36 none 75.94 76.95 76.62 77.63 UVA 72.49 73.46 74.73 73.14 UVB 70.99 70.70 69.89 70.23 Vis light 74.73 76.05 75.68 75.69 398 exposure Chem_Charge high low medium Very high full 67.67 67.94 66.88 67.38 I.R. 75.21 75.70 74.18 73.69 none 76.68 75.61 77.61 77.25 UVA 74.02 73.64 73.30 72.85 UVB 71.00 70.41 70.04 70.35 Vis light 75.93 75.32 75.55 75.34 treatment Chem_Charge high low medium Very high acetic acid 72.91 72.80 73.00 71.24 carpropamid 73.34 72.56 72.95 73.49 tinuvin 73.86 73.89 72.63 73.86 water 73.56 73.17 73.13 72.65 exposure treatment Chem_Charge high low medium Very high full acetic acid 68.51 67.17 67.85 64.43 carpropamid 67.00 66.27 65.84 67.59 tinuvin 66.29 69.66 68.34 69.70 water 68.89 68.66 65.50 67.80 I.R. acetic acid 74.07 75.31 73.02 72.77 carpropamid 75.64 74.33 74.41 74.37 tinuvin 76.72 77.37 74.73 74.95 water 74.40 75.81 74.57 72.68 none acetic acid 77.05 74.21 77.70 74.81 carpropamid 77.42 76.40 76.08 77.89 tinuvin 75.59 76.12 77.59 77.18 water 76.65 75.71 79.06 79.12 UVA acetic acid 72.02 72.31 73.25 72.37 carpropamid 72.94 73.50 73.67 73.72 tinuvin 76.79 75.65 72.97 73.49 water 74.33 73.11 73.33 71.81 UVB acetic acid 70.09 73.54 70.78 69.55 carpropamid 72.11 68.51 71.59 70.59 tinuvin 71.74 69.58 66.88 71.35 water 70.07 70.00 70.92 69.92 Vis light acetic acid 75.75 74.26 75.43 73.50 carpropamid 74.96 76.36 76.11 76.78 tinuvin 76.02 74.93 75.26 76.50 water 77.00 75.74 75.42 74.59 399 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 0.597 0.413 0.242 1.060 d.f. 20 72 288 86.92 Except when comparing means with the same level(s) of exposure 1.012 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 0.788 0.589 1.477 d.f. 58.43 234.97 258.38 Except when comparing means with the same level(s) of exposure 0.594 1.443 d.f. 288 234.97 treatment 0.485 d.f. 288 exposure.treatment 1.188 d.f. 288 exposure.Chem_Charge 1.443 d.f. 234.97 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.845 0.584 0.343 1.500 d.f. 20 72 288 86.92 Except when comparing means with the same level(s) of exposure 1.431 d.f. 72 400 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.115 0.833 2.089 d.f. 58.43 234.97 258.38 Except when comparing means with the same level(s) of exposure 0.840 2.040 d.f. 288 234.97 treatment 0.686 d.f. 288 exposure.treatment 1.680 d.f. 288 exposure.Chem_Charge 2.040 d.f. 234.97 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.762 1.164 0.675 2.981 d.f. 20 72 288 86.92 Except when comparing means with the same level(s) of exposure 2.852 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 2.231 1.641 4.114 d.f. 58.43 234.97 258.38 Except when comparing means with the same level(s) of exposure 1.653 4.020 d.f. 288 234.97 treatment 1.350 d.f. 288 exposure.treatment 3.306 d.f. 288 exposure.Chem_Charge 4.020 d.f. 234.97 401 Analysis of variance Variate: a Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 45.592 11.398 3.43 block.board stratum exposure 5 1667.440 333.488 100.49 <.001 Residual 20 66.375 3.319 0.98 block.board.area stratum treatment 3 10.706 3.569 1.06 0.372 exposure.treatment 15 42.379 2.825 0.84 0.633 Residual 72 242.610 3.370 2.91 block.board.area.Strip stratum Chem_Charge 3 0.533 0.178 0.15 0.927 exposure.Chem_Charge 15 17.233 1.149 0.99 0.463 treatment.Chem_Charge 9 21.491 2.388 2.06 0.033 exposure.treatment.Chem_Charge 45 80.316 1.785 1.54 0.020 Residual 286 (2) 331.019 1.157 Total 477 (2) 2503.762 Message: the following units have large residuals. block 2 board 4 -0.870 s.e. 0.372 block 3 board 1 0.780 s.e. 0.372 block 3 board 2 -0.755 s.e. 0.372 block 1 board 3 area 1 1.948 s.e. 0.711 block 1 board 5 area 3 2.015 s.e. 0.711 block 2 board 5 area 2 1.989 s.e. 0.711 block 1 board 5 area 3 Strip 1 -2.548 s.e. 0.830 block 1 board 5 area 3 Strip 2 2.472 s.e. 0.830 block 2 board 5 area 1 Strip 3 2.545 s.e. 0.830 block 4 board 3 area 2 Strip 3 2.924 s.e. 0.830 block 5 board 6 area 2 Strip 3 2.775 s.e. 0.830 402 Tables of means Variate: a Grand mean 7.209 exposure full I.R. none UVA UVB Vis light 9.825 6.036 5.335 6.894 9.672 5.492 treatment acetic acid carpropamid tinuvin water 7.453 7.126 7.059 7.198 Chem_Charge high low medium Very high 7.267 7.186 7.192 7.190 exposure treatment acetic acid carpropamid tinuvin water full 9.542 10.348 9.483 9.925 I.R. 6.810 5.582 5.711 6.042 none 5.624 5.522 5.320 4.873 UVA 7.242 7.003 6.447 6.882 UVB 9.654 9.290 9.817 9.925 Vis light 5.846 5.009 5.572 5.539 exposure Chem_Charge high low medium Very high full 9.895 9.790 9.925 9.688 I.R. 6.320 5.745 5.928 6.151 none 5.371 5.855 4.944 5.169 UVA 6.779 6.870 6.885 7.041 UVB 9.845 9.422 9.872 9.548 Vis light 5.389 5.435 5.598 5.544 treatment Chem_Charge high low medium Very high acetic acid 7.703 7.192 7.155 7.763 carpropamid 7.215 7.424 7.081 6.783 tinuvin 7.029 7.076 7.321 6.810 water 7.119 7.054 7.212 7.407 403 exposure treatment Chem_Charge high low medium Very high full acetic acid 9.260 9.406 9.326 10.178 carpropamid 10.516 10.608 10.536 9.732 tinuvin 10.172 9.216 9.448 9.098 water 9.632 9.932 10.392 9.746 I.R. acetic acid 8.306 5.402 7.052 6.478 carpropamid 5.512 6.496 4.826 5.494 tinuvin 5.540 5.420 5.902 5.984 water 5.922 5.664 5.932 6.650 none acetic acid 5.040 6.604 4.724 6.130 carpropamid 5.338 5.856 5.728 5.168 tinuvin 5.638 5.804 4.846 4.994 water 5.468 5.158 4.480 4.386 UVA acetic acid 7.412 7.326 6.848 7.382 carpropamid 7.160 7.070 7.018 6.766 tinuvin 6.450 5.810 6.990 6.538 water 6.094 7.274 6.684 7.478 UVB acetic acid 10.412 8.698 9.302 10.204 carpropamid 9.234 9.610 9.236 9.082 tinuvin 9.030 10.158 11.154 8.926 water 10.704 9.222 9.796 9.980 Vis light acetic acid 5.786 5.716 5.678 6.204 carpropamid 5.532 4.904 5.144 4.456 tinuvin 5.342 6.046 5.584 5.318 water 4.896 5.074 5.988 6.200 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 0.2037 0.1676 0.0982 0.4097 d.f. 20 72 286 91.52 Except when comparing means with the same level(s) of exposure 0.4105 d.f. 72 404 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 0.2914 0.2388 0.5843 d.f. 77.79 234.23 282.17 Except when comparing means with the same level(s) of exposure 0.2406 0.5849 d.f. 286 234.23 treatment 0.1964 d.f. 286 exposure.treatment 0.4811 d.f. 286 exposure.Chem_Charge 0.5849 d.f. 234.23 (Not adjusted for missing values) Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.2880 0.2370 0.1389 0.5794 d.f. 20 72 286 91.52 Except when comparing means with the same level(s) of exposure 0.5805 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.4120 0.3377 0.8264 d.f. 77.79 234.23 282.17 Except when comparing means with the same level(s) of exposure 0.3402 0.8272 d.f. 286 234.23 treatment 0.2778 d.f. 286 exposure.treatment 0.6804 d.f. 286 exposure.Chem_Charge 0.8272 d.f. 234.23 (Not adjusted for missing values) 405 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 0.6008 0.4724 0.2734 1.1508 d.f. 20 72 286 91.52 Except when comparing means with the same level(s) of exposure 1.1572 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 0.8203 0.6653 1.6267 d.f. 77.79 234.23 282.17 Except when comparing means with the same level(s) of exposure 0.6696 1.6296 d.f. 286 234.23 treatment 0.5467 d.f. 286 exposure.treatment 1.3393 d.f. 286 exposure.Chem_Charge 1.6296 d.f. 234.23 (Not adjusted for missing values) Analysis of variance Variate: b Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 88.173 22.043 1.38 block.board stratum exposure 5 8606.887 1721.377 107.47 <.001 Residual 20 320.337 16.017 2.38 block.board.area stratum treatment 3 4.003 1.334 0.20 0.897 exposure.treatment 15 110.708 7.381 1.10 0.376 Residual 72 485.073 6.737 3.08 406 block.board.area.Strip stratum Chem_Charge 3 9.754 3.251 1.48 0.219 exposure.Chem_Charge 15 33.599 2.240 1.02 0.432 treatment.Chem_Charge 9 24.747 2.750 1.26 0.261 exposure.treatment.Chem_Charge 45 106.004 2.356 1.08 0.353 Residual 285 (3) 624.300 2.191 Total 476 (3) 10288.922 Message: the following units have large residuals. block 1 board 1 -1.733 s.e. 0.817 block 1 board 6 1.763 s.e. 0.817 block 4 board 6 -1.725 s.e. 0.817 block 1 board 1 area 4 Strip 4 3.445 s.e. 1.140 block 1 board 5 area 3 Strip 2 4.988 s.e. 1.140 block 1 board 5 area 3 Strip 3 -4.762 s.e. 1.140 block 1 board 6 area 1 Strip 3 4.193 s.e. 1.140 block 1 board 6 area 4 Strip 1 3.874 s.e. 1.140 block 2 board 6 area 1 Strip 1 3.728 s.e. 1.140 Tables of means Variate: b Grand mean 28.663 exposure full I.R. none UVA UVB Vis light 32.811 24.776 24.569 28.865 35.572 25.386 treatment acetic acid carpropamid tinuvin water 28.693 28.508 28.742 28.710 Chem_Charge high low medium Very high 28.874 28.655 28.653 28.471 exposure treatment acetic acid carpropamid tinuvin water full 31.830 33.419 33.124 32.872 I.R. 24.981 24.163 24.861 25.097 none 24.460 25.154 24.503 24.159 UVA 29.381 29.138 28.088 28.852 UVB 35.717 34.845 36.020 35.705 Vis light 25.787 24.329 25.857 25.571 407 exposure Chem_Charge high low medium Very high full 32.938 32.806 32.904 32.595 I.R. 25.260 24.666 24.469 24.708 none 24.652 25.013 24.247 24.364 UVA 28.641 28.959 28.828 29.031 UVB 36.341 35.159 35.632 35.154 Vis light 25.409 25.327 25.833 24.975 treatment Chem_Charge high low medium Very high acetic acid 29.132 28.593 28.403 28.643 carpropamid 28.861 28.818 28.514 27.839 tinuvin 28.636 28.626 29.069 28.639 water 28.866 28.584 28.624 28.764 exposure treatment Chem_Charge high low medium Very high full acetic acid 31.946 31.934 31.692 31.748 carpropamid 33.890 33.098 33.724 32.964 tinuvin 33.278 32.896 33.412 32.910 water 32.640 33.298 32.790 32.760 I.R. acetic acid 26.176 23.766 25.466 24.518 carpropamid 24.432 25.942 22.512 23.766 tinuvin 24.668 24.434 25.114 25.230 water 25.766 24.522 24.784 25.318 none acetic acid 23.754 25.508 23.724 24.854 carpropamid 24.684 26.052 25.134 24.746 tinuvin 25.024 24.200 24.430 24.360 water 25.146 24.294 23.700 23.496 UVA acetic acid 29.596 29.398 29.098 29.432 carpropamid 29.280 29.226 29.328 28.720 tinuvin 27.524 27.542 28.826 28.462 water 28.166 29.670 28.062 29.512 UVB acetic acid 37.116 35.388 34.500 35.864 carpropamid 35.874 34.326 35.398 33.782 tinuvin 36.022 36.248 36.398 35.412 water 36.352 34.676 36.234 35.558 Vis light acetic acid 26.204 25.564 25.940 25.442 carpropamid 25.008 24.262 24.990 23.056 tinuvin 25.298 26.436 26.232 25.462 water 25.126 25.046 26.172 25.942 408 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 0.4474 0.2369 0.1351 0.6729 d.f. 20 72 285 70.94 Except when comparing means with the same level(s) of exposure 0.5804 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 0.5314 0.3330 0.8840 d.f. 39.31 226.52 186.77 Except when comparing means with the same level(s) of exposure 0.3309 0.8157 d.f. 285 226.52 treatment 0.2702 d.f. 285 exposure.treatment 0.6619 d.f. 285 exposure.Chem_Charge 0.8157 d.f. 226.52 (Not adjusted for missing values) 409 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.6328 0.3351 0.1911 0.9517 d.f. 20 72 285 70.94 Except when comparing means with the same level(s) of exposure 0.8208 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.7515 0.4710 1.2501 d.f. 39.31 226.52 186.77 Except when comparing means with the same level(s) of exposure 0.4680 1.1536 d.f. 285 226.52 treatment 0.3821 d.f. 285 exposure.treatment 0.9361 d.f. 285 exposure.Chem_Charge 1.1536 d.f. 226.52 (Not adjusted for missing values) Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.3200 0.6680 0.3761 1.8976 d.f. 20 72 285 70.94 Except when comparing means with the same level(s) of exposure 1.6362 d.f. 72 410 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.5196 0.9280 2.4662 d.f. 39.31 226.52 186.77 Except when comparing means with the same level(s) of exposure 0.9212 2.2732 d.f. 285 226.52 treatment 0.7522 d.f. 285 exposure.treatment 1.8425 d.f. 285 exposure.Chem_Charge 2.2732 d.f. 226.52 (Not adjusted for missing values) Analysis of variance week 4 Variate: L4 Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 206.440 51.610 1.86 block.board stratum exposure 5 4924.346 984.869 35.55 <.001 Residual 20 554.127 27.706 1.32 block.board.area stratum treatment 3 70.698 23.566 1.12 0.345 exposure.treatment 15 187.691 12.513 0.60 0.868 Residual 72 1508.248 20.948 2.86 block.board.area.strip stratum Chem_Charge 3 22.726 7.575 1.03 0.378 exposure.Chem_Charge 15 93.926 6.262 0.85 0.617 treatment.Chem_Charge 9 66.939 7.438 1.01 0.428 exposure.treatment.Chem_Charge 45 336.982 7.488 1.02 0.441 Residual 288 2111.840 7.333 Total 479 10083.961 411 Message: the following units have large residuals. block 2 board 5 2.48 s.e. 1.07 block 5 board 1 area 4 4.84 s.e. 1.77 block 1 board 4 area 4 strip 2 -6.74 s.e. 2.10 block 1 board 5 area 2 strip 3 -6.24 s.e. 2.10 block 5 board 3 area 1 strip 2 6.44 s.e. 2.10 Tables of means Variate: L4 Grand mean 72.37 exposure full I.R. none UVA UVB Vis. light 67.07 75.15 76.37 72.60 69.49 73.53 treatment acetic acid carpropamid tinuvin water 71.79 72.65 72.77 72.25 Chem_Charge high low medium very high 72.71 72.14 72.23 72.39 exposure treatment acetic acid carpropamid tinuvin water full 66.42 66.69 68.26 66.89 I.R. 74.63 75.93 74.70 75.33 none 75.89 76.46 76.35 76.77 UVA 71.85 72.46 73.81 72.30 UVB 70.22 69.73 68.80 69.20 Vis. light 71.75 74.66 74.71 73.00 exposure Chem_Charge high low medium very high full 67.29 67.04 66.31 67.63 I.R. 75.56 74.74 75.46 74.84 none 75.80 75.63 77.18 76.87 UVA 73.44 72.47 72.53 71.97 UVB 70.05 69.37 69.20 69.33 Vis. light 74.13 73.56 72.73 73.70 treatment Chem_Charge high low medium very high acetic acid 72.04 71.87 72.10 71.16 carpropamid 73.02 71.96 72.63 73.01 tinuvin 73.28 72.64 71.81 73.35 water 72.50 72.06 72.39 72.04 412 exposure treatment Chem_Charge high low medium very high full acetic acid 67.02 65.66 66.02 66.98 carpropamid 67.72 66.16 66.25 66.65 tinuvin 66.92 68.29 67.54 70.28 water 67.48 68.05 65.44 66.59 I.R. acetic acid 75.06 74.54 75.87 73.05 carpropamid 75.01 75.92 76.07 76.71 tinuvin 75.18 73.42 74.51 75.70 water 76.98 75.08 75.37 73.90 none acetic acid 75.42 74.89 77.71 75.54 carpropamid 76.44 75.95 76.12 77.33 tinuvin 75.59 75.61 77.30 76.89 water 75.74 76.04 77.58 77.74 UVA acetic acid 72.28 71.24 73.11 70.75 carpropamid 71.86 71.99 73.06 72.93 tinuvin 75.37 74.72 72.32 72.82 water 74.25 71.94 71.61 71.38 UVB acetic acid 69.59 72.76 70.32 68.20 carpropamid 71.27 67.73 70.04 69.86 tinuvin 71.43 68.02 66.15 69.58 water 67.89 68.97 70.29 69.66 Vis. light acetic acid 72.84 72.14 69.59 72.42 carpropamid 75.80 74.03 74.22 74.59 tinuvin 75.17 75.78 73.07 74.81 water 72.69 72.30 74.04 72.98 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 0.588 0.418 0.247 1.064 d.f. 20 72 288 87.94 Except when comparing means with the same level(s) of exposure 1.023 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 0.788 0.598 1.494 d.f. 61.67 237.22 265.39 Except when comparing means with the same level(s) of exposure 0.606 1.465 d.f. 288 237.22 treatment 0.494 d.f. 288 exposure.treatment 1.211 d.f. 288 exposure.Chem_Charge 1.465 d.f. 237.22 413 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.832 0.591 0.350 1.505 d.f. 20 72 288 87.94 Except when comparing means with the same level(s) of exposure 1.447 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.115 0.846 2.113 d.f. 61.67 237.22 265.39 Except when comparing means with the same level(s) of exposure 0.856 2.072 d.f. 288 237.22 treatment 0.699 d.f. 288 exposure.treatment 1.713 d.f. 288 exposure.Chem_Charge 2.072 d.f. 237.22 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.736 1.178 0.688 2.990 d.f. 20 72 288 87.94 Except when comparing means with the same level(s) of exposure 2.885 d.f. 72 414 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 2.229 1.667 4.160 d.f. 61.67 237.22 265.39 Except when comparing means with the same level(s) of exposure 1.685 4.083 d.f. 288 237.22 treatment 1.376 d.f. 288 exposure.treatment 3.371 d.f. 288 exposure.Chem_Charge 4.083 d.f. 237.22 Analysis of variance Variate: a_4 Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 52.178 13.044 3.01 block.board stratum exposure 5 1903.812 380.762 87.97 <.001 Residual 20 86.571 4.329 1.20 block.board.area stratum treatment 3 8.538 2.846 0.79 0.503 exposure.treatment 15 51.071 3.405 0.95 0.518 Residual 72 258.856 3.595 2.74 block.board.area.strip stratum Chem_Charge 3 5.731 1.910 1.46 0.227 exposure.Chem_Charge 15 22.106 1.474 1.12 0.334 treatment.Chem_Charge 9 21.041 2.338 1.78 0.071 exposure.treatment.Chem_Charge 45 78.753 1.750 1.33 0.085 Residual 287 (1) 376.406 1.312 Total 478 (1) 2840.669 415 Message: the following units have large residuals. block 3 board 1 1.326 s.e. 0.425 block 1 board 3 area 1 1.851 s.e. 0.734 block 3 board 4 area 2 -1.854 s.e. 0.734 block 1 board 4 area 3 strip 3 -2.838 s.e. 0.886 block 3 board 2 area 4 strip 4 2.725 s.e. 0.886 block 5 board 6 area 2 strip 3 2.954 s.e. 0.886 Tables of means Variate: a_4 Grand mean 7.585 exposure full I.R. none UVA UVB Vis. light 10.295 5.835 5.316 7.440 10.205 6.420 treatment acetic acid carpropamid tinuvin water 7.759 7.440 7.472 7.670 Chem_Charge high low medium very high 7.575 7.752 7.568 7.445 exposure treatment acetic acid carpropamid tinuvin water full 9.948 10.617 9.881 10.733 I.R. 6.118 5.316 6.010 5.894 none 5.522 5.492 5.246 5.005 UVA 7.737 7.681 6.859 7.484 UVB 10.080 9.888 10.449 10.402 Vis. light 7.148 5.647 6.385 6.498 exposure Chem_Charge high low medium very high full 10.287 10.451 10.546 9.896 I.R. 5.706 5.957 5.763 5.912 none 5.630 5.641 4.898 5.097 UVA 7.050 7.523 7.460 7.728 UVB 10.242 10.143 10.310 10.124 Vis. light 6.537 6.796 6.430 5.915 treatment Chem_Charge high low medium very high acetic acid 7.888 7.760 7.525 7.862 carpropamid 7.454 7.914 7.320 7.073 tinuvin 7.290 7.585 7.922 7.089 water 7.669 7.748 7.504 7.757 416 exposure treatment Chem_Charge high low medium very high full acetic acid 9.702 10.290 10.198 9.602 carpropamid 10.692 11.156 10.752 9.870 tinuvin 10.352 9.538 10.222 9.414 water 10.402 10.820 11.012 10.698 I.R. acetic acid 6.316 6.064 5.492 6.600 carpropamid 5.636 5.414 5.426 4.790 tinuvin 5.554 6.806 5.962 5.718 water 5.318 5.546 6.172 6.540 none acetic acid 5.690 6.252 4.658 5.488 carpropamid 5.338 5.880 5.450 5.298 tinuvin 5.682 5.382 4.942 4.980 water 5.808 5.050 4.542 4.622 UVA acetic acid 7.438 7.848 7.190 8.472 carpropamid 7.814 7.958 7.628 7.324 tinuvin 6.408 6.256 7.692 7.080 water 6.540 8.030 7.332 8.036 UVB acetic acid 10.858 9.110 9.540 10.812 carpropamid 9.618 10.122 10.078 9.736 tinuvin 9.294 11.162 11.724 9.616 water 11.198 10.178 9.900 10.334 Vis. light acetic acid 7.324 6.998 8.074 6.198 carpropamid 5.624 6.956 4.588 5.420 tinuvin 6.452 6.368 6.992 5.728 water 6.750 6.864 6.066 6.314 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 0.2326 0.1731 0.1045 0.4347 d.f. 20 72 287 89.50 Except when comparing means with the same level(s) of exposure 0.4240 d.f. 72 417 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 0.3214 0.2505 0.6210 d.f. 68.92 242.86 278.69 Except when comparing means with the same level(s) of exposure 0.2561 0.6136 d.f. 287 242.86 treatment 0.2091 d.f. 287 exposure.treatment 0.5122 d.f. 287 exposure.Chem_Charge 0.6136 d.f. 242.86 (Not adjusted for missing values) Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.3290 0.2448 0.1478 0.6147 d.f. 20 72 287 89.50 Except when comparing means with the same level(s) of exposure 0.5996 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.4545 0.3543 0.8782 d.f. 68.92 242.86 278.69 Except when comparing means with the same level(s) of exposure 0.3621 0.8677 d.f. 287 242.86 treatment 0.2957 d.f. 287 exposure.treatment 0.7243 d.f. 287 exposure.Chem_Charge 0.8677 d.f. 242.86 (Not adjusted for missing values) 418 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 0.6862 0.4880 0.2910 1.2213 d.f. 20 72 287 89.50 Except when comparing means with the same level(s) of exposure 1.1953 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 0.9067 0.6978 1.7288 d.f. 68.92 242.86 278.69 Except when comparing means with the same level(s) of exposure 0.7128 1.7093 d.f. 287 242.86 treatment 0.5820 d.f. 287 exposure.treatment 1.4256 d.f. 287 exposure.Chem_Charge 1.7093 d.f. 242.86 (Not adjusted for missing values) Analysis of variance Variate: b_4 Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 140.897 35.224 1.67 block.board stratum exposure 5 7529.344 1505.869 71.50 <.001 Residual 20 421.207 21.060 3.11 block.board.area stratum treatment 3 22.932 7.644 1.13 0.343 exposure.treatment 15 121.155 8.077 1.19 0.297 Residual 72 487.453 6.770 2.72 419 block.board.area.strip stratum Chem_Charge 3 27.929 9.310 3.74 0.012 exposure.Chem_Charge 15 51.769 3.451 1.39 0.153 treatment.Chem_Charge 9 22.259 2.473 0.99 0.446 exposure.treatment.Chem_Charge 45 127.222 2.827 1.13 0.267 Residual 287 (1) 714.918 2.491 Total 478 (1) 9630.811 Message: the following units have large residuals. block 1 board 1 -1.983 s.e. 0.937 block 3 board 1 2.038 s.e. 0.937 block 2 board 1 area 2 2.700 s.e. 1.008 block 4 board 1 area 2 -2.647 s.e. 1.008 block 1 board 4 area 3 strip 1 4.267 s.e. 1.220 block 3 board 2 area 4 strip 4 3.827 s.e. 1.220 block 4 board 4 area 3 strip 4 3.710 s.e. 1.220 block 5 board 2 area 1 strip 1 -3.701 s.e. 1.220 Tables