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Growth, nutrition and genetic factors that affect pigmentation of wood-sapstain fungi Fleet, Carlos Antonio 2001

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GROWTH, NUTRITION AND GENETIC FACTORS THAT AFFECT PIGMENTATION OF WOOD-SAPSTAIN FUNGI by CARLOS ANTONIO FLEET B.Sc, University of British Columbia, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Wood Science, Faculty of Forestry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 2001 © Carlos Antonio Fleet, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date Abstract In this thesis, we examine several factors, including growth, nutrition and genetic identity, which affect pigmentation of sapstain fungi that discolour softwood in Canada from both chemical and molecular approaches. The presented results suggest that there are some chemical and physical factors to living host tissue that may stimulate growth and pigmentation by Ceratocystis resinifera. It was observed that reduced fungal growth on wood with closed border pits was concatenate with reduced consumption of wood nutrients. Nutrients in wood (such as mannose or TG-bound glycerol and fatty acids) play an important role in pigmentation and growth, but it appears that other factors, such as changes to wood ultrastructure or other biochemical factors, are also critical. Thus, some explanation for the differences in fungal distribution between logs and lumber may lie in the access that fungal species have to the host nutrients. Additionally, the presence of the D H N melanin biosynthesis pathway was demonstrated in all tested sapstain fungi using both chemical inhibitors (including tricyclazole, carpropamid and cerulenin) and molecular techniques. Furthermore, since no fungus has ever been found, to our knowledge, to have more than one melanin synthesis pathway, we can speculate with some confidence that the tested species only use the DHN pathway for melanin production. In addition, partial D N A sequences for the genes encoding scytalone dehydratase (SD), 1,3,8-trihyhydroxynaphthalene reductase (3HNR), 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR) and polyketide synthase (PKS) were obtained from species of Ceratocystis and Ophiostoma and found to have homology with known respective D H N biosynthesis gene sequences. Sequence analysis of the partial SD amino acid sequences showed greater than 80% similarity among the sapstain species, and corresponded well with known parsimony analyses of sapstain fungi based on rDNA sequences. Sequence analysis for the genes encoding 3HNR and PKS showed that these sequences had lower interspecies similarities than the gene encoding SD. It is anticipated that this information will contribute to ii the development of safe and effective means to control sapstain by both researchers and the forest products industry. iii Table of Contents Abstract ii Table of Contents iv List of Figures vii List of Tables ix List of Abbreviations x Acknowledgements xiv Chapter 1 General Introduction / 1.1 Forestry in Canada and British Columbia 1 1.2 Sapstain 2 1.2.1 Sapstain in Wood 2 1.2.2 Wood Protection 4 1.2.3 Sapstain Fungi 6 1.3 Melanin in Fungi 14 1.4 Project Objectives 18 Chapter 2 Pigmentation and Nutrition of Sapstain Fungi in Wood and Nutrient Media—19 2.1 Introduction 19 2.2 Materials and Methodology 22 2.2.1 Selection of sapstain fungi 22 2.2.2 Inoculation of lodgepole pine 22 2.2.3 Assessment of Fungal Growth in Wood 29 iv 2.2.4 Moisture Content 3 0 2.2.5 Sugar and Starch Analysis 30 2.2.6 Lipid Analysis 31 2.2.7 Nutrition and Pigmentation 32 2.2.8 Microscopy 33 2.2.9 Statistics 33 2.3 Results 34 2.3.1 Moisture content, fungal growth and stain in billets 34 2.3.2 Moisture content, fungal growth and stain in sawnwood 37 2.3.3 Soluble sugars and starch in wood 44 2.3.4 Lipids in wood 50 2.3.5 Pigmentation and growth in defined media 53 2.4 Discussion 56 2.5 Conclusions 60 Chapter 3 Genetic Analysis of DHN Melanin Genes in Sapstain Fungi 62 3.1 Introduction 62 3.2 Materials and Methodology 66 3.2.1 Fungal strains and growth conditions 66 3.2.2 Bacterial strains and growth conditions 67 3.2.3 DHN Pathway Inhibitor Studies 67 3.2.4 Purification of DNA molecules 68 3.2.5 Primer Design 69 3.2.6 Polymerase Chain Reaction conditions 71 3.2.7 Restriction Digests 71 v 3.2.8 Ligation, Cloning and Transformation of PCR Products 72 3.2.9 Gel electrophoresis 72 3.2.10 Southern Blotting — 72 3.2.11 Sequencing . 73 3.2.12 Phylogenetic analysis 73 3.3 Results 74 3.3.1 D H N inhibitors 74 3.3.2 PCR amplification and sequencing of D H N genes from fungal genomic D N A — 76 3.4 Discussion 93 3.5 Conclusions 99 Chapter 4 General conclusions and future work 101 References 105 vi List of Figures Figure 1-1: Examples of sapstain in Pinus contorta (A) log cross section and (B) lumber 3 Figure 1-2: D H N Melanin Pathway 16 Figure 2-1: Lodgepole pine billet experiment 25 Figure 2-2: Lodgepole pine sawnwood experiment methodology 27 Figure 2-3: Composite image of cross sections taken from P. contorta logs after 28 day infection with sapstain fungi 36 Figure 2-4: Testing for viable host parenchyma in cross section of P. contorta infected with sapstain fungi for 28 days 38 Figure 2-5A: Comparison of Merrit sawnwood blocks infected with A. pullulans (Ap), C. resinifera (Cc) and Leptographium spp. (L) for 28 days 39 Figure 2-6: Comparison of kiln-conditioned Edson sawnwood blocks infected with sapstain fungi for 28 days 42 Figure 2-7: Photomicrographs of P. contorta infected with C. resinifera C 43 Figure 2-8: Photomicrograph of bordered pits (indicated by red arrows) in P. contorta 45 Figure 2-9: Glucose (A) and mannose (B) concentrations in glucose:mannose (1:1, w/w) liquid media during incubation with selected sapstain fungi 55 Figure 3-1: Feature maps of C. lagenarium DHN melanin biosynthesis genes PKS1 (A), THR1 (B) and SCD1 (C) 64 Figure 3-2: Examples of effect of DHN melanin synthesis pathway inhibitors on different sapstain fungi 75 Figure 3-3: Agarose gel electrophoresis of PCR amplification of genomic D N A using primers PKS3 and PKS6 7 7 vii Figure 3-4: C L U S T A L W alignment of partial amino, acid sequence of the gene encoding PKS from different fungi 78 Figure 3-5: Unrooted phylogram of partial amino acid sequence alignment of the gene encoding PKS from different fungi 80 Figure 3-6: Agarose gel electrophoresis of PCR amplification of genomic D N A using primers T29F and T14R 82 Figure 3-7: Agarose gel electrophoresis of PCR-RFLP of selected colonies that hybridized in Southern blotting experiment 83 Figure 3-8: C L U S T A L W alignment of partial amino acid sequence of the gene encoding 3HNR from different fungi 85 Figure 3-9: Rooted phylogram of partial amino acid sequence alignment of the genes encoding 3HNR and 4HNR from different fungi 86 Figure 3-10: Agarose gel electrophoresis of PCR amplification of genomic D N A using primers SD1 and SD2 88 Figure 3-11: MultAlin alignment of D N A sequence of the intron from the gene encoding SD from different fungi 89 Figure 3-12: C L U S T A L W alignment of partial amino acid sequence of the gene encoding SD from different fungi 90 Figure 3-13: Rooted phylogram of partial amino acid sequence alignment of the gene encoding SD from different fungi 92 viii List of Tables Table 1-1: Summary of growth, mature conidial production and mature perithecia production in different nutrients by sapstain fungi in vitro (adapted from Kaarik 1960) 11 Table 2-1: Colour of fungal mycelia after 8 days of growth in liquid culture supplemented with different carbon and nitrogen sources. (Reproduced from Eagen et. al. 1997) 20 Table 2-2: Codes and source information of fungal species isolates 23 Table 2-3: Average longitudinal growth of fungi on P. contorta (Merrit) billets after 28 days....35 Table 2-4: Concentration (ppm) of soluble sugars in P. contorta (Merrit) infected with fungi. ...46 Table 2-5: Concentration (ppm) of soluble sugars in kiln-conditioned P. contorta (Edson) sawnwood infected with fungi for 14 and 28 days 48 Table 2-6: Concentration (ppm) of starch-derived glucose in kiln conditioned P. contorta (Edson) sawnwood infected with fungi for 14 and28 days 49 Table 2-7: Concentration (mg g"1 freeze-dried wood) of fatty/resin acid fractions in P. contorta (Merrit) sawnwood infected with fungi for 28 days 51 Table 2-8: Concentration (mg g"1 FD wood) of fatty/resin acids and triglycerides in kiln-conditioned P. contorta (Edson) sawnwood infected with fungi for 14 and 28 days 52 Table 2-9: Pigment score and growth diameter of selected isolates on tested carbon sources 54 Table 3-1: Oligonucleotide sequences and intended use 70 ix List of Abbreviations Numbers & Symbols 15-80MC sawnwood kiln dried to 15% moisture content, then re-hydrated to 80% moisture content 3HN 1,3,8-trihydroxynaphthalene 3HNR 1,3,8-trihydroxynaphthalene reductase 4HN 1,3,6,8-tetrahydroxynaphthalene 4HNR 1,3,6,8-tetrahydroxynaphthaIene reductase 80MC sawnwood kiln conditioned to 80% moisture content y gamma (radiation) pL microlitre A t o C A adenine aa amino acid A R A arabinose B L A S T basic local alignment search tool B L A S T N standard nucleotide-nucleotide B L A S T B L A S T X nucleotide query - protein database B L A S T bp base pair C degrees Celsius C cytosine Cc Ceratocystis resinifera cm centimetre D to F d day DG diglyceride D H N 1,8-dihydroxynaphthalene DMSO dimethyl sulfoxide D N A deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate DOPA dihydroxyphenylalanine x EtBr ethidium bromide F A fatty acid FA1 fatty alcohol G to K g gram G guanine G A L galactose GC gas chromatography GHB glutaiminyl-4-hydroxybenzene G L U glucose h hour HPLC high performance liquid chromatograp kg kilogram kPa kilopascal L to N L litre L B Luria-Bertani medium Lept Leptographium m meter M molar M13F pCR2.1 plasmid insert forward primer M13R pCR2.1 plasmid insert reverse primer M A N mannose M C moisture content M E A malt extract agar mg milligram M G monoglyceride min minute mL milliltre mm millimetre mM milllimolar Mrad megarad M T B E methyl-tert-butylether xi N A D P H P-Nicotinamide adenine dinucleotide phosphoric acid N A D P + P-Nicotinamide adenine dinucleotide phosphate O t o R Oflo Ophiostoma floccosum Opic Ophiostoma piceae Opil Ophiostoma piliferum Oset Ophiostoma setosum PCR polymerase chain reaction PKS polyketide synthase (general); pentaketide synthase (strict) PKS3 fungal polyketide synthase gene forward primer #3 PKS4 fungal polyketide synthase gene reverse primer #4 PKS6 fungal polyketide synthase gene reverse primer #6 ppm parts per million RA resin acid rDNA ribosomal deoxyribonucleic acid RFLP restriction fragment length polymorphism R H relative humidity rpm revolution per minute S t o T s sterol SD scytalone reductase SD1 fungal scytalone dehydratase gene primer # 1 SD2 fungal scytalone dehydratase gene primer #2 SE steryl ester sp species (singular) SPE solid phase extraction spp species (plural) T thymine T14R fungal 1,3,8-trihydroxynaphthalene reductase gene reverse primer #14 T29F fungal 1,3,8-trihydroxynaphthalene reductase gene forward primer #29 T31 fungal 1,3,8-trihydroxynaphthalene reductase gene forward primer #31 T33 fungal 1,3,8-trihydroxynaphthalene reductase gene forward primer #33 T42 fungal 1,3,8-trihydroxynaphthalene reductase gene reverse primer #42 xii T44 fungal 1,3,8-trihydroxynaphthalene reductase gene reverse primer #44 Taq Taq D N A polymerase TE total acetone extractives (in Chapter 2); Tris-EDTA buffer (in Chapter 3) TG triglyceride TTC 2,3,5-triphenyl-2H-tetrazolium chloride U t o Z U unit W wax X Y L xylose xiii Acknowledgements I would like to thank all those helped me along the way to make this possible. First, I would like to thank my supervisor and mentor, Dr. Colette Breuil. I would also like to thank the rest of my committee, Drs. Brian Ellis and Louis Bernier. Many thanks are also owed to the staff at Forintek Canada, especially Tony Byrne and Adnan Uzunovic. I would never have been able to finish this degree without the help and friendship of the members of the FPB group. I would especially like to thank my friends Rob Leone, Derrick Stebbing, Peter Loppnau and last but not least, Brad Hoffman, who aside from being one of my best friends, helped me a great deal with the molecular aspect of this project. I would also like to thank the PDFs, Seong Kim, Tao Chen and Alessio Serreqi. Aside from teaching me everything I know about GC and HPLC, Alessio also taught me one of the most important skills for grad school... how to play Windows Solitaire. I also owe thanks to Dominik Domanski, Jennifer Evans and Isabella Gadawski who assisted me in my work. I would also like to thank my friend, Jason Malloff, for all the needed breaks. A huge thanks goes to my parents, who always encouraged my desire for education and taught me most of what I needed to know for living life. Finally, my greatest thanks goes to my wife, Judy, who has endured my student lifestyle of the last few years and taught me some very important lessons of love. This work was funded by a Natural Science and Engineering Research Council of Canada strategic grant. Personal financial support was funded by a B.C. Science Council Graduate Research Engineering and Technology award with industrial support from Forintek Canada Corporation. Chapter 1 General Introduction In this thesis, we examined several factors, including growth, nutrition and genetic identity, which affect pigmentation of sapstain fungi that discolour softwood in Canada from both chemical and molecular approaches. This chapter introduces the problem of sapstain and its impact on the forest products industry. It then follows with a description of the fungi that cause sapstain and the known metabolic pathways for pigment production. The second chapter describes the chemical analysis of nutrient consumption by sapstain fungi in wood substrates and artificial media, followed by an examination of the impact of different nutrients on pigmentation. Chapter 3 describes the investigation of the pigmentation pathways of sapstain fungi. First, inhibitor studies were carried out to demonstrate the existence of a known melanin pathway in sapstain fungi. This is followed by the molecular analysis of three genes that code for enzymes involved in melanin synthesis in these fungi. The fourth chapter summarizes the main outcomes of this thesis and proposes some considerations for future work. 1.1 Forestry in Canada and British Columbia The forest products industry plays a critical role in the national and provincial economies. In 1999, Canada produced 68.2 x IO6 m 3 of softwood lumber, which amounted to 13.5% of the total world softwood harvest. British Columbia produced 31.8 x 106 m 3 of softwood lumber or 6.6% of world harvest. Canada's total wood products production was valued at $62.8 billion of which $44.2 billion were exports. Canada is also the largest single softwood exporter in the world, accounting for 47.8% of the world softwood export volume while BC alone accounted for 25.5% of world export volume in 1999. In BC, lumber (non- pulp & paper) exports accounted for $12.2 V 1 billion toward the balance of trade. A l l data were obtained from Council of Forest Industries Fact Book 2000 (COFI 2000). The largest customers for the BC wood product market (non- pulp & paper) are the USA at 59%, the rest of Canada at 22.1%, Japan at 14.4%, European Union at 2.6% and others at 1.9% (COFI 2000). The size of the export market emphasizes the need for sustaining a steady source of wood products while maintaining a high quality product free of defects. These values underscore the enormous importance of the softwood industry to the citizens of B C and Canada. 1.2 Sapstain 1.2.1 Sapstain in Wood Sapstain, also known as blue stain, is a cosmetic discolouration of sapwood caused by wood-inhabiting fungi that can affect both logs and lumber (Figure 1-1). The discolouration is typically black, grey or blue depending on the host wood and infecting fungal species, and can vary widely in intensity and subjective appearance (Seifert 1993). Sapstain and the fungi that cause it, pose some important problems for the forest products industry, both in Canada and worldwide. Most importantly, when wood is discoloured by sapstain, its economic value can be reduced significantly simply based on its cosmetic appearance. Although the wood is usually structurally sound, its appearance may not suit its final use or the customer may incorrectly assume that the wood is infected with wood decay fungi. Further, some sapstain species have also been implicated in contributing to the decline and death of standing trees by bark beetle attacks. (Seifert 1993). 2 Figure 1-1: Examples of sapstain in Pinus contorta (A) log cross section and (B) lumber. White bars indicate 10cm. 3 Furthermore, there is an emerging global awareness of the problems associated with the international trade of goods; namely, the import of foreign species, pathogens and diseases (Filip and Morrell 1996, CFS 1999). Sometimes, when foreign species are introduced into a new environment, they may fill an ecological niche resulting in damage to the existing ecosystem. One example includes the accidental introduction of Ophiostoma ulmi, the causative agent of Dutch elm disease, to North America in the early 1930s on European elm logs infected with a bark beetle vector. Over the decades, this has lead to the near elimination of the American elm, which possesses little resistance to O. ulmi, in North America (Alexopolous et. al. 1996). With this and many other recent examples in mind, some wood-importing nations, including Canada and the United States, are attempting to place strict controls and rules regarding the import of wood infected with fungi or other parasites (USDA 1995, CFS 1999). Thus, shipments of sapstained wood may be unacceptable to a foreign customer and thus unmarketable. The economic losses due to the cosmetic and trade problems associated with sapstain are difficult to estimate due to the complexities of the global wood market. However, one benchmark is the capital spent on the prevention of sapstain by the industry. In 1995, the estimated worth of the anti-sapstain chemical market in Canada was between $16 and $20 million with about 3 billion board feet of lumber (valued at $2.9 billion) treated in BC alone (Abraham 1995). One recent study by Forintek Canada Corp estimated that some BC coast mills each spend as much as $5 million per year in anti-sapstain chemicals and treatments (A. Byrne, Personal Communication). 1.2.2 Wood Protection Of the protective methods currently in use to prevent wood discoloration in lumber, the two most common are kiln drying and chemical treatments. Ki ln drying is used to reduce the moisture content of wood to below 20%, the critical limit for fungal growth. However, kiln drying is 4 costly; it can cause wood defects in large timbers and ih certain species; and thus it is only feasible where wood species and markets factors permit (Nielson 1978, Seifert 1993). Further, while kiln dried wood is considered protected from stain and decay, that protection is only effective if the dried wood is not re-wet during storage and transport. Large proportions of softwood exports are green (non- kiln dried) and are commonly protected against fungi by chemical treatments. With the termination of some chemicals due to their broad toxicity, such as pentachlorophenol (PCP), narrow-spectrum fungicides have been substituted. Six fungicides are currently registered for sapstain prevention by Agriculture Canada: azaconazole, borax or borate, sodium carbonate (Na2CC>3), didecyldimethylammonium chloride (DDAC), 3-iodo-2-propynylbutylcarbamate (IPBC), and 2-(thiocyanomethylthio) benzothiazole (TCMTB). In 1995, the major anti-sapstain active ingredients identified by the BC Ministry of Environment Lands and Parks were DDAC, borax or borate, Na2C03, and IPBC. The active ingredients TCMTB and azaconazole constituted a minor portion of total use due to worker safety concerns (BC Ministry of Environment 1997). While safer than PCP, these alternative chemicals are less effective than PCP against sapstain and still present significant environmental persistence and toxicity concerns towards microbial, invertebrate and aquatic life (Bull 1997 Chapter 1). Further, due to these concerns, none of these chemical treatments can be applied at the most effective step in the wood handling process, i.e. the harvest site. Maintaining future sales and exports will require addressing new government regulations on anti-sapstain chemicals and public antipathy towards chemical treatments of products. These issues can be addressed by developing more environmentally acceptable products and technologies that target fungal growth or fungal pigmentation. A more recent approach to sapstain protection has been the use of biological control agents and perhaps coupling these agents with chemical treatments (Seifert et. al. 1988, Freitag et. al. 1991, 5 Behrendt et. al. 1995a, Yang and Rossignol 1999). It is important that the biological control agent only consumes non-structural wood nutrients and does not lead to wood decay. In the case of sapstain, biological control has been tested with a pigment-free mutant of a sapstain fungus (Cartapip97™ described in section 1.2.3.4.2 below) that when inoculated onto wood, establishes itself in the wood ahead of the detrimental sapstain fungi. Beyond laboratory or field settings however, studies have rarely demonstrated biological control to work as effectively as conventional chemical control methods (Behrendt et. al. 1995b, Whitemcdougall et. al. 1998, Dawson-Andoh and Lovell 2000). This has been attributed to the lack of information about the complex interactions between the biological control agent, the pest organisms, and the wood substrate on which they must compete (Uzunovic et. al. 1999a). In addition, wood purposely infected with a fungal biological control agent may be subject to import/export restrictions described in section 1.2.1 above. 1.2.3 Sapstain Fungi Sapstain, or blueing, fungi were first described nearly one hundred years ago by Munch in 1907 and have since been studied over the last century due to their importance to the wood products industry (Munch 1907, Lagerberg et. al. 1927, Nisikado and Yamauti 1935, Kaarik 1960, Wingfield et. al. 1993). As noted by Siefert (1993), sapstain is generally caused by three groups of fungi. These are: 1. Species of Ceratocystis, Ceratocystiopsis, and Ophiostoma. 2. Black yeasts including Aureobasidiumpullulans, Hormonema dematiodes and Phialophora spp. 3. Dark moulds including Alternaria alternata and species of Cladosporium. 