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The role of extractive depletion in the fungal colonization of Western redcedar (Thuja plicata Donn) Chedgy, Russell James 2006

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The Role of Extractive Depletion in the Fungal Colonization of Western Redcedar {Thuja plicata Donn) by RUSSELL JAMES CHEDGY B.Sc, University of Exeter, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE / THE FACULTY OF GRADUATE STUDIES (Forestry) The University of British Columbia October 2006 © Russell James Chedgy, 2006 Abstract Western redcedar (Thujaplicata Donn) (WRC) is a naturally durable softwood species native to British Columbia, Canada, as well as Washington, Oregon and California in the U S A . W R C wood products are valued for their durability conferred by anti-microbial extractive compounds. However, such products are still susceptible to fungal colonization that can result in decay and discoloration. The main objective of this thesis was to provide information on the relationship between durability, the change in extractives, and micro-organisms on W R C products in-service in order to help the industry to develop strategies to improve product service life. We first developed protocols to extract, separate and quantify extractives by ultra-sonication and reverse phase high performance liquid chromatography. We developed techniques to screen a range of fungi commonly isolated from W R C in service products for extractive-tolerance in vitro. Results indicated that the Basidiomycete Pachnocybe ferruginea exhibited the highest extractive-tolerance of the range of fungi tested. The next section of this thesis focuses on black stain of W R C siding by Aureobasidium pullulans and the role of weathering. We characterized the effect of weathering on extractives at the surface and correlated this with ability of A. pullulans to colonize. U V plus water spray treatments substantially reduced extractives but did not promote fungal colonization. In contrast, UV-only treatments reduced extractive contents less but stimulated fungal colonization. A. pullulans exhibited high tolerance to the tropolone /?-thujaplicin in vitro; thus loss of tropolone content may not be required for colonization. In the final part of this thesis we investigated the relationship between extractive depletion caused by leaching and how this influenced decay. Leaching resulted in an 80% reduction of extractives, ii which generally resulted in a greater degree of decay by six commonly isolated fungal species. Fungi which exhibited low tolerance to W R C leachate in vitro were able to decay leached W R C blocks more readily than non-leached W R C blocks. Extractive-tolerant species did not require leaching of extractives for decay to occur. The Basidiomycetes P. ferruginea, and to a lesser extent Acanthophysiwn lividocaeruleum and Heterobasidion annosum consistently exhibited high extractive tolerance and could decay non-leached W R C to a similar extent as leached. Such species are candidate 'pioneer' species that may detoxify extractives in wood products, paving the way for decay by less specialized fungi to occur. iii Table of Contents Page Abstract " Table of Contents iv List of Tables viii List of Figures ix List of Abbreviations xi Acknowledgments • xiii Co-authorship Statement xiv Chapter 1 General Introduction and Research Objectives 1 1.1 Introduction 1 1.2 The Importance of WRC 2 1.3 The Chemistry of WRC Extractives 2 1.4 Biological Activity of WRC Extractives 11 1.5 Weathering of WRC Products 14 1.6 Fungal Colonization and Decay of WRC Products 16 1.7 Biosynthesis of WRC Extractives 17 1.7.1 Tropolone Biosynthesis 18 1.7.2 Lignan Biosynthesis 24 1.8 References 29 iv 1.9 Objectives 43 Chapter 2 Extracting and Quantifying Western Redcedar Heartwood Extractives Using Ultrasonication and Reverse Phase H P L C 44 2.1 Abstract 44 2.2 Introduction 45 2.3 Materials and Methods 46 2.3.1 Chemicals and Solvents 46 2.3.2 Separation of Extractives by H P L C 48 2.3.3 Confirmation of Extractive Chemical Structures by N M R 48 2.3.4 Chromatographic sensitivity and quantification of extractives 48 2.3.5 Sample Preparation and Optimization of Extraction 51 2.3.6 Method Evaluation 52 2.3.7 Statistical Analysis 53 2.4 Results and Discussion 53 2.5 Conclusions 60 2.6 References 61 Chapter 3 Isolating and Testing Fungi Tolerant to Western redcedar (Thuja plicata Donn) Extractives 65 3.1 Abstract 65 3.2 Introduction 66 3.3 Materials and Methods 67 3.3.1 Fungal Isolations and Identification 67 3.3.2 Inhibition of Fungal Growth by WRC Feeder Strips (WRC-FSs) 68' 3.3.3 Extractive Analysis of WRC-FSs 70 3.3.3 Statistical Analysis 70 3.4 Results and Discussion 71 3.4.1 Fungal Identification and Extractive Resistance Tests 71 3.4.2 Extractive Analyses of WRC-FSs 74 3.5 Conclusions 78 3.6 References 79 Chapter 4 Black Stain if Western Redcedar (Thuja plicata Donn) by Aureobasidium pullulans: The Role of Weathering 83 4.1 Abstract ' 83 4.2 Introduction 83 4.3 Materials and Methods : 85 4.3.1 Isolation and Identification of Black Stain Fungi 85 4.3.2 /i-Thujaplicin Resistance 86 4.3.3 Weathering and Fungal Effects on Wood Chemistry 86 4.3.4 Inoculation of A. pullulans on Weathered Siding 91 4.3.5 Statistical Analysis 92 4.4 Results and Discussion 92 4.5 Summary 99 4.6 References 100 vi Chapter 5 The Effect of Leaching on the Decay of Western Redcedar (Thuja plicata Donn) 104 5.1 Abstract 104 5.2 Introduction 105 5.3 Materials and Methods 106 5.3.1 Wood Materials 106 5.3.2 Leaching of WRC Blocks and Chemical Analysis 107 5.3.3 Fungal Growth with WRC Leachate 107 5.3.4 Soil Block Decay Tests 110 5.3.5 Statistical Analysis I l l 5.4 Results and Discussion 112 5.5 Conclusions 117 5.5 References 119 Chapter 6 Concluding Remarks and Future Work 122 Appendices Table of Contents 127 Appendix I Fungal Diversity from Western Redcedar Fences and Their Resistance to /^•Thujaplicin 132 Appendix II Morphology of Black Staining Fungi: Aureobasidium pullulans and Hormonema dematioides 153 Appendix III ANOVA Source Tables 154 vii List of Tables Page Table 1.1: Chemical composition of Western redcedar, Western hemlock and Douglas-fir wood (Lewis, 1950) 3 Table 1.2: Common names, chemical structures and relevant literature for extractive compounds 6 Table 2.1: HPLC program for the separation of WRC heartwood extractives (Daniels and Russell, 2006) 49 Table 2.2: A Summary of NMR Data for the Extractives of Interest 50 Table 2.3: Linear regression analysis from mean detector response data for calibration standard solutions over specified concentration ranges 55 Table 2.4: The effect of various extraction methods on extractive concentration (pg/gDW) 57 Table 2.5: Recovery and repeatability data for compounds 2-acetonapthone and methoxyhydroquinone 58 Table 3.1: Fungal isolates from WRC deck in service and fungal growth inhibition by WRC-FSs 69 Table 3.2: Concentration of five major extractive compounds that leached out in the media and accumulated in 25 ml of 1% MEA 77 Table 4.1: Weathering treatments .88 Table 4.2: Total fungal growth (mm) of black staining isolates after 21 days on MEA containing various concentrations of /7-thujaplicin 94 viii Table 4.3.a: A. pullulans growth on weathered wood surfaces estimated qualitatively .97 Table 4.3.b: Spectrophotometer measurements of weathered wood discoloration before and after colonization by A. pullulans 97 Table 5.1: The mean fungal growth rate (mm/day) on media containing various concentrations (ppm) of leachate 114 ix List of Figures Page Figure 1.1: Common extractive compounds of the Cupressaceae family (Barton and MacDonald, 1971) 8 Figure 1.2: Secretion of heartwood extractives (Krahmer and Cote, 1963) 17 Figure 1.3: The proposed biosynthetic pathway of tropolones in W R C (Lai, 1971) 20 Figure 1.4: Schematic illustration of signal transduction and metabolic flux of elicitor-induced /?-thujapIicin and other monoterpenes in Cupressus lusitanica cell cultures (Zhao et al. 2006) 23 Figure 1.5: The proposed biosynthetic pathway of plicatic acid (Gang et al. 1998) 25 Figure 1.6: The proposed Iignan biosynthesis pathways in the sapwood vs. the heartwood of W R C (Swan and Jiang, 1970) 27 Figure 2.1: Chemical structure and nomenclature used for W R C heartwood extractive compounds of interest 47 Figure 2.2: Chromatogram obtained from the analysis of W R C heartwood extractives. 54 Figure 3.1: Pachnocybe ferruginea and Phellinus ferreus growth on 1% M E A (control) vs. on 1% M E A with W R C feeder strips 73 Figure 3.2: Extractive concentrations (u.g/g) vs. W R C feeder strip storage conditions 75 Figure 4.1: Chemical structure and nomenclature used for W R C heartwood extractive compounds of interest (Barton and MacDonald, 1971) 90 Figure 4.2: Weathering treatment effects on the concentrations (u.g/g DW) of WRC extractive compounds of interest 95 Figure 4.3: A. pullulans colonization of weathered wood surfaces 98 Figure 5.1: Chemical structure and nomenclature used for WRC heartwood extractive compounds of interest (Barton and MacDonald, 1971) 109 Figure 5.2: The concentration of extractives (jig/g DW) in non-leached and leached WRC heartwood blocks 113 Figure 5.3: Estimation of the degree of decay (% mass loss) of wood by six fungal species of interest 116 xi List of Abbreviations °C degrees Celsius ug microgram uL microliter pm micrometer cm centimetres DW dry weight FD freeze-dried FS feeder strips g grams h hour HPLC high performance liquid chromatograpghy IS internal standard ITS internal transcribed spacer Kg kilogram kHz kilohertz Kj/m 2 kilojoules per square meter L litre LSU large subunit M E A malt extract agar mins minutes mL millilitre x i i mm millimeter nm nanometers PCR polymerase chain reaction UV ultraviolet WRC Western redcedar WS water spray Acknowledgements I would like to thank, first and foremost, my graduate supervisor, Dr. Colette Breuil, for her guidance and support throughout this work. I would also like to thank members of my graduate committee, Dr. John F. Kadla and Dr. Paul I. Morris, for their advice and help with this project. Thanks also to Dr. Rod Stirling and Dr. Bob Daniels of the Forintek Canada Corporation for their contributions to- and assistance with this research. Thank you to Dr. Batia Bar-Nir for her assistance with NMR analysis. I would like to thank my family, friends and colleagues for their years of support. This work was funded by the Natural Sciences and Engineering Research Council of Canada xiv Co-authorship Statement I hereby declare that I undertook the following contributions to the work reported in this thesis: Chapter 2 Extracting and Quantifying Western Redcedar {Thuja plicata Donn) Heartwood Extractives Using Ultrasonication and Reverse Phase H P L C . Authors: Russell J. Chedgy, C. R. Daniels, John F. Kadla and Colette Breuil . Submitted to the journal of Holzforschung, submission # HOLZ-D-06-00099. For this chapter I was the primary contributor, and took full writing responsibilities. Dr C. R. Daniels contributed by providing a method for the separation of W R C extractives using reverse phase H P L C that he had previously developed. Dr John F. Kadla and Dr Colette Breuil had supervisory roles. Chapter 3 Isolating and Testing Fungi Tolerant to Western redcedar (Thuja plicata Donn) Extractives. Authors: Young Woon L i m , Russell J. Chedgy, Sabarish Amirthalingam, and Colette Breuil. Submitted to the journal of Holzforschung, submission # HOLZ-D-06-00100. For this work Dr. Young Woon L i m and I were the main contributors. I conducted all duties related to the chemical analysis of W R C extractives. Specifically, I conducted H P L C analysis and quantification of W R C extractives that had leached from W R C - F S s that were inoculated on media. I tested individual extractive compounds for fungicidal activity on selected fungal strains. In addition I took approximately half of the writing responsibilities. Mr . Sabarish Amirthalingam xv participated by conducting screening of fungal strains on media inoculated with W R C - F S s . This work was supervised by Dr. Colette Breuil. Chapter 4 Black Stain of Western Redcedar (Thuja plicata Donn) by Aureobasidium pullulans: the Role of Weathering. Authors: Russell J. Chedgy, Paul I. Morris, Young Woon L i m and Colette Breuil. Submitted to the journal of Wood and Fiber Science, currently under review. For this chapter I was the primary contributor, and took full writing responsibilities. Dr. Young Woon L i m provided assistance with fungal isolation and identification. Dr. Paul I. Morris and Dr. Colette Breuil had supervisory roles. Chapter 5 The Effect of Leaching on the Decay of Second-growth Western Redcedar (Thuja plicata Donn). Authors: Russell J. Chedgy, Young Woon L i m , and Colette Breuil . For this work I was the primary contributor, and took full writing responsibilities. Dr. Young Woon L i m provided assistance with fungal isolation and identification and Dr. Colette Breuil had supervisory roles. Appendix I Fungal Diversity from Western Redcedar Fences and Their Resistance to fi-Thujaplicin. Authors: Young Woon L i m , Jae-Jin K i m , Russell J. Chedgy, Paul I. Morris , and Colette Breuil. For this work Dr. Young Woon L i m was the primary contributor. Dr. L i m and Dr. Jae-Jin K i m under took the majority of the fungal isolation in this work and shared writing responsibilities. xvi My role was to screen isolates for /J-thujaplicin resistance, as well as an editorial role during manuscript preparation. Dr. Paul Morris and Dr. Colette Breuil had supervisory roles. xvii Chapter 1 General Introduction and Research Objectives 1.1 Introduction The emphasis of this project is on the depletion of extractives from western redcedar (Thuja plicata Donn) ( W R C ) wood products caused by weathering and how this affects the ability of fungi to colonize and cause discoloration or decay. This research is relevant to product service life. This chapter begins by assessing the chemistry and occurrence of W R C extractives and their biological activity. Also discussed are the modern day applications of several key extractive compounds that have roles in the wood protective coating, pharmaceutical, and cosmetic industries. Then the effect of weathering on W R C extractives is explored, followed by the fungal colonization and decay of W R C wood products. Finally, I have included a brief review on extractive biosynthesis that touches on their biological and ecological roles in members of the Cupressaceae family. Chapter 1 focuses on extractive compounds that confer natural durability to W R C wood products due to anti-microbial activity. Chapter 2 describes the development of protocols to extract, separate and accurately quantify extractives. These protocols were then utilized for the work described in Chapter 3 that describes a novel method to screen fungi isolated from W R C products for tolerance to specific W R C extractive constituents. Chapter 4 investigates the effect o f various types of weathering on extractives and how this influences the colonization by black staining fungi of W R C siding products. This chapter also summarizes data on the resistance of several species of black staining fungi to W R C extractives. The relationship between extractive depletion by leaching and decay are examined in Chapter 5. The growth and decay ability of six frequently isolated fungi from W R C products are assessed on leached versus 1 non-leached WRC heartwood. Finally, the implications of the work conducted in this thesis as well as future research are discussed in Chapter 6. 1.2 The Importance of WRC WRC is a naturally durable softwood species native to British Columbia, Canada, as well as Washington, Oregon, and California in the USA. The heartwood of this species contains an array of antimicrobial extractive compounds which act. as a natural chemical defense shield against pathogen invasion in standing trees (Rennerfelt, 1948; Barton & MacDonald, 1971; Van der Kamp, 1986; Johnson & Croteau, 1987; Zaprometov, 1992; Belanger et al, 1997; DeBell et al, 1997) and confer natural durability in WRC products (Rudman, 1962; Barton & MacDonald, 1971). WRC is utilized in the manufacture of wood products with exterior residential applications which account for a significant portion of Canada's forest products industry (Gonzalez, 2004). Despite this, WRC products can still fail in-service due to fungal decay as well as discoloration by staining fungi which can lead to premature replacement of wood products. Such failure may be attributable in part to 1) colonization by extractive-tolerant micro-organisms, or 2) depletion of anti-microbial extractives by weathering, as well as 3) biodegradation of extractives by micro-organisms paving the way for colonization by less specialized wood-destroying micro-organisms. 1.3 The Chemistry of WRC Extractives The chemical composition of WRC heartwood is homologous to other softwood species such as douglas-fir (Pseudotsuga menziesii) and black spruce {Picea mariana) in terms of the relative abundance of major structural constituents such as alpha-cellulose, hemicelluloses and lignin (Lewis, 1950) (Table 1.1). Unlike other North American softwood species like true firs (Abies 2 Table 1.1 Chemical composition of western redcedar, western hemlock and douglas-fir heartwood (Lewis, 1950) Species Alpha cellulose Hemi-celluloses Lignin Total extractives Ash Western redcedar 47.5 (52.8) 13.2(14.7) 29.3 (32.6) 10.2 0.2 Western hemlock 48.8(51.6) 14.7(15.5) 28.8 (30.4) 5.3 0.5 Douglas-fir 53.8 (57.2) 13.3 (14.1) 26.7 (28.4) 5.9 0.3 Note: numbers represent the percentage content on a moisture-free basis; figures in parenthesis represent percentage composition on a moisture-free extractive-free basis. 3 spp.), western larch (Larix occidentalis), Colorado blue spruce (Piceapungerns), douglas-fir and Pinus species, W R C produces only trace amounts of oleoresin (Penhallow, 1907; Fahn, 1979; Johnson and Croteau, 1987; Lewinsohin et al., 1991, 1994) as a chemical deterrent and a physical barrier against pathogen attack (Johnson and Croteau, 1987; Zaprometov, 1992). However, W R C has an unusually high proportion of extractives that are aromatic and polyphenolic in nature (Barton and MacDonald, 1971). Several of these compounds exhibit antimicrobial and insecticidal activities, (Inamori et al., 1999, 2000; Ar ima et al., 2003; Morita et al., 2004a, 2004b) as well as herbicidal properties (Sakagami et al., 2000), with efficacy comparable to commercial pest control agents (See Section 1.4). Such extractives also contribute to many of cedar's desirable qualities such as its distinctive red-brown color, pleasant odor and excellent finishing quality (Barton and MacDonald, 1971). Conversely, their presence can adversely affect wood-pulping processes, leading to increased production costs (Wethern, 1959). The concentration of extractives is subject to wide variation throughout the tree. They increase radially, reaching a maximum at the heartwood-sapwood border before rapidly declining in the sapwood region; and decrease longitudinally from the tree base to the crown (MacLean and Gardner, 1956; Nault, 1988; DeBel l et al., 1999). W R C extractives are a mixture of tropolone and lignan compounds. Tropolones are 2-hydroxy-2,4,6-cycloheptatrien-l-one molecules and their derivatives which possess special characteristic properties due to the 1,2 arrangement of the carbonyl and hydroxyl groups on an unsaturated seven-membered carbon ring (Dewar, 1945). Lignans are nonstructural, dimeric, phenolic metabolites, many of which are complex molecules of unusual structure (Gang et al., 1998). To date, at least eight tropolone and eleven lignan compounds have been identified and characterized in W R C . Many of these compounds occur in species throughout the Cupressaceae family (Zavarin et al., 1967). Most research has been 4 centered on tropolones extracted from W R C of North America and a species native to Japan: Thujopsis dolabrata (Sieb. et Zucc. var. hondai Makino), also know as 'aomori hiba', and the 'dwarf hiba cedar'. The tropolone content of W R C heartwood is estimated at 1-1.5% (w/w) while the lignan portion comprises 5-15% (w/w) (Barton and Macdonald, 1971). Within the group of tropolone compounds are three isomeric isopropyl-tropolones/known as the 'thujaplicins'. These are specifically: (i) a-thujaplicin, (ii) /?-thujaplicin, and (iii) y-thujaplicin. Table 1.2 shows the common name, chemical description and relevant literature for each of the extractive compounds cited, and Figure 1.1 displays their chemical structures. a-Thujaplicin is normally present in trace amounts only at 0-1% (w/w) (Barton and MacDonald, 1971; Jones and Falk, 2005). y-Thujaplicin is marginally more abundant than /?- thujaplicin. Analysis of the methanol extractive of W R C heartwood shows that these two compounds account for approximately 17% (w/w) of the total tropolone content (Jones and Falk, 2005). The other tropolone compounds identified are (iv) /i-thujaplicinol, (v) /J-dolabrin, (vi) thujic acid, (vii) methyl thujate, and finally, (viii) nezukone. y5-Thujaplicinol is estimated to account for approximately one tenth of the total thujaplicin content. Thujic acid is the most abundant tropolone comprising 26% (w/w) of the total tropolone portion (Jones and Falk, 2005), while /?-dolabrin, methyl thujate and nezukone are present only in trace amounts (Barton and MacDonald, 1971). The non-tropolone compound (ix) carvacrol methyl ether is also found in trace amounts (MacLean, 1970). Also noteworthy is the additional tropolone compound (x) 4-acetyltropolone which has not been reported in W R C but is known to be common in other Cupressaceous species (Zavarin et al, 1967). 5 Table 1.2 Common names, chemical structures and relevant literature for extractives Compound Name Chemical Structure Relevant Literature (i) a-Thujaplicin 2-Hydroxy-3-isopropyl-2,4,6-cycloheptatrien-1 -one Gripenberg, 1948 (ii) /i-Thujaplicin 2-Hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1 -one Erdtman and Gripenberg, 1948 (iii) y-Thujaplicin 2-Hydroxy-5-isopropyl-2,4,6-cycloheptatrien-1-one Erdtman and Gripenberg, 1948 (iv) yff-Thujaplicinol 2,7-Dihydroxy-4-isopropyl-2,4,6-cycloheptatrein-1 -one Gardner et al., 1957 (v) /?-Dolabrin 2-Hydroxy-4-isopropenyl-2,4,6-cycloheptatrien-1 -one Nozoe et al., 1957; Gardner and Barton, 1958 (vi) Thujic acid 5,5-Dimethyl-l,3,6-Cycloheptatriene-l-carboxylic acid Gripenberg, 1956; Davis and Tulinsky, 1962 (vii) Methyl thujate 7,7-Dimethyl-3-carbomethoxy-l,3,5-cycloheptatriene Barton and Gardner, 1954 (viii) Nezukone 4-Isopropyl-2,4,6-cycloheptatrien-1 -one Hirose et al, 1966, 1967, 1968 (ix) Carvacrol methyl ether 5-Isopropyl-2-methylanisole MacLean, 1970 (x) 4-Acetyltropolone 4-Acetyl-2-hydroxy-2,4,6-cycloheptatrien-1 -one Zavarin et al, 1967 (xi) (-)-Plicatic acid 2,3,6-Trihydroxy-7-methoxy-2-hydroxymethyl-4-(3' ,4' -dihydroxy-5' -methoxyphenyl)-tetralin-3-carboxylic acid Gardner et al, 1960, 1966; Swan et al, 1967 (xii) (-)-Plicatin Naphtho [2,3 -c] furan-1 (3 H)-one MacDonald and Swan, 1970 (xiii) (-)-Thujaplicatin methyl ether 2-(4"-Hydroxy-3",5"-dimethoxybenzyl)-3 -(4' -hydroxy-3 'methoxybenzyl)-butyro-lactone MacLean and Murakami, 1966a; Nishibe et al, 1974 (xiv) (-)-Thujaplicatin 2-(3",4"-Dihydroxy-5"-methoxybenzyl)-3 -(4' -hydroxy-3' -methoxybenzyl)-butyrolactone MacLean and Murakami, 1966a; Nishibe et al, 1974 (xv) (-)-Hydroxythuja -plicatin methyl ether Dihydro-3 -hydroxy-3 - [(4-hydroxy-3,5 -dimethoxyphenyl)methyl] -4- [(4-hy droxy- 3 -methoxypheny l)methy 1] -2(3H)-furanone MacLean and Murakami, 1966b. 6 Table 1.2 (Continued) Common names, chemical structures and relevant literature for extractives Compound Name Chemical Structure Relevant Literature (xvi) (-)-Dihydroxy-thujaplicatin methyl ether 2,3-dihydroxy-2-(4"-hydroxy-3",5"-dimethoxybenzyl)-3 -(4' -hydroxy-3' -methoxybenzyl)-butyrolactone MacLean and Murakami, 1966c. (xvii) (-)-Dihydroxy -thujaplicatin 2,3-dihydroxy-2-(3",4"-dihydroxy-5"-methoxybenzyl)-3 -(4' -hydroxy-3' -methoxybenzyl)-butyrolactone MacLean and MacDonald, 1966 (xviii) Plicatinaphthol l,6-dihydroxy-2-(hydroxymethyl)-7-methoxy-4-(3' ,4'-dihydroxy-5'-methoxyphenyl)-3-naphthoic acid lactone MacLean and MacDonald, 1969a (xix) Plicatinaphthalene 6-hydroxy-2(hydroxyl-methyl)-7-methoxy-4-(3' ,4'-dihydroxy-5'-methoxyphenyl)-3-naphthoic acid lactone MacLean and MacDonald, 1969b (xx) y-Thujaplicatene 2-(3",4"-dihydroxy-5"-methoxybenzylidene)-3 -(4' -hydroxy-3 '-methoxybenzyl)-butyrolactone MacDonald and Barton, 1970 (xxi) /?-Apoplicatitoxin l,4-dihydronaphthalene-6-hydroxyl-2-(hydroxymethyl)-7-methoxy-4(3' ,4' -dihydroxy-5 '-methoxyphenyl)-3-carboxylic acid lactone MacDonald and Barton, 1973 7 CH-i C H 3 O O H (i) cc-TIiujaplidii C H 3 X- C H , O H Qi) ^-Tl iujapl ici i i H 3 C ^ ^ C H 3 O O H (iii) y-Thujapl ic i i i C H 2 H,C -4. HO HO (iv) /7-HiujapIiciiiuI HO O (v)/?-DoIabiiii H 3 C ^ X H 3 f 1 O H H 3 C ^ ^ C H 3 n O o A \ — / C H 3 O 1 C H ? ) - C H 3 C H 3 ^ p C H 3 C H 3 O V O H , C ,c-o (vi) Thu j ic A c i d (vii) M e t h y l Thujate (viii) Nezukone (ix) Ca rvac ro l (x) 4-Acetyl tropolone* M e t h y l E ther H,CO H O ' X ^ C H 2 O H T "C0 2 H OH HO ^ O C H 3 OH (xi) (-)-Plicatic Acid H 3CO O H O HO" V ' 1 ° H O Y O C H 3 O H (xii) (-)-Plicatin H 3 C v » H 2 1 T r % O n (xiii) (-)-Thujaplicatin Me thy l Ether H 3 C O. HO' H 2 b .1 1> H O ' O O H (xiv) (-)-Thujapl icat in ' Not. present in Thuja plicate C H 3 H 3 C H 2 1 /IHOJ^ O H O - A ^ V O H H 3 C H 3 C . O OH IHO C H 3 P T Q. H 3 C OH C H 3 (xv) (-)-Hydroxythujapl icat in (xvi) (-)-Dihydroxy-thujapl icat in Me thy l E ther Me thy l E the r Figure 1.1 Common extractive compounds of the Cupressaceae fami ly (Barton and M a c D o n a l d , 1971) 8 OH C H 3 H 3 C' (xx) 7-Thujaplicatene (xxi) /?-ApopUcatitoxin Figure 1.1 (Continued) Common extractive compounds of the Cupressaceae family (Barton and MacDonald, 1971) 9 O f the lignan extractive compounds, (xi) (-)-plicatic acid is the most abundant, accounting for 40-50% of this portion of extractives (MacDonald and Swan, 1970; Barton and MacDonald, 1971; Jones and Falk, 2005). It is a reactive, polyoxyphenolic, amorphous acid, which is optically active as well as heat and light sensitive. It is a very strong acid because of its highly hydroxylated side chain. K a i and Swan (1990) suggested that lignans such as plicatic acid are responsible for the red-brown color of W R C heartwood. Further to this, Johansson et al. (2000) isolated an insoluble polymeric fraction that accounted for 34% by weight of the methanol extractive but 72% of the color. Analysis suggested that these polymers were less related to lignin and more closely related to the highly phenolic lignans such as plicatic acid and plicatin. Plicatic acid has been reported to cause occupational asthma - an issue amongst forestry workers exposed to cedar dust particles on a regular basis (Chan-Yeung et al., 1980; Frew et al., 1993, 1998; Chan-Yeung, 1994). The second lignan in the series is (xii) (-)-plicatin which is a plicatic acid lactone. Other lignans include (xiii) (-)-thujaplicatin methyl ether, the first syringyl derivatives found in softwood and the first lignan known to possess both guaiacyl and syringyl rings, and (xiv) (-)-thujaplicatin. Together, plicatin, thujaplicatin methyl ether, and thujaplicatin make up approximately 30-40% of the total lignan content (Barton and MacDonald, 1971). The remaining 10% of the lignan portion comprises of seven other lignan compounds: (xv) (-)-hydroxythujaplicatin methyl ether, (xvi) (-)-dihydroxy-thujaplicatin methyl ether, (xvii) (-)-dihydroxythujaplicatin, (xviii) plicatinaphthol, (ixx) plicatinaphthalene, (xx) y-thujaplicatene, and finally (xxi) /J-apoplicatitoxin (see Table 1.2 for appropriate references). 