of means Variate: b_4 Grand mean 29.156 exposure full I.R. none UVA UVB Vis. light 32.869 26.467 24.735 29.888 35.461 25.514 treatment acetic acid carpropamid tinuvin water 28.993 28.897 29.432 29.302 Chem_Charge high low medium very high 29.369 29.384 29.072 28.798 exposure treatment acetic acid carpropamid tinuvin water full 31.703 32.990 33.303 33.481 I.R. 26.895 25.362 27.058 26.552 none 24.494 25.356 24.651 24.442 UVA 30.035 30.206 29.202 30.108 UVB 35.168 34.758 36.343 35.576 Vis. light 25.662 24.709 26.033 25.652 420 exposure Chem_Charge high low medium very high full 32.850 33.222 33.117 32.289 I.R. 26.426 26.689 26.561 26.191 none 25.202 24.965 24.288 24.488 UVA 29.548 29.930 29.836 30.236 UVB 36.272 35.237 35.365 34.972 Vis. light 25.918 26.261 25.266 24.611 treatment Chem_Charge high low medium very high acetic acid 29.274 29.275 28.704 28.717 carpropamid 29.086 29.514 28.770 28.216 tinuvin 29.502 29.345 29.789 29.090 water 29.614 29.400 29.025 29.167 exposure treatment Chem_Charge high low medium very high full acetic acid 31.242 32.430 31.808 31.332 carpropamid 33.564 33.786 33.234 31.374 tinuvin 33.644 32.396 33.982 33.190 water 32.948 34.274 33.442 33.260 I.R. acetic acid 27.222 26.906 26.576 26.876 carpropamid 25.738 25.532 25.700 24.478 tinuvin 26.320 28.122 26.892 26.896 water 26.424 26.196 27.074 26.514 none acetic acid 24.728 25.396 23.826 24.024 carpropamid 24.630 26.082 24.860 25.850 tinuvin 25.322 24.262 24.690 24.330 water 26.126 24.118 23.776 23.746 UVA acetic acid 29.746 29.982 29.518 30.892 carpropamid 30.284 30.312 30.696 29.532 tinuvin 28.808 28.548 29.920 29.532 water 29.354 30.878 29.210 30.988 UVB acetic acid 36.664 34.822 33.980 35.206 carpropamid 35.404 34.414 35.216 33.998 tinuvin 36.436 36.594 36.866 35.476 water 36.582 35.116 35.398 35.206 Vis. light acetic acid 26.044 26.112 26.518 23.972 carpropamid 24.896 26.960 22.916 24.064 tinuvin 26.484 26.150 26.382 25.116 water 26.248 25.820 25.250 25.290 421 Standard errors of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 e.s.e. 0.5131 0.2375 0.1441 0.7191 d.f. 20 72 287 61.33 Except when comparing means with the same level(s) of exposure 0.5818 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 e.s.e. 0.5972 0.3445 0.9438 d.f. 36.39 244.07 163.72 Except when comparing means with the same level(s) of exposure 0.3529 0.8439 d.f. 287 244.07 treatment 0.2882 d.f. 287 exposure.treatment 0.7058 d.f. 287 exposure.Chem_Charge 0.8439 d.f. 244.07 (Not adjusted for missing values) Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.7256 0.3359 0.2038 1.0170 d.f. 20 72 287 61.33 Except when comparing means with the same level(s) of exposure 0.8228 d.f. 72 422 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.8446 0.4872 1.3348 d.f. 36.39 244.07 163.72 Except when comparing means with the same level(s) of exposure 0.4991 1.1934 d.f. 287 244.07 treatment 0.4075 d.f. 287 exposure.treatment 0.9982 d.f. 287 exposure.Chem_Charge 1.1934 d.f. 244.07 (Not adjusted for missing values) Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.5136 0.6696 0.4010 2.0334 d.f. 20 72 287 61.33 Except when comparing means with the same level(s) of exposure 1.6402 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.7123 0.9597 2.6356 d.f. 36.39 244.07 163.72 Except when comparing means with the same level(s) of exposure 0.9824 2.3508 d.f. 287 244.07 treatment 0.8021 d.f. 287 exposure.treatment 1.9647 d.f. 287 exposure.Chem_Charge 2.3508 d.f. 244.07 (Not adjusted for missing values) 423 Analysis of variance week 6 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 205.531 51.383 1.62 block.board stratum exposure 5 5110.868 1022.174 32.14 <.001 Residual 20 636.106 31.805 1.50 block.board.area stratum treatment 3 66.188 22.063 1.04 0.381 exposure.treatment 15 172.792 11.519 0.54 0.908 Residual 72 1531.343 21.269 2.82 block.board.area.Strip stratum Chem_Charge 3 21.694 7.231 0.96 0.413 exposure.Chem_Charge 15 113.209 7.547 1.00 0.455 treatment.Chem_Charge 9 94.317 10.480 1.39 0.192 exposure.treatment.Chem_Charge 45 395.825 8.796 1.17 0.228 Residual 288 2172.563 7.544 Total 479 10520.435 Message: the following units have large residuals. block 2 board 4 2.33 s.e. 1.15 block 5 board 1 area 4 4.88 s.e. 1.79 block 5 board 3 area 1 Strip 2 7.77 s.e. 2.13 Tables of means Variate: L Grand mean 71.34 exposure full I.R. none UVA UVB Vis light 66.35 73.42 75.89 70.78 68.23 73.35 treatment acetic acid carpropamid tinuvin water 70.95 71.64 71.77 70.98 Chem_Charge high low medium Very high 71.66 71.09 71.22 71.38 424 exposure treatment acetic acid carpropamid tinuvin water full 65.56 66.03 67.93 65.89 I.R. 72.62 74.39 74.45 72.21 none 75.30 75.91 76.08 76.25 UVA 70.01 70.95 71.79 70.37 UVB 69.00 68.57 67.37 67.98 Vis light 73.23 73.97 73.00 73.21 exposure Chem_Charge high low medium Very high full 67.00 66.23 65.75 66.43 I.R. 73.64 74.13 72.76 73.13 none 75.22 75.16 76.82 76.35 UVA 71.82 70.05 70.33 70.91 UVB 68.66 67.76 67.95 68.55 Vis light 73.59 73.19 73.70 72.93 treatment Chem_Charge high low medium Very high acetic acid 71.05 71.28 70.95 70.53 carpropamid 72.03 70.40 71.65 72.48 tinuvin 72.52 71.74 71.06 71.76 water 71.03 70.92 71.22 70.77 exposure treatment Chem_Charge high low medium Very high full acetic acid 66.26 64.84 64.91 66.22 carpropamid 66.89 64.92 65.59 66.73 tinuvin 67.05 68.45 67.82 68.41 water 67.81 66.71 64.69 64.34 I.R. acetic acid 73.01 74.28 70.51 72.66 carpropamid 75.37 73.89 73.89 74.42 tinuvin 75.54 75.23 72.79 74.22 water 70.64 73.12 73.87 71.23 none acetic acid 74.67 74.40 77.37 74.78 carpropamid 76.37 74.32 75.81 77.16 tinuvin 75.09 76.18 77.21 75.85 water 74.74 75.75 76.88 77.62 UVA acetic acid 69.76 69.62 70.39 70.27 carpropamid 70.70 69.32 71.30 72.49 tinuvin 74.05 71.51 70.59 71.02 water 72.78 69.75 69.06 69.88 UVB acetic acid 68.43 71.17 68.73 67.68 carpropamid 70.54 66.04 68.89 68.82 tinuvin 69.68 66.92 64.80 68.07 water 66.00 66.90 69.39 69.63 Vis light acetic acid 74.18 73.37 73.80 71.56 carpropamid 72.29 73.91 74.41 75.28 tinuvin 73.69 72.17 73.16 72.97 water 74.19 73.33 73.43 71.89 425 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.892 0.595 0.355 1.546 d.f. 20 72 288 85.34 Except when comparing means with the same level(s) of exposure 1.458 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.167 0.855 2.157 d.f. 56.60 239.07 255.55 Except when comparing means with the same level(s) of exposure 0.869 2.095 d.f. 288 239.07 treatment 0.709 d.f. 288 exposure.treatment 1.737 d.f. 288 exposure.Chem_Charge 2.095 d.f. 239.07 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.860 1.187 0.698 3.074 d.f. 20 72 288 85.34 Except when comparing means with the same level(s) of exposure 2.907 d.f. 72 426 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 2.336 1.685 4.248 d.f. 56.60 239.07 255.55 Except when comparing means with the same level(s) of exposure 1.709 4.127 d.f. 288 239.07 treatment 1.396 d.f. 288 exposure.treatment 3.419 d.f. 288 exposure.Chem_Charge 4.127 d.f. 239.07 Analysis of variance Variate: a Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 61.548 15.387 2.88 block.board stratum exposure 5 1848.915 369.783 69.15 <.001 Residual 20 106.952 5.348 1.63 block.board.area stratum treatment 3 12.257 4.086 1.25 0.298 exposure.treatment 15 35.428 2.362 0.72 0.755 Residual 72 235.552 3.272 2.29 block.board.area.Strip stratum Chem_Charge 3 5.929 1.976 1.38 0.249 exposure.Chem_Charge 15 18.526 1.235 0.86 0.606 treatment.Chem_Charge 9 31.965 3.552 2.48 0.010 exposure.treatment.Chem_Charge 45 99.745 2.217 1.55 0.019 Residual 288 412.130 1.431 Total 479 2868.948 427 Message: the following units have large residuals. block 3 board 1 1.320 s.e. 0.472 block 3 board 5 area 1 2.029 s.e. 0.701 block 1 board 5 area 4 Strip 2 3.904 s.e. 0.927 block 5 board 3 area 1 Strip 1 3.037 s.e. 0.927 block 5 board 6 area 2 Strip 3 3.151 s.e. 0.927 Tables of means Variate: a Grand mean 8.005 exposure full I.R. none UVA UVB Vis light 10.082 6.437 5.492 8.423 10.871 6.726 treatment acetic acid carpropamid tinuvin water 8.109 7.850 7.849 8.212 Chem_Charge high low medium Very high 7.913 8.129 8.101 7.878 exposure treatment acetic acid carpropamid tinuvin water full 9.921 10.322 9.554 10.532 I.R. 6.909 5.890 6.087 6.862 none 5.639 5.650 5.253 5.428 UVA 8.562 8.508 7.997 8.623 UVB 10.749 10.490 11.218 11.026 Vis light 6.876 6.239 6.987 6.801 exposure Chem_Charge high low medium Very high full 9.825 10.213 10.311 9.980 I.R. 6.499 6.380 6.818 6.052 none 5.736 5.757 5.299 5.177 UVA 7.980 8.716 8.622 8.373 UVB 10.859 10.943 10.999 10.682 Vis light 6.577 6.769 6.556 7.001 treatment Chem_Charge high low medium Very high acetic acid 8.142 7.883 8.268 8.144 carpropamid 7.736 8.530 7.872 7.261 tinuvin 7.441 7.995 8.184 7.777 water 8.332 8.109 8.079 8.328 428 exposure treatment Chem_Charge high low medium Very high full acetic acid 9.594 9.940 10.780 9.370 carpropamid 10.128 11.114 10.302 9.742 tinuvin 9.786 9.292 9.506 9.634 water 9.792 10.504 10.658 11.176 I.R. acetic acid 7.072 6.276 8.264 6.024 carpropamid 5.538 6.568 6.200 5.252 tinuvin 5.644 6.254 6.690 5.760 water 7.742 6.420 6.116 7.170 none acetic acid 5.610 6.270 4.932 5.742 carpropamid 5.430 6.382 5.620 5.166 tinuvin 5.646 5.286 4.998 5.080 water 6.258 5.088 5.644 4.720 UVA acetic acid 8.408 8.620 8.602 8.620 carpropamid 8.642 9.324 8.544 7.522 tinuvin 7.300 7.786 8.580 8.322 water 7.570 9.134 8.762 9.028 UVB acetic acid 11.298 9.544 10.592 11.560 carpropamid 9.950 11.418 10.356 10.236 tinuvin 9.802 11.840 12.602 10.626 water 12.384 10.968 10.446 10.306 Vis light acetic acid 6.868 6.648 6.440 7.548 carpropamid 6.726 6.374 6.208 5.648 tinuvin 6.468 7.514 6.728 7.240 water 6.248 6.540 6.848 7.568 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.3656 0.2335 0.1544 0.6157 d.f. 20 72 288 83.06 Except when comparing means with the same level(s) of exposure 0.5720 d.f. 72 429 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.4909 0.3551 0.8991 d.f. 62.22 269.10 275.74 Except when comparing means with the same level(s) of exposure 0.3783 0.8697 d.f. 288 269.10 treatment 0.3089 d.f. 288 exposure.treatment 0.7566 d.f. 288 exposure.Chem_Charge 0.8697 d.f. 269.10 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 0.7627 0.4655 0.3040 1.2245 d.f. 20 72 288 83.06 Except when comparing means with the same level(s) of exposure 1.1402 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 0.9813 0.6991 1.7699 d.f. 62.22 269.10 275.74 Except when comparing means with the same level(s) of exposure 0.7446 1.7124 d.f. 288 269.10 treatment 0.6079 d.f. 288 exposure.treatment 1.4891 d.f. 288 exposure.Chem_Charge 1.7124 d.f. 269.10 430 Analysis of variance Variate: b Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 92.598 23.149 0.89 block.board stratum exposure 5 4477.607 895.521 34.44 <.001 Residual 20 520.072 26.004 3.31 block.board.area stratum treatment 3 37.018 12.339 1.57 0.204 exposure.treatment 15 102.226 6.815 0.87 0.602 Residual 72 565.765 7.858 2.49 block.board.area.Strip stratum Chem_Charge 3 21.034 7.011 2.22 0.086 exposure.Chem_Charge 15 32.221 2.148 0.68 0.803 treatment.Chem_Charge 9 88.312 9.812 3.11 0.001 exposure.treatment.Chem_Charge 45 207.015 4.600 1.46 0.037 Residual 288 908.560 3.155 Total 479 7052.427 Message: the following units have large residuals. block 1 board 1 -2.780 s.e. 1.041 block 1 board 6 2.152 s.e. 1.041 block 3 board 1 2.377 s.e. 1.041 block 2 board 1 area 2 2.753 s.e. 1.086 block 5 board 1 area 1 3.711 s.e. 1.086 block 5 board 1 area 4 -3.219 s.e. 1.086 block 1 board 2 area 1 Strip 1 4.369 s.e. 1.376 block 1 board 5 area 4 Strip 2 4.618 s.e. 1.376 block 1 board 6 area 4 Strip 1 4.032 s.e. 1.376 block 3 board 2 area 4 Strip 4 4.714 s.e. 1.376 block 4 board 1 area 1 Strip 3 -4.475 s.e. 1.376 block 5 board 1 area 3 Strip 4 4.247 s.e. 1.376 block 5 board 6 area 2 Strip 3 4.075 s.e. 1.376 431 Tables of means Variate: b Grand mean 28.707 exposure full I.R. none UVA UVB Vis light 29.410 25.647 25.091 30.448 34.067 27.581 treatment acetic acid carpropamid tinuvin water 28.497 28.370 28.993 28.970 Chem_Charge high low medium Very high 28.793 28.869 28.819 28.348 exposure treatment acetic acid carpropamid tinuvin water full 28.829 29.164 29.503 30.146 I.R. 25.936 25.139 25.343 26.171 none 24.707 25.612 24.809 25.236 UVA 29.972 30.441 30.470 30.910 UVB 33.716 33.169 35.422 33.964 Vis light 27.824 26.696 28.413 27.392 exposure Chem_Charge high low medium Very high full 29.044 29.657 29.853 29.088 I.R. 25.873 25.921 25.813 24.981 none 25.561 25.158 25.120 24.524 UVA 30.021 30.769 30.567 30.436 UVB 34.728 33.895 33.962 33.685 Vis light 27.534 27.815 27.602 27.374 treatment Chem_Charge high low medium Very high acetic acid 28.865 28.289 28.749 28.085 carpropamid 28.297 29.519 28.458 27.205 tinuvin 28.599 28.947 29.261 29.165 water 29.412 28.720 28.809 28.937 432 exposure treatment Chem_Charge high low medium Very high full acetic acid 28.418 28.964 29.962 27.972 carpropamid 28.928 30.910 29.862 26.954 tinuvin 30.222 28.180 29.314 30.294 water 28.606 30.572 30.274 31.132 I.R. acetic acid 26.250 26.022 27.190 24.280 carpropamid 24.782 26.608 25.164 24.002 tinuvin 24.798 25.844 25.660 25.068 water 27.662 25.210 25.238 26.574 none acetic acid 25.052 24.982 24.562 24.230 carpropamid 25.210 26.682 25.304 25.250 tinuvin 25.198 24.698 24.810 24.528 water 26.784 24.268 25.802 24.088 UVA acetic acid 29.710 29.860 30.274 30.044 carpropamid 30.558 31.582 30.598 29.024 tinuvin 29.828 29.954 31.038 31.060 water 29.988 31.680 30.356 31.614 UVB acetic acid 35.554 32.148 32.682 34.478 carpropamid 33.214 34.266 32.910 32.284 tinuvin 33.752 35.976 36.726 35.232 water 36.390 33.190 33.530 32.746 Vis light acetic acid 28.206 27.760 27.824 27.504 carpropamid 27.090 27.066 26.910 25.718 tinuvin 27.796 29.032 28.016 28.806 water 27.042 27.402 27.656 27.466 433 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.8063 0.3619 0.2293 1.1133 d.f. 20 72 288 59.19 Except when comparing means with the same level(s) of exposure 0.8864 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.9416 0.5373 1.4785 d.f. 36.87 256.76 164.39 Except when comparing means with the same level(s) of exposure 0.5617 1.3161 d.f. 288 256.76 treatment 0.4586 d.f. 288 exposure.treatment 1.1233 d.f. 288 exposure.Chem_Charge 1.3161 d.f. 256.76 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.6819 0.7214 0.4513 2.2276 d.f. 20 72 288 59.19 Except when comparing means with the same level(s) of exposure 1.7671 d.f. 72 434 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.9082 1.0581 2.9192 d.f. 36.87 256.76 164.39 Except when comparing means with the same level(s) of exposure 1.1055 2.5918 d.f. 288 256.76 treatment 0.9026 d.f. 288 exposure.treatment 2.2110 d.f. 288 exposure.Chem_Charge 2.5918 d.f. 256.76 Analysis of variance week 8 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 604.751 151.188 2.96 block.board stratum exposure 5 6238.411 1247.682 24.45 <.001 Residual 20 1020.494 51.025 1.99 block.board.area stratum treatment 3 39.023 13.008 0.51 0.679 exposure.treatment 15 155.713 10.381 0.40 0.974 Residual 72 1850.658 25.704 3.09 block.board.area.Strip stratum Chem_Charge 3 4.310 1.437 0.17 0.915 exposure.Chem_Charge 15 173.374 11.558 1.39 0.152 treatment.Chem_Charge 9 41.208 4.579 0.55 0.837 exposure.treatment.Chem_Charge 45 459.497 10.211 1.23 0.165 Residual 288 2398.378 8.328 Total 479 12985.816 435 Message: the following units have large residuals. block 2 board 1 3.30 s.e. 1.46 block 1 board 1 area 3 -5.32 s.e. 1.96 block 1 board 1 area 4 5.15 s.e. 1.96 block 4 board 2 area 4 Strip 1 -7.91 s.e. 2.24 Tables of means Variate: L Grand mean 69.97 exposure full I.R. none UVA UVB Vis light 64.22 72.75 75.35 69.18 67.38 70.96 treatment acetic acid carpropamid tinuvin water 69.50 70.23 70.17 70.00 Chem_Charge high low medium Very high 70.00 70.11 69.85 69.93 exposure treatment acetic acid carpropamid tinuvin water full 63.12 63.59 64.83 65.35 I.R. 72.18 73.31 73.64 71.85 none 74.72 75.59 75.54 75.55 UVA 68.89 69.12 70.02 68.70 UVB 67.47 68.38 66.22 67.47 Vis light 70.63 71.37 70.74 71.08 exposure Chem_Charge high low medium Very high full 63.60 65.01 64.08 64.19 I.R. 73.08 73.95 72.14 71.81 none 74.93 74.51 76.07 75.88 UVA 70.09 69.15 68.75 68.75 UVB 66.98 68.07 66.95 67.54 Vis light 71.29 69.98 71.14 71.42 treatment Chem_Charge high low medium Very high acetic acid 69.21 69.78 69.80 69.21 carpropamid 70.31 70.05 69.81 70.73 tinuvin 70.34 70.34 69.67 70.31 water 70.12 70.27 70.13 69.47 436 exposure treatment Chem_Charge high low medium Very high full acetic acid 63.51 62.24 63.84 62.89 carpropamid 63.39 64.68 61.96 64.31 tinuvin 61.92 65.90 66.19 65.30 water 65.60 67.24 64.32 64.24 I.R. acetic acid 72.08 73.82 71.46 71.36 carpropamid 73.87 73.53 73.02 72.81 tinuvin 74.65 74.85 71.55 73.53 water 71.71 73.61 72.53 69.55 none acetic acid 74.98 72.71 76.45 74.73 carpropamid 75.81 74.97 74.92 76.65 tinuvin 74.12 75.68 76.92 75.45 water 74.82 74.68 76.00 76.68 UVA acetic acid 68.73 68.67 70.06 68.07 carpropamid 68.75 68.63 69.09 70.03 tinuvin 72.12 70.94 67.90 69.11 water 70.76 68.34 67.93 67.77 UVB acetic acid 64.59 70.94 66.26 68.08 carpropamid 69.85 67.05 68.33 68.29 tinuvin 67.91 65.98 63.98 67.03 water 65.56 68.31 69.24 66.76 Vis light acetic acid 71.36 70.32 70.76 70.10 carpropamid 70.21 71.47 71.52 72.28 tinuvin 71.31 68.68 71.50 71.47 water 72.28 69.45 70.77 71.83 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.129 0.655 0.373 1.790 d.f. 20 72 288 77.17 Except when comparing means with the same level(s) of exposure 1.603 d.f. 72 437 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.378 0.919 2.388 d.f. 43.65 226.49 210.22 Except when comparing means with the same level(s) of exposure 0.913 2.251 d.f. 288 226.49 treatment 0.745 d.f. 288 exposure.treatment 1.825 d.f. 288 exposure.Chem_Charge 2.251 d.f. 226.49 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 2.356 1.305 0.733 3.564 d.f. 20 72 288 77.17 Except when comparing means with the same level(s) of exposure 3.196 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 2.779 1.811 4.707 d.f. 43.65 226.49 210.22 Except when comparing means with the same level(s) of exposure 1.796 4.436 d.f. 288 226.49 treatment 1.467 d.f. 288 exposure.treatment 3.592 d.f. 288 exposure.Chem_Charge 4.436 d.f. 226.49 438 Analysis of variance Variate: a Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 65.085 16.271 1.21 block.board stratum exposure 5 1517.003 303.401 22.59 <.001 Residual 20 268.570 13.429 2.50 block.board.area stratum treatment 3 4.462 1.487 0.28 0.842 exposure.treatment 15 86.348 5.757 1.07 0.396 Residual 72 386.064 5.362 2.94 block.board.area.Strip stratum Chem_Charge 3 2.106 0.702 0.39 0.764 exposure.Chem_Charge 15 22.437 1.496 0.82 0.654 treatment.Chem_Charge 9 19.403 2.156 1.18 0.305 exposure.treatment.Chem_Charge 45 139.945 3.110 1.71 0.005 Residual 286 (2) 520.835 1.821 Total 477 (2) 2994.412 Message: the following units have large residuals. block 1 board 1 -1.892 s.e. 0.748 block 3 board 1 1.908 s.e. 0.748 block 3 board 2 -1.782 s.e. 0.748 block 4 board 1 area 3 2.424 s.e. 0.897 block 5 board 1 area 1 3.107 s.e. 0.897 block 5 board 1 area 4 -2.374 s.e. 0.897 block 1 board 1 area 3 Strip 1 4.114 s.e. 1.042 block 2 board 5 area 2 Strip 2 -3.450 s.e. 1.042 block 3 board 2 area 4 Strip 4 4.043 s.e. 1.042 439 Tables of means Variate: a Grand mean 8.186 exposure full I.R. none UVA UVB Vis light 8.129 6.680 5.665 9.453 11.138 8.053 treatment acetic acid carpropamid tinuvin water 8.347 8.115 8.109 8.174 Chem_Charge high low medium Very high 8.243 8.260 8.136 8.107 exposure treatment acetic acid carpropamid tinuvin water full 7.921 9.406 7.669 7.520 I.R. 7.213 6.104 6.567 6.838 none 6.015 5.664 5.391 5.588 UVA 9.484 9.540 9.043 9.743 UVB 11.195 10.430 11.642 11.283 Vis light 8.256 7.543 8.342 8.072 exposure Chem_Charge high low medium Very high full 8.329 8.243 7.768 8.176 I.R. 6.911 6.451 6.679 6.680 none 5.782 6.069 5.499 5.309 UVA 9.058 9.494 9.683 9.575 UVB 11.427 10.911 11.045 11.167 Vis light 7.951 8.389 8.141 7.731 treatment Chem_Charge high low medium Very high acetic acid 8.374 8.441 8.179 8.396 carpropamid 8.362 8.336 8.137 7.623 tinuvin 7.978 8.107 8.396 7.957 water 8.260 8.155 7.831 8.451 440 exposure treatment Chem_Charge high low medium Very high full acetic acid 7.036 9.260 7.744 7.644 carpropamid 10.128 9.160 10.148 8.190 tinuvin 8.546 6.658 7.116 8.358 water 7.608 7.894 6.065 8.514 I.R. acetic acid 7.716 6.436 7.834 6.866 carpropamid 6.496 6.762 5.187 5.970 tinuvin 6.294 6.522 7.052 6.400 water 7.140 6.086 6.642 7.484 none acetic acid 5.690 7.266 5.378 5.726 carpropamid 5.490 6.164 5.694 5.308 tinuvin 5.878 5.352 5.190 5.146 water 6.070 5.492 5.732 5.056 UVA acetic acid 9.390 9.526 9.154 9.868 carpropamid 9.910 9.858 9.628 8.766 tinuvin 8.366 8.390 10.104 9.312 water 8.568 10.204 9.846 10.356 UVB acetic acid 12.092 9.950 10.826 11.912 carpropamid 10.192 10.476 10.362 10.690 tinuvin 10.938 12.230 12.868 10.532 water 12.488 10.988 10.124 11.534 Vis light acetic acid 8.320 8.206 8.140 8.358 carpropamid 7.956 7.596 7.806 6.814 tinuvin 7.844 9.488 8.044 