6 The first group (also known as Ophiostomatoids) are generally regarded as deep stainers since their hyphae penetrate deep into the sapwood, causing significant discolouration throughout the wood. The Ophiostomatoids Ceratocystis and Ophiostoma are considered the most economically important to the wood products industry and are thus the main emphasis of this thesis. A detailed and thorough review of the taxonomy, ecology and pathogenicity of these two genera is presented in the book, Ceratocystis and Ophiostoma (Wingfield et. al. 1993) The final two groups (black yeasts and dark moulds) are generally regarded as surface stainers because they grow on exposed sapwood and their superficial stain damage can be removed by planing a few millimetres off the wood surface (Uzunovic and Webber 1998). 1.2.3.1 Taxonomy The taxonomy of Ophiostomatoid fungi has been debated over the decades with two main camps of thought based on morphological characteristics. The first camp categorized Ceratocystis and Ophiostoma as congeneric while the second camp considered them as two distinct genera (Samuels 1993, Upadhyay 1993). Since the 1970s, numerous studies have supported the separation of the two genera. Carbohydrate analysis of the cell walls of these fungi showed that in Ceratocystis, rhamnose and cellulose were absent, whereas these carbohydrates were present in Ophiostoma (Weijman and de Hoog 1975). Further, a study of antibiotic resistance of sapstain fungi showed that Ceratocystis species do not grow in the presence of cycloheximide while Ophiostoma species are resistant to the antibiotic (Harrington 1981). Finally, genetic analyses of the rDNA sequences of these species support the phylogenetic distinction of these genera (Hausner et. al. 1993, Spatafora and Blackwell 1994, Hausner et. al. 2000). Today, the consensus taxonomic structure for the genus Ceratocystis is: family Incertae sedis, order Microascales, class Incertae sedis, phylum Ascomycota, kingdom Fungi. The consensus taxonomic structure for the genus Ophiostoma is: family Ophiostomataceae, order 7 Ophiostomatales, class Incertae sedis, phylum Ascomycota, kingdom Fungi (CABI FUNINDEX 2001). The anamorph taxonomy of these genera is also a controversial area and has recently met re-examination by several researchers. Currently, Chalara is considered an anamorph of Ceratocystis while Graphium and Ambrosiella are considered anamorphs of both Ceratocystis and Ophiostoma. However, recent phylogenetic studies using 18S rDNA have refuted Graphium as an anamorph ic group of Ophiostoma (Okada et. al. 1998, Okada et. al. 2000). Leptographium is also considered an anamorph of Ophiostoma and possibly other species. Other anamorphs of Ophiostoma include Pesotum and Sporothrix. Other anamorphs of Ceratocystis include Acremonium (Wingfield 1993a & 1993b, Harrington and Wingfield 1998, Hausner et. al. 2000, CABI FUNINDEX 2001). In the sexual cycle, both Ophiostoma and Ceratocystis possess spherical ascomata with extended perithecial stems and asci produced in a scattered manner from an ill-defined hymenium. When the ascus wall liquefies, the ascospores are released into the gelatinous matrix within the central cavity of the perithecium. When mature, the asci are then discharged through the ostiole of the perithecium to form a spore droplet (adapted from Spatafora and Blackwell 1994). The dimensions, shapes and colours of the morphological features vary between the species and are often used to distinguish the species groups. 1.2.3.2 D i s p e r s a l Species of Ceratocystis and Ophiostoma include both pathogens and saprobes. Historically, most sapstain species were regarded as saprophytic and only associated with harvested wood and not with live, standing trees. This was given since most diseased trees are generally sorted at the harvest site and not introduced to downstream sawmills or other manufacturing sites. However, recent examinations have shown that some, if not most sapstain species are capable of growing in 8 standing trees, generally in conjunction with other pests. Suspected species include O. clavigerum and O. minus. It should also be noted that the debate of saprophytic versus pathogenic sapstain fungi also depends on the strictness of the definition of these terms and the degree of overlap between them. At both the tree harvest site and sawmills, the major vectors for infection of wood tissue are: beetle species and other insects, which carry asexual and/or sexual fungal spores on their exoskeletons; air or water dispersal; harvesting damage to the bark; and manual post-harvest contamination (Harrington 1993, Malloch and Blackwell 1993, Siefert 1993, Harrington and Wingfield 1998, Uzunovic et. al. 1999a). Some pathogenic species have strong symbiotic associations with bark beetles. For example, the fungus C. polonica assists the European spruce bark beetle (Ips typographus) to kill healthy trees of Norway spruce (Piceae abies) (Harrington and Wingfield 1998, Krokene and Solheim 1997 and 1998). It is suggested that sapstain fungi also have symbiotic, albeit weak, relationships with their arthropod vectors (Malloch and Blackwell 1993, Klepzig 1998, Krokene and Solheim 1998). Fungal spores may also be spread by other animal vectors such as mites and nematodes, though there is currently little information in this area. 1.2.3.3 Distribution Detailed and systematic surveys of the prevalence and distribution of sapstain fungi in different geographical regions are generally scarce (Campbell 1960, Griffin 1968, Olchowecki and Reid 1974, Kaarik 1980, Seifert and Grylls 1993). This has been attributed to confusing taxonomy and difficulty in carrying out an extensive and organized sampling survey, especially for large and ecologically diverse countries like Canada. Recent collaborative work between the University of British Columbia, Universite Laval and Forintek Canada Corporation examined in more detail which sapstaining fungi were involved in staining logs and lumber of different softwoods in Canada (Uzunovic et. al. 1999b). The investigators surveyed seven sawmills across Canada and 9 in each mill, sawn timber and fresh logs were set aside, and sampled after one month. Among the 1863 fungal isolates, 13 different sapstain species were recognised. The study demonstrated that no sapstain species was specific to any of the geographical regions in Canada, or to specific wood species. However, C. resinifera was almost exclusively isolated from fresh logs, whereas other species such as O. piceae were isolated more frequently from sawn lumber. 1.2.3.4 Nutrition Most mycelial fungi acquire nutrients from their environment by secreting extracellular enzymes to break down larger molecules, then absorbing the smaller units. Sapstain fungi generally only utilize non-structural wood components such as lipids, soluble carbohydrates, starch and proteins. They do not cause bio-deterioration of wood since they generally have no or incomplete celluloytic and/or lignolytic activities (Nilsson 1973, Siefert 1993). A limited amount of research has investigated nutrient consumption of sapstain fungi, both in vitro and in vivo. 1.2.3.4.1 In vitro Several pioneering studies have been carried out on the growth of sapstain fungi in artificial media, though most were mainly concerned with culture maintenance and sporulation and focused on a narrow range of species on rich media. Kaarik carried out an extensive investigation of the effects of various vitamins, carbon & nitrogen sources and pH on 24 species of wood-inhabiting fungi (Kaarik I960). Generally, it was found that most species grew and produced conidia on a wide range of carbon sources, though the production of perithecia was more particular to certain carbon sources depending on the species and/or strain. A summary of growth and sporulation for three selected species on different carbon sources is shown in Table l -1. Furthermore, it was found that Ophiostoma spp.: grew and sporulated best on high 10 Table 1-1: Summary of growth, mature conidial production and mature perithecia production in different nutrients by sapstain fungi in vitro1 (adapted from Kaarik 1960) Nutrient C. virescens2 O. floccosum 0. piceae Growth Conidia Perithecia Growth Conidia Perithecia Growth Conidia Perithecia D-xylose + + - + + - + + -L-arabinose + + + + + - + + -D-ribose + + - + + - + + -L-rhamnose + + + + + - + + -D-glucose + + - + + - + + + D-fructose + + - + + - + + + D-mannose + + - + + - + + + D-galactose + + - + + - + + + L-sorbose + + - + + - + + -Maltose + + - + + - + + + Saccharose + + + + + - + + + Lactose + + + + + - + + + Cellobiose + + - + + - + + -Raffinose + + - + + - + + -Starch + + + + + - + + -Cellulose + + + + + - + + -Inulin + + - + + - + + -Glycogen - - - + + - + + -D-sorbitol + + + + + - + + + D-mannitol + + + + + - + + -Inositol + + + + + - + + -Erythritol + + - + + - + + + Glycerol + + - + + - + + -Citrus pectin + + - n/a3 n/a n/a + + -L-malic acid - - - n/a n/a n/a + + -Citric acid - - - n/a n/a n/a + + -Oleic acid + + — n/a n/a n/a + + -1. All growth media included vitamin stock. 2. Formerly O. coerulescens 3. +: present; -: absent; n/a: no experiment 11 carbon/nitrogen ratio media; had an optimum pH between 3.5 and 6.5; and were generally deficient in one or more of the vitamins thiamine, pyridoxine and biotin (Kaarik 1960). Lagerberg et. al. (1927) determined the optimal temperatures for growth for Ceratocystis coerulea (formerly Ceratostomella coerulea) and Ophiostomapiceae (formerly Ceratostomella piceae) to be 22C with observable growth between the tested ranges of 6 - 27C. Free oxygen is also a critical requirement for sapstain growth, as evidenced by most sapstain fungi's inability to grow in aerobic conditions, including water-saturated logs (Lagerberg et. al. 1927, Nisikado and Yamauti 1935). 1.2.3.4.2 In vivo Most in vivo studies have been limited to observations of fungal growth and host cell penetration and the factors that affect them (Lagerberg et. al. 1927, Siefert 1993). One of the most important factors for in vivo growth is the moisture content of the wood. Depending on the species, fungal growth will occur between 20 and 160 percent moisture content (based on oven-dry method), with an optimum of 60 to 80 percent. However, growth is greatly reduced below 35% and above 120% moisture content due to the low amount of free water and low concentrations of oxygen, respectively (Lagerberg et. al. 1927, Siefert 1993). Since sapstain fungi are mesophiles, temperature is also a critical parameter for growth, with optimum growth in wood between 15 and 30C, though slow growth may occur at lower and higher temperatures. Furthermore, most sapstain species cannot grow at temperatures above 40 to 50C. Another important factor is ambient humidity. It has been found that sapstain growth is enhanced at relative humidity above 90%, though they can survive a wide range of humidity for long periods of time (Lagerberg et. al. 1927, Gibbs 1993, Siefert 1993). 12 Little work has been carried out on nutrient utilization by sapstain fungi in wood tissue and this has lead to a serious deficiency in understanding the biology of these organisms (Gibbs 1993, Siefert 1993). Pioneering studies found that Ceratocystis coerulea (formerly Ceratostomella coerulea) and Ophiostoma piceae (formerly Ceratostomella piceae) growing on Pinus sylvestris (Scots pine) and Picea abies (Norway Spruce) could consume starch and fats in wood. The information was sparse and the tests were done qualitatively with specific dyes for starch and fats (Lagerberg et. al. 1927). However, some recent studies have lead to some insights. In a strain of O. floccosum (strain renamed from O. piceae) triglycerides and fatty acids decreased by 75 and 60 percent, respectively, after a two-week incubation on P. contorta sapwood (Gao et. al. 1994). This study also found that the fatty acid content was temporarily increased due to the decrease of triglycerides. In further studies, it was found that this strain also produced an extra-cellular lipase that was shown to degrade wood triglycerides (Gao and Breuil 1995, Gao 1996). This species was also shown to produce an extra-cellular serine protease that was purified and shown to cleave proteins isolated from wood. Polyclonal antibodies were raised against the protease and used to localize the enzyme to the cell wall and extra-cellular sheath (10 daltons thick) of the fungus when grown on four different softwood species (Abraham 1995, Abraham and Breuil 1996, Gharibian et. al. 1996). There have also been some investigations on lipid consumption by a mutant strain of O. piliferum. These studies have been concerned with the prevention of pitch deposits (sticky colloidal lipid particles that bind to machinery and paper products) in pulp mills using an albino mutant of the sapstain fungus Ophiostoma piliferum, commercialized as Cartapip97™ (also described in section 1.2.2 above). In these experiments, after incubation of Southern Yellow pine (Pinus taeda and P. virginiana) wood chips for two weeks with Cartapip97™, triglycerides and fatty acids were reduced by 75 to 100 percent, resulting in downstream pitch reductions of 20 to 40% (Blanchette et. al. 1992, Brush et. al. 1994). 13 1.3 Melanin in Fungi Melanin is a ubiquitous, dark coloured macromolecule composed of various phenolic or indolic polymers, often complexed with proteins and carbohydrates. The conditions for defining a pigment as a melanin include: black colour, insolubility in water (cold or hot), resistance to acidic degradation, bleachability by oxidizers such as H2O2, and solubility & degradation in hot alkali solutions (Butler and Day 1998). Such recalcitrant properties have lead to significant difficulties in chemical analysis of these pigments from their natural sources. The corollary of this is that melanins are generally difficult to define in a precise manner in their natural and intact state. It is known that certain fungi, such as Ophiostoma spp., produce darkly pigmented cell walls when growing in pine or aspen sapwood, and little or no pigmentation when growing in other woods, such as Western hemlock (Tsuga heterophylla) (Kreber and Byrne 1996). However, very little work has been done to evaluate the actual process of fungal pigmentation in wood of economic importance to the forest products industry. Most of the pigments have been described as melanins, but their mechanism of production and means of attachment to the fungal cell wall remain unknown (Zink and Fengel 1988, 1989, 1990; Brisson et. al. 1996,). In microscopic and chemical studies of C. coerulescens, it was shown that melanin was deposited in the hyphal walls in the form of globular granules. This occurred as early as one day into the growth of the fungus and as the cells aged, their hyphal wall structure became lumpy in appearance due to the melanin globules (Zink and Fengel 1988, 1989, 1990). These melanin globules are thought to arise from fungal cell organelles since there is no evidence of melanin synthesis in the cytoplasm of sapstain fungi (Zink and Fengel 1989). Today, the consensus is that sapstain fungi produce melanin as their main source of pigmentation, though other pigmentation pathways may exist (Butler and Day 1998). 14 There are three known pathways for melanin synthesis in fungi: GHB (glutaniminyl-4-hydroxybenzene), Catechol, and D H N (1,8 dihydroxynaphthalene) (Bell and Wheeler 1986, Butler and Day 1998). For many years, it was believed that there was another fungal melanin synthesis pathway, the DOPA (dihydroxyphenylalanine) pathway found in mammals. This was because many studies showed that fungi could produce melanin if the precursor, DOPA, was supplied to their growth media. However, DOPA is known to spontaneously polymerize to melanin in alkaline conditions or in the presence of many fungal enzymes such as laccases and polyphenoloxidases (Butler and Day 1998). Thus, evidence has eroded regarding the presence of the DOPA pathway in the fungal kingdom. However, most melanin research has been focussed on mammalian DOPA melanin (due to its role in human diseases such as Parkinson's and skin cancer), though little information on the physical and chemical properties of DOPA melanin would be applicable to microbial melanins (Butler and Day 1998). The GHB pathway has only been found in the reproductive cell walls of the basidiomycetes, whereas the Catechol pathway has only been described in the basidiomycetes smut fungi, Ustilago sp. (Butler and Day 1998). In Ascomycetes and related Deuteromycetes, melanins are generally synthesised via a pentaketide pathway, with DHN as the intermediate precursor to melanin (Wheeler 1983, Kubo et. al. 1986). The DHN melanin pathway has been targeted for research in phytopathogenic fungi due its role in appressorial development of the hyphae. It appears that melanin concentrates turgor pressure (to as high as 8000 kPa) in the penetration peg of the appressorium, allowing the hyphae to penetrate host plant cell walls (Butler and Day 1998). D H N melanin also seems to play a role in the virulence of the human pathogen fungi, though information in this area is vague (Butler and Day 1998). 15 NADPH NADP+ / v O H o r 4HNR /KstK SD H O ^ ^ ^ ^ ^ O H H O ' H Z ° O H O H 4HN A Scytalone r Ti I 3HN PKS o o C H 3 O ^ ^ C O O H o Pentaketide PKS H-C. .CoA Y o Acetyl CoA NADPH NADP+ 3HNR I O O H H O Vermelone SD MELANIN t O H O H DHN Figure 1-2: D H N Melanin Pathway. Enzymes are indicated in italics. PKS: polyketide synthase; 4HNR: 1,3,6,8-tetrahydroxynaphthalene reductase; SD: scytalone dehydratase; 3HNR: 1,3,8-trihydroxynaphthalene reductase; DHN: 1,8-dihydroxynaphthalene; NADPH: P -Nicotinamide adenine dinucleotide phosphate; NADP+: P-Nicotinamide adenine dinucleotide phosphoric acid. (Adapted from Thompson et. al., 2000) 16 To demonstrate that the DHN pathway is used by fungi to produce melanin, inhibitors to enzymes of this pathway or mutants that accumulate intermediates have been used. However, none of the shunt products or branch products resulting from mutations are available commercially and must be purified as required, resulting in considerable challenges to research (Wheeler 1983, Kubo et. al. 1986, Butler and Day 1998). The pathway, shown in Figure 1-2, is thought to utilize four key enzymes: polyketide synthase (PKS); 1,3,6,8-tetrahydroxynapthalene reductase (4HNR); scytalone dehydratase (SD); and 1,3,8- trihydroxynapthalene reductase (3HNR). There has been some confusion over the nomenclature of the reductases due to the fact that they have overlapping activities thus leading to the conclusion that there was only one reductase (Thompson et. al. 2000). It is currently unclear whether species that were described as having a single known reductase, have in fact, two. This document adopts the standard nomenclature set forth by Thompson et. al. (2000). The pathway starts with the enzyme PKS, which polymerizes and cyclizes acetyl-CoA to generate 1,3,6,8-tetrahydroxynapthalene (4HN). PKS is inhibited by the compound cerulenin, which also inhibits other polyketide synthase enzymes such as fatty acid synthase (Butler and Day 1998). The enzyme 4HNR reduces 4HN to scytalone and is inhibited by the compounds tricyclazole, pyroquilon and phthalide, among others (Butler and Day 1998, Thompson et. al. 2000). SD converts scytalone to 1,3,8- trihydroxynapthalene (3HN) and is inhibited by the compounds carpropamid, 4-amino quinazoline, and salicylamide (Butler and Day 1998). In a second reduction step, 3HNR converts 3HN to vermelone, which is subsequently converted to dihydroxynapthalene by SD (Thompson et. al. 2000). The presently understood pathway is shown in Figure 1-2. 17 1.4 Project Objectives It has been observed that Ceratocystis is a rapid colonizer and intense stainer of fresh logs and is rarely isolated from sawnwood. Conversely, Ophiostoma is more commonly found in sawnwood and grows slower and with less intensity in logs (Uzunovic and Webber 1998, Uzunovic et. ai. 1999b). Since there was little further information on the impact of wood types on the growth of different sapstain fungi, the first objective of this research was to determine the degree to which logs versus lumber affects fungal pigmentation and growth. The associated objective of this research was to also examine the nutrient consumption and pigmentation of sapstain fungi growing in different wood substrates and defined artificial media, in order to gain insight into the differences in nutrient utilization between deep and shallow stain fungi. The results of this work are discussed in Chapter 2. Furthermore, there has been little information on the biochemical pathways of pigmentation in different sapstain fungi. Thus, the next objective was to determine the biochemical pathway(s) of fungal cell pigmentation using known inhibitors of melanin production in fungi and then to use this information to assess the presence of melanin synthesis enzymes. Once the enzymes were demonstrated to be present, the subsequent objective was to sequence portions of the genes encoding for these enzymes and analyze them. The goal was to gain insight into the relationship of the fungi on a molecular level. The results of this work are described in Chapter 3. It is anticipated that this information can help both researchers and the forest products industry develop safe and effective means to control sapstain. 