MOLECULAR ANALYSIS OF THE DRY ROT FUNGUS Serpula lacrymans♦ Anne Vigrow B.Sc., P.G.C.E. This thesis is presented to the Council for National Academic Awards in partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy. Department of Molecular and Life Sciences, Dundee Institute of Technology. September, 1992. I certify that this thesis is the true and accurate version of the thesisvapproved by the examiners. S i g n e d D a t e i e s ) Sarah, John and I wish to dedicate this thesis to P e t e r . 1971 - 1985. Acknowledgements. I would like to express my gratitude to my supervisors Professor Bernard King and Drs David Button and John Palfreyman for their encouragement, supervision and guidance throughout this project and for constructive criticism of this manuscript. I hope that completion of this work is adequate recompense for the debt which I undoubtedly owe them, the last named in particular, for having faith in my scientific abilities at a time when I had neither faith nor ability. I am grateful to Professor W. Emond for enabling me to have a year's secondment in order to complete this work; and I also wish to thank my external advisor, Dr. J.A. Carey of the Building Research Establishment, for her advice. Other members of staff at DIT who deserve thanks are Mr. George Hunter and Mr. Charles Craig, for the preparation of photographs; Miss Margot Dunnachie, for the maps and drawings; and Miss Moira Malcolm and the operations' staff of the computer centre, who guided me in the use of Massll. Thanks must go to those who ensured that a room was always available in which to write up this thesis, viz Mr. Duncan MacLeod, other members of the janitorial staff and, especially, Miss Doris Kinnison, Canteen Manageress. I am very appreciative of the advice, encouragement and acceptance of my fellow research students. I feel privileged to have been part of their company! Drs Heather Glancy, Elspeth Henry and Sandra Hainey, Mrs. Una Henry and other friends in many departments at DIT, especially those amongst the technical staff of MLS, will always be especially valued for their support and friendship. Finally, special thanks are reserved for my daughter Sarah and my husband John. Whilst the former typed the references and the latter checked the references, stuck on photographs and collated this thesis, more importantly both have shared the trials and tribulations of the past seven years. Thanks you two! DUNDEE INSTITUTE OF TECHNOLOGY LIBRARY Reproduction of Project Report A u t h o r Anne Vigrow T i t l e Molecular Analysis of the Dry Rot Fungus, Serpula lacrymans. Qualification Ph.D. Y e a r 1 9 9 2 I agree that a copy may be made of the whole, or any part, of the above mentioned project report by the Library of Dundee Institute of Technology at the request of any one of its readers without further reference to the undersigned on completion of a Copyright Declaration Form, and on payment of the fee currently in force. S i g n a t u r e A d d r e s s September 16th, 1992. MOLECULAR ANALYSIS OF THE DRY ROT FUNGUS S erp u la lacrym an s. Anne Vigrow B.Sc., P.G.C.E. A b s t r a c t . SDS-PAGE/silver staining and western blotting were used to investigate the molecular profile of a PBS soluble extract from standard mycelium of S . lacrymans FPRL 12C. Both techniques enabled a reference profile for this organism to be established. A polyclonal antiserum raised against standard mycelium of S. lacrymans FPRL 12C was used as the immunological probe. The profiles of other isolates of S . lacrymans and different Basidiomycetes were compared with FPRL 12C. Protein and antigenic similarity indices enabled differences between these organisms to be quantified. 