10 1.4 Biological Act iv i ty of W R C Extractives O f the range of extractive chemicals present, tropolones have warranted a great deal of scientific interest because of their biological activity. The tropolones cc-, /?-, y-thujaplicin, /J-dolabrin, 4-acetyltropolone and their derivatives have anti-fungal activity against a range wood destroying and plant pathogenic fungal species in vitro. Their minimum inhibitory concentrations (MICs) range from 0.2-50.0pg/ml (Raa and Goksoeyr, 1965; Inamori et al., 2000; Baya et al., 2001; Inamori and Morita, 2001; Morita et al., 2004a, 2004b). This range is comparable to biocides such as Amphotericin B and Pentachlorophenol. In addition, several of these tropolones exhibit anti-bacterial activity against species such as Legionella pnuemophila (causal agent of Legionnaires' disease) with M I C s in the range of 6.3-50.0u.g/ml (Morita et al. 2004a, 2004b). The tropolone /?-thujaplicin has been used as a preservative of vegetables, flowers, and mushrooms because of its strong antibacterial activity, as well as it being a plant growth stimulator. The antimicrobial activity of tropolones has been used in commercial products ranging from aqueous ink (Yatake, 2005) to cosmetics (Pillai et al., 2005). In addition to anti-microbial activity, insecticidal and acaricidal properties have also been reported for several of these tropolones. They have been demonstrated to be highly effective against species such as Coptoterm.es formosanus (formosan subterranean termite), Reticulitermes speratus (Japanese termite), Dermatophagoides farinae (house mite), and Tyrophagus putrescentiae (mould mite) with a 50%-leathal concentrations (LC50) ranging from 0.02-0.66g/m 2 (Inamori et al, 2000; Inamori and Morita, 2001; Morita et al, 2004a). This is comparable to commercially available insecticides such as Chlorpyrifos and D E E T ( N , N -diethyl-m-toluamide). Given this, a W R C oil-containing miticide for domestic use was developed by Ishibashi et al. (1992). The researchers soaked porous ceramic granules (0.1-3.0 11 mm) in 5% W R C oil which resulted in a mite mortality rate o f 95% in 24 hours. There is also some evidence that the W R C tropolone thujic acid acts as insect juvenile hormone in species such as the Ye l low Mealworm beetle (Tenebrio molitor) (Barton, et al, 1972). Tropolones also have phytogrowth-inhibitory activity. Sakagami et al. (2000) showed that y- and ft-thujaplicin, and /i-dolabrin inhibited germination in seeds of field mustard (Brassica campestris) and sesame (Sesamum indicum) at concentrations as low as 1 Oppm. This phytogrowth-inhibitory activity was as high as the herbicide sodium 2,4-dichlorophenoxyacetate. These tropolones were also found to significantly decrease the amount of chlorophyll in cotyledons of the two plant species. This phytogrowth-inhibitory action might be a common biological activity of tropolone compounds. From a pharmaceutical perspective, several of the tropolone compounds present in heartwood of W R C and other members of the Cupressaceae family have been shown to have cytotoxic activity against a range of cancerous cell lines in vitro (Matsumura et al., 2001; Inamori et al., 2004; Morita et al., 2004a, 2004b). The tropolones a-, /?-, y-thujaplicin, /?-dolabrin, and 4-acetyltropolone are effective against cell lines such as murine P388 lymphocytic leukemia, human stomach cancer K A T O - I I I and Ehrlich's ascites carcinoma. The inhibition within 24 hours is >70-95% at concentrations ranging from 0.3-5.0u.g/ml (Matsumura et al., 2001; Morita et al, 2001, 2002, 2004a, 2004b; Inamori et al, 2003, 2004). In addition, several tropolone compounds have potential applications in the field of sunburn protection as they have an inhibitory effect on ultraviolet B-induced apoptosis in keratinocytes (also known as sunburn cells) (Baba et al, 1998; Ar ima et al. 1997; Nakano et al. 2006). The thujaplicins compounds have strong antioxidant abilities and protect cultured cells from oxidative stress-mediated 12 damaged (Paschalis-Thomas et al., 2005), and may have pharmaceutical applications for the prevention of UV-induced photo-damage in skin cells. Overall, the pharmaceutical utilization of tropolones may be feasible given that in preliminary trials in mice suggested that they showed low toxicity. The ability of tropolones to chelate b i - and trivalent metal ions has been implicated in their anti-microbial and cytotoxic activity, although the exact mechanism is still subject to investigation (Miyamoto et al., 1998). Tropolones are known to form stable chelates with various metals ions I which include: F e 3 + , F e 2 + , C u 2 + , N i 2 + , Z n 2 + , C o 2 + , and M n 2 + (Oka and Matsuo, 1958; Oka et al. 1964, 1968; Oka and Yanai, 1965; Hirai and Oka, 1970). Raa and Goksoeyr (1965) demonstrated that the /?-thujapIicin-copper chelate inhibited respiration in Yeast (Saccharomyces cerrivasae) at concentrations >10" 6M. They also suggested that the keto-enol group of the tropolone molecule is essential for the inhibition since the methyl ether of /?-thujaplicin was not toxic. Inamori et al. (1999) postulated that at least part of this activity is due to metal chelation between the carbonyl group at C - l and the hydroxyl group at C-2 in the tropolone skeleton since y8-thujaplicin-acetate, which was unable to chelate divalent metals ions, did not show any antimicrobial activity. Budihas et al, (2005) explored the role of /^thujaplicinol as a potent and selective inhibitor of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT) . They concluded that the ability of thujaplicins to chelate divalent metals such as C u 2 + and Z n 2 + via their /?-diketone moiety (Endo et al, 1988) may play a role. They suggested that metal chelation and/or altering the co-ordination geometry of the divalent metal in the RNase H catalytic center, which is essential for catalysis, would seem a plausible mechanism of action. Based on this hypothesis of inhibition by metal chelation, Wakabayashi et al. (1997) developed wood preservatives containing troponoid metal complexes. 13 An a-thujaplicin-Cu/+ complex at 200ppm inhibited the lignin-decomposing fungus Coriolus versicolor, and the cellulose-decomposing fungus Tyromyces palustris. It was effective at inhibiting fungal growth and was non-toxic to humans and showed good fixation to wood. Given the strong and broad-spectrum of antimicrobial activity (Sakai, 2004), and the many other biological activities of tropolone compounds, there is an increasing demand for these compounds in the industry (Sakai, 2004). 1.5 Weathering of WRC Products WRC products are utilized heavily for exterior residential applications given their natural durability and offer an alternative perceived as more environmentally friendly than wood products treated with biocides such as CCA (chromated copper arsenate). However, exposure to weathering can cause depletion of extractives which confer durability. Several fungicidal tropolone extractive compounds are prone to photo-degradation caused by ultra-violet (UV) radiation from sunlight. Coombs and Trust (1973) observed that exposure of aqueous solutions of /^-thujaplicin to UV radiation led to a measurable loss of antibacterial activity. This compound is known to have a maximum absorption peak at 244 nm with smaller absorption peaks in the near-UV region (280-380 nm). They noted that photochemical decomposition of /i-thujaplicin was found to occur following shortwave irradiation (210-280 nm) and that this possibly caused a % —> 7i* electronic transition. A longer wavelength source (280-380 nm) possibly caused an n—* 7t* electron transition. Shibata et al. (2003) noted similar photochemical decomposition patterns of/i-thujaplicin but also suggested that during this degradation process a reactive oxygen species was generated that also had some bactericidal properties. The possible decomposition product under laboratory light has been suggested to be isopropyl-substituted 4-oxo-cyclopentane-l-acetic acid. 14 In addition to the reported photo-instability of W R C tropolone extractives, l ignin is also susceptible to photo-degradation whereas pure cellulose and hemicelluloses absorb little U V (Crestini, 1996; Chang, 2002). Oxidative cleavage of Ca-CB and /3-0-4 bonds that link lignin precursor guaiacyl phenylpropanoid molecules together in softwoods is known to occur as a result of U V exposure. A progressive destruction of l ignin aromaticity, demethoxylation and the formation of carboxyl groups, soluble carbohydrates and lignin fragments have also been observed fol lowing irradiation of wood, pulp and paper (Chang, 2002). This is relevant for certain black staining fungal species such as Aureobasidium pullulans which has been reported to colonize the surface of wood products exposed to weathering (including W R C ) and utilize lignin photo-degradation products as a carbon source (Dickinson, 1972; Bourbonnais and Paice, 1987; Sharpe and Dickinson, 1992a, 1993; Schoeman and Dickinson, 1997). However, U V radiation from sunlight only penetrates the upper 1mm of exposed surfaces (Hon, 1991) and may have a limited effect on the total extractive content of W R C products. Leaching of extractives by rain, which penetrates deep, is more l ikely to affect extractive concentration, especially considering that the majority of W R C extractives are water soluble (Barton and MacDonald, 1971). Johnson and Cserjesi (1980) investigated the depletion of B- and y-thujaplicin in W R C shakes exposed to natural weather conditions in Vancouver, British Columbia, Canada. They established that a one year exposure resulted in a 25% depletion of B-and y-thujaplicin. This increased to 90% after three years. Although this may have been due in-part to biodegradation. Chedgy et al. (2005) monitored the thujaplicin content of W R C siding exposed to various simulated weathering treatments using a Weather-Ometer® and reported - 4 7 % decrease in thujaplicins following U V weathering, and a 100% loss following water spray, and water spray and U V combined. This suggested that leaching of extractives may lead to 15 depletion of fungicidal extractives which may leave W R C products susceptible to colonization by decay and staining fungi leading to a potentially reduced service life. 1.6 Fungal Colonization and Decay of W R C Products L i m et al. (2005) investigated the fungal community inhabiting in-service W R C fence material with a focus on species colonizing wood below the surface. They reported twenty-three different fungal species which included thirteen ascomycetous and ten basidiomycetous fungi. They tested isolates for their resistance to /J-thujaplicin - one of the principle fungicidal agents of W R C heartwood extractives. Generally, ascomycetous fungi exhibited greater resistance to (5-thujaplicin than basidiomycetous fungi. Interestingly, three soft-rot ascomycetous species, Oidiodendron sp., Phialophorafastigiata and Phialophora sp. 3, and two basidiomycetous species, Pachnocybe ferruginea and Acanthophysium lividocaeruleum, were frequently isolated and had high tolerance to this compound. The researchers concluded that these species could be 'pioneer' species that invade and detoxify W R C extractives, paving the way for colonization by brown-rot and white-rot fungi. Extractive content is often used to predict service life of W R C products although little literature focuses on the relationship between extractive content and rate of decay of W R C . Wood product manufacture increasingly relies on second growth trees that typically contain 6.2-9.8% total extractive by weight, compared with 11.4-22.8% in old growth trees (Barton and MacDonald, 1971; Nault, 1988). This may lead to potentially less durable W R C wood products given the lower extractive content in second growth W R C . This issue was explored by Freitag and Morrel l (2001) who examined the durability of the changing W R C stock. In standard soil block tests 16 using the decay fungus Postia placenta, they compared the durability of W R C blocks from young and older trees. They concluded that, in fact, the durability of younger W R C material has not changed from that of older stocks. Despite these encouraging observations, a greater understanding of the chemical nature of heartwood extractives and their ability to confer natural durability is imperative in order to develop systems that may enhance product service life in the future. 1.7 Biosynthesis of WRC Extractives The major proportion of W R C extractives are formed in situ at the sapwood-heartwood boundary in phloem parenchyma cells and are carried to the heartwood via ray parenchyma cells (Swan and Jiang, 1970). Cedar heartwood is red-brown in color whereas the sapwood is a pale yellow, off-white color. Krahmer and Cote (1963) conducted electron microscopy photography of W R C tracheids; they noted that heartwood tracheids were heavily encrusted with extractives, whereas those in the sapwood were devoid of extractives. Across the sapwood-heartwood transition, the concentration of most extractive compounds increase by a factor of 100 times (Swan and Jiang, 1970). Metabolites are released into the M i l transformation of lignans and other extractive heartwood from specialized ray parenchyma cells via pit apertures into the lumen of adjacent, dead (lignified) cells and then diffuse into neighboring, pre-lignified cells (tracheids) (Figure 1.2). It is reported that further components continues well after their release into the Figure 1.2 Secretion of heartwood heartwood, beyond the visible heartwood-sapwood constituents by ray parenchyma cells into the lumen of neighboring cells appears to occur through pit apertures (Krahmer and CfiteT196.T>. 17 boundary, and that this process can continue for many years (Swan et al., 1969; Swan and Jiang, 1970). W R C extractives are classed as terpenoids which are multicyclic structures, derived by repetitive fusion of a branched five carbon monomer termed isoprene which can polymerize further. Biosynthesis of the fundamental terpenoid precursor isopentenyl diphosphate (IPP) occurs principally via the acetate/mevalonate ( M V A ) pathway active in the cytosol and endoplasmic reticulum of cells, and via the 2C-methyl-D-erythritol 4-phosphate ( M E P ) pathway operating in the plastids (Lichtenthaler, 1999). Following condensation of IPP monomers, the action of terpene synthases (TPS) leads to the formation of geranyl diphosphate (GPP) —» monoterpenes (Cio) (volatile); IPP —> farnesyl diphosphate (FPP) —> sesquiterpenes ( C 1 5 ) (volatile); and IPP —> geranyl geranyl diphosphate (GGPP) -> Diterpenes (C20) and so on (Croteau et al, 2000; Croteau and Johnson, 1985). A l l of these pyrophosphates are the common precursors for all monoterpenes and their derivatives. These precursor pools and various terpenoid products markedly increase in plant response to biotic or abiotic stresses (Bohlmann et al:, 1998). 1.7.1 Tropolone Biosynthesis Among the range of secondary metabolites present in W R C are tropolones such as /^thujaplicin which are major phytoalexins - metabolites produced by a plant in response to infection by a fungus or other pathogens, or by abiotic factors such as wounding. Several lines of evidence show that biosynthesis is regulated by wounding, microorganism elicitor molecules, by methyl jasmonate (MeJA) and oxidative stress (Yin et al, 1997; Steele et al, 1998; Mandujano-Chavez et al, 2000; Martin et al. 2002; Zhao et al. 2004a, 2004b, 2005a, 2005b). Cel l cultures from 18 several Cupressaceae trees such as mexican cypress (Cupressus lusitanica), Florin (Calocedrus formosana) and white cedar (Thuja occidentalis) can produce /J-thujaplicin and a number of monoterpenes (Witte et al, 1983; Ono et al, 1998; Matsunga et al, 2003). They have been used to explore the possible biosynthetic pathways involved in tropolone production because they are easy to manipulate in the laboratory environment. Sakai et al. (1997) demonstrated that /?-thujaplicin is synthesized via the M V A pathway using C 1 4 radioactive /i-thujaplicin precursors as well as by selective inhibition of one of the key regulatory enzymes in the mevalonate pathway, 3-hydroxy-3-methylglutaryl coenzyme A ( H M G - C o A ) reductase. This enzyme inhibition resulted in significant suppression of /i-thujaplicin synthesis. Alternatively, Fujita et al. (2000), also working with Cupressus lusitanica, demonstrated in tracer experiments that geraniol and glucose were efficiently incorporated into ^-thujaplicin via the M E P pathway through an intermediate with menthane-type skeleton. This suggests that M E P pathway, rather than M V A pathway, acts as a main synthesis pathways for /?-thujaplicin, although a M V A pathways also contributes a minor portion of /^-thujaplicin biosynthesis (Yamaguchi et al., 1997). To date, these pathways are speculative, and enzymes involved in the biosynthesis o f tropolones have yet to be characterized. A biosynthetic pathway for tropolones in the Cupressaceae family was proposed by La i (1971). These pathways are illustrated in Figure 1.3. The pathways shown are only those relevant to W R C . The initial step in the pathway is the condensation of acetyl Co A (A) and a-ketoglutaric acid (B) to produce homocitric acid ( Q . Both of these starting materials are available from the metabolic pool and the compound acetyl C o A is a common precursor of the M V A pathway. Homocitric acid is then converted into /i-ketoadipic acid (E) by subsequent decarboxylation and oxidation. Further isopentenylation of /?-ketoadipic acid produces the intermediate (F) which 19 C02H H2C C ^ O H2C H2C C0 2H H3C C A Acetyl-CoA B a-Ketoglutaric acid C Homocitric acid D lntennediate E yff-Ketoadipic acid F Intermediate G lntennediate H Intermediate I Intermediate J Intermediate K Nezukone L yS-Thujaplicin M Intermediate N lntennediate O lntennediate P yfi-Dolabrin Q Intermediate R lntennediate S 1-Isopropyltropolone T a-Thujaplicin U y-Thujaplicin V yS-Thujaplicinol Figure 1.3 The proposed biosynthetic pathway of tropolones in W R C (Lai, 1971) 20 undergoes several reactions that include cyclization followed by carbonium ion rearrangement, and elimination of a hydrogen ion, reduction, dehydration, oxidative hydroxylation and finally, dehydration reactions to form the compound nezukone (K). Synthesis of the tropolone ot-thujaplicin (7) is initiated from an intermediate compound (H), which is present in the pathways leading up to the formation of nezukone. This compound also undergoes cyclization followed by similar reactions as described above to form 1 -isopropyltropolone (S). Compound (S) then undergoes a simple hydroxylation to form a-thujaplicin (7). The remaining thujaplicins can be synthesized from nezukone (K). Nezukone undergoes hydroxylation to form y-thujaplicin (U), and /i-thujaplicin (L), a hypothesis also supported by Swan et al. (1969). The tropolone /?-thujaplicinol (V) is derived from /i-thujaplicin by further hydroxylation, isopentenylation, hydration, and methylation processes. La i (1971) also postulated that /i-dolabrin (P), which is a tropolone with an unsaturated side chain, is synthesized from intermediate compound (G) shown in the diagram. This can undergo a direct elimination of a hydrogen ion and a series of reactions as described above to produce /i-dolabrin. Again, the enzymes involved in the biosynthetic pathway have yet to be established, and these are speculative pathways. In W R C , nezukone is a precursor of the thujaplicins, and hydroxylation is required for this to occur. The hydroxylation mechanism is perhaps by the direct reaction of the precursor with oxygen as suggested by Frey-Wyssling and Bossard (1959) who studied the cytology of the ray cells in the sapwood and heartwood of 12 tree species. They concluded that the transition between sapwood and heartwood was characterized by a semi-anaerobic metabolism. In the heartwood, a slow oxidation and polymerization of extractives occurred by direct reaction with oxygen. Such oxidations may account for the high concentrations of thujaplicins in the outer heartwood, and may explain the decrease in extractives that is observed in mature heartwood 21 closer to the pith, where conditions become increasingly anaerobic. It is also probable that this observed decrease in extractives from the outer heartwood to the pith is due to lower amounts of extractives laid down in the juvenile tree and biodegradation of extractives over the long natural life of W R C trees (MacLean and Gardner, 1956; Nault, 1988). Little is known about the enzymes and genes that are involved in /^thujaplicin biosynthesis. Recent work also suggests that both methylation and oxidation of /i-thujaplicin are responsible for /i-thujaplicin transformation in C. lusitanica cell cultures, since a certain level of /?-thujaplicin is toxic to plant cells (Yamada et al., 2002; Zhao and Sakai, 2003a, 2003b). A l l these data show that biosynthesis of /i-thujaplicin in C. lusitanica cell culture is highly regulated and that its metabolism is also tightly controlled. Figure 1.4 illustrates a proposed biosynthetic pathway for monoterpenes including /i-thujaplicin in C. lusitanica (taken from Zhao et al., 2006). 22 Cell wall Extracellular . Elicitor Vacuole monoterpene derivatives GPP < t IPP < t Mevalonate t 3-Hydroxy-3-methyl Olutaryl-CoA t Acetoacetyl-CoA t Acety l -CoA Monoterpenes f Monoterpene synthases >-GPP t > IPP <=> DMAPP \ IPP isomerase Isopentenyl monophosphate t 2-C-methyl-D-eryhthritoM-phosphate t 1 -Deoxy-D-xylulose-5-phosphate t. Pyruvate - D-Glyceraldehyde-3-phosphate Multiple signaling: Fungal elicitor molecules, Ca 2 + ,H 2 0 2 , MeJA... Transcription factors Cytosol Plastid Ribosomes Figure 1.4 Schematic illustration of signal transduction and metabolic flux of elicitor-induced /Mhujapl icin and other monoterpenes in Cupressus lusitanica cell cultures (Zhao et al. 2006). Note: Multiple elicitor signals are incorporated into transcriptional factors, which selectively activate or inactivate metabolic gene expression and downstream metabolic pathways. B-Thujaplicin biosynthesis involves two separate pathways and compartmentation regulation by the plasmid, cytosol, and vacuole. Transport of metabolic substrates and products across through these compartments may require different transporters, which could also be regulated by elicitors. Taken from Zhao et al. (2006). 23 1.7.2 Lignan Biosynthesis Lignan formation and accumulation differs profoundly from that leading to lignins. First, lignans are transported through specialized cells (such as ray parenchyma) and are infused into surrounding pre-lignified cells. Second, they are formed via distinct biochemical pathways. In contrast, lignification results via direct monomer transport from the cytoplasm of a lignifying cell into its polysaccharide-rich cell wall with subsequent polymerization. This process represents the first and final committed step of lignification, being primarily initiated and completed in maturing cell walls not far from the cambial zone. Gang et al. (1998) reviewed lignan biosynthesis in several softwood species including cedar (Figure 1.5). Much of the initial work on the lignan biosynthetic pathway had been done using Forsythia intermedia, also known as 'Border Forsythia'. It was suggested that the same pathway must also operate in the WRC heartwood metabolite forming process, and that these precursors undergo further transformation to give lignans such as plicatic acid. In cedar, coniferyl alcohol serves as the initial precursor, being subsequently metabolized with precise regio- and stereochemical control to produce the first species-specific lignans. Two monoligols of E-coniferyl alcohols are coupled via a dirigent protein to form the furofuran lignan (+)-pinoresinol. This represents the entry point into the pathway. (+)-Pinoresinol is subsequently reduced, first to the tetrahydrofuran, (+)-lariciresinol, and then to the dibenzylbutane lignan, (-)-secoisolariciresinol. This subsequently undergoes a two-step dehydrogenation to form the dibenzylbutyrolactone lignan, (-)-matairesinol. These precursors can then be further transformed to give the lignan (-)-plicatic acid (Davin and Lewis, 1995; Davin et al., 1997). 24 iT-Coniferyl alcohol Dirigent Protein Oxidase/ OCH, Oxidant OH HO OCH 3 (+)-Pinoresinol plr-Tpl OCH HO N A D P H .OH OCH 3 (+)-Lariciresinol plr-Tp2 H 3 CO HO N A D P H OCH3 OH (-)-Secoisolariciresinol (-)-secoisolariciresinol Dehydrogenase N A D P + OH (-)-Matairesinol H 3 C . OH (-)-Thujaplicatin CH, H^CO HO HO y ~OCH 3 OH (-)-Plicatin I H3CO HO °HCH->OH CO,H OCH3 OH (-)-Plicatic acid = 0 = Known conversion : Probable conversion Figure 1.5 The proposed biosynthetic pathway of plicatic acid (Gang et ai, 1998) 2 5 Swan and Jiang (1970) compared the sapwood and heartwood phenolic extractive content. They estimated the concentration of individual extractive compounds for each growth ring along a transect spanning from the outer sapwood to the pith of two W R C trees. From this data they postulated a biosynthetic pathway. Their first observation was that the concentrations of most extractive compounds increased by a factor of around 100 times across the sapwood to heartwood boundary. In the sapwood region the thujaplicatin methyl ethers (T.M.E.s) had the highest concentration of all compounds at approximately 0.1 % D W . They noted that T . M . E . concentration only increased by an order of 7-10 times into the heartwood yielding an average of 0.73% D W . However, there is a much greater increase in (-)-thujaplicatin concentration from sapwood to heartwood reaching a level 1.44 % D W . They also noted that the thujaplicins were almost nonexistent in the sapwood, but become rapidly more abundant in the heartwood, reaching approximately 0.2 % D W . They concluded that in the heartwood, lignans are formed in the sequence of (-)-thujaplicatin to (-)-dihydroxythujaplicatin to (-)-plicatin to (-)-plicatic acid by a series of hydroxylation reactions. However, in the sapwood, the starting material thujaplicatin is preferably converted into T . M . E ' s by O-methylation. It seems that thujaplicins in the heartwood act as enzyme inhibitors that prevent O-methylation, thereby promoting hydroxylation of (-)-thujaplicatin leading to the formation of (-)-plicatic acid rather than the T.M.E.s . Swan and Jiang's proposed biosynthetic pathways are shown in Figure 1.6. The rapid decrease in (-)-plicatic acid content toward the pith after its maximum content in the outer heartwood is reached might be attributed either to the lesser ability of the tree to synthesize it when younger, or to its self-polymerization due to its strongly acidic nature, or to further oxidation of it to other lignans. 