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THE Etoi,'ECTS of CERTAIN WOOD PRESERVATIVES and THEIR DISTRIBUTION on SOME FUNGI CAUSING TIMBER DECAY

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THE Etoi,'ECTS of CERTAIN WOOD PRESERVATIVES and THEIR DISTRIBUTION on SOME FUNGI CAUSING TIMBER DECAY THE Etoi,'ECTS OF CERTAIN WOOD PRESERVATIVES AND THEIR DISTRIBUTION ON SOME FUNGI CAUSING TIMBER DECAY A thesis submitted in part fulfilment of the requirements for the Diploma of Membership of the Imperial College of Science and Technology by Anthony Frederick Bravery BSc (Southampton) Department of Botany Imperial College of Science and Technology London SW 7 October 1972 ABSTRACT The present study is primarily concerned with determining the fungitoxic significance of a new principle in the preservation of wood, that of introducing organic-solvent type preservatives into wood in a solvent system which penetrates the cell walls. The need for greater research effort on the inter-relationships between wood destroying fungi, wood preserving chemicals and the wood substrate itself is identified and discussed, together with the potential theoretical and practical significance of the principle forming the basis of the present study. The few previous studies in the specific field are reviewed. The study employs water-free diethylene dioxide as the non wall- penetrating solvent and hydrated diethylene dioxide as the wall- penetrating system. The three common, commercial, preservatives, pentachlorophenol, tri butyl tin oxide and copper naphthenate have been employed. The effect of these chemicals with and without wall- swelling has been studied by sequential observation on 3 permeable timbers representing hardwoods (beech - Fagus sylvatica, and birch - Betula sp) and softwoods (Scots pine - Pinus sylvestris) attacked by 3 fungi representing white rot (Polystictus versicolor) brown rot (Coniophora cerebella) and soft-rot (Chaetomium globosum). From histochemical staining techniques, scanning electron microprobe analysis, measurements of physical swelling characteristics, and chemical extraction experiments, it has been concluded that, although the particular swelling solvent system may lead to more even gross distribution of the chemicals, it does not deposit them evenly at depth across the thickness of the cell walls. Instead they are left as more complete and even internal films, keyed into the lumen/wall interface by physical and/or chemical forces. A small-scale, petri -dish/agar wood-block comparative toxicity test has been developed. Tri -butyl tin oxide was found to be more effective when deposited from the swelling system in beech and birch against all 3 test fungi, but only against P versicoZ.or in,Scots pine. Copper naphthenate in all 3 timbers showed improved toxicity only against ii P versicolor, No differences in toxic effect were observed with pentachlorophenol. Common pathways were established in the timbers for both preservative penetration and hyphal invasion. Micro-•morphological studies using light and scanning reflection electron microscopic techniques revealed essentially similar patterns of hyphal colonisation and wall lysis regardless of preservative treatment or solvent. The presence of the preservatives was shown to cause certain slight changes in the relative importance of the different pathways for hyphal penetration. The most important effect of the chemicals lay in reducing the rates of colonisation and proliferation by the fungi in treated wood. It is suggested that the presence and composition of the S wall layer is of 3 particular significance in limiting both extra—cellular enzyme activity and penetration of the chemicals into the cell walls. Simple techniques were developed to investigate the action mechanisms of the chemicals and