2 isolates of S . lacrymans differed from FPRL 12C but to a lesser extent than species in other genera. Lectin staining confirmed these observations and indicated that the majority of antigens were glycoproteins. SDS-PAGE/silver staining and western blotting allied with similarity indices were found to aid in identification studies which established the identity of one of the anomalous isolates of S . lacrymans. Variation in basic parameters of culture caused minor changes of profile but potentially lethal factors had great effect on profiles. Antigenic differences attributable to growth phase were noted in young and aged mycelium of S . lacrymans. Protein and antigenic profiles indicated that S . lacrymans in pine and lime sapwood blocks was more similar to young mycelium at low weight loss and to aged mycelium at high weight losses. Protein and antigenic profiles of field samples of S . lacrymans varied markedly from those of the reference isolate. Protein profiles allied with similarity indices were only appropriate for investigation of a few field mycelial and basidiocarp samples but western blotting identified antigens in most field samples. Identification was made of antigens (a) common to S. lacrymans FPRL 12C and fresh field samples and (b) associated with either fresh or desiccated field samples. The use of these antigens for development of more specific immunological probes is d i s c u s s e d . CONTENTS. Page CHAPTER 1. INTRODUCTION. 1 1.1 Timber and decay. 2 1.1.1 The importance of wood. 2 1.1.2 World requirements of wood. 2 1.1.3 Source of wood. 3 1.1.4 Commercial wood. 4 1.1.5 Wood for construction. 5 1.1.6 Wood anatomy. 6 1.1.7 Wood microstructure. 8 1.1.8 Wood cell walls. 8 1.1.9 Durability of wood. 11 1.1.10 The polymeric nature of the wood cell. 12 1.1.11 Tree pathogens. 13 1.1.12 Biodegradation of wood. 14 1.1.13 Biodeterioration. 15 1.1.14 Fungal hyphae. 16 1.1.15 Fungal cell walls. 17 1.1.16 Wall architecture. 17 1.1.17 Cell wall biochemistry. 19 1.1.18 Decay fungi. 20 1.1.19 The Basidiomycetes. 20 1.1.20 White rot fungi. 23 1.1.21 Degradation of cellulose by white rot fungi. 24 1.1.22 Brown rot fungi. 25 1.1.23 Brown rot fungal decay of cellulose. 26 1.1.24 Timber preservation. 27 1.1.25 Alternatives to chemical preservation. 28 1.2 The dry rot fungus. 3 0 1.2.1 Serpula lacrymans. 30 1.2.2 Nomenclature. 30 1.2.3 Distribution. 31 1.2.4 Growth characteristics. 32 1.2.5 Economic importance. 34 1.2.6 Initial infection of building timber. 34 1.2.7 Morphogenesis of the mycelium. 36 1.2.8 Stage I mycelium. 37 1.2.9 Stage II mycelium. 37 1.2.10 Stage III strand. 38 1.2.11 Strand initiation. 38 1.2.12 Stage IV strand. 39 1.2.13 Fruit body formation. 40 1.2.14 Historical records. 40 1.2.15 History of morphological studies. 43 1.2.16 Recent physiological studies. 43 1.2.17 Studies on prevention and cure. 44 1.2.18 Novel aspects of control. 45 1.2.19 Treatment of infected buildings. 45 1.2.20 Detection of S. lacrymans. 46 1.2.21 Early detectionof S. lacrymans. 47 1.3 Molecular analysis. 48 1.3.1 Protein electrophoresis. 48 1.3.2 Immunological detection techniques. 49 1.3.3 Immunoassays. 50 1.3.4 Immunoanalysis. 52 1.4 Description and aims of the project. 53 CHAPTER 2. MATERIALS AND METHODS. 54 Source of equipment and materials. 55 1. Fungal isolates. 55 2. Culture of organisms. 55 2.1 Temperature for growth. 55 2.2 Routine maintenance of fungal organisms. 55 2.3 Growth of mycelium for analysis. 59 2.3.1 Agar grown mycelium. 59 2.3.2 Standard mycelium. 59 2.3.3 Young mycelium. 60 2.3.4 Aged mycelium. 60 2.3.5 Variation in cultural parameters. 60 2.3.6 Mycelium in low nitrogen medium. 61 2.3.7 Mycelium exposed to 40°C. 61 2.3.8 Mycelium exposed to other fungal organisms. 62 2.4 Infection of wood with S. lacrymans FPRL 12C. 62 2.4.1 Preparation of wood. 62 2.4.2 Infection of blocks. 63 2.4.3 Uplift of blocks. 63 2.4.4 Assessment of infection. 64 2.4.4.1 Microscopical assessment. 64 2.4.4.2 Weight loss. 64 3. Field studies. 65 3.1 Sites of collection of field samples. 65 3.2 Field notes. 65 3.3 Sample collection. 65 3.3.1 Fungal samples. 65 3.3.2 Wood samples. 67 4. Further sample processing and storage. 67 5. Antiserum production. 68 5.1 Immunogen preparation and immunisation. 68 5.2 Antisera. 68 5.2.1 Preparation of specific antiserum. 68 5.2.2 Commercial antisera. 69 5.3 Absorbtion of antiserum. 69 6. Reagents for immunological techniques. 70 6.1 Membranes. 70 6.2 Buffers. 70 6.3 Chromogenic substrates. 70 7. Dot immuno-binding assay. 71 7.1 Titre assessment. 71 7.1.1 Antigen preparation. 71 7.1.2 Initial protocol. 71 7.1.3 Detection of HRP-Ab 2 binding. 72 7.1.4 Non-specific binding to nitrocellulose. 72 7.1.5 Binding of control antiserum to antigen. 72 7.1.6 The effect of sample preparation. 72 7.2 Cross reactivity. 73 8. Protein separation by electrophoresis. 73 8.1 Sample preparation prior to electrophoresis. 73 8.1.1 Initial experiments.