26 In the Sapwood (O-methylation is favored) (-)-Thujaplicatin OH CH. H-,C, J O HO H 2 b (-)-Thujaplicatin Methyl Ether ,o \ o OH C H 3 H 3 C (-) -Hy droxythuj ap 1 i c atin Methvl Ether OH C H 3 H 3 C - ^ HO (-)-Dihydroxythujaplicatin Methyl Etlier 1 OH P T o s H 3 C OH C H 3 —k> Indicates site of hydroxylation = > Indicates site of O-methylation In the Heartwood (Hydroxylat ion is favored) H 3 c . HO (-)-Thujaplicatin OH C H 3 HoC (-)-Dihydroxy -thujaplicatin (-)-Plicatin O C H 3 OH CH 2 OH 'CQ 2 H (-)-Plicatic Acid OCH. OH Figure 1.6 The proposed lignan biosynthesis pathways in the sapwood vs. the heartwood of W R C (Swan & Jiang, 1970) 27 Given the biological activity of several of the extractive compounds of W R C there is an increasing demand for them in an ever-growing natural products industry. Their antimicrobial activity may play an important physiological role in protecting standing W R C trees from pathogen invasion and subsequently confer durability to W R C wood products. In the forest products sector, use of naturally durable woods such as W R C offer a perceived environmentally friendly alternative to biocide treated products. Presently, the high production costs of compounds such as y5-thujaplicin from callus cell cultures prevent their large scale application to non-durable woods such as pine but this may be a possibility i f such tropolones can be artificially synthesized on an industrial scale. Such production may be driven by the fact that such compounds also have potential pharmaceutical applications that range from U V skin protection, to anti-cancer and anti-HIV treatments. Given the small molecular size of tropolone compounds such as ^-thujaplicin in the field of cancer treatments may offer an attractive alternative to expensive, present day treatments such as Piclataxel, know commercially as 'Taxol ' , a large, complex molecule extracted from Taxus cuspidate, o f the genus Taxus (Yews). 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Oxidative stress in plant cell culture: a role in production of /i-thujaplicin by Cupressus lusitanica suspension culture. Biotechnol. Bioeng. 90: 621-631. 41 Zhao, J., Guo, T. Q., Kosaihira, A . , and Sakai, K . 2004b. Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated Cupressus lusitanica cell cultures. Planta. 219: 121-131. Zhao, J., Matsunaga, Y . , Fujita, K . , and Sakai, K . 2006. Signal transduction and metabolic flux of /i-thujaplicin and monoterpene biosynthesis in elicited Cupressus lusitanica cell cultures. Met. Eng. 8: 14-29. Zhao, J., Zheng, S., Fujita, K . , and Sakai, K . 2004a. Jasmonate and ethylene signaling and their interaction are integral parts of the elicitor signaling pathway leading to P-thujaplicin biosynthesis in Cupressus lusitanica cell cultures. J. Exp. Bot. 55: 1003-1012. 42 1.9 Objectives The overall goal of this work was to provide information on the relationship between durability, the change in extractive content, and microorganisms on W R C products in service in order to help the industry to develop strategies to improve the service life of W R C products. The main objective was to determine whether depletion or modification of natural extractives by microorganisms or weathering can explain the premature failure of W R C products I will test the following hypotheses: (a) Extractives are chemical ly modif ied / detoxified by extractive tolerant, 'p ioneer ' micro-organisms, paving the way for decay to occur. (b) Extractives are depleted by weathering making products susceptible to decay. (c) A combinat ion of (a) and (b). In reference to hypothesis (a), given the time limitations of this work, the primary focus was on examining extractive-tolerance of micro-organisms as being indicative that chemical modification of extractives may occur. Exploring the possible extractive detoxification mechanisms by micro-organisms wi l l be examined in future work. 43 Chapter 2 Extracting and Quantifying Heartwood Extractives of Western Redcedar {Thujaplicata Donn) Using Ultrasonication and Reverse Phase HPLC 2.1 Abstract Western redcedar {Thuja plicata Donn) ( W R C ) heartwood samples were extracted in methanol and reverse phase high performance liquid chromatography ( H P L C ) with an ultraviolet (UV) detection system was used for extractive separation and analysis. Six major extractives were quantified by comparing analyte response with the response factor of an internal standard by a single point calibration. The method's limits of detection were 0.6ug/ml for (-)-plicatic acid, 3.0ug/ml for y-thujaplicin, 3.0ug/ml for /^thujaplicin, 3.0pg/ml for yS-thujaplicinol, 0.6pg/ml for thujic acid and 1.2ug/ml for methyl thujate. Yields were 36% higher for powdered samples than for sliced samples. A temperature of 4°C during ultrasonication yielded 16% more (-)-plicatic acid than in non-cooled extractions but did not significantly increase yields for the remaining five compounds. We assessed the recovery and repeatability of the extraction method by adding the aromatic compounds methoxyhydroquinone and 2-acetonaphthone to heartwood samples. Recovery yield was - 9 0 % with - 5 % variability. Key words: extractives; high performance liquid chromatograpghy; lignan; quantification; tropolone; western redcedar (Thujaplicata Donn). 44 2.2 Introduction Western redcedar (Thuja plicata Donn) (WRC) is a naturally durable softwood species native to British Columbia in Canada as well as in Washington, Oregon and California in the U S A . Its heartwood extractives are comprised of a mixture of tropolones (2-hydroxy-2,4,6-cycloheptatrien-1-one) (Dewar, 1945) and lignan (dimeric, phenolic metabolites) compounds (Gang et al, 1998) with high commercial value (Barton and Macdonald, 1971; Jones and Falk, 2005). Several of these extractives, namely the 'thujaplicins' (a-,/?-, and y-thujaplicin) exhibit strong antimicrobial, insecticidal, and herbicidal activities with efficiency comparable to commercial pest control agents (Inamori et al., 1999, 2000; Sakagami et al., 2000; Ar ima et al., 2003; Morita et al., 2004). These and other structurally similar compounds also have cytotoxic activity against cancer cell lines in vitro (Baba et al., 1998; Matsumura et al., 2001; Inamori et al., 2003, 2004) and can inhibit human immunodeficiency virus (HIV) replication (Budihas et al., 2005). To date, only /i-thujaplicin is synthesized on a large scale using callus cultures of species such as Mexican cypress (Cupressus lusitanica). The remaining compounds are still obtained by extracting heartwood of W R C and other members of the Cupressaceae family. Formerly, wood extraction has been achieved using hot water or steam distillation (Barton and MacDonald, 1971; Mitsuhiko et al. 2002), soxhlet apparatus with organic solvents (Johansson et al., 2000), or supercritical fluid extraction using carbon dioxide (Terauchi et al., 1993; Ohira et al, 1994, 1996; Eller and K i n g , 2000). These methods are performed at temperatures ranging from 40-100°C. Other quantitative methods have been developed for analyzing W R C wood and foliage extractives using gas chromatograpghy (Nault, 1987; Kimbal l et al, 2005) that inherently require temperatures in the range of 100-300°C for the volatilization of analytes. However, 45 several of the compounds of interest such as (-)-plicatic acid, y- and /Mhujaplicin are thermo- or photo-sensitive (Coombs and Trust, 1973; Shibata et al., 2003). We describe a method to extract and quantify six major W R C extractives without exposing them to excess heat. We used reverse phase H P L C to separate extractives. Using extractive standards and an ultraviolet ( U V ) detection system we constructed calibration equations for quantifying six relevant extractives: (-)-plicatic acid, y-thujaplicin, /?-thujaplicin, /^thujaplicinol, thujic acid, and methyl thujate (Figure 2.1). We tested the effect of a) sample wood preparation and b) temperature on extractive yield using a methanol extraction by ultrasonication. Finally, we evaluated how method parameters affect extractive detection, recovery, and repeatability. 2.3 Materials and Methods 2.3.1 Chemicals and Solvents H P L C grade methanol and acetonitrile were purchased from the Fisher Scientific Company (Nepean, O N , Canada). Reagents/standards ethyl 4-hydroxy 3-mefhoxycinnamate (99% pure), methoxyhydroquinone (99% pure), 2-acetonaphthone (99% pure) and /^thujaplicin (99 % pure) were purchased from Sigma-Aldrich Canada Ltd (Oakville, Ontario, Canada). y-Thujaplicin (96% pure), /?-thujaplicinol (96% pure), and methyl thujate (96% pure) were provided by Forintek Canada Corp. (Vancouver, British Columbia, Canada). (-)-Plicatic acid (96% pure) and thujic acid (96% pure) were supplied by X y l o n Biotechnologies Ltd. , (Vancouver, British Columbia, Canada). A l l solvents and compounds were used as received. 46 CH2OH "CQ2H OH OCH3 H 3 C. X H 3 OH 2,3,6-Trihychoxy-7-methoxy2hydioxy^ 2-Hydioxy-5-isopropyl-2,4.,6--4-(3' ,4' -dihydroxy-5' -methoxyphenyl)- cycloheptatrien-1 -one tetralin-3-carboxylic acid (-)-Plicatic A c i d 2-Hydroxy-4-isopropyl-2,4,6-cycloheptatrien- 1-one /^-Thujapl icin HO HO 2,7-Dihydroxy-4-isopropyl-2,4,6-cycloheptatrien-1 -one /?-Thujapl ic inol OCH-7J-Dimethylcycloheptatriene-3-carboxyhc acid Thuj ic A c i d 7,7-Dimethyl-3-carbomethoxy-1,3,5- cycloheptatriene Methy l Thujate Figure 2.1 Chemica l structure and nomenclature used for W R C heartwood extractive compounds of interest. 47 2.3.2 Separation of Extractives by H P L C Extractive compounds were separated by reverse phase H P L C as described in Daniels and Russell (2006) using a Waters 2695 H P L C from Waters Corp. (Mississauga, Ontario, Canada) equipped with a ODS3 C-18 reverse phase column (3pm, 4.6mm x 150mm) from Intersil Corp. (Milpitas, California, U S A ) and a Dionex A D 2 0 ultraviolet ( U V ) detector from Dionex Corp. (Oakville, Ontario, Canada). MassLynx analytical software from Micromass Ltd (Manchester, U K ) was utilized for chromatographic analysis. Mixed extracts were injected (15ul) on to the column and readily separated using the method described in Table 2.1. 2.3.3 Confirmation of Extractive Chemical Structures by N M R The molecular structures of selected extractives were confirmed from pure, crystalline standards 1 13 using nuclear magnetic resonance ( N M R ) . H , and C N M R spectra were measured using a Bruker A V A N C E - 3 0 0 spectrometer from Bruker (Billerica, Massachusetts, U S A ) at 40 °C in either M e O D (deuterated methanol) or D M S O - d 6 (deuterated dimethyl sulfoxide). Chemical shifts were referenced to tetramethyl silane ( T M S ; 0.0 ppm), summary in Table 2.2. N M R data is consistent with structures reported in literature sources (Erdtman and Gripenberg, 1948; Barton and Gardner, 1954; Gripenberg, 1956; Gardner et al., 1957, 1966). A mixed extract sample was then spiked with each of the standards and subject to H P L C analysis in order to confirm which peak corresponded to which extractive compound. 2.3.4 Chromatographic Sensitivity and Quantification of Extractives Pure crystalline extractive standards were used to estimate the method limit of detection ( M L O D ) for the six extractives. M L O D was defined as the concentration of analyte (pg/ml) required to produce a chromatographic signal equal to three times the peak-to-peak noise. A 48 Table 2.1 HPLC program for the separation of WRC heartwood extractives (Daniels and Russell, 2006). Time (min) Solvent A (%) Solvent B (%) Flow Rate (ml/min) 0 10 90 1 50 60 40 1 60 10 90 1.5 62 10 90 1 Note: solvent A = 0.1% formic acid, 99.9% nano pure H 2 0 . Solvent B = 99.9% acetonitrile with 0.1% formic acid. Column temperature was 50°C, and a U V wavelength of 230nm was used. A total elution was typically reached at 48 min. 49 Table 2.2 A Summary of N M R Data for the Extractives of Interest. Compound NMR Data (-)-Plicatic acid ' H N M R (DMSO-d 6 , 300 MHz): 5 = 2.75 (d, J= 6.9 Hz, 2H, CH 2 ) , 3.44 (s, 2H, CH 2 OH), 3.70 (s, 6H, OCH 3 ) , 4.30 (s, 1H, CH), 6.06 (s, 1H, CH), 6.21 (s, 1H, CH), 6.30 (s, 1H, CH), 6.56 (s, 1H, CH). 1 3 C N M R (DMSO-d 6 , 75.4 MHz): 5 = 19.0, 34.62, 56.1, 56.5, 62.2, 93.2, 97.8, 102.4, 112.8, 116.4, 126.1, 132.1, 132.9, 144.1, 144.7, 145.9, 147.4, 177.5. y-Thujaplicin *H N M R (MeOD, 300 MHz): 5 = 1.26 (d, J= 6.9 Hz, 6H, CH 3 ) , 2.93 (q, J = 6.9 Hz, 1H, CH), 7.32 (d,J= 11.7 Hz, 2H, CH), 7.45 (d,J= 11.7 Hz, 2H, CH). 1 3 C N M R (MeOD, 75.4 MHz): 5 = 22.5(2C), 37.5 (IC), 124.9 (2C), 136.5 (2C), 150.0 (IC), 171.0 (2C). ^-Thujaplicin ' H N M R (MeOD, 300 MHz): 5 = 1.27 (d, J= 6.9 Hz, 6H, CH 3 ) , 2.93 (q, J= 6.9 Hz, 1H, CH), 7.08 (d, J= 10.2 Hz, 1H, CH), 7.22 (d, J= 10.8 Hz, 1H, CH), 7.33 (s, 1H, CH), 7.43 (t,J= 10.5 Hz, 1H, CH). > 1 3 C N M R (MeOD, 75.4 MHz): 8 = 22.3, 38.7, 122.7, 123.7, 127.7, 137.5, 160.4, 171.6. ^-Thujaplicinol 'H N M R (MeOD, 300 MHz): 8 = 1.26 (d, J= 6.9 Hz, 6H, CH 3 ) , 2.92 (q, J= 6.9 Hz, 1H, CH), 7.12 (dd,J= 10.8, 1.5 Hz, 1H, CH), 7.39 (d, J= 11.1 Hz, 1H, CH), 7.43 (d,J= 1.5 Hz, 1H, CH). 1 3 C N M R (MeOD, 75.4 MHz): 8 = 22.7, 38.1, 120.6, 121.5, 127.2, 151.2, 158.8, 160.6, 166.9. Thujic Acid ! H N M R (MeOD, 300 MHz): 5 = 1.01 (s, 6H, CH 3 ) , 5.25 (d, J= 10.5 Hz, 1H, CH), 5.49 (d, J = 9.9 Hz, 1H, CH), 6.28 (dd, J = 9.9, 6.9 Hz, 1H, CH), 6.67 (d, J= 10.2 Hz, 1H, CH), 7.56 (d, J= 6.9 Hz, 1H, CH). 1 3 C N M R (MeOD, 75.4 MHz): 8 = 25.2, 34.7, 122.8, 123.2, 131.7, 132.1, 135.5, 138.3, 169.4. Methyl thujate 'H N M R (MeOD, 300 MHz): 8 = 1.00 (s, 6H, CH 3 ) , 3.81 (s, 3H, OCH 3 ) , 5.24 (d, J= 10.2 Hz, 1H, CH), 5.49 (d, J= 10.2 Hz, 1H, CH), 6.27 (dd, J= 9.9, 6.9 Hz, 1H, CH), 6.66 (d, J= 10.2 Hz, 1H, CH), 7.54 (d, J= 6.6 Hz, 1H, CH). 1 3 C N M R (MeOD, 75.4 MHz): 8 = 25.5, 34.7, 51.2, 122.5, 123.2, 131.3, 132.2, 135.4, 138.3, 168.0. 50 1 mg/ml stock solution prepared for each standard in methanol was diluted to make solutions of 5, 4, 3, 2, 1 pg/ml. Following chromatographic analysis an additional set of solutions was prepared with concentration points of smaller, more directed increments until the M L O D was estimated to within 0.1 pg/ml. Calibration equations were constructed relative to the internal standard ethyl 4-hydroxy 3-methoxycinnamate whish possesses a chromatographic retention time ( /R ) proximal to extractives of interest and its ?R does not overlap with extractive peaks. From stock standard solutions, a six-point concentration gradient was prepared that reflected the natural abundance of each compound in heartwood; the lowest point was the M L O D in each case. To the six solutions the IS was added at a concentration of lOOug/ml. Then six additional solutions with the compound of interest at lOOug/ml were spiked with the IS at the same concentrations. From these data a twelve-point scatter plot was constructed, where the y axis was defined as area ratio (extractive peak area divided by IS peak area); and the x axis as concentration (pg/ml) ratio ([extractive] divided by [IS]). This process was repeated three times and a scatter plot was constructed from the mean data obtained. The detector responses for each compound were subjected to linear regression analysis, and response factors were calculated. Statistical analysis was performed of the response factors to distinguish between significantly different values. 2.3.5 Sample Preparation and Optimization of Extraction W R C cubes of 19mm on each dimension were manufactured from the outer heartwood of a sound 136yr old second growth W R C tree felled in the U B C Malco lm Knapp research forest, Maple Ridge, British Columbia, Canada. Samples originated from a single longitudinal axis parallel with growth rings. Blocks were sequentially numbered relative to their position. Even-51 numbered blocks were utilized for chemical analysis. Odd-numbered blocks were oven dried at 105°C for 24 hours to estimate the dry weight (DW) of the neighboring even-numbered blocks. Each sample was typically 2.0 ± 0.2g D W . O f twenty four samples, eight were sliced as thinly as possible; slices were typically 2mm in width. Sixteen samples were ground to a fine powder (<150 microns) using a freezer mi l l equipped with a coolant circulation chamber from Bel-art products (Pequannock, New Jersey, U S A ) . A temperature of ~ 4°C was maintained during grinding to avoid generation of excess heat caused by friction. W R C Samples were steeped in 15ml methanol and exposed to ultrasonic frequency (40 kHz) for 120 minutes using a Branson 8510 ultrasonic bath (Danbury, Connecticut, U S A ) . Samples were extracted once only in each case. Eight ground samples and eight sliced samples were extracted in a 4°C bath with a l id and cooled with ice. Another eight ground samples were extracted in a non-cooled bath. Extracts were filtered using a 0.2pm nylon syringe filter to remove any wood particles and stored at 4°C in the dark. Typically 12ml of filtered extract was recovered from each sample. This experiment followed a completely randomized design (CRD) with three treatments: grinding (cooled), slicing (cooled), and grinding (non-cooled) (k - 3), Eight replicate samples were used in each case {n = 8). 2.3.6 Method Evaluat ion Method recovery and repeatability was assessed by spiking heartwood samples with two aromatic compounds, methoxyhydroquinone and 2-acetonapthone not typically present in W R C but which have «R near to peaks of interest. lOOpl of a solution containing 1.5mg/ml methoxyhydroquinone and 2-acetonapthone in methanol was added to ground samples prior to extraction. Eight spiked samples and eight control samples (not spiked) extracted and analyzed. 52 Response factors were calculated for methoxyhydroquinone and 2-acetonaphthone as stated previously. The mean recovery value and relative standard deviation (RSD) was calculated for both compounds as a measure of the repeatability. 2.3.7 Statistical Analysis A l l experiments followed a completely randomized design (CRD) with treatments denoted as k and replicates as n. A one-way analysis of variance ( A N O V A ) (a = 0.05) and Tukey's test for comparison of means (Tukey, 1949) were performed on data. Statistical analysis was performed using J M P I N software (version 4.0.3 (academic), S A S Institute Inc., North Carolina, U S A ) . 2.4 Results and Discussion A typical chromatogram of W R C heartwood extractives is shown in Figure 2.2. Chromatographic analysis using this method was highly sensitive, detecting extractive compounds at concentrations ranging from 0.6 to 3.0pg/ml depending on the compound. The method's limit of detection was estimated at 0.6ug/ml for (-)-plicatic acid, y-thujaplicin (3.0pg/ml), ^thujapl icin (3.0ug/ml), /Mhujaplicinol (3.0ug/ml), thujic acid (0.6pg/ml) and methyl thujate (1.2ug/ml). The best fits by regression analysis of the detector responses for each of the extractive compounds were linear (R 2 > 0.96) (Table 2.3) over the range of concentrations investigated, indicating that single point calibrations could be used. A N O V A (a=0.05) followed by Tukey's test of multiple comparisons (Tukey, 1949) suggested that mean response factor values for extractives could be separated into four groups. Response factor values for the isomeric isopropyl-tropolones y- and /^thujaplicin were similar and it is common practice to assume equivalent response factors among compounds of identical carbon number and similar structure (Kimball et al, 2005). 53 E c o CO CM CD O £Z co .£ *\ o CO -Q < > u 1. 2-Acetonapthone 2. (-)-Plicatic Ac id 3. y-Thujaplicin 4. /J-Thujaplicin 5. y?-Thujaplicinol 6. Internal Standard 7. Thujic Ac id 8. Methoxyhydroquinone| 9. Methyl Thujate 5.00 ' 7.5)) 'VoW ' Time (min) Figure 2.2 Chromatogram obtained from the analysis of W R C heartwood extractives. 54 Table 2.3 Linear regression analysis from mean detector response data for calibration standard solutions over specified concentration ranges. Extractive compound Range (pg ml" 1) Linear Formula R 2 (-)-Plicatic acid ^ 0.6-1000 y = 0.4496x - 0.0074 0.97 y-Thujaplicin * 3.0-200 y = 2.4223x-0.5015 0.96 /^-Thujaplicin * 3.0-200 y = 2.3401x-0.5642 0.96 y5-Thujaplicinol 1 1 3.0-200 y = 1.171x-0.3653 0.97 Thujic acid t 0.6-200 y = 0.121 l x +0.0083 0.98 Methyl thujate * 1.2-200 y = 0.185x- 0.0079 0.97 2-Acetonaphthone * 1.0-200 y = 0.443x- 0.0116 0.99 Methoxyhydroquinone s 1.0-200 y = 3.2781x +0.0563 0.99 y- & /?-Thujaplicin* 3.0-200 y = 2.3812x-0.5329 0.96 Note: compounds followed by the same symbol indicate that their mean response factor values were not significantly different according to Tukey's test of multiple comparisons. R 2 indicates correlation coefficient. Compounds 2-acetonapthone and methoxyhydroquinone are used for sample fortification and are not extractives of W R C heartwood. *Mean response factor and linear equation for isomers y- and /^-thujaplicin. 55 We compared two wood preparations, powders and slices. Grinding heartwood made extraction more efficient and uniform. During a cooled extraction, ground samples yielded ~ 36% more extractives than sliced (Table 2.4). The increased yield was statistically significant (a = 0.05). Further, extractive concentration variability was higher for sliced than for ground samples (mean RSDs for pg/g D W were 9.2 and 4.0%, respectively). Grinding wood increased the ratio of surface area to volume, providing a greater contact area for the solvent and so improving the extraction efficiency. Grinding may also disrupt cells, promoting solvent penetration between cells and cell fragments, and into lignified xylem cells with aspirated pits. Because some extractives are not thermostable, we examined the effect o f cooling samples during ultrasonication. Without cooling, we recorded temperatures of up to 42°C during the ultrasonic extraction cycle. On average, ground samples extracted in a cooled ultrasonic bath yielded ~ 16% more (-)-plicatic acid than in ground samples extracted in an un-cooled bath. However, low temperatures (4°C) did not significantly increase the yield for the other five compounds. Estimating the efficiency of extraction is difficult given that a majority of heartwood extractives are synthesized in situ at the sapwood/heartwood border and infuse into lignified cells (Swan and Jiang, 1970). However, recovery of spiked compounds gives a good indication of losses that may occur during the extraction process. To assess such losses we spiked eight samples with compounds methoxyhydroquinone and 2-acetonaphthone (Table 2.5). A 100% yield recovery equated to a final concentration of lOOug/ml when samples were extracted in 15ml of solvent. Recovery of methoxyhydroquinone from spiked samples was 95.5% with a R S D of 5.3%. Recovery of 2-acetonaphthone was marginally lower at 83.1%) with a R S D of 5.9%. 56 Table 2.4 The effect of various extraction methods on extractive concentration (pg/g DW). Extractive Finely ground Sliced Finely ground compound (cooled) RSD* (cooled) RSD (non-cooled) RSD (-)-Plicatic Acid 11041.7 '(393.1)2a 3.6 7033.3 (406.7)° 5.8 9182.3 (479.8)b 5.2 y-Thujaplicin 1842.9 (45.8)a 2.5 1269.1 (83.6)b 6.6 1806.5 (54.1)a 3 /?-Thujaplicin 1828.5 (45.1)a 2.5 1175.2 (64.5)b 5.5 1717.4 (73.8)a 4.3 /?-Thujaplicinol 243.4 (2.7)a 1.1 154.3 (25.0)b 16.2 240.7 (23.5)a 9.8 Thujic Acid 5963.2 (248.5)a 4.2 4112.2 (171.4)b 4.2 5751.4 (246.3)a 4.3 Methyl Thujate 69.2 (7.1)a 10.3 35.8 (6.1)b 17 54.3 (13.4)a 24.7 Note: 'numbers is parenthesis indicate standard deviation. A N O V A generated the following critical F values for extractive of interest: (-)-plicatic acid (F(2,2i)= 279.6), y-thujaplicin (F(2,2i) = 133.9),^-thujaplicin (F ( 2 ,2i)= 341.3),jff-thujaplicinol (F ( 2 ,2i)= 71.4), thujic acid (F ( 2 ; 2 i )= 162.3), and methyl thujate ( F ( 2 j 2 i ) = 31.9). Values were considered significant i f greater than the tabular value of F(2,2i) = 2.57 (a=0.05). Numbers followed by the same letter were not significantly different (a=0.05) according to Tukey's test of multiple Comparison of means. * R S D = relative standard deviation expressed as a percentage. 57 Table 2.5 Recovery and repeatability data for compounds 2-acetonapthone and methoxyhydroquinone. Concentration of Recovered Compounds (pg ml"1) 2-Acetonapthone Methoxyhydroquinone Sample 1 84.1 99.4 Sample 2 84.8 99.7 Sample 3 86.5 96.8 Sample 4 88.4 99.5 Sample 5 79.1 92 Sample 6 75.3 88.1 Sample 7 87.9 99.6 Sample 8 78.3 89 Mean Recovery 83.1 '(4.9) 95.5 (5.0) * R S D % 5.9 5.3 Note: numbers is parenthesis indicate standard deviation. * R S D = relative standard deviation expressed as a percentage. 58 We estimated that the total extractive content of W R C heartwood was approximately 3.3% (w/w D W ) . This was achieved by conducting an exhaustive extraction of eight W R C heartwood samples in methanol, filtering the extract and then using a rotovaporator to bring extracts to dryness. Among the six extractives analyzed (-)-plicatic acid was the most abundant at 11708.7pg/g D W (RSD = 4.1%), accounting for 35.7% of the total extractives (w/w). Consistent with the literature (Barton and MacDonald, 1971) y-thujaplicin (~1971.4pg/g D W ; R S D = 2.9%) was slightly more abundant than /?-thujaplicin (~1893.1ug/g D W ; R S D = 2.9%), both chemicals accounting for 6% and 5.8% (w/w) of the total extractives respectively. /i-Thujaplicinol at 244.6ug/g D W (RSD = 1.3%) (0.8% w/w) was equivalent to 12% of y- and /i-thujaplicin concentration. Thujic acid was the most abundant non-lignan extractive at 6094ug/g D W (RSD = 4.8%) (18.6% w/w), while methyl thujate was present in trace amounts 74.5pg/g D W (RSD = 11.1%) (0.2% w/w). Our data were consistent with those of Jones and Falk (2005) who compared extractive yields from W R C heartwood using various extraction techniques. Except for methyl thujate (RSD o f 11.05%), very little variation was observed in the estimated concentration (ug/g D W ) of the compounds of interest with RSDs generally 5% or lower. Variability in the data were minimized by a) processing the samples with the freezer mi l l , b) protecting experimental samples from excess heat and light and c) using heartwood experimental samples in close proximity to one another within the tree. Only six extractive compounds have been examined in the work reported here, many more have been characterized with several more uncharacterized. However, we estimated that overall these six extractive compounds accounted for 67% of the total extractive content of W R C used in this work. 59 2.5 Conclusions Extraction of finely ground wood samples using methanol under ultra-sonic frequency provided yield of extractives and compounds with a broad range of polarity. The method itself is capable of detecting individual extractives at very low concentrations (0.6-3.0 pg/ml). The extraction and analysis process showed good efficiency and repeatability with recovery two aromatic compounds which were added to samples prior to extraction of at ~ 90% and an R S D of ~ 5%. It is difficult to estimate the recovery yield of heartwood extractives given that they are synthesized in situ and secreted with specialized cells. Quantitative analysis of six analytes was achieved by employing single-point calibrations using a single internal standard which is commercially available. This method was subsequently used for screening extractive tolerant fungal species (part 2) (see L i m et al., 2006). The method described is straightforward and could be applied to almost any situation where such extractive compounds are required to be quantitated, particularly given the interest in using such compounds for pharmaceutical applications. 