7.994 water 7.686 8.268 8.576 7.760 441 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.5794 0.2989 0.1742 0.8590 d.f. 20 72 286 69.08 Except when comparing means with the same level(s) of exposure 0.7323 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.6872 0.4248 1.1332 d.f. 39.13 232.66 184.78 Except when comparing means with the same level(s) of exposure 0.4267 1.0404 d.f. 286 232.66 treatment 0.3484 d.f. 286 exposure.treatment 0.8535 d.f. 286 exposure.Chem_Charge 1.0404 d.f. 232.66 (Not adjusted for missing values) Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.2086 0.5959 0.3429 1.7136 d.f. 20 72 286 69.08 Except when comparing means with the same level(s) of exposure 1.4597 d.f. 72 442 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.3899 0.8369 2.2357 d.f. 39.13 232.66 184.78 Except when comparing means with the same level(s) of exposure 0.8400 2.0499 d.f. 286 232.66 treatment 0.6858 d.f. 286 exposure.treatment 1.6799 d.f. 286 exposure.Chem_Charge 2.0499 d.f. 232.66 (Not adjusted for missing values) Analysis of variance Variate: b Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 88.335 22.084 0.34 block.board stratum exposure 5 3886.552 777.310 12.10 <.001 Residual 20 1284.675 64.234 5.30 block.board.area stratum treatment 3 19.512 6.504 0.54 0.659 exposure.treatment 15 197.737 13.182 1.09 0.383 Residual 72 872.297 12.115 2.57 block.board.area.Strip stratum Chem_Charge 3 7.013 2.338 0.50 0.685 exposure.Chem_Charge 15 39.080 2.605 0.55 0.908 treatment.Chem_Charge 9 27.982 3.109 0.66 0.744 exposure.treatment.Chem_Charge 45 291.794 6.484 1.38 0.064 Residual 288 1355.383 4.706 Total 479 8070.359 443 Message: the following units have large residuals. block 1 board 1 -4.031 s.e. 1.636 block 4 board 1 3.873 s.e. 1.636 block 2 board 1 area 2 3.524 s.e. 1.348 block 5 board 1 area 1 5.106 s.e. 1.348 block 5 board 1 area 4 -3.713 s.e. 1.348 block 1 board 1 area 3 Strip 1 6.881 s.e. 1.680 block 1 board 5 area 4 Strip 2 7.062 s.e. 1.680 block 2 board 2 area 1 Strip 1 -4.955 s.e. 1.680 block 2 board 2 area 3 Strip 3 5.296 s.e. 1.680 block 3 board 2 area 4 Strip 4 5.534 s.e. 1.680 block 4 board 1 area 1 Strip 2 8.528 s.e. 1.680 block 4 board 1 area 1 Strip 3 -5.571 s.e. 1.680 block 5 board 1 area 1 Strip 2 -5.306 s.e. 1.680 Tables of means Variate: b Grand mean 27.395 exposure full I.R. none UVA UVB Vis light 23.512 25.941 25.160 30.092 31.736 27.929 treatment acetic acid carpropamid tinuvin water 27.263 27.320 27.742 27.255 Chem_Charge high low medium Very high 27.544 27.435 27.393 27.209 exposure treatment acetic acid carpropamid tinuvin water full 22.660 25.106 23.509 22.775 I.R. 25.906 25.602 26.331 25.926 none 25.099 25.738 24.704 25.101 UVA 29.732 30.119 29.993 30.522 UVB 31.923 30.431 33.278 31.313 Vis light 28.261 26.926 28.635 27.895 exposure Chem_Charge high low medium Very high full 23.530 23.528 23.339 23.652 I.R. 26.101 25.876 26.316 25.473 none 25.204 25.377 25.105 24.957 UVA 29.847 30.057 30.247 30.216 UVB 32.561 31.532 31.160 31.691 Vis light 28.022 28.239 28.190 27.265 444 treatment Chem_Charge high low medium Very high acetic acid 27.474 27.484 27.090 27.005 carpropamid 27.512 27.618 27.518 26.632 tinuvin 27.707 27.616 27.979 27.664 water 27.483 27.020 26.983 27.534 exposure treatment Chem_Charge high low medium Very high full acetic acid 21.210 25.076 22.170 22.184 carpropamid 26.092 24.730 26.472 23.128 tinuvin 24.760 21.494 22.478 25.302 water 22.056 22.812 22.236 23.994 I.R. acetic acid 26.574 25.384 27.282 24.384 carpropamid 25.282 26.904 25.320 24.902 tinuvin 25.734 26.546 27.278 25.766 water 26.814 24.668 25.384 26.838 none acetic acid 24.796 26.022 24.796 24.782 carpropamid 24.916 26.404 25.028 26.602 tinuvin 25.058 24.588 24.970 24.200 water 26.044 24.494 25.624 24.242 UVA acetic acid 29.722 29.508 29.698 30.000 carpropamid 30.548 30.664 30.198 29.066 tinuvin 29.696 28.982 30.850 30.444 water 29.420 31.072 30.242 31.354 UVB acetic acid 33.766 30.736 30.480 32.708 carpropamid 30.928 29.890 30.372 30.532 tinuvin 32.786 34.450 33.866 32.008 water 32.764 31.052 29.920 31.514 Vis light acetic acid 28.776 28.178 28.116 27.972 carpropamid 27.306 27.118 27.716 25.562 tinuvin 28.208 29.636 28.434 28.262 water 27.798 28.024 28.492 27.264 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.2672 0.4494 0.2801 1.5857 d.f. 20 72 288 45.03 Except when comparing means with the same level(s) of exposure 1.1007 d.f. 72 445 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.3996 0.6612 1.9815 d.f. 29.66 252.03 104.64 Except when comparing means with the same level(s) of exposure 0.6860 1.6197 d.f. 288 252.03 treatment 0.5601 d.f. 288 exposure.treatment 1.3720 d.f. 288 exposure.Chem_Charge 1.6197 d.f. 252.03 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 2.6434 0.8958 0.5512 3.1937 d.f. 20 72 288 45.03 Except when comparing means with the same level(s) of exposure 2.1942 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 2.8597 1.3022 3.9291 d.f. 29.66 252.03 104.64 Except when comparing means with the same level(s) of exposure 1.3502 3.1898 d.f. 288 252.03 treatment 1.1025 d.f. 288 exposure.treatment 2.7005 d.f. 288 exposure.Chem_Charge 3.1898 d.f. 252.03 446 Analysis of variance week 10 Variate: L Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 1599.351 399.838 2.58 block.board stratum exposure 5 13901.431 2780.286 17.97 <.001 Residual 20 3093.703 154.685 4.99 block.board.area stratum treatment 3 165.881 55.294 1.78 0.158 exposure.treatment 15 334.734 22.316 0.72 0.756 Residual 72 2231.307 30.990 3.11 block.board.area.Strip stratum Chem_Charge 3 110.679 36.893 3.71 0.012 exposure.Chem_Charge 15 131.662 8.777 0.88 0.585 treatment.Chem_Charge 9 89.308 9.923 1.00 0.442 exposure.treatment.Chem_Charge 45 390.873 8.686 0.87 0.703 Residual 287 (1) 2855.697 9.950 Total 478 (1) 24381.991 Message: the following units have large residuals. block 1 board 1 -5.33 s.e. 2.54 block 3 board 6 -6.72 s.e. 2.54 block 2 board 1 area 3 5.73 s.e. 2.16 block 4 board 5 area 1 Strip 1 -7.37 s.e. 2.44 Tables of means Variate: L Grand mean 65.87 exposure full I.R. none UVA UVB Vis light 54.95 70.48 71.60 65.77 65.94 66.50 treatment acetic acid carpropamid tinuvin water 66.08 66.05 64.90 66.47 Chem_Charge high low medium Very high 66.04 65.43 65.44 66.59 447 exposure treatment acetic acid carpropamid tinuvin water full 54.96 54.25 52.82 57.76 I.R. 71.09 70.27 70.44 70.11 none 70.99 72.14 71.96 71.32 UVA 67.07 65.69 64.17 66.14 UVB 65.96 66.68 64.05 67.09 Vis light 66.39 67.25 65.94 66.41 exposure Chem_Charge high low medium Very high full 55.17 54.46 54.57 55.60 I.R. 70.74 70.63 70.41 70.13 none 70.86 71.89 71.12 72.54 UVA 66.30 64.86 65.16 66.75 UVB 65.79 66.03 65.20 66.76 Vis light 67.38 64.72 66.16 67.74 treatment Chem_Charge high low medium Very high acetic acid 66.49 65.59 65.55 66.69 carpropamid 66.12 65.82 65.12 67.13 tinuvin 65.13 65.17 64.26 65.03 water 66.42 65.14 66.82 67.50 exposure treatment Chem_Charge high low medium Very high full acetic acid 56.29 52.27 55.03 56.26 carpropamid 53.29 55.33 54.18 54.22 tinuvin 52.31 54.19 50.54 54.24 water 58.78 56.02 58.55 57.67 I.R. acetic acid 70.57 71.17 72.65 69.97 carpropamid 70.90 71.42 68.88 69.87 tinuvin 71.97 70.78 69.96 69.05 water 69.54 69.14 70.13 71.62 none acetic acid 69.03 71.29 70.08 73.57 carpropamid 70.99 72.46 71.48 73.62 tinuvin 72.52 72.79 72.06 70.46 water 70.89 71.02 70.87 72.51 UVA acetic acid 69.15 65.69 65.50 67.95 carpropamid 65.72 64.48 64.64 67.91 tinuvin 64.86 63.70 64.32 63.80 water 65.48 65.58 66.19 67.33 UVB acetic acid 66.16 67.36 65.72 64.60 carpropamid 67.35 65.10 65.41 68.85 tinuvin 62.65 65.25 62.29 66.01 water 67.01 66.39 67.36 67.58 Vis light acetic acid 67.73 65.73 64.31 67.77 carpropamid 68.49 66.11 66.11 68.31 tinuvin 66.46 64.34 66.38 66.60 water 66.84 62.69 67.85 68.28 448 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.967 0.719 0.407 2.488 d.f. 20 72 287 46.59 Except when comparing means with the same level(s) of exposure 1.760 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.148 1.007 3.029 d.f. 28.39 225.11 98.63 Except when comparing means with the same level(s) of exposure 0.998 2.467 d.f. 287 225.11 treatment 0.814 d.f. 287 exposure.treatment 1.995 d.f. 287 exposure.Chem_Charge 2.467 d.f. 225.11 (Not adjusted for missing values) Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 4.102 1.433 0.802 5.007 d.f. 20 72 287 46.59 Except when comparing means with the same level(s) of exposure 3.509 d.f. 72 449 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 4.397 1.984 6.011 d.f. 28.39 225.11 98.63 Except when comparing means with the same level(s) of exposure 1.963 4.861 d.f. 287 225.11 treatment 1.603 d.f. 287 exposure.treatment 3.927 d.f. 287 exposure.Chem_Charge 4.861 d.f. 225.11 (Not adjusted for missing values) Analysis of variance Variate: a Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 93.043 23.261 2.02 block.board stratum exposure 5 2221.254 444.251 38.60 <.001 Residual 20 230.156 11.508 1.76 block.board.area stratum treatment 3 66.787 22.262 3.40 0.022 exposure.treatment 15 77.459 5.164 0.79 0.685 Residual 72 470.941 6.541 2.76 block.board.area.Strip stratum Chem_Charge 3 5.338 1.779 0.75 0.523 exposure.Chem_Charge 15 29.486 1.966 0.83 0.646 treatment.Chem_Charge 9 26.700 2.967 1.25 0.264 exposure.treatment.Chem_Charge 45 82.429 1.832 0.77 0.853 Residual 288 683.439 2.373 Total 479 3987.031 450 Message: the following units have large residuals. block 4 board 1 1.566 s.e. 0.692 block 5 board 1 -1.431 s.e. 0.692 block 1 board 3 area 1 2.442 s.e. 0.991 block 2 board 1 area 2 2.489 s.e. 0.991 block 4 board 2 area 3 2.987 s.e. 0.991 block 1 board 1 area 1 Strip 1 3.729 s.e. 1.193 block 1 board 2 area 1 Strip 3 3.865 s.e. 1.193 block 4 board 1 area 1 Strip 2 3.959 s.e. 1.193 Tables of means Variate: a Grand mean 7.667 exposure full I.R. none UVA UVB Vis light 4.713 6.149 6.135 9.597 10.792 8.617 treatment acetic acid carpropamid tinuvin water 7.673 7.431 8.269 7.295 Chem_Charge high low medium Very high 7.716 7.650 7.795 7.508 exposure treatment acetic acid carpropamid tinuvin water full 5.114 4.560 5.256 3.923 I.R. 6.500 5.257 6.667 6.173 none 6.547 6.326 5.924 5.741 UVA 8.572 9.758 10.625 9.433 UVB 10.648 10.485 11.869 10.163 Vis light 8.658 8.198 9.274 8.338 exposure Chem_Charge high low medium Very high full 4.553 4.656 5.119 4.525 I.R. 6.616 5.894 5.698 6.389 none 6.019 6.321 6.226 5.973 UVA 9.509 9.811 9.735 9.333 UVB 11.022 10.458 11.361 10.324 Vis light 8.575 8.759 8.633 8.502 treatment Chem_Charge high low medium Very high acetic acid 7.909 7.171 7.853 7.761 carpropamid 7.513 7.890 7.459 6.861 tinuvin 8.248 8.251 8.240 8.337 water 7.194 7.288 7.628 7.072 451 exposure treatment Chem_Charge high low medium Very high full acetic acid 5.254 5.156 5.398 4.648 carpropamid 4.746 5.028 4.422 4.044 tinuvin 5.218 4.718 5.516 5.574 water 2.996 3.722 5.140 3.836 I.R. acetic acid 7.242 5.690 6.198 6.872 carpropamid 5.898 5.204 4.220 5.706 tinuvin 6.564 6.946 6.498 6.660 water 6.762 5.736 5.876 6.318 none acetic acid 7.360 6.688 6.026 6.116 carpropamid 6.418 7.198 6.265 5.424 tinuvin 4.984 5.572 6.186 6.954 water 5.314 5.828 6.426 5.398 UVA acetic acid 8.098 7.998 9.392 8.802 carpropamid 9.552 10.700 9.564 9.216 tinuvin 10.584 10.692 10.460 10.764 water 9.804 9.856 9.524 8.550 UVB acetic acid 10.916 9.728 11.070 10.880 carpropamid 10.602 10.720 11.544 9.076 tinuvin 12.786 11.206 12.566 10.918 water 9.786 10.178 10.266 10.424 Vis light acetic acid 8.582 7.766 9.036 9.248 carpropamid 7.860 8.490 8.742 7.700 tinuvin 9.354 10.372 8.216 9.154 water 8.504 8.406 8.536 7.906 452 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.5364 0.3302 0.1989 0.8822 d.f. 20 72 288 80.97 Except when comparing means with the same level(s) of exposure 0.8088 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.6824 0.4771 1.2207 d.f. 51.04 242.27 240.31 Except when comparing means with the same level(s) of exposure 0.4871 1.1688 d.f. 288 242.27 treatment 0.3977 d.f. 288 exposure.treatment 0.9743 d.f. 288 exposure.Chem_Charge 1.1688 d.f. 242.27 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.1189 0.6582 0.3914 1.7553 d.f. 20 72 288 80.97 Except when comparing means with the same level(s) of exposure 1.6122 d.f. 72 453 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.3700 0.9399 2.4047 d.f. 51.04 242.27 240.31 Except when comparing means with the same level(s) of exposure 0.9588 2.3022 d.f. 288 242.27 treatment 0.7829 d.f. 288 exposure.treatment 1.9176 d.f. 288 exposure.Chem_Charge 2.3022 d.f. 242.27 Analysis of variance Variate: b Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 72.034 18.008 0.40 block.board stratum exposure 5 8093.225 1618.645 35.85 <.001 Residual 20 902.954 45.148 2.59 block.board.area stratum treatment 3 280.390 93.463 5.36 0.002 exposure.treatment 15 284.302 18.953 1.09 0.383 Residual 72 1255.187 17.433 2.57 block.board.area.Strip stratum Chem_Charge 3 7.471 2.490 0.37 0.777 exposure.Chem_Charge 15 76.946 5.130 0.75 0.727 treatment.Chem_Charge 9 105.982 11.776 1.73 0.081 exposure.treatment.Chem_Charge 45 222.243 4.939 0.73 0.902 Residual 288 1957.093 6.795 Total 479 13257.826 454 Message: the following units have large residuals. block 4 board 1 3.65 s.e. 1.37 block 2 board 1 area 2 5.66 s.e. 1.62 block 4 board 1 area 2 -4.15 s.e. 1.62 block 4 board 1 area 4 4.45 s.e. 1.62 block 4 board 2 area 3 5.47 s.e. 1.62 block 1 board 1 area 1 Strip 1 7.87 s.e. 2.02 block 1 board 1 area 1 Strip 2 -8.19 s.e. 2.02 block 1 board 1 area 2 Strip 3 5.96 s.e. 2.02 block 2 board 2 area 1 Strip 3 -6.11 s.e. 2.02 block 4 board 1 area 1 Strip 2 9.63 s.e. 2.02 Tables of means Variate: b Grand mean 25.17 exposure full I.R. none UVA UVB Vis light 16.56 25.02 25.49 27.86 29.29 26.81 treatment acetic acid carpropamid tinuvin water 25.13 24.65 26.43 24.47 Chem_Charge high low medium Very high 25.37 25.03 25.16 25.13 exposure treatment acetic acid carpropamid tinuvin water full 17.06 16.01 18.27 14.90 I.R. 25.72 23.26 25.96 25.14 none 25.97 25.88 25.10 25.01 UVA 26.39 27.87 29.29 27.88 UVB 28.89 28.54 32.03 27.69 Vis light 26.77 26.38 27.91 26.19 exposure Chem_Charge high low medium Very high full 16.41 16.54 17.22 16.05 I.R. 25.80 24.82 24.15 25.31 none 25.33 25.77 25.27 25.59 UVA 27.95 27.98 27.67 27.83 UVB 29.80 28.67 29.95 28.73 Vis light 26.92 26.39 26.67 27.26 455 treatment Chem_Charge high low medium Very high acetic acid 25.58 24.31 25.10 25.53 carpropamid 25.25 25.41 24.13 23.83 tinuvin 26.42 26.17 26.26 26.86 water 24.22 24.22 25.13 24.30 exposure treatment Chem_Charge high low medium Very high full acetic acid 17.48 17.57 17.33 15.84 carpropamid 16.76 17.29 15.70 14.28 tinuvin 18.31 16.82 18.91 19.03 water 13.10 14.47 16.94 15.07 I.R. acetic acid 26.28 24.72 25.65 26.21 carpropamid 25.06 23.26 20.83 23.90 tinuvin 26.02 26.37 25.42 26.03 water 25.84 24.93 24.70 25.10 none acetic acid 27.14 25.57 24.49 26.68 carpropamid 26.21 27.39 25.03 24.87 tinuvin 23.41 25.03 25.40 26.56 water 24.57 25.07 26.17 24.23 UVA acetic acid 26.36 25.36 26.80 27.06 carpropamid 27.72 29.40 26.79 27.55 tinuvin 29.22 29.02 29.16 29.76 water 28.50 28.12 27.93 26.97 UVB acetic acid 29.28 27.56 29.61 29.09 carpropamid 29.61 28.67 29.80 26.07 tinuvin 33.85 31.06 32.14 31.07 water 26.46 27.38 28.23 28.69 Vis light acetic acid 26.96 25.08 26.72 28.32 carpropamid 26.15 26.42 26.63 26.30 tinuvin 27.70 28.69 26.55 28.70 water 26.87 25.37 26.79 25.71 456 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.062 0.539 0.337 1.561 d.f. 20 72 288 67.87 Except when comparing means with the same level(s) of exposure 1.320 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.280 0.794 2.115 d.f. 41.55 252.52 196.56 Except when comparing means with the same level(s) of exposure 0.824 1.945 d.f. 288 252.52 treatment 0.673 d.f. 288 exposure.treatment 1.649 d.f. 288 exposure.Chem_Charge 1.945 d.f. 252.52 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 2.216 1.075 0.662 3.115 d.f. 20 72 288 67.87 Except when comparing means with the same level(s) of exposure 2.632 d.f. 72 457 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 2.584 1.564 4.172 d.f. 41.55 252.52 196.56 Except when comparing means with the same level(s) of exposure 1.623 3.830 d.f. 288 252.52 treatment 1.325 d.f. 288 exposure.treatment 3.245 d.f. 288 exposure.Chem_Charge 3.830 d.f. 252.52 Analysis of variance week 12 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 3366.18 841.54 4.99 block.board stratum exposure 5 11796.16 2359.23 14.00 <.001 Residual 20 3370.98 168.55 4.72 block.board.area stratum treatment 3 51.43 17.14 0.48 0.697 exposure.treatment 15 259.11 17.27 0.48 0.941 Residual 72 2568.72 35.68 2.36 block.board.area.Strip stratum Chem_Charge 3 0.33 0.11 0.01 0.999 exposure.Chem_Charge 15 355.45 23.70 1.56 0.083 treatment.Chem_Charge 9 131.81 14.65 0.97 0.468 exposure.treatment.Chem_Charge 45 761.35 16.92 1.12 0.291 Residual 288 4361.95 15.15 Total 479 27023.47 458 Message: the following units have large residuals. block 2 board 4 5.94 s.e. 2.65 block 4 board 4 -6.15 s.e. 2.65 block 2 board 3 area 3 5.97 s.e. 2.31 block 1 board 1 area 4 Strip 3 12.49 s.e. 3.01 block 4 board 6 area 1 Strip 4 -9.20 s.e. 3.01 block 5 board 3 area 1 Strip 2 9.63 s.e. 3.01 Tables of means Variate: L Grand mean 62.38 exposure full I.R. none UVA UVB Vis light 52.10 65.70 67.83 62.50 62.93 63.21 treatment acetic acid carpropamid tinuvin water 62.23 62.33 62.92 62.04 Chem_Charge high low medium Very high 62.40 62.41 62.35 62.36 exposure treatment acetic acid carpropamid tinuvin water full 52.61 49.85 53.16 52.78 I.R. 65.62 65.83 66.31 65.02 none 67.71 67.37 68.93 67.32 UVA 61.78 63.47 63.58 61.18 UVB 62.90 63.82 62.20 62.83 Vis light 62.77 63.61 63.33 63.13 exposure Chem_Charge high low medium Very high full 52.69 52.75 50.65 52.30 I.R. 66.65 66.41 65.19 64.55 none 65.80 67.49 69.64 68.40 UVA 63.77 61.65 62.67 61.91 UVB 63.06 62.85 62.41 63.42 Vis light 62.45 63.29 63.53 63.56 treatment Chem_Charge high low medium Very high acetic acid 61.44 62.88 62.85 61.76 carpropamid 62.95 61.85 62.25 62.26 tinuvin 63.59 63.07 61.84 63.18 water 61.63 61.85 62.46 62.24 459 exposure treatment Chem_Charge high low medium Very high full acetic acid 53.34 52.41 52.08 52.61 carpropamid 52.81 50.59 47.60 48.42 tinuvin 51.60 54.43 51.12 55.48 water 53.02 53.59 51.81 52.70 I.R. acetic acid 66.44 67.27 65.39 63.40 carpropamid 66.88 65.82 63.78 66.84 tinuvin 68.14 67.29 65.86 63.97 water 65.12 65.25 65.74 63.98 none acetic acid 65.60 66.75 70.48 68.02 carpropamid 66.90 66.19 69.57 66.82 tinuvin 65.99 70.49 69.93 69.33 water 64.71 66.55 68.59 69.44 UVA acetic acid 61.01 61.94 62.92 61.24 carpropamid 62.96 64.01 63.11 63.81 tinuvin 67.31 61.77 61.97 63.26 water 63.80 58.89 62.69 59.34 UVB acetic acid 61.09 65.93 62.86 61.71 carpropamid 67.62 60.43 64.28 62.94 tinuvin 64.75 60.62 59.33 64.09 water 58.79 64.43 63.15 64.94 Vis light acetic acid 61.17 62.95 63.37 63.56 carpropamid 60.54 64.04 65.13 64.72 tinuvin 63.77 63.81 62.84 62.92 water 64.32 62.36 62.78 63.06 460 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 2.053 0.771 0.502 2.625 d.f. 20 72 288 48.08 Except when comparing means with the same level(s) of exposure 1.889 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.313 1.163 3.381 d.f. 32.07 264.80 123.45 Except when comparing means with the same level(s) of exposure 1.231 2.848 d.f. 288 264.80 treatment 1.005 d.f. 288 exposure.treatment 2.461 d.f. 288 exposure.Chem_Charge 2.848 d.f. 264.80 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 4.282 1.537 0.989 5.277 d.f. 20 72 288 48.08 Except when comparing means with the same level(s) of exposure 3.765 d.f. 72 461 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 4.711 2.289 6.693 d.f. 32.07 264.80 123.45 Except when comparing means with the same level(s) of exposure 2.422 5.608 d.f. 288 264.80 treatment 1.978 d.f. 288 exposure.treatment 4.845 d.f. 288 exposure.Chem_Charge 5.608 d.f. 264.80 Analysis of variance Variate: a Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 295.715 73.929 3.86 block.board stratum exposure 5 2305.684 461.137 24.07 <.001 Residual 20 383.150 19.158 3.73 block.board.area stratum treatment 3 12.897 4.299 0.84 0.478 exposure.treatment 15 95.710 6.381 1.24 0.261 Residual 72 369.422 5.131 2.32 block.board.area.Strip stratum Chem_Charge 3 10.855 3.618 1.63 0.182 exposure.Chem_Charge 15 28.341 1.889 0.85 0.617 treatment.Chem_Charge 9 24.195 2.688 1.21 0.286 exposure.treatment.Chem_Charge 45 123.594 2.747 1.24 0.152 Residual 288 637.596 2.214 Total 479 4287.158 462 Message: the following units have large residuals. block 4 board 4 -1.860 s.e. 0.893 block 1 board 3 area 1 3.499 s.e. 0.877 block 1 board 5 area 1 Strip 1 3.689 s.e. 1.153 block 2 board 3 area 3 Strip 2 3.674 s.e. 1.153 block 3 board 2 area 4 Strip 4 4.047 s.e. 1.153 block 4 board 2 area 1 Strip 3 -3.449 s.e. 1.153 block 4 board 2 area 4 Strip 1 3.752 s.e. 1.153 block 4 board 4 area 3 Strip 4 3.581 s.e. 1.153 block 4 board 5 area 4 Strip 2 -3.417 s.e. 1.153 Tables of means Variate: a Grand mean 5.874 exposure full I.R. none UVA UVB Vis light 3.215 