18 Chapter 2 Pigmentation and Nutrition of Sapstain Fungi in Wood and Nutrient Media 2.1 Introduction The conditions under which logs or lumber become infected with sapstain and even the fungal species causing the stain are only partially understood. As noted in section 1.2.3.3 above, recent collaborative work between our research group, the group of Dr. L. Bernier (Universite Laval), and Forintek Canada Corporation examined the diversity of sapstain fungi present in logs and lumber of different Canadian softwoods (Uzunovic et. al. 1999b). In each of seven sawmills across Canada, sawn timber and fresh logs were set aside, and sampled after one month. Thirteen different sapstain species were recognised among the 1863 fungal isolates obtained. It was found that the fungi isolated from logs were generally not found in the same frequency as those on lumber. The deep stain species C. resinifera was almost exclusively isolated from fresh logs, whereas other species such as O. piceae (which do not penetrate as deep into the sapwood) were isolated more frequently from lumber (Uzunovic et. al. 1999b). It has also been observed that the deep stain species can colonize fresh logs more efficiently than Ophiostoma (Uzunovic and Webber 1998, Strong 1999). This divergent behaviour has lead to questions on whether the presence of living host tissue may in fact stimulate Ceratocystis and other deep stain genera, or whether some sapstain fungi can kill host tissue more efficiently. It has been suggested that the deep stain species Leptographium wingfieldii can colonize Pinus sylvestris before triggering the host defence mechanism (Strong 1999). One hypothesis for rapid colonisation and stain of fresh logs by deep stain fungi is that they can utilise nutrients that other sapstain species cannot, and so can quickly grow and overwhelm host defence mechanisms. Sapstain fungi generally do not utilize wood structural 19 compounds (such as cellulose, hemicellulose or lignin) for nutrition since they generally produce no (or partial) celluloytic or lignolytic activity. Further, sapstain fungi generally only utilize non-structural wood components such as lipids, soluble carbohydrates, starch and proteins. (Nilsson 1973, Siefert 1993). Beyond this, however, the impact of nutrients on pigmentation and growth of deep stain fungi in host tissue is not known. Previous work by our lab has showed that nutrients play an important role in O. floccosum pigment production in vitro (Eagen et. al. 1997). Fungal pigmentation varied from white, grey, brown to black when the fungus was grown in nutrient media supplemented with different carbon and nitrogen sources (Table 2-1). Another hypothesis is that some physical or chemical events may occur in the wood during the period from log harvesting to lumber processing. Physical events include closing of the bordered pits due to wood moisture loss. Chemical events could include the production of antifungal agents or the consumption of nutrients during the dying process of the host tissue (Strobel and Sugawara 1985). Table 2-1: Colour of fungal mycelia after 8 days of growth in liquid culture supplemented with different carbon and nitrogen sources. (Reproduced from Eagen et. al. 1997) Nitrogen Source Carbon Source Globulins L-asparagine L-tyrosine NH4C1 NH4OH Rhamnose Pale brown Black Beige Grey Black Glycerol Brown Brown Pale brown Grey Brown Ethanol Brown Black Beige Grey Black Starch Brown Light beige White White Brown Raffinose Brown Beige Beige White White 20 In this chapter, the results of infection of Pinus contorta (lodgepole pine) logs and sawnwood with sapstain fungi and the effect of fungal growth on wood nutrient depletion, is discussed. Lodgepole pine was selected due to its importance to the forest products industry in B C 1 . Initially, it was important to verify whether the isolated organisms from the survey (described above) would grow and produce discolouration when re-inoculated on wood. The role of host tissue viability on the growth of deep and surface staining fungi was also examined by testing the ability of sapstain fungi to kill host tissue in logs. The effect of changing moisture content was examined by growing fungi on both kiln-dried sawnwood that had been re-wet to 80% moisture content and sawnwood that had been conditioned to 80% moisture content. Further, since there is little information on the in vivo nutrient consumption of sapstain fungi, chemical analysis of the host tissue nutrients was carried out throughout the infection experiments. The nutrients included lipids, simple sugars and starch. Sapstain nutrition and pigmentation on defined media with selected carbon sources was also investigated. ' P. contorta is the second most abundant tree species by volume in British Columbia accounting for 1.61 x 109 m3 (22.3% of total) of the mature standing timber volume in the entire province. The bulk of this volume is in the BC Interior region with 1.581 x 109 m 3 (COFI 2000). Lodgepole pine is also the most harvested species in BC accounting for 18.5 x 106 m3 or 27.3 % of the total harvest volume. The species ranges from southeastern Alaska, central Yukon and southwestern Mackenzie District, south into Alberta and British Columbia, through Washington, central Montana, northeastern Utah and southern Colorado, south along the Sierra Nevada and Pacific Coast into northern Mexico (Alden 1997). 21 2.2 Materials and Methodology Except where noted, all chemicals were supplied by Sigma Chemical Company (St. Louis, USA). 2.2.1 Selection of sapstain fungi Fungal strains with high frequency of isolation were selected from the U B C Department of Wood Science culture collection of over 1300 strains isolated from four western Canadian sawmills as described in Uzunovic et. al. (1999b). The tested strains belong to the following species: Aureobasidium pullulans, Ceratocystis resinifera (also known as C. coerulescens type C), Leptographium spp, Ophiostoma coronatum (Doug McNew, Iowa State University, Personal communication), O. floccosum, O. minus, O. piceae, O. piliferum, and O. setosum. The isolate codes and source information are shown in Table 2-2. The isolates were selected to represent a variety of geographical locations and host wood types, and because they produce a range of light to dark pigmentation on malt extract agar (MEA). Working cultures were maintained on 2% M E A (Oxoid Ltd. Basingstoke, England) for no more than three subcultures to maintain physiological characteristics of the culture. Stock cultures were stored in 10% glycerol at -80°C. 2.2.2 Inoculation of lodgepole pine 2.2.2.1 Wood source Two geographical and seasonal sources of wood were used in this study. The bulk of the wood was derived from the first source: two Pinus contorta (lodgepole pine) trees that were gently felled on March 6, 1998 in the Merritt Forest District of British Columbia, Canada. Although the trees were of similar height (20m) and diameter (26cm at breast height), one was 58 years old and the other was 88 years old. Both were growing in a mixed conifer/aspen forest at an elevation of 22 Table 2-2: Codes and source information of fungal species isolates1 Species Isolate Code Short Code Source Host Host Type Source Location A. pullulans 72 K Lodgepole pine Log Okanagan Falls A. pullulans 123-436 HH Jack pine Log Big River A. pullulans 156-127 A Lodgepole pine Log Prince George C. resinifera 123-22-12 L Jack pine Log Big River C. resinifera 125-214 C White spruce Log Big River C. resinifera 157-152 X White spruce Log Prince George Leptographium spp. 55-5 D Lodgepole pine Sawnwood Okanagan Falls Leptographium spp. 71-15 Q Lodgepole pine Sawnwood Okanagan Falls Leptographium spp. 123-239 E Jack pine Log Big River Leptographium spp. 156-234 R Lodgepole pine Log Prince George Leptographium spp. 157-253 U White spruce Log Prince George 0. floccosum 55-1 AA Lodgepole pine Sawnwood Okanagan Falls 0. floccosum 82-1-1 V Jack pine Sawnwood Big River 0. floccosum 156-211 DD Lodgepole pine Log Prince George 0. floccosum 197-3 FF Lodgepole pine Sawnwood Blainmore O. minus 58-4 P Lodgepole pine Sawnwood Okanagan Falls 0. minus 123-151 J Jack pine Log Big River 0. minus 123-43-13 T Jack pine Log Big River 0. minus 198-4 I Lodgepole pine Sawnwood Blainmore 0. piceae 55-3 II Lodgepole pine Sawnwood Okanagan Falls 0. piceae 123-142 CC Jack pine Log Big River O. piceae 153-5 F Lodgepole pine Sawnwood Prince George 0. piceae 157-241 Y White spruce Log Prince George 0. piceae 187-1 EE Lodgepole pine Sawnwood Blainmore 0. piliferum 55-2 M Lodgepole pine Sawnwood Okanagan Falls 0. piliferum 80-3 W Jack pine Sawnwood Big River 0. piliferum 156-112 H Lodgepole pine Log Prince George 0. piliferum 199-4 BB Lodgepole pine Sawnwood Blainmore 0. setosum 55-6-1 0 Lodgepole pine Sawnwood Okanagan Falls 0. setosum 160-21 B Hemlock Log Vancouver Island 0. setosum 160-38 S Hemlock Log Vancouver Island 0. coronatum 125-238 GG White spruce Log Big River 0. coronatum 57-2 N Lodgepole pine Sawnwood Okanagan Falls 0. coronatum 195-7 G Lodgepole pine Sawnwood Blainmore Codes: Storage Code number refers to stock culture collection location; Isolate Code is shorthand code used during experiments. Source information indicates from where fungi isolates were harvested including host species, host wood type and geographical location (Okanagan Falls, BC; Prince George, BC; Vancouver Island, BC; Blainmore, AB; Big River, SK.) 23 approximately 1200 m. Felling took place as flushing after the winter was about to occur. The second wood source was derived from a lodgepole pine tree that was gently felled near Edson, Alberta, Canada on August 31, 1999. The tree came from an evenly aged stand of 85 to 100 years old. These two wood sources are henceforth referred to as Merritt pine and Edson pine, respectively. The trees were carefully de-limbed in the field to avoid damaging the bark and logs brought back to the laboratory (Figure 2-1A). The day after felling, sawnwood blocks (25x25x75 mm; radial x tangential x longitudinal) were cut from the sapwood of a log from each tree and stored at -20°C (Figure 2-2A). The remaining logs were wrapped and sealed in 0.5 mm polyethylene sheets and stored at -20°C. Samples (27 cm3each) were taken from various locations in the sapwood ring on each tree for time zero chemical analyses. Within six weeks, stem logs were recovered from storage and cut into inter-nodal billets (branch rings cut out) 40-80 cm long. The ends and any branch scars were sealed with a bituminous paint (Black Shield #7510; Insulmastic Ltd., Delta, B.C.) and the billets stored overnight in an unheated shed away from ground contact (Figure 2-1B). 24 Figure 2-1: Lodgepole pine billet experiment. (A) Early spring harvesting in Merrit, BC during spring 1998, (B) billet ends-sealed with bitminous paint and (C) inoculation with fungi grown on wheat grain. 25 2.2.2.2 Billet inoculation Each billet was randomly inoculated with infected wheat grain following the procedure of Uzunovic and Webber (1998). Prior to infection on wood billets, two or three M E A cores (6mm diameter) of actively growing fungi were inoculated onto autoclaved wheat grains (approximately 20 grains) and incubated for 14 days at room temperature. On the wood billets, the infection points were flamed lightly in order to sterilize the area. A 5-mm diameter x 10-mm deep hole was drilled with a flame-sterilised drill (cooled to room temperature). Three infected wheat grains were aseptically placed in each hole (Figure 2-1C). Fungal isolates were infected in triplicate, essentially at random, although care was taken to ensure that each isolate was inoculated onto at least one billet originating from each tree, and that each isolate was infected only once per billet. Each billet contained between four to six inoculation points, depending on the size of the billet. To conserve moisture and protect the experiment, the holes were covered and wrapped with electrical tape. Infected billets and two non-infected control billets were incubated at 20 C, 85 % relative humidity (RH) for 28 days. Only Merritt pine trees were used in this experiment. 26 27 2.2.2.3 Sawnwood inoculation For sawnwood infection, both Merrit and Edson pine sapwood blocks described in section 2.2.2.1 above, were y -sterilised for 96 hours with a total dose of 2.5 Mrads (Gamma Cell Facility, U B C Biomedical Research Centre, Vancouver, BC). For experiments with Edson pine, wood blocks were kiln dried to target moisture contents (see section 2.2.2.4 below) prior to infection. The blocks were aseptically placed on a 2mm thick plastic mesh above water-saturated fdter paper contained within a sterilised plastic box (Figure 2-2A). Fungal cores (6mm diameter, taken from the actively growing edge of a fungal isolate growing on M E A ) were placed on the tangential surface and incubated for up to 28 days at 18C. Moisture was maintained in the box by periodic addition of sterile water to the filter paper. Three wood blocks were infected for each fungal isolate. 2.2.2.4 Sawnwood kiln drying Kiln drying of y-sterilized sawnwood blocks (derived from Edson pine) was carried out as follows. Hollow boxes (25 x 30 x 20 cm) were assembled from breathable 2.5 cm thick polyurethane foam, autoclaved (121C, 103kPa, 20 min) and tarred with the glass rods described in the next sentence. The box was then aseptically filled with y-sterilized Edson pine blocks plus sterile glass rod stickers placed between each block to allow airflow, and then tarred. The box was then placed in a 0.127m3 mobile batch kiln (model CL-4521, Parameter Generation Control Inc, Black Mountain, NC) set to 60.0 ± 0.5 C air temperature and 45 ± 0.5 C water temperature, thus establishing a 50 ± 1 % R H (USDA 1991). The mass of the box was then measured daily without disturbing the contents. Prior to kiln drying, the average oven dry weight per block was determined (as described in section 2.2.4 below) and these values were then used to estimate the 28 target weight of a target moisture content (MC) of the sawnwood during the kiln drying procedure without destructive sampling (Equation 2-1). %MC, (arg et mass I arg el 100% x mass total OD + mass, total OD + mass, box( 2 - 1 ) where : masslolal 0D - estimated oven dry weight of all blocks based on previously determined average massbox = mass of foam box plus glass rod stickers Two moisture contents were targeted for the kiln experiments: 80% and 15%. After drying, the 15% M C batch was re-saturated by submersion in sterile de-ionized water for 48 hours, then re-kiln dried to 80% M C , as described. A l l blocks were inspected after kiln drying to ensure that no contamination took place, then stored at -20C until required for the experiments. In order to confirm the estimated moisture content, sample blocks were removed after kiln drying and their moisture content determined as described in section 2.2.4 below. 2.2.3 Assessment of Fungal Growth in Wood After incubation, the wood billets and blocks were analysed for fungal growth. With a fine-toothed band saw, the billets were cross-sectioned into discs at 2-cm intervals above and below the infection point. Each slice was rinsed with de-ionized water and photographed with a digital camera. Longitudinal growth was measured as the distance of the last disc with visible sapstain measured from the inoculation point. The first disc of each billet was incubated at 20 C, 80% R H for two days and fungi were carefully re-isolated from the wood surface of each disc. Fungal identities were confirmed by microscopic examination of mycelia outgrowth and by re-isolation of cultures. If contamination was present, the data were not used for further testing. The second disc of each billet was soaked overnight in a 1 % solution of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) to distinguish living host plant cells from dead ones (Feist et. al. 1971). The third 29 disc was further cut into small blocks, frozen and milled through a 60-mesh (0.5 mm) screen. After freeze-drying, the samples were sealed in polyethylene bags and stored at -20 C for further analysis. After incubation, sections of the top two 6.25mm layers of the sawnwood blocks (labelled layers one and two, respectively; shown in Figure 2-2B) were photographed, milled and stored as described for the billet samples, above. 2.2.4 Moisture Content Wood moisture content was measured by sampling at least a 3cm3 block for each test. Each sample was tarred before and after drying in an oven (105C for 16 hours). Moisture content percentage (%MC) was calculated according to Equation 2-2. %MC = maSSf>^<><>« ~ m a s s ^ y x l 0 0 o / o ( 2 _ 2 ) maSSovendry 2.2.5 Sugar and Starch Analysis Soluble sugar extraction of freeze-dried groundwood was based on the protocol of Saranpaa and H611 (1989). Freeze-dried wood powder (500mg) was extracted for 2 hours with 3.0mL de-ionized water in a disposable 3cc syringe cartridge fitted with a 9 mm glass microfibre filter (Whatman 934-AH; Whatman Paper, Kent, England) and a layer of Parafilm M (Pechiney Plastics Inc., Neenah, WI) to prevent leaking. Extracts were vacuum eluted into glass vials and rinsed with 1 mL de-ionized water. Next, 900 uL of the filtrate was syringed through a membrane filter (0.45 um pore size, Chromatographic Specialties, Brockville, ON, Canada) and spiked with 100 pL of 5 mg/mL fucose internal standard. Carbohydrates were separated and detected by Anion Exchange HPLC equipped with a Carbopac™ PA1 analytical (4x250 mm) and 30 guard (4x50 mm) column (Dionex Corporation, Sunnyvale, CA) by the method of Mansfield (1997). The sugars arabinose, galactose, glucose, xylose and mannose were used as standards. Starch extraction was based on the protocol of Saranpaa and Holl (1989). This required boiling 50 mg of freeze-dried groundwood at 100C for 10 min to destroy endogenous enzymes. The starch was then extracted with 1 mL 0.05M citrate buffer at 100C for 4 hours. After centrifugation, 500 pL of the supernatant was added to 450 pL citrate buffer and 50 U.L of amyloglucosidase (10 mg/mL; Boehringer Mannheim GmbH, Germany), and then incubated at 37C for 16 hours. The resulting glucose was measured as described above, then corrected for the free glucose measured earlier. The amount of starch present was reported in units of starch-bound glucose. 2.2.6 Lipid Analysis Lipids analysis of control and infected wood was carried out using a modification of Orsa and Holmbom (1994). One to two grams of freeze-dried milled wood was extracted with acetone in a soxhlet apparatus overnight and vacuum dried. Acetone extracts were re-extracted with methyl-tert-butyl ether (MTBE), dried down under nitrogen, tarred, and stored under nitrogen at 4°C. This total lipid extract (TE) fraction was spiked with four internal standards (40pg heneicosanoic acid, 4ug betulin, 40ug cholesteryl heptadecanoate, and 40|ig l,2-dipalmitoyl-2-oleoyl glycerol) dissolved in 1.0 mL M T B E , then dried under nitrogen and vacuum desiccated for 15 min. Spiked samples were silated by the addition of 80pL bis(trimethylsilyl)-triflouro-acetamide and 40uL trimethylchlorosilane, with subsequent incubation at 70C for 20min. Silated samples were dissolved to 1.0 mL in M T B E and subsequently resolved by GC (HP Series II 5890 GC with HP 7673 Auto-injector, Hewlett-Packard Ltd., Mississauga, ON). For each sample, 2pL was injected (splitless) onto a 5m x 0.25 mm DB-5 (0.25pm film thickness) fused-silica capillary column (J & 31 W Scientific, Agilent Technologies, Mississauga, ON). Helium was the carrier gas at a flow rate of 20mL min"'. The injector temperature was initially set to 90C for 1.5 min, then increased to 320C at a rate of 200C min"'. The column temperature was initially 100C for 1.5 min, then increased to 115C at a rate of 30C min"', held for 1 min, then increased to 320C at a rate of IOC min"' and held for 7 min. TE samples were also fractionated by solid phase extraction (SPE) using the method of Chen et. al. (1994) and analysed by GC to determine the retention times of the lipid fractions. These retention times were used to identify and quantify the major fractions in the total extracted samples. The steryl ester/ wax (SE/W) fraction did not resolve well by GC and thus was not examined in this work. However, the remaining fractions resolved as follows: 96-98% of fatty acid/ resin acid (FARA) peaks occurred between 6.050-9.500 minutes; 12-15% of monoglyceride (MG) peaks occurred between 10.500-12.400 minutes; 60%) of sterol/ fatty alcohol/ diglyceride (S/FA1/DG) peaks occurred between 15.300-21.000 minutes; 72-75% of triglyceride (TG) peaks occurred between 25.500-30.000 minutes. 2.2.7 Nutrition and Pigmentation Seven carbon sources found in wood were tested with representative isolates of four species of sapstain fungi. The carbon sources included arabinose, glucose, mannose, xylose, starch, glycerol, olive oil, and linoleic acid. The nutrient medium (designated as B-media) previously described (Abraham 1995) was supplemented with 2% (w/v) of filter-sterilised carbon source, except for linoleic acid, which was included at 0.5 %. Colony colour, morphology and radial size were measured every three to four days. Colony colour was determined by scoring each plate from 1 (cream/beige) to 5 (black). Five replicates of each isolate were plated and analyzed. 32 For glucose versus mannose experiments on defined media, fungi were inoculated onto lOmL liquid B-media (containing 1% mannose and 1% glucose w/v) in a 5cm diameter Petri dish. Cultures were incubated at 18C for up to 196 hours and analyzed for sugar composition as described above. 2.2.8 Microscopy Microscopic images were prepared from selected wood samples to examine fungal growth in vivo. Samples (5mm x 20mm x 10mm) were cut from fresh and infected sawnwood blocks and immersed in sterile water overnight in order to saturate the wood fibres to allow efficient microtome cuts. Sections (20um thick) were cut across the radial, longitudinal, and tangential planes with a microtome (model 860, American Optical Co., Buffalo, N Y ) and were delicately transferred to a wet mount glass slide. The sections were stained with lactophenol cotton blue (VWR, West Chester, PA) and then rinsed with distilled water. Samples were examined with a Zeiss Axioplan microscope (Carl Zeiss Inc., Toronto, ON) using phase contrast, differential interference contrast or U V fluorescence. Images were captured with a MDS 100 digital video camera (Eastman Kodak SIS, New Haven, CT). 2.2.9 Statistics Except where noted, reported error values for all tables and figures were determined at the a=0.05 confidence interval of the standard deviation. Sample sizes (n) are indicated with the table or figure. Significance of variance between means was determined by A N O V A (single factor) with a significance of F (a) <0.05. 33 2.3 Results 2.3.1 Moisture content, fungal growth and stain in billets Moisture content (MC) was determined in freshly felled logs, and after six weeks storage at -20C. No differences in moisture content were observed along the length of the trees, and consequently, all samples were pooled and averaged. At the start of the experiments, the mean moisture content was 170 ± 12% and 131 ± 20% for the Merrit and Edson pines, respectively. 