60 2.6 References Arima, Y. , Nakai, Y. , Hayakawa, R., and Nishino, T. 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Shibata, H . , Nagamine, T., Wang, Y . , and Ishikawa, T. (2003) Generation of reactive oxygen species from Hinokitol under near-UV irradiation. Biosci . Biotechnol. Biochem. 67:1996-1998. Swan, E . P., Jiang, K . S., and Gardner, J. A . F. (1969) The lignans of Thuja plicata and the sapwood-heartwood transformations. Phytochem. 8:345-351. Terauchi, F., Ohira, T, Yatagai, M , Ohgama, T, A o k i , H , and Suzuki, T. (1993) Extraction of volatile compounds from coniferous woods with supercritical carbon dioxide. Mokuzai Gakkaishi 39:1421-1430. Tukey, J. W. (1949) Comparing individual means in the analysis of variance. Biometrics 5:99-114. 64 Chapter 3 Isolating and Testing Fungi Tolerant to Western Redcedar {Thujaplicata Donn) Extractives 3.1 Abstract Western redcedar (Thuja plicata Donn) (WRC) is a naturally durable softwood species native to British Columbia in Canada, as well as Washington, Oregon and California in the U S A . Untreated W R C products are durable because they contain extractive compounds that have strong anti-microbial activity. However, products can still fail in service due to fungal colonization initiated by extractive tolerant species and to depletion of extractives caused by weathering. To screen for extractive tolerant species we developed a WRC-feeder strip assay. When WRC-feeder strips were placed on malt extract agar, extractives from the wood accumulated in the media and strongly inhibited growth of non-tolerant fungal strains. Extractives remaining in feeder strips following incubation on media were characterized. O f the many compounds leached out, y- and /i-thujaplicin, /i-thujaplicinol, plicatic acid and thujic acid were quantified. The growth of selected fungal strains was not affected by plicatic acid; however, it was inhibited to different degrees by /?- and y-thujaplicin. Pachnocybe ferruginea was extractive tolerant and may play an important role in the initial stages of degradation of W R C products. Key words: y-thujaplicin, /J-thujaplicin, Extractives, Pioneer fungi, Western redcedar, Decks 3.2 Introduction Western redcedar (Thuja plicata Donn) (WRC) is a naturally durable softwood species native to British Columbia, Canada as well as Washington, Oregon and California in the U S A . Its heartwood contains higher concentrations of aromatic and polyphenolic extractives than other softwood species (Barton and MacDonald, 1971). Several of these compounds exhibit strong antimicrobial activity (Inamori et al., 1999, 2000; Ar ima et al., 2003; Morita et al., 2004). Because these compounds are highly effective against wood decay fungi, they may protect standing trees from being weakened and prone to wind-throw (Van der Kamp, 1986; DeBel l et al., 1997) and make W R C wood products naturally durable. Given its durability, cedar is extensively utilized for wood products for exterior residential applications and accounts for a significant proportion of Canada's forest products industry (Gonzalez, 2004). W R C products can still fail in service due to a) depletion of extractives caused by weathering or b) colonization by extractive-tolerant fungal species. Weathering involves photo-degradation of extractives by U V radiation in sunlight and precipitation leaching out chemicals from the surfaces or ends of wood boards causing depletion of the overall extractive content (Coombs and Trust, 1973; Johnson and Cserjesi, 1980). This may permit colonization by decay fungi that are inhibited by the original extractive content. Pioneer fungi have been isolated from standing W R C trees and products in service, frequently from the inner regions of W R C products where extractives may be less affected by weathering (Van der Kamp, 1975; L i m et al, 2005). Such species do not decay wood, but may detoxify fungicidal extractives, facilitating wood colonization by decay fungi, which are less tolerant to extractives (Jin et al., 1988). Preventing colonization by pioneer fungi may increase the service life of W R C products. To date only a few pioneer fungi have been isolated and characterized. In this work, we described a method for screening W R C extractive tolerant fungi. Agar media with extractives or pure compounds at different concentrations have been used to screen microorganism tolerance to W R C extractives (Jin et al., 1988, L i m et al, 2005). Synthesis or isolation o f total or specific extractive compounds is costly and using a single compound may not reflect the effects of total extractives. Given this, we used W R C - feeder strips ( W R C - F S ) to detect decay and pioneer fung that are tolerant to a mixture o f naturally occurring W R C extractives. Results for fungal growth with W R C - F S were compared to data obtained with single extractives. 3.3 Materials and Methods 3.3.1 Fungal Isolations and Identification W R C boards were collected from house decks and experimental test sites located in Vancouver, British Columbia, Canada. The service life of decks ranged from 20-100 years. We sampled twelve boards from deck A (100 years), five boards from deck B (20 years) and seven boards from deck C (25 years). Two sections about 2 cm in thickness were cut from each board using a circular saw..Fungi were isolated within and near decay pockets as described by L i m et al. (2005). A 1% malt extract agar ( M E A ) was used for isolating general fungal flora and 1% M E A with benomyl ( B M E A ) was used for basidiomycetes fungi (Clubbe and Levy, 1977). Isolates were grouped by macro- and micro-morphological characteristics using taxonomic guides and standard procedures (Nobles, 1965; Stalpers, 1978; Wang and Zabel, 1990). This initial identification was complemented by molecular techniques. D N A was extracted from mycelia and the internal transcribed spacer (ITS) region was amplified using the primers ITS5 and ITS4 ( L i m et al, 2005; White et al, 1990). P C R products were purified using a Qiaquick P C R Purification K i t (Qiagen Inc., Mississauga, Ontario, Canada). Sequencing was performed on an A B I 3700 automated sequencer (Perkin-Elmer Inc., Foster City, California, U S A ) at the D N A synthesis and Sequencing Facility, M A C R O G E N (Seoul, Korea). A l l the nucleotide sequences presented in this work have been deposited at Genbank and their accession numbers are shown in Table 3.1. 3.3.2 Inhibition of Fungal Growth by WRC Feeder Strips (WRC-FSs). W R C - F S s , about 5 x 3 x 0.2cm in size, were obtained from the outer heartwood of a sound 80 year old W R C tree harvested at U B C Malcolm Knapp Research Forest, Maple Ridge, British Columbia. A l l wood samples originated from a single longitudinal axis parallel with growth rings. Blocks were sequentially numbered relative to their position. Samples were immediately placed into individual sealable bags, labeled and stored at -20°C until further use to minimize volatilization of extractives. The FSs used in this study were sterilized by irradiation using electron beam technology (Iontron Industries Canada, Port Coquitiam, British Columbia, Canada) which prevents alteration of extractives that may occur through sterilization by alternative methods such as autoclaving. To measure fungal growth rate (mm/day), an agar plug (5 mm in diameter) from a freshly grown fungal isolate on M E A , was transferred onto 1 % M E A with a W R C - F S . The inoculum and the W R C - F S were placed on opposite sites of the M E A plate. Fungi were also grown on 1% M E A without FS (control). A l l plates (three replicates per isolate) were incubated at 20 °C in the dark to prevent extractive photo-degradation. Fungal growth from each culture was measured every 3 days by taking two perpendicular measurements from the inoculum to the maximum and minimum edge of the colony. The two measurements were averaged. The average growth rate (mm/day) was calculated after 21 days Growth inhibition tests were performed onto 1% M E A supplemented with individual extractives. The added concentration for each compound was calculated from the W R C - F S experiment (see below). Table 3.1. Fungal isolates from W R C deck in service and fungal growth inhibition by WRC-FSs. Source Growth (mm/day)a Fungal ID Acc. No for ITS A B C Location Con FS Inhibition (%) Acanthophysium lividocaeruleum AY618666 5 3 4 D / S 2.4 0.9 64.4 Coniophora puteana DQ516523 2 1 - D 2.6 1.1 56 Dacrymyces stillatus DQ516524 1 2 2 D 0.6 0.1 83.5 Hyphoderma praetermissum AY618668 3 - 2 D 1.7 0.1 94.5 Pachnocybe ferruginea AY618669 9 14 7 D / S 0.8 0.8 8.1 Phellinus ferreus DQ516525 2 - 5 D 2.2 0.1 95.6 Aureobasidium pullulans DQ516526 2 - 2 D 0.3 0.1 69.4 Exophiala heteromorpha DQ516527 5 - 5 D 1.4 0.4 71.4 Phialocephala dimorphospora AY618688 12 8 9 D 2.1 0.3 86.6 Rhinocladiella atrovirens AY618683 20 10 12 D 0.6 0.2 71.7 WRCF-A1 AY618686 22 5 12 D 1.3 0.2 82.1 Umbelopsis autotrophica DQ516528 2 3 1 D 2.4 0.2 90.3 Note: Numbers below the source represent isolate frequency for A, 100 year old deck; B, 20 year old deck; C, 25 year old deck. From each source approximately 150 isolations were made. Site of isolation: D (decay areas), S (sound inner areas). aAverage growth rate (mm day'1) calculated after 21 days on 1% MEA without (control = con) or with WRC-FS (FS). Values were mean of three replicates. 3.3.3 Extractive Analysis of WRC-FSs W R C - F S s were placed onto 1% M E A and then examined in order to estimate the amount of extractives that leached out of the W R C - F S and accumulated in the media. Five W R C extractive compounds of interest were extracted, separated and quantified (ug/g dry weight, D W ) by reverse phase high performance chromatography ( H P L C ) equipped with an ultraviolet (UV) detection system as described in Chedgy et al. (2006) and Daniels and Russell (2006). Extractives of interest were (-)-plicatic acid, y-thujaplicin, B-thujaplicin, /Mhujaplicinol, and thujic acid. These compounds were selected on the basis that they are: (a) known to have antimicrobial activity; and (b) are major constituents of W R C heartwood. W R C - F S s were cut into two equal sections and weights were recorded for each section. One section was oven dried at 105°C for 24 hours to calculate the dry weight (DW). The second section was finely ground, and subjected to chemical analysis. This experiment followed a completely randomized design (CRD) with three treatments: W R C -FSs (i) frozen at -20°C (control), (ii) placed in empty plates and stored at 20°C for 21 days; (iii) stored on 1% M E A at 20°C for 21 days (k = 3). Six replicates were made in each case (n = 6), and the mean concentration of extractives was then calculated. 3.3.4 Statistical analysis A l l experiments followed a C R D with treatments denoted as k and replicates as n. A one-way analysis of variance ( A N O V A ) (a = 0.05) and Tukey's test for multiple comparisons of means (Tukey, 1949) were performed on data. Treatment' effects were considered significant i f the resulting critical F value was greater than the appropriate tabular value (F[(k-i)],[(k(n-\)])- Statistical analysis was performed using J M P IN software (version 4.0.3 (academic), S A S Institute Inc., North Carolina, U S A ) . 3.4 Results and Discussion 3.4.1 Fungal Identification and Extractive Resistance Tests A total of 242 fungal isolates were recovered from 24 boards taken from three W R C decks. Using macro- and microscopic characteristics, as well as molecular D N A data, 12 fungal taxa were isolated more than three times. Six species isolated on B M E A were identified as basidiomycetes. One zygomycete and five ascomycetes were identified because of their sporangia and asexual structures, respectively (Table 3.1). The initial fungal identification was complemented by ITS sequence analyses. ITS sequences often diverge at the species level, and are preferentially used for identification (Schmidt and Moreth, 2002, 2003; Hogberg and Land, 2004). Except for one ascomycete species ( W R C F - A 1 ) , sequence analyses confirmed morphological identifications, and allowed fungi that could not be identified by morphology to be grouped with known species (Table 3.1). W R C F - A 1 had no distinct asexual morphology on M E A media and its ITS sequence showed 95% sequence similarity with unknown endophytic fungi and leaf litter ascomycetes. The basidiomycetes most frequently isolated were Acanthophysium lividoeaeruleum and Pachnocybe ferruginea. They were present in both decay areas and sound inner areas which tend to have a higher extractive content as extractives in the inner regions are less prone to photo-degradation from U V and depletion by leaching (Coombs and Trust, 1973; Hon, 1991; Shibata et al, 2003). The ascomycetes, Phialocephala dimorphosphora, Rhinocladiella atrovirens, and the species W R C F - A 1 were commonly isolated but were present mainly in decay areas. It is likely that these species occupy decay areas because they have low to moderate extractive resistance and decay pockets contained lower extractive concentrations compared to sound areas. The four known fungal species reported here, have been also isolated from W R C products in service (Scheffer et al., 1984; Wang and Zabel, 1990). One or two representatives from each of the 12 taxa were used to determine tolerance to W R C extractives. When grown on 1% M E A with W R C - F S , most isolates showed less growth than the controls (Figure 3.1). Several of the extractives are water-soluble and diffuse from the W R C - F S into the media when placed in contact. In addition, W R C - F S were manufactured such that the transverse wood face was in contact with the media promoting infusion of moisture into the vesicles and tracheids of the wood, allowing more extractives to diffuse from the wood into the media. P. ferruginea exhibited the highest tolerance to W R C - F S extractives, displaying a growth rate comparable to controls. This species was the most common one in this work and has frequently been reported on W R C fences and creosote-treated W R C poles (Wang and Zabel, 1990; L i m et al., 2005). It has also been reported to be commonly isolated from the heartwood of Douglas-fir and was characterized by Kropp and Corden (1986). Two basidiomycetes, A. lividoeaeruleum and Coniophoraputeana, and three ascomycetes, Aureobasidium pullulans, Exophiala heteromorpha, and R. atrovirens, had growth of 28 to 44% relative to controls, which represented moderate tolerance to W R C extractives (Table 3.1). Although C. puteana had higher W R C extractive tolerance than A. lividoeaeruleum, it was found in or near decay pockets and was less frequently isolated. A. lividoeaeruleum, one of the most frequently isolated species from W R C fences, was suggested as being a pioneer species with P. ferruginea, based on their tolerance to /i-thujaplicin (L im et al, 2005). A. pullulans, E. heteromorpha and R. atrovirens were detected near or in decay pockets. A. pullulans is able to colonize weathered wood surfaces from a variety of tree species, including W R C , and to cause black stain (Schoeman and Dickinson, 1997; Chedgy et al, 2005). It may play an important role in colonizing and modifying wood surfaces rather than inner areas of W R C products. The growth of other fungi was significantly reduced on plates with W R C - F S . The only zygomycete Umbelopsis autotrophica showed very low tolerance to extractives and thus is unlikely to have ability to Figure 3.1. Fungal Growth on media with or without a WRC-FS. A : Pachnocybe ferruginea, B: Phellinus ferreus growths on 1% M E A (control) and on 1% M E A with W R C feeder strips after 21 days incubation at room temperature. modify extractives and is unlikely to cause decay of wood. On W R C product surfaces, it has been reported that the pattern of fungal colonization consisted of staining fungi followed by soft-rot and decay fungi (Banerjee and Levy, 1971; Clubbe, 1980). The inner sound areas of W R C boards contained no staining or soft-rot fungi, only extractive tolerant basidiomycetes (e.g. P. ferruginea), suggesting that these species may play a pivotal role in the degradation of the inner parts of W R C products. 3.4.2 Extractives Analyses of WRC-FSs Wood is a naturally variable material and its extractive concentrations vary within and between trees. We observed a similar trend in our control W R C - F S s . The total extractive content of W R C - F S was approximately 3.3% (w/w D W ) , and the five compounds quantified in this work represent about 67% (w/w D W ) of this total. The overall concentration of the five extractives varied from 16.86-19.24 mg/g D W accounting for a variation of 14%. We determined that 57.5% of the extractives that we measured diffused into the media based o f the amount of extractives recovered from the W R C - F S s placed on M E A . Among the five compounds measured, y-thujaplicin and /?-thujaplicin leached out more than plicatic and thujic acids or /j-thujaplicinol from the W R C - F S s (Figure 3.2). Data analyses by A N O V A showed that the treatment effect was significant for each of the five extractives of interest. Incubation of W R C - F S on media caused a significant loss (a = 0.05) of extractives from the wood to the media. We examined the effects of the above five pure compounds on fungal growth on M E A . Each compound was used at a concentration equivalent to its loss from the FS (Figure 3.2, Table 3.2). • Control ttffl 21 Days on Empty Plates ^ 2 1 Days on MEA Plates Plicatic Acid v-Thujaplicin P-Thujaplicin p-Thujaplicinol Thujic Acid Figure 3.2 Extractive concentrations (pg/g DW) versus W R C feeder strip storage conditions. Note: A N O V A was performed on the amount of individual extractive compounds lost on W R C -FSs on M E A compared to controls. Critical F values (a = 0.05) were as follows: plicatic acid (F(2,i5) 35.97); y-thujaplicin ( F ( 2 > i 5 ) 79.34); ^-thujaplicin (F ( 2,i5) 87.90); /tohujaplicinol (F( 2 > i 5 ) 28.35); and thujic acid (Fp.is) 44.57). Values are considered significant i f the critical F value is greater than the tabular value of F ( 2 ; i 5 ) = 3.68. 10000 t Q 8000 H D) 3 c .2 6000 id c a o c O 4000 «l .2: u n . x 2000 Three fungi with high, moderate and low tolerance to W R C extractives were selected: P. ferruginea, R. atrovirens and Phellinus ferreus, respectively (Table 3.2). P. ferruginea showed high tolerance to the five compounds. R. atrovirens' growth was inhibited by y-thujaplicin, reduced to level similar to FSs by /i-thujaplicin and thujic acid. P. ferreus did not grow on M E A with y-thujaplicin, /i-thujaplicin and thujic acid, and its growth was slightly inhibited by plicatic acid and /i-thujaplicinol. /i-thujaplicinol, a very effective natural fungicide (Barton and MacDonald 1971), affected slightly the growth of the three fungi; however, its concentration in M E A resulting from FS leaching was much lower than the concentration previously tested (Rennerfelt, 1948; Roff and Whittaker, 1959; L i m et al., 2005). Plicatic and thujic acid have been reported as having low antimicrobial activity (Rennerfelt, 1948; Barton and MacDonald, 1971). Consistent with this, relatively high concentrations (up to 150 ppm) of plicatic acid did not affect fungal growth significantly; however, high concentrations of thujic acid (107 ppm) inhibited growth of the less tolerant basidiomycetes. Many other chromatographic peaks were present in H P L C chromatograms from FS, and some of these compounds diffused into M E A (data not shown). Although more compounds than the five tested have been identified (Barton and MacDonald, 1971) and several remain uncharacterized, the purification of such compounds is difficult, and they are not available commercially. Given this, the microbial toxicity of these compounds and their synergistic effects could not be assessed. Extractives such as the thujaplicins also occur in plant species throughout the Cupressaceae family (Zavarin et al., 1967). Similar antimicrobial activity has been reported for tropolone compounds in species such as aomori hiba cedar (Thujopsis dolabrata) (Inamori and Morita, 2001), Mexican cypress (Cupressus lusitanica) (Zhao et al., 2006) and Taiwan incense-cedar (Calocedrus formosana) (Ono et al, 1998). Table 3.2 Concentration of extractives that leached into media and their effect of fungal growth. Treatment Chemical Concentration Growth rate (mm/day)a in M E A (ppm) P. ferruginea R. atrovirens P. ferreus Control - 0.9(0.1) 0.6 (0.0) 2.2 (0.3) Plicatic A c i d 149.4 0.9 (0.0) 0.6 (0.0) 1.9(0.1)* y-Thujaplicin 34.0 0.8 (0.1) 0.0 (0.0)* 0.0 (0.0)* /i-Thujaplicin y 27.6 0.7 (0.0) 0.2 (0.0)* 0.0 (0.0)* /J-Thujaplicinol 5.6 0.8 (0.0) 0.5 (0.0)* 1.8(0.1)* Thujic A c i d 107.0 0.8 (0.0) 0.2 (0.1)* 0.0 (0.0)* W R C - F S 0.8 (0.0) 0.2 (0.0)* 0.0 (0.0)* Note: 1% M E A plates were infused with extractives at the concentration that they accumulated in media having leached from W R C - F S s . a Media were then inoculated with three representative fungal species and the growth rate (mm/day) was calculated. Asterisk indicates that growth rate was significantly different from the control (a = 0.05) following A N O V A . While ascomycetes may appear from the literature to be more tolerant than basidiomycetes to /?-thujaplicin, basidiomycetes tolerance has been characterized for relatively few species (Morita et al.i 2004). The basidiomycete P. ferruginea often has been misidentified because its morphology is graphium-like. In this work we showed that this species was highly tolerant to antimicrobial W R C extractives. Currently, there is no information on the mechanism by which it detoxifies W R C extractives. The only work reporting fungal detoxification of thujaplicins is Jin et al. (1988). They suggested that two species that they isolated from red cedar heartwood, a Sporothrix species, Kirschsteiniella thujina, and a Phialophora species, could convert thujaplicins into a non-toxic lactone termed 'thujin'. However, many groups have failed to isolate similar species from W R C . In future work we intend to characterize the ability of 'pioneer' species like P. ferruginea to detoxify W R C extractives. 3.5 Conclusions Using FSs from sound W R C heartwood permits screening fungi for tolerance to the mixture of extractive compounds that they would encounter in trees or wood products, and overcomes the limited availability and cost of pure extractive compounds. Results indicated that P. ferruginea is an extractives-tolerant pioneer fungus that may play an important role in the initial modification or detoxification of W R C extractives in wood products. These issues are being addressed in ongoing work. 3.6 References Arima, Y., Nakai, Y , Hayakawa, R., and Nishino, T. (2003) Antibacterial effect of P-thujapicin on Staphylococci isolated from atopic dermatitis: relationship between changes in the number of viable bacterial cells and clinical improvement in an eczematous lesion of atopic dermatitis. J. Antimicrob. Chemother. 51:113-122. Banerjee, A . K. , and Levy, J. R. (1971) Fungal succession in wooden fence posts. Material und Organismen 6:199-211. Barton G. F., and MacDonald, B. F. (1971) The chemistry and utilization of western redcedar. Department of Fisheries and Forestry Canadian Forestry Service (publication No. 1023). Chedgy, R. J, Morris, R I., Lim, Y. M . , and Breuil, C. (2006b) Black stain of western redcedar {Thuja plicata Donn) by Aureobasidium pullulans: the role of weathering. Wood Fiber Sci. Under review. Chedgy, R. J., Daniels, C. R., Morris, R I., and Breuil, C. (2005) Black stain of western redcedar by Aureobasidium pullulans and its relationship with tropolone depletion. International Research Group on Wood Preservation IRG (Document No. IRG/WP 05-10564). Clubbe, C. P. (1980) The colonization and succession of fungi in wood. International Research Group on Wood Preservation Document No. IRG/WP/1107. Clubbe, C. P., and Levy, J. F. (1977) Isolation and identification of the fungal flora in treated wood. Revised technique. International Research Group on Wood Preservation Document No. IRG/WP/159. Coombs, R. W., and Trust, T. J. (1973) The effect of light on the antibacterial activity of /?-thujaplicin. Can. J. Microbiol. 19:1177-1180. Daniels, C. R., and Russell, J. H . (2006) Analysis of western redcedar (Thuja plicata Donn) heartwood components by HPLC as a possible screening tool for trees with enhanced natural durability. J. Chromatog. Sci . Under review. DeBel l , J. D . , Morrel l , J. J, and Gartner, B . L . (1997) Tropolone content of increment cores as an indicator of decay resistance in western redcedar. Wood Fiber Sci . 29:364-369. Gonzalez, J. S. (2004) Growth properties and uses of western redcedar (Thuja plicata Donn ex D . Don.). Forintek Canada Corp. Special Publication No. SP-37R. ISSN No. 0824-2119. Hogberg, N . , and Land, C. J. (2004) Identification of Serpula lacrymans and other decay fungi in construction timber by sequencing of ribosomal D N A - a practical approach. Holzforschung 58:199-204. Hon, D . N . S. (1991) Wood and cellulosic chemistry. Ed. Shiraishi N . Marcel Dekker, Inc. N e w York, N . Y . 1020 pp. Inamori, Y , and Morita, Y. (2001) Physiological activities and prospects of oil of aomori hiba. Aroma Res. 2:137-143. Inamori, Y , Sakagami, Y , Morita, Y , Shibata, M . , Sugiura, M . , Kumeda, Y , Okabe, T., Tsujibo, H . , and Ishida, K . (2000) Antifungal activity of hinokitiol-related compounds on wood-rotting fungi and their insecticidal activities. B io l . Pharm. B u l l . 23:995-99.7. Inamori, Y. , Shinohara, S., Tsujibo, H . , Shibata, M . , Okabe, T., Morita, Y , Sakagami, Y , Kumeda, Y , and Ishida, K . (1999) Antimicrobial activity and metalloprotease inhibition of hinokitol-related compounds, the constituents of Thujopsis dolabrata S. and Z . hindai mak. B io l . Pharm. Bu l l . 22:990-993. Jin, L . , Van der Kamp, B . J., Wilson, J., and Swan, E. P. (1988) Biodegradation of thujaplicins in living western red cedar. Can. J. For. Res. 18:782-86. Johnson, E . L . , and Cserjesi, A . J. (1980) Weathering effect on thujaplicin concentration in western redcedar shakes. For. Prod. J. 30:52-53. Kropp, B . R., and Corden, M . E . (1986) Morphology and taxonomy o f Pachnocybe ferruginea. Mycologia. 78:334-342. Lim, Y. W, Kim J. J. Chedgy, R. J., Morris, P. I., and Breuil, C. (2005)'Fungal diversity from western redcedar fences and their resistance to /i-thujaplicin. Ant. van Leeuwen. 87:109-117. Morita, Y., Matsumura, E., Okabe, T., Fukui, T., Shibata, M , Sugiura, M , Ohe, T., Ishida, N . , and Inamori, Y. (2004) Biological activity of /?-dolabrin, y-thujaplicin, and 4-acetyltropolone, hinokitiol-related compounds. Biol. Pharm. Bull. 27:1666-1669. Nobles, M . K. (1965) Identification of cultures of wood inhabiting Hymenomycetes. Can. J. Bot. 43:1097-1139. Ono, M . , Asai, T., and Watanabe, H. (1998) Hinokitol production in a suspension culture of Calocedrus formosana. Florin. Biosci. Biotechnol. Biochem. 