4.492 3.619 8.016 8.828 7.076 treatment acetic acid carpropamid tinuvin water 6.059 5.618 5.862 5.960 Chem_Charge high low medium Very high 5.789 5.735 6.126 5.848 exposure treatment acetic acid carpropamid tinuvin water full 3.064 3.743 2.752 3.302 I.R. 5.266 3.970 3.864 4.870 none 4.173 3.539 3.447 3.319 UVA 7.969 8.019 7.851 8.226 UVB 8.517 7.706 9.756 9.334 Vis light 7.365 6.729 7.501 6.710 exposure Chem_Charge high low medium Very high full 3.125 2.902 3.711 3.124 I.R. 4.776 4.105 4.748 4.341 none 3.395 4.007 3.627 3.449 UVA 7.774 7.826 8.382 8.083 UVB 8.964 8.226 9.269 8.854 Vis light 6.702 7.345 7.018 7.240 treatment Chem_Charge high low medium Very high acetic acid 5.839 5.731 6.180 6.485 carpropamid 5.654 5.892 5.726 5.198 tinuvin 5.698 5.772 6.299 5.678 water 5.966 5.544 6.297 6.033 463 exposure treatment Chem_Charge high low medium Very high full acetic acid 2.216 2.834 3.810 3.396 carpropamid 3.588 3.940 4.062 3.382 tinuvin 3.334 2.288 2.544 2.842 water 3.360 2.546 4.426 2.874 I.R. acetic acid 5.440 4.472 6.500 4.652 carpropamid 4.090 4.428 3.424 3.938 tinuvin 3.806 3.592 4.468 3.590 water 5.766 3.928 4.600 5.184 none acetic acid 3.848 5.198 3.650 3.996 carpropamid 3.408 3.488 4.232 3.026 tinuvin 3.300 3.646 3.292 3.548 water 3.022 3.696 3.332 3.226 UVA acetic acid 7.378 7.956 7.884 8.656 carpropamid 8.558 8.810 7.712 6.996 tinuvin 7.866 6.542 8.756 8.238 water 7.294 7.994 9.174 8.442 UVB acetic acid 9.230 6.956 8.376 9.504 carpropamid 7.864 7.934 7.638 7.386 tinuvin 8.772 9.640 11.812 8.798 water 9.988 8.372 9.248 9.728 Vis light acetic acid 6.920 6.972 6.860 8.706 carpropamid 6.414 6.754 7.288 6.460 tinuvin 7.108 8.924 6.922 7.050 water 6.366 6.730 7.002 6.742 464 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.6921 0.2924 0.1921 0.9294 d.f. 20 72 288 55.16 Except when comparing means with the same level(s) of exposure 0.7163 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.8031 0.4430 1.2361 d.f. 35.97 267.14 155.04 Except when comparing means with the same level(s) of exposure 0.4705 1.0850 d.f. 288 267.14 treatment 0.3842 d.f. 288 exposure.treatment 0.9410 d.f. 288 exposure.Chem_Charge 1.0850 d.f. 267.14 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.4436 0.5829 0.3781 1.8624 d.f. 20 72 288 55.16 Except when comparing means with the same level(s) of exposure 1.4279 d.f. 72 465 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.6288 0.8721 2.4417 d.f. 35.97 267.14 155.04 Except when comparing means with the same level(s) of exposure 0.9261 2.1363 d.f. 288 267.14 treatment 0.7561 d.f. 288 exposure.treatment 1.8522 d.f. 288 exposure.Chem_Charge 2.1363 d.f. 267.14 Analysis of variance Variate: b Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 663.050 165.762 2.46 block.board stratum exposure 5 7830.401 1566.080 23.22 <.001 Residual 20 1348.633 67.432 4.67 block.board.area stratum treatment 3 40.053 13.351 0.92 0.433 exposure.treatment 15 273.387 18.226 1.26 0.248 Residual 72 1039.288 14.435 2.23 block.board.area.Strip stratum Chem_Charge 3 42.893 14.298 2.21 0.087 exposure.Chem_Charge 15 77.772 5.185 0.80 0.676 treatment.Chem_Charge 9 53.484 5.943 0.92 0.509 exposure.treatment.Chem_Charge 45 329.979 7.333 1.13 0.270 Residual 288 1863.679 6.471 Total 479 13562.620 466 Message: the following units have large residuals. block 4 board 4 -3.60 s.e. 1.68 block 1 board 3 area 1 4.85 s.e. 1.47 block 1 board 5 area 1 Strip 1 6.40 s.e. 1.97 block 3 board 2 area 4 Strip 4 5.86 s.e. 1.97 block 4 board 1 area 1 Strip 2 5.95 s.e. 1.97 block 4 board 4 area 3 Strip 4 6.00 s.e. 1.97 block 4 board 5 area 4 Strip 2 -6.46 s.e. 1.97 Tables of means Variate: b Grand mean 21.47 exposure full I.R. none UVA UVB Vis light 12.93 21.75 21.18 24.69 24.39 23.89 treatment acetic acid carpropamid tinuvin water 21.56 21.01 21.80 21.50 Chem_Charge high low medium Very high 21.44 21.15 21.95 21.33 exposure treatment acetic acid carpropamid tinuvin water full 12.27 13.89 12.47 13.07 I.R. 22.87 20.82 20.99 22.30 none 21.70 21.04 21.15 20.81 UVA 24.48 24.57 24.93 24.77 UVB 23.84 22.40 26.56 24.75 Vis light 24.21 23.33 24.72 23.31 exposure Chem_Charge high low medium Very high full 12.64 12.34 13.94 12.79 I.R. 22.43 21.29 21.97 21.29 none 20.62 21.46 21.71 20.91 UVA 24.61 24.21 25.23 24.69 UVB 25.02 23.30 24.92 24.32 Vis light 23.33 24.28 23.96 24.00 treatment Chem_Charge high low medium Very high acetic acid 21.22 21.15 21.98 21.90 carpropamid 21.04 21.43 21.21 20.35 tinuvin 21.87 21.43 22.28 21.63 water 21.64 20.57 22.34 21.46 467 exposure treatment Chem_Charge high low medium Very high full acetic acid 10.29 11.98 13.60 13.23 carpropamid 13.55 14.73 14.46 12.80 tinuvin 13.75 11.12 12.18 12.84 water 12.96 11.52 15.51 12.31 I.R. acetic acid 23.23 22.37 24.88 21.00 carpropamid 21.11 21.68 19.39 21.11 tinuvin 21.51 20.63 21.66 20.16 water 23.89 20.49 21.95 22.89 none acetic acid 20.84 22.76 21.85 21.37 carpropamid 20.71 20.55 22.62 20.28 tinuvin 20.69 21.63 20.87 21.40 water 20.26 20.89 21.48 20.60 UVA acetic acid 23.61 24.72 24.54 25.06 carpropamid 25.21 25.73 24.07 23.25 tinuvin 25.71 22.32 26.03 25.65 water 23.90 24.09 26.28 24.81 UVB acetic acid 25.84 21.56 23.20 24.78 carpropamid 23.13 22.45 22.29 21.74 tinuvin 25.37 26.28 29.04 25.56 water 25.75 22.90 25.15 25.18 Vis light acetic acid 23.52 23.54 23.84 25.94 carpropamid 22.55 23.44 24.43 22.92 tinuvin 24.19 26.62 23.89 24.17 water 23.05 23.53 23.67 22.97 Standard errors of differences of means Tableexposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.298 0.490 0.328 1.664 d.f. 20 72 288 48.39 Except when comparing means with the same level(s) of exposure 1.201 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.473 0.751 2.170 d.f. 32.98 272.62 129.37 Except when comparing means with the same level(s) of exposure 0.804 1.840 d.f. 288 272.62 treatment 0.657 d.f. 288 exposure.treatment 1.609 d.f. 288 exposure.Chem_Charge 1.840 d.f. 272.62 468 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 2.708 0.978 0.646 3.345 d.f. 20 72 288 48.39 Except when comparing means with the same level(s) of exposure 2.395 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 2.998 1.479 4.294 d.f. 32.98 272.62 129.37 Except when comparing means with the same level(s) of exposure 1.583 3.622 d.f. 288 272.62 treatment 1.293 d.f. 288 exposure.treatment 3.167 d.f. 288 exposure.Chem_Charge 3.622 d.f. 272.62 Analysis of variance week 14 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 3383.04 845.76 3.41 block.board stratum exposure 5 12653.41 2530.68 10.19 <.001 Residual 20 4966.51 248.33 5.65 block.board.area stratum treatment 3 314.43 104.81 2.38 0.076 exposure.treatment 15 485.25 32.35 0.74 0.741 Residual 72 3166.01 43.97 2.07 469 block.board.area.Strip stratum Chem_Charge 3 2.82 0.94 0.04 0.988 exposure.Chem_Charge 15 624.44 41.63 1.96 0.018 treatment.Chem_Charge 9 144.15 16.02 0.76 0.658 exposure.treatment.Chem_Charge 45 1023.51 22.74 1.07 0.357 Residual 288 6107.76 21.21 Total 479 32871.33 Message: the following units have large residuals. block 2 board 3 -6.99 s.e. 3.22 block 4 board 4 -7.02 s.e. 3.22 block 2 board 2 area 4 -6.49 s.e. 2.57 block 2 board 3 area 3 6.91 s.e. 2.57 block 3 board 1 area 1 -6.49 s.e. 2.57 block 1 board 1 area 4 Strip 3 12.63 s.e. 3.57 block 1 board 6 area 4 Strip 2 12.54 s.e. 3.57 block 2 board 1 area 4 Strip 1 13.77 s.e. 3.57 block 5 board 3 area 1 Strip 2 10.59 s.e. 3.57 Tables of means Variate: L Grand mean 60.36 exposure full I.R. none UVA UVB Vis light 49.46 63.80 65.32 60.61 61.94 61.01 treatment acetic acid carpropamid tinuvin water 60.00 60.54 61.55 59.34 Chem_Charge high low medium Very high 60.44 60.26 60.30 60.43 exposure treatment acetic acid carpropamid tinuvin water full 49.65 48.33 50.22 49.65 I.R. 62.96 64.91 65.84 61.48 none 66.22 64.14 67.25 63.68 UVA 60.07 60.39 63.10 58.87 UVB 62.05 62.79 61.23 61.68 Vis light 59.08 62.67 61.64 60.67 470 exposure Chem_Charge high low medium Very high full 50.01 50.42 48.20 49.22 I.R. 65.69 64.06 62.07 63.37 none 62.18 64.83 67.93 66.35 UVA 61.37 59.52 61.31 60.24 UVB 62.61 61.83 61.12 62.19 Vis light 60.76 60.91 61.18 61.20 treatment Chem_Charge high low medium Very high acetic acid 59.49 60.01 60.81 59.71 carpropamid 61.04 60.34 60.59 60.18 tinuvin 62.47 61.70 60.68 61.34 water 58.76 59.00 59.11 60.48 exposure treatment Chem_Charge high low medium Very high full acetic acid 50.29 46.96 50.61 50.74 carpropamid 50.26 52.65 45.86 44.54 tinuvin 49.11 51.66 48.18 51.94 water 50.38 50.39 48.15 49.67 I.R. acetic acid 66.18 63.66 60.34 61.66 carpropamid 65.62 65.36 62.15 66.49 tinuvin 67.91 66.94 64.29 64.25 water 63.06 60.27 61.49 61.08 none acetic acid 62.93 64.44 70.08 67.43 carpropamid 63.00 62.33 67.60 63.63 tinuvin 63.75 69.09 69.72 66.45 water 59.04 63.47 64.32 67.88 UVA acetic acid 58.29 60.66 62.07 59.27 carpropamid 60.32 60.15 60.35 60.73 tinuvin 67.05 60.29 62.70 62.38 water 59.83 56.98 60.11 58.57 UVB acetic acid 60.81 64.80 61.85 60.72 carpropamid 66.03 59.19 63.83 62.12 tinuvin 65.13 59.51 58.03 62.26 water 58.49 63.83 60.76 63.63 Vis light acetic acid 58.41 59.54 59.94 58.42 carpropamid 60.99 62.34 63.77 63.58 tinuvin 61.88 62.71 61.20 60.77 water 61.74 59.06 59.82 62.04 471 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 2.492 0.856 0.595 3.083 d.f. 20 72 288 43.48 Except when comparing means with the same level(s) of exposure 2.097 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.793 1.339 3.984 d.f. 31.42 282.98 113.49 Except when comparing means with the same level(s) of exposure 1.456 3.280 d.f. 288 282.98 treatment 1.189 d.f. 288 exposure.treatment 2.913 d.f. 288 exposure.Chem_Charge 3.280 d.f. 282.98 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 5.197 1.707 1.170 6.216 d.f. 20 72 288 43.48 Except when comparing means with the same level(s) of exposure 4.180 d.f. 72 472 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 5.693 2.636 7.892 d.f. 31.42 282.98 113.49 Except when comparing means with the same level(s) of exposure 2.866 6.457 d.f. 288 282.98 treatment 2.340 d.f. 288 exposure.treatment 5.733 d.f. 288 exposure.Chem_Charge 6.457 d.f. 282.98 Analysis of variance Variate: a Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 236.968 59.242 2.17 block.board stratum exposure 5 2314.830 462.966 16.95 <.001 Residual 20 546.418 27.321 4.51 block.board.area stratum treatment 3 44.234 14.745 2.43 0.072 exposure.treatment 15 128.586 8.572 1.41 0.164 Residual 72 436.453 6.062 3.00 block.board.area.Strip stratum Chem_Charge 3 6.819 2.273 1.13 0.339 exposure.Chem_Charge 15 23.236 1.549 0.77 0.713 treatment.Chem_Charge 9 56.138 6.238 3.09 0.001 exposure.treatment.Chem_Charge 45 145.102 3.224 1.60 0.013 Residual 286 (2) 577.236 2.018 Total 477 (2) 4485.939 473 Message: the following units have large residuals. block 1 board 2 2.458 s.e. 1.067 block 1 board 3 area 1 3.465 s.e. 0.954 block 2 board 2 area 2 2.775 s.e. 0.954 block 4 board 2 area 1 Strip 3 -3.582 s.e. 1.097 block 4 board 4 area 3 Strip 4 3.748 s.e. 1.097 block 5 board 2 area 1 Strip 1 -3.550 s.e. 1.097 Tables of means Variate: a Grand mean 5.376 exposure full I.R. none UVA UVB Vis light 2.468 4.168 3.170 7.356 8.178 6.918 treatment acetic acid carpropamid tinuvin water 5.498 4.920 5.755 5.333 Chem_Charge high low medium Very high 5.322 5.211 5.450 5.522 exposure treatment acetic acid carpropamid tinuvin water full 2.843 2.792 1.963 2.274 I.R. 4.678 3.584 4.049 4.362 none 3.475 3.050 3.332 2.821 UVA 7.225 7.221 7.763 7.216 UVB 7.748 6.456 9.652 8.857 Vis light 7.022 6.414 7.768 6.465 exposure Chem_Charge high low medium Very high full 2.298 2.516 2.622 2.437 I.R. 4.473 3.940 4.190 4.069 none 2.884 3.400 3.347 3.047 UVA 7.333 6.830 7.398 7.864 UVB 8.204 7.631 8.352 8.525 Vis light 6.740 6.951 6.788 7.190 treatment Chem_Charge high low medium Very high acetic acid 5.311 5.293 5.143 6.247 carpropamid 4.999 5.219 5.064 4.397 tinuvin 5.602 5.672 6.150 5.594 water 5.377 4.661 5.441 5.851 474 exposure treatment Chem_Charge high low medium Very high full acetic acid 2.212 3.090 2.954 3.114 carpropamid 2.608 2.994 2.882 2.682 tinuvin 2.372 1.932 1.516 2.034 water 1.998 2.046 3.134 1.918 I.R. acetic acid 4.638 4.392 5.204 4.476 carpropamid 3.568 4.422 2.916 3.428 tinuvin 4.178 3.852 4.576 3.588 water 5.508 3.093 4.064 4.784 none acetic acid 2.998 4.104 3.424 3.374 carpropamid 2.624 2.914 3.930 2.732 tinuvin 3.050 3.866 3.298 3.116 water 2.866 2.716 2.736 2.968 UVA acetic acid 6.968 7.492 5.807 8.632 carpropamid 7.914 7.574 7.122 6.274 tinuvin 8.074 5.684 9.222 8.072 water 6.376 6.570 7.442 8.476 UVB acetic acid 8.402 6.206 7.144 9.240 carpropamid 6.762 6.822 6.648 5.594 tinuvin 8.226 9.858 11.272 9.252 water 9.426 7.640 8.346 10.016 Vis light acetic acid 6.646 6.472 6.326 8.644 carpropamid 6.516 6.586 6.884 5.672 tinuvin 7.712 8.842 7.018 7.502 water 6.088 5.904 6.926 6.944 475 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.8265 0.3179 0.1834 1.0666 d.f. 20 72 286 49.41 Except when comparing means with the same level(s) of exposure 0.7786 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.9135 0.4494 1.3203 d.f. 29.75 229.92 110.58 Except when comparing means with the same level(s) of exposure 0.4493 1.1008 d.f. 286 229.92 treatment 0.3668 d.f. 286 exposure.treatment 0.8985 d.f. 286 exposure.Chem_Charge 1.1008 d.f. 229.92 (Not adjusted for missing values) Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.7239 0.6336 0.3610 2.1430 d.f. 20 72 286 49.41 Except when comparing means with the same level(s) of exposure 1.5521 d.f. 72 476 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.8662 0.8854 2.6163 d.f. 29.75 229.92 110.58 Except when comparing means with the same level(s) of exposure 0.8843 2.1689 d.f. 286 229.92 treatment 0.7220 d.f. 286 exposure.treatment 1.7685 d.f. 286 exposure.Chem_Charge 2.1689 d.f. 229.92 (Not adjusted for missing values) Analysis of variance Variate: b Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 513.312 128.328 1.32 block.board stratum exposure 5 9896.041 1979.208 20.40 <.001 Residual 20 1940.548 97.027 6.31 block.board.area stratum treatment 3 194.994 64.998 4.23 0.008 exposure.treatment 15 366.835 24.456 1.59 0.098 Residual 72 1106.978 15.375 2.19 block.board.area.Strip stratum Chem_Charge 3 18.965 6.322 0.90 0.442 exposure.Chem_Charge 15 94.886 6.326 0.90 0.564 treatment.Chem_Charge 9 119.720 13.302 1.89 0.053 exposure.treatment.Chem_Charge 45 376.011 8.356 1.19 0.202 Residual 287 (1) 2016.661 7.027 Total 478 (1) 16600.051 477 Message: the following units have large residuals. block 1 board 2 4.06 s.e. 2.01 block 4 board 4 -4.31 s.e. 2.01 block 1 board 3 area 1 5.07 s.e. 1.52 block 1 board 3 area 3 -3.88 s.e. 1.52 block 2 board 2 area 2 4.17 s.e. 1.52 block 4 board 4 area 3 Strip 4 6.28 s.e. 2.05 block 4 board 5 area 4 Strip 2 -6.37 s.e. 2.05 block 5 board 2 area 1 Strip 1 -6.45 s.e. 2.05 Tables of means Variate: b Grand mean 20.09 exposure full I.R. none UVA UVB Vis light 10.39 20.65 19.89 23.66 22.70 23.22 treatment acetic acid carpropamid tinuvin water 20.24 19.31 21.03 19.75 Chem_Charge high low medium Very high 20.06 19.78 20.30 20.21 exposure treatment acetic acid carpropamid tinuvin water full 11.18 11.00 9.55 9.84 I.R. 21.06 19.90 21.07 20.59 none 20.37 19.46 20.45 19.30 UVA 23.53 23.23 24.70 23.16 UVB 22.03 19.69 25.81 23.27 Vis light 23.30 22.61 24.61 22.36 exposure Chem_Charge high low medium Very high full 9.96 10.55 10.62 10.45 I.R. 21.56 20.40 20.30 20.35 none 18.94 20.05 20.74 19.84 UVA 23.68 22.65 24.18 24.11 UVB 23.08 21.88 22.97 22.87 Vis light 23.11 23.12 22.98 23.66 treatment Chem_Charge high low medium Very high acetic acid 19.75 19.97 20.13 21.13 carpropamid 19.42 19.79 19.58 18.47 tinuvin 21.07 20.82 21.46 20.77 water 19.99 18.52 20.03 20.48 478 exposure treatment Chem_Charge high low medium Very high full acetic acid 9.68 12.11 11.07 11.86 carpropamid 10.79 11.69 11.06 10.48 tinuvin 10.23 9.04 8.76 10.16 water 9.13 9.36 11.58 9.29 I.R. acetic acid 21.33 21.40 21.45 20.04 carpropamid 19.94 21.61 18.08 19.95 tinuvin 21.79 21.13 21.38 19.99 water 23.16 17.48 20.29 21.43 none acetic acid 18.60 20.90 21.46 20.51 carpropamid 18.37 18.91 21.52 19.03 tinuvin 19.80 21.23 20.73 20.03 water 19.00 19.16 19.23 19.81 UVA acetic acid 22.27 23.51 23.66 24.67 carpropamid 24.12 23.76 23.25 21.80 tinuvin 25.91 21.24 26.56 25.08 water 22.42 22.09 23.25 24.88 UVB acetic acid 23.48 19.71 20.95 24.00 carpropamid 20.57 20.27 20.15 17.77 tinuvin 24.20 26.08 27.82 25.14 water 24.08 21.45 22.98 24.55 Vis light acetic acid 23.12 22.19 22.17 25.71 carpropamid 22.71 22.52 23.44 21.78 tinuvin 24.49 26.21 23.48 24.25 water 22.13 21.55 22.83 22.91 479 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.557 0.506 0.342 1.892 d.f. 20 72 287 40.96 Except when comparing means with the same level(s) of exposure 1.240 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.718 0.779 2.385 d.f. 29.54 275.07 98.55 Except when comparing means with the same level(s) of exposure 0.838 1.909 d.f. 287 275.07 treatment 0.684 d.f. 287 exposure.treatment 1.677 d.f. 287 exposure.Chem_Charge 1.909 d.f. 275.07 (Not adjusted for missing values) Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 3.249 1.009 0.674 3.821 d.f. 20 72 287 40.96 Except when comparing means with the same level(s) of exposure 2.472 d.f. 72 480 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 3.512 1.534 4.732 d.f. 29.54 275.07 98.55 Except when comparing means with the same level(s) of exposure 1.650 3.759 d.f. 287 275.07 treatment 1.347 d.f. 287 exposure.treatment 3.300 d.f. 287 exposure.Chem_Charge 3.759 d.f. 275.07 (Not adjusted for missing values) Analysis of variance week 16 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 3444.14 861.03 3.99 block.board stratum exposure 5 12408.31 2481.66 11.49 <.001 Residual 20 4321.14 216.06 4.99 block.board.area stratum treatment 3 216.92 72.31 1.67 0.181 exposure.treatment 15 466.23 31.08 0.72 0.758 Residual 72 3116.25 43.28 2.41 block.board.area.Strip stratum Chem_Charge 3 4.55 1.52 0.08 0.968 exposure.Chem_Charge 15 319.56 21.30 1.19 0.280 treatment.Chem_Charge 9 139.26 15.47 0.86 0.559 exposure.treatment.Chem_Charge 45 800.77 17.79 0.99 0.491 Residual 288 5163.58 17.93 Total 479 30400.70 481 Message: the following units have large residuals. block 2 board 4 7.29 s.e. 3.00 block 2 board 2 area 4 -6.96 s.e. 2.55 block 2 board 3 area 3 6.53 s.e. 2.55 block 5 board 1 area 4 7.07 s.e. 2.55 block 1 board 1 area 4 Strip 3 10.67 s.e. 3.28 block 1 board 6 area 1 Strip 3 -13.90 s.e. 3.28 block 1 board 6 area 4 Strip 2 11.98 s.e. 3.28 Tables of means Variate: L Grand mean 58.95 exposure full I.R. none UVA UVB Vis light 48.32 62.54 64.13 58.72 60.27 59.71 treatment acetic acid carpropamid tinuvin water 58.55 58.47 60.11 58.66 Chem_Charge high low medium Very high 59.06 58.81 59.00 58.91 exposure treatment acetic acid carpropamid tinuvin water full 48.96 45.67 49.79 48.84 I.R. 61.74 63.02 64.31 61.09 none 64.15 63.32 66.07 62.96 UVA 58.30 58.72 60.52 57.34 UVB 59.91 59.21 60.19 61.76 Vis light 58.24 60.87 59.75 59.97 exposure Chem_Charge high low medium Very high full 48.94 48.02 47.97 48.33 I.R. 63.93 63.20 61.89 61.14 none 61.93 63.83 66.01 64.72 UVA 59.50 57.90 58.88 58.61 UVB 60.09 60.28 59.85 60.84 Vis light 59.98 59.60 59.41 59.84 treatment Chem_Charge high low medium Very high acetic acid 58.32 58.63 59.06 58.18 carpropamid 59.03 57.62 58.52 58.72 tinuvin 61.13 60.38 59.37 59.54 water 57.77 58.59 59.07 59.21 482 exposure treatment Chem_Charge high low medium Very high full acetic acid 49.39 46.85 50.32 49.31 carpropamid 47.65 45.12 45.16 44.75 tinuvin 48.80 51.37 47.88 51.11 water 49.94 48.74 48.53 48.17 I.R. acetic acid 64.22 62.75 60.85 59.13 carpropamid 63.81 63.58 60.78 63.93 tinuvin 65.59 65.36 64.64 61.68 water 62.12 61.12 61.29 59.82 none acetic acid 63.87 62.95 66.43 63.33 carpropamid 62.51 61.73 65.88 63.16 tinuvin 63.25 67.69 68.06 65.27 water 58.10 62.97 63.68 67.11 UVA acetic acid 57.04 58.72 59.37 58.08 carpropamid 58.57 57.95 58.71 59.66 tinuvin 64.79 57.92 59.61 59.78 water 57.59 57.00 57.85 56.93 UVB acetic acid 56.55 61.57 60.70 60.82 carpropamid 62.34 56.68 58.82 59.02 tinuvin 63.30 59.26 56.86 61.31 water 58.18 63.61 63.02 62.22 Vis light acetic acid 58.89 58.94 56.71 58.42 carpropamid 59.28 60.68 61.77 61.77 tinuvin 61.04 60.69 59.15 58.11 water 60.71 58.08 60.03 61.05 Standard errors of differences of means Tableexposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 2.324 0.849 0.547 2.941 d.f. 20 72 288 46.59 Except when comparing means with the same level(s) of exposure 2.080 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.597 1.272 3.745 d.f. 31.06 261.27 115.35 Except when comparing means with the same level(s) of exposure 1.339 3.116 d.f. 288 261.27 treatment 1.093 d.f. 288 exposure.treatment 2.678 d.f. 288 exposure.Chem_Charge 3.116 d.f. 261.27 483 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 4.848 1.693 1.076 5.917 d.f. 20 72 288 46.59 Except when comparing means with the same level(s) of exposure 4.147 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 5.297 2.505 7.418 d.f. 31.06 261.27 115.35 Except when comparing