34 Table 2-3: Average longitudinal growth of fungi on P. contorta (Merrit) billets after 28 days. Species Isolate Code Longitudinal growth (cm) stained Kill zone1 A. pullulans 72 123-436 156-127 <1.00±0.00 2.33 ± 1.53 2.50 ± 2.12 C. resinifera 123-22-12 125-214 157-152 >30.00± 8.49 >33.00±4.24 >27.00 ± 4.24 Leptographium spp 55-5 14.67 ±2.31 18.00 ±0.00 71-15 18.00 ±4.00 20.00 ±3.46 123-239 >18.00±0.00 23.33 ±4.62 156-234 >16.00±3.46 18.67 ±7.02 157-253 3.50 ±3.54 2 O. minus 58-4 123-43-13 123-151 198-4 4.00 ±2.00 12.67 ± 4.16 11.00 ± 1.41 6.00 ±2.00 O. piceae 55-3 123-142 153-5 157-241 187-1 1.00 ±0.00 3.67 ±2.52 2.67± 1.15 2.50 ±2.12 2.00 ±0.00 O. piliferum 55-2 80-3 156-112 199-4 4.00 ±0.00 2.50±2.12 5.33 ± 1.15 5.33 ± 1.15 0. floccosum 55-1 82-1-1 156-211 197-3 2.50±2.12 4.00 ±0.00 3.00 ± 1.41 2.67± 1.15 0. setosum 55-6-1 160-2 A 160-38 2.67± 1.15 2.67 ±1.15 3.00 ± 1.41 0. pluriannulatum 57-2 125-238 195-7 1.50 ± 0.71 1.33 ±0.58 2.67 ± 1.15 For Leptographium spp., k: non-stained dead host tissue. —: not present 35 Infection point Longitudinal growth Figure 2-3: Composite image of cross sections taken from P. contorta logs after 28 day infection with sapstain fungi. Elevated disks in bottom row indicate corresponding image above. Cc: C. resinifera C, L-k: Leptographium sp. Q kill zone, L-s: Leptographium sp. Q stain zone, Omin: O. minus P, Opic: O. piceae EE, Opil: O. piliferum H, Oset: O. setosum S. 36 No change in moisture content was observed between the time-0 logs and the logs sampled 6 weeks later. Table 2-3 shows the results of the growth experiment on lodgepole pine billets. In the billets, the fastest growing species was C. resinifera (>30cm longitudinal stain in 28 days), followed by Leptographium spp (14-18 cm), O. minus (4-13 cm), O. piliferum (2-5 cm), O. piceae (1-4 cm), O. floccosum (2-4 cm), O. setosum (2-3 cm), O. pluriannulatum (1-3 cm) and Aureobasidium pullulans (1-2 cm). Figure 2-3 shows the typical progress of selected isolates through Merrit logs after 28 days. Viability of host parenchyma was easily visualised by TTC immersion (Figure 2-4). Non-infected sapwood turned a deep red colour (viable parenchyma) whereas heartwood and infected sapwood did not. Leptographium isolates typically showed a non-pigmented, dead host cell zone, approximately two to five cm ahead of the stained area within the logs. Leptographium mycelia were isolated from this dead-host area shortly after cutting and synnemata grew in the same zone after two days of post-cut incubation. Smaller dead-host zones (0 to 0.5 cm) were observed for C. resinifera and Ophiostoma spp. 2.3.2 Moisture content, fungal growth and stain in sawnwood Prior to incubation, the mean M C for fresh Merrit pine sawnwood blocks was 150 + 12% (n=6). In general, moisture content of infected wood did not differ by more than ± 20% from the control samples. In fresh sawnwood blocks, visual inspection showed that most fungal species colonized all parts of the sapwood wood blocks, and stain was observed throughout the blocks. However, two fungal species, O. piceae and A. pullulans showed a different pattern and only the top layer, where inoculation had occurred, was darkly pigmented (Figure 2-5). Wood that had been kiln dried to 80% M C (80MC wood) had similar infection patterns as the fresh sawnwood blocks. However, wood that had been kiln dried to 15% M C then re-wet to 80% M C (15-80MC wood) had a much different infection pattern (Figure 2-6). The deep stain species, C. resinifera did not 37 Figure 2-4: Testing for viable host parenchyma in cross section of P. contorta infected with sapstain fungi for 28 days. Disk was taken 4cm (longitudinal) from the infection point and immersed in 1% TTC (2,3,5-triphenyl-2H-tetrazolium chloride) dye overnight. The viable sapwood tissue (SW) is stained red (dark grey in picture), whereas non-viable heartwood tissue (HW) is unstained. Cc: C. resinifera C, L-k: Leptographium sp. Q kill zone, L-s: Leptographium sp. Q stain zone, Omin: O. minus P, Opic: O. piceae EE. 38 Ap K Ap HH ApA CcL CcC CcX LU LD L Q L E L R Figure 2-5A: Comparison of Merrit sawnwood blocks infected with A. pullulans (Ap), C. resinifera (Cc) and Leptographium spp. (L) for 28 days. Each image shows four sequential slices of one sawnwood block. The top slice is layer 1 (where inoculation occurred) followed by layers 2, 3 and 4 as shown in Figure 2-2B. Each slice measures 25mm x 75mm. Each image is indicated with the corresponding inoculated fungus species and isolate short code. 39 Omin P Omin T Omin J Omin I Opic II Op/'c CC Op/cF Op/c EE Op/7M 0p/7W 0p/7H Op/7BB Figure 2-5B: Comparison of Merrit sawnwood blocks infected with O. minus (Omin), O. piceae (Opic) and O. piliferum (Opil) for 28 days. Each image shows four sequential slices of one sawnwood block. The top slice is layer 1 (where inoculation occurred) followed by layers 2, 3 and 4 as shown in Figure 2-2B. Each slice measures 25mm x 75mm. Each image is indicated with the corresponding inoculated fungus species and isolate short code. 40 Oflo A A 0/7o V Of /o D D 0/7o F F Ocor G G O c o r G Figure 2-5C: Comparison of Merrit sawnwood blocks infected with O. floccosum (Oflo), O. setosum (Oset) and O. coronatum (Ocor) for 28 days. Each image shows four sequential slices of one sawnwood block. The top slice is layer 1 (where inoculation occurred) followed by layers 2, 3 and 4 as shown in Figure 2-2B. Each slice measures 25mm x 75mm. Each image is indicated with the corresponding inoculated fungus species and isolate short code. 41 Control CcC LeptoQ Opic EE Op/'/H 80% MC 14 day 15-80% MC 14 day 15-80% MC 28 day Figure 2-6: Comparison of kiln-conditioned Edson sawnwood blocks infected with sapstain fungi for 28 days. Sterile wood blocks were conditioned to either 80%MC or 15% then 80%MC, infected with different fungal species and sampled after 14 and 28 days. The top row indicates the tested species and isolate. The left column indicates the wood conditioning regime and incubation time. Each image shows four sequential slices of one sawnwood block. The top slice is layer 1 (where inoculation occurred) followed by layers 2, 3 and 4 as shown in Figure 2-2B. Each slice measures 25mm x 75mm. 42 Figure 2-7: Photomicrographs of P. contorta infected with C. resinifera C. A : Light microscopy image of tangential section. White triangles indicate hyphae growing in tracheids. B : UV illumination of longitudinal section. White triangles indicate lactophenol blue stained hyphae growing through parenchyma cells. C : UV illumination of radial cross section. White triangles indicate lactophenol blue stained hyphae growing through ray cells. 43 colonize any part of the 15-80MC wood blocks, whereas other surface stain species, such as O. piceae were able to colonize the blocks, though to a lesser degree than the 80MC wood. The colonization of fresh sawnwood was also examined with visible and U V microscopy. The typical hyphal penetration of tracheids, parenchyma and rays by C. resinifera C is shown in Figure 2-7. Other tested fungi were observed to be present in their respective wood block and resembled the colonization pattern of C. resinifera C under the microscope (data not shown). C. resinifera C hyphae were not observed in 15-80MC wood. The intactness of the bordered pits of fresh Merrit sawnwood, 80MC sawnwood and 15-80MC sawnwood was examined using light and U V microscopy (Figure 2-8). Both fresh Merrit pine sawnwood and 80MC sawnwood had intact border pits throughout their xylem tissue, whereas the bordered pits throughout the 15-80MC sawnwood were closed or damaged. Similar observations were made in both infected and control wood samples. 2.3.3 Soluble sugars and starch in wood During the 28-day seasoning period of non-inoculated Merrit logs, glucose, mannose and arabinose concentrations changed significantly (Table 2-4). Xylose decreased by 100% from a starting concentration of 140 ± 20 ppm. Galactose increased by about 180 % from a starting concentration of 51 ± 2 ppm. In non-inoculated, sterile (y-treated), Merrit sawnwood, sugar concentrations did not change significantly during the incubation period. Sugar levels of selected fungal inoculated wood samples from Merrit are shown in Table 2-4. When compared to unseasoned wood, fungal-depletion of mannose averaged 99 ± 1% and 92 ± 4%, in logs and sawnwood, respectively. Glucose depletion averaged about 70 + 10% in logs and 66 ± 17% in sawnwood. In fungal-inoculated logs, xylose was not detected in significant 44 Figure 2-8: Photomicrograph of bordered pits (indicated by white triangles) in P. contorta. A: Fresh, unseasoned sawnwood with intact bordered pits. B: Sawnwood conditioned to 80%MC, with intact bordered pits. C: Sawnwood kiln dried to 15%MC, then re-wet to 80%MC, with closed bordered pits. All samples were stained with 1% safranine dye prior to visualization. 45 Table 2-4: Concentration1'2 (ppm) of soluble sugars in P. contorta (Merrit) infected with fungi. Logs Sawnwood Species ARA 3 G L U 3 M A N 3 ARA GLU MAN Unseasoned Control 19.9 ±0.6 921 ± 82 1063'±72 19.9 ±0.6 921.± 81 1063 ±72 Seasoned Control 240 ±3 348 ± 10 335 ±20 20.9 ±0.6 860 ± 81 997 ± 72 C. resinifera C 251 ±22 298 ± 15 16± 15 3 7 ± 6 3 371 ±36 99 ± 4 C. resinifera X 398 ±35 434 ± 20 3 ±20 36 ± 5 143 ±51 83 ± 5 Leptographium Q n.a.4 n.a. n.a. 33 ±4 500 ± 15 86 ± 5 Leptographium D n.a. n.a. n.a. 20 ±2 327 ±74 86 ± 3 Leptographium R-k 5 896 ± 70 205 ± 20 4± 15 n.a. n.a. n.a. Leptographium R-s 5 1026 ±83 248 ± 18 4 ± 8 n.a. n.a. n.a. Leptographium E-k 1165 ±84 246 ± 22 3 ± 6 n.a. n.a. n.a. Leptographium E-s 1112 ±90 262 ± 26 4± 10 n.a. n.a. n.a. 0. piliferum W n.a. n.a. n.a. 36 ± 1 434± 140 132 ± 9 0. piceae M 270 ±35 234 ± 20 3 ± 5 n.a. n.a. n.a. O. piceae EE n.a. n.a. n.a. 70 ±7 498 ±51 120 ± 1 0. piceae F n.a. n.a. n.a. 76 ± 19 267± 45 0 ± 9 0. minus J 395 ±32 418 ± 30 4 ± 7 31 ±7 59 ± 8 112 ± 6 (9. floccosum DD 513 ± 40 145 ± 15 0± 10 33 ± 3 250 ±33 81 ± 5 Sugar concentration was not significantly different in unseasoned logs and unseasoned sterile sawnwood. Reported values are averages of all samples. Error values equal 95% confidence. Control n=12, log fungi n=3, sawnwood fungi n=7 or 8. ARA: arabinose, GLU: glucose, MAN: mannose n.a.: not measured k: non-stained dead host tissue, s: stained portion. 46 quantities and decreased by an average of 88% (125 + 10 ppm) in sawnwood. In logs, arabinose increased approximately 50-fold (1000 ± 100 ppm) for Leptographium spp. and over 17-fold (350 ± 100 ppm) for the remaining fungi. In sawnwood, arabinose increased by 100% (21 ± 19 ppm). Galactose increased by about 330%) (170 ± 50 ppm) and 40%> (18 ± 13 ppm) in logs and sawnwood, respectively. Further, there was little difference in sugar content between the stained and kill zones of Leptographium spp. on logs. No significant levels of starch-bound glucose were found in control or fungal inoculated samples from the Merrit pine samples (data not shown). The sugar contents for control and fungal inoculated kiln-conditioned sawnwood is shown in Table 2-5. Results are separated by kiln-conditioning (15-80MC wood and 80MC wood) and incubation time (14 and 28 days). Sugar concentrations were identical between layers 1 and 2 of 80MC control sawnwood and did not change significantly during the incubation period. The starting concentration for xylose and galactose in 80MC wood was 63 ± 9 and 165 + 21 ppm, respectively. In 15-80MC wood, sugar concentrations were generally higher in layer 1 than in layer 2 (Table 2-5). In 15-80MC sawnwood, the concentration for: xylose was 57 ± 6 ppm in layer 1 and 51 ± 3 ppm in layer 2; and galactose was 165 ± 12 ppm in layer 1 and 115 ± 8 ppm in layer 2. Mannose depletion in 80MC pine by the fungi was similar to that for fresh Merrit sawnwood with 100% reduction of free mannose after 28 days. Glucose depletion was between 70 to 80% for the tested fungi after 28 days in 80MC wood. Unlike the Merrit pine batch, arabinose decreased by 100% in fungal inoculated 80MC Edson pine after 28 days, with the exception of Leptographium sp Q (Table 2-5). 47 '3 If a (ZJ e 3 t u 3 w w <3 VJ s s u a .s c o U cs C/5 -I IU -1 -1 1 1 . d „ Tt" — ' rt — m -H CN m -H vo oo -H o CN OS -H VO Tf OS +1 vo 2 < -H o m in m 00 VO CN m +1 -H -H OS CN cn T f o CN T f CN -H -H +1 OS in m T f CN o m CN CN CN ON o T f CN o T f T f VO cn O vo >n -H T f vo vo OS -H vo OS +1 p o TT CN cn m T f CN 00 +1 T f -H CN CN OS +1 VO T f OS -H VO T f o 00 -H VO in cn T f CN -H O cn cn CN CN -H OS T f -H oo m +1 vo -H CN CN T f CN CN CN CN VO CN VO OS -H VO Os -H P a oo CN 00 +1 T f +1 cn -H vo cn -H CN CN 00 -H CN CN T f +1 OS vo CN o T f in 00 cn CN cn CN Os cn CN in vo o T f CN o 00 cn in CN -H -H +1 T f OS CN CN cn CN r~- 00 rt cn un f --fl -H -H T f Os oo CN cn cn r-- cn Z < P —1 2 < a VO CN cn CN +1 vo T f CN OO -fl r-oo cn c-~ vo o T f o CN -H -H -H vo O cn vo T f oo -H r~ vo T f CN VO 00 VO < T f in CN -H -H -H cn cn in 00 in r-cn 00 m « -H vo cn 503 ± -H cn OS T f cn <n -f! -H -H VO T f T f OS in CN CN m - 00 -H -H T f CN r~-Os CN oo Os VO +1 -H 1 CN CN CN cn -fl -H j 00 CN oo 00 >n VO cn i n o -H -H -H C"~ cn cn oo r- 1398 r-+1 OS -H OS oo CN CN CN CN 00 OO cn cn P .—I a 00 CN VO Os T f CN VO oo vo T f CN vo oo vo o oo U "S, E CO 1 1 M O P ?. OJ „ O CJ .5 o < : 00 T f The results were much different for 15-80MC wood infected with fungi, where mannose depletion was much slower for Leptographium sp Q and the Ophiostoma spp. O. piceae EE and O. piliferum H depleted about 60 to 80% and 30%) of glucose in layer 1 and 2, respectively. Leptographium sp. Q depleted 85% and 65% of the glucose in layer 1 and 2, respectively. C. resinifera C did not affect glucose whatsoever. After 28 days in 15-80MC wood, arabinose decreased by almost 20 ppm due to infection with'Leptographium sp. Q, though remained relatively constant for the remaining fungi. Starch levels for the infected Edson pine sawnwood samples are shown in Table 2-6. In the Edson pine samples, the starting concentrations of starch-derived glucose did not change significantly between kiln drying regimes or over incubation time. Starch was eventually reduced to zero by the four tested fungi after 28 days incubation on 80%MC kiln wood. In the 15-80%MC kiln batch, starch was reduced by 85%> to 99%) by Leptographium sp. Q and the two tested Ophiostoma spp., whereas C. resinifera C did not significantly reduce the starch content (Table 2-6). Table 2-6: Concentration1'2 (ppm) of starch-derived glucose in kiln conditioned P. contorta (Edson) sawnwood infected with fungi for 14 and28 days. Fungal Species Time Wood type Control C. resinifera C Leptographium O. piceae EE O. piliferum sp. Q H 80 MC 14 569± 16 90 ± 6 136±11 48 ± 7 n.a. 28 575 ±22 0±11 0±10 0 ± 9 0±15 15-80 MC 14 560 ± 32 550±24 150± 18 206±11 37±9 28 540 ±23 544 ± 35 73 ± 14 51 ± 7 2.2 ± 0.8 1 Starch-derived glucose concentration was not significantly different in layers 1 and 2. Reported values are averages of all samples. 2 Error values equal 95% confidence (n=6). 49 2.3.4 Lipids in wood Lipid analysis of P. contorta (Merrit) revealed significant changes in the F A R A and TG fractions during the 28-day incubation period of non-inoculated controls (Table 2-7). In the control logs, TG decreased by around 60% with a concomitant increase in F A R A of about 280%. In y-sterilised Merrit sawnwood, TG decreased by approximately 25% whereas F A R A increased by about 130%). In kiln-conditioned sawnwood, lipids did not change significantly between control samples over the 28 day incubation period (Table 2-8). For infected wood samples, all reported changes are relative to the fresh, unseasoned control. Because the surface stain fungi grew slowly on logs and samples from logs were initially tested for sugar content, only enough groundwood from the infection areas of C. resinifera and Leptographium spp. remained for lipid analysis. In logs, C. resinifera and Leptographium spp. reduced TG content to very low or undetectable amounts and increased the F A R A content by 4 to 9 mg g"1 OD wood (Table 2-7). In Merrit sawnwood, C. resinifera, Leptographium spp. and O. piliferum reduced TG by an average of 80 ± 10% in both layers. The remaining Ophiostoma fungi reduced TG by an average of 70 ± 10%o. TG depletion was not statistically different between layers one and two for most fungi except fox A. pullulans (86 ± 6% and 71 ±8%) and an isolate of O. piceae (74 ± 6% and 56 ± 9%). A l l tested fungi increased the level of F A R A of the Merrit sawnwood blocks. However, the F A R A content of layer two was usually greater than layer one (Table 2-7). Results for the 80MC kiln-conditioned sawnwood (Edson) was similar to the Merrit sawnwood (Table 2-8). In 15-80MC wood though, TG consumption was significantly reduced after 28 days. Leptographium sp. Q and O. pliferum H decreased TG by about 65%), while O. piceae EE decreased TG by about 50%). C. resinifera C did not significantly reduce TG in 15-80MC sawnwood after 28 days. 50 Table 2-7: Concentration1'2 (mg g"1 freeze-dried wood) of fatty/resin acid fractions in P. contorta (Merrit) sawnwood infected with fungi for 28 days Logs ' Sawnwood FARA 3 T G 3 FARA TG Species Layer 1 Layer 2 Layer 1 Layer 2 Unseasoned control 2.210.3 3 9.210.6 2.2 ±0.3 2 2 ± 0 3 9.2 ±0.6 9.2 ±0.6 Seasoned control 8.4 ±0.8 4.0 ±0.6 5.0 ±0.8 5 0 ± 0 8 6.8 ±0.9 6.8 ±0.9 C. resinifera L 8.7 ±0.8 3 0.210.1 n.a.4 n.a. n.a. n.a. C. resinifera C 7 ± 1 0.13 10.07 6.1 ±0.8 6 9 ± 0 4 0.7 ±0.4 1.2 ±0.2 C. resinifera X 11 ± 1 0.810.2 5.5 ±0.7 7 2 ± 0 2 0.7 ±0.5 1.0 ±0.2 Leptographium sp. Q 7± 1 4 6.5 ±0.5 8± 1 3± 1 3.7 ±0.5 Leptographium sp. D 6± 1 3.3 ±0.7 5± 1 0.7 ±0.5 0.9 ±0.4 0. piliferum H n.a. n.a. 5 ±0.7 5 6 ± 0 3 0.8 ±0.5 1 ± 2 0. piliferum W n.a. n.a. 6.7±0.8 9± 1 2± 1 3± 1 0. piceae EE n.a. n.a. 4.4 ±0.7 5 ± 2 3.6 ±0.5 3.9±0.3 O. piceae F n.a. n.a. 2.5 ±0.7 3± 1 2.4 ±0.5 3.9 ±0.2 O. floccosum DD n.a. n.a. 6 ± 3 4 8 ± 0 6 1.9 ±0.1 2.2 ±0.8 O. floccosum V n.a. n.a. 2± 1 4 7 ± 0 8 1.2 ±0.5 2.0 ±0.1 0. minus P n.a. n.a. 3.8 ±0.7 4 7 ± 0 6 3.6 ±0.5 3.3 ±0.2 A. pullulans A n.a. n.a. 6.4 ±0.7 7± 1 0.9 ±0.5 2.1 ±0.5 A. pullulans HH n.a. n.a. 5.8 ±0.7 5 ± 1 1.6 ±0.4 3.2 ±0.5 Lipid concentration was not significantly different in unseasoned logs and unseasoned sterile sawnwood. Reported values are averages of all samples. Error values equal 95% confidence. Control n=12, log fungi n=3, sawnwood fungi n=7 or 8. FARA: fatty and resin acids, TG: triglycerides —: not present, n.a.: not measured 51 u <J c/J "a. 3 fc 5 is s, Q. I a •2 , « .2 Si u e •a o o a o o a OS n.a. © +1 T f n.a. 0±0. 1 CN 00 VO d d ,7±0. n.a. 8±0 m VD Os os r-© © © © -H -H -H -H T f oo O oo T f CN >n CN in Os OS Os © © © © -H +1 -H -H CN 00 —< r-; vd r~ vd OS T f CN CN © O © O +1 -H -H -H CN vo T f ' '—' T f CN OS OS T f © © © © -H -H -H -H CN rn — T f t-^ vo in vd VO r~ Os m O o o © -H +1 +1 +1 p VO r-; OS rn d in in vo CN T f r-; © © © © +1 +1 •M •» CN 00 os vq in T f >n rn m 00 00 O © d d +1 -H -H -H o o o o vd vd vd vd T f T f T f T f © © d d -H -H •H -H rn rn rn m in >n >n in days days days days T f oo CN T f 00 _ cN MC -80 MC o oo wn _0J "H. I on o a ) <D 00 03 "3 > -a <u t o a. a) CD > s > > T f II c c CO a u 43 o 60 0X) <o N T >n os O c/> CD >- T3 •a g 3 ca cu ca ca > 3 fe .£• fc CN i n •2 H ca FA consumption is more complex. To examine FA consumption, we performed a mass balance of TG and FA. First, the difference in TG between unseasoned controls and fungal or seasoned control samples was calculated (Equation 2-3). Since FA comprise about 95% of the weight of TG of non-infected lodgepole pine (Gao 1996), the theoretical mass of FA produced by TG hydrolysis in a sample could be derived and subtracted from die difference between F A s a m p | e and F A R A u n s e a s o n e d c o n I r o | (Equation 2-4). = TGumeasnnillkontrol — TGsampk (2 — 3) FAconsumed = FARA.san,ple ~ FARAun_dclmtml - 0.95 X ATG (2 - 4) On both sawnwood and logs, there was often large variability between isolates of a single species and no clear relationship between species and consumption of TG-derived FA. Consumption of TG-derived F A averaged 30% (0 to 60%) in logs, 60% (25 to 100%)in Merrit sawnwood layer one and 40% (0 to 80%>) in sawnwood layer two. In kiln-conditioned Edson sawnwood, TG-derived FA consumption ranged between 20% to 85% in 80MC wood and 0%> to 10% in 15-80MC wood (Table 2-8). No significant changes were seen for M G and S/FA1/DG (data not shown). The steryl ester/ wax (SE/W) fractions did not resolve well by GC and thus were not examined in this work. 2.3.5 Pigmentation and growth in defined media The results of the pigment colour scores and growth diameters after 14 days are shown in Table 2-9 for four selected isolates that were representative of their respective species (all isolates presented in Table 2-2, with the exception of Aureobasidium pullulans, were tested on defined media). Mannose consistently yielded the densest growth and the darkest colour in all tested fungi. Leptographium and O. piliferum had identical colour scoring on all carbon sources. O. piceae had a very similar colour ranking except for glucose and linoleic acid. Interestingly, 53 C. resinifera has an almost reversed ranking order compared to the other species. Linoleic acid yielded dense growth and dark brown colour in C. resinifera but light growth and light pigmentation with the other fungi. Furthermore, C. resinifera had negligible growth on glycerol. However, this carbon source yielded good growth and dark pigmentation in the other fungi. Table 2-9: Pigment score 1 and growth diameter of selected isolates on tested carbon sources. Species C-source C. resinifera C Leptographium R O. piceae EE O. piliferum H Colour score 1 Diameter (mm) Colour score Diameter (mm) Colour score Diameter (mm) Colour score Diameter (mm) Mannose 5 50 5 50 4 45 4 45 Linoleic Acid 4.5 50 3 30 2.5 25 3 40 Starch 4.5 45 4.5 45 3 50 4 45 Glucose 4 45 4.5 50 2 30 4 30 Arabinose . 3.5 50 4.5 45 3.5 20 4 35 Olive Oil 3.5 50 4.5 50 3 20 4 30 Glycerol +/- +/- 5 50 3.5 50 4 45 5: black/very dark brown, 4: dark brown, 3: brown, 2: light brown, 1: beige, 0: white, +/-: negligible To test mannose or glucose preference of the fungi, several species were grown on defined liquid media (containing 1:1 glucose:mannose) for up to 196 hours without agitation (to promote hyphal versus yeast-like growth). Growth and pigmentation patterns were similar to the defined solid media described above and HPLC analysis of sugar consumption showed no difference in the utilization of both sugars by the tested fungal isolates after 196 hours incubation. The results of sugar consumption for selected species are shown in Figure 2-9. 54 Figure 2-9: Glucose (A) and mannose (B) concentrations in glucose:mannose (1:1, w/w) liquid media during incubation with selected sapstain fungi. Fungi were inoculated onto shallow liquid media (in triplicate) and incubated at 18C without agitation in order to promote hyphal (versus yeast-like) growth. Samples were analysed for sugar content by HPLC as described in Materials and Methods. Cc C: C. resinifera C; Lepto Q: Leptographium sp. Q; Opil H: O. piliferum H ; Opic EE: O. piceae EE. Similar results were obtained for other tested fungi (not shown). 