62:1653-1659. Rennerfelt, E. (1948) Investigations of thujaplicin, a fungicidal substance in the heartwood of Thuja plicata D. Don. Physiologia Plantarum 1:245-254. Roff, J. W., and Whittaker, E. I. (1959) Toxicity tests of a new tropolone, /i-thujaplicinol (7-hydroxy-4-isopropyltropolone) occurring in western red cedar. Canadian journal of Botany. 37:1132-1134. Scheffer, T. C , Goodell, B. S., and Lombard, F. F. (1984) Fungi and decay in western redcedar utility poles. Wood Fiber Sci. 16:543-548. Schmidt, O., and Moreth, U . (2002) Data bank of rDNA-ITS sequences from building-rot fungi for their identification. Wood Sci. Tech. 36:429-433. Schmidt, O., and Moreth, U . (2003) Molecular identity of species and isolates of internal pore fungi, Antrodia spp. and Oligoporusplacenta. Holzforschung 57:120-126 Schoeman, M . , and Dickinson, D. J. (1997) Growth of Aureobasidium pullulans on lignin breakdown products at weathered wood surfaces. Mycologist 11:168-172. Shibata, H. , Nagamine, T., Wang, Y., and Ishikawa, T. (2003) Generation of reactive oxygen species from Hinokitol under near-UV irradiation. Biosci . Biotechnol. Biochem. 67:1996-1998. Stalpers, J. A . (1978) Identification of wood-inhabiting fungi in pure culture. Stud. Myco l . 16:1-248. Tukey, J. W. (1949) Comparing individual means in the analysis of variance. Biometrics 5:99-114. Van der Kamp, B . J. (1975) The distribution of microorganisms associated with decay of western redcedar. Can. J. For. Res. 1:61-67. Van der Kamp B . J. (1986) Effects of heartwood inhabiting fungi on thujaplicin content and decay resistance of western redcedar (Thuja plicata D O N N ) . Wood Fiber Sci. 18:421-427. Wang, C. J. K . , and Zabel, R . A . (1990) Identification manual for fungi from utility poles in the eastern united states. A l l en Press, Inc., Lawrence, K . S . U . S . A . White, T. J., and Bruns, T. D . , Lee, S. B . , and Taylor, J. W. (1990) Amplification and direct sequencing of fungal ribosomal R N A genes for phylogenetics., Innis, M . A , Gelfand, D . H , Sninisky, J.J., White, T.J. (eds.). Zavarin, E . , Smith, L . V . , and Bicho, J. G . (1967) Tropolones of Cupressaceae-III. Phytochem. 6:1387-1394. Zhao, J., Matsunaga, Y . , Fujita, K . , and Sakai, K . (2006). Signal transduction and metabolic flux of /?-thujaplicin and monoterpene biosynthesis in elicited Cupressus lusitanica cell cultures. Met. Eng. 8:14-29. Chapter 4 Black Stain of Western Redcedar {Thujaplicata Donn) by Aureobasidiumpullulans: the Role of Weathering 4.1 Abstract Western redcedar (Thuja plicata Donn) (WRC) is valued for its natural durability conferred by fungicidal extractive chemicals. However, weathered surfaces of W R C products are susceptible to black stain caused by fungi such as Aureobasidium pullulans. The effect of weathering on extractive concentrations at the wood surface was characterized and correlated with the ability of this fungal species to colonize weathered surfaces. U V plus water spray treatments substantially reduced extractives but did not promote fungal colonization. In contrast, UV-on ly treatments reduced extractive contents less than the other treatments but stimulated fungal colonization. A. pullulans exhibited high tolerance to the tropolone /i-thujaplicin in vitro; thus loss in tropolone content may not be required for colonization. Water spray most l ikely washed away products of lignin photo-degradation, which resulted in decreased fungal colonization. Key words: Aureobasidium pullulans, black stain, extractive resistance, weathering, western redcedar (Thujaplicata Donn). 4.2 Introduction W R C is utilized in the manufacture of wood products for exterior residential applications. It is valued for its natural durability conferred by fungicidal extractive compounds (Barton and MacDonald, 1971). However, W R C products are susceptible to black stain caused by staining 83 fungi which can significantly reduce wood aesthetic qualities and lead to premature replacement. Aureobasidium pullulans (de Bary) G . Arnaud causes black stain and is a major colonizer of weathered wood surfaces (Schoeman and Dickinson, 1997) and painted wood (Bardage and Bjurman, 1998; Jakubowsky et al.,1983; O'Niel , 1986; Shirikawa et al., 2002). It can also penetrate many protective coatings (Sharpe and Dickinson, 1992b). The black coloration is attributed to the presence of melanin in the fungal hyphae (Yurlova et a l , 1999), which protects fungal cells from the damaging effects of U V radiation (Kawamura, 1999). Weathering of the wood surface promotes A. pullulans' growth since the fungus metabolizes the complex aromatic organic molecules that are formed as a result of lignin photo-degradation (Bourbonnais and Paice, 1987; Dickinson, 1972; Schoeman and Dickinson, 1997; Sharpe and Dickinson, 1992a, 1993). This may provide a significant competitive advantage to this fungal species and may explain why A. pullulans is predominantly isolated from weathered surfaces (Dickinson, 1972). Many of the fungicidal extractive compounds present in W R C are aromatic and polyphenolic in nature (Barton and MacDonald, 1971). O f the array of W R C extractive compounds characterized to date are a series of compounds known as the tropolones. Several of these compounds, namely the thujaplicins (a-, B-, and y-thujaplicin) are reported to exhibit strong antimicrobial activity against a range of wood-inhabiting and plant pathogenic fungi (Inamori et al., 2000; Morita et al., 2004a, 2004b). Extractive contents at the surface of W R C products may form the first line of defense in preventing fungal spore germination. At this early developmental stage, microorganisms may be less resistant to the toxic effect of extractives. However, extractives near the wood surface may be prone to leaching from precipitation (Chedgy et al., 2005) and are degraded by ultra-violet 84 (UV) radiation from the sun (Coombs and Trust, 1973; Shibata et al., 2003) that penetrates the upper 0.75mm of the wood surface (Hon, 1991). It is not known whether A. pullulans is tolerant to W R C extractives or i f they are simply depleted by weathering at exposed surfaces, paving the way for colonization. To address W R C black stain, it is necessary to understand the interactions between W R C extractives, weathering and fungal colonization. Therefore, the aims of this research were to 1) establish whether or not A. pullulans has resistance to fungicidal tropolone compounds present in W R C in vitro, and 2) assess the ability of this fungal species to colonize weathered wood surfaces. 4.3 Materials and Methods 4.3.1 Isolation and Identification of Black Staining Fungi Black staining fungi were isolated from in service W R C siding located in Vancouver, British Columbia, Canada. Wood flecks that exhibited visible signs of black stain were removed from the surface of siding and placed onto 1% malt extract agar ( M E A ) plates then incubated at 20°C for several weeks. Fungi growing on M E A were routinely sub-cultured from mycelial margins to new M E A plates to obtain pure cultures. Fungal identification was achieved by macro- and micro-morphological analyses using taxonomic guides (de Hoog and Yurlova, 1994). This was complemented by molecular technique for species identification as described by L i m et al. (2005). The internal transcribed spacer (ITS) region was used for molecular identification and amplified using P C R with the primers ITS5 and ITS4 (Schmidt and Moreth, 2002; White et al., 1990). Sequencing from three representative strains of each isolated taxon was performed on an A B I 3700 automated sequencer (Perkin-Elmer Inc., Wellesley, Massachusetts) at the D N A synthesis and Sequencing Facility, M A C R O G E N (Seoul, Korea). 85 4.3.2 yff-ThujapIicin Resistance Two isolated black stain fungal species, A. pullulans and Hormonema dematioides Lagerb & Mel in , were tested. Mycel ia l growth was measured on 25ml M E A plates containing various concentrations of ^-thujaplicin (99% pure, Sigma-Aldrich Ltd, Oakville, Ontario). A 10 mg ml" 1 stock solution prepared in 50% ethanol was filter-sterilized and kept in the dark at 4°C. Concentrations of 0, 2, 8, 16, 32 and 64 ppm (parts per million) were added to M E A . Control M E A plates with and without ethanol showed that ethanol had no effect on fungal growth at the low concentrations used to prepare the /i-thujaplicin plates. Media were inoculated with a 5 mm plug of agar taken from the edge of actively growing isolate colonies. The cultures were maintained in the dark at 20°C and the growth (mm) was evaluated by measuring two perpendicular diameters of the colony after 21 days. This experiment followed a completely randomized design ( C R D ) with treatments defined as the various /i-thujaplicin concentrations (k = 6). Three replicate plates were used per strain at each concentration, and three strains of each species were used (n = 9). Statistical analysis was performed on data obtained with this experimental design. 4.3.3 Weathering and Fungal Effects on Wood Chemistry Wood samples - W R C outer heartwood was obtained from a 136 year old standing tree harvested at the U B C Malcolm Knapp research forest, Maple Ridge, British Columbia. A l l wood samples originated from a single longitudinal axis parallel with growth rings. Siding pieces were manufactured of dimensions 160mm x 65mm x 10mm with the radial face on the largest face. Ponderosa pine (Pinus ponderosa P. & C. Lawson) sapwood was also used as a control. The sapwood of this species contains small amounts of extractives that are not fungicidal like those of W R C . 86 Weathering of siding material - W R C and pine siding pieces were exposed to simulated weather conditions using a Weather-Ometer® (Ci65A, Atlas Material Testing Technology L L C , Chicago, Illinois) located at the Forintek Canada Corp. laboratory, British Columbia. Four treatments were used: i) water spray (WS), ii) U V , iii) WS and U V , and iv) no weathering (Table 4.1). Each treatment was run continuously for a period of 200 hrs, at a temperature of 50°C to prevent mould growth and a relative humidity ranging from 40-95% depending on treatment to provide some moisture to facilitate chemical reactions. Eighteen W R C and pine siding pieces were subjected to each of the weathering treatments. Each treatment was repeated three times with a new set of samples. Following the weathering process twelve W R C and pine pieces from each treatment were used for chemical analyses. For chemical analysis the upper 1 mm of the weathered surface was removed using a computer controlled Precix 3600 router (Precix, Surrey, British Columbia). Four pieces were processed each time and the shavings from these samples were combined. This reduced the number of replicates from twelve to three for each weathering treatment. Samples were placed into clean glass vials in methanol and extracted for 120 minutes at an ultrasonic frequency of 40 kHz using a Branson 8510 ultrasonic bath (Branson Ultrasonics Corp., Connecticut). A temperature of 4°C was maintained throughout the ultrasonication process by the addition of ice to the water bath, and a lid was used to shield samples from incandescent light to avoid thermal and photo-degradation of extractive compounds of interest. Extract solutions were filtered using a 25mm 0.2pm nylon syringe filter to remove any wood particles and stored at 4°C in the dark. 87 Table 4.1 Weathering treatments. Program cycle 1 2 3 4 Control cycle - - - -WS only 30 mins WS* (dark) 30 mins (dark) 30 mins WS (dark) 30 mins (dark) UV only 30 mins U V * 30 mins (dark) 30 mins U V 30 mins (dark) UV and WS 30 mins U V 30 mins WS (dark) 30 mins U V and WS 30 mins (dark) *WS = water spray; U V = ultraviolet. The duration of each weathering regime was 200hrs. WS rate was approximately 9.08 liters per hour. The U V wavelength was set at 340nm, and we measured 120Kj/m 2 total for each of the 200 hours weathering cycles with U V . 88 Reverse phase HPLC analysis - Separation and quantification of extractive compounds was carried out by reverse phase high performance liquid chromatography ( H P L C ) coupled with an ultraviolet detection system as described in Chedgy et al. (2006a). Five extractives of interest were quantified (pg/g D W ) by comparing analyte response with the response factor of an internal standard by a single point calibration. Extractives of interest were (-)-plicatic acid, y-thujaplicin, ^-thujaplicin, y9-thujaplicinol, and thujic acid (Figure 4.1). A Waters 2695 H P L C separation module (Waters Corp., Milford, Massachusetts) equipped with an Intersil ODS3 C-18 (3pm, 4.6mm x 150mm) reverse phase separation column (Intersil Corp., Milpitas, California) was used for extractive separation. Mixed extracts were injected (15ul) on to the column and separated based on their hydrophobic character using a mobile phase of 0.1% formic acid, 10% acetonitrile, and 89.9%) nano-pure H2O which run against an increasing linear gradient of 99.9% acetonitrile with 0.1 % formic acid. The column chamber was heated to temperature of 50°C, and a Dionex A D 2 0 U V absorbance detector (Dionex Corp., Sunnyvale, California) at wavelength of 230nm was used for the extractive detection. A total elution of extractives was typically reached after approximately 48 minutes and MassLynx analytical software (version 4.0, Micromass Ltd, Manchester, England) was utilized for chromatographic analysis. A C R D was used to assess the effect of weathering on extractive concentration. Experimental treatments were defined as the different weathering treatments (k = 4). The surface wood was analyzed from twelve replicate siding pieces from each weathering treatment. Surface wood was removed from four pieces simultaneously using the automated router and the dust was pooled together. This reduced the replicate number from twelve to three. Therefore, n = 9 (3 x analytical surface wood samples, 3 x weathering treatments). 89 23,6-Trmydroxy-7-me1ho 2-Hydroxy-5-isopropyl-2,4,6--4-(3\4'-diiiyd^oxy-5'-metJioxyphenyl)- cycloheptatrien- 1-one tetralin-3-carboxylic acid ^-Thujaplicin (-)-PIicatic Acid 2,7-Dihydroxy-4-isopropyl-2,4,6-cycloheptatrien-1 -one /?-Thujaplicinol 2-Hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1 -one /?-Thujapiicin OH 7,7-Dimethylcycloheptatriene-3-carboxylic acid Thujic Acid Figure 4.1 Chemical structure and nomenclature used for W R C heartwood extractive compounds of interest (Barton and MacDonald, 1971). 90 4.3.4 Inoculation of A. pullulans on Weathered Siding Weathered and un-weathered (control) W R C and pine siding pieces were sprayed with a liquid culture of A. pullulans and incubated in growth chambers. Three A. pullulans strains were grown in 250ml flasks containing 100ml of 1% malt extract media. The inoculum was approximately l x l O 6 spores. Shake cultures were incubated at 22°C, 200rpm, for fourteen days. Cells were collected by centrifugation and re-suspended in nano-pure H 2 O . The culture suspension from three A. pullulans strains were mixed together in equal parts. The resulting solution was sprayed homogeneously over all o f the weathered siding pieces. Six W R C and pine siding pieces from each weathering treatment were completely randomized and divided among four growth chambers. Samples were suspended over a layer of water. The incubation was carried out for 6 months at 20°C and close to 100% relative humidity. To provide a surface fdm of water, the samples were sprayed with a distilled water mist every 3-4 days. The degree of colonization was assessed in two ways: i) qualitatively, by scoring siding pieces to reflect the degree of black staining; ii) quantitatively, using a Minolta CM-2600d portable integrated sphere spectrophotometer (Konica-Minolta Ltd, Ontario) which yields an index of white-black coloration ranging from 0-100 (0 = Black, 100 = White). Discoloration of wood after exposure to A. pullulans could be calculated by taking readings before and after colonization at six regions of each siding piece. A s before, a C R D was used to assess the effect o f weathering on the colonization ability of A. pullulans on weathered surfaces. Experimental treatments were defined as the different weathering treatments (k = 4). Six siding pieces were analyzed for the degree of discoloration from each weathering run, and each weathering treatment was repeated three times in = 18) (6 replicate siding pieces x 3 weathering treatments). 91 4.3.5 Statistical Analysis A l l experiments followed a completely randomized design ( C R D ) with treatments denoted as k and replicates as n. Analysis of variance ( A N O V A ) (a = 0.05) and Tukey's test for comparison of means (Tukey, 1949) were performed with this experimental design. Treatment effects were considered significant at the 95% significance level i f the resulting critical F value was greater than the appropriate tabular value (F[(£-i)], [(k(n-\)])- A l l statistical analyses were performed using J M P IN software (version 4.0.3 (academic), S A S Institute Inc., Cary, North Carolina). 4.4 Results and Discussion Macro- and microscopic characterization of the fungal isolates, along with the ITS sequences allowed us to recognize two fungal taxa, Aureobasidium pullulans and Hormonema dematioides. Aureobasidium and Hormonema species are related to the bitunicate Ascomycete-family Dothideaceae and form black, yeast-like cells (Yurlova et al., 1999). Because both species have similar morphological characteristics and cannot be easily differentiated (Takeo and de Hoog, 1991), we sequenced ITS regions as suggested by Ray et al. (2004) to confirm their identification. See Appendix 2 for light microscope photographic images of A. pullulans and H. dematioides. Sequence dissimilarity was not observed within the species, but was found between the two species at a level of 12.38%. A. pullulans was the most commonly isolated species and our results agreed with previous research (Dickinson, 1972, Bardage and Bjurman, 1998), but only a few strains (three) of H. dematioides were isolated from W R C siding. The ITS sequences of our isolates have been deposited in GenBank with the accession numbers DQ787427 {A. pullulans) and DQ787428 (H. dematioides). 92 Resistance to /?-thujaplicin in vitro was assessed for both A. pullulans and H. dematioides. Statistical analysis suggested that ^-thujaplicin concentration had a significant effect on the growth of both A. pullulans and H. dematioides (Table 4.2). A. pullulans isolates exhibited high tolerance to /^-thujaplicin. They were able to grow at 32 ppm (5.5 ± 1.1 mm), although growing more slowly than the controls (33.3 ± 1.5 mm). Similar tolerance was observed with some pioneer and decay fungi identified in W R C standing trees and other wood products (Lim et al., 2005). H. dematioides showed little /^thujaplicin resistance, with growth completely inhibited at 8 ppm and severely impaired at 2 ppm (14.7 ± 2.1 mm) compared to the control (31.9 ± 1.8 mm) The five extractives of interest were quantified following analysis of the upper 1mm of W R C wood surfaces following different weathering treatments (figure 4.2). Weathering was found to have a significant effect on extractive concentration (see figure 3 for critical F values). Overall losses for the five extractives measured were 29.8%, 79.9% and 89.4% for U V , WS and U V + WS treatments, respectively. U V alone had the least effect on extractive content. Our data were consistent with the results of Johnson & Cserjesi (1980) for the depletion of /?-and y-thujaplicin in W R C shakes exposed in natural conditions in Vancouver, British Columbia, Canada. They established that /?- and y-thujaplicin depletion was 25%> after a year of exposure and 90% after three years, although this may have been due in part to biodegradation. Extractive loss in our U V + W S treatment using a weather-Ometer was approximately equivalent to one year's weathering loss in natural conditions. Johnson & Cserjesi (1980) also noted that the B- to y-thujaplicin concentration ratio (average 1:1.5) remained constant during weathering. Compounds such as ^thujapl icin are prone to photo-degradation. Shibata et al. (2003) 93 Table 4.2 Tota l fungal growth (mm) of black staining isolates after 21 days on MEA containing various concentrations of /?-thujaplicin. /^-Thujaplicin concentration (ppm) 0 2 8 16 32 64 A. pullulans 1 33.33 '(1.53)2a 31.00 (0.93)a 26.67 (2.3 l) b 9.33 (2.08)° 5.00(1.00)d A. pullulans 2 32.33 (2.52)a 32.33 (2.67)a 22.67 (5.03)b 11.50 (0.5)c 5.17 (1.04)d A. pullulans 3 34.33 (0.58)a 30.33 (2.47)a 25.33 (2.52)b 10.67 (0.58)c 6.33 (1.15)d Mean 33.33 (1.54)a 31.22 (2.03)a . 24.89 (3.29)b 10.50 (1.05)c 5.50 (1.07)d H. dematioides 1 30.00 (2.00)a 15.00 (3.25)b - - -H. dematioides 2 32.33 (0.58)a 14.67 (2.09)b - - -H. dematioides 3 33.33 (2.87)a 14.33 (0.9)b • - - -Mean 31.89 (1.82)" 14.66 (2.08)b - - -Note: 'numbers is parenthesis indicate standard deviation (mm). A N O V A indicated that treatment effect was significant for bothyl pullulans ( F ^ g ^ 324.6) and for H. dematioides (F(5,48) = 559.6). Values were considered significant i f greater than the tabular value of F ^ s ) = 2.41 (a=0.05). ^Numbers followed by the same letter were not significantly different (a=0.05) according to Tukey's test of multiple comparison of means. 94 Q -SP DJO c o 1 u o c o o > Xi u 10000 9000 8000 i 7000 6000 -5000 -4000 3000 2000 1000 0 oo • Control • UV M Water Spray • UV+ Water Spray c> oo C > ON i - l oo fiffitaiiiL C V ( N c v C ) H Plicatic Acid y-Thujaplicin p-Thujaplicin p-Thujaplicinol Thujic Acid Extractive compounds of interest Figure 4.2 Weathering treatment effects on the concentrations ( n g / g DW) of W R C extractive compounds of interest. Note: Y error bars indicate standard deviation. Numbers above error bars represent concentration expressed as a percentage of the control. Critical F values are as follows for each extractive of interest: plicatic acid (F ( 3 > 3 2 )= 68.2); y-thujaplicin (F ( 3 j 3 2 ) = 253.5); /i-thujaplicin ( F ( 3 ; 3 2 ) = 615.8); /i-thujaplicinol (F( 3 ; 3 2) = 292.3); and thujic acid (F( 3 > 3 2) = 397.8). Values were considered significant i f greater than the tabular value of F( 3 : 3 2 ) = 2.90 (a=0.05). 95 demonstrated that irradiation (210-380 nm) caused photochemical decomposition of B-thujaplicin and a measurable loss of antibacterial activity. We assessed whether A. pullulans was likely to colonize W R C when extractives were depleted by weathering compared to un-weathered wood. Growth of A. pullulans isolates on weathered siding pieces after six months incubation was estimated qualitatively and quantitatively. Table 4.3.a shows the mean qualitative growth scores that were assigned by visual inspection following different weathering treatments. In addition we recorded photograph images of A. pullulans growing on weathered surfaces using a stereo microscope (Figure 4.3). Growth of A, pullulans was greatest on surfaces weathered by U V . Quantitative assessment of growth using spectrophotometer readings to calculate the mean degree of discoloration of weathered wood before and after colonization by A. pullulans agreed with these observations (Table 4.3.b). Statistical analysis suggested that treatment effects had a significant impact on the discoloration of weathered surfaces by A. pullulans on pine and on cedar. In every case, A. pullulans was successfully re-isolated and identified from all siding pieces used in the trial. A s expected, A. pullulans grew more vigorously on pine siding than on cedar. Weathering by U V - o n l y gave the greatest degree of fungal colonization and discoloration on both cedar and pine, while treatment by U V + W S gave the least. U V consistently promoted fungal colonization. Lignin is highly susceptible to photo-degradation (Chang, 2002; Crestini and Auria, 1996). A. pullulans can metabolize products of lignin photo-degradation (Bourbonnais and Paice, 1987; Schoeman and Dickinson, 1997; Sharpe and Dickinson, 1992a, 1993). Our data suggested that UV-on ly caused less decrease in extractive concentrations in contrast to other weathering treatments. A. pullulans showed a high tolerance to the fungicidal 96 Table 4.3.a A. pullulans growth on weathered wood surfaces estimated qualitatively. Weathering Treatment No weathering UV UV+ water spray Water spray Ponderosa Pine 3.58 4.15 2.21 2.01 Cedar 2.25 3.71 1.51 1.47 0 = no visible growth; 1 = trace growth, some mild grey/black coloration; 2 = obvious coloration; 3 = significant black stain; 4 = some regions completely covered; 5 = total coverage, no unstained wood. Table 4.3.b Spectrophotometer measurements of weathered wood discoloration before and after colonization by A. pullulans. Weathering Treatments Control U V U V + WS WS Ponderosa Pine 35.66 ;(4.47)2a WRC 8.49 (3.37)" 31.94 (5.92)a 5.15 (2.69)c 18.98 (7.92)a 8.68 (3.37)b 21.17 (0.88)b 8.77 (3.49)" Note: 'numbers in parenthesis indicate standard deviation from the three replications of the experiment. Critical F values were as follows: on pine (F(3,68) = 194.4) and on cedar (Fp^s) = 25.7). These values were much greater than the tabular value of F ^ s ) = 2.7 (d = 0.05).2Numbers followed by the same letter in each row are not significantly different (a=0.05) according to Tukey's test for multiple comparison of means. 97 WRC Un-weathered UV UV+WS Figure 4.3 A. pullulans colonization of weathered wood surfaces. 98 effects of /^thujaplicin; this suggests that extractive depletion is not necessary for fungal colonization. A. pullulans could also colonize un-weathered wood surfaces. Furthermore, the depletion of extractives was greater when water spray was introduced into the treatments and resulted in less colonization than on un-weathered wood surfaces. 4.5 Conclusions Weathering treatments of W R C siding caused significant changes in the extractive content of exposed surface. U V plus water spray severely reduced extractives but did not lead to increased fungal colonization compared to un-weathered wood. Water spray most likely washed away products of lignin photo-degradation, leaving the wood surface void of accessible carbon sources resulting in decreased fungal growth. In contrast, UV-on ly treatments reduced extractive contents less than the other treatments but stimulated fungal colonization. A. pullulans exhibited high tolerance to the tropolone /Mhujaplicin in vitro, suggesting that tropolone reductions by weathering may not be required for colonization. It is likely that A. pullulans may have competitive advantages in colonizing exposed W R C surfaces because it can use lignin breakdown products as a carbon source, it is resistant to U V due to its melanized cells and it tolerates tropolones. 99 4.6 References Bardage, S. L . , and J. Bjurman. 1998. Isolation of an Aureobasidium pullulans polysaccharide that promotes adhesion of blastospores to water-borne paints. Can. J. Microbiol . 44: 954-958. Barton, G . F., and MacDonald, B . F. 1971. The chemistry and utilization of western redcedar. Department of Fisheries and Forestry Canadian Forestry Service (publication No. 1023). Bourbonnais, R., and Paice, M . G . 