means with the same level(s) of exposure 2.635 6.135 d.f. 288 261.27 treatment 2.152 d.f. 288 exposure.treatment 5.271 d.f. 288 exposure.Chem_Charge 6.135 d.f. 261.27 Analysis of variance Variate: a Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 191.028 47.757 2.15 block.board stratum exposure 5 1991.722 398.344 17.90 <.001 Residual 20 445.171 22.259 3.45 block.board.area stratum treatment 3 26.781 8.927 1.38 0.255 exposure.treatment 15 101.698 6.780 1.05 0.416 Residual 72 464.537 6.452 3.16 484 block.board.area.Strip stratum Chem_Charge 3 6.360 2.120 1.04 0.375 exposure.Chem_Charge 15 26.278 1.752 0.86 0.611 treatment.Chem_Charge 9 43.923 4.880 2.39 0.013 exposure.treatment.Chem_Charge 45 122.493 2.722 1.33 0.085 Residual 288 587.291 2.039 Total 479 4007.282 Message: the following units have large residuals. block 1 board 2 2.333 s.e. 0.963 block 4 board 4 -2.000 s.e. 0.963 block 1 board 3 area 1 3.979 s.e. 0.984 block 1 board 3 area 3 -2.821 s.e. 0.984 block 2 board 2 area 2 3.217 s.e. 0.984 block 2 board 3 area 3 Strip 2 3.466 s.e. 1.106 block 3 board 2 area 4 Strip 3 -3.696 s.e. 1.106 block 3 board 2 area 4 Strip 4 3.565 s.e. 1.106 block 5 board 2 area 2 Strip 1 3.631 s.e. 1.106 Tables of means Variate: a Grand mean 5.223 exposure full I.R. none UVA UVB Vis light 2.546 4.102 3.142 7.134 7.763 6.651 treatment acetic acid carpropamid tinuvin water 5.302 4.878 5.533 5.181 Chem_Charge high low medium Very high 5.060 5.178 5.362 5.293 exposure treatment acetic acid carpropamid tinuvin water full 2.816 2.636 2.223 2.509 I.R. 4.388 3.825 3.723 4.472 none 3.302 3.106 3.255 2.905 UVA 7.634 6.994 7.149 6.760 UVB 7.254 6.528 9.212 8.056 Vis light 6.414 6.177 7.634 6.380 485 exposure Chem_Charge high low medium Very high full 2.483 2.480 2.695 2.526 I.R. 4.366 4.055 4.051 3.936 none 2.812 3.430 3.456 2.869 UVA 6.736 6.996 7.587 7.218 UVB 7.518 7.323 7.975 8.234 Vis light 6.441 6.782 6.408 6.973 treatment Chem_Charge high low medium Very high acetic acid 5.081 5.103 5.433 5.589 carpropamid 4.809 5.445 4.945 4.313 tinuvin 5.247 5.439 5.951 5.495 water 5.102 4.725 5.121 5.775 exposure treatment Chem_Charge high low medium Very high full acetic acid 2.330 3.048 2.830 3.058 carpropamid 2.508 2.912 3.010 2.116 tinuvin 2.686 1.756 1.894 2.556 water 2.410 2.204 3.048 2.374 I.R. acetic acid 4.344 3.782 5.304 4.124 carpropamid 4.192 4.610 2.842 3.656 tinuvin 3.540 3.674 4.390 3.290 water 5.388 4.156 3.670 4.674 none acetic acid 2.890 3.942 3.514 2.862 carpropamid 2.524 3.082 4.030 2.788 tinuvin 2.916 3.870 3.308 2.926 water 2.920 2.828 2.974 2.900 UVA acetic acid 7.140 7.728 7.996 7.674 carpropamid 7.294 7.940 7.040 5.704 tinuvin 7.062 5.626 8.484 7.424 water 5.450 6.690 6.830 8.070 UVB acetic acid 7.696 5.766 7.266 8.290 carpropamid 6.166 7.468 6.392 6.086 tinuvin 7.718 9.116 10.832 9.184 water 8.494 6.944 7.412 9.376 Vis light acetic acid 6.088 6.354 5.688 7.526 carpropamid 6.168 6.660 6.354 5.526 tinuvin 7.560 8.590 6.800 7.588 water 5.950 5.526 6.792 7.254 486 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.7460 0.3279 0.1844 1.0200 d.f. 20 72 288 57.77 Except when comparing means with the same level(s) of exposure 0.8032 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.8423 0.4577 1.2853 d.f. 32.33 223.12 136.24 Except when comparing means with the same level(s) of exposure 0.4516 1.1211 d.f. 288 223.12 treatment 0.3687 d.f. 288 exposure.treatment 0.9032 d.f. 288 exposure.Chem_Charge 1.1211 d.f. 223.12 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.5561 0.6537 0.3629 2.0419 d.f. 20 72 288 57.77 Except when comparing means with the same level(s) of exposure 1.6012 d.f. 72 487 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.7149 0.9020 2.5418 d.f. 32.33 223.12 136.24 Except when comparing means with the same level(s) of exposure 0.8888 2.2094 d.f. 288 223.12 treatment 0.7257 d.f. 288 exposure.treatment 1.7776 d.f. 288 exposure.Chem_Charge 2.2094 d.f. 223.12 Analysis of variance Variate: b Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 517.811 129.453 1.57 block.board stratum exposure 5 8579.239 1715.848 20.75 <.001 Residual 20 1653.538 82.677 5.08 block.board.area stratum treatment 3 166.528 55.509 3.41 0.022 exposure.treatment 15 278.724 18.582 1.14 0.337 Residual 72 1171.336 16.269 2.31 block.board.area.Strip stratum Chem_Charge 3 13.262 4.421 0.63 0.597 exposure.Chem_Charge 15 95.812 6.387 0.91 0.556 treatment.Chem_Charge 9 101.273 11.253 1.60 0.115 exposure.treatment.Chem_Charge 45 345.714 7.683 1.09 0.327 Residual 288 2025.951 7.035 Total 479 14949.188 488 Message: the following units have large residuals. block 1 board 2 3.78 s.e. 1.86 block 4 board 4 -4.03 s.e. 1.86 block 1 board 3 area 1 5.50 s.e. 1.56 block 1 board 3 area 3 -4.30 s.e. 1.56 block 2 board 2 area 2 4.71 s.e. 1.56 block 1 board 5 area 1 Strip 1 7.23 s.e. 2.05 block 1 board 6 area 1 Strip 3 -6.82 s.e. 2.05 block 3 board 2 area 4 Strip 4 6.35 s.e. 2.05 Tables of means Variate: b Grand mean 19.46 exposure full I.R. none UVA UVB Vis light 10.26 20.40 19.72 22.49 21.59 22.28 treatment acetic acid carpropamid tinuvin water 19.37 18.79 20.40 19.27 Chem_Charge high low medium Very high 19.24 19.41 19.70 19.49 exposure treatment acetic acid carpropamid tinuvin water full 10.75 10.18 9.85 10.27 I.R. 20.51 19.83 20.45 20.81 none 19.90 19.53 20.30 19.15 UVA 22.63 22.13 23.33 21.88 UVB 20.73 19.19 24.74 21.70 Vis light 21.69 21.87 23.76 21.81 exposure Chem_Charge high low medium Very high full 10.07 10.12 10.55 10.31 I.R. 21.10 20.57 20.13 19.79 none 18.70 20.03 20.85 19.30 UVA 22.02 22.35 22.96 22.64 UVB 21.51 20.91 21.80 22.14 Vis light 22.00 22.46 21.90 22.75 treatment Chem_Charge high low medium Very high acetic acid 19.06 19.34 19.52 19.56 carpropamid 18.65 19.71 18.91 17.89 tinuvin 20.23 20.15 20.91 20.33 water 19.01 18.44 19.46 20.18 489 exposure treatment Chem_Charge high low medium Very high full acetic acid 9.72 11.41 10.40 11.46 carpropamid 10.25 10.89 10.91 8.69 tinuvin 10.86 8.51 9.23 10.79 water 9.47 9.66 11.66 10.29 I.R. acetic acid 20.76 20.36 21.72 19.17 carpropamid 20.05 21.68 17.82 19.77 tinuvin 20.87 20.37 21.31 19.23 water 22.72 19.88 19.65 20.99 none acetic acid 18.97 20.93 21.19 18.53 carpropamid 18.32 19.03 21.58 19.19 tinuvin 19.34 21.36 20.76 19.74 water 18.18 18.82 19.87 19.74 UVA acetic acid 21.62 23.25 22.77 22.88 carpropamid 22.48 23.63 22.00 20.40 tinuvin 24.09 20.69 24.96 23.60 water 19.91 21.82 22.10 23.68 UVB acetic acid 21.89 18.42 20.58 22.03 carpropamid 19.07 20.69 18.84 18.16 tinuvin 22.67 24.62 26.66 25.02 water 22.43 19.92 21.10 23.34 Vis light acetic acid 21.38 21.68 20.44 23.24 carpropamid 21.73 22.33 22.30 21.13 tinuvin 23.56 25.32 22.53 23.62 water 21.32 20.52 22.35 23.02 Standard errors of differences of means Tableexposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.438 0.521 0.342 1.813 d.f. 20 72 288 46.12 Except when comparing means with the same level(s) of exposure 1.275 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.611 0.789 2.323 d.f. 31.37 267.44 116.65 Except when comparing means with the same level(s) of exposure 0.839 1.933 d.f. 288 267.44 treatment 0.685 d.f. 288 exposure.treatment 1.677 d.f. 288 exposure.Chem_Charge 1.933 d.f. 267.44 490 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 2.999 1.038 0.674 3.649 d.f. 20 72 288 46.12 Except when comparing means with the same level(s) of exposure 2.543 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 3.284 1.554 4.601 d.f. 31.37 267.44 116.65 Except when comparing means with the same level(s) of exposure 1.651 3.806 d.f. 288 267.44 treatment 1.348 d.f. 288 exposure.treatment 3.302 d.f. 288 exposure.Chem_Charge 3.806 d.f. 267.44 Analysis of variance week 18 Variate: L Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 4 4018.45 1004.61 5.20 block.board stratum exposure 5 10259.75 2051.95 10.62 <.001 Residual 20 3863.08 193.15 5.14 block.board.area stratum treatment 3 139.81 46.60 1.24 0.302 exposure.treatment 15 416.26 27.75 0.74 0.738 Residual 72 2706.75 37.59 2.02 491 block.board.area.Strip stratum Chem_Charge 3 60.46 20.15 1.08 0.357 exposure.Chem_Charge 15 263.56 17.57 0.94 0.516 treatment.Chem_Charge 9 110.14 12.24 0.66 0.747 exposure.treatment.Chem_Charge 45 764.38 16.99 0.91 0.635 Residual 286 (2) 5325.80 18.62 Total 477 (2) 27825.87 Message: the following units have large residuals. block 2 board 3 -5.80 s.e. 2.84 block 2 board 4 6.43 s.e. 2.84 block 2 board 3 area 3 6.44 s.e. 2.37 block 5 board 1 area 4 5.97 s.e. 2.37 block 1 board 5 area 3 Strip 1 10.02 s.e. 3.33 block 1 board 6 area 4 Strip 2 11.76 s.e. 3.33 Tables of means Variate: L Grand mean 58.12 exposure full I.R. none UVA UVB Vis light 48.67 61.48 63.23 57.62 59.11 58.57 treatment acetic acid carpropamid tinuvin water 57.83 57.62 59.02 57.99 Chem_Charge high low medium Very high 58.65 57.65 58.09 58.07 exposure treatment acetic acid carpropamid tinuvin water full 49.62 46.61 49.73 48.71 I.R. 60.50 62.40 63.01 60.03 none 63.15 61.62 65.27 62.88 UVA 57.28 57.52 58.91 56.79 UVB 58.74 58.06 58.84 60.82 Vis light 57.70 59.50 58.37 58.70 492 exposure Chem_Charge high low medium Very high full 49.90 48.18 47.92 48.67 I.R. 63.07 61.59 60.70 60.57 none 61.85 62.27 64.95 63.87 UVA 58.80 56.70 57.87 57.13 UVB 59.69 58.59 58.56 59.62 Vis light 58.59 58.58 58.57 58.54 treatment Chem_Charge high low medium Very high acetic acid 57.61 57.96 58.17 57.58 carpropamid 58.57 56.43 57.88 57.60 tinuvin 60.45 58.56 58.34 58.73 water 57.97 57.65 57.98 58.35 exposure treatment Chem_Charge high low medium Very high full acetic acid 50.53 48.28 49.83 49.83 carpropamid 49.92 45.89 45.29 45.35 tinuvin 49.11 49.93 48.26 51.61 water 50.06 48.62 48.28 47.89 I.R. acetic acid 62.66 60.65 59.44 59.25 carpropamid 63.87 61.93 60.47 63.32 tinuvin 64.74 62.99 63.16 61.16 water 61.02 60.80 59.73 58.56 none acetic acid 62.18 61.69 64.16 64.59 carpropamid 60.62 59.58 65.53 60.77 tinuvin 64.04 65.78 67.15 64.13 water 60.55 62.01 62.95 66.00 UVA acetic acid 55.49 57.57 59.67 56.38 carpropamid 57.44 57.01 57.83 57.81 tinuvin 62.91 55.90 57.66 59.18 water 59.36 56.31 56.32 55.17 UVB acetic acid 57.08 61.40 58.71 57.75 carpropamid 62.08 54.29 58.09 57.79 tinuvin 61.54 58.29 55.41 60.10 water 58.04 60.39 62.04 62.82 Vis light acetic acid 57.69 58.20 57.20 57.71 carpropamid 57.50 59.89 60.07 60.56 tinuvin 60.39 58.48 58.43 56.20 water 58.78 57.77 58.58 59.68 493 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 2.197 0.792 0.557 2.766 d.f. 20 72 286 45.83 Except when comparing means with the same level(s) of exposure 1.939 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.495 1.248 3.638 d.f. 33.05 285.99 126.43 Except when comparing means with the same level(s) of exposure 1.365 3.057 d.f. 286 285.99 treatment 1.114 d.f. 286 exposure.treatment 2.729 d.f. 286 exposure.Chem_Charge 3.057 d.f. 285.99 (Not adjusted for missing values) Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 4.584 1.578 1.097 5.567 d.f. 20 72 286 45.83 Except when comparing means with the same level(s) of exposure 3.865 d.f. 72 494 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 5.076 2.457 7.199 d.f. 33.05 285.99 126.43 Except when comparing means with the same level(s) of exposure 2.686 6.017 d.f. 286 285.99 treatment 2.193 d.f. 286 exposure.treatment 5.372 d.f. 286 exposure.Chem_Charge 6.017 d.f. 285.99 (Not adjusted for missing values) Analysis of variance Variate: a Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 115.697 28.924 1.71 block.board stratum exposure 5 1740.227 348.045 20.62 <.001 Residual 20 337.576 16.879 3.11 block.board.area stratum treatment 3 13.883 4.628 0.85 0.469 exposure.treatment 15 83.259 5.551 1.02 0.442 Residual 72 390.416 5.422 3.11 block.board.area.Strip stratum Chem_Charge 3 4.360 1.453 0.83 0.477 exposure.Chem_Charge 15 11.031 0.735 0.42 0.972 treatment.Chem_Charge 9 37.250 4.139 2.37 0.013 exposure.treatment.Chem_Charge 45 106.705 2.371 1.36 0.073 Residual 288 502.795 1.746 Total 479 3343.197 495 Message: the following units have large residuals. block 1 board 2 2.234 s.e. 0.839 block 4 board 4 -1.854 s.e. 0.839 block 1 board 3 area 1 3.333 s.e. 0.902 block 1 board 3 area 3 -2.346 s.e. 0.902 block 2 board 2 area 2 2.943 s.e. 0.902 block 2 board 2 area 3 -2.404 s.e. 0.902 block 1 board 3 area 1 Strip 2 -3.155 s.e. 1.023 block 1 board 3 area 1 Strip 4 3.696 s.e. 1.023 block 2 board 3 area 3 Strip 2 3.744 s.e. 1.023 block 3 board 2 area 1 Strip 2 3.046 s.e. 1.023 block 3 board 2 area 4 Strip 3 -3.181 s.e. 1.023 Tables of means Variate: a Grand mean 4.896 exposure full I.R. none UVA UVB Vis light 2.396 3.782 3.006 6.587 7.335 6.268 treatment acetic acid carpropamid tinuvin water 4.990 4.632 5.087 4.874 Chem_Charge high low medium Very high 4.814 4.896 5.051 4.822 exposure treatment acetic acid carpropamid tinuvin water full 2.497 2.578 1.918 2.593 I.R. 4.495 3.459 3.497 3.677 none 2.945 3.035 3.167 2.879 UVA 6.927 6.543 6.685 6.193 UVB 7.030 6.076 8.474 7.761 Vis light 6.048 6.101 6.783 6.140 exposure Chem_Charge high low medium Very high full 2.304 2.330 2.505 2.447 I.R. 4.006 3.690 3.877 3.553 none 2.765 3.265 3.174 2.822 UVA 6.307 6.573 6.708 6.761 UVB 7.462 7.162 7.668 7.049 Vis light 6.040 6.354 6.376 6.301 496 treatment Chem_Charge high low medium Very high acetic acid 4.860 4.784 5.091 5.225 carpropamid 4.400 5.211 4.784 4.132 tinuvin 5.027 5.105 5.504 4.713 water 4.969 4.482 4.826 5.218 exposure treatment Chem_Charge high low medium Very high full acetic acid 2.066 2.588 2.518 2.814 carpropamid 2.456 2.760 2.844 2.250 tinuvin 2.284 1.578 1.726 2.084 water 2.410 2.392 2.932 2.638 I.R. acetic acid 4.598 4.034 5.192 4.154 carpropamid 3.426 4.330 2.732 3.348 tinuvin 3.622 3.374 3.896 3.094 water 4.380 3.024 3.690 3.616 none acetic acid 2.654 3.356 2.798 2.972 carpropamid 2.448 3.178 3.796 2.716 tinuvin 2.992 3.622 3.254 2.800 water 2.966 2.902 2.848 2.798 UVA acetic acid 6.546 7.234 7.026 6.902 carpropamid 6.490 7.424 6.456 5.802 tinuvin 6.594 5.548 7.592 7.006 water 5.596 6.086 5.756 7.334 UVB acetic acid 7.694 5.312 7.398 7.718 carpropamid 5.860 6.914 6.302 5.230 tinuvin 7.328 9.136 10.142 7.290 water 8.966 7.286 6.832 7.960 Vis light acetic acid 5.602 6.182 5.614 6.792 carpropamid 5.718 6.660 6.576 5.448 tinuvin 7.344 7.370 6.412 6.004 water 5.494 5.204 6.900 6.960 497 Standard errors of differences of means Tableexposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.6496 0.3006 0.1706 0.9103 d.f. 20 72 288 61.31 Except when comparing means with the same level(s) of exposure 0.7364 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.7436 0.4215 1.1629 d.f. 34.11 225.63 150.50 Except when comparing means with the same level(s) of exposure 0.4178 1.0325 d.f. 288 225.63 treatment 0.3412 d.f. 288 exposure.treatment 0.8357 d.f. 288 exposure.Chem_Charge 1.0325 d.f. 225.63 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.3550 0.5993 0.3357 1.8201 d.f. 20 72 288 61.31 Except when comparing means with the same level(s) of exposure 1.4679 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.5110 0.8306 2.2978 d.f. 34.11 225.63 150.50 Except when comparing means with the same level(s) of exposure 0.8224 2.0345 d.f. 288 225.63 treatment 0.6715 d.f. 288 exposure.treatment 1.6448 d.f. 288 exposure.Chem_Charge 2.0345 d.f. 225.63 498 Analysis of variance Variate: b Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 394.740 98.685 1.41 block.board stratum exposure 5 8528.227 1705.645 24.31 <.001 Residual 20 1403.458 70.173 4.50 block.board.area stratum treatment 3 104.567 34.856 2.23 0.092 exposure.treatment 15 282.581 18.839 1.21 0.287 Residual 72 1123.477 15.604 2.56 block.board.area.Strip stratum Chem_Charge 3 10.114 3.371 0.55 0.647 exposure.Chem_Charge 15 73.753 4.917 0.81 0.670 treatment.Chem_Charge 9 96.364 10.707 1.76 0.076 exposure.treatment.Chem_Charge 45 297.242 6.605 1.08 0.340 Residual 288 1755.910 6.097 Total 479 14070.433 Message: the following units have large residuals. block 1 board 2 3.51 s.e. 1.71 block 4 board 4 -3.98 s.e. 1.71 block 1 board 3 area 1 4.69 s.e. 1.53 block 1 board 3 area 3 -3.86 s.e. 1.53 block 2 board 2 area 2 4.31 s.e. 1.53 block 5 board 1 area 1 4.16 s.e. 1.53 block 1 board 3 area 4 Strip 1 5.92 s.e. 1.91 block 1 board 5 area 1 Strip 1 6.12 s.e. 1.91 block 1 board 6 area 1 Strip 3 -5.62 s.e. 1.91 block 3 board 2 area 1 Strip 2 5.90 s.e. 1.91 block 4 board 5 area 4 Strip 2 -5.62 s.e. 1.91 499 Tables of means Variate: b Grand mean 18.57 exposure full I.R. none UVA UVB Vis light 9.34 19.64 19.14 21.41 20.47 21.43 treatment acetic acid carpropamid tinuvin water 18.53 18.00 19.30 18.46 Chem_Charge high low medium Very high 18.49 18.58 18.80 18.41 exposure treatment acetic acid carpropamid tinuvin water full 9.31 9.56 8.57 9.91 I.R. 20.37 19.12 19.57 19.50 none 19.00 18.90 19.96 18.72 UVA 21.52 21.26 22.28 20.58 UVB 20.06 17.83 23.03 20.94 Vis light 20.93 21.31 22.36 21.14 exposure Chem_Charge high low medium Very high full 8.94 9.28 9.56 9.57 I.R. 20.49 19.73 19.37 18.97 none 18.27 19.44 19.91 18.95 UVA 21.15 21.44 21.45 21.58 UVB 21.05 20.04 20.81 19.97 Vis light 21.07 21.56 21.70 21.41 treatment Chem_Charge high low medium Very high acetic acid 18.36 18.32 18.67 18.78 carpropamid 17.56 19.00 18.27 17.16 tinuvin 19.48 19.17 19.85 18.69 water 18.58 17.85 18.41 19.02 500 exposure treatment Chem_Charge high low medium Very high full acetic acid 8.33 9.60 9.13 10.16 carpropamid 9.17 10.24 10.26 8.57 tinuvin 9.25 7.69 8.12 9.23 water 8.99 9.57 10.72 10.35 I.R. acetic acid 21.05 20.15 21.22 19.07 carpropamid 18.97 21.01 17.37 19.12 tinuvin 20.31 19.40 19.91 18.67 water 21.61 18.38 18.97 19.04 none acetic acid 18.14 19.45 19.14 19.26 carpropamid 17.62 18.94 20.90 18.13 tinuvin 19.79 20.64 20.35 19.07 water 17.55 18.73 19.25 19.34 UVA acetic acid 20.63 22.22 21.84 21.38 carpropamid 21.04 22.95 20.87 20.16 tinuvin 22.96 19.98 23.36 22.81 water 19.98 20.61 19.75 21.98 UVB acetic acid 21.61 17.29 20.28 21.05 carpropamid 17.90 18.92 18.11 16.40 tinuvin 21.64 23.91 25.29 21.29 water 23.05 20.03 19.55 21.15 Vis light acetic acid 20.41 21.18 20.39 21.74 carpropamid 20.67 21.92 22.08 20.57 tinuvin 22.92 23.39 22.07 21.07 water 20.27 19.77 22.25 22.25 Standard errors of differences of means Tableexposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.325 0.510 0.319 1.710 d.f. 20 72 288 49.47 Except when comparing means with the same level(s) of exposure 1.249 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.487 0.752 2.180 d.f. 31.64 252.87 122.47 Except when comparing means with the same level(s) of exposure 0.781 1.841 d.f. 288 252.87 treatment 0.638 d.f. 288 exposure.treatment 1.562 d.f. 288 exposure.Chem_Charge 1.841 d.f. 252.87 501 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 2.763 1.017 0.627 3.436 d.f. 20 72 288 49.47 Except when comparing means with the same level(s) of exposure 2.490 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 3.031 1.480 4.316 d.f. 31.64 252.87 122.47 Except when comparing means with the same level(s) of exposure 1.537 3.626 d.f. 288 252.87 treatment 1.255 d.f. 288 exposure.treatment 3.074 d.f. 288 exposure.Chem_Charge 3.626 d.f. 252.87 Analysis of variance week 20 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 3810.63 952.66 5.30 block.board stratum exposure 5 11414.69 2282.94 12.70 <.001 Residual 20 3595.57 179.78 4.38 block.board.area stratum treatment 3 62.43 20.81 0.51 0.679 exposure.treatment 15 466.57 31.10 0.76 0.719 Residual 72 2958.15 41.09 2.14 502 block.board.area.Strip stratum Chem_Charge 3 22.09 7.36 0.38 0.765 exposure.Chem_Charge 15 412.96 27.53 1.43 0.130 treatment.Chem_Charge 9 217.58 24.18 1.26 0.258 exposure.treatment.Chem_Charge 45 846.24 18.81 0.98 0.513 Residual 288 5525.28 19.18 Total 479 29332.19 Message: the following units have large residuals. block 2 board 3 -5.49 s.e. 2.74 block 2 board 4 6.20 s.e. 2.74 block 2 board 2 area 4 -6.23 s.e. 2.48 block 2 board 3 area 3 7.34 s.e. 2.48 block 5 board 1 area 4 6.13 s.e. 2.48 block 1 board 1 area 4 Strip 3 10.35 s.e. 3.39 block 1 board 5 area 2 Strip 3 10.80 s.e. 3.39 block 1 board 6 area 1 Strip 3 -12.10 s.e. 3.39 block 1 board 6 area 4 Strip 2 13.14 s.e. 3.39 Tables of means Variate: L Grand mean 57.47 exposure full I.R. none UVA UVB Vis light 47.31 60.93 62.39 56.96 59.07 58.17 treatment acetic acid carpropamid tinuvin water 57.39 57.25 58.08 57.17 Chem_Charge high low medium Very high 57.55 57.14 57.73 57.47 exposure treatment acetic acid carpropamid tinuvin water full 48.62 45.20 47.42 47.99 I.R. 60.42 61.65 62.44 59.22 none 62.57 61.25 64.12 61.61 UVA 56.64 57.13 58.56 55.53 UVB 58.58 59.06 58.31 60.33 Vis light 57.51 59.18 57.64 58.34 503 exposure Chem_Charge high low medium Very high full 48.42 47.06 46.29 47.47 I.R. 62.36 61.03 60.29 60.05 none 59.96 61.72 64.65 63.22 UVA 57.52 55.66 58.15 56.53 UVB 59.08 59.16 58.64 59.40 Vis light 57.95 58.22 58.37 58.13 treatment Chem_Charge high low medium Very high acetic acid 57.25 57.35 58.01 56.95 carpropamid 57.75 56.33 57.55 57.36 tinuvin 59.19 58.36 56.98 57.80 water 56.01 56.52 58.39 57.75 exposure treatment Chem_Charge high low medium Very high full acetic acid 49.67 47.27 