55 2.4 Discussion C. resinifera grew more rapidly in fresh logs than the other sapstain fungi species. Similar results were reported by Uzunovic and Webber (1998) who noted that this species and Leptographium wingfieldii were able to colonize Scots pine logs rapidly. C. resinifera typically caused a deep radial stain, often reaching the heartwood boundary, while other species such as Ophiostoma spp. only penetrated a short radial distance (Figure 2-3 and Figure 2-4). However, on fresh y-sterilised wood most fungi were able to fully colonize and stain the entire block with the exception of O. piceae and A. pullulans which stained only the top 6.25 mm-layer closest to the point of inoculation (Figure 2-5). No significant trends were found between growth, stain, and moisture content in Merrit pine samples (data not shown). Infection of 15-80MC kiln-dried wood showed that the physical changes to sawnwood that occur during drying, such as the closure of bordered pits (Figure 2-8) may play a significant role in preventing C. resinifera to colonize wood. The closure of the bordered pits in 15-80MC sawnwood also reduced, but did not prevent other tested species from colonizing the blocks (Figure 2-6). Such physical changes do not rule out, though, other chemical changes that may occur to reduce or prevent fungal growth. The production of a chemical during the drying process has been previously noted. Strobel and Sugawara (1985) grew Ceratocystis montia (a sapstain fungus associated with the Dendroctonus ponderosae bark beetle) on media comprised of fresh Lodgepole pine sawdust, and dried-and-re-wet sawdust. They also extracted each type with water or ethanol for use as a media. They noted that the fungus had superior growth on dried sawdust that was extracted with ethanol, whereas dried sawdust extracted with water had the least amount of growth. The authors postulated that the drying process results in the production of ethanol soluble inhibitor(s), though no follow up studies have been found. Such chemical changes might include modification or increased production of resin acids (due to host defence mechanisms in dying tissue), or possibly yet unexamined phenomenon. Testing host viability of infected logs showed that C. resinifera 56 usually had a very small region of dead host tissue at the stain boundary. This was also true for most other species except for Leptographium spp., which caused host cell death far ahead of the stain boundary (Figure 2-4). Tissue viability of the sapwood in the logs, coupled with aggressive growth by the deep stainers support previous studies on Pinus sylvestris suggesting that living host tissue may stimulate C. resinifera (Uzunovic and Webber 1998). However, none of the fungi actually required living host tissue for growth and pigmentation as demonstrated by their growth and pigmentation on y-sterilized wood. Further research examined the changes in wood nutrients of non-infected control and infected wood samples. The absence of starch in the Merrit pine samples was probably due to seasonal fluctuation of the food reserves as the trees would have been about to flush and these reserves mobilized for bud and leaf growth. Depletion of starch reserves by Pinus trees over the winter and early spring season followed by replenishment over summer and fall has been well documented (Fischer and Holl 1992; Sauter and van Cleve 1994). Saranpaa and Holl (1989) noted that hemicelluloses could be a food reserve in trees. In non-infected fresh logs, the changes in soluble sugars might be due to hydrolysis of cell wall hemicellulose for use as a food source by the host. Therefore, the depleted sugars, mannose and glucose, were probably consumed as a food source, whereas the increased arabinose was not utilised by the host tissue. Some migration of free sugars towards the surface layers during kiln drying was observed. This trend has been well documented during both air and kiln drying of sawnwood Pinus species and further, has been shown to promote mould growth (Terziev 1996). The consequences of sugar migration would reduce the ability of most sapstain fungi to penetrate host tissue. Changes in the lipid concentrations of non-infected control logs were likely due to the presence of host lipases that hydrolyse the TG fraction into glycerol and free fatty acids. In addition, during storage, wood extractives can undergo abiotic volatization and oxidation (Nugent et. al. 1977). Further, plant triglycerides can undergo oxidative degradation during long-term storage or short term heating 57 (Hannan 1991, O'Keefe et. al. 1993). We observed this seasoning effect in y-sterilised wood, though to a lesser degree than in logs. Thus, small changes in sawnwood extractives were most likely due to abiotic processes. Further investigation examined whether wood nutrients were consumed differentially by C. resinifera versus other fungi such as O. piceae. Due to the 28-day incubation period, it was impossible to separate concurrent changes in nutrients due to fungal infection and natural seasoning. This is due to the fact that fungi may consume or produce some of the products of the natural seasoning process or they may alter the rate of seasoning. Moreover, the 28-day control logs and sawnwood samples had different levels of seasoning, thus creating an unstable baseline for comparison and complicating the presentation of the results. Consequently, all comparisons were made to fresh, unseasoned controls. In both billets and sawnwood infected with fungi, mannose was overall the most depleted monosaccharide, followed by glucose (Table 2-4 and Table 2-5). This may be due to easier accessibility of the mannose in wood or a genuine preference for mannose by sapstain fungi. Further study of sapstain fungi in vitro, though, showed that there was no preference for glucose or mannose when both sugars were freely available in a liquid media (Figure 2-9). The high degree of consumption of mannose in wood by fungi combined with consistent yield of dark pigment and excellent growth with mannose as a sole carbon source in defined media indicates that mannose may have a significant impact on stain production by fungi. It has been previously shown that carbon and nitrogen sources can affect the pigmentation and melanin type of O. floccosum (Eagen et. al. 1997). Kaarik (1960) found that mannose was a superior carbon source for growth and sporulation of many sapstain fungi, including C. virescens (formerly O. coerulescens) and O. piceae. However, no correlation between in vivo mannose and sapstain has been previously reported. In a study on chemical composition of fungal-decayed wood, Worrall et. al. (1997) noted that decay fungi that selectively delignify (preferentially attack hemicelluloses and lignin) birch also had a larger 58 decrease of total-wood mannose versus other sugars. These researchers also noted that total-mannose utilisation usually exceeded total-glucose uptake by decay fungi on pine. This similarity between decay and sapstain fungi may imply that accessibility to mannose is the key factor. Conversely, arabinose generally increased in fungal-infected wood. However, the changes were much larger in the fresh logs when compared to the very small changes seen in sterilised sawnwood. This was especially true for Leptographium spp. where arabinose increased in logs by over 50 fold (1000 ± 100 ppm). This points toward either a host defence mechanism or the combined effect of hemicellulolytic activities of sapstain fungi and host tissue. Lipids can comprise 1-10% of the dry weight of standing P. contorta sapwood and consist of triglycerides, diglycerides, monoglycerides, fatty acids, resin acids, sterols, steryl esters, fatty alcohols, waxes, phenolic compounds and terpenes (Shrimpton 1972). Their exact compositions vary depending on the wood species, tissue type and season. It is important to note that in wood, lipids are present in at least ten times greater amounts than the soluble sugars, are a more concentrated form of energy and are thus a more important food source. Previous studies have examined lipid consumption in softwoods by sapstain fungi (Blanchette et. al. 1992; Brush et. al. 1994; Chen et. al. 1994; Gao et. al. 1994; Martinez-Inigo et. al. 1999). These studies focussed on O. piliferum or O. floccosum and found near complete reduction of TG and FA. Further, it has been demonstrated that some sapstain fungi produce extracellular lipases that hydrolyse wood TG (Gao and Breuil 1995). In logs, C. resinifera and Leptographium spp. reduced TG to very low or undetectable amounts (Table 2-7). On fresh and 80MC sawnwood, these two species were also the most effective at TG reduction, though to a lesser degree than on fresh logs (Table 2-8). As noted above, most fungi had reduced growth on wood conditioned to 15% M C then re-wet to 80%MC. This was concatenate with reduced TG and FA consumption. A l l of the remaining species reduced TG content of sawnwood. Infection with O. piceae and O. minus resulted in the least TG reduction. This might indicate that deep stainers produce larger amounts or more robust 59 extracellular lipases than the other fungi. Most fungi consumed around half of the F A derived from TG hydrolysis though there was wide variability among isolates. Moreover, there were no obvious trends in consumption between the species. On defined media, the dense growth on linoleic. acid-supplemented medium by C. resinifera C supported the results observed in sawnwood and logs (20 to 50% reduction of TG-derived FA). On agar glycerol media, C. resinifera C did not show a dense growth, indicating that this carbon source was poorly assimilated (Table 2-9). The reason for this is unknown. Similar results were obtained for the other C. resinifera isolates (data not shown). However, the growth media must be optimized and glycerol liquid media should be tested before this result can be confirmed. The other fungi can apparently utilise both the glycerol and fatty acid portion of the triglyceride, though it is unclear if either portion is preferred. On defined carbon source media, the ranking of colour scoring was reversed (aside from mannose) for C. resinifera compared to the other fungi (Table 2-9). Similar results were obtained for the other C. resinifera and Ophiostoma spp. isolates (data not shown). This further indicates differences in wood nutrient preference between the fungi. 2.5 Conclusions The presented results suggest that there is (are) some aspect(s) to living host tissue that may stimulate growth and pigmentation by C. resinifera. Uzunovic and Webber (1998) previously noted that living host tissue might somehow stimulate colonization by deep stain fungi. Conversely, it may simply be that the closure of bordered pits during the wood products manufacturing process may reduce or arrest growth by the deep stain fungi. It was observed that reduced fungal growth on wood with closed border pits was concatenate with reduced consumption of wood nutrients. Nutrients in wood (such as mannose or TG-bound glycerol and fatty acids) may play an important role in pigmentation and growth, but it appears that other factors, such as changes to wood ultrastructure or other biochemical factors, are also critical. 60 Thus, some explanation for the differences in fungal distribution between logs and lumber may lie in the access that fungal species have to the host nutrients. 61 Chapter 3 Genetic Analysis of DHN Melanin Genes in Sapstain Fungi 3.1 Introduction It is well documented that sapstain fungi, including Ceraratocystis spp. and Ophiostoma spp., produce melanin for pigmentation (Zink and Fengel 1988, 1999, 1990; Brisson et. al. 1996; Butler and Day 1998). However, until recently, little was known of the biosynthetic pathways used by these species to produce melanin. As described in Section 1.3 above, there are three known pathways for melanin biosynthesis in fungi: GHB (glutaniminyl-4-hydroxybenzene), Catechol, and D H N (1,8 dihydroxynaphthalene). However, only the D H N pathway (Figure 1-2) has been described in Ascomycetes (Bell and Wheeler 1986, Butler and Day 1998). D H N melanin has been relatively well-studied in phytopathogenic fungi (including Alternaria alternata, Cochliobolus heterostrophus, Colletotrichum lagenarium and Magnaporthe [=Pyricularia] griseae) in addition to human pathogens (including Aspergillus fumigatus, Sporothrix schenckii, and Wangiella dermatitidis) (Wheeler 1983, Wheeler and Stipanovic 1985, Shimizu et. al. 1997, Butler and Day 1998, Kawamura et. al. 1999, Tsai et. al. 1999, Romero-Martinez et. al. 2000). Several genes encoding the DHN synthesis proteins polyketide synthase (PKS), scytalone dehydratase (SD) and the naphthalene reductases (1,3,8- trihydroxynapthalene reductase and 1,3,6,8-tetrahydroxynapthalene reductase; 3HNR and 4HNR, respectively) have been characterized from many of these species (Kubo et. al. 1989, Kimura and Tsuge 1993, Vidal-Cros et. al. 1994, Takano et. al. 1995, Perpetua et. al. 1996, Thompson et. al. 2000). In C. lagenarium, the PKS1 gene was found to contain a 6701 bp open reading frame, consisting of three exons separated by two short introns and yielded a primary transcript of 6561 bp. A gene map for PKS1 is shown in Figure 3-1 A. The deduced polypeptide of PKS1 consists of 2187 amino acids and 62 shows significant similarities with other polyketide synthases, particularly with wA in Aspergillus nidulans, involved in conidial pigmentation. The PKS1 gene contains highly conserved B-ketoacyl synthase, acetyl/malonyl transferase, and acyl carrier protein domains, though there is currently no structural information on the active site residues of the native enzyme (Takano et. al. 1995). In M. grisea and C. lagenarium, the gene encoding 3HNR contains a 1503 bp open reading frame, consisting of six exons separated by five introns and yields a primary transcript of 846 bp (Andersson et. al. 1996, Perpetua et. al. 1996). The gene map for THR1 from C. lagenarium is shown in Figure 3-1B. The protein crystal structure consists of a 120 kDa tetramer comprised of four identical monomers (282 amino acids each). The nine naphthol-binding site residues that have been identified for 3HNRare: Ser-164, He-165, Tyr-178, Met-215, Tyr-216, Cys-220, Tyr-223, Trp-243 and Met-283. The catalytic triad is comprised of Ser-164, Tyr-178 and Lys-182 (Andersson et. al. 1996). In M. grisea, the 825 bp cDNA sequence encoding 4HNR was recently published and the deduced 274 amino acid sequence was found to have a 46% identity to that of 3HNR from the same species (Thompson et. al. 2000). Polypeptide sequence analysis showed that five of the nine active site residues were conserved between 4ITNR and 3HNR. Of the remaining residues; Tyr-216 changed to Phe, Cys-220 changed to Ser, Trp-243 changed to Met, and Met-283 was missing in 4HNR. The 4HNR protein was produced and purified to homogeneity via transformation of Escherichia coli with cDNA that encoded 4HNR. Substrate competition experiments indicated that 4HNR prefers 4HN to 3HN by a factor of 310; and that 3HNR prefers 3HN to 4HN by a factor of 4.2. 4HNR had a 200-fold larger K; for the fungicide tricyclazole than that of 3HNR, and this accounts for the latter enzyme being the primary physiological target of the fungicide. Further modelling also suggested that the absence of Met-283 (in 4HNR) was responsible for the preference of 4HNR for 4HN (Thompson et. al. 2000). 63 Figure 3-1: Feature maps of C. lagenarium DHN melanin biosynthesis genes PKS1 (A), THR1 (B) and SCD1 (C). The open reading frames are indicated as boxes. TATA signal indicated in (A) and (C) at 255 and 117, respectively; exon sequences indicated in hatched boxes; intron sequences indicated in white boxes; polyA signal indicated in (A), (B) and (C) at 7604, 1828/1835/1848, and 1054/1073, respectively. Annotated numbers indicate starting nucleotide positions of different features. Bottom ruler units in nucleotide bases. The arrows indicate PCR primer amplification sites for PKS3/PKS6 (A), T29F/T14R (B) and SD1/SD2 (C). Data adapted as follows: PKS1 (GenBank D83643, Takano et. al. 1995), THR1 (GenBank D83988, Perpetua et. al. 1996) and SCD1 (GenBank D86079, Kubo et. al. 1996). THR1 based on cDNA and thus TATA signal is unknown. 64 The SCD1 gene of C. lagenarium encoding SD (shown in Figure 3-1C) contains a 690 bp open reading frame, consisting of three exons separated by two introns, and yields a primary transcript of 564 bp. The deduced SD polypeptide consists of 188 amino acids (Kubo et. al. 1996). Studies of the SD crystal structure from M. grisea, showed that the final enzyme is a 69 kDa trimer comprised of identical 23 kDa subunits and functions without metal ions or cofactors. The hydrophobic active site was found to be buried within the interior of the enzyme and consists of eight residues: Tyr-30, Asp-31, Tyr-50, Lys-73, His-85, His-110, Ser-129 and Asn-131 (Lundqvist et. al. 1994). Computer modelling has showed that the tyrosine residues may be responsible in binding and polarization of a critical water molecule that in turn stabilizes the transition state and acts as a general acid (Zheng and Bruice 1998). Further, site-directed mutagenesis has shown that all residues, with the exception of Lys-73, are important for enzymatic activity and that the two histidines were most critical (Basarab et. al. 1999). Eagen first showed that melanin was produced in the sapstain fungus O. floccosum 387N via the D H N melanin synthesis pathway by using known inhibitors and molecular techniques (Eagen et. al. 2001). Our group has since isolated, purified, and sequenced genes encoding SD, 3HNR and 4HNR in O. floccosum 387N and has shown functional homology to known D H N pathway genes (Eagen et. al. 2001, Wang and Breuil in press, Wang et. al. 2001). However, until now, there has been no data on the melanin synthesis pathway(s) of other sapstain fungi, or the genetic relationships of the genes in these pathways among sapstain species. There are several reasons for targeting the D H N melanin synthesis pathway in sapstain fungi. First, this biosynthetic pathway is the source of the cosmetic defect caused by these fungi; thus it is prudent to gain knowledge of this important feature of sapstain. Second, because D H N melanin synthesis is a critical parameter for phytopathogenicity in many fungi, it is a target for fungicides to control many plant diseases. Therefore, it is possible that this biosynthetic pathway 65 could be a target for sapstain control as well. Third, the biocontrol agent Cartapip97™ (an albino strain of the sapstain fungus O. piliferum described in Sections 1.2.2 and 1.2.3.4.2) is currently being tested for control of sapstain in the lumber industry. However, little information is known about this organism and the reason(s) for its lack of pigmentation. To ensure safe and responsible use of this biocontrol agent, the organism should be well studied and described. This includes a thorough description of the pigmentation pathway used by members of this species. In this chapter, we first demonstrate the presence of the D H N melanin synthesis pathway in species of Ceratocystis and Ophiostoma, using known inhibitors of the D H N pathway. This was followed by the isolation and sequencing of partial DNA sequences coding for PKS, 3HNR and SD enzymes. These sequences were then compared to other known, respective sequences from other fungal species and the results discussed. 3.2 Materials and Methodology Except where noted, all chemicals were supplied by Sigma Chemical Company (St. Louis, MO), and methods were derived from Current Protocols in Molecular Biology (Ausubel et. al. 1994). 3.2.1 Fungal strains and growth conditions The fungal strains Ceratocystis resinifera 125-214 (also known as C. coerulescens type C), Ophiostoma minus 58-4, O. piceae 187-1, O. piliferum 198-2gf and O. setosum 160-38 origintated from Canada (see Table 2-2) and were obtained from the UBC Department of Wood Science culture collection. O. floccosum 387N originated from Mason, Quebec and was obtained from the Forintek Canada Corporation culture collection (Ste. Foy, PQ). C. pinicola 0901 (also known as C. coerulescens type A), O. floccosum GrlO, and O. minus OM3 originated from 66 Thetford Forest, U K , and were courtesy of Dr. Adnan Uzunovic (Forintek Canada Corporation, Vancouver, BC). O. piceae W5, and O. piliferum 201-1A originated from Oldenberg, Austria and were courtesy of Susanne Schroeder (University of Oldenberg, Oldenberg, Germany). O. setosum NZFS3734 originated from New Zealand and was courtesy of Dr. Bernard Kreber (Forest Research Institute, Rotorua, New Zealand). Working cultures were maintained on 2% malt extract agar (MEA; Oxoid Ltd., Basingstoke, UK) for no more than three subcultures to maintain physiological characteristics of the culture. Stock cultures were stored in 10% glycerol at -80°C. 3.2.2 Bacterial strains and growth conditions Escherichia coli strain TOP 10 (Invitrogen Corporation; Carlsbad, CA) was used for all transformation reactions. The transformed bacterial strains were grown at 37C on Luria-Bertani (LB) agar plates containing 1 u.g mL"' of ampicillin and 80 ug mL"1 of X-Gal (5-Bromo-4-chloro-3-indolyl beta-D-galactopyranoside) according to supplied instructions. Selected E. coli colonies were sub-cultured in 5 mL of liquid L B media with 1 ug mL"1 of ampicillin overnight with shaking at 37C in a G24 Environmental Incubator Shaker (New Brunswick Scientific Co., Inc.; Edison, NJ, USA). 3.2.3 DHN Pathway Inhibitor Studies The presence of the D H N melanin pathway was tested by inoculating selected fungi on mannose B-media (described in section 2.2.7) spiked with increasing concentrations of the DHN inhibitors tricyclazole, carpropamid or cerulenin. Tricyclazole (donated by DOW Elanco, Midland, MI) and cerulenin were diluted in ethanol solvent (0.05%) final solvent concentration) while 67 carpropamid (donated by Bayer A G , Montreal, PQ) was supplied in solution (300 g L" 1). Inhibitors were added to sterile molten media prior to setting in 60mm diameter Petri dishes. 