1987. Oxidation and reduction of lignin-related aromatic compounds by Aureobasidium pullulans. App l . Microbiol . Biotechnol. 26: 164-169. Chang, H . T., Yeh, T. F., and Chang, S. T. 2002. Comparisons of chemical characteristic variation for photo-degraded softwood and hardwood with/without polyurethane clear coating. Polymer Degradation and Stability 77: 129-135. Chedgy, R. J., Daniels, C. R., Kadla, J. , and C. Breuil . 2006a. Screening for extractive resistant fungi, part 1: extraction and quantitation of western red cedar (Thuja plicata) heartwood extractives by ultrasonication and reverse phase H P L C . Holzforschung. Under review, Submission # HOLZ-D-06-00099. Chedgy, R. J., Daniels, C. R., Morris, P. I., and Breuil , C. 2005. Black stain of western redcedar by Aureobasidium pullulans and its relationship with tropolone depletion. International Research Group on Wood Preservation document No. I R G / W P 05-10564. Coombs, R. W. , and Trust, T. J. 1973. The effect of light on the antibacterial activity of /?-thujaplicin. Can. J. Microbiol . 19: 1177-1180. Crestini, C , and Auria , M . D. 1996. Photodegradation of lignin: the role of singlet oxygen. Journal of Photochemistry and Photobiology A : Chemistry 101:69-73. Dickinson, D. J. 1972. Disfigurement of decorative timbers by blue stain fungi. Int. Pest Control 14:21-25. 100 Hon, D. N . S. 1991. Wood and cellulosic chemistry. Shiraishi N , N e w York, N . Y . , Marcel Dekker, Inc: 1020 pp. Hoog de, G . S., and Yurlova, N . A . 1994. Conidiogenesis, nutritional physiology and taxonomy of Aureobasidium and Hormonema. Antonie van Leeuwenhoek 65: 41-54. Inamori, Y . , Sakagami, Y . , Morita, Y . , Shibata, M . , Sugiura, M . , Kumeda, Y . , Okabe, T., Tsujibo, H . , and Ishida, K . 2000. Antifungal activity of hinokitiol-related compounds on wood-rotting fungi and their insecticidal activities. B i o l . Pharm. B u l l . 23: 995-997. Jakubowsky, J. A . , Gyris, J., and Simpson, S. L . 1983. Microbiology of modern coating systems. J. Coat. Technol. 55: 49-53. Johnson, E . L . , and Cserjesi, A . J. 1980. Weathering effect on thujaplicin concentration in western redcedar shakes. For. Prod. J. 30: 52-53. Kawamura, C , Tsujimoto, T., and Tsuge, T. 1999. Targeted disruption of a melanin biosynthesis gene affects conidial development and U V tolerance in the Japanese pear pathotype of Alternaria alternata. M o l . Plant-Microbe Interact. 12: 59-63. L i m , Y . W, K i m , J. J., Chedgy, R. J., Morris, P. I., and Breuil , C . 2005. Fungal diversity from western redcedar fences and their resistance to ^thujaplicin. Ant. van Leeuwen. 87: 109-117. Morita, Y . , Matsumura, E . , Okabe,T., Fukui, T., Shibata, M . , Sugiura, M . , Ohe, T., Tsujibo, H . , Ishida, N . , and Inamori, Y . 2004a. Biological activity of a-thujaplicin, the isomer of hinokitol. B i o l . Pharm. B u l l . 27: 899-902. Morita, Y . , Matsumura, E . , Okabe,T., Fukui, T., Shibata, M . , Sugiura, M . , Ohe, T., Ishida, N . , and Inamori, Y . 2004b. Biological activity of /i-dolabrin, y-thujaplicin, and 4-acetyltropolone, hinokitol-related compounds. B i o l . Pharm. Bu l l . 27: 1666-1669. O'Niel , T. B . 1986. Succession and interrelationships of microorganisms on painted surfaces. J. Coat. Technol. 58: 51-56. 101 Ray, M . J., Dickinson, D . J., and Buck, M . 2004. Aureobasidium or Hormonema? A genetic approach. The International Research Group on Wood Preservation Document No: IRG/WP 04-10529. I R G , Stockholm,Sweden. Schmidt, O., and Moreth, U . 2002. Data bank of r D N A - I T S sequences from building-rot fungi for their identification. Wood Sci. Technol. 36: 429-433. Schoeman, M . , and Dickinson, D . 1997. Growth of Aureobasidium pullulans on lignin breakdown products at weathered wood surfaces. Mycologist 11: 168-172. Sharpe, P. R., and Dickinson D . J. 1992a. Blue stain in service on wood surface coatings part 1; The nutritional requirements of Aureobasidium pullulans. The International Research Group on Wood Preservation document No : IRG/WP/1556-92. I R G , Stockholm, Sweden. Sharpe, P. R., and Dickinson, D . J. 1992b. Blue stain in service on wood surface coatings part 2; The ability of Aureobasidium pullulans to penetrate wood surface coatings. The International Research Group on Wood Preservation document No : IRG/WP/1557-92. IRG, Stockholm, Sweden. Sharpe, P. R., and Dickinson, D . J. 1993. Blue stain in service on wood surface coatings, part 3: The nutritional capability of Aureobasidium pullulans compared to other fungi commonly isolated from wood surface coating. The International Research Group on Wood Preservation Document No : IRG/WP/93-10035. IRG, Stockholm, Sweden. Shibata, H . , Nagamine, T., Wang, Y . , and Ishikawa, T. 2003. Generation of reactive oxygen species from hinokitol under near-UV irradiation. Biosci . Biotechnol. Biochem. 67: 1996-1998. Shirikawa, M . A . , Gaylarde, C. C. , Gaylarde, P. M . , John, V . , and Gambale, W . 2002. Fungal colonization and succession on newly painted buildings and the effect of biocide. F E M S Microbiology Ecology 3^ 9: 165-173. 102 Takeo, K . , and de Hoog, G . S. 1991. Karyogamy and hyphal characters as taxanomic criteria in ascomycetous black yeasts and related fungi. Antonie van Leeuwenhoek 60: 35-42. Tukey, J. W . 1949.Comparing individual means in the analysis of variance. Biometrics 5: 99-114. White, T. J., Bruns, T. D . , Lee, S. B . , and Taylor, J. W . 1990. Amplification and direct sequencing of fungal ribosomal R N A genes for phylogenetics. Pages 315-322 in Innis, M . A . , Gelfand, D. FL, Sninisky, J. J., and T. J. White, eds. P C R Protocols: Guide Methods Appl . 1990. Academic, San Diego, C A . Yurlova, N . A . , Hoog de, G . S., and Gerrits van den Ende, A . H . G . 1999. Taxonomy of Aureobasidium and Al l i ed Genera. Studies in Mycology 43: 63-69. 103 Chapter 5 Effects of Leaching on Fungal Growth and Decay of Western Redcedar {Thujaplicata Donn) 5.1 Abstract We tested the effect of leaching on the concentration of second-growth Western redcedar (Thuja plicata Donn) ( W R C ) heartwood extractives and compared the ability of six commonly isolated fungal species to decay leached versus non-leached W R C in standard soil block decay tests. We leached W R C blocks and used reverse phase high performance liquid chromatograpghy ( H P L C ) and ultra-violet ( U V ) detection to separate and quantify five key extractives compounds: (-)-plicatic acid, y-thujaplicin, /i-thujaplicin, /i-thujaplicinol, and thujic acid. Leaching reduced the concentration of extractives by -80%. We assessed the extractive-tolerance in vitro of fungal species: Acanthophysium lividoeaeruleum, Coniophoraputeana, Heterobasidion annosum, Pachnocybe ferruginea, Phellinus sulphurascens, and Phellinus weirii by measuring growth rate (mm/day) on media infused with W R C leachate compared to controls. These data were correlated with the ability of species to decay pine, leached W R C and non-leached W R C . P. sulphurascens exhibited the lowest extractive-tolerance and caused minimal decay in non-leached W R C but could decay pine and to a lesser degree, leached W R C . C. puteana, H. annosum, and P. weirii displayed moderate to high tolerance to leachate and caused decay in non-leached as well as in leached W R C , but decay was always greatest on leached W R C and pine suggesting that depletion of extractives promotes decay in these fungi. A. lividoeaeruleum and P. ferruginea exhibited high tolerance to leachate and did not require depletion of 104 extractives for decay to occur. While A. lividocaeruleum clearly caused decay on all types of wood, only small but consistent amounts of decay were observed with P. ferruginea, perhaps due to its slow growth rate or its inability to decay wood. Key words: extractives, extractive-tolerance, decay, fungi, leaching, western redcedar (Thuja plicata Donn). 5.2 Introduction W R C wood products are valued for their natural durability conferred by fungicidal extractive compounds (Barton & MacDonald, 1971) and are used heavily in the manufacture of wood products with exterior residential applications which account for a significant portion of Canada's forest products industry (Gonzalez, 2004). However, such products are still prone to decay and this may be attributable in part to extractive depletion caused by weathering (Chedgy et al., 2005, 2006b). Ultraviolet ( U V ) radiation from sun light is known to cause photo-degradation of extractive compounds (Coombs and Trust, 1973; Shibata et al., 2003) but can only penetrate the upper 1mm of exposed surfaces (Hon, 1991) and may have a limited effect on the extractive content. Leaching of extractives by precipitation that can penetrate deep within W R C products is more likely affecting extractive concentration. This could result in an increased susceptibility to wood-destroying micro-organisms and a potentially reduced service life. The extractives of W R C are comprised of a mixture of lignans and tropolone compounds. Several of the tropolones, namely the 'thujaplicins' are reported to exhibit strong antimicrobial activity against a range of wood-inhabiting and plant pathogenic fungi in vitro (Inamori et al., 2000; Morita et al., 2004a, 2004b). Tropolones are 2-hydroxy-2,4,6-cycloheptatrien-l-one molecules and their derivatives which possess special characteristic properties due to the 1,2 105 arrangement of the carbonyl and hydroxyl groups on an unsaturated seven-membered carbon ring (Dewar, 1945). These compounds act as a natural chemical defense shield against pathogen invasion in standing trees (DeBell et al., 1997; Van der Kamp, 1986) and confer durability in cedar wood products (Barton & MacDonald, 1971; Rennerfelt, 1948; Rudman, 1962). Johnson and Cserjesi (1980) reported that in W R C shakes, the two most abundant compounds with anti-microbial activity, y- and /i-thujaplicin were depleted by 25% after one year exposure to natural weather conditions, and by 90% after three years. Biodegradation of extractives by microorganisms may have also contributed to this depletion. Premature replacement of W R C wood products resulting from extractive depletion may be further compounded by the fact that W R C product manufacture increasingly relies on second growth lumber which contains approximately half the extractive content of the best old growth lumber (Barton & MacDonald, 1971; Nault, 1988). This may lead to potentially less durable W R C wood products. To address the decay of W R C , it is necessary to understand the interactions between W R C extractives, leaching and fungal decay. Therefore, the aims of this research were to 1) characterize the effect of leaching on the concentration of extractives in second-growth W R C , and 2) compare the ability of several decay fungal species to decay leached versus non-leached W R C . 5.3 Materials and Methods 5.3.1 Wood Materials W R C blocks of 19mm on each dimension were manufactured from the outer heartwood of an 80yr old second growth W R C tree which was felled in the U B C Malcolm Knapp research forest, Maple Ridge, British Columbia. Ponderosa pine (Pinus ponderosa P. & C. Lawson) sapwood blocks were used as a control species which does not contain any known fungicidal extractives similar to those present in W R C . 106 5.3.2 Leaching of W R C Blocks and Chemical Analysis In total, 84 W R C blocks were leached and an additional 84 were not leached, and frozen at -20°C until further use. We followed the American Wood-Preservers' Association ( A W P A ) standard method of testing wood preservatives by laboratory soil-block cultures ( E l 0-01) ( A W P A , 2004) which includes guidelines for leaching of wood samples. W R C blocks were submerged in a volume 50ml of distilled H2O per block which was replaced daily with fresh water for a period of 14 days. Wood samples were extracted and extractive compounds were quantified as described in Chedgy et al. (2006a). Samples were sliced into 2mm thick sections , then finely ground under liquid nitrogen and extracted in methanol with ultrasonication. Separation and analysis of extractives were carried out by reverse phase high performance liquid chromatography coupled with an ultraviolet (UV) detection system. Extractives were quantified (ug/g dry weight, D W ) by comparing analyte response with the response factor of an internal standard by a single point calibration. The five compounds of interest were (-)-plicatic acid, y-thujaplicin, ^thujaplicin, ^ thujaplicinol, and thujic acid (Figure 5.1). This experiment followed a completely randomized design (CRD) with two treatments: no leaching and leaching (k=2). Six replicates W R C blocks were used in each case (n=6). 5.3.3 Fungal Growth with W R C Leachate The growth and tolerance of six fungal species to W R C leachate was tested. The fungal species examined were Acanthophysium lividocaeruleum, Coniophora puteana, Heterobasidion annosum, Pachnocybe ferruginea, Phellinus sulphurascens, and Phellinus weirii. Two strains of each species were used, except for P. sulphurascens and P. weirii where only one strain was available. The strains used were isolated from in-service W R C products (L im et al., 2005), and were the most frequently isolated species. 107 Following the leaching of W R C blocks, the resulting leachate was retained and stored at 4°C in the dark. Approximately 25% of this volume was freeze-dried using an Edwards Modulyo freeze dryer equipped ( B O C Edwards Pharmaceutical Systems, Wilmington, Massachusetts) with a ThermoSavant V L P 200 vacuum pump (Thermo Electron Corp., Waltham, Massachusetts). This was accomplished by filling multiple 50ml falcon tubes with 35ml of leachate and frozen at -80°C in a horizontal position. Once frozen, a small hole was pieced in the cap of the falcon tubes" to allow air flow. Tubes were then placed into a pre-cooled freeze dryer (-45°C) under a vacuum of lmbar to freeze-dry for a period of 48 hours. The resulting leachate powder was re-suspended to a concentration of 50mg ml" 1 in 50% ethanol and filter sterilized. The agar dilution method was used for the antifungal activity tests. Mycel ia l growth was measured on 25ml 1% malt extract agar ( M E A ) plates containing various concentrations of W R C leachate. Concentrations of 0, 16, 32, 64, 128 and 256ppm (parts per million) were used and prepared by homogenously spreading an appropriate volume of the stock solution onto the surface of each media plate. Control plates were simultaneously prepared containing 50% ethanol only at the same volumes used to prepare the various leachate containing plates. The medium was then inoculated with a 5 mm plug of agar taken from the edge of actively growing isolate colonies. The cultures were maintained in the dark at 20°C and the growth was evaluated by measuring two perpendicular diameters of the colony every three days. The growth rate (mm/day) was then 108 H 3 C O . C H 2 O H t ' ' C 0 2 H O H H O y O C H 3 O H 2,3,6-Trmydroxy-7-methoxy2hydroxyxn 2-Hydroxy-5-isopropyl-2,4,6--4-(3' ,4' -dihydroxy-5' -methoxyphenyl)-tetralin-3-carboxylic acid (-)-Plicatic Acid cycloheptatrien-1 -one ^Thujaplicin H ^ C H O 2,7-Dihydioxy-4-isopropyl-2,4,6-cycloheptatrien-1 -one /7-ThujapIicinol 2-Hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1 -one ^-Thujaplicin 7,7-Dimethylcycloheptatriene-3-carboxyhc acid Thujic Acid Figure 5.1 Chemical structure and nomenclature used for W R C heartwood extractive compounds examined (Barton and MacDonald, 1971). 109 estimated for each isolate during its exponential growth phase. Three replicate plates were prepared for each isolate at each leachate concentration. This trial followed a C R D with six treatments defined as the varying concentrations of W R C leachate (k=6). Three replicates cultures were established for each species strain, at each leachate concentrations, and two strains were used per species. Statistical analysis was performed the mean growth rates (mm/day) calculated from both strains together (n=6), apart for species P. sulphur ascens, and P. weirii were only one strain was used in which case separate statistical analysis was performed (k=6, «=3). . ' . 5.3.4 Soil Block Decay Tests The decay ability of the six fungal species was compared on leached and non-leached W R C blocks, as well as on blocks of ponderosa pine sapwood. The extent of decay was measured by estimating the percentage mass loss (dry. weight, D W ) of blocks compared to controls after a period of incubation with the various fungal isolates. We followed the standard procedure for laboratory soil block cultures (El0-01) as outlined by the A W P A (2004). A l l wood blocks used were free of knots, with 2 to 4 rings per cm. Blocks showed no visible evidence of infection by mold, stain or wood-destroying fungi. In the case of pine, no visible concentrations of resin were observed. Before use, pine and W R C blocks were placed in a conditioning room at 20°C and a relative humidity of 65% for 48 hours then sorted by weight into narrow weight range groups. Pine blocks used in the experiment were 3.3g ± O.lg, and all W R C blocks used in the experiment were approximately 2.1 g ± O.lg. Pine blocks were numbered and the dry weight (DW) of each was calculated by oven drying blocks at 105°C for 24 h. For W R C blocks it was only possible to estimate the D W as several of the extractive compounds of interest are prone to thermal as well 110 as photo-degradation (Johnson & Cserjesi, 1980; Shibata et al. 2003). In this instance, all W R C blocks originated from heartwood on a single longitudinal axis parallel with growth rings. Blocks were sequentially numbered relative to their position. Prior to use, all blocks (pine, leached and non-leached W R C ) were ion beam sterilized (Iotron Industries Canada Ltd., Port Coquitlam, British Columbia). Glass jars containing soil were prepared as described in the A W P A standard protocol. Feeder strips of dimensions 3 x 28 x 34mm were manufactured from lodgepole pine (Pinus contorta) sapwood and placed on the soil surface (one per jar). Jars were autoclaved (with lids) at 103.4 K P a for 30 minutes on two consecutive days. Jars were then inoculated with fungal colonies by placing 10mm x 30mm agar blocks removed from near the leading edge of the mycelium of actively growing colonies. Agar blocks were placed in contact with one edge o f the feeder strip and in contact with the soil. Jar lids were loosened by lA turn from the fully tightened position to allow limited oxygen flow and placed in an incubation room (25°C with a relative humidity of 70%) until feeder strips were completely covered with mycelium. Test blocks were bought to a moisture content ( M c ) of 40% by placing sterilized blocks at 100%+ M c into a sterile fume hood and allowing them to air dry until the appropriate M c had been reached. Blocks were then placed on the surface of feeder strips with the tracheids in the vertical orientation to encourage mycelial penetration. A l l samples were incubated for a period of sixteen weeks. Six replicates were used for each fungal isolate and for each wood type: W R C leached, W R C non-leached and pine sapwood. In addition, for each wood type six controls were used, which were not inoculated with fungal cultures. To reduce the number of jars used in the experiment, two blocks were used per jar. I l l 5.3.5 Statistical Analysis A l l experiments followed a completely randomized design (CRD) with treatments denoted as k and replicates as n. A one-way analysis of variance ( A N O V A ) (a = 0.05) and Tukey's test for comparison of means (Tukey, 1949) were performed on data. Statistical analysis was performed using J M P I N software (version 4.0.3 (academic), S A S Institute Inc., North Carolina). 5.4 Results and Discussion Leaching of W R C blocks resulted in significant losses of the five key extractives. Leached W R C contained - 8 0 % less extractives than non-leached W R C (Figure 5.2). The ability of fungal species A. lividocaeruleum, C. puteana, H. annosum, P. ferruginea, P. sulphurascens, and P. weirii to grow on media supplemented with varying concentrations of W R C leachate was examined. Table 5.1 shows the mean growth rate (mm/day) of fungal species with different W R C leachate concentrations (ppm) in vitro. Statistical analysis suggested that the presence of W R C leachate had a significant effect on the mycelial growth of A. lividocaeruleum, C. puteana, H. annosum, P. sulphur ascens, and P. weirii. However, the presence of W R C leachate did not significantly affect the growth of P. ferruginea, even at the highest concentration of 256ppm. The growth rate of P. ferruginea seemed to marginally increase with extractive concentration suggesting that it may have the ability to utilize extractives as a carbon source. This wi l l be further explored in future work. A. lividocaeruleum and H. annosum were also able to grow on plates with 256ppm leachate but at a slower growth rate than on the control plates. Soil block test experiments showed that, in most cases, the six fungal species were able to decay pine sapwood to a greater degree than second growth W R C heartwood. Overall, leached W R C wood was decayed more readily than non-leached W R C (Figure 5.3). 112 8000 7000 H 6000 -Sf on 3 5000 4000 3 3000 u a o 2000 1000 0\ •^2 T f • Non-Icachcd • Leached (-)-Plicatic A d d y-Thujaplicin P Thujaplicin P-Thujaplicinol Thujic A d d Figure 5.2 Concentration of extractives (jig/g DW) in non-leached and leached W R C heartwood blocks. Note: Y error bars indicate standard deviation. Numbers above error bars represent concentration expressed as a percentage of the non-leached W R C blocks. A N O V A generated the following critical F values for extractives of interest: (-)-plicatic acid (F(i jio)850.3); y-thujaplicin (F(i jio)85.1); /i-thujaplicin (F(ii 0)76.4); /?-thujaplicinol (F(iio)36613.7); and thujic acid (F(ijo)63.9). Treatment effect is significant i f F value is greater than tabular value of F(iio)4.96. 113 Table 5.1. Mean fungal growth rate (mm/day) on media containing various concentrations (ppm) of leachate. Leachate Concentration (ppm) Species 0 16 32 64 128 256 A. lividocaeruleum 1 2 Mean 4.17 '(0.30) 3.94 (0.24) 4.06 (0.27)a 4.31 (0.27) 4.72 (0.19) 4.51 (0.23)a 4.22 (0.19) 4.89 (0.10) 4.56(0.14)" 3.67 (0.33) 4.64 (0.27) 4.15 (0.30)a 2.64 (0.13) 1.96 (0.13) 2.30 (0.13)b 1.63 (0.15) 0.99 (0.28) 1.31 (0.22)c C. puteana 1 2. Mean 4.06 (2.34) 3.86(1.71) 3.96 (2.02)a 3.00(1.67) 3.22 (1.39) 3.11 (1.53)a 2.06(1.75) 2.83 (0.17) 2.44 (0.96)b 1.69 (0.34) 2.19(0.76) 1.94 (0.55)c 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)d 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)d H. annosum 1 2 Mean 9.58 (0.30) 5.94 (0.24) 7.76 (0.27)a 8.94 (0.63) 5.81 (0.13) 7.38 (0.38)a 9.39 (0.54) 5.86 (0.13) 7.63 (0.33)a 9.28 (0.54) 4.86 (0.19) 7.07 (0.36)a 3.53 (0.42) 3.81 (0.46) 3.67 (0.44)b 0.69 (0.6) 2.61 (0.53) 1.65 (0.57)c P. ferruginea 1 2 Mean 0.51 (0.30) 0.47 (0.38) 0.49 (0.34)a 0.69 (0.13) 0.69 (0.05) 0.69 (0.09)a 0.61 (0.10) 0.68 (0.31) 0.65 (0.2 l)a 0.64 (0.02) 0.52 (0.17) 0.58(0.10)a 0.66 (0.15) 0.67 (0.12) 0.67 (0.14)b 0.67 (0.11) 0.67 (0.1) 0.67 (0.1 l) b P. sulphurascens 6.00 (0.17)a 1.36 (0.42)b 0.0 (0.0)c 0.0 (0.0)c 0.0 (0.0)c 0.0 (0.0)c P. weirii 6.22 (0.35)a 4.78 (0.75)\ 4.47 (0.05)b 3.33 (0.17)c 0.0 (0.0)d 0.0 (0.0)d Note: numbers in parenthesis indicate standard deviation (mm). A N O V A generated the following critical F values for the six species examined: A. lividocaeruleum (F(5 ;30)56.87), C. puteana (F(5>30)13.26), H. annosum (F(5;3o)21.51), P. ferruginea (F(5 ;30)2.12), P. sulphurascens (F(5,12)509.35), and P. weirii (F(5 ji2)169.68). Treatments are considered to have a significant effect i f the critical F values are greater than the appropriate tabular values, these were F(5 ;30)2.53 and F(5>i2)3.11 (a=0.05) for this experiment. Numbers followed by the same letter were not significantly different (a=0.05) according to Tukey's test of multiple comparison of means. 114 For C. puteana, H. annosum, P. sulphurascens, and P. weirii statistical analysis suggested that leaching treatments had a significant effect on the amount of decay observed, in this case, loss of extractives resulted in greater decay of W R C . For these species the greatest amount of decay occurred on pine, followed by leached W R C and the least amount of decay was recorded in non-leached W R C . For example, C. puteana produces a mean mass loss of - 5 7 % for pine, - 3 7 % for leached W R C , and - 1 3 % for on non-leached W R C . Tukey's test for multiple comparison of means also showed that for C. puteana the degree of decay was significantly different (a = 0.05) on the three wood types tested. A similar, but less defined pattern was observed for H. annosum and P. weirii with no significant difference between pine and leached W R C wood with mean % mass loss in the ranges o f 30-40% for pine and leached W R C . Non-leached W R C blocks were decayed to a lesser extent at approximately 18-26% mass loss with both fungal species. P. sulphur ascens only caused decay on pine (-32% loss) and leached W R C (-12%) but could barely grow on non-leached W R C (-2%). However, treatment effects were not significant for A. lividoeaeruleum and P. ferruginea suggesting that loss of extractives is not necessary for decay to occur. A. lividoeaeruleum provided conclusive results from the decay test as it grew well on the three wood types causing mass loss between 18-24%. P. ferruginea caused small amounts of decay (-5%) on all three wood types suggesting that extractive content does not affect the growth and decaying ability of this species. However, this result is inconclusive given the low amounts of decay observed, perhaps a function of its slow growth rate. Also noteworthy was the fact that aggressive wood decayers: A. lividoeaeruleum and H. annosum caused mass losses in the range of 25-30% were observed in pine blocks, lower that would have been expected. This may be due to the fact that each fungal species requires different M c for optimal growth. In the work reported here a M c of 115 Figure 5.3 Estimation of the degree of decay (% mass loss) of wood blocks by six fungal species of interest. Note: Y error bars indicate standard deviation. Critical F values are as follows for each of the fungal species: C. puteana (Fp ; 15)= 94.14); A. lividoeaeruleum (F(2 ,i5)= 1.39); H. annosum (F(2, i5)= 2.25); P. weirii (F(2,\s) = 7.39); P. ferruginea ( F (2 j i 5 )= 0.83).; and P. sulphurascens (F(2,i5) = 49.43). Values were considered significant i f greater than the tabular value of F ( 2, i5) = 3.68 (a=0.05). Bars with the same letter were not significantly different according to Tukey's test for multiple comparison of means (a = 0.05). 116 40% was created which may be lower than the M c encountered in natural conditions. H. annosum for example is typically causes rot in standing trees, Neilson et al. (1985) reported a typical M c of 58%) in freshly cut W R C . To this end, we intend to repeat the decay test at a slightly higher M c in order to further explore this aspect. 