48.90 48.65 carpropamid 48.12 44.95 43.74 44.01 tinuvin 46.55 48.22 44.95 49.96 water 49.35 47.79 47.56 47.26 I.R. acetic acid 63.40 60.38 59.37 58.54 carpropamid 62.17 61.43 60.22 62.77 tinuvin 64.16 63.19 61.79 60.60 water 59.71 59.10 59.77 58.28 none acetic acid 61.58 60.64 64.84 63.19 carpropamid 59.54 59.99 64.18 61.30 tinuvin 61.84 65.67 65.30 63.66 water 56.89 60.55 64.26 64.74 UVA acetic acid 54.81 55.77 60.01 55.96 carpropamid 57.05 55.71 57.68 58.06 tinuvin 61.87 56.96 57.06 58.35 water 56.34 54.18 57.84 53.76 UVB acetic acid 56.62 60.97 58.30 58.42 carpropamid 63.29 56.02 58.90 58.05 tinuvin 61.17 58.44 54.91 58.70 water 55.25 61.21 62.46 62.40 Vis light acetic acid 57.39 59.07 56.62 56.96 carpropamid 56.30 59.86 60.56 59.99 tinuvin 59.55 57.66 57.84 55.53 water 58.55 56.28 58.47 60.05 504 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 2.120 0.827 0.565 2.752 d.f. 20 72 288 50.26 Except when comparing means with the same level(s) of exposure 2.027 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.436 1.282 3.651 d.f. 34.61 278.42 141.40 Except when comparing means with the same level(s) of exposure 1.385 3.141 d.f. 288 278.42 treatment 1.131 d.f. 288 exposure.treatment 2.770 d.f. 288 exposure.Chem_Charge 3.141 d.f. 278.42 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 4.422 1.650 1.113 5.528 d.f. 20 72 288 50.26 Except when comparing means with the same level(s) of exposure 4.041 d.f. 72 505 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 4.947 2.524 7.218 d.f. 34.61 278.42 141.40 Except when comparing means with the same level(s) of exposure 2.726 6.183 d.f. 288 278.42 treatment 2.226 d.f. 288 exposure.treatment 5.452 d.f. 288 exposure.Chem_Charge 6.183 d.f. 278.42 Analysis of variance Variate: a Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 131.962 32.990 1.75 block.board stratum exposure 5 1493.529 298.706 15.82 <.001 Residual 20 377.611 18.881 3.76 block.board.area stratum treatment 3 11.515 3.838 0.77 0.517 exposure.treatment 15 88.784 5.919 1.18 0.307 Residual 72 361.073 5.015 2.85 block.board.area.Strip stratum Chem_Charge 3 1.966 0.655 0.37 0.773 exposure.Chem_Charge 15 14.333 0.956 0.54 0.915 treatment.Chem_Charge 9 42.120 4.680 2.66 0.006 exposure.treatment.Chem_Charge 45 122.833 2.730 1.55 0.019 Residual 288 507.448 1.762 Total 479 3153.174 506 Message: the following units have large residuals. block 1 board 2 2.407 s.e. 0.887 block 4 board 4 -1.936 s.e. 0.887 block 1 board 2 area 3 2.367 s.e. 0.867 block 1 board 3 area 1 3.494 s.e. 0.867 block 1 board 3 area 3 -2.401 s.e. 0.867 block 2 board 2 area 2 2.543 s.e. 0.867 block 3 board 5 area 2 -2.151 s.e. 0.867 block 1 board 3 area 1 Strip 2 -3.268 s.e. 1.028 block 1 board 3 area 1 Strip 4 3.237 s.e. 1.028 block 2 board 3 area 3 Strip 2 3.820 s.e. 1.028 block 3 board 2 area 4 Strip 4 4.371 s.e. 1.028 block 4 board 2 area 1 Strip 3 -3.738 s.e. 1.028 block 4 board 2 area 4 Strip 1 3.030 s.e. 1.028 Tables of means Variate: a Grand mean 4.769 exposure full I.R. none UVA UVB Vis light 2.286 3.984 2.990 6.400 6.806 6.151 treatment acetic acid carpropamid tinuvin water 4.886 4.508 4.885 4.799 Chem_Charge high low medium Very high 4.743 4.704 4.875 4.755 exposure treatment acetic acid carpropamid tinuvin water full 2.332 2.436 2.042 2.334 I.R. 4.569 3.466 3.535 4.366 none 3.118 2.996 2.892 2.953 UVA 6.755 6.592 6.351 5.905 UVB 6.435 5.534 7.958 7.298 Vis light 6.106 6.027 6.532 5.938 exposure Chem_Charge high low medium Very high full 2.282 2.215 2.416 2.231 I.R. 4.129 4.095 3.869 3.844 none 2.864 3.121 3.175 2.800 UVA 6.116 6.179 6.649 6.658 UVB 7.115 6.361 6.932 6.816 Vis light 5.956 6.252 6.211 6.185 507 treatment Chem_Charge high low medium Very high acetic acid 4.702 4.676 4.972 5.193 carpropamid 4.301 5.071 4.537 4.123 tinuvin 4.781 4.797 5.318 4.643 water 5.189 4.271 4.673 5.062 exposure treatment Chem_Charge high low medium Very high full acetic acid 1.934 2.348 2.528 2.518 carpropamid 2.454 2.512 2.654 2.122 tinuvin 2.460 1.862 1.670 2.174 water 2.278 2.138 2.812 2.108 I.R. acetic acid 4.212 4.286 5.072 4.704 carpropamid 3.560 4.148 2.840 3.316 tinuvin 3.278 4.094 3.840 2.928 water 5.464 3.850 3.724 4.426 none acetic acid 2.778 3.440 3.182 3.072 carpropamid 2.382 3.302 3.568 2.730 tinuvin 2.704 3.114 3.104 2.646 water 3.590 2.628 2.844 2.750 UVA acetic acid 6.312 6.768 6.890 7.050 carpropamid 6.490 7.682 6.548 5.646 tinuvin 6.488 4.562 7.478 6.874 water 5.174 5.704 5.678 7.062 UVB acetic acid 7.216 4.840 6.682 7.002 carpropamid 5.308 6.364 5.012 5.450 tinuvin 6.782 7.990 9.722 7.336 water 9.154 6.250 6.312 7.476 Vis light acetic acid 5.760 6.374 5.478 6.812 carpropamid 5.614 6.418 6.602 5.474 tinuvin 6.972 7.160 6.094 5.902 water 5.476 5.054 6.670 6.552 508 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.6870 0.2891 0.1714 0.9209 d.f. 20 72 288 54.89 Except when comparing means with the same level(s) of exposure 0.7082 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.7773 0.4143 1.1733 d.f. 32.59 237.74 134.66 Except when comparing means with the same level(s) of exposure 0.4198 1.0149 d.f. 288 237.74 treatment 0.3427 d.f. 288 exposure.treatment 0.8395 d.f. 288 exposure.Chem_Charge 1.0149 d.f. 237.74 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.4331 0.5763 0.3373 1.8457 d.f. 20 72 288 54.89 Except when comparing means with the same level(s) of exposure 1.4117 d.f. 72 509 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.5821 0.8163 2.3206 d.f. 32.59 237.74 134.66 Except when comparing means with the same level(s) of exposure 0.8262 1.9994 d.f. 288 237.74 treatment 0.6746 d.f. 288 exposure.treatment 1.6524 d.f. 288 exposure.Chem_Charge 1.9994 d.f. 237.74 Analysis of variance Variate: b Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 433.112 108.278 1.48 block.board stratum exposure 5 8458.490 1691.698 23.18 <.001 Residual 20 1459.843 72.992 4.76 block.board.area stratum treatment 3 69.942 23.314 1.52 0.216 exposure.treatment 15 298.505 19.900 1.30 0.226 Residual 72 1103.630 15.328 2.42 block.board.area.Strip stratum Chem_Charge 3 9.438 3.146 0.50 0.685 exposure.Chem_Charge 15 75.739 5.049 0.80 0.680 treatment.Chem_Charge 9 92.774 10.308 1.63 0.107 exposure.treatment.Chem_Charge 45 308.978 6.866 1.08 0.339 Residual 288 1823.109 6.330 Total 479 14133.558 510 Message: the following units have large residuals. block 1 board 2 4.04 s.e. 1.74 block 4 board 4 -4.15 s.e. 1.74 block 1 board 3 area 1 5.10 s.e. 1.52 block 1 board 3 area 3 -4.24 s.e. 1.52 block 2 board 2 area 2 4.06 s.e. 1.52 block 5 board 1 area 1 4.18 s.e. 1.52 block 1 board 3 area 1 Strip 2 -6.06 s.e. 1.95 block 1 board 5 area 1 Strip 1 5.76 s.e. 1.95 block 2 board 2 area 4 Strip 4 -6.17 s.e. 1.95 block 3 board 2 area 4 Strip 4 7.42 s.e. 1.95 block 4 board 2 area 1 Strip 3 -6.12 s.e. 1.95 block 4 board 5 area 4 Strip 2 -5.78 s.e. 1.95 Tables of means Variate: b Grand mean 18.23 exposure full I.R. none UVA UVB Vis light 9.00 19.81 19.00 21.04 19.43 21.08 treatment acetic acid carpropamid tinuvin water 18.34 17.76 18.78 18.02 Chem_Charge high low medium Very high 18.22 18.09 18.46 18.14 exposure treatment acetic acid carpropamid tinuvin water full 9.01 9.14 8.81 9.04 I.R. 20.66 19.20 19.25 20.15 none 19.35 18.92 19.16 18.55 UVA 21.31 21.29 21.59 19.95 UVB 18.82 16.92 22.09 19.92 Vis light 20.89 21.10 21.80 20.52 exposure Chem_Charge high low medium Very high full 8.95 8.87 9.21 8.97 I.R. 20.37 20.08 19.40 19.40 none 18.17 19.08 19.84 18.89 UVA 20.76 20.68 21.39 21.31 UVB 20.27 18.58 19.63 19.27 Vis light 20.78 21.25 21.27 21.01 511 treatment Chem_Charge high low medium Very high acetic acid 18.10 18.11 18.62 18.53 carpropamid 17.36 18.61 17.88 17.19 tinuvin 18.80 18.61 19.32 18.41 water 18.61 17.03 18.00 18.44 exposure treatment Chem_Charge high low medium Very high full acetic acid 8.28 9.17 9.17 9.41 carpropamid 9.27 9.36 9.60 8.34 tinuvin 9.60 8.33 7.97 9.34 water 8.65 8.63 10.08 8.79 I.R. acetic acid 20.49 20.91 21.16 20.06 carpropamid 19.10 20.77 17.70 19.23 tinuvin 19.74 19.60 19.50 18.18 water 22.16 19.07 19.24 20.12 none acetic acid 18.26 19.52 20.39 19.23 carpropamid 17.50 19.19 20.42 18.56 tinuvin 18.46 19.72 19.68 18.80 water 18.47 17.88 18.86 18.99 UVA acetic acid 20.46 21.34 21.84 21.61 carpropamid 21.14 23.00 21.01 20.03 tinuvin 22.41 18.78 22.86 22.32 water 19.05 19.59 19.88 21.28 UVB acetic acid 20.65 16.08 19.13 19.42 carpropamid 16.88 17.86 16.35 16.57 tinuvin 20.32 22.23 24.56 21.24 water 23.23 18.13 18.47 19.84 Vis light acetic acid 20.48 21.65 20.00 21.44 carpropamid 20.29 21.49 22.23 20.41 tinuvin 22.25 23.00 21.37 20.58 water 20.11 18.87 21.49 21.61 512 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.351 0.505 0.325 1.725 d.f. 20 72 288 47.86 Except when comparing means with the same level(s) of exposure 1.238 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.516 0.756 2.208 d.f. 31.61 260.83 120.33 Except when comparing means with the same level(s) of exposure 0.796 1.853 d.f. 288 260.83 treatment 0.650 d.f. 288 exposure.treatment 1.591 d.f. 288 exposure.Chem_Charge 1.853 d.f. 260.83 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 2.818 1.008 0.639 3.468 d.f. 20 72 288 47.86 Except when comparing means with the same level(s) of exposure 2.468 d.f. 72 513 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 3.090 1.489 4.371 d.f. 31.61 260.83 120.33 Except when comparing means with the same level(s) of exposure 1.566 3.648 d.f. 288 260.83 treatment 1.279 d.f. 288 exposure.treatment 3.132 d.f. 288 exposure.Chem_Charge 3.648 d.f. 260.83 Analysis of variance week 24 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 3894.60 973.65 4.56 block.board stratum exposure 5 7941.62 1588.32 7.44 <.001 Residual 20 4271.26 213.56 5.23 block.board.area stratum treatment 3 210.35 70.12 1.72 0.171 exposure.treatment 15 389.00 25.93 0.64 0.836 Residual 72 2938.99 40.82 2.68 block.board.area.Strip stratum Chem_Charge 3 12.60 4.20 0.28 0.843 exposure.Chem_Charge 15 260.58 17.37 1.14 0.319 treatment.Chem_Charge 9 144.25 16.03 1.05 0.398 exposure.treatment.Chem_Charge 45 829.55 18.43 1.21 0.180 Residual 288 4385.50 15.23 Total 479 25278.32 514 Message: the following units have large residuals. block 2 board 3 -6.29 s.e. 2.98 block 2 board 4 6.62 s.e. 2.98 block 2 board 3 area 3 6.98 s.e. 2.47 block 4 board 3 area 3 -6.18 s.e. 2.47 block 1 board 6 area 1 Strip 3 -8.98 s.e. 3.02 block 1 board 6 area 4 Strip 2 10.65 s.e. 3.02 block 5 board 3 area 1 Strip 2 9.16 s.e. 3.02 Tables of means Variate: L Grand mean 56.86 exposure full I.R. none UVA UVB Vis light 48.70 59.93 61.57 56.22 57.66 57.08 treatment acetic acid carpropamid tinuvin water 56.55 56.31 57.99 56.60 Chem_Charge high low medium Very high 57.10 56.74 56.69 56.91 exposure treatment acetic acid carpropamid tinuvin water full 48.65 46.73 50.44 48.99 I.R. 58.88 60.86 61.57 58.43 none 61.81 59.85 64.02 60.60 UVA 55.72 56.14 57.64 55.41 UVB 57.75 56.61 57.52 58.76 Vis light 56.49 57.67 56.78 57.40 exposure Chem_Charge high low medium Very high full 49.66 48.90 47.70 48.54 I.R. 61.14 60.34 58.68 59.57 none 60.22 60.57 62.95 62.55 UVA 57.29 55.68 56.07 55.87 UVB 57.21 57.60 57.47 58.35 Vis light 57.11 57.37 57.27 56.58 treatment Chem_Charge high low medium Very high acetic acid 56.55 56.69 56.75 56.20 carpropamid 57.04 55.53 56.07 56.60 tinuvin 59.17 58.12 57.13 57.54 water 55.66 56.62 56.81 57.30 515 exposure treatment Chem_Charge high low medium Very high full acetic acid 49.64 47.46 48.91 48.59 carpropamid 49.87 46.50 45.02 45.54 tinuvin 49.76 51.85 48.62 51.51 water 49.37 49.77 48.26 48.54 I.R. acetic acid 61.46 58.45 57.79 57.81 carpropamid 61.11 61.86 57.57 62.88 tinuvin 63.10 61.79 61.77 59.61 water 58.87 59.27 57.60 57.96 none acetic acid 62.10 60.54 62.47 62.15 carpropamid 59.54 57.28 63.03 59.54 tinuvin 63.00 65.03 64.75 63.29 water 56.23 59.43 61.55 65.21 UVA acetic acid 54.45 56.53 56.48 55.41 carpropamid 56.63 55.27 56.30 56.35 tinuvin 60.54 56.21 55.94 57.84 water 57.52 54.69 55.55 53.86 UVB acetic acid 55.36 59.77 58.20 57.66 carpropamid 59.59 53.68 56.21 56.96 tinuvin 59.81 57.01 54.18 59.08 water 54.10 59.94 61.28 59.71 Vis light acetic acid 56.28 57.41 56.67 55.60 carpropamid 55.46 58.59 58.28 58.34 tinuvin 58.83 56.84 57.54 53.90 water 57.86 56.64 56.60 58.50 516 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 2.311 0.825 0.504 2.898 d.f. 20 72 288 45.37 Except when comparing means with the same level(s) of exposure 2.020 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.546 1.201 3.601 d.f. 29.38 246.23 103.32 Except when comparing means with the same level(s) of exposure 1.234 2.941 d.f. 288 246.23 treatment 1.008 d.f. 288 exposure.treatment 2.468 d.f. 288 exposure.Chem_Charge 2.941 d.f. 246.23 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 4.820 1.644 0.992 5.836 d.f. 20 72 288 45.37 Except when comparing means with the same level(s) of exposure 4.028 d.f. 72 517 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 5.204 2.365 7.142 d.f. 29.38 246.23 103.32 Except when comparing means with the same level(s) of exposure 2.429 5.793 d.f. 288 246.23 treatment 1.983 d.f. 288 exposure.treatment 4.858 d.f. 288 exposure.Chem_Charge 5.793 d.f. 246.23 Analysis of variance Variate: a Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 110.494 27.623 1.56 block.board stratum exposure 5 1402.918 280.584 15.81 <.001 Residual 20 354.842 17.742 3.96 block.board.area stratum treatment 3 11.560 3.853 0.86 0.466 exposure.treatment 15 83.596 5.573 1.24 0.261 Residual 72 322.622 4.481 2.70 block.board.area.Strip stratum Chem_Charge 3 0.637 0.212 0.13 0.943 exposure.Chem_Charge 15 17.343 1.156 0.70 0.786 treatment.Chem_Charge 9 32.613 3.624 2.19 0.023 exposure.treatment.Chem_Charge 45 107.965 2.399 1.45 0.039 Residual 288 477.212 1.657 Total 479 2921.803 518 Message: the following units have large residuals. block 1 board 2 2.315 s.e. 0.860 block 1 board 3 area 1 2.950 s.e. 0.820 block 1 board 3 area 3 -2.128 s.e. 0.820 block 2 board 2 area 2 2.582 s.e. 0.820 block 3 board 5 area 1 2.147 s.e. 0.820 block 3 board 5 area 2 -2.153 s.e. 0.820 block 1 board 1 area 1 Strip 4 3.169 s.e. 0.997 block 1 board 2 area 2 Strip 4 -2.990 s.e. 0.997 block 1 board 3 area 1 Strip 4 3.211 s.e. 0.997 block 3 board 2 area 4 Strip 3 -3.881 s.e. 0.997 block 3 board 2 area 4 Strip 4 3.496 s.e. 0.997 block 5 board 2 area 4 Strip 3 4.891 s.e. 0.997 Tables of means Variate: a Grand mean 4.664 exposure full I.R. none UVA UVB Vis light 2.248 3.865 2.980 6.121 6.682 6.089 treatment acetic acid carpropamid tinuvin water 4.697 4.414 4.704 4.841 Chem_Charge high low medium Very high 4.624 4.638 4.677 4.717 exposure treatment acetic acid carpropamid tinuvin water full 2.393 2.389 1.633 2.576 I.R. 4.480 3.301 3.395 4.282 none 3.004 2.927 2.961 3.029 UVA 6.364 6.126 6.149 5.843 UVB 6.139 5.614 7.652 7.321 Vis light 5.802 6.128 6.433 5.993 exposure Chem_Charge high low medium Very high full 2.020 2.158 2.365 2.448 I.R. 3.985 3.964 3.825 3.685 none 2.955 3.144 3.085 2.737 UVA 5.893 5.921 6.364 6.304 UVB 7.066 6.446 6.379 6.834 Vis light 5.823 6.195 6.043 6.295 519 treatment Chem_Charge high low medium Very high acetic acid 4.577 4.567 4.686 4.958 carpropamid 4.242 4.900 4.383 4.133 tinuvin 4.566 4.765 4.960 4.525 water 5.111 4.320 4.679 5.254 exposure treatment Chem_Charge high low medium Very high full acetic acid 2.056 2.494 2.470 2.552 carpropamid 2.118 2.466 2.640 2.330 tinuvin 1.834 1.492 1.358 1.848 water 2.072 2.178 2.992 3.062 I.R. acetic acid 4.124 4.404 4.866 4.526 carpropamid 3.178 4.058 2.764 3.206 tinuvin 3.302 3.668 3.746 2.864 water 5.336 3.726 3.924 4.144 none acetic acid 2.866 3.496 2.990 2.664 carpropamid 2.354 2.942 3.438 2.976 tinuvin 3.102 3.362 2.920 2.462 water 3.500 2.776 2.992 2.848 UVA acetic acid 6.268 6.148 6.470 6.572 carpropamid 5.976 7.186 6.098 5.244 tinuvin 6.078 4.828 7.060 6.632 water 5.250 5.524 5.830 6.770 UVB acetic acid 7.012 4.760 5.910 6.874 carpropamid 5.858 6.320 5.038 5.240 tinuvin 6.214 8.408 8.748 7.240 water 9.182 6.298 5.820 7.984 Vis light acetic acid 5.138 6.100 5.410 6.560 carpropamid 5.966 6.430 6.318 5.800 tinuvin 6.864 6.834 5.930 6.104 water 5.326 5.416 6.514 6.718 520 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.6660 0.2733 0.1662 0.8830 d.f. 20 72 288 53.29 Except when comparing means with the same level(s) of exposure 0.6694 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.7535 0.3969 1.1299 d.f. 32.60 244.98 132.91 Except when comparing means with the same level(s) of exposure 0.4071 0.9722 d.f. 288 244.98 treatment 0.3324 d.f. 288 exposure.treatment 0.8141 d.f. 288 exposure.Chem_Charge 0.9722 d.f. 244.98 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.3892 0.5448 0.3271 1.7708 d.f. 20 72 288 53.29 Except when comparing means with the same level(s) of exposure 1.3344 d.f. 72 521 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.5338 0.7818 2.2349 d.f. 32.60 244.98 132.91 Except when comparing means with the same level(s) of exposure 0.8012 1.9149 d.f. 288 244.98 treatment 0.6542 d.f. 288 exposure.treatment 1.6024 d.f. 288 exposure.Chem_Charge 1.9149 d.f. 244.98 Analysis of variance Variate: b Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 471.081 117.770 1.65 block.board stratum exposure 5 8648.067 1729.613 24.19 <.001 Residual 20 1429.840 71.492 5.27 block.board.area stratum treatment 3 67.167 22.389 1.65 0.185 exposure.treatment 15 285.940 19.063 1.41 0.168 Residual 72 976.059 13.556 2.37 block.board.area.Strip stratum Chem_Charge 3 0.145 0.048 0.01 0.999 exposure.Chem_Charge 15 76.705 5.114 0.89 0.573 treatment.Chem_Charge 9 77.452 8.606 1.50 0.146 exposure.treatment.Chem_Charge 45 315.358 7.008 1.22 0.167 Residual 288 1649.174 5.726 Total 479 13996.990 522 Message: the following units have large residuals. block 1 board 2 3.70 s.e. 1.73 block 4 board 4 -3.95 s.e. 1.73 block 1 board 3 area 1 4.40 s.e. 1.43 block 1 board 3 area 3 -3.65 s.e. 1.43 block 2 board 2 area 2 4.04 s.e. 1.43 block 5 board 1 area 1 3.93 s.e. 1.43 block 1 board 2 area 1 Strip 1 5.88 s.e. 1.85 block 3 board 2 area 4 Strip 3 -6.66 s.e. 1.85 block 3 board 2 area 4 Strip 4 5.73 s.e. 1.85 block 5 board 2 area 4 Strip 3 8.55 s.e. 1.85 Tables of means Variate: b Grand mean 17.52 exposure full I.R. none UVA UVB Vis light 8.16 19.20 18.56 20.04 18.67 20.50 treatment acetic acid carpropamid tinuvin water 17.48 16.98 18.03 17.58 Chem_Charge high low medium Very high 17.52 17.53 17.49 17.54 exposure treatment acetic acid carpropamid tinuvin water full 8.45 8.51 7.19 8.47 I.R. 19.97 18.39 18.84 19.60 none 18.61 18.11 19.17 18.33 UVA 20.14 19.94 20.82 19.28 UVB 17.76 16.38 21.01 19.53 Vis light 19.98 20.55 21.17 20.29 exposure Chem_Charge high low medium Very high full 7.69 8.04 8.52 8.37 I.R. 19.64 19.73 18.66 18.76 none 18.24 18.63 19.05 18.31 UVA 19.91 19.81 20.26 20.19 UVB 19.57 18.15 17.98 18.98 Vis light 20.09 20.82 20.46 20.61 523 treatment Chem_Charge high low medium Very high acetic acid 17.41 17.32 17.48 17.71 carpropamid 16.63 17.89 16.87 16.54 tinuvin 18.01 18.12 18.25 17.75 water 18.04 16.79 17.36 18.15 exposure treatment Chem_Charge high low medium Very high full acetic acid 7.87 8.67 8.41 8.85 carpropamid 7.81 8.77 9.11 8.37 tinuvin 7.46 6.63 6.63 8.04 water 7.63 8.10 9.94 8.21 I.R. acetic acid 19.77 20.40 20.42 19.27 carpropamid 18.04 20.31 16.51 18.69 tinuvin 19.13 19.47 19.09 17.66 water 21.61 18.75 18.63 19.41 none acetic acid 18.34 19.06 18.95 18.08 carpropamid 17.05 17.83 19.71 17.86 tinuvin 19.60 19.88 19.03 18.19 water 17.96 17.76 18.50 19.10 UVA acetic acid 19.65 20.04 20.43 20.42 carpropamid 19.76 21.95 19.53 18.51 tinuvin 21.34 18.54 21.91 21.48 water 18.86 18.71 19.18 20.36 UVB acetic acid 19.70 15.34 17.14 18.86 carpropamid 16.91 17.35 15.44 15.83 tinuvin 18.85 22.15 22.24 20.79 water 22.80 17.75 17.11 20.45 Vis light acetic acid 19.14 20.43 19.54 20.80 carpropamid 20.23 21.12 20.90 19.95 tinuvin 21.64 22.08 20.62 20.33 water 19.35 19.64 20.80 21.36 524 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.337 0.475 0.309 1.675 d.f. 20 72 288 45.17 Except when comparing means with the same level(s) of exposure 1.164 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.489 0.716 2.126 d.f. 30.64 264.08 110.93 Except when comparing means with the same level(s) of exposure 0.757 1.753 d.f. 288 264.08 treatment 0.618 d.f. 288 exposure.treatment 1.513 d.f. 288 exposure.Chem_Charge 1.753 d.f. 264.08 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 2.789 0.948 0.608 3.372 d.f. 20 72 288 45.17 Except when comparing means with the same level(s) of exposure 2.321 d.f. 72 525 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 3.038 1.409 4.214 d.f. 30.64 264.08 110.93 Except when comparing means with the same level(s) of exposure 1.489 3.452 d.f. 288 264.08 treatment 1.216 d.f. 288 exposure.treatment 2.979 d.f. 288 exposure.Chem_Charge 3.452 d.f. 264.08 Analysis of variance week 32 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 4801.66 1200.41 5.19 block.board stratum exposure 5 8825.57 1765.11 7.63 <.001 Residual 20 4626.46 231.32 5.92 block.board.area stratum treatment 3 154.91 51.64 1.32 0.274 exposure.treatment 