3.2.4 Pur i f i ca t ion of D N A m o l e c u l e s 3.2.4.1 Pur i f i ca t ion of G e n o m i c D N A f r o m F u n g a l S t r a i n s Purification of genomic D N A from fungal strains was adapted from the method of Kim et. al. (1999). Fungal stock plugs (4 mm diameter) were inoculated and spread with sterile distilled water onto 2% M E A plates overlaid with cellophane membrane (BioRad ; Hercules, CA) and incubated at 18C for 2-5 days. Approximately 200mg of mycelia was collected from M E A cellophane plates, placed in a 2ml cryo tube (Eppendorf A G , Mississauga, ON.), and then frozen at -80C until required. For D N A extraction, 300pl of extraction buffer (50mM Tris-Cl, 50mM EDTA, 3%SDS, pH 8.5) was added to each tube. A custom-made drill bit was then used to disrupt the mycelia with 5 minutes of drilling (200 rpm) in one-minute on/off intervals; the mycelia were kept on ice for the entire process. After drilling, the resulting suspension was mixed with 150pL sodium acetate (3M, pH 5.2) then stored at -20C for 20 min. The mixture was then melted at 4C, micro-centrifuged at 13000 rpm for 15 minutes and the supernatant transferred to a new cryo-tube. If the supernatant was turbid, it was centrifuged again. The supernatant was mixed 1:1 with phenol chloroform and vortexed for 30 seconds, and then centrifuged (13000 rpm, 5 minutes). The supernatant was recovered and another cycle of phenol chloroform extraction was then performed. This step was added to obtain high purity DNA. The supernatant was then recovered and mixed with an equal volume of isopropanol, stored at room temperature for 5 minutes and centrifuged (13000 rpm, 20min). The supernatant was discarded and the resulting pellet was washed briefly with 400pL of 70% ethanol, and then gently re-dissolved in 50-100 pL TE buffer (lOmM Tris-Cl, ImM EDTA, pH 8.0). D N A concentration was measured via 68 absorbance at 260/280 nm with a GeneQuant II R N A / D N A calculator (Amersham Pharmacia Biotech, Baie d'Urfe, PQ) and then diluted to 200 ng uL/ 1 with sterile distilled water. 3.2.4.2 DNA purification from agarose gel D N A was purified from agarose gels by using a QIAquick Gel Extraction Kit (QIAGEN Inc., Missisauga, ON) following the supplied protocol. If the D N A was to be used for sequencing, then all optional steps as indicated in the manual were included, otherwise these steps were not used. 3.2.5 Primer Design Primer sequences were based on previously published primers and sequence data for target genes. A summary of the tested primers is shown in Table 3-1. 69 Table 3-1: Oligonucleotide sequences and intended use. Name 1 Sequence 2 U s e 3 P K S 3 4 5 ' - T T C T T C A A C A T G T C Y C C Y CGIGA-3' PKS P K S 4 4 5 ' - C G Y G C G G T I G T ^ G T ^ T A £ G C - 3 ' PKS P K S 6 4 5 ' - C T G ^ G T A C C y GJj C C ^ T G C A - 3 ' PKS S D 1 5 5 ' - G A ^ T G G G C I G A Y f § I T A Y G A - 3 " SD S D 2 5 5 ' - G C I G C ^ A A Y T T C C A I A C I C C - 3 ' SD T 2 9 F 5 5 ' - G G - ^ - A A ^ G T - § - G C I Y T I G T - § - A C I G G ^ - G C I G G - 3 ' T T T T 3 H N R T 1 4 R 5 5 ' - § T A I G C Y T C I C Y I G C I A C ^ A A ^ A A Y T G I C C - 3 ' 3 H N R T H N 4 4 5 ' - T T G A C C G G T G C C G T C T T G - 3 ' 3 H N R T H N 4 2 5 ' - T T ^ A C C G G C G C § § ^ G T T G - 3 ' 3 H N R T H N 3 3 5 ' - C G C ^ A A C G T T G T T G T C A A C - 3 ' 3 H N R T H N 3 1 5 ' - C G C ^ A A G G T C ^ T T G T C A A C - 3 ' 3 H N R M 1 3 F 6 5 ' - G T A A A A C G A C G C C C A G - 3 ' TOPO M 1 3 R 6 5 ' - C A G G A A A C A G C T A T G A C - 3 ' TOPO Forward (coding) primers indicated by ' F ' or odd numbering. Reverse (non-coding) primers indicated by 'R ' or even numbering. Degenerate base positions indicated by fractional positions or ' I ' (inosine). PKS: Polyketide synthase gene target (-720 bp). SD: Scytalone dehydratase gene target (-420 bp). 3HNR: 1,3,8- trihydroxynapthalene reductase gene target (-366 bp). TOPO: Used for PCR amplification of cloned D N A fragments from pCR 2.1 TOPO vector. Courtesy of H.Wang. Courtesy of R. Eagen. Ausubel et.al. 1994. 70 3.2.6 Polymerase Chain Reaction conditions A l l polymerase chain reactions (PCR) were carried out in a Hybaid TouchDown Thermocycler (InterScience; Markham, ON). A l l PCR reactions were performed in 50 pL volumes. Each reaction contained 40 pmoles of each of the two desired primers, 200 to 1000 ng of template D N A , 4 mmoles of dNTPs and IU of Thermostable Taq D N A polymerase (Rose Scientific; Edmonton, A B ) all in IX Thermo D N A polymerase reaction buffer (lOmM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 m M M g C l 2 , 0.001% gelatin). In some circumstances, Taq was added after the initial denaturing step (Hot start PCR) or the PCR reaction also contained 2% to 10% dimethylsulfoxide (DMSO). PCR reactions started with a 4-minute denaturing step (or 10 minutes when carrying out PCR directly from re-suspended transformed E. coli) at 94°C. Then 35 cycles of: a 94°C denaturing step for 50 seconds, followed by a 50-64C (depending on the primers and specificity required) annealing step for 50 seconds, and finally a 72C extension step for 50 seconds. After completion of the 35 cycles, the PCR reaction ended with a 72C step for 10 minutes to ensure complete synthesis of all D N A fragments. 3.2.7 Restriction Digests In some circumstances, restriction digests were carried out on genomic and PCR-amplified D N A . Samples of genomic D N A (200ng) were digested with 2U of Sail (Amersham Pharmacia Biotech) in I X buffer at 37C for two hours. PCR-RFLP of T29F/T14R PCR amplicons was carried out after the final PCR elongation step by addition of 2U of Haell (Amersham Pharmacia Biotech) to the 50 uL PCR reaction and incubation at 37C for one hour. 71 3.2.8 Ligation, Cloning and Transformation of PCR Products Freshly purified PCR products were cloned using a pCR-II® TOPO TA Cloning® Kit with TOP10 E. coli (Invitrogen Corporation, Carlsbad, CA) following the supplied protocol. Transformed colonies (white) were selected for further analysis. 3.2.9 Gel electrophoresis D N A fragments were resolved using agarose gel electrophoresis. Aqueous D N A samples were mixed 5:1 with 6X loading buffer (20% glycerol, 0.25% xylene cyanol) and loaded into 1 to 2 % agarose gels (depending on the expected size of the DNA fragment) containing 0.004% EtBr solution (supplied as a lOmg mL"' solution, Bio-Rad Incorporated). Gels were electrophoresed at 6V cm"1 for 10 to 30 minutes. D N A fragments were visualized under U V light and photographed with an Ultra-liim 6000D image analysis system (Ultra-Lum, Claremont, CA). 3.2.10 Southern Blotting In some circumstances, transformed E. coli were screened for the correct insert using a Southern blotting technique. White colonies from the TOPO TA Cloning® reaction were streaked in a grid pattern onto L B agar plates containing ampicillin and X-Gal (as in Section 3.2.2 above) and incubated overnight at 37C. Each plate was blotted directly with a Hybond N + nylon membrane (Amersham Pharmacia Biotech) and the membranes were then washed according to the supplied instructions and fixed at 80C for 2 hours. The probe was prepared from E. coli transformant 387N PCB' (courtesy of H. Wang) as follows. The transformant was PCR amplified with T29F/T14R, and the resulting amplicon was purified by gel electrophoresis and gel extraction as described above. lOOng of purified D N A was initially random primed with the Odyssey Random ' E. coli transformant 387N PCB contained the full length DNA sequence of the gene encoding 3HNR in O. floccosum 387N (Wang and Breuil submitted). 72 Priming Kit (#928-13200, Li-Cor Biosciences, Lincoln, NE) according to the manufacturer's instructions. Unincorporated nucleotides were removed with supplied G F X columns. The D N A was then labelled with Odyssey IRDye 700™ (#928-10020) according to manufacturer's instructions. Blotted membranes were pre-hybridized for one hour and then probed at 65C overnight using the Odyssey DNA Hybridization Solution Southern Blotting Kit (#927-10000). The membranes were then washed with Odyssey D N A Wash Solutions 1, 2, and 3 (#926-10400) according to the manufacturer's instructions. Membranes were then scanned at 680nm with an Odyssey Infrared Imaging System model 9120 (Li-Cor Biosciences). 3.2.11 Sequencing PCR fragments were cloned into the pCR-II® vector (Invitrogen Corporation) as above and then amplified directly from the resulting transformed E. coli using the M l 3 forward and reverse primers. The resulting products were gel purified and l00-300ng of the extracted DNA was sequenced. Sequencing reactions were carried out using an ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA) according to the supplied protocol. Nucleotide sequences were analyzed at the UBC Nucleic Acid and Protein Service Laboratory (University of British Columbia, Vancouver, BC) on an ABI 373 D N A sequencer (PE Applied Biosystems). 3.2.12 Phylogenetic analysis Sequences were analyzed for homology with other known sequences via the B L A S T N and B L A S T X search engines at the National Centre of Biotechnology Information website (National Library of Medicine; Bethesda, M D , http://www.ncbi.nlm.nih.gov/, Altschul et. al. 1997). The sequences were then translated and proof read using GeneTool Lite 1.0 software (BioTools Incorporated, Edmonton, AB). As well, online ClustalW (European Bioinformatics Institute,' 73 http://www.ebi.ac.uk/clustalw/, Thompson et. al. 1994) and MultAlin (Institut National de la Recherche Agronomique, http://www.toulouse.inra.fr/multalin.html, Corpet 1988) multiple sequence alignment servers were used for further analysis. ClustalW alignments were formatted with BioEdit 5!0.6 software (Hall 2001). Protein motif analysis was carried out with the ScanProsite software at the ExPASy proteomics server (http://ca.expasy.org/). Neighbor-joining analyses were carried out on the ClustalW alignments with with optimitality criterion set to distance using PAUP* 4.0b6 for Windows/DOS (Sinauer Associates Inc., Sunderland, MA. ) . Bootstrap percentages were calculated (n=1000 replications) based on neighbour-joining searches. Consensus trees were generated from PAUP* 4.0b6 ire output files with TREEVIEW software (Page 1996). 3.3 Results 3.3.1 DHN inhibitors Three D H N pathway inhibitors were tested at several concentrations in mannose B-media. These were: tricyclazole, carpropamid and cerulenin. Figure 3-2 shows typical results observed for this experiment. In C. resinifera and Leptographium spp, tricyclazole at 10 ppm reduced pigmentation from black/brown to medium brown, but little or no growth was observed at 100 ppm. In O. piliferum and O. piceae, reduced pigment and growth were observed at 100 ppm. No change was seen at 10 ppm. Carpropamid reduced pigmentation to grey in C. resinifera at 10 ppm, but no growth was observed at 100 ppm. For Leptographium spp, reduced growth was observed at 10 ppm and 100 ppm. For O. piliferum and O. piceae, we observed growth and a reduction of pigment from brown to light brown at 100 ppm. The inhibitor cerulenin was highly toxic to most fungi and prevented growth and pigmentation at the tested concentrations (1 ppm and 10 ppm). Other tested Ophiostoma spp. gave similar results to O. piliferum and O. piceae for all three inhibitors (data not shown). 74 C c C LeptoQ Opic EE Op//H CER 10ppm • • Figure 3-2: Examples of effect of DHN melanin synthesis pathway inhibitors on different sapstain fungi. Fungal isolates (indicated in top row) were grown for 14 days on 2% mannose B-media agar plates (60mm diameter) spiked with increasing amounts of inhibitors: carpropamid (CARP), tricyclazole (TCZ) or cerulenin (CER). Final inhibitor concentrations are given in parts per million (ppm). Cc C: C resinifera C, Lepto Q: Leptographium sp. Q, Opic EE: O. piceae EE, Opil H: O. piliferum H. 75 3.3.2 PCR amplification and sequencing of DHN genes from fungal genomic DNA 3.3.2.1 Polyketide synthase (PKS) PKS amplicons of about 720 bp were successfully amplified with the primer pair PKS3/PKS6 from fungal genomic D N A of C. pinicola 0901, C. resinifera 125-214, O. floccosum 387N, O. floccosum GrlO, O. minus OM3, O. minus 58-4, O. piceae W5 and O. setosum 3T54 (Figure 3-3). Despite attempts with different annealing temperatures of 45C, 48C, 52C, 55C, 58C, 62C and 65C combined with the addition of 2%, 3%, 5% and 10% DMSO in the PCR reaction, no correctly-sized PCR products were obtained for O. piceae 187-1, O. piliferum 198-2gf, O. piliferum 201-1 A, Cartapip, and O. setosum 160-38 with PKS3/PKS6. The primer pair PKS3/PKS4 did not yield any correctly-sized bands in any of the tested fungi. Two control reactions (each lacking one of the primers) were always processed alongside the tested primer pairs and generally did not yield background bands. Most of the obtained PKS sequences were found to have excellent B L A S T X E-scores (E < lxl0" 4 0 ) with known fungal polyketide synthases. Sequences with poor (E > 0.001) B L A S T N or B L A S T X E-scores were not included in further analyses. A C L U S T A L W alignment of the inferred amino acid sequences and the amino acid sequences of other known fungal PKS sequences from D H N melanin and toxin pathways is presented in Figure 3-4. The deduced partial amino acid sequences for PKS corresponded to residues 455 to 694 of the C. lagenarium PKS protein (Takano et. al. 1995). Motif analysis of the partial PKS sequences using ScanProsite at the ExPASy proteomics server indicated that the sequences contained a B-ketoacyl synthase motif found in other Type I PKS enzymes (Takano et. al. 1995). The active site cysteine residue of the motif was conserved in all sequences (Figure 3-4). 76 1600 1000 500 100 Cc 0901 Oflo387N Cc 125214 Omin 584 P3 P6 P3/6 P3 P6 P3/6 P3 P6 P3/6 P3 P6 P3/6 •• w ~720bp Oset 3734 Oflo Gr10 Opic W5 Omin OM3 P3 P6 P3/6 P3 P6 P3/6 P3 P6 P3/6 P3 P6 P3/6 1600 1000 500 -720bp 100 Figure 3-3: Agarose gel electrophoresis of PCR amplification of genomic DNA using primers PKS3 and PKS6. 20pL of fungal genomic DNA PCR reaction was electrophoresed in a 1.5% agarose gel (spiked with ethidium bromide) at 6V cm'1 for 20 minutes, and then visualized under U V light. Each fungal isolate comprised of three lanes: one reaction with both primers (P3/6) and two controls, each with either PKS 3 (P3) or PKS 6 (P6). Exterior lanes contained 100 bp ladder. Isolates included Cc0901: C. pinicola 0901, Oflo387N: O. floccosum 387N, Ccl25214: C. resinifera 125-214, Omin584: O. minus 58-4, Oset: O. setosum 3734, OfloGrlO: O. floccosum GrlO, OpicW5: O. piceae W5, and OminOM3: O. minus OM3. Fungal isolates that did not amplify were not included in this figure. 77 C. lagenarium i FFNMSPREAFQTDPMQRMALTTAYEAL E M C G Y V P N R T P S T R L D R I G T F Y G Q T S D D W R E I N AAQ - - E VD TYY I TG G - - V R A Nodulisporium sp. i L . . S . . . . Phomasp.WA i . Y . . . . . . S . . . . ... A F . . L . 0.setosum3734 i . L . . . . . I S . . F . . S . . . . . S . 0. piceae W5P i GL . . . . K . I S . . F . . S . . . . . S . 0. floccosum OilO i . L . . . . . I . . . F . . S . . . . . S . 0. floccosum 387N i . L . . . . . I . . . F . . S . . . . . S . C.piiucolaOPOl i . . S . . . . S L C. resinifera 125211 i . . S . . . . L . . . R . A. parasiticus_WA major i - . KNS SK. . L V . . . . . S . . . G ..R..T.S Y.DV . D I F . . I . 0. minus 0M3 i . A . . . . Q . L L . L . T . . . A . . S . D AAR ...S.V Y.DV . . S . . . D . F . . I . 0. minus 58-4 i . A . . . . Q . LL . L . T . . . A . . A . D AAR ...S.V Y.DV . . S . . . D . F . . I . A. nidulans WA i . L . A . . A . . L L . . . . G A . F . . DS....QR..V.I...M....Y..V . S G . . D I F P . . N . A. fumigatus Alblp i . L . A . . A . . L L S . . . . A . F . . .SS...QR..V.I.M.M....Y... . S G . . D I F P . . N . A. parasiticus WAminor i . L . A . . A . . L L . . . . . A . F I . DS....QR..V.L...M....Y... . S G . . D I F P . . N . P. patulum WA i . L . A . . A . . L L . . . . . A . F I . D S . . . . QKN.V.V...M....Y..V . SG . . . D I F P . . N . A. parasiticus PKSL1 i . . G I . K . .P.M.. A . . MST . . . M . R A . L . . DT....QR....V.H.V..N..M.T . T . . . . N I F . . N . GA. nidulans PKSST i . . S I . . K . .P.M.. A . . MST . . . M . R G . I . . DT. . . . Q RN. . .V.H.V. .N. . M . T . T . . .  N I F . . N . GG. moniliformis PKStox i . . S . GMT . V S D I . . Q • • L . E V . . . CMQ S S . Q - -TNWRGSN CYV.VWGE..LDLHSKDLYDSG.. R V S . . HD F C. hetenjstrophusPKSl i V . . A . . AAL. . Q ; : L . ECS. . . F .NS.TPMSKIVG.D- - - T S V . V S S F A T . Y TDMLWRD PES. PM. QC NSGF S . S Clustai Consensus 1 * * . * * * . : * : . . . * : : * 100 1 . 1 . . \ . 110 130 130 MO 150 160 17t 1 C. lagenarium 87 GF S U P S L N V D T A C S S S A A A L N V A C N S L W Q K D C D T A I V G G L S C M T N P D I F A G L S R G Q F L S K T G P C A T F D N G A D G Y C R A D G C Noduhsporiumsp. 87 I Q. . . T RA . E s . . . s , EHN . N , , . , D Phomasp.WA 87 I V . QI . . .V . . . . S O.setosum 3734 87 I M T . A R . . . S . , n . G . . 0. piceae W5P 87 I . M T . , .AR.... 0. floccasumOrlO 87 I M T . A R . . . . . . I S A . G . . 0. floccosum 387N 87 I M T . AR. . . . . . I s A . G . . C. pinicola0901 87 I Q. . . TA . . AQE .V s . . . S . K . . . n . C. resinifera 125214 87 I Q. . . T A . . AQE .V s . . . S . K . . , n , A. parasiticus_WAmajor 86 K . E . F S I. I QL. . T . . . S G . . . . . V T . . . . V L . S . . L . S Q. . S . K . . . K A N V 0. minus 0M3 87 . WE . YS S I QL . . S . . L SRE . . . V A . . AN F L . S S . L S... . . G . K . . AD V V 0. minus 58-4 87 . WE . YS S I QL . . S . . L ARE . . . VA . . AN F L . S S . L S... . . G . K . . . AD V V A. nidulans WA 87 K V S . . I, I HL . . . . I . RN. . . . . T . . V N I L . . . . N H . . . D . . H . . . N . . V A. fumigaius Alblp 87 K V S . . L HL . . . A I .RN.... . . S . . VN L L .... NH... D .. H.. . R . . N . N . . D V A. parasiticus WAminor 87 K . . V S , , I. I HM. . . . I .RN...A . . A . . VNIL . . . . NH. . . D . . H. . . R . . N . N . . . n . V P. patulum WA 87 K . . . V S . . I. . I H . . . , RNES . S .VA..VNIL....NH...D..H... R . . N . T . . n , I A. parasiticus PKSL1 87 E . A . YTN , . I. . I HL . , . RG. . . . V A . . T N M I Y T . . G H T . . D K . F . . . R . . N . KP Y . D K , , E . VA. nidulans PKSST 87 E . Y SN I. I HL . RG. . . . V A . . T N M I F T . . G H T . . D K . F . . . R . . N . K A . . D A E . V G. moniliformis PKStox 84 D LK . FT IK A G. . . . L I H E.VRAIRAG...G . . . A. T N L V F S. TMS VAMTEQGV. . PDAS. K . . . AN N . A . GE A I C. heterostrophus PKS1 88 DLK . VL GGLT HL . . Q • • L VG. VRQ . L AA . S.LILG.EMMVTM.MMK. . . PD . R . YA . . E R N . A . GE . V Clustai Consensus * * * * * * * * * 190 200 310 330 330 340 C. lagenarium 177 ALADKDNVLAVI LGTATNHSADAI S ITHPHGPTQS IL SRAILDDAGVDPLDVDYVEMHGTG 237 Nodulisporium sp. 177 . . I • Q • . . . . S S . . . E . . 237 Phoma sp.WA 177 . I . . . . . S . . . . E D . . 237 O.setosum 3734 177 . E . . G . . I . . I . M . .... S .... E . . 237 0. piceae W5P 177 . E . . G . . I . . I . . . .... S .... E . . 237 0. floccosum GrlQ 177 . E . . G . . I . . . . S I . . 237 0. floccosum 387N 177 . E . . G . . I . . . . S I . . 237 C. pinicola09Ql 177 . E . . N . R . . . . V .V C . E . . I . . 237 C. resinifera 125214 177 . E . . N . R . . . . V .V C.E . . 237 A. parasiticus_WAmajor 176 . EL . N A . . . Q. V . . . AE . . . K . Y . E..QQS....F..G . . 236 0. minus 0M3 177 . I . . N . . I ...VRSAV. . E . V . . . A A . . E R . F . . A . N R . . L H . H . . . . A . L . . . . 237 0. minus 58-4 177 . I . . N . . I ...VRSAV. . E . V . . . A A . . E R . F . .A. NR. .LH.H. I. . A. L. . . . 237 A. nidulans WA 177 . N . P I . G . . N . AY . . E . V R . . VGA. A F I F K K L . N E . N . . .KNIS. I. . . . . . 237 A. fumigatus Alblp 177 . E . . N . P I .G..NAAY. . E . V R.-VGA. A F I F N K F F N . T N T N . H E I G . . 237 A. parasiticus WAminor 177 • QV . N . P 1 . G . . N . AY . . E . V R..VGA. A F I F N K L . N . . N I . . K . . S . . 237 P. patulum WA 177 • Q • . N . P I V G 1 . G . AY . . E . V R..VGA. . F I F D K L . N E S N S . . K E . S . I . . . . . . 237 A. parasiticus PKSL1 177 . N . P I .G...DAK. . MSE . M. R..VGA. I D N M T . A . N T T . L H . N . F S . I . . . . . . 237 A. nidulans PKSST 177 EN . P I . . T . . D I K . . MSD . M . R.FK.A. I D N M S . L . S T . . I S . . . L S . I . . . . . . 237 G. rnoruJJformis PKStox 174 ..REG . P I R. L VRA . S S SDGKTPGMSM. SSESHEA. I . RAYGEVFL . . K . T C F . .A. . . . 234 C. heterostrophus PKS1 178 . N . T I R . . . R . . GC QDGKTPG.. M . N S V S . EA. I. S V Y K K . A L . . . .TT. . .C. . . . 238 Clustai Consensus 56 * : * * : : * * * * * 74 Figure 3-4: CLUSTALW alignment of partial amino acid sequence of the gene encoding PKS from different fungi. Identical residues within aligned sequences indicated by periods (.). Amino acid sequences aligned are; A.fumigatus Alb lp : Aspergillus fumigatus B-5233 (GenBank AF025541), A.nidulansPKSST: A. nidulans (AAA81586), A.nidulans WA: A. nidulans (X65866), A.parasiticusPKSLl: A. parasiticus (L42766), A.parasiticus_WAmajor: A. parasiticus (AJ132275), A.parasiticus_WAminor: A. parasiticus (AJ 132276), C.lagenarium: Colletotrichum lagenarium 104T (D83643), C.heterostrophusPKSl: Cochliobolus heterostrophus (AAB08104), C.pinicola0901: Ceratocystispinicola 0901, C.resiniferal25214: C. resinifera 125-214, G.moniliformisPKStox: Gibberella moniliformis fumonisin mycotoxin PKS, Nodulisporium sp: Nodulisporium sp. ATCC74245 (AF151533), O.floccosumGrlO: O. floccosum GrlO, 0.floccosum387N: O. floccosum 387N, O.minusOIVG: O. minus OM3, 0.minus58-4: O. minus 58-4, 0.piceaeW5P: O. piceae W5, 0.setosum3734: O. setosum 3734, Phoma sp. WA: Phoma sp. C2932 (AJ132278), and P.patulumWA: Penicilliumpatulum (AJ 132274). Amino acid residue similarity in the consensus sequence is indicated in decreasing order from identical (*) to similar (:) to less similar (.). Arrow indicates active site cysteine residue within B-ketoacyl synthase motif (box). 78 A phylogram of the partial PKS alignment is presented in Figure 3-5. In the phylogram, members from each species grouped with one another and had intraspecies similarity >97%. A l l obtained PCR products grouped within a single monophyletic group, together with other known WA-type PKS sequences. This clade was further split into two sub-clades. The first consisted of known WA-type PKS genes involved in non-DHN melanin pigmentation with the exception of A. fumigatus Alb lp , which is involved in DHN melanin synthesis (Tsai et. al. 2001). The second sub-clade consisted of known WA-type PKS sequences involved in DHN-melanin synthesis and the obtained sapstain PKS sequences. Within this sub-clade, the obtained O. minus sequences grouped separately from the remaining sequences. The remaining Ophiostoma spp. sequences grouped within a single clade. Sequences for Ceratocystis spp. grouped separately from the remaining Ophiostoma spp., Colletotrichum lagenarium, Phoma sp. and Nodulisporium sp. DHN-melanin PKS sequences. 79 75 100 84 931 C. heterostrophus PKS1 — G . monilformis PKStoxin 100 66 86 100 P. patulum WA — A. fumigatus Alblp • A. parasiticus WAminor A. nidulans WA 100 A. parasiticus PKSL1 A. nidulans PKSST 100 A. parasiticus WAmajor — Nodulisporium sp. g6^— O. piceae W5 I L O. setosum 3734 O. floccosum GM0 O. floccosum 387N Phoma sp. WA 100 j~ ioo r* C. lagenarium C. pinicola 0901 C. resinifera 125-214 O. minus OM3 O. minus 58-4 0.1 Figure 3-5: Rooted phylogram of partial amino acid sequence alignment of the gene encoding PKS from different fungi. Amino acid sequences aligned are the same as in Figure 3-4. Neighbor-joined phylogram was generated using PAUP 4.0b6 with branch lengths proportional to genetic distances and C. heterostrophus PKS1 as the outgroup. Bootstrap percentages based on 1000 replications are shown above branch nodes. Scale bar represents 0.1 steps. Sequences determined in this thesis highlighted in boldtype. 