5.5 Conclusions We observed that in laboratory conditions, leaching of second-growth W R C heartwood blocks resulted in an 80%) loss of extractives. Extractive loss generally resulted in a greater degree of decay of W R C in a standard decay block tests using six of the most commonly isolated fungal species from in-service W R C wood products.. This was typified by species such as P. sulphurascens, which only caused decay on pine and leached W R C but could barely grow on non-leached W R C . This pattern was apparent to a lesser extent with species C. puteana, H. annosum, and P. weirii which were able to decay non-leached W R C blocks but to a lesser degree than leached W R C and pine blocks. This suggests that for these species leaching of extractives promotes fungal decay of W R C . However, this was not the case for species A. lividocaeruleum and P. ferruginea, which were able to decay pine, leached W R C and non-leached W R C to a statistically similar degree. Wood moisture content may have influenced the amount of decay observed by the various fungal species in this work, with each species requiring different moisture levels for optimal growth. Several aggressive wood decaying species such as H. annosum and A. lividocaeruleum caused less decay than expected. We intent to re-explore these aspects of the experiment to ensure that we have a clear representation of the nature of decay of W R C wood products. However, we did establish a clear correlation between tolerance to W R C leachate in vitro and the ability to decay leached versus non-leached second growth W R C in soil block decay tests. 117 P. ferruginea is of particular interest as it exhibited high tolerance to W R C leachate in vitro and could decay non-leached W R C blocks. This species is a candidate 'pioneer' species that may detoxify extractives in wood products, paving the way for colonization by less extractive-tolerant decay fungi. We observed low amounts of decay by this species, perhaps due to its slow growth rate. To our knowledge there is no evidence in the literature to suggest that this species can cause wood decay by degrading cellulose, hemicellulose or lignin. We also observed that the growth rate of this species marginally increased when grown on media supplemented with W R C leachate compared with controls. To this end, it is a possibility that it may utilize such extractives as a carbon source. The small mass losses observed our decay test may be attributable to loss of simple sugars or extractives. Further work is required to better understand its role in the decay of W R C products. 118 5.6 References American Wood-Preservers' Association Standards (2004). Standard method of testing wood preservatives by laboratory soil-block cultures (El0-01). American Wood-Preservers' Association Standards 2004: 406-414. Barton, G . F. , and MacDonald, B . F. 1971. The chemistry and utilization of western redcedar. Department of Fisheries and Forestry Canadian Forestry Service (publication No. 1023). Chedgy, R. J, Morris, P. I., L i m , Y . M . , and Breuil , C. 2006b. Black stain of western redcedar (Thuja plicata Donn) by Aureobasidium pullulans: the role of weathering. Wood Fiber Sci. Under review. Chedgy, R. J., Daniels, C. R., Kadla, J., and C. Breuil. 2006a. Screening for extractive resistant fungi, part 1: extraction and quantitation of western redcedar (Thuja plicata) heartwood extractives by ultrasonication and reverse phase H P L C . Holzforschung. Under review, Submission # HOLZ-D-06-00099. Chedgy, R. J., Daniels, C. R., Morris, P. I., and Breuil , C . 2005. Black stain of western redcedar by Aureobasidium pullulans and its relationship with tropolone depletion. International Research Group on Wood Preservation document No . I R G / W P 05-10564. I R G , Stockholm, Sweden. Coombs, R. W. , and Trust, T. J. 1973. The effect of light on the antibacterial activity of B-thujaplicin. Can. J. Microbiol . 19: 1177-1180. DeBel l , J. D. , Morrel l , J. J, and Gartner, B . L . 1997. Tropolone content of increment cores as an indicator of decay resistance in western redcedar. Wood Fiber Sci . 29: 364-369. Dewar, M . J. S. 1945. Structure of stipitatic acid. Nature (London United Kingdom) 155: 50-51. Gonzalez, J. S. 2004. Growth properties and uses of western redcedar (Thuja plicata Donn). Vancouver : Forintek Canada Corp. Special publication N o SP-37R. ISSN No. 0824-2119. 119 Hon, D. N . S. 1991. Wood and cellulosic chemistry. Shiraishi N , N e w York, N . Y . , Marcel Dekker, Inc: 1020 pp. Inamori, Y . , Sakagami, Y . , Morita, Y . , Shibata, M . , Sugiura, M . , Kumeda, Y . , Okabe, T., Tsujibo, H . , and Ishida, K . 2000. Antifungal activity of hinokitiol-related compounds on wood-rotting fungi and their insecticidal activities. B i o l . Pharm. B u l l . 23: 995-997. Johnson, E. L . , and Cserjesi, A . J. 1980. Weathering effect on thujaplicin concentration in western redcedar shakes. For. Prod. J. 30: 52-53. L i m , Y . W, K i m , J. J., Chedgy, R. J., Morris, P. I., and Breuil , C . 2005. Fungal diversity from western redcedar fences and their resistance to /i-thujaplicin: Ant. van Leeuwen. 87: 109-117. L i m , Y . W. , Chedgy, R. J., Amirthalingam, S., and Breuil , C . 2006. Screening for extractive resistant micro-organisms, part 2: isolation of extractive resistant fungi from western redcedar (Thuja plicata Donn) decks. Holzforschung Under review, submission # H O L Z - D -06-00100. Morita, Y . , Matsumura, E . , Okabe,T., Fukui, T., Shibata, M . , Sugiura, M . , Ohe, T., Tsujibo, H . , Ishida, N . , and Inamori, Y . 2004a. Biological activity of a-thujaplicin, the isomer of hinokitol. B i o l . Pharm. B u l l . 27: 899-902. Morita, Y . , Matsumura, E . , Okabe,T., Fukui, T., Shibata, M . , Sugiura, M . , Ohe, T., Ishida, N . , and Inamori, Y . 2004b. Biological activity of /i-dolabrin, y-thujaplicin, and 4-acetyltropolone, hinokitol-related compounds. B io l . Pharm. Bu l l . 27: 1666-1669. Nault, J. 1988. Radial distribution of thujaplicins in old growth and second growth western redcedar. Wood Sci . Technol. 22: 73-80. Nielson, R .W. , J. Dobie, and Wright, D . M . 1985.Conversion factors of the forest product industry in western Canada. Forintek Canada Corp., Western Laboratory Special Publication SP-24R. Vancouver,B.C. 120 Rennerfelt, E . 1948. Investigations of thujaplicin, a fungicidal substance in the heartwood of Thuja plicata Donn. Physiol. Plantarum 1: 245-254. Rudman, P. 1962. Causes of natural durability in timber. IX . The anti-fungal activity of heartwood extractives in a wood substrate. Holzforschung 16: 74-77. Shibata, H . , Nagamine, T., Wang, Y . , and Ishikawa, T. 2003. Generation of reactive oxygen species from hinokitol under near-UV irradiation. Biosci . Biotechnol. Biochem. 67: 1996-1998. Tukey, J. W . 1949.Comparing individual means in the analysis of variance. Biometrics 5: 99-114. Van der Kamp, B . J. 1986. Effects of heartwood inhabiting fungi on thujaplicin content and decay resistance of western redcedar {Thujaplicata Donn). Wood Fiber Sci . 18: 421-427. 121 Chapter 6 General Conclusions and Future Work The aim of this work was to explore the relationship between extractive content of W R C wood products and colonization by micro-organisms that may reduce service life either through decay or discoloration. We investigated the role of weathering in causing depletion of anti-microbial extractives and how this affected colonization by fungi. In addition, we identified extractive-tolerant fungi that did not require depletion of extractives for colonization. These 'pioneer' species may play an important role in the initial modification or detoxification of W R C extractives in wood products, paving the way for colonization by less specialized decay fungi. We successfully modified a method to extract, separate and accurately quantify extractive compounds using ultra-sonication and reverse phase H P L C . Six compounds that accounted for approximately 67% (w/w) of the total extractive content of W R C heartwood were quantified by comparing analyte response with the response factor of an internal standard by a single point calibration. This method was highly sensitive with limits of detection at 0.6ug/ml for (-)-plicatic acid, 15.6ug/ml for y-thujaplicin, 15.6ug/ml for /i-thujaplicin, 15.6pg/ml for/i-thujaplicinol, l.Opg/ml for thujic acid and 1.8ug/ml for methyl thujate. We showed that grinding heartwood samples under liquid nitrogen produced a 36% increase in extractive recovery yield compared to sliced samples. Finally, extraction using methanol with ultra-sonic frequency provided good recovery of extractives (-90%) with high repeatability (RSD -5%) . This approach enabled multiple samples to be analyzed simultaneously and provided a controlled environment in which a low temperature could be maintained and samples shielded from incandescent light. This 122 approach was straightforward, highly sensitive and detected extractives present at very low concentrations with a high level of precision. This method could be applied to almost any situation where W R C extractives are required to be quantitated, particularly given the interest in using W R C extractives for pharmaceutical applications ranging from anti-cancer and H I V treatments. We then developed a novel technique to screen fungi for extractive-tolerance in vitro. This was achieved by monitoring growth rates of isolated fungal strains on media inoculated with W R C -FSs. This enabled fungal strains to be exposed to a mixture of extractives that they would encounter in W R C wood products. Furthermore, researchers can overcome the limited availability and cost of pure extractive compounds. From the twelve species screened, results indicated that the basidiomycetes, Acanthophysium lividocaeruleum and Coniophora puteana, and three ascomycetes, Aureobasidium pullulans, Exophiala heteromorpha, and Rhinocladiella atrovirens had moderate tolerance to W R C extractives. The basidiomycetous species Pachnocybe ferruginea exhibited a high level of tolerance to W R C extractives. The five fungal species were commonly isolated from W R C wood products. In order to determine which of the extractive constituents were affecting fungal growth we estimated the concentrations of individual extractives that leached from W R C - F S s and accumulated into the media using our chemical analytical method. We prepared media supplemented with individual extractive compounds at concentrations corresponding to amount leached from W R C - F S s and inoculated this media with three representative species, P. ferruginea (high extractive-tolerance), R. atrovirens (moderate extractive-tolerance), and Phellinus ferreus (low extractive-tolerance). We concluded that (-)-plicatic acid even at high 123 concentrations had little fungicidal activity, and /i-thujaplicinol also had a minimal effect on growth at the low concentrations leached from WRC-FSs. y-Thujaplicin and /i-thujaplicin exhibited the highest fungicidal activity, while thujic acid also decreased fungal growth at high concentrations. Again, P. ferruginea consistently showed a high tolerance to all extractive compounds. Weathered surfaces of WRC products are also susceptible to black stain caused by fungi such as Aureobasidium pullulans which is reported to utilize products of lignin photo-degradation as a carbon source. Extractive contents at the surface of WRC products may form the first line of defense in preventing fungal spore germination. The effect of weathering on extractive concentrations at the surface was characterized and correlated with the ability of this fungal species to colonize weathered surfaces. Simulated weathering treatments using a Weather-Ometer® caused significant changes in the extractive content of exposed surface. UV-only treatments reduced extractive contents less than the other treatments but stimulated fungal colonization. However, U V plus water spray severely reduced extractives but did not lead to increased fungal colonization compared to un-weathered wood. Water spray most likely washed away products of lignin photo-degradation, leaving the wood surface void of accessible carbon sources resulting in decreased fungal growth. A. pullulans exhibited moderate to high tolerance to the tropolone /i-thujaplicin in vitro, suggesting that tropolone reductions by weathering may not be required for colonization. It is likely that A. pullulans may have competitive advantages in colonizing exposed WRC surfaces because it can use lignin breakdown products as a carbon source, it is resistant to U V due to its melanized cells and it tolerates tropolones. 124 Finally, in standard laboratory tests we determined that the effect of leaching on the extractive content of second growth W R C was an - 8 0 % reduction of the five key compounds (-)-plicatic acid, y-thujaplicin, /^thujaplicin, /Mhujaplicinol, and thujic acid. For six fungal species that had been commonly isolated from in service W R C wood products, we observed a correlation between tolerance to W R C leachate in vitro and the ability to decay leached versus non-leached second growth W R C in soil block decay tests. Phellinus sulphurascens exhibited the lowest tolerance to W R C leachate and caused minimal decay on non-leached W R C but was able to decay leached W R C and Ponderosa pine blocks. C. puteana, H. annosum, and Phellinus weirii exhibited moderate to high tolerance to extractives were able to decay non-leached W R C blocks but to a lesser degree than leached W R C and pine blocks. This suggests that for these species leaching of extractives promotes fungal decay of W R C . A. lividocaeruleum and P. ferruginea were the most tolerant to W R C extractives and could grow on all types of wood to an equal degree suggesting that these species do not require depletion of extractives by leaching for decay to occur. However, we observed little wood decay by P. ferruginea in this work, perhaps due to the slow growth rate of this species. Extractive tolerant micro-organisms such as P. ferruginea are of particular interest for future work as they may be a 'pioneer' species playing an important role in the initial modification or detoxification of W R C extractives in wood products, paving the way for colonization by less specialized decay fungi. In this work we have only demonstrated their tolerance to extractives, yet to date there is no evidence to suggest that they can detoxify fungicidal extractives. In . addition, to our knowledge there is no evidence in the literature that this species is capable of decaying wood. The mass loss recorded could be attributable to loss of extractives or simple sugars present in wood. The next step wi l l be to develop techniques in which candidate pioneer , 1 2 5 fungal species can be exposed to individual extractive compounds in vitro which enable us to monitor the any chemical modifications that may occur to extractives over time. This wi l l provide the industry with a better understand the role of this species and others in causing decay of W R C wood products. 126 Appendicies Table of Contents Page Appendix I Fungal Diversity from Western Redcedar Fences and Their Resistance to /^-Thujaplicin 132 AI . l Abstract 132 AI.2 Introduction ..133 AI.3 Materials and Methods 134 AI.3.1 Collection Sites and Fungal Isolations 134 AI.3.2 D N A Extraction, P C R and Sequencing 136 AI.3.3 Inhibition of Fungal Growth by /7-Thujaplicin 137 AI.4 Results and Discussion 137 AI.4.1 Isolation and Morphological Grouping of Fungi 137 AI.4.2 Molecular Analysis of Fungal Isolates 141 AI.4.3 /^-Thujaplicin Resistance and Fungal Colonization 143 AI.5 Conclusions 145 AI.6 References 147 Appendix II Morphology of Black Staining Fungi: Aureobasidium pullulans and Hormonema dematioides 153 Appendix III ANOVA Source Tables.... 154 ALU. 1 A N O V A Source Tables Relating to Chapter 2 155 127 A I H . 1.1 Comparing Response Factors Among Extractive Compounds 155 AIII. 1.2 The Effect of Various Extraction Methods on Extractive Concentration ( u g / g D W ) : 155 AIII.2 A N O V A Source Tables Relating to Chapter 3 156 AIII.2.-1 Extractive Concentrations (pg/g) Versus W R C Feeder Strip Storage Conditions 156 AIII.3 A N O V A Source Tables Relating to Chapter 4 157 AIII.3.1 The Effect of /i-Thujaplicin on the Growth of Black Staining Fungi 157 AIII.3.2 The Effect of Weathering on the Concentration of Individual Extractives 158 AIII.3.3. The Degree of Colonization of Weathered Wood Surfaces by A. pullulans (Quantitative Assessment) 159 AIII.4 A N O V A Source Tables Relating to Chapter 5 159 AIII.4.1 The Effect of Leaching on Extractive Concentration 159 AIII.4.2 The Effect of W R C Leachate on Fungal Growth In Vitro 160 AIII.4.3 Estimation of the Degree of Decay (% mass loss) of Wood Blocks by Six Fungal Species of Interest 161 128 List of Tables Page Table A I . l : List of fungi isolated in this work; source, their characteristics and GenBank accession numbers 139 Table AI.2: Effect of the various concentrations of /^-thujaplicin on the fungi isolated . 144 Table AIII . l : Comparing response factors among extractive compounds (Chpt 2)......155 Table AIII.2: (-)-Plicatic acid (Chpt 2. extraction method vs. concentration) 155 Table AIII.3: y-Thujaplicin (Chpt 2. extraction method vs. concentration) 155 Table AIII.4: /?-Thujaplicin (Chpt 2. extraction method vs. concentration) 155 Table AIII.5: /?-Thujaplicinol (Chpt 2. extraction method vs. concentration) 155 Table AIII.6: Thuj ic acid (Chpt 2. extraction method vs. concentration) 156 Table AIII.7: Methyl thujate (Chpt 2. extraction method vs. concentration) 156 Table AIII.8: (-)-Plicatic acid (Chpt 3. W R C - F S storage vs. concentration) 156 Table AIII.9: ^Thujapl ic in (Chpt 3. W R C - F S storage vs. concentration) 156 Table AIII.10: ^-Thujaplicin (Chpt 3. W R C - F S storage vs. concentration) 156 Table AIII.11: /?-Thujaplicinol (Chpt 3. W R C - F S storage vs. concentration) 157 Table AIII.12: Thujic acid (Chpt 3. W R C - F S storage vs. concentration) 157 Table AIII.13: Aureobasidium pullulans (Chpt 4. [/?-thujaplicin] vs. growth) 157 Table AIII.14: Hormonema dematioides (Chpt 4. [/^-thujaplicin] vs. growth) 157 Table AIII.15: (-)-Plicatic acid (Chpt 4. weathering vs. concentration) 158 Table AIII.16: y-Thujaplicin (Chpt 4. weathering vs. concentration) 158 129 Table AIII.17: /?-Thujaplicin (Chpt 4. weathering vs. concentration) 158 Table AIII.18: /?-Thujaplicinol (Chpt 4. weathering vs. concentration) 158 Table AIII.19: Thujic acid (Chpt 4. weathering vs. concentration) 158 Table AIII.20: Ponderosa pine sapwood (Chpt 4. A.pullulans colonization vs. weathering) 159 Table AIII.21: W R C heartwood (Chpt 4. A.pullulans colonization vs. weathering) 159 Table AIII.22: (-)-Plicatic acid (Chpt 5. leaching vs. concentration) 159 Table AIII.23: y-Thujaplicin (Chpt 5. leaching vs. concentration) 159 Table AIII.24: /^-Thujaplicin (Chpt 5. leaching vs. concentration) 160 Table AIII.25: /?-Thujaplicinol (Chpt 5. leaching vs. concentration) 160 Table AIII.26: Thujic acid (Chpt 5. leaching vs. concentration) 160 Table AIII.27: Acanthophysium lividocaeruleum (Chpt 5. [ W R C leachate] vs. growth) 160 Table AIII.28: Coniophora puteana (Chpt 5. [ W R C leachate] vs. growth) 160 Table AIII.29: Heterobasidion annosum (Chpt 5. [ W R C leachate] vs. growth) 161 Table AIII.30: Pachnocybe ferruginea (Chpt 5. [ W R C leachate] vs. growth) 161 Table AIII.31: Phellinus sulphurascens (Chpt 5. [ W R C leachate] vs. growth) 161 Table AIII.32: Phellinus weirii (Chpt 5. [ W R C leachate] vs. growth)... 161 Table AIII.33: Acanthophysium lividocaeruleum (Chpt 5. wood type vs. decay) 161 Table AIII.34: Coniophora puteana (Chpt 5. wood type vs. decay) 161 Table AIII.35: Heterobasidion annosum (Chpt 5. wood type vs. decay) 162 Table AIII.36: Pachnocybe ferruginea (Chpt 5. wood type vs. decay) 162 Table AIII.37: Phellinus sulphurascens (Chpt 5. wood type vs. decay) ...162 Table AIII.38: Phellinus weirii (Chpt 5. wood type vs. decay) ; 162 130 Table of Figures Page Figure A I . l : Schematic diagram for fungal isolation from W R C fences .135 Figure A I I . l : Morphology of Aureobasidium pullulans 153 Figure AII.2: Morphology of Hormonema dematioides 153 131 Appendix I Fungal Diversity from Western Redcedar Fences and Their Resistance to /^-Thujaplicin. ALI Abstract The work reported here investigated the fungal community inhabiting western redcedar fence material with a focus on species colonizing wood below the surface of which little is known. From seven pieces of fence material, twenty-three different fungal species were isolated and characterized using both traditional morphology and molecular identification methods. The species identified included thirteen ascomycetous and ten basidiomycetous fungi. Isolates were tested for their resistance to /^-thujaplicin - one of the principle fungicidal agents of western redcedar heartwood extractives. Generally, ascomycetous fungi exhibited greater resistance to P-thujaplicin than basidiomycetous fungi. Interestingly, three ascomycetous and two basidiomycetous species frequently isolated had high tolerance to this compound. These species could be candidate 'pioneer' species that invade and detoxify W R C extractives, paving the way for colonization by decay fungi. Key words: ascomycota, basidiomycota, /^-thujaplicin, pioneer fungi, western redcedar (Thuja plicata Donn.), wood products. 132 AI.2 Introduction Canada's and B C ' s value-added forest product industries depend in part on the unique qualities of some of this country's native wood species. Western redcedar ( W R C ) (Thuja plicata Donn) is a well-known and commercially important coniferous tree species common in the Pacific northwest. Its heartwood is valued for the natural durability conferred by fungicidal agents in its extractives (Wethern, 1959); in particular, by a group of tropolone compounds known as 'thujaplicins' (Rennerfelt, 1948). O f the several classes of thujaplicins characterized, p-thujaplicin (2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-l-one) appears to be the most prevalent and effective against decay fungi (Arima et al., 2003; Erdtman and Gripenberg, 1948; Inamori et al., 1999; 2000; Trust and Coombs, 1973). Despite such extractives, decay fungi are still a major factor in product failure in service. Furthermore, products manufactured from second growth W R C may have lower extractive contents than the best of the old growth (Nault, 1986). Optimizing the service life and value of products that rely on such natural protection requires an understanding of how extractives and fungal communities interact and evolve in service. In W R C trees 'pioneer' fungal species can detoxify fungicidal extractives, clearing the way for less specialized fungi to colonize and decay wood freely (Jin, 1987; Van der Kamp, 1975). However, to our knowledge, the fungal succession in biodeterioration has not been documented for W R C products, and little is known about the microbial communities that these products harbor. While research groups have reported a limited number o f decay fungi from W R C utility poles (Eslyn and Highlery, 1976; Morrell et al., 2001; Scheffer et al., 1984) and shingles/shakes (Smith and Swan, 1975), these studies relied on species identification by morphology only. This approach has two major limitations. Firstly, fungi in artificial cultures often exhibit fewer 133 morphological features than in their natural environments. This impedes identification, especially for fungi that lack asexual spores; e.g. Homobasidiomycetes. Secondly, a fungal species' characteristics can vary when it is grown on different media or under different culture conditions. The wealth of sequence information that has been compiled in databases means that it is now possible to identify fungi at a far higher resolution using molecular techniques than can be achieved using morphological methods. However, databases contain sequences from only a fraction of all known species to date, in this case morphological methods are still being relied on heavily (Allen et al., 2003; Wirsel et al., 2001). AI.3 Materials and Methods AI.3.1 Collection Sites and Fungal Isolations > W R C fence material was collected in Vancouver, B . C , Canada. Five pieces (125 X 19 X 4.5 cm) were from a fence in service between 1960 to 2001 and two (166.5 X 23 X 8 cm) from a fence in service between (1970 to 2001). Each piece was sliced horizontally into 2 cm blocks. Each block was further divided into five lateral sections. Wood flecks taken from within and near decay pockets were aseptically detached, briefly flamed to remove contaminating surface microflora and plated (Figure A 1.1). Eight decay areas from seven fence pieces were labeled fence A - G. A 1% malt extract agar ( M E A ) was used for isolating the general microflora and 1% M E A with benomyl ( B M E A ) was used for the basidiomycetous fungi (Clubbe and Levy, 1977). The plates were incubated at room temperature for several weeks with fungi routinely sub-cultured from mycelial margins to new plates in order to obtain pure cultures. Species identification via classical methodology was achieved by macro- and micro-morphological analyses using taxonomic guides and standard procedures (Arx, 1981; Barnett and Hunter, 1987; Carmichael et 134 Slices 19 and 20 Fence B Figure AI . l Schematic diagram for fungal isolation from W R C fences. In the example shown, fence material (Fence B) was cut horizontally into 2 cm slices and then divided further into five lateral sections. Wood flecks (shown by bold arrows) from the central regions were aseptically detached and inoculated onto plates. 135 al., 1980; Cole and Kendrick, 1973; El l is , 1971; Nobles, 1965; Schol-Schwarz, 1970; Stalpers, 1978; Wang and Zabel, 1990). This was complemented by molecular techniques for species identification. AI.3.2 D N A Extrac t ion , P C R and Sequencing D N A was extracted from mycelium scraped from the fungal colonies and placed into micro-centrifuge tubes with 300 pi of extraction buffer [100 m M Tr i s -HCl (pH 8.0), 1 m M E D T A (pH 8.0), 100 m M N a C l and 2% SDS]. The mixture was vortexed for 10 s, incubated at 75°C for 30 min. 4/5 volumes of glass beads were then added into the tube and vortexed for a further 10 min. D N A was purified via a two step phenol-chloroform extraction and precipitated with one volume of iso-propanol then centrifuged immediately at 12,000 rpm at room temperature for 10 min. After removing the supernatant, the pellet was washed with 70% ethanol, allowed to air dry and resuspended in 40 p i of distilled water. The extracted D N A was stored at -20°C until further use. To achieve P C R amplification of the internal transcribed spacer (ITS) regions, fungal universal primers (ITS5 and ITS4) and the basidiomycetous specific reverse primer (ITS4B) with ITS5 primer (Gardes and Bruns, 1993; White et al., 1990) were used. Amplification was performed as described by Lee et al. (2000). Usually 3 pi of each P C R product was used for the electrophoresis on 0.5% agarose gel containing EtBr in Tris-acetate E D T A (TAE) buffer. The P C R product sizes were determined by comparison to a 1 kb D N A marker ( G I B C O B R L , U.S .A. ) . The P C R products were purified using a Qiaquick P C R Purification K i t (Qiagen Inc.). Sequencing was performed on an A B I 3700 automated sequencer (Perkin-Elmer Inc. U S A ) at the D N A synthesis and Sequencing Facility, M A C R O G E N (Seoul, Korea). A l l of the nucleotide sequences determined in this work have been deposited in the GenBank, their accession numbers 136 are shown in Table A l . 