15 328.81 21.92 0.56 0.895 Residual 72 2814.78 39.09 2.42 block.board.area.Strip stratum Chem_Charge 3 19.26 6.42 0.40 0.755 exposure.Chem_Charge 15 350.99 23.40 1.45 0.123 treatment.Chem_Charge 9 172.91 19.21 1.19 0.300 exposure.treatment.Chem_Charge 45 832.61 18.50 1.15 0.252 Residual 288 4646.64 16.13 Total 479 27574.61 526 Message: the following units have large residuals. block 2 board 4 7.05 s.e. 3.10 block 2 board 2 area 4 -5.99 s.e. 2.42 block 2 board 3 area 3 6.04 s.e. 2.42 block 4 board 2 area 3 -7.26 s.e. 2.42 block 5 board 1 area 4 6.09 s.e. 2.42 block 1 board 1 area 4 Strip 3 9.55 s.e. 3.11 block 1 board 5 area 2 Strip 3 10.52 s.e. 3.11 block 1 board 6 area 1 Strip 3 -10.40 s.e. 3.11 block 1 board 6 area 4 Strip 2 11.39 s.e. 3.11 block 2 board 2 area 1 Strip 3 9.31 s.e. 3.11 block 5 board 3 area 1 Strip 2 9.57 s.e. 3.11 Tables of means Variate: L Grand mean 56.25 exposure full I.R. none UVA UVB Vis light 47.47 59.19 60.82 55.64 58.07 56.31 treatment acetic acid carpropamid tinuvin water 55.85 56.22 57.18 55.74 Chem_Charge high low medium Very high 56.59 56.08 56.13 56.21 exposure treatment acetic acid carpropamid tinuvin water full 48.13 45.63 48.52 47.60 I.R. 58.58 59.89 60.34 57.97 none 60.55 59.80 63.36 59.57 UVA 55.12 56.27 56.79 54.37 UVB 57.21 58.48 57.89 58.70 Vis light 55.53 57.26 56.21 56.24 exposure Chem_Charge high low medium Very high full 48.57 46.96 46.97 47.38 I.R. 60.71 59.95 58.22 57.90 none 58.77 60.34 62.23 61.93 UVA 56.55 54.84 56.00 55.15 UVB 58.92 57.78 57.28 58.30 Vis light 56.00 56.59 56.07 56.57 527 treatment Chem_Charge high low medium Very high acetic acid 55.84 56.33 56.42 54.82 carpropamid 57.10 55.36 56.15 56.28 tinuvin 58.23 57.03 55.99 57.49 water 55.18 55.59 55.96 56.23 exposure treatment Chem_Charge high low medium Very high full acetic acid 49.25 45.40 49.49 48.39 carpropamid 48.80 44.80 44.92 44.00 tinuvin 47.66 49.91 46.16 50.37 water 48.58 47.74 47.30 46.78 I.R. acetic acid 60.90 60.33 57.57 55.52 carpropamid 60.90 60.82 57.20 60.64 tinuvin 62.38 59.88 60.17 58.93 water 58.64 58.77 57.93 56.53 none acetic acid 60.46 58.51 61.99 61.24 carpropamid 59.53 58.40 61.68 59.61 tinuvin 60.57 64.67 64.89 63.30 water 54.52 59.79 60.37 63.60 UVA acetic acid 54.52 55.41 57.47 53.06 carpropamid 55.41 55.94 56.49 57.22 tinuvin 60.08 55.68 54.51 56.89 water 56.20 52.31 55.52 53.45 UVB acetic acid 55.16 60.90 56.94 55.85 carpropamid 62.23 54.84 58.58 58.29 tinuvin 60.77 56.18 54.50 60.11 water 57.53 59.23 59.09 58.94 Vis light acetic acid 54.76 57.42 55.03 54.89 carpropamid 55.73 57.33 58.04 57.94 tinuvin 57.93 55.88 55.68 55.35 water 55.59 55.73 55.54 58.10 528 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 2.405 0.807 0.519 2.952 d.f. 20 72 288 42.39 Except when comparing means with the same level(s) of exposure 1.977 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.644 1.208 3.682 d.f. 29.16 260.73 98.11 Except when comparing means with the same level(s) of exposure 1.270 2.958 d.f. 288 260.73 treatment 1.037 d.f. 288 exposure.treatment 2.540 d.f. 288 exposure.Chem_Charge 2.958 d.f. 260.73 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 5.016 1.609 1.021 5.956 d.f. 20 72 288 42.39 Except when comparing means with the same level(s) of exposure 3.942 d.f. 72 529 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 5.407 2.378 7.306 d.f. 29.16 260.73 98.11 Except when comparing means with the same level(s) of exposure 2.500 5.825 d.f. 288 260.73 treatment 2.041 d.f. 288 exposure.treatment 5.000 d.f. 288 exposure.Chem_Charge 5.825 d.f. 260.73 Analysis of variance Variate: a Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 88.399 22.100 1.25 block.board stratum exposure 5 1219.149 243.830 13.78 <.001 Residual 20 353.978 17.699 5.89 block.board.area stratum treatment 3 19.400 6.467 2.15 0.101 exposure.treatment 15 79.938 5.329 1.77 0.056 Residual 72 216.440 3.006 2.30 block.board.area.Strip stratum Chem_Charge 3 6.000 2.000 1.53 0.207 exposure.Chem_Charge 15 13.202 0.880 0.67 0.811 treatment.Chem_Charge 9 24.663 2.740 2.09 0.030 exposure.treatment.Chem_Charge 45 95.029 2.112 1.61 0.011 Residual 288 377.065 1.309 Total 479 2493.262 530 Message: the following units have large residuals. block 1 board 2 2.631 s.e. 0.859 block 1 board 2 area 3 1.708 s.e. 0.672 block 1 board 3 area 1 2.978 s.e. 0.672 block 2 board 2 area 2 1.717 s.e. 0.672 block 1 board 2 area 1 Strip 3 3.108 s.e. 0.886 block 1 board 2 area 2 Strip 4 -2.599 s.e. 0.886 block 2 board 3 area 3 Strip 2 3.700 s.e. 0.886 block 2 board 5 area 1 Strip 3 2.655 s.e. 0.886 block 4 board 2 area 2 Strip 1 -3.085 s.e. 0.886 block 5 board 2 area 1 Strip 2 -2.690 s.e. 0.886 Tables of means Variate: a Grand mean 4.405 exposure full I.R. none UVA UVB Vis light 1.785 3.918 3.140 5.923 5.547 6.116 treatment acetic acid carpropamid tinuvin water 4.527 4.061 4.559 4.472 Chem_Charge high low medium Very high 4.278 4.311 4.534 4.497 exposure treatment acetic acid carpropamid tinuvin water full 1.886 1.850 1.553 1.851 I.R. 4.606 3.385 3.474 4.208 none 3.170 3.149 3.052 3.192 UVA 5.979 5.934 6.012 5.766 UVB 5.323 4.177 6.678 6.012 Vis light 6.200 5.871 6.589 5.806 exposure Chem_Charge high low medium Very high full 1.714 1.790 1.851 1.785 I.R. 3.926 3.778 3.961 4.009 none 3.077 3.296 3.235 2.954 UVA 5.729 5.441 6.349 6.172 UVB 5.463 5.370 5.754 5.604 Vis light 5.763 6.189 6.055 6.460 531 treatment Chem_Charge high low medium Very high acetic acid 4.364 4.348 4.461 4.936 carpropamid 4.062 4.350 4.055 3.776 tinuvin 4.277 4.434 5.070 4.457 water 4.410 4.111 4.550 4.818 exposure treatment Chem_Charge high low medium Very high full acetic acid 1.464 2.146 1.862 2.070 carpropamid 1.790 1.946 1.938 1.726 tinuvin 1.936 1.310 1.494 1.470 water 1.664 1.758 2.108 1.874 I.R. acetic acid 4.390 4.288 5.128 4.618 carpropamid 3.446 3.558 2.770 3.766 tinuvin 3.196 3.082 4.212 3.406 water 4.670 4.184 3.732 4.244 none acetic acid 3.034 3.488 3.006 3.150 carpropamid 2.786 3.270 3.722 2.816 tinuvin 2.990 3.104 3.170 2.942 water 3.496 3.322 3.040 2.908 UVA acetic acid 5.664 5.774 6.056 6.420 carpropamid 6.408 6.324 5.960 5.044 tinuvin 6.066 4.502 6.948 6.532 water 4.778 5.164 6.432 6.690 UVB acetic acid 6.026 4.050 5.270 5.946 carpropamid 4.164 4.854 3.854 3.836 tinuvin 5.176 7.522 8.326 5.686 water 6.484 5.054 5.564 6.946 Vis light acetic acid 5.604 6.340 5.442 7.414 carpropamid 5.780 6.150 6.084 5.470 tinuvin 6.298 7.082 6.268 6.708 water 5.370 5.182 6.424 6.246 532 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.6652 0.2238 0.1477 0.8173 d.f. 20 72 288 42.51 Except when comparing means with the same level(s) of exposure 0.5483 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.7353 0.3399 1.0299 d.f. 29.76 268.48 102.00 Except when comparing means with the same level(s) of exposure 0.3618 0.8327 d.f. 288 268.48 treatment 0.2954 d.f. 288 exposure.treatment 0.7237 d.f. 288 exposure.Chem_Charge 0.8327 d.f. 268.48 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.3876 0.4462 0.2907 1.6487 d.f. 20 72 288 42.51 Except when comparing means with the same level(s) of exposure 1.0930 d.f. 72 533 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.5022 0.6693 2.0428 d.f. 29.76 268.48 102.00 Except when comparing means with the same level(s) of exposure 0.7122 1.6394 d.f. 288 268.48 treatment 0.5815 d.f. 288 exposure.treatment 1.4244 d.f. 288 exposure.Chem_Charge 1.6394 d.f. 268.48 Analysis of variance Variate: b Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 549.088 137.272 1.69 block.board stratum exposure 5 9791.420 1958.284 24.10 <.001 Residual 20 1625.091 81.255 8.23 block.board.area stratum treatment 3 121.507 40.502 4.10 0.010 exposure.treatment 15 290.766 19.384 1.96 0.030 Residual 72 710.443 9.867 1.84 block.board.area.Strip stratum Chem_Charge 3 12.599 4.200 0.78 0.504 exposure.Chem_Charge 15 63.313 4.221 0.79 0.692 treatment.Chem_Charge 9 53.239 5.915 1.10 0.360 exposure.treatment.Chem_Charge 45 291.265 6.473 1.21 0.183 Residual 288 1543.885 5.361 Total 479 15052.616 534 Message: the following units have large residuals. block 1 board 2 4.71 s.e. 1.84 block 1 board 2 area 3 3.14 s.e. 1.22 block 1 board 3 area 1 5.13 s.e. 1.22 block 1 board 3 area 3 -3.09 s.e. 1.22 block 1 board 2 area 1 Strip 3 5.68 s.e. 1.79 block 1 board 2 area 1 Strip 4 -5.40 s.e. 1.79 block 1 board 5 area 1 Strip 1 5.96 s.e. 1.79 block 2 board 3 area 3 Strip 2 6.03 s.e. 1.79 block 4 board 2 area 2 Strip 1 -6.43 s.e. 1.79 block 5 board 2 area 1 Strip 4 5.55 s.e. 1.79 block 5 board 2 area 2 Strip 1 5.37 s.e. 1.79 Tables of means Variate: b Grand mean 16.36 exposure full I.R. none UVA UVB Vis light 6.64 18.64 18.24 18.94 15.83 19.87 treatment acetic acid carpropamid tinuvin water 16.39 15.66 17.08 16.31 Chem_Charge high low medium Very high 16.16 16.24 16.54 16.49 exposure treatment acetic acid carpropamid tinuvin water full 6.83 6.66 6.32 6.76 I.R. 19.49 17.89 18.37 18.82 none 18.22 18.06 18.82 17.86 UVA 18.78 18.84 19.60 18.54 UVB 15.23 12.97 18.61 16.51 Vis light 19.79 19.56 20.79 19.35 exposure Chem_Charge high low medium Very high full 6.45 6.60 6.72 6.81 I.R. 19.10 18.76 18.30 18.39 none 17.59 18.53 18.70 18.14 UVA 18.79 18.10 19.71 19.15 UVB 15.83 15.40 16.12 15.97 Vis light 19.19 20.09 19.71 20.49 535 treatment Chem_Charge high low medium Very high acetic acid 16.20 16.25 16.33 16.77 carpropamid 15.61 16.18 15.66 15.20 tinuvin 16.85 16.83 17.69 16.96 water 15.97 15.72 16.50 17.03 exposure treatment Chem_Charge high low medium Very high full acetic acid 6.02 7.39 6.54 7.38 carpropamid 6.56 6.85 6.91 6.31 tinuvin 7.13 5.58 6.01 6.57 water 6.09 6.59 7.40 6.97 I.R. acetic acid 19.64 19.65 20.26 18.40 carpropamid 18.00 18.82 16.05 18.70 tinuvin 18.73 17.58 19.31 17.85 water 20.05 19.00 17.60 18.63 none acetic acid 17.86 18.45 18.29 18.29 carpropamid 17.20 18.28 19.50 17.25 tinuvin 18.15 19.23 19.36 18.55 water 17.16 18.15 17.66 18.47 UVA acetic acid 18.07 18.73 19.40 18.91 carpropamid 19.50 19.42 18.87 17.57 tinuvin 20.44 16.92 20.57 20.46 water 17.15 17.31 20.01 19.67 UVB acetic acid 16.93 12.88 14.95 16.15 carpropamid 13.31 13.79 12.46 12.32 tinuvin 16.16 20.05 20.90 17.32 water 16.93 14.86 16.18 18.07 Vis light acetic acid 18.69 20.41 18.56 21.50 carpropamid 19.09 19.89 20.19 19.06 tinuvin 20.50 21.64 19.97 21.04 water 18.47 18.41 20.13 20.39 536 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.425 0.406 0.299 1.665 d.f. 20 72 288 35.90 Except when comparing means with the same level(s) of exposure 0.993 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.560 0.658 2.093 d.f. 28.62 299.24 86.05 Except when comparing means with the same level(s) of exposure 0.732 1.611 d.f. 288 299.24 treatment 0.598 d.f. 288 exposure.treatment 1.464 d.f. 288 exposure.Chem_Charge 1.611 d.f. 299.24 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 2.973 0.808 0.588 3.377 d.f. 20 72 288 35.90 Except when comparing means with the same level(s) of exposure 1.980 d.f. 72 537 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 3.192 1.294 4.160 d.f. 28.62 299.24 86.05 Except when comparing means with the same level(s) of exposure 1.441 3.170 d.f. 288 299.24 treatment 1.177 d.f. 288 exposure.treatment 2.882 d.f. 288 exposure.Chem_Charge 3.170 d.f. 299.24 Analysis of variance week 40 Variate: L Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 6832.59 1708.15 7.64 block.board stratum exposure 5 9144.39 1828.88 8.18 <.001 Residual 20 4472.72 223.64 7.05 block.board.area stratum treatment 3 100.59 33.53 1.06 0.373 exposure.treatment 15 367.88 24.53 0.77 0.702 Residual 72 2283.97 31.72 2.44 block.board.area.Strip stratum Chem_Charge 3 33.42 11.14 0.86 0.464 exposure.Chem_Charge 15 290.32 19.35 1.49 0.109 treatment.Chem_Charge 9 125.30 13.92 1.07 0.385 exposure.treatment.Chem_Charge 45 801.50 17.81 1.37 0.068 Residual 288 3748.45 13.02 Total 479 28201.14 538 Message: the following units have large residuals. block 2 board 3 -6.26 s.e. 3.05 block 2 board 4 6.69 s.e. 3.05 block 2 board 3 area 3 5.57 s.e. 2.18 block 4 board 3 area 3 -5.62 s.e. 2.18 block 5 board 1 area 4 6.40 s.e. 2.18 block 1 board 5 area 2 Strip 3 9.37 s.e. 2.79 block 1 board 6 area 4 Strip 2 10.49 s.e. 2.79 block 5 board 3 area 3 Strip 3 -8.48 s.e. 2.79 Tables of means Variate: L Grand mean 54.64 exposure full I.R. none UVA UVB Vis light 45.18 56.39 58.00 54.62 57.53 56.10 treatment acetic acid carpropamid tinuvin water 54.15 54.52 55.39 54.49 Chem_Charge high low medium Very high 54.89 54.23 54.58 54.84 exposure treatment acetic acid carpropamid tinuvin water full 45.20 44.33 45.41 45.78 I.R. 55.63 57.28 57.81 54.84 none 58.02 57.29 59.89 56.78 UVA 53.10 54.98 56.35 54.04 UVB 57.23 57.13 56.40 59.37 Vis light 55.73 56.09 56.48 56.10 exposure Chem_Charge high low medium Very high full 45.94 44.94 44.28 45.56 I.R. 57.95 56.65 55.37 55.59 none 56.34 57.16 59.92 58.56 UVA 55.04 54.14 54.82 54.46 UVB 58.05 56.56 57.03 58.50 Vis light 56.03 55.91 56.08 56.39 treatment Chem_Charge high low medium Very high acetic acid 54.14 54.41 54.12 53.95 carpropamid 54.87 53.58 54.57 55.06 tinuvin 56.30 55.55 54.47 55.24 water 54.27 53.38 55.17 55.12 539 exposure treatment Chem_Charge high low medium Very high full acetic acid 46.99 43.22 45.02 45.58 carpropamid 46.43 43.53 43.52 43.85 tinuvin 43.99 47.63 42.73 47.30 water 46.36 45.39 45.86 45.51 I.R. acetic acid 58.02 55.80 54.65 54.05 carpropamid 58.31 58.05 54.52 58.24 tinuvin 60.06 57.63 57.47 56.07 water 55.40 55.11 54.84 54.02 none acetic acid 57.35 56.79 59.80 58.15 carpropamid 56.04 55.11 60.46 57.56 tinuvin 58.17 61.13 61.84 58.44 water 53.82 55.61 57.59 60.11 UVA acetic acid 52.15 54.20 53.34 52.73 carpropamid 52.56 55.16 55.31 56.90 tinuvin 60.62 55.40 53.93 55.44 water 54.85 51.82 56.69 52.79 UVB acetic acid 55.02 59.83 57.00 57.08 carpropamid 61.34 53.12 57.09 56.98 tinuvin 57.51 54.76 54.05 59.27 water 58.31 58.54 59.98 60.65 Vis light acetic acid 55.29 56.60 54.91 56.13 carpropamid 54.52 56.48 56.50 56.84 tinuvin 57.46 56.76 56.82 54.90 water 56.86 53.80 56.07 57.68 540 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 2.365 0.727 0.466 2.823 d.f. 20 72 288 38.70 Except when comparing means with the same level(s) of exposure 1.781 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 2.563 1.086 3.446 d.f. 27.54 259.90 83.22 Except when comparing means with the same level(s) of exposure 1.141 2.660 d.f. 288 259.90 treatment 0.932 d.f. 288 exposure.treatment 2.282 d.f. 288 exposure.Chem_Charge 2.660 d.f. 259.90 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 4.932 1.449 0.917 5.712 d.f. 20 72 288 38.70 Except when comparing means with the same level(s) of exposure 3.550 d.f. 72 541 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 5.253 2.139 6.854 d.f. 27.54 259.90 83.22 Except when comparing means with the same level(s) of exposure 2.245 5.238 d.f. 288 259.90 treatment 1.833 d.f. 288 exposure.treatment 4.491 d.f. 288 exposure.Chem_Charge 5.238 d.f. 259.90 Analysis of variance Variate: a Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 23.3843 5.8461 0.37 block.board stratum exposure 5 865.9996 173.1999 11.04 <.001 Residual 20 313.6738 15.6837 8.19 block.board.area stratum treatment 3 8.4121 2.8040 1.46 0.231 exposure.treatment 15 27.4774 1.8318 0.96 0.508 Residual 72 137.8261 1.9143 2.23 block.board.area.Strip stratum Chem_Charge 3 4.1611 1.3870 1.61 0.186 exposure.Chem_Charge 15 10.9747 0.7316 0.85 0.619 treatment.Chem_Charge 9 12.6021 1.4002 1.63 0.106 exposure.treatment.Chem_Charge 45 53.5583 1.1902 1.39 0.061 Residual 288 247.4463 0.8592 Total 479 1705.5157 542 Message: the following units have large residuals. block 1 board 2 2.450 s.e. 0.808 block 1 board 3 area 1 2.058 s.e. 0.536 block 1 board 3 area 3 -1.464 s.e. 0.536 block 3 board 5 area 2 -1.424 s.e. 0.536 block 4 board 3 area 3 1.531 s.e. 0.536 block 1 board 2 area 1 Strip 3 2.935 s.e. 0.718 block 1 board 2 area 2 Strip 2 2.254 s.e. 0.718 block 1 board 3 area 1 Strip 2 -2.429 s.e. 0.718 block 1 board 3 area 1 Strip 4 2.733 s.e. 0.718 block 2 board 3 area 3 Strip 2 2.661 s.e. 0.718 block 2 board 5 area 1 Strip 3 2.119 s.e. 0.718 block 4 board 2 area 4 Strip 1 2.128 s.e. 0.718 block 5 board 2 area 1 Strip 4 2.227 s.e. 0.718 Tables of means Variate: a Grand mean 3.751 exposure full I.R. none UVA UVB Vis light 1.202 3.851 3.108 5.255 4.106 4.983 treatment acetic acid carpropamid tinuvin water 3.836 3.540 3.886 3.741 Chem_Charge high low medium Very high 3.591 3.790 3.819 3.804 exposure treatment acetic acid carpropamid tinuvin water full 1.213 1.242 1.189 1.165 I.R. 4.316 3.610 3.415 4.064 none 3.268 3.035 3.046 3.085 UVA 5.257 4.992 5.601 5.170 UVB 4.202 3.417 4.652 4.150 Vis light 4.759 4.944 5.414 4.816 exposure Chem_Charge high low medium Very high full 1.075 1.197 1.312 1.226 I.R. 3.934 3.732 3.890 3.848 none 2.825 3.481 3.191 2.936 UVA 4.960 5.120 5.419 5.521 UVB 4.008 3.925 4.231 4.258 Vis light 4.742 5.287 4.871 5.034 543 treatment Chem_Charge high low medium Very high acetic acid 3.613 3.724 3.943 4.063 carpropamid 3.505 3.783 3.541 3.331 tinuvin 3.695 3.860 4.222 3.769 water 3.550 3.794 3.570 4.052 exposure treatment Chem_Charge high low medium Very high full acetic acid 1.038 1.188 1.312 1.312 carpropamid 1.254 1.252 1.360 1.102 tinuvin 1.250 0.908 1.324 1.274 water 0.756 1.438 1.252 1.214 I.R. acetic acid 3.966 4.158 4.934 4.206 carpropamid 3.704 3.772 3.146 3.818 tinuvin 3.388 3.246 3.806 3.220 water 4.680 3.754 3.674 4.148 none acetic acid 2.982 3.748 3.184 3.156 carpropamid 2.744 3.134 3.434 2.830 tinuvin 2.778 3.348 3.134 2.924 water 2.798 3.696 3.012 2.834 UVA acetic acid 4.962 5.006 5.616 5.444 carpropamid 5.106 5.726 5.010 4.124 tinuvin 5.426 4.468 6.322 6.188 water 4.346 5.278 4.728 6.326 UVB acetic acid 4.290 3.306 4.358 4.856 carpropamid 3.394 3.782 3.244 3.248 tinuvin 4.008 4.716 5.704 4.182 water 4.338 3.898 3.618 4.746 Vis light acetic acid 4.442 4.938 4.252 5.406 carpropamid 4.826 5.034 5.054 4.862 tinuvin 5.318 6.474 5.040 4.824 water 4.380 4.700 5.136 5.046 544 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 0.6262 0.1786 0.1197 0.7319 d.f. 20 72 288 35.99 Except when comparing means with the same level(s) of exposure 0.4375 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 0.6757 0.2736 0.8907 d.f. 27.06 272.79 76.73 Except when comparing means with the same level(s) of exposure 0.2931 0.6702 d.f. 288 272.79 treatment 0.2393 d.f. 288 exposure.treatment 0.5862 d.f. 288 exposure.Chem_Charge 0.6702 d.f. 272.79 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 1.3062 0.3561 0.2355 1.4844 d.f. 20 72 288 35.99 Except when comparing means with the same level(s) of exposure 0.8722 d.f. 72 545 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 1.3862 0.5387 1.7738 d.f. 27.06 272.79 76.73 Except when comparing means with the same level(s) of exposure 0.5769 1.3194 d.f. 288 272.79 treatment 0.4711 d.f. 288 exposure.treatment 1.1539 d.f. 288 exposure.Chem_Charge 1.3194 d.f. 272.79 Analysis of variance Variate: b Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 301.434 75.358 0.84 block.board stratum exposure 5 12379.447 2475.889 27.53 <.001 Residual 20 1798.434 89.922 11.87 block.board.area stratum treatment 3 93.004 31.001 4.09 0.010 exposure.treatment 15 127.254 8.484 1.12 0.355 Residual 72 545.474 7.576 2.00 block.board.area.Strip stratum Chem_Charge 3 8.948 2.983 0.79 0.502 exposure.Chem_Charge 15 61.849 4.123 1.09 0.367 treatment.Chem_Charge 9 35.524 3.947 1.04 0.407 exposure.treatment.Chem_Charge 45 181.090 4.024 1.06 0.374 Residual 288 1091.413 3.790 Total 479 16623.870 546 Message: the following units have large residuals. block 1 board 2 5.284 s.e. 1.936 block 1 board 3 area 1 4.031 s.e. 1.066 block 1 board 3 area 3 -3.094 s.e. 1.066 block 2 board 2 area 3 -2.654 s.e. 1.066 block 2 board 5 area 3 2.757 s.e. 1.066 block 1 board 2 area 1 Strip 3 6.143 s.e. 1.508 block 1 board 2 area 1 Strip 4 -4.583 s.e. 1.508 block 1 board 3 area 1 Strip 2 -4.805 s.e. 1.508 block 1 board 5 area 1 Strip 1 5.331 s.e. 1.508 block 1 board 6 area 4 Strip 2 4.445 s.e. 1.508 block 2 board 3 area 3 Strip 2 4.990 s.e. 1.508 block 5 board 2 area 1 Strip 4 5.425 s.e. 1.508 Tables of means Variate: b Grand mean 14.978 exposure full I.R. none UVA UVB Vis light 4.577 18.063 17.453 18.003 12.715 19.057 treatment acetic acid carpropamid tinuvin water 14.948 14.461 15.674 14.830 Chem_Charge high low medium Very high 14.750 14.998 15.102 15.061 exposure treatment acetic acid carpropamid tinuvin water full 4.441 4.654 4.602 4.609 I.R. 18.701 17.768 17.668 18.116 none 17.765 17.025 17.784 17.239 UVA 17.377 17.296 19.557 17.784 UVB 12.782 11.164 14.351 12.562 Vis light 18.620 18.857 20.081 18.667 exposure Chem_Charge high low medium Very high full 4.498 4.450 4.745 4.614 I.R. 18.558 18.095 17.733 17.868 none 16.575 17.908 18.062 17.268 UVA 17.535 17.683 18.435 18.360 UVB 12.725 12.203 12.720 13.213 Vis light 18.612 19.649 18.920 19.045 547 treatment Chem_Charge high low medium Very high acetic acid 14.637 14.765 15.155 15.233 carpropamid 14.268 14.859 14.550 14.166 tinuvin 15.602 15.584 16.143 15.367 water 14.495 14.784 14.560 15.479 exposure treatment Chem_Charge high low medium