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 DHN 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 DNA 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 DHN 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 DHN inhibitors 74 3.3.2 PCR amplification and sequencing of DHN genes from fungal genomic DNA— 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: DHN 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 DNA using primers PKS3 and PKS6 77 vii Figure 3-4: CLUSTALW 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 DNA 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: CLUSTALW 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 DNA using primers SD1 and SD2 88 Figure 3-11: MultAlin alignment of DNA sequence of the intron from the gene encoding SD from different fungi 89 Figure 3-12: CLUSTALW 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 AtoC A adenine aa amino acid ARA arabinose BLAST basic local alignment search