1. The ITS region sequences were analyzed using B L A S T in order to find the most similar available database sequences. The closest matched sequence for each species was shown in Table A1 .1 . AI.3.3 Inhibition of Fungal Growth by yff-Thujaplicin The agar dilution method was used for the antifungal activity tests. Mycel ia l growth was measured on M E A plates with various concentrations of /?-thujaplicin. A 10 mg ml" 1 stock solution was prepared in 50% ethanol and kept in the dark at 4°C. Concentrations of 0, 2, 8, and 32 ppm were prepared. Vigorous mixing of the medium prevented the precipitation of the chemical. Ethanol had no effect on the hyphal growth at the low concentrations present in the growth medium. The medium was inoculated with a 5 mm plug of agar taken from the edge of actively growing isolate colonies. The cultures (three replicates used) were maintained in the dark at 20°C and growth was evaluated after 12 days by measuring two perpendicular diameters of the colony. AI.4 Results and Discussion AI.4.1 Isolation and Morphological Grouping of Fungi Regions of wood decay generally occurred in or around the ends of fence panels. A total of 144 fungal isolates were recovered from 303 sampling sites in 8 regions of decay present on 7 W R C fence sources. The highest count of fungal isolates was recorded in the decay area of fence G (39 isolates: 27.1%), while the lowest was recorded in fence A and B (7 isolates each : 4.7%). The fungal diversity was highest in decay area F l (10 different types) and lowest in decay area A (3 types). The macro- and microscopic characterization of the isolates allowed us to recognize 23 137 different fungal taxa, including thirteen ascomycetous fungi and ten basidiomycetous fungi. The basidiomycetes were identified using three characteristics: growth on B M E A , presence of clamp connection, and P C R amplification using forward primer ITS5 and basidio-specific reverse primer ITS4B, (Gardes and Bruns, 1993). However, P C R using ITS5 and ITS4B failed to amplify the ITS regions of two basidiomycetous isolates, Cerinosterus luteoalba and W R C F - B 2 . B M E A permits isolation of basidiomycetous fungi and prevents the growth of most microfungi (Clubbe and Levy, 1977). Two ascomycetous fungi, W R C F - A 2 and Phialophora sp. 1, grew on B M E A while a basidiomycetous fungus, W R C F - B 2 , did not. Among the ascomycetous species we recognized Oidiodendron griseum, Phialophora spp., Rhinocladiella atrovirens, Sporothrix spp., and three unidentified Ascomycota ( W R C F - A 1 , W R C F - A 2 , and W R C F - A 3 ) . O. griseum, Phialophora spp., Sporothrix spp., and W R C F - A 2 were isolated from a limited number of sites, while three species, R. atrovirens, W R C F - A 1 , and W R C F - A 3 were isolated from a broad range of decay areas. These ascomycetous fungi were . easily identified by their asexual structures. The most commonly isolated species was from the genus Phialophora (Table A L I ) . In this genus six species, including P. lignicola, P. versicola and four unidentified, were recognized. Two Sporothrix species were also isolated but their specific identification was not pursued. Most ascomycetous fungi found in this study were ubiquitous; this is concurrent with other research groups' findings. For example, O. griseum has been isolated from soil of cedar and spruce bogs, wood pulp (Barron, 1962), and pulp and paper samples (Wang. 1965), it causes a superficial discoloration of wood (Kaarik, 1980). Phialophora species are recognized as staining agents of wood products in service and also as important soft rot fungi (Eaton and Hale, 1993). 138 Table A I . l L is t of fungi isolated in this work ; source, their characteristics and GenBank accession numbers. Fungal Species GenBank Source Isolation B M E A C C ITS5 / 1TS5 / Closest match in B L A S T Identity [%]3 Acc No. (no. of isolates)' site2 Growth ITS4B ITS4 Basidiomvcota Acanthophysium lividoeaeruleum AY618666 C(2), D(2), G(9) B G P A A Acanthophysium lividoeaeruleum [AF506400] 282/289 (97%) Cerinosterus luteoalba AY618667 D(1),F1(1) D G N P N A A Sporobolomyces symmetricus [AY364836] 119/123 (96%) Hyphoderma praetermissum AY618668 A(2), E(3), Fl(4) D G P A A Hyphodontia flavipora [AF455399] 168/173 (97%) Pachnocybe ferruginea AY618669 A(2), B( l ) , D( l ) , Fl(10), F2(3), G(2) B G NP A A Septobasidium sp. [AB043972] 211/229(92%) Stereum sanguinolentum AY618670 G(2) D G P A A Stereum sanguinolentum [AY089730] 443/466 (95%) W R C F - B 2 AY618671 Fl(6), G(2) D N G P N A A Rhodotorula nothofagi [AF444641] 151/155 (97%) W R C F - B 4 AY618672 B(2),F1(1) D G N P A A Hyphodontiaradula [AF145580] 555/557 (99%) W R C F - B 5 AY618673 C(2), G(5) D G P A A Butlerelfia eustacei [U85800] 386/413 (93%) W R C F - B 7 AY618674 F2(2) D G NP A A Phanerochaete sordida [AY219381] 580/594 (97%) W R C F - B 9 AY618675 F2(2) D G NP A A Phlebia livida [AB084618] 363/391 (92%) Ascomvcota Oidiodendron griseum AY618676 E( l ) , Fl(4), F2(l) B N G NP N A A Oidiodendron griseum [AF062794] 345/345 (100%) Phialophora lignicola AY618677 E(3) 0 N G NP N A A Salal root associated fungus [AF149081] 482/485 (99%) Phialophora versicola AY618678 B(1),F1(1) B N G N P N A A Ectomycorrhizal isolate [AJ430410] 464/465 (99%) Phialophora sp. 1 AY618679 E(4) D G N P N A A Ascomycete sp. [AY3 54276] 335/350 (95%) Phialophora sp. 2 AY618680 E(3), Fl(2) B N G NP N A A Cadophora fastigiata [AY249073] 497/497 (100%) Phialophora sp. 3 AY618681 F2(3), G(3) B N G NP N A A Phialophora sp. [AY465463] 469/471 (99%) Phialophora sp. 4 AY618682 D(2) D N G NP N A A Phialophora sp. [AY465462] 467/500 (93%) Rhinocladiella atrovirens AY618683 E(2), Fl(6), F2(l), G(9) B N G NP N A A Rhinocladiella atrovirens [AB091215] 563/567 (99%) Sporothrix sp. 1 AY618684 Fl(2), F2(l) D N G N P N A A Sporothrix schenckii [AF484468] 424/440 (96%) Sporothrix sp. 2 AY618685 C(3) D N G N P N A A Ophiostoma grandicarpum [AJ293884] 171/174 (98%) W R C F - A 1 AY618686 A(3), B( l ) , C( l ) , D(l ) , E(2) D N G NP N A A Leaf litter ascomycete [AF502745] 422/441 (95%) W R C F - A 2 AY618687 C(3), G(4) 0 G NP N A A Oidiodendron myxotrichoides [AJ635314] 267/288 (92%) W R C F - A 3 AY618688 B(2), Fl(2), F2(3), G(3) B N G N P N A A Phialocephala dimorphospora [AF486121] 486/495 (98%) Note: G, growth; NG, no growth; CC, Clamp connection; P, present; NP, not present; A, amplification; and NA, no amplification. 1 Number of sampling point (isolates) collected from each fence: A, 16(7); B, 35(7); C, 33(11); D, 26(7); E, 30(18); F l , 56(39); F2, 25(16); and G, 82(39). 2 D, decay pocket; O, outside of decay pocket; and B, both regions, identity [% similarity] was derived from matched nucleotide/compared nucleotide in GenBank. 139 R. atrovirens originally isolated from material on decayed wood was also found in wood products (Barnett and Hunter, 1987). Many Sporothrix species are the anamorphs of Ophiostoma and Ceratocystis (Domsch et al., 1980), some of them are commonly found in creosote-treated wood products (Wang and Zabel, 1990). Among the Basidiomycetes five isolates were identified to the genus or species level by morphological features. The most frequentlylsolated species was Pachnocybe ferruginea; it was present in most of the decay areas except in fence C and E . This species has also been reported on creosote-treated western redcedar poles by Warren and Marshall (1986). Two other species, Acanthophysium lividoeaeruleum, easily recognized by its scattered clamp connections, and gloeocystidia (Nakasone, 1990), and Hyphoderma praetermissum, with its white mat with subtomentous to short-woolly, nodous septate, and spathulate cystidia, were also frequently isolated. One isolate of Stereum sanguinoleritum was identified by its simple septate, scattered single, double, or multiple clamps, and cystidium-like structures. This species is the only Stereum species that occurs primarily on gymnosperms. A l l of the above basidiomycetous fungi have been associated with white rot of various softwoods and wood products in North America (Eslyn, 1970; Gilbertson, 1974; Ginns, 1986; Lemke, 1964; Scheffer et al., 1984; Zabel et al., 1985). A n orange Sporothrix colony found in D and F l fences that we initially identified as Sporothrix luteoalba based on its morphology, but using D N A sequence analysis it was renamed Cerinosterus luteoalba. Some Sporothrix species are reported as the anamorphs of the basidiomycetous genus Cerinomyces (Dacrymycetaceae), and Moore (1987) erected the new genus Cerinosterus to accommodate species of Sporothrix having dolipores and imperfect 140 parenthesome septa. Finally, some unknown Basidiomycetes WRCF-B2, and WRCF-B5 were isolated with high frequencies but rarely from the decay sites, while WRCF-B7 and WRCF-B9 were isolated only from a decay site (Table A l . 1). AI.4.2 Molecular Analysis of Fungal Isolates In order to discriminate the isolates to the species level, the ITS regions were amplified. The amplified products ranged from 640 to 770 bp for the Basidiomycota and from 570 to 660 bp for the Ascomycota. However, for two isolates, Phialophora versicola and WRCF-A3, the amplified products were larger than the other species reported in this work, at about 1100 bp and 927 bp, respectively. The two fungi have introns of 520 bp for P. versicola and 340 bp for WRCF-A3 located near the 3' end of the 18S rDNA (data not shown). These introns contained four conserved regions, a characteristic of group I introns (Cech, 1988; Dujon, 1989). Blast searches revealed high similarity between the intron sequences of WRCF-A3 and a Phialographium-Wke fungus (AB038422), while the intron sequence of P. versicola matched closely those of Lachnum sclerotii (AF505520), Rhabdoclineparkeri (AF462428), Cadophora gregata f. sp. adzukicola (AF056487), and Hymenoscyphus ericae (AY394907). Although phylogenetically distant, these species are coniferous pathogens or ectomycorrhizal fungi. Sequences of 18S and L S U rDNA regions have been used for fungal identification in many ecological studies (Hunt et al., 2004; Kernaghan et al., 2003; Tedersoo et al., 2003). In addition to these regions, ITS sequence comparison is regarded as an excellent tool for identifying unknown fungi to broad species groups or genera (Horton and Bruns, 2001). The ITS sequence data enabled the linkage of most morphologically unidentifiable fungi to established genera. For example, WRCF-B5 belongs to the genus Butierelfia, WRCF-B7 to Phanerochaete, WRCF-B9 141 to Phlebia, W R C F - A 2 to Oidiodendron, and W R C F - A 3 to Phialocephala (Table A 1.1). A t this stage of analysis, W R C F - B 4 was identified as Hyphodontia radula since both sequences were 99% similar. Phialophora sp. 2 was identified as Phialophora fastigiata (telemorph -Cadophora fastigiata) with 100% sequence similarity. Five Phialophora species were positioned in five distantly related clades in the ITS phylogenetic tree (data not shown). This result is consistent with previous work that suggested that the genus Phialophora is clearly polyphyletic (Gams, 2000). Interestingly P. versicola and P. lignicola were closely related to salal root associated fungi, which were isolated from Vancouver Island (Allen et al., 2003). Some Phialophora species are known to form ectendomycorrhizal relationships with Pinus and Larix, and have also been observed forming ericoid mycorrhizal with Gaultheria shallon (Monreal et al., 1999; Y u et al., 2001), though their ecological functions are not well understood. It is important to note that Phialophora species have different phylogenetic histories and ecological roles. Pachnocybe ferruginea, Cerinosterus luteoalba and W R C F - B 2 were matched to members of a primitive order of Basidiomycota. These results might explain why C. luteoalba and W R C F - B 2 were not amplified by basidio-specific primers. P. ferruginea was closely related to Septobasidium sp. which was classified as Urediniomycetes. This is consistent with previous results on 5S r D N A and large subunit r D N A sequence analysis (McLaughlin, et al., 1995; Walker, 1984). P. ferruginea described first as a Hyphomycetes with reddish brown synnemata by Ell is (1971) was later transferred to Heterobasidiomycetes because of its simple septal pore structure (Kropp and Corden, 1986; Oberwinkler and Bandoni, 1982). The ITS sequence of C. 142 luteoalba confirmed its position within the Basidiomycota instead of Ascomycota, which coincided with Moore's (1987) suggestion. Overall there was a good agreement between morphological and ITS-sequence based approaches. However, due to the limited ITS sequence data within the database the closest matches of some fungal isolates could not be established. Specifically, ITS sequences of C. luteoalba, H. praetermissum and W R C F - B 2 had matches to only 5.8S r D N A region sequence of Sporobolomyces symmetricus (AY364836), uncultured fungus (AY241671) and Rhodotorula nothofagi (AF444641), respectively. AI.4.3 /^-Thujaplicin Resistance and Fungal Colonization Although /^-thujaplicin concentration varies within W R C trees (MacLean and Gardner, 1956), it inhibits many decay fungi at concentrations between 10 to 20 ppm (Inamori et al., 2000; Rennerfelt, 1948). In our work, p-thujaplicin showed some antifungal activity against most of the fungi examined (Table A1.2). Most basidiomycetous fungi tested were inhibited by concentrations between 2 and 8 ppm, while most of the ascomycetous fungi tested were affected at concentrations between 8 and 32 ppm. Our results were consistent with those of Rennerfelt (1948), who also showed that some ascomycetous fungi had higher resistance to thujaplicin than decay fungi. However, two basidiomycetous species, R ferruginea and A. lividocaeruleum, and three ascomycetous species, Oidiodendron sp. ( W R C F - A 2 ) , Phialophora fastigiata (Phialophora sp. 2) and Phialophora sp. 3, had high tolerance to this compound (Table A l .2). That these fungi were isolated both outside and in the center of decay areas suggests that they may be pioneer fungi in W R C fences. Such fungi may tolerate high concentrations of inhibitors, 143 Table AI.2 Effect of the various concentrations of /^-thujaplicin on fungi isolated. Fungi Fungal growth1 (mm) (identification by ITS sequence) Q ( C ( m t r o l ) 2 p p m ( m g / L ) g p p m ^ ^ ^ Basidiomycota Acanthophysium lividocaeruleum 31.5 (1.3)'a2 • 27.0 (1.3) b 23.8 (0.3) c 15.5 (0.9) d Cerinosterus luteoalba 5.7 (0.6) a 0.0 (0.0) b 0.0 (0.0) b 0.0 (0.0) b Hyphoderma praetermissum 13.8 (0.8) a 3.8 (0.8) b 0.0 (0.0) c 0.0 (0.0) c Pachnocybe ferruginea 7.1 (0.5) a 6.9 (0.6) a 7.0 (0.3) a 5.1(0.3)a Stereum sanguinolentum 39.0 (1.0) a 14.3 (1.2) b 0.8 (0.3) c 0.0 (0.0) c WRCF-B2 9.8 (0.3) a 8.1 (0.4) a 1.8 (0.3) b 0.0 (0.0) b WRCF-B4 (Hyphodontia raduld) 12.3 (0.6) a 0.0 (0.0) b 0.0 (0.0) b 0.0 (0.0) b WRCF-B5 (Butlerelfia sp.) 15.3 (0.6) a 0.8 (0.3) b 0.0 (0.0) b 0.0 (0.0) b WRCF-B7 (Phanerochaete sp.) 85.3 (0.6) a 23.0 (1.0) b 4.8 (0.3) c 0.0 (0.0) d WRCF-B9 (Phlebia sp.) 35.7 (0.6) a 32.7 (1.2) b • 2.8 (0.3) c 0.0 (0.0) d Ascomycota Oidiodendron griseum 3.2 (0.3) a 2.3 (0.3) a 2.0 (0.0) ab 0.0 (0.0) b Phialophora lignicola 10.7 (1.2) a 9.8 (1.0) ab 7.7 (1.3) b 0.0 (0.0) c Phialophora versicola 6.7 (0.3) a 5.2 (0.8) a 3.8 (0.3) b 0.0 (0.0) c Phialophora sp. 1 16.0 (1.0) a 15.7 (1.2) a 7.3 (1.5) b 0.0 (0.0) c Phialophora sp. 2 (P. fastigiata) 17.8 (0.3) a 1.7.2 (0.3) a 16.5 (0.5) ab 14.3 (0.6) b Phialophora sp. 3 7.3 (0.3) a 7.0 (0.0) ab 7.0 (0.5) ab 4.8 (0.3) b Phialophora sp. 4 9.7 (0.6) a 7.2 (0.3) b 0.0 (0.0) c 0.0 (0.0) c Rhinocladiella atrovirens 3.7 (0.6) a 2.8 (0.3) a 1.4 (0.4) b 0.0 (0.0) b Sporothrix sp. 1 11.7 (0.6) a 10.8 (0.8) ab 9.7 (0.6) b 0.0 (0.0) c Sporothrix sp. 2 13.0 (0.5) a 11.8 (0.3) a 9.8 (0.3) b 0.0 (0.0) c WRCF-A1 8.7 (0.6) a 7.2 (0.3) ab 5.0 (0.5) b 0.0 (0.0) c WRCF-A2 (Oidiodendron sp.) 31.7 (0.6) a 31.2 (0.3) a 26.8 (0.3) b 8.8 (0.3) c WRCF-A3 (Phialocephala sp.) 10.8 (0.8) a 6.8 (0.3) b 1.8 (0.3) c 0.0 (0.0) c Fungal growth was measured 12 days after exposure on M E A . Values are mean of three replicates and standard error in parenthesis;2 Numbers followed by the same letter in each row are not significantly different (a = 0.05) according to the Duncan's method. 144 and, by detoxifying them, permit other ascomycetous and basidiomycetous fungi to become established. These suggestions agree with the conclusions of Findlay (1966), Chesters (1950), Meredith (1960) and Shigo (1967) on W R C logs and fallen trees, and of Van der Kamp (1975) on standing trees. They showed that heartwood is invaded by a succession of fungi that allow decay to occur. Later, Jin et al. (1988) demonstrated that a pioneer Sporothrix fungus that was consistently isolated from the outer heartwood in W R C tree transformed thujaplicins into thujin, which was nontoxic to decay fungi. AI.5 Conclusions Complementing molecular techniques with traditional morphology based methods greatly increased the accuracy and speed of fungal species identification. Compared to earlier studies on W R C wood products, a variety of different types of wood-rotting basidiomycetous fungi were isolated from W R C fence materials. Identified Homobasidiomycetes in the present study were corticioid fungi, which cause white rot, and most of them were not commonly associated with W R C products. The isolates' tolerance to /^thujaplicin, as well as location and frequency of isolation may provide evidence of pioneer species involved in a succession of fungi that ends in decay of W R C products. Two basidiomycetous species: Pachnocybe ferruginea and Acanthophysium lividocaeruleum; three ascomycetous, soft-rot fungal species: Oidiodendron sp. ( W R C F - A 2 ) , Phialophora fastigiata (Phialophora sp. 2), and Phialophora sp. 3 might be pioneer fungi in W R C fence decay. These findings contrast with previous research that suggested 145 basidiomycetes generally followed ascomycetous, soft-rot fungi (Butcher, 1968; Corbett and Levy, 1963; Duncan, 1960). 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Aureobasidium pullulans A. conidia; B. dark arthroconidia; C. dark hyphae; D. conidial apparatus. Hormonema dematioides Hormonema dematioides; A. dark hyphae; B. conidia; C. fertile hyphae. 153 Appendix III ANOVA Source Tables Displayed below are the analysis of variance ( A N O V A ) tables following statistical analysis of data reported in each chapter of this thesis. Each table is accompanied by a brief description of contents. A l l experiments followed a completely randomized design (CRD) with treatments denoted as k and replicates as n. A one-way analysis of variance ( A N O V A ) (a = 0.05) and Tukey's test for comparison of means (Tukey, 1949) were performed with this experimental design. Treatment effects were considered significant at the 95% significance level i f the resulting critical F value was greater than the appropriate tabular value (Fp.i)], [(k(n-\)])- A l l statistical analyses were performed using J M P IN software (version 4.0.3 (academic), S A S Institute Inc., Cary, North Carolina). Note: D F = degrees of freedom; W R C - F S = western redcedar feeder strips. 154 AIII. 1 A N O V A Tables Relating to Chapter 2: Extracting and Quantifying Western Redcedar (Thuja plicata Donn) Heartwood Extractives Using Ultrasonication and Reverse Phase H P L C AIII. 1.1 Comparing Response Factors Among Extractive Compounds Source D F Sum of Squares Mean Square F Ratio Extractive compound 3 31.005 4.429 205.6 Error. 16 0.344 0.022 Total 23 31.349 AIII. 1.2 The Effect of Various Extraction Methods on Extractive Concentration (p.g/g DW) Table A3.2: (-)-Plicatic acid Source D F Sum of Squares Mean Square F Ratio Extraction Method 2 Error 21 Total 23 102553112 3850608 106403721 51276556 183362.3 279.6 Table AIII.3: y-Thujaplicin Source D F Sum of Squares Mean Square F Ratio Extraction Method 2 Error 21 Total 23 1072062 84057.5 1156119.5 536031 4003 133.9 Table AIII.4: /?-ThujapIicin Source D F Sum of Squares Mean Square F Ratio Extraction Method 2 Error 21 Total 23 2646601.5 81432.1 2728033.6 1323301 3878 341.3 Table AIII.5: /?-ThujapIicinoI Source D F Sum of Squares Mean Square F Ratio Extraction Method 2 Error 21 Total 23 56513.2 8313.1 64826.2 28256.6 395.9 71.4 155 Table AIII.6: Thujic acid Source D F Sum of Squares Mean Square F Ratio Extraction Method 2 . 16421174 8210587 162.3 Error 21 1062525 50596 Total 23 17483699 Table AIII.7: Methyl thujate Source D F Sum of Squares Mean Square F Ratio Extraction Method 2 5686.2 2843.1 31.9 Error 21 1867.8 88.9 Total 23 7554 AIII.2 A N O V A Tables Relating to Chapter 3: Isolating and Testing Fungi Tolerant to Western Redcedar (Thujaplicata Donn) Extractives AIII.2.1 Extractive Concentrations (pg/g) Versus W R C Feeder Strip Storage Conditions Table AIII.8: (-)-Plicatic acid Source D F Sum of Squares Mean Square F Ratio W R C - F S Storage Condition 2 76732233 Error 15 15999464 Total 17 92731697 38366117 1066630.9 35.9 Table AIII.9: y-ThujapIicin Source D F Sum of Squares Mean Square F Ratio W R C - F S Storage Condition 2 4458827.4 Error 15 421494.2 Total 17 4880321.6 22294.1 28100 79.3 Table AIII.10: /7-Thujaplicin Source D F Sum of Squares Mean Square F Ratio W R C - F S Storage Condition 2 3136438.7 Error 15 267602.8 Total 17 3404041.5 1568219 17840 87.9 156 TableAIII.il; /7-Thujaplicinol Source D F Sum of Squares Mean Square F Ratio W R C - F S Storage Condition 2 138023.51 69011.8 28.4 Error 15 36507.1 2433.8 Total 17 174530.6 le AIII.12: Thujic acid Source D F Sum of Squares Mean Square F Ratio W R C - F S Storage Condition 2 43566181 21783090 44.6 Error 15 7331562 488770.8 Total 17 50897743 AIII.3 A N O V A Tables Relating to Chapter 4: Black Stain of Western Redcedar (Thuja plicata Donn) by Aureobasidium pullulans: the Role of Weathering AIII.3.1 The Effect of /?-Thujaplicin on the Growth of Black Staining Fungi: Table AIII. 13: Aureobasidium pullulans Source D F Sum of Squares Mean Square F Ratio /J-Thujaplicin concentration 5 10402.926 2080.59 324.6 Replicates 2 26.037 13.02 2.0 Error 46 294.852 6.41 Total 53 10723.815 D F Sum of Squares Mean Square F Ratio Table AIII.14: Hormonema dematioides Source /5-Thujaplicin concentration 5 Replicates 2 Error 46 Total 53 10999.648 2199.93 559.6 51.37 25.69 6.5 180.852 3.93 11231.87 157 AIII.3.2 The Effect of Weathering on the Concentration of Individual Extractives AIIL15: (-)-Plicatic acid Source D F Sum of Squares Mean Square F Ratio Weathering effect 3 324347588 108115863 68.2 Replicates 2 6347507 3173753.6 2.0 Error 30 47528915 1584297.2 Total 35 378224011 1.16: y-Thujaplicin Source D F Sum of Squares . Mean Square F Ratio Weathering effect 3 38801060 12933687 253.5 Replicates 2 73496 36747.927 0.7 Error 30 1530787 51026.249 Total 35 40405343 AIII.17: /7-Thujaplicin Source D F Sum of Squares Mean Square F Ratio Weathering effect 3 10203725 . 3401242 615.8 Replicates 2 1076 538 0.1 Error 30 165698 5523 Total 35 10370499 AIII.18: /7-Thujaplicinol Source D F Sum of Squares , Mean Square F Ratio Weathering effect 3 110846.4 36948.8 292.3 Replicates 2 195.58 97.8 0.8 Error 30 3792.78 126.4 Total 35 114834.75 AIIL19: Thujic acid Source D F Sum of Squares Mean Square F Ratio Weathering effect 3 146329522 48776507 397.8 Replicates 2 189457 94728.251 0.8 Error 30 3678423 122614.09 Total 35 150197401 158 AIII.3.2 The Degree of Colonization of Weathered Wood Surfaces by A. pullulans (Quantitative Assessment) AIII.20: Ponderosa pine sapwood Source D F Sum of Squares Mean Square F Ratio Weathered surfaces 3 10102.02 3367.34 194.4 Replicates 2 20.236 10.12 0.6 Error 66 1143 17.32 Total 71 11265.256 AIIL21: W R C heartwood Source D F Sum of Squares Mean Square F Ratio Weathered surfaces 3 1444.2095 481.403 25.7 Replicates 2 43.6553 21.828 1.2 Error 66 1235.157 18.714 Total 71 2723.0218 AIII.4 A N O V A Tables Relating to Chapter 5: Effects of Leaching on Fungal Growth and Decay of Western Redcedar (Thuja plicata Donn) AIII.4.1 The effect of leaching on extractive concentration: Table AIII.22: (-)-Plicatic acid Source D F Sum of Squares Mean Square F Ratio Leaching effect 1 122669190 122669190 850.3 Error 10 1442675 144267.53 Total 11 124111865 Table AIII.23: y-Thujaplicin Source D F Sum of Squares Mean Square F Ratio Leaching effect 1 967317.2 967317 85.1 Error 10 113638.8 11364 Total 11 1080956.1 159 Table AIII.24: /7-Thujaplicin Source D F Sum of Squares Mean Square F Ratio Leaching effect Error Total 1 10 11 754914 98812.69 853726.69 754914 9881 76.4 Table AIII.25: /?-Thujaplicinol Source D F Sum of Squares Mean Square F Ratio Leaching effect Error Total 1 10 11 911153.61 248.86 911402.48 911154 25 36612.7 Table AIII.26: Thujic acid Source D F Sum of Squares Mean Square F Ratio Leaching effect Error Total 1 10 11 10204156 1597652 11801809 10204156 159765.24 63.9 AIII.4.2 The Effect of Leachate on Fungal Growth In Vitro Table AIII.27: Acanthophysium lividocaeruleum Source D F Sum of Squares Mean Square F Ratio Leachate concentration 5 52.007215 , 10.4014 56.9 Block (strains) 1 0.066736 0.0667 0.4 Error 29 5.30375 0.1829 Total 35 57.377701 e AIII.28: Coniophora puteana Source D F Sum of Squares Mean Square F Ratio Leachate concentration 5 79.32813 15.8656 13,3 Block (strains) 1 0.42612 0.4261 0.4 Error 29 34.69541 1.1964 Total 35 114.44965 160 Table AIII.29: Heterobasidion annosum Source D F Sum of Squares Mean Square F Ratio Leachate concentration 5 198.05652 39.6113 21.5 Block (strains) 1 39.2363 39.2363 Error 29 53.39911 1.8413 Total 35 290.69194 Table AIII.30: Pachnocybe ferruginea Source D F Sum of Squares Mean Square F Ratio Leachate concentration 5 1.997772 0.399554 12.1 Block (strains) 1 0.0054186 0.005419 0.2 Error 29 0.9557504 0.032957 Total 35 2.958941 Table AIII.31: Phellinus sulphurascens Source D F Sum of Squares Mean Square F Ratio Leachate concentration 5 86.464892 17.293 509.4 Error 12 0.407407 0.034 Total 17 86.872299 ) Table AIII.32: Phellinus weirii Source D F Sum of Squares Mean Square F Ratio Leachate concentration 5 101.14082 20.2282 169.7 Error 12 1.43056 0.1192 Total 17 102.57137 AIII.4.3 Estimation of the Degree of Decay (% mass loss) of Wood Blocks by Six Fungal Species of Interest Table AIII.33 Acanthophysium lividoeaeruleum Source D F Sum of Squares Mean Square F Ratio % Decay 2 117.9 58.9 1.39 Error 15 632.7 42.2 Total 17 750.7 111.34 Coniophora puteana Source D F Sum of Squares Mean Square F Ratio % Decay 2 5942.4 2971.2 94.14 Error 15 473.4 31.6 Total 17 6415.8' 161 Table AIII.35 Heterobasidion annosum Source D F Sum of Squares Mean Square F Ratio % Decay 2 361.3 180.7 2.25 Error 15 1200.3 80 Total 17 1561.7 Table AIII.36 Pachnocybe ferruginea Source D F Sum of Squares Mean Square F Ratio % Decay 2 21.1 10.5 0.83 Error 15 190.8 12.7 Total 17 211.8 Table AIII.37 Phellinus sulphurascens Source D F Sum of Squares Mean Square F Ratio % Decay 2 2900.2 1450.1 49.43 Error 15 440.1 29.3 Total 17 3340.3 Table AII1.38 Phellinus weirii Source D F Sum of Squares Mean Square F Ratio % Decay 2 767.4 383.7 7.39 Error 15 778 51.9 Total 17 1545.4 162 

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