Very high full acetic acid 4.086 4.440 4.574 4.662 carpropamid 4.794 4.448 5.060 4.314 tinuvin 4.894 3.624 4.854 5.038 water 4.218 5.288 4.490 4.442 I.R. acetic acid 18.432 18.704 19.452 18.216 carpropamid 17.904 18.534 16.226 18.408 tinuvin 18.450 17.430 17.978 16.814 water 19.446 17.710 17.274 18.034 none acetic acid 17.034 18.420 18.162 17.442 carpropamid 16.094 16.662 18.600 16.744 tinuvin 17.048 18.610 18.338 17.140 water 16.124 17.938 17.146 17.748 UVA acetic acid 16.524 16.970 18.336 17.676 carpropamid 17.032 18.800 17.330 16.020 tinuvin 19.994 17.456 20.352 20.428 water 16.590 17.508 17.722 19.316 UVB acetic acid 13.618 10.854 12.698 13.960 carpropamid 11.436 11.556 10.746 10.920 tinuvin 13.214 14.488 15.770 13.933 water 12.632 11.914 11.666 14.038 Vis light acetic acid 18.128 19.200 17.710 19.444 carpropamid 18.346 19.156 19.340 18.588 tinuvin 20.014 21.894 19.568 18.850 water 17.960 18.348 19.064 19.298 548 Standard errors of differences of means Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 s.e.d. 1.4993 0.3553 0.2513 1.6782 d.f. 20 72 288 30.84 Except when comparing means with the same level(s) of exposure 0.8704 d.f. 72 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 s.e.d. 1.5913 0.5619 1.9882 d.f. 25.35 288.06 59.72 Except when comparing means with the same level(s) of exposure 0.6156 1.3764 d.f. 288 288.06 treatment 0.5026 d.f. 288 exposure.treatment 1.2312 d.f. 288 exposure.Chem_Charge 1.3764 d.f. 288.06 Least significant differences of means (5% level) Table exposure treatment Chem_Charge exposure treatment rep. 80 120 120 20 l.s.d. 3.1276 0.7084 0.4947 3.4234 d.f. 20 72 288 30.84 Except when comparing means with the same level(s) of exposure 1.7351 d.f. 72 549 Table exposure treatment exposure Chem_Charge Chem_Charge treatment Chem_Charge rep. 20 30 5 l.s.d. 3.2751 1.1060 3.9775 d.f. 25.35 288.06 59.72 Except when comparing means with the same level(s) of exposure 1.2116 2.7091 d.f. 288 288.06 treatment 0.9893 d.f. 288 exposure.treatment 2.4233 d.f. 288 exposure.Chem_Charge 2.7091 d.f. 288.06 550 Appendix 4: Images of fungal colonization evolution in southern pine samples exposed under filter transmitting different wavelengths of solar radiation (Chapter 5) 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 40 551 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 40 552 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 40 553 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 40 554 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 40 555 Appendix 5: Result for reciprocal Simpson index (Chapter 5) Table A5.1: reciprocal diversity Simpson index for fungi isolated from weathered southern pine samples exposed outdoors under different filters for 40 weeks Simpson index Rack UVB+UVA+Vis.light+IR transmitted UVA+Vis.light+IR transmitted Vis. light+IR transmitted IR transmitted No light transmitted 1 4 5 4 5 4 2 4 5 5 4 4 3 9 4 5 5 5 4 4 5 4 7 2.3 5 3 4 7 5 2.3 Ave. 3 4.8 4.6 5 5.2 SD 1.6 2.4 0.5 1.2 1.1 556 Appendix 6: Statistical analysis Chapter 6 Analysis of variance fungal biomass Variate: grams of biomass Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 5 2.851E-05 5.701E-06 7.34 block.*Units* stratum exposure 2 1.607E-04 8.035E-05 103.38 <.001 fungi 5 1.754E-04 3.508E-05 45.14 <.001 exposure.fungi 10 1.460E-04 1.460E-05 18.79 <.001 Residual 83 (2) 6.451E-05 7.772E-07 Total 105 (2) 5.724E-04 Message: the following units have large residuals. block 2 *units* 5 0.002343 s.e. 0.000773 block 2 *units* 14 0.001943 s.e. 0.000773 block 4 *units* 15 0.002004 s.e. 0.000773 Tables of means Variate: grams Grand mean 0.003421 exposure 1 2 3 0.001781 0.003778 0.004704 fungi 1 2 3 4 5 6 0.003985 0.005117 0.002556 0.001466 0.004656 0.002744 exposure fungi 1 2 3 4 5 6 1 0.003350 0.004050 0.000000 0.001000 0.002283 0.000000 2 0.003850 0.007417 0.003250 0.001416 0.003717 0.003017 3 0.004756 0.003883 0.004417 0.001983 0.007967 0.005217 557 Standard errors of differences of means Table exposure fungi exposure fungi rep. 36 18 6 d.f. 83 83 83 s.e.d. 0.0002078 0.0002939 0.0005090 (Not adjusted for missing values) Least significant differences of means (5% level) Table exposure fungi exposure fungi rep. 36 18 6 d.f. 83 83 83 l.s.d. 0.0004133 0.0005845 0.0010124 (Not adjusted for missing values) Analysis of variance lightness fungal mycelia Variate: L Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 5 270.36 54.07 2.19 block.*Units* stratum exposure 2 4078.05 2039.03 82.41 <.001 fungi 5 3346.93 669.39 27.05 <.001 exposure.fungi 8 (2) 1893.98 236.75 9.57 <.001 Residual 75 (10) 1855.79 24.74 Total 95 (12) 11331.83 Message: the following units have large residuals. block 1 *units* 13 14.46 s.e. 4.15 block 2 *units* 13 -10.48 s.e. 4.15 block 3 *units* 13 -14.48 s.e. 4.15 558 Tables of means Variate: L Grand mean 20.70 exposure 1 2 3 12.86 21.39 27.86 fungi 1 2 3 4 5 6 17.89 13.39 25.32 17.67 19.61 30.34 exposure fungi 1 2 3 4 5 6 1 13.83 9.00 17.47 7.50 6.83 22.51 2 18.50 6.83 33.00 16.67 19.00 34.33 3 21.33 24.33 25.50 28.83 33.00 34.17 Standard errors of differences of means Table exposure fungi exposure fungi rep. 36 18 6 d.f. 75 75 75 s.e.d. 1.172 1.658 2.872 (Not adjusted for missing values) Least significant differences of means (5% level) Table exposure fungi exposure fungi rep. 36 18 6 d.f. 75 75 75 l.s.d. 2.336 3.303 5.721 (Not adjusted for missing values) Mean fungi 1 2 3 4 5 exposure 1 13.83 9.00 * 7.50 6.83 2 18.50 6.83 33.00 16.67 19.00 3 21.33 24.33 25.50 28.83 33.00 fungi 6 exposure 1 * 2 34.33 3 34.17 559 Analysis of variance melanin concentration Variate: mg_melanin_mg_biomass_new Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 5 0.087432 0.017486 3.81 block.*Units* stratum exposure 2 0.033081 0.016540 3.60 0.032 fungi 5 0.320606 0.064121 13.96 <.001 exposure.fungi 10 0.123496 0.012350 2.69 0.007 Residual 81 (4) 0.372158 0.004595 Total 103 (4) 0.901307 Message: the following units have large residuals. block 2 *units* 10 -0.1708 s.e. 0.0587 block 2 *units* 15 0.1633 s.e. 0.0587 Tables of means Variate: mg_melanin_mg_biomass_new Grand mean 0.0826 exposure 1 2 3 0.0983 0.0913 0.0581 fungi 1 2 3 4 5 6 0.0861 0.0961 0.0447 0.1900 0.0606 0.0180 exposure fungi 1 2 3 4 5 6 1 0.1089 0.1455 0.0000 0.2067 0.1285 0.0000 2 0.0906 0.1070 0.0373 0.2325 0.0456 0.0346 3 0.0586 0.0357 0.0967 0.1307 0.0077 0.0194 Standard errors of differences of means Table exposure fungi exposure fungi rep. 36 18 6 d.f. 81 81 81 s.e.d. 0.01598 0.02259 0.03913 (Not adjusted for missing values) 560 Least significant differences of means (5% level) Table exposure fungi exposure fungi rep. 36 18 6 d.f. 81 81 81 l.s.d. 0.03179 0.04496 0.07787 (Not adjusted for missing values) Analysis of variance radial growth Variate: ln_mm (natural logarithm radial growth (mm)) Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 5 0.86397 0.17279 2.75 block.*Units* stratum exposure 2 23.72496 11.86248 188.65 <.001 fungi 5 2.34376 0.46875 7.45 <.001 exposure.fungi 10 4.76523 0.47652 7.58 <.001 Residual 85 5.34476 0.06288 Total 107 37.04268 Message: the following units have large residuals. block 2 *units* 1 0.687 s.e. 0.222 block 5 *units* 5 0.723 s.e. 0.222 block 6 *units* 13 -0.708 s.e. 0.222 Tables of means Variate: ln_mm Grand mean 1.004 exposure 1 2 3 0.344 1.385 1.283 fungi 1 2 3 4 5 6 0.972 0.819 1.045 1.288 0.892 1.008 561 exposure fungi 1 2 3 4 5 6 1 0.359 0.548 0.000 0.877 0.278 0.000 2 1.289 0.898 1.566 1.587 1.358 1.609 3 1.270 1.009 1.569 1.399 1.039 1.415 Standard errors of differences of means Table exposure fungi exposure fungi rep. 36 18 6 d.f. 85 85 85 s.e.d. 0.0591 0.0836 0.1448 Least significant differences of means (5% level) Table exposure fungi exposure fungi rep. 36 18 6 d.f. 85 85 85 l.s.d. 0.1175 0.1662 0.2879 Analysis of variance spore concentration Variate: ln_spore (natural ogarithm spore concentration) Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. block stratum 5 5.5762 1.1152 4.57 block.*Units* stratum exposure 2 73.4410 36.7205 150.62 <.001 fungi 5 107.6042 21.5208 88.27 <.001 exposure.fungi 10 79.3314 7.9331 32.54 <.001 Residual 84 (1) 20.4787 0.2438 Total 106 (1) 286.2279 562 Tables of means Variate: ln_spore Grand mean 3.432 exposure 1 2 3 2.281 4.170 3.845 fungi 1 2 3 4 5 6 2.987 2.834 2.390 5.204 4.346 2.830 exposure fungi 1 2 3 4 5 6 1 2.543 2.305 0.000 4.869 3.968 0.000 2 3.153 2.251 4.140 5.411 5.307 4.758 3 3.265 3.946 3.030 5.332 3.762 3.734 Standard errors of differences of means Table exposure fungi exposure fungi rep. 36 18 6 d.f. 84 84 84 s.e.d. 0.1164 0.1646 0.2851 (Not adjusted for missing values) Least significant differences of means (5% level) Table exposure fungi exposure fungi rep. 36 18 6 d.f. 84 84 84 l.s.d. 0.2314 0.3273 0.5669 (Not adjusted for missing values) 563 Appendix 7: Calibration curves for calculation of fungal melanin concentration (Chapter 6) Table A7.1: UV-VIS light absorbance and concentration of fungal melanin produced by C. cladiosporiodes [R2F33] Fungi 1 Clad. [R2F33] Concentration ABS mg/g 0.0004 6.3073 0.063419 (1:1) 0.1495 0.063419 (1:2) 0.081467 0.031709 (1:5) 0.031133 0.012684 (1:10) 0.015 0.006342 (1:20) 0.007233 0.003171 (1:50) 0.004667 0.001268 (1:70) 0.003667 0.000906 (1:100) 0.0016 0.000634 (1:200) 0.0009 0.000317 Figure A7.1: Calibration curve absorbance vs concentration C. cladiosporioides y = 0.4195x - 0.0004 R\u00C2\u00B2 = 0.9982 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 564 Table A7.2: UV-VIS light absorbance and concentration of fungal melanin produced by A. pullulans [R2F32.2] Fungi 2 A. pull. [R2F32.2 ] Concentration ABS mg/g 0.0004 6.3073 0.063419 (1:1) 0.098667 0.063419 (1:2) 0.048167 0.031709 (1:5) 0.0199 0.012684 (1:10) 0.012067 0.006342 (1:20) 0.005967 0.003171 (1:50) 0.002867 0.001268 (1:70) 0.001833 0.000906 (1:100) 0.001233 0.000634 (1:200) 0.0009 0.000317 Figure A7.2: Calibration curve absorbance vs concentration A. pullulans [R2F32.2] y = 0.6507x - 0.0005 R\u00C2\u00B2 = 0.9994 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0 0.02 0.04 0.06 0.08 0.1 0.12 565 Table A7.3: UV-VIS light absorbance and concentration of fungal melanin produced by O. piliferum [TAB28] Fungi 3 O. pilif. [TAB28] Concentration ABS mg/g 0.0002 6.3073 0.031709 (1:1) 0.028433 0.031709 (1:2) 0.011667 0.015855 (1:5) 0.006 0.006342 (1:10) 0.003167 0.003171 (1:20) 0.0009 0.001585 (1:50) 0.000167 0.000634 (1:70) 6.67E-05 0.000453 (1:100) 0.000667 0.000317 (1:200) 0.0004 0.000159 Figure A7.3: Calibration curve absorbance vs concentration O. piliferum [TAB28] y = 1.1353x + 0.0002 R\u00C2\u00B2 = 0.9911 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0 0.005 0.01 0.015 0.02 0.025 0.03 566 Table A7.4: UV-VIS light absorbance and concentration of fungal melanin produced by A. pullulans [ATCC 42371] Fungi 4 A. pull. [ATCC 42371] Concentration ABS mg/g 0.0003 6.3073 0.047564 (1:1) 0.067767 0.047564 (1:2) 0.0341 0.023782 (1:5) 0.0153 0.009513 (1:10) 0.009033 0.004756 (1:20) 0.0058 0.002378 (1:50) 0.003933 0.000951 (1:70) 0.002933 0.000679 (1:100) 0.003233 0.000476 (1:200) 0.002133 0.000238 Figure A7.4: Calibration curve absorbance vs concentration A. pullulans [ATCC 42371] y = 0.7298x - 0.0017 R\u00C2\u00B2 = 0.9997 -0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 567 Table A7.5: UV-VIS light absorbance and concentration of fungal melanin produced by A. pullulans [R1F22W] Fungi 5 A. pull. [R1F22W] Concentration ABS mg/g 0.0008 6.3073 0.126837 (1:1) 0.474367 0.126837 (1:2) 0.239033 0.063419 (1:5) 0.0966 0.025367 (1:10) 0.048633 0.012684 (1:20) 0.027267 0.006342 (1:50) 0.012433 0.002537 (1:70) 0.008133 0.001812 (1:100) 0.0059 0.001268 (1:200) 0.0034 0.000634 Figure A7.5: Calibration curve absorbance vs concentration A. pullulans [R1F22W] y = 0.2683x - 0.0005 R\u00C2\u00B2 = 1 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0 0.1 0.2 0.3 0.4 0.5 568 Table A7.6: UV-VIS light absorbance and concentration of fungal melanin produced by O. piliferum [Cartapip97] Fungi 6 O. pilif. [Cartapip97] Concentration ABS mg/g 0.0002 6.3073 0.031709 (1:1) 0.0157 0.031709 (1:2) 0.009 0.015855 (1:5) 0.0047 0.006342 (1:10) 0.0026 0.003171 (1:20) 0.0026 0.001585 (1:50) 0.002333 0.000634 (1:70) 0.0003 0.000453 (1:100) 0.0002 0.000317 (1:200) 0.002 0.000159 Figure A7.6: Calibration curve absorbance vs concentration O. piliferum [Cartapip97] y = 2.1052x - 0.0025 R\u00C2\u00B2 = 0.9764 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0 0.005 0.01 0.015 0.02 569 Appendix 8: Statistical analysis melanin biosynthesis inhibitors tested in artificial media (Chapter 7) Analysis of variance fungal colonies in plates after exposure artificial media Variate: Number of colonies Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Block stratum 4 80919. 20230. 9.86 Block.*Units* stratum Fungi 1 116459. 116459. 56.77 <.001 Exposure 1 71344. 71344. 34.78 <.001 chemical 4 106128. 26532. 12.93 <.001 Fungi.Exposure 1 32781. 32781. 15.98 <.001 Fungi.chemical 4 17082. 4270. 2.08 0.092 Exposure.chemical 4 54044. 13511. 6.59 <.001 Fungi.Exposure.chemical 4 12783. 3196. 1.56 0.195 Residual 72 (4) 147692. 2051. Total 95 (4) 609273. Message: the following units have large residuals. Block 1 *units* 1 96.1 s.e. 38.4 Block 1 *units* 7 99.7 s.e. 38.4 Block 1 *units* 12 98.3 s.e. 38.4 Block 5 *units* 5 102.3 s.e. 38.4 Tables of means Variate: Colonies Grand mean 118.2 Fungi A pull Clad 84.1 152.3 Exposure uv v 91.5 144.9 chemical Carp Cer Control Quin Tricy 98.4 106.9 157.7 74.1 154.0 570 Fungi Exposure uv v A pull 75.5 92.7 Clad 107.5 197.2 Fungi chemical Carp Cer Control Quin Tricy A pull 66.8 87.1 99.9 48.7 117.9 Clad 130.0 126.6 215.5 99.5 190.1 Exposure chemical Carp Cer Control Quin Tricy uv 53.3 87.7 160.0 12.8 143.7 v 143.5 126.0 155.4 135.4 164.3 Fungi Exposure chemical Carp Cer Control Quin Tricy A pull uv 50.4 95.0 110.4 13.0 108.6 v 83.2 79.2 89.4 84.4 127.2 Clad uv 56.2 80.4 209.6 12.6 178.8 v 203.8 172.8 221.4 186.4 201.4 Standard errors of differences of means Table Fungi Exposure chemical Fungi Exposure rep. 50 50 20 25 d.f. 72 72 72 72 s.e.d. 9.06 9.06 14.32 12.81 Table Fungi Exposure Fungi chemical chemical Exposure chemical rep. 10 10 5 d.f. 72 72 72 s.e.d. 20.25 20.25 28.64 (Not adjusted for missing values) Least significant differences of means (5% level) Table Fungi Exposure chemical Fungi Exposure rep. 50 50 20 25 d.f. 72 72 72 72 l.s.d. 18.06 18.06 28.55 25.54 Table Fungi Exposure Fungi chemical chemical Exposure chemical rep. 10 10 5 d.f. 72 72 72 l.s.d. 40.38 40.38 57.10 (Not adjusted for missing values) 571 Appendix 9: Statistical analysis melanin biosynthesis inhibitors tested in wood veneers (Chapter 7) Analysis of variance color differences veneers inoculated Variate: Color difference (Delta E) fungi inoculated veneers Source of variation d.f. s.s. m.s. v.r. F pr. Block stratum 4 10.134 2.534 2.35 Block.*Units* stratum exposure 1 2.243 2.243 2.08 0.160 Chemical 1 16.814 16.814 15.59 <.001 Concentration 1 1.379 1.379 1.28 0.268 exposure.Chemical 1 0.490 0.490 0.45 0.506 exposure.Concentration 1 0.975 0.975 0.90 0.350 Chemical.Concentration 1 0.174 0.174 0.16 0.691 exposure.Chemical.Concentration 1 0.013 0.013 0.01 0.913 Residual 28 30.192 1.078 Total 39 62.414 Message: the following units have large residuals. Block 1 *units* 8 -1.97 s.e. 0.87 Block 2 *units* 6 -1.85 s.e. 0.87 Tables of means Variate: Delta_E_F Grand mean 2.10 exposure UV V 1.87 2.34 Chemical Carp Qui 2.75 1.45 Concentration 3000 6000 1.92 2.29 572 exposure Chemical Carp Qui UV 2.40 1.33 V 3.10 1.58 exposure Concentration 3000 6000 UV 1.52 2.21 V 2.31 2.37 Chemical Concentration 3000 6000 Carp 2.50 3.00 Qui 1.34 1.57 Chemical Carp Qui exposure Concentration 3000 6000 3000 6000 UV 1.98 2.83 1.07 1.59 V 3.02 3.18 1.60 1.56 Standard errors of differences of means Table exposure Chemical Concentration exposure Chemical rep. 20 20 20 10 d.f. 28 28 28 28 s.e.d. 0.328 0.328 0.328 0.464 Table exposure Chemical exposure Concentration Concentration Chemical Concentration rep. 10 10 5 d.f. 28 28 28 s.e.d. 0.464 0.464 0.657 573 Analysis of variance color differences veneers inoculated vs not inoculated Variate: Color differences (Delta E) veneers inoculated vs not inoculated Source of variation d.f. s.s. m.s. v.r. F pr. Block stratum 4 30.194 7.548 6.09 Block.*Units* stratum exposure 1 1.619 1.619 1.31 0.259 Chemical 1 54.507 54.507 43.96 <.001 Concentration 2 23.633 11.817 9.53 <.001 exposure.Chemical 1 0.688 0.688 0.55 0.460 exposure.Concentration 2 5.527 2.764 2.23 0.120 Chemical.Concentration 2 26.156 13.078 10.55 <.001 exposure.Chemical.Concentration 2 1.410 0.705 0.57 0.570 Residual 44 54.560 1.240 Total 59 198.295 Tables of means Variate: Delta_E_F_vs_NF Grand mean 3.12 exposure UV V 3.29 2.96 Chemical Carp Qui 2.17 4.07 Concentration 0 3000 6000 4.00 2.76 2.60 exposure Chemical Carp Qui UV 2.23 4.35 V 2.11 3.80 exposure Concentration 0 3000 6000 UV 3.98 3.35 2.53 V 4.03 2.16 2.68 Chemical Concentration 0 3000 6000 Carp 3.87 1.78 0.85 Qui 4.14 3.73 4.36 574 Chemical Carp Qui exposure Concentration 0 3000 6000 0 3000 6000 UV 3.63 2.16 0.88 4.33 4.53 4.17 V 4.11 1.41 0.82 3.95 2.92 4.54 Standard errors of differences of means Table exposure Chemical Concentration exposure Chemical rep. 30 30 20 15 d.f. 44 44 44 44 s.e.d. 0.288 0.288 0.352 0.407 Table exposure Chemical exposure Concentration Concentration Chemical Concentration rep. 10 10 5 d.f. 44 44 44 s.e.d. 0.498 0.498 0.704 Least significant differences of means (5% level) Table exposure Chemical Concentration exposure Chemical rep. 30 30 20 15 d.f. 44 44 44 44 l.s.d. 0.579 0.579 0.710 0.819 Table exposure Chemical exposure Concentration Concentration Chemical Concentration rep. 10 10 5 d.f. 44 44 44 l.s.d. 1.004 1.004 1.419 575 Analysis of variance color differences veneers not inoculated Variate: Color differences (delta E) veneer not inoculated Source of variation d.f. s.s. m.s. v.r. F pr. Block stratum 4 1.0189 0.2547 0.33 Block.*Units* stratum exposure 1 0.2412 0.2412 0.31 0.583 Chemical 1 0.0147 0.0147 0.02 0.892 Concentration 1 0.2225 0.2225 0.28 0.598 exposure.Chemical 1 0.0231 0.0231 0.03 0.865 exposure.Concentration 1 0.0914 0.0914 0.12 0.735 Chemical.Concentration 1 0.0470 0.0470 0.06 0.808 exposure.Chemical.Concentration 1 0.0081 0.0081 0.01 0.920 Residual 28 21.9259 0.7831 Total 39 23.5928 Message: the following units have large residuals. Block 1 *units* 2 -1.63 s.e. 0.74 Block 1 *units* 5 1.87 s.e. 0.74 Block 1 *units* 7 1.82 s.e. 0.74 Tables of means Variate: Delta_E_NF Grand mean 1.33 exposure UV V 1.41 1.26 Chemical Carp Qui 1.35 1.31 Concentration 3000 6000 1.26 1.41 exposure Chemical Carp Qui UV 1.41 1.42 V 1.30 1.21 576 exposure Concentration 3000 6000 UV 1.29 1.53 V 1.23 1.28 Chemical Concentration 3000 6000 Carp 1.31 1.39 Qui 1.20 1.42 Chemical Carp Qui exposure Concentration 3000 6000 3000 6000 UV 1.33 1.48 1.24 1.59 V 1.29 1.30 1.17 1.26 Standard errors of differences of means Table exposure Chemical Concentration exposure Chemical rep. 20 20 20 10 d.f. 28 28 28 28 s.e.d. 0.280 0.280 0.280 0.396 Table exposure Chemical exposure Concentration Concentration Chemical Concentration rep. 10 10 5 d.f. 28 28 28 s.e.d. 0.396 0.396 0.560 577 Analysis of variance fungal stain ratio Variate: natural logarithm ratio of fungal stains (lnrat) Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Block stratum 4 0.81069 0.20267 4.11 Block.*Units* stratum exposure 1 0.03632 0.03632 0.74 0.398 Chemical 1 7.97230 7.97230 161.75 <.001 Concentration_ppm 1 0.02397 0.02397 0.49 0.492 exposure.Chemical 1 0.13816 0.13816 2.80 0.106 exposure.Concentration_ppm 1 0.01718 0.01718 0.35 0.560 Chemical.Concentration_ppm 1 0.09800 0.09800 1.99 0.170 exposure.Chemical.Concentration_ppm 1 0.00936 0.00936 0.19 0.666 Residual 27 (1) 1.33073 0.04929 Total 38 (1) 10.23561 Message: the following units have large residuals. Block 2 *units* 8 0.398 s.e. 0.182 Block 4 *units* 7 -0.513 s.e. 0.182 Tables of means Variate: lnrat Grand mean 0.597 exposure UV V 0.627 0.567 Chemical Carp Qui 0.151 1.043 Concentration_ppm 3000 6000 0.572 0.621 exposure Chemical Carp Qui UV 0.239 1.015 V 0.062 1.072 578 exposure Concentration_ppm 3000 6000 UV 0.623 0.631 V 0.522 0.612 Chemical Concentration_ppm 3000 6000 Carp 0.176 0.125 Qui 0.969 1.117 Chemical Carp Qui exposure Concentration_ppm 3000 6000 3000 6000 UV 0.270 0.209 0.977 1.053 V 0.081 0.042 0.962 1.182 Standard errors of differences of means Table exposure ChemicalConcentration_ppm exposure Chemical rep. 20 20 20 10 d.f. 27 27 27 27 s.e.d. 0.0702 0.0702 0.0702 0.0993 Table exposure Chemical exposure Concentration_ppm Concentration_ppm Chemical Concentration_ppm rep. 10 10 5 d.f. 27 27 27 s.e.d. 0.0993 0.0993 0.1404 (Not adjusted for missing values) "@en . "Thesis/Dissertation"@en . "2012-11"@en . "10.14288/1.0073211"@en . "eng"@en . "Forestry"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "Attribution-NonCommercial 3.0 Unported"@en . "http://creativecommons.org/licenses/by-nc/3.0/"@en . "Graduate"@en . "Role of non-decay fungi on the weathering of wood"@en . "Text"@en . "http://hdl.handle.net/2429/43298"@en .