80 3.3.2.2 1,3,8- trihydroxynapthalene reductase (3HNR) and 1,3,6,8-tetrahydroxynapthalene reductase (4HNR) The primer pair T29F/T14R produced a -360 bp band for most of the tested fungi. However, after sequencing, most of these PCR fragments showed no significant similarity (BLAST Escores >0.0001) to known 3HNR or 4HNR gene sequences. Further, significant background bands of various sizes were usually present in control PCR reactions lacking one or the other primers (Figure 3-6). Many attempts were made to optimize the PCR and screening conditions to isolate the correct sequences. Attempted PCR conditions included annealing temperatures of 45C, 48C, 52C, 55C, 58C, 62C and 65C combined with the addition of 2%, 3%, 5% and 10% D M S O in the PCR reaction. Further, partial restriction enzyme digest (with Sail) of genomic D N A prior to PCR as well as secondary PCR reactions of harvested bands were also attempted with little success. While many of these conditions did produce bands of around 360 bp, subsequent sequencing usually showed that meaningless sequences were obtained. In order to overcome these difficulties, the more specific primers THN31, THN33, THN42 and THN44 were designed based on the obtained sequences for O. floccosum 387N, C.pinicola 0901, O. minus OM3, O. minus 58-4 and Cartapip97 (O. piliferum). These primers were also tried in different primer pair combinations with little success. Finally, high-stringency (65C) Southern blotting of transformed E. coli with a 3HNR-specific probe (derived from O. floccosum 387N) was also used to screen transformants with vectors containing T29F/T14R amplicons. Transformants that hybridized were further screened using PCR-RFLP with HaeII and then grouped into different classes based on their digestion pattern (Figure 3-7). Upon sequencing and B L A S T analysis of the clones from different restriction digest classes, only one sequence (for C. resinifera 125-214 clone 10, which corresponded to 4HNR) with an excellent B L A S T X score was obtained. 81 Oflo387N Cc0901 OminOM3 Omin584 Cart Figure 3-6: Agarose gel electrophoresis of PCR amplification of genomic DNA using primers T29F and T14R. 20pL of fungal genomic D N A PCR reaction was electrophoresed in a 1.8% agarose gel (spiked with ethidium bromide) at 6V cm"1 for 20 minutes, and then visualized under U V light. Each fungal isolate comprised of three lanes: one reaction with both primers (T29/14) and two controls, each with either T29F (T29) or T M R (T14). Exterior lanes contained 100 bp ladder. Isolates included Cc0901: C. pinicola 0901, Oflo387N: O. floccosum 387N, OminOM3: O. minus OM3, Omin584: O. minus 58-4, and Cart: Cartapip97. Fungal isolates that did not amplify or that were not successfully sequenced were not included in this figure. 82 Ofio Oset16038 Cc125214 387N 2 14 30 27 55 57 58 59 10 25 3 19 P C B ^nfl i»»s;iM niii1 agiAgui^  jnng^ gn^  SRHRHr HHHRP 98X99 JUU flw^a^ ^ + *w"'-*>*t *w ntpjpHM fpRHP -: • - ' 200 100 * * • Opic Oset3734 1871 Of loGr lO 8 12 13 19 20 23 26 32 44 1 0 , 2 7 6 10 J 5 20 * ^ J P H i p m v MOTMRHUI '^PRPIMPi :vHR|P0EI' ' ••+tw^ww^'' ™ J U U -' , w w , p :3pj8HEpWBy 200 ~ ' 100 fl*M>-MMI s^PiUfe 4tMH9 MMfel . a'Wm < W » .^ .fr-/.,'. SiSsf; a is, # i i p | | | » Figure 3-7: Agarose gel electrophoresis of PCR-RFLP of selected colonies that hybridized in Southern blotting experiment. E. coli transformants that hybridized with the 3//A7?-specific probe were further screened via direct PCR of the colonies using the primer pair T29F/T14R followed by restriction digestion with Haell for one hour at 37C. 40pL of each PCR-RFLP reaction was electrophoresed in a 1.8% agarose gel (spiked with ethidium bromide) at 6V cm"1 for 20 minutes, and then visualized under U V light. Fungal names; Ccl25214: C. resinifera 125-214, Oflo387N PCB: O. floccosum 387N (positive control), OfloGrlO: O.floccosum GrlO, Opicl871: O. piceae 187-1, Osetl6038: O. setosum 160-38. Transformant code numbers are indicated below each respective fungal species. 83 Only some of the obtained sequences derived from the primer pair T29F/T14R were found to have good B L A S T scores with known fungal 3HNR or 4HNR sequences. Typically, sequences that had poor B L A S T results were within ± 20 bp of the correct sequence length and included the correct primer sequences. Sequences with poor (E score > 0.0001) B L A S T N or B L A S T X homology were not included in further analyses. Correct sequences for O. floccosum GrlO, O. piceae, O, setosum, O. piliferum 198-2gf, and O. piliferum 201-1A could not be obtained. The deduced partial amino acid sequences for 3HNR corresponded to residues 29 to 150 of the C. lagenarium 3HNR protein (Perpetua et. al. 1996). Figure 3-8 shows the C L U S T A L W alignment of the inferred amino acid sequences from the obtained 3FINR and 4HNR sequences and the amino acid sequences of other known fungal melanin 3FFNR/4PINR sequences. The partial sequence f o r M grisea 3HNR contained eight (Arg-39, Ile-41, Ala-61, Asn-62, Ser-63, Val-88, Asn-114 and Ser-115) of ten residues previously identified in forming the N A D P H binding pocket of the native protein (Andersson et. al. 1996). Arg-39 (Arg-1 lof the partial sequence), which has been identified via X-ray crystallography as responsible for N A D P H specificity, was conserved in all sequences (Figure 3-8) as well as in the versicolorin reductase and verA proteins from fungal aflotoxin synthesis pathway of Aspergillus parasiticus and Emericella nidulans, respectively (Andersson et. al. 1996). Three of the remaining seven residues were identical in all reductase sequences, two were very similar and two were somewhat similar (Figure 3-8). Val-118 of the native 3HNR in M. grisea (Val-90 of the partial sequence), which was noted for tricyclazole inhibitor binding, was conserved in most sequences with the exception of M. griseae 4HNR, Ceratocystis spp. and O. minus sequences. 84 M. grisea 3HNR C. lagenarium 3HNR 0. floccosum 387N 0. piliferum Cartapip C. heterostrophus 3HNR A.altemata3HNR M. grisea 4HNR 0. minus 58-4 0. minus 0M3 C. pinicok 0901 C. resinifera 125-214 E. nidulans stcll Clustal Consensus 10 . . . . I . . . . I GKVAL VTGAGI v v, * * * + 20 30 | . . . . | . . . . | . . . . | . GREMAMELGRRGCKV I VN Y|A A A . A . K A . . I . . A K KA . . AK . G I . I . . . . T . . . . T . . . . L . L . V A . G I . G I . G I . G I A A I A . . A . A . . A , A S . V . A . . V , A . . V . . K. . AN. V. . K. . AN. V. N S s . s . s . s . s . TE S A . T |D S . D S . V . G VG -SKA S G A S G A AK . AK . R . A 40 • I • • AE E I 50 • I Q Q . . s . . s . . K V V A A IK • • Q • • • . . D . . . . . D . . . . . KE . . . . KK . . . E . . . S . . TN . . . E . . . E . . D E + + AL 47 47 47 47 49 39 47 47 47 47 47 47 25 M. grisea 3HNR 48 C. lagenarium 3HNR 48 0. floccosum 387N 48 O. piliferum Cartapip 48 C. heterostrophus 3HNR 50 A. alternate 3HNR 40 M. grisea 4HNR 48 O. minus 58-4 48 0. minus OM3 48 C. pinicola0901 48 C. resinifera 125-214 48 £. nidulans stcU 48 Clustal Consensus KN G . S . A A . A S . G . . G . . . L . AL . AL . S Y . S Y . S . A 60 . . . | . . . . | SD A AC VK AN . . S I . . . A I . . . A I . . H AF . . . A - . AQGV A I Q T..VAMQ T..VAMQ TK S IAL Q TK S IAL Q QT . I S I Q 70 80 90 ! . . . | . . . . | . . . I . . . . I G V V E D I V RMF E E A V K I F G K L D IVCS| SD.DQ. .K. .G. .KQ.W.R SD.DQ. .TL. . KTKQQW. . SD.DQ. .TL. .KTK.QW. . . E S EKLMDD V. . H. . . . SKMDD V. AH - . -SKP S E V . A L . D K . . S H . SKPAEV.DL.DR..SH. DR..SH. DKVKAQY DKVKAQY . N . . N • SKPAEV.DL SKP . E V IAL SKP . E V IAL G G I G I R I R I . . C KD C . F . DPDAVTKLMDQ..EH..Y....S N S GV 100 • I FG VWCD VWA S VWA S VWCP VWCP 93 93 93 93 95 76 94 94 94 94 94 93 uo 120 • I M. grisea 3HNR 94 H V K D V T P E E F D R V F T I N T R G Q F F V A R E A Y 122 C lagenarium 3HNR 94 . A . 122 O. floccosum 387N 94 . SV 122 O. piliferum Cartapip 94 . SV 122 C. heterostrophus 3HNR 96 . F . N . KA: . 124 A. altemata3HNR 77 T - - K A . . 95 M. grisea 4HNR 95 E - L E . . Q . L . . K . . NL QQG- 121 0. minus 58-4 95 E - L . . . Q . L . . K . . NL . c 122 O. minus OM3 95 E - L . . .Q.L. .K. . NL . c 122 C. pinicok 0901 95 E - E K . . . . L . . . I . NL . c 122 C resinifera 125-214 95 E - E K . . . . L . . . I . NL . c 122 E. nidulans stcU 94 D . RV 122 Clustal Consensus 35 * * * : : * * * . * * . 47 Figure 3-8: C L U S T A L W alignment of partial amino acid sequence of the genes encoding 3HNR and 4HNR from different fungi. Identical residues and gaps within aligned sequences indicated by periods (.) and dashes (-), respectively. Amino acid sequences aligned are; A.alternata3HNR: Alternaria altemata 15A (GenBank ABO 15743), E.nidulans stcU: Emericella nidulans sterigmatocystin putative ketoreductase (AAC49205.1), C.heterostrophus3HNR: Cochliobolus heterostrophus HIT07711 (AB001564), C.lagenarium3HNR: Colletotrichum lagenarium 104T 3HNR(D83988), C.pinicola0901: Ceratocystispinicola 0901, C.resiniferal25-214: C. resinifera 125-214, M.grisea3HNR: Magnaporthe grisea (AF290182), M.grisea4HNR: M. grisea 4HNR (1127197), 0.floccosum387N: O. floccosum 387N, 0.minusOM3: O. minus OM3, 0.minus58-4: O. minus 58-4, and O.piliferum Cartapip: O. piliferum Cartapip97.. Amino acid residue similarity in the consensus sequence is indicated in decreasing order from identical (*) to similar (:) to less similar (.). Boxes indicate sequences features; hollow triangle: conserved NADPH-specific binding residue; filled triangle: conserved/similar nucleotide-forming-pocket residues; filled star: site of a tricyclazole-inhibitor-binding residue in C. lagenarium 3HNR. 85 E. nidulans stall 100 85 M. grisea 4HNR 100 O. minus 58-4 O. minus OM3 59 I 100 C. pinicola 0901 C. resinifera 125-214 M. grisea 3HNR 99 I C. lagenarium 3HNR 85 i — O. floccosum 387N 100 I— 0. piliferum Cartapip 0.1 100 • A. altemata C. heterostrophus F igure 3-9: Rooted phylogram of part ia l amino acid sequence al ignment of the genes encoding 3HNR and 4HNR f rom different fungi. Amino acid sequences aligned are the same as in Figure 3-8. Neighbor-joined phylogram was generated using PAUP 4.0b6 with branch lengths proportional to genetic distances and E. nidulans stcU as the outgroup. Bootstrap percentages based on 1000 replications are shown above branch nodes. Scale bar represents 0.1 steps. Sequences determined in this thesis highlighted in boldtype. 86 Figure 3-9 shows a bootstrapped phylogram based on the C L U S T A L W alignment using E. nidulans stcU as the outgroup. Members from each species grouped together and intraspecies similarity was >97%. Sequences from M. griseae 4HNR, O. minus and Ceratocystis spp. comprised a single clade where Ceratocystis spp. grouped separately. The second main clade comprised of M. griseae 3HNR, C. lagenarium 3HNR, O. floccosum, O. piliferum, Alternaria alternata, and Cochliobolus heterostrophus. Within this clade, A. alternata and C. heterostrophus grouped separately from the other sequences. Bootstrap values for most branches were between 85% and 100% with the exception of the branch of C lagenarium 3HNR from O. floccosum and O. piliferum. 3.3.2.3 Scytalone dehydratase (SD) PCR amplification with SD1/SD2 yielded strong bands of about 420 bp in all tested fungi (Figure 3-10). Control PCR lacking one or the other primers did not produce any background bands (data not shown). A l l of the obtained sequences were found to have excellent BLAST E-scores (BLASTN E < lxlO" 7 , B L A S T X E < lxlO" 4 0) with known fungal scytalone dehydratases. Further analysis showed that each obtained SD sequence contained an intron of approximately 58 ± 10 bp beginning after around 69 ± 2 bp. A MultAlin alignment for the intron sequences is shown in Figure 3-11. The signal sequences in the presumptive introns matched the consensus sequences for fungal 5' splice sites [GT(A/G/T)(A/C/T)G(C/T)], the 3' splice sites [(C/T)AG] and internal splicing signals [(G/A)CT(A/G)AC] of Neurospora crassa, the P-tubulin genes of C. graminicola, and the THR1 gene of C. lagenarium (Panaccione and Hanau 1990, Bruchez et. al. 1993, Perpetua et. al. 1996). A C L U S T A L W alignment of the inferred amino acid sequences (exon only) from the obtained sequences and the amino acid sequences of other known fungal SD is presented in Figure 3-12. 87 o T— Im o o in u CL O co o "SL O < 1— t o CN 'SL O CM o. O Q . CL ro r ro O CO CO 6 V) O CO co O CO c 1 O CO O c '£ O CNI US CM T -O O o o o o 1600 1000 JO 200 100 -424bp Figure 3-10: Agarose gel electrophoresis of PCR amplification of genomic DNA using primers SD1 and SD2. 20pL of fungal genomic DNA PCR reaction was electrophoresed in a 1.8% agarose gel (spiked with ethidium bromide) at 6V cm"1 for 20 minutes, and then visualized under U V light. Neighbouring control reactions, each with eitherSDl or SD2, did not produce any bands in any sample and are not shown. Exterior lanes contained 100 bp ladder. Isolates included OfloGrlO: O. floccosum GrlO, OpicW5: O. piceae W5, Opic 187-1: O. piceae 187-1, Opil201-1 A: O. piliferum 201-1 A, Opill98-2gf: O. piliferum 198-2gf, Cartapip: Cartapip 97, Osetl60-38: O. setosum 160-38, Oset3734: O. setosum 3734, Omin584: O. minus 58-4, OminOM3: O. minus OM3, Ccl25214: C. resinifera 125-214, and Cc0901: C. pinicola 0901. 88 ClagINT . 1 GT A C G r c C - T C A C A A C C GC A - - - - A C T C A C A A C A T G T c G c - - - - G A GC A A Cc0901INT 1 . . . A . c A T C C T C G A GT A C A G G A G A T T A T A T C A TT c T T K "C T A c Cc 1252 HINT 1 . . GA . c A T C T C T C G T . AT A T A G C A G A T T A T C T A TT C T T A C T A c Qpill982gfINT 1 . . . A . c A - - - C T C T - - - - T C T C T C C G A C . GCT Opil2011AINT 1 . . . A . c A - - - C T C T - - - - T C T C T . A C C G A C A GCT Cart 1 . . . A . c A - - - C C TAG - - - - C T C T A G . A C c A c . T T T OfloGrlOINT 1 T A T A c A C T G T T T C T T A A c . c A T A A .ATC OfloAUlINT 1 T A T A c A C T G T T T C T T A A c . C A T A A . A T C Oflo387NINT 1 T A T A c A C T G T T T C T T A A C . c A T A A .ATC OrainOM3INT 1 . . . T . c A A T T A c T T T T G G G T G C A A . A C A G A c C G c 0min584INT 1 . . . T . c A G C T c A T T C A C A T T T T C T G GA C G A c A . 0set3734INT 1 . . . T . A G A T C T C G T T C A G G A G A C A A G C A G A A C T . A A C G |A "c T A" "c OsetieOINT 1 . . . T . A G A T C T C G T T C A G G A G A C A A G C A G A A C T . A A C G A c T A c OpicW5INT 1 . . . T . G G A T T T C T GG G A G A C A A G - - G A c A . A A T A A c T A c Opic 1871 INT 1 . . . T . G A A T T T C T GG G A G A C A A G C A G A c A . A A T A A c T A c ClagINT 42 TJGCTG- AC fc T - - C T T C|C A~G~ 57 CcQ901INT 5D A A . ATATAT GT T T . G . . | 68 Cc 1252 MINT 50 A A . ATATAT GTTT . G .IT . . j 68 0pill982gfINT 38 :G - . . . . . . . . GA :C GA . . | 49 Opil2011AINT 39 |G - . . . . . . . . GA .iC GA1. . . , 50 Cart 39 ir, - . . . . . . . ^ A A ^ J G G A 1 . . . 50 OfloGrlOINT 46 . T . . T . - J G " " X A " | A GA1. 62 OfloAUlINT 46 . T . . T . -!G . . A A .-AGA'. . . 1 62 0fb387NINT 46 . T . . T . T G . . AA .JAGAl. . . 1 63 QminOM3INT 46 A C T . G3A . GA j . G Al. . . 1 63 0min584INT 50 A A . A . C G|A . A A j . GA| . . 1 68 0set3734INT 51 A - GAP- . . 1 58 Osetl60INT 51 A - - . . . . GA|T . . I 58 OpicW5INT 49 A - - . . A . A Aj. . . | 56 Opic 1871 INT 51 A - - . . . . CAj. . . i 58 Figure 3-11: MultAlin alignment of DNA sequence of the intron from the gene encoding SD from different fungi. Identical residues and gaps within aligned sequences indicated by periods (.) and dashes (-), respectively. Intron sequences aligned are; Cart: Cartapip97 (O. piliferum), ClaglNT: Colletotrichum lagenarium 104-T (GenBank D86079), Cc0901INT: C. pinicola 0901, Ccl25214INT: C. resinfera 125-214, OfloGrlOINT: O. floccosum GrlO, OfloAUlINT: O. floccosum AU1 (courtesy of SH Kim), Oflo387NINT: O. floccosum 387N (courtesy of H. Wang), OminOM3INT: O. minus OM3, Omin584INT: O. minus 58-4, OpicW5INT: O. piceae W5, Opicl871INT: O. piceae 187-1, Opil 1982gfINT: O. piliferum 198-2gfand Opil2011AINT: O. piliferum 201-1A, Oset37341NT: O. setosum 3734, Osetl60INT: O. setosum 160-38. Boxes indicated consensus sequences for 5' splice sites (solid line), 3' splice sites (dashed line) and internal splice sites (dotted line). 89 O. floccosum GrlO O. flocccsum AU1 0. floccosum 387N O. piliferum 198-2gf 0. piliferum 201-1A O. pMezumCartapip O. rnirius OM3 O. minus 58-4 O. setosum 3734 0. setosum 1(50-38 0. piceae W5 O. piceae 187-1 C. lagenarium C.piiucola 0901 C. resinifera 125-214 M. griseae A. fumigatus Clustai Consensus Spom4ACD_OUT _ _ 10 30 _ 30 40 ,50 I IT - - I . . . I . . . . I . . . . I T ... I . . . I . . . . I . . . . I | E W A D R | Y | E | S K D W D R L R K C I A P T L R I D[\] R S F L N K L W E A M P A E E F I G M I S D|EJ S 50 50 . . . . S D . . . S . + + + R T . R T . . V . S I . T V Q I G L R K . D D A . K . K . . . V . . V . S D Y M A . . . : : : * : * . . 3 5 M N T S A . A V S K . - - K N N . C D T K K - - . R K N 24 m . . . . I . . . . I I . | 70 . . | . . • • 1 so . . I . . . • I f • 90 . | . . . . 1 100 . . . I O. floccosum GrlQ 51 V L G N P L L R T Q F F G A S R W E R I S D T E V V G Y H Q L R V P Q V Y T D T T L T O V A V K 1D0 O. floccosum AU1 51 100 O. floccosum 3S7N 51 100 O. piliferum 198-2gf 51 . H R . . . . . A . . S 100 O. piliferum 201-1A 51 . H . . . . . . A . . s 100 0. piliferum Cartapip 51 . H . . . . . . A . . 100 0. minus OM3 51 S . . G . V . , I . . . A S . S T 100 0. minus 58-4 51 . . . V . . 1 . . . A S . S T 100 0. setosum 3734 51 . . . V . . 1 . . . A S . S T 100 O.setosum 160-38 51 . . . V . . . V I G A S . S T 100 0. piceae W5 51 . . . V . . 1 . . . A S . S T 100 0. piceae 187-1 51 . . . V . . 1 . , . A S . S T 100 C. lagenarium 51 K . . . I . G . . . . K V . . 1 . H . . . . K . , . A S R . E 100 C. pnucolatWOl 51 . . . D . . . K . . . V . . . V . . D . . W . . . . R . . . A . K T . K . . 100 C. resinifera 125-214 51 . . . D . . . K . . V . . . V . n . W . . . R . . . A . K T . K . . 100 M. griseae 51 . . . D . T . . . . . I . G T . . . K V . E D . . I . R . K . . . M K E . T M . 100 A. fumigatus 51 F . . D . T V K . . L L . E . W . . K . . . 1 . H . . . . A A . . . S . . Q T . K L . 100 Clustai Consensus 35 * * . + . . + + i + * . . + + . * : * * * * * . . : : •k . * 66 Spom4ACD_OUT 25 A W . M S C V A R A N - - - - H H . N K V D T . T H D T . 53 O. floccosum GrlO 101 G H A H S A F T H W Y R K VD G V W K F 120 0. floccosum AU1 101 A 121 0. floccosum 387N 101 A 121 0. piliferum 198-2gf 101 A 121 0. piliferum 201-1A 101 A 121 0. piliferum Cartapip 101 A 121 0. minus OM3 101 Q • A 121 0. minus 58-4 101 Q • A 121 0. setosum 3734 101 Q - A 121 0. setosum 160-38 101 D Q - A 121 0. piceae W5 101 Q • A 121 0. piceae 187-1 101 Q • A 121 C. lagenarium 101 Y M N A 121 C. pmkolaOOOl 101 K . . K . A 121 C. resinifera 125-214 101 K . . K . A 121 M. griseae 101 I. . . . K . 1 , A 121 A. fumigatus 101 . . G . A T F . Y . . . A 121 Clustai Consensus 67 * + + . i . * . * * * * * * 83 Spom4ACD_OUT 54 . T K D K . D A K . N - - - - 65 Figure 3-12: C L U S T A L W alignment of partial amino acid sequence of the gene encoding SD from different fungi. Amino acid aligned are: A.fumigatus: Aspergillus fumigatus B-5233 (GenBank U95042), C.lagenarium: Colletotrichum lagenarium 104-T (D86079), C.pinicola0901 C. pinicola 0901, C.resiniferal25-214: C. resinifera 125-214, O.floccosumGrlO: O. floccosum GrlO, O.floccosumAUl: O. floccosum AU1 (courtesy of SH Kim), 0.floceosum387N: O. floccosum 387N (courtesy of H. Wang), 0.minusOM3: O. minus OM3, 0.minus58-4: O. minus 58-4, 0.piceaeW5: O. piceae W5, 0.piceael87-l: O. piceae 187-1, 0.piliferuml98-2gf: O. piliferum 198-2gf, O.piliferum201-1 A: O. piliferum 201-1A, O.piliferumCartapip: O. piliferum Cartapip97, 0.setosum3734: O. setosum 3734, O.setosuml6038: O. setosum 160-38, M.griseae: Magnaporthe griseae (AB004741), and Spom4ACD_OUT: Schizosaccharomyces pombe pterin-4-alpha-carbinolamine dehydratase.. Amino acid residue similarity in the consensus sequence is indicated in decreasing order from identical (*) to similar (:) to less similar (.). Boxes indicate known active site residues: conserved/similar (filled triangle) and not conserved (filled star). 90 The partial sequence for M. grisea SD (which corresponded to residues 22 to 142 of the native protein) contained all eight residues (Tyr-30, Asp-31, Tyr-50, Lys-73, His-85, His-110, Ser-129 and Asn-131 of the native protein) identified in the active site (Lundqvist et. al. 1994). Five residues were conserved in all other SD sequences, two had high similarity and one was not conserved (Figure 3-12). A phylogram based on the C L U S T A L W alignment with Schizosaccharomycespombe P4ACD as the outgroup is shown in Figure 3-13. Members within each species grouped together and typically had 97% to 100%> intraspecies sequence similarity. A. fumigatus branched separately from the remaining SD sequences which together, formed a single clade. Within this clade, M. griseae branched separately from the remaining sequences. These remaining sequences split (61%o confidence) into a Ceratocystis spp. group and a third branch. The third branch split (56% confidence) into C. lagenarium and a fourth branch. This branch comprised exclusively of Ophiostoma spp. SD sequences and split into two groups: O. floccosum and O. piliferum in the first; and O. minus, O. setosum, and O. piceae in the second. Branch bootstrap values were >95% except the second and third branches. Interspecific similarity between the two most distant groups was 83%. 91 S. pombe P4ACD A. fumigatus 95 61 M. griseae 56 C. lagenarium 91 78 97 89 100 r O. floccosum 387N O. floccosum Gr10 76 O. floccousum AU1 O. piliferum Cartapip 66 |p O. piliferum 198-2gf 68 O. piliferum 201-1A f O. minus OM3 J O. minus 58-4 84 O. setosum 3734 II I 64 98 | O. setosum 160-38 O. piceae W5 O. piceae 187-1 • C. pinicola 0901 C. resinifera 125-214 0.1 Figure 3-13: Rooted phylogram of partial amino acid sequence alignment of the gene encoding SD from different fungi. Amino acid sequences aligned are the same as in Figure 3-12. Neighbor-joined phylogram was generated using PAUP 4.0b6 with branch lengths proportional to genetic distances and S. pombe P4ACD as the outgroup. Bootstrap percentages based on 1000 replications are shown above branch nodes. Scale bar represents 0.1 steps. Sequences determined in this thesis highlighted in boldtype. 92 3.4 Discussion The presence of the D H N pathway was demonstrated in the tested fungi by the inhibition or reduction of pigmentation with the compounds tricyclazole and carpropamid (Figure 3-2). The compound cerulenin inhibited the growth of all fungi at all tested concentrations. Aside from inhibiting the PKS step of DHN synthesis, cerulenin also inhibits the enzyme, fatty acid synthetase — a physiologically critical enzyme (Kubo et. al, 1986). Thus, cerulenin was a poor indicator of DHN melanin synthesis in the tested sapstain fungi. Tricyclazole has been acknowledged as an inhibitor of 3HNR and 4HNR in the DHN pathway (Wheeler and Greenblatt, 1988), though a recent study demonstrated that tricyclazole has a 200 fold higher affinity for 3HNR from M. grisea than 4HNR (Thompson et. al. 2000). If tricyclazole has similar affinity for naphthalene reductases of other fungi, including sapstain species, then pigment reduction by the addition of tricyclazole may only indicate the presence of 3HNR. Pigment reduction by the compound carpropamid was indicative of the presence of SD, which unlike other enzymes of the DHN pathway, is unique to the DHN pathway and has no known crossover activity into other metabolic activities (Tsuji et. al. 1997). It appeared that these two DHN pathway inhibitors also reduced the growth of the tested fungi at higher concentrations, though this result did not interfere with the conclusion that the DHN melanin pathway is present in these sapstain fungi. Furthermore, the reduction of pigmentation by the addition of carpropamid to growth media combined with the successful sequencing of fragments of the gene encoding SD from all tested fungi confirmed the presence and expression of the SD gene of the D H N melanin synthesis pathway in these sapstain fungi. In the SD partial-amino acid sequences (which comprised approximately 70% of the full amino acid sequence), all Pyrenomycetes SD sequences formed a monophyletic group. Within this group, species of Ophiostoma formed a single clade (Figure 3-13). This clade, however, did not branch significantly from C. lagenarium and Ceratocystis spp. branches. These three groups, 93 though, did branch significantly from M. griseae. Furthermore, two sub-branches of the Ophiostoma clade were observed: the first comprised O. minus, O. piceae and O. setosum; and the second comprised O. floccosum and O. piliferum. This result agreed with previous rDNA alignment results of Ophiostoma spp. (Uzunovic et. a/.2000). This suggests that the functional evolution of SD is indicative of the evolution of the Ophiostoma species in general. Another interesting aspect is that M. grisea branched outside of the C. lagenarium, Ceratocystis spp., and Ophiostoma spp. group rather than within the Ophiostoma spp. clade. ( M griseae is considered more related to Ophiostoma than to Ceratocystis or Glomerella [Colletotrichum] [Alexopolous 1996, p. 329]) However, this branching topology had low bootstrap support and these results might change if different amino acid substitutions were weighted differently in further analysis. It should also be noted that the obtained SD sequences contained all eight known active site residues from C. lagenarium (Lundqvist et. al. 1994) and seven of these residues were conserved in all obtained and previously published sequences (Figure 3-12). Four of the conserved residues, Tyr-30, Tyr-50, His 50 and Hisl 18 of the native protein (Tyr-6, Tyr-26, His-61 and His 86 of the partial sequence) are considered essential for enzymatic activity, whereas the other three are considered beneficial (Zheng and Bruice 1998, Basarab et. al. 1999). The eighth residue, Lys-73 of the native protein (Lys-49 of the partial sequence) was substituted with proline in all Ophiostoma and Ceratocystis sequences. The substitution was not surprising because a previous study showed that Lys73Ala site-directed mutagenesis did not affect enzymatic activity. It is also interesting to note that the introns of the obtained partial SD sequences were all of similar size and occurred in the same location. Further, the signal sequences in the presumptive introns were conserved with the fungal 5' splice sites, the 3' splice sites and internal splicing signals of other known fungal genes, (Figure 3-11) including: those of Neurospora crassa, the P-tubulin genes of C. graminicola, and the THR1 gene of C. lagenarium (Panaccione and Hanau 1990, Bruchez et. al. 1993, Perpetual, al. 1996). 94 Unsuprisingly, the parsimony analyses for the reductases grouped 3HNR and 4HNR sequences separately (Figure 3-9). The partial 3FTNR and 4FTNR sequence alignments (which comprised approximately 43% of the respective full length amino acid sequences) did support the seperation of Ophiostoma species from other species as shown by the alignments of the partial SD sequences. However, it should be cautioned that the obtained sequences for 3HNR do not span the nine active site residues known in M. grisea (Andersson et. al. 1996). Nevertheless, they did span the eight nucleotide-pocket forming residues, including the conserved NADPH-specific residue Arg-39 (Arg-11 of the partial sequence). A previous biochemical study of M. grisea showed that 4HNR was more specific for 4HN than 3HN by a factor of 310. In contrast, 3HNR was only slightly more specific for 3HN than 4HN by a factor of 4.2 (Thompson et. al. 2000). The authors also determined that the catalytic triad and eight of nine active sites residues of 3HNR were conserved or similar in 4HNR. Subsequent computer modelling based on the known crystal structure for 3HNR suggested that the absence of the ninth residue (Met-283) was responsible for the specificity of 4HNR for 4HN. Unfortunately, the obtained sequences here do not span this residue, so this suggestion cannot be confirmed. Further, the authors determined 3HNR was required for pathogenicity, but 4HNR was not. Considered together, the biochemical, structural and partial genetic data suggests that 3HNR may be a functional ancestor to 4HNR. It is difficult, though, to answer these questions with only a few short-range sequences from a few species. Further study, including elucidation of the full-length sequences of these reductase genes in multiple species, should be carried out to help answer these hypotheses. The partial PKS sequence alignments (which comprised approximately 11% of the full length amino acid sequences) did not ratify the seperation of Ophiostoma species from other species as shown by the alignments of the partial SD sequences. A search of the PROSITE database showed that the obtained PKS sequences contained a P-ketoacyl synthase (P -KAS) motif found in other Type I PKS proteins (Figure 3-4). P-ketoacyl synthase is found as a component of a number of enzymatic systems including: fatty acid synthetase, which catalyzes the formation of 95 long-chain fatty acids from acetyl-CoA, malonyl-CoA and N A D P H ; polyketide antibiotic synthase systems (6-methysalicylic acid synthase) from Penicillium patulum; and the conidial green pigment biosynthesis protein Wa of Emericella nidulans (Kauppinen et. al. 1988, Beck et. al. 1990). Further, the active site cysteine and surrounding residues were conserved in all obtained sequences. The overall topology of the PKS phylogram (Figure 3-5) agreed with a previous study on the ketosynthase domains of several fungal PKS genes (Bingle et. al. 1999). These authors found that these domains could be classified into two major types: M S A S (encoding 6-methylsalicylic acid synthase) and W A (involved in pigment and aflotoxin biosynthetic pathways). The partial PKS sequences presented here fell into the WA-type due to their grouping within the WA sub-class (Figure 3-5). Further, all obtained sequences for Ophiostoma spp. and Ceratocystis spp. grouped with other sequences considered to encode for PKS in the D H N melanin pathway (Takano et. al. 1995, Bingle et. al. 1999). This further supports that these sapstain fungi utilise the D H N pathway to produce melanin. It should be noted, though, that O. minus sequences grouped outside of the clade comprising Ceratocystis, Ophiostoma and other WA-DHN-type sequences, thus indicating that the O. minus sequences are possible paralogues and may not represent true D H N melanin PKS genes. Further functional studies, such as gene knockouts, may be employed to determine whether these O. minus sequences are homologues or paralogues to DHN melanin PKS genes. Thus, the phylograms for PKS and 3HNR/4HNR might reflect a functional divergence or unrecovered paralogues of the enzymes, whereas SD may reflect the overall evolution of the fungi. It should be cautioned, though, that the sizes of the PKS and 3HNR/4HNR sequences comprised a smaller proportion of the respective whole length genes compared to SD. In Colletotrichum lagenarium, and other fungal species, the genes for SD, PKS and 3HNR have full length coding regions of 740 bp, 6561 bp and 846 bp, respectively (Takano et. al. 1995, Perpetua 96 et. al. 1996, Butler and Day 1998). However, it may still be possible that these enzymes (3HNR/4HNR and PKS) have different selective pressures for their evolution than SD and may approach the biochemistry of DHN melanin synthesis differently. This is supported by a recent study that showed the D H N melanin synthesis pathway of the fungus Aspergillus fumigatus, rather than using just a pentaketide synthase, uses a heptaketide synthase (Alblp) to synthesize heptaketide naphthpyrone from Acetyl-CoA, followed by enzymatic shortening to 4HN by a novel protein, Ayglp (Tsai et. al. 2001). Alignments were incomplete in the number of species compared to SD. This was especially true for 3HNR, which proved to be very problematic to isolate and sequence and in some cases, resulted in the non-target amplification of 4HNR. Most likely, this was due to the weak nucleotide sequence identity among different species for 3HNR and 4HNR versus SD. The nucleotide identity for 3HNR and 4HNR was 59% to 79% while for SD it was 69% to 98%, with the exception of M. grisea. Conversely, the relative ease of amplification and sequencing of the gene encoding SD was largely due to the highly conserved nature of the target sequences (Figure 3-12). The low sequence identity of the gene for 3HNR might indicate that the target sequence for the primers T29F and T M R in the PCR reactions was absent or variable in fungal isolates that could not be amplified. Further, the low specificity of the primers to the target gene lead to significant background amplicons during PCR, thus reducing successful amplification of the desired sequence. This was observed in the form of multiple bands when PCR was attempted at a range of temperatures from 45C to 60C (data not shown). We tried to circumvent this by designing and testing new primers with higher specificity (THN31, THN 33, T H N 42, T H N 44; see Table 3-1), but we did not succeed in amplifying the correct sequences from problematic species. Another possibility is that the tertiary structure of the genomic DNA in this region may prevent D N A synthesis by the Taq enzyme. We tried to circumnavigate this by adding DMSO, at concentrations up to 10%, in order to disrupt the tertiary structure and aid polymerization (Ausubel et. al. 1994). This was also, not successful in amplifying correct sequences from 97 problematic species. Considered together, this suggests that the target sequences are highly variable and cannot be amplified without specific sequence information. High stringency Southern blotting and subsequent PCR-RFLP of transformed E. coli containing T29F/T14R inserts were attempted to retrieve correct clones. Only one correct sequence was obtained from several candidate clones and it emitted strong probe hybridization signals (this sequence corresponded to C. resinifera 125-214 4HNR). Since the clones had inserts containing the primer sequences (the primers comprised ~60 bp out of a total of 360 bp amplicon), it was likely that the probe hybridized with the primer sites of the query clones. Thus, Southern blot screening may exclude some, but not all, transformed clones containing incorrect sequences. Another suggested reason for the lack of successful sequences includes the disengagement of Taq enzyme from the template DNA during polymerization due to either high GC content of the target sequences or complex tertiary structure of the DNA. We also unsuccessfully attempted to circumvent this problem by trying multiple PCR conditions including annealing temperatures from 45C to 65C, addition of 2% to 10% DMSO and adding Taq after the initial 94C denaturing step (Hot start PCR). It was also suggested that the genomic DNA may not have been purified sufficiently, though this was refuted since the same genomic D N A samples did yield background PCR amplicons and were used for successful SD gene amplification. One possible solution that would likely yield success would be to repeat the initial approaches of the pioneer researchers in this area. Namely, create genomic libraries of the target species, screen them with heterologous probes for either PKS or 3HNR and sequence the positive sub-clones (Kubo et. al. 1989, Kimura and Tsuge 1993, Vidal-Cros et. al. 1994, Takano et. al. 1995, Perpetua et. al. 1996). This approach was also used to isolate and characterize the gene encoding 4HNR from M. grisea (Thompson et. al. 2000). A second possible approach would be to purify the 3HNR enzyme from a target species, sequence the N-terminus region and design degenerate PCR primers based on the derived amino acid sequence. This, however, would be a very tedious 98 and difficult procedure due to the instabilities of the substrates and products, and would require significant chemistry expertise (Tajima et. al. 1989). Lastly, another approach is the design of new specific PCR primers based on the known, full-length sequences of genes encoding D H N melanin synthesis genes in O. floccosum 387N and O. ulmi. The full-length sequences of 3HNR and 4HNR have been recently obtained from O. floccosum 387N (Eagen et. al. 2001, Wang and Breuil submitted) and a joint project was recently undertaken to partially sequence the genome of O. ulmi, the causative agent of Dutch elm disease (C. Breuil; personal communication). 3.5 Conclusions The presence of the D H N melanin biosynthesis pathway was demonstrated in all tested sapstain fungi using both chemical inhibitors and molecular techniques. Furthermore, since no fungus has ever been found, to our knowledge, to have more than one melanin synthesis pathway, we can state that these species likely only use the D H N pathway for melanin production. At low concentrations, the inhibitor compounds tricyclazole and carpropamid effectively reduced pigmentation in all tested fungal species, but also lead to growth inhibition at higher concentrations. The inhibitor cerulenin prevented fungal growth in all tested fungi at all tested concentrations, likely due to its inhibitory effect on another enzyme, the metabolically critical fatty acid synthase. Further, partial D N A sequences for the genes encoding SD, 3HNR, 4HNR and PKS were obtained from species of Ceratocystis and Ophiostoma and found to have homology with known respective DHN biosynthesis gene sequences. Aside from the work carried out on the isolate O. floccosum 387N, this is the first known documentation of the pigmentation pathway used by species of the sapstain fungi Ceratocystis, Leptographium and Ophiostoma (Eagen et. al. 2001, Wang and Breuil submitted, Wang et. al. in press). Sequence analysis of the partial SD amino acid sequences showed greater than 80% similarity among the sapstain species, and corresponded well with known parsimony analyses of sapstain fungi based on rDNA sequences. Analysis for the partial sequences encoding 3HNR, 4HNR and PKS 99 showed that these sequences had lower interspecies similarities than the sequence encoding SD. Further, the data suggests significant divergence of the 3HNR/4HNR and PKS proteins from the species level evolutionary taxonomy. However, these results were less conclusive due to the lack of sequences (in the case of 3HNR/4HNR) and the relatively lower obtained proportion of the whole gene (in the cases of 3HNR/4HNR and especially PKS). 100 Chapter 4 General conclusions and future work The work presented in this document sheds some light on why certain sapstain species, such as those of Ceratocystis, are generally found on logs versus species of Ophiostoma, which are generally found on lumber, and the metabolic pathway these species use to produce their pigmentation. Prior to this work, little was known on the role of wood nutrients in sapstain fungal pigmentation and growth. We have found that nutrients in wood (such as mannose or TG-bound glycerol and fatty acids) do play an important role in pigmentation and growth by sapstain fungi, but it appears that other factors, such as changes to wood ultrastructure or other biochemical factors, are also critical. For example, we observed that reduced fungal growth on wood with closed border pits was corelated with reduced consumption of wood nutrients. In addition, it was unknown which pigmentation pathway was used for the production of melanin in these fungal species. We found that the inhibitor compounds tricyclazole and carpropamid effectively reduced pigmentation in all tested fungal species and we obtained partial D N A sequences for the genes encoding SD, 3HNR, 4HNR and PKS from the tested species. Thus, we can confidently state that the sapstain fungi tested in this work utilise the DHN melanin pathway for pigment production. Further, this is the first known documentation of the pigmentation pathway used by species of the sapstain fungi Ceratocystis, Leptographium and Ophiostoma, aside from the work carried out on the isolate O. floccosum 387N (Eagen et. al. 2001, Wang and Breuil submitted, Wang et. al. 2001). Some explanation for the differences in fungal species distribution between logs and lumber may lie in the access that different species have to the host tissue nutrients. It has been previously shown that these sapstain fungi produce negligible amount of celluloytic and lignolytic enzymes that would assist in the breakdown of wood cell wall components and permit access to wood 101 nutrients (Nilsson 1973). However, for brown rot fungi, there is evidence that degradation is initiated by the production and release of extracellular organic acids and/or peroxides, that serve to cleave and partially depolymerise hemicellulose, thus increasing wood cell wall porosity and allowing the enzymes to penetrate into the wood (Green and Highley 1997). It is unknown whether any sapstain species also have any of these capabilities, though it may explain the differences in the growth of C. resinifera and other sapstain fungi in previously dried wood. Future work should attempt to answer the question whether different species of sapstain fungi produce such degrading compounds, even in miniscule amounts. If they did, then the impact of such a capability on the fitness of the organism could also be studied. In addition, there has been reported evidence of production of ethanol-soluble inhibitor(s) of Ceratocystis montia during the drying process in Lodgepole pine (Strobel and Sugawara 1985). Further work should be carried out to replicate this effect on species of Ceratocystis and Ophiostoma described in this document. If such a natural inhibitor could be isolated (likely a resin acid) and shown to prevent sapstain fungal growth in vitro, then it may be feasible to test the application of such a compound to green lumber or logs in the field. Large-scale production of such a compound may also be feasible via purification from pulp mill tall oil (Maloney 1978, Bratt 1979, Koebner 1983, Ustun 1996). It is also interesting to note that tall oils have been shown to prevent the attack of mountain pine beetles on Lodgepole pine and other softwoods (Nijholt et. al. 1981, Richmond 1985). This is certainly an area worth exploring. Further work should also be carried out on the nutrient consumption by these fungi. Although several different nutrients were examined in this work, there is much more to be done. For example, for in vivo studies, we examined sugar monomers, starch and lipids; three very important wood nutrient classes. Further work could look at the consumption of other monomers (such as fructose), dimeric sugars (such as sucrose), other polymeric sugars and proteins. 102 Additionally, more work should be done to quantify the consumption of other nutrients in vitro as we did for glucose and mannose. Further work should also attempt to carry out full length sequencing of the genes encoding D H N melanin synthesis proteins in order to draw a better understanding of the relationships between species and their biosynthetic pathways. This would be especially interesting to pursue not just in the fungal isolates tested in this work, but in other species of Ceratocystis and Ophiostoma as well the black yeasts (Aureobasidium pullulans, Hormonema dematiodes and Phialophora spp.) and the surface moulds {Alternaria altemata and species of Cladosporium). One approach for carrying out full-length sequencing entails the use of RACE-PCR (Rapid Amplification of cDNA Ends) of cDNA isolated from sapstain fungi with specific primers based on the known partial D N A sequences. This method may also be successful with the degenerate primers used in this study to isolate full-length sequences from fungal isolates that were unsuccessful in this work. Another approach to gain the full-length sequences (from both successfully and unsuccessfully sequenced fungal isolates) would be to create genomic libraries of the target species, screen them with heterologous probes and sequence the positive sub-clones. Yet another approach would be to design new specific PCR primers based on recent and future sequence information for O. floccosum 387N and O. ulmi. Another aspect to examine is (are) the role(s) of 3HNR and 4HNR in different sapstain species. A full-length gene encoding 4HNR has been recently published and further work should be carried out to elucidate the presence of 4HNR in other sapstain fungi (Eagen et. al. 2001). This could answer some questions on the functional versus species evolution of sapstain fungi. It should not be forgotten that one of the major objectives of this research is to gain information that may help to control sapstain. Consequently, since they have been shown to reduce pigmentation 103 and reduce growth, the inhibitors carpropamid, cerulenin and tricyclazole should be tested on sapstain fungi in vitro. It is anticipated that the presented work in this document will stimulate further research into the biochemistry and molecular genetics of sapstain fungi. It is further hoped that this research will serve the forest products industry to develop safe and effective means of controlling sapstain in the future. 104 References Abraham, L . 1995. 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