these indicated that the major toxic effects of all the chemicals are exerted within the fungus cells or at the cell membranes and that extra—cellular action is probably limited and of lesser significance. iii INDEX Title page Abstract ii Index iv I INTRODUCTION 1 1.1 General 1 1.2 Historical, technical and scientific background 2 1.3 Previous studies 12 II PRESERVATIVE STUDIES 19 2.1 Treatment process 19 2.1.1 Introduction 19 2.1.2 General materials and methods 19 2.1.2.1 Preservatives 19 2.1.2.1.1 copper naphthenate 20 2.1.2.1.2 pentachlorophenol 21 2.1.2.1.3 tri—n—butyl tin oxide 21 2.1.2.2 Timber samples 22 2.1.2.g Treating solutions 24 2.1.2.3.1 range of concentrations 24 2.1.2.3.2 solvents 24 2.1.2.3.3 preparation 25 2.1.2.4 Impregnation 25. 2.1.2.5 Soaking 25 2.1.2.6 Absorptions and preservative retentions . 27 2.1.2.7 Solvent removal 28 2.2 Distribution studies 33 2.2.1 General introduction 33 2.2.2 Histochemical staining techniques 37 2.2.2.1 Introduction 37 2.2.2.1.1 Copper tests 38 2.2.2.1.2 pentachlorophenol tests 39 2.2.2.1.3 tri—n—butyl tin oxide tests 40 iv 2.2.2.2 Experimental 41 2.2.2.2.1 General preparation of samples 41 2.2.2.2.2 Tests for Cut 2.2.2.2.2.1 methods 42 2.2.2.2.2.2 results 45 2.2.2.2.3 Tests for PCP 47 2.2.2.2.3.1 methods 47 2.2.2.2.3.2 results 49 2.2.2.2.4 Tests for TBTO 50 2.2.2.2.4.1 methods 50 2.2.2.2.4.2 results 51 2.2.2.3 Discussion of the methods 51 2.2.3 Scanning electron probe microanalysis 52 2.2.3.1 Introduction 52 2.2.3.2 Principles of the technique 53 2.2.3.3 Application 55 2.2.3.4 Experimental 59 2.2.3.4.1 Equipments 59 2.2.3.4.2 Sample preparation 59 2.2.3.4.3 Data recording 61 2.2.3.5 Results 62 2.2.3.5.1 Geoscan BRS 62 2.2.3.5.2 Jeol JXA 3A 64 2.2.3.5.3 Geoscan IC 66 2.2.3.5.4 Cambridge Geoscan 68 2.2.3.6 Discussion of the method 70 2.2.4 Physical swelling characteristics 72 2.2.4.1- Introduction 72 2.2.4.2 Experimental 73 2.2.4.2.1 Preliminary test 1 73 2.2.4.2.2 Preliminary test 2 74 2.2.4.2.3 Main tests 74 2.2.4.2.3.1 preparation of wood blocks 74 2.2.4.2.3.2 measurement of swelling 75 2.2.4.3 Results 77 2.2.4.3.1 Records 77 2.2.4.3.2 Copper naphthenate 79 2.2.4.3.3 Tri—n—butyltin oxide 81 2.2.5.4 Discussion 95 2.3 Discussion of Distribution Studies 101 III MICROBIOLOGICAL STUDIES 112 3.1 Toxicity determinations 112 3.1.1 Introduction 112 3.1.2 Materials and methods 114 3.1.2.1 Preparation of wood blocks 114 3.1.2.2 Test fungi 115 3.1.2.3 Culture vessels and growth media 115 3.1.2.4 Preparation of cultures 116 3.1.2.5 Infection of blocks 116 3.1.2.5.1 Agar tests 117 3.1.2.5.2 Mixed—culture soil burial tests 119 3.1.2.6 Assessment of decay 120 3.1.3 Results 121 3.1.3.1 Records 121 3.1.3.2 Pentachlorophenol 121 3.1.3.3 Copper naphthenate 123 3.1.3.4 Tri—butyl tin oxide 123 3.1.3.5 Soil burial tests 125 3.1.4 Discussion 125 3.2 Micromorphological studies 132 3.2.1 Introduction 132 3.2.2 Methods 133 3.2.2.1 Exposure to fungus 133 3.2.2.2 Fixing and storage 134 3.2.3 EXamination 134 3.2.3.1 Gross visual examination 134 3.2.3.2 Optical microscopy 138 3.2.3.2.1 Sectioning 138 3.2.3.2.2 Staining 140 3.2.3.2.3 Observation 141 3.2.3.2.4 Results 142 3.2.3.2.5 Discussion 175 3.2.3.3 Scanning reflection electron microscopy . 185 3.2.3.3.1 Introduction 185 3.2.3.3.2 Sample preparation 187 vi 3.2.3.3.3 Results — untreated timber 188 TBTO treated timber 206 3.2.3.3.4 Discussion — untreated timber 219 TBTO treated timber 225 3.3 Action mechanisms 231 3.3.1 Introduction 231 3.3.2 Experimental 234 3.3.2.1 Cellulose agar growth medium 234 3.3.2.2 Inoculation 236 3.3.2.3 Cellulose enzyme assay medium 236 3.3.2.4 Preparation of cell free culture filtrates 238 3.3.2.5 Harvesting of filtrates 238 3.3.2.6 Plate assay technique 239 3.3.2.7 Tube assay technique 239 3.3.2.8 Gel electrophoresis 242 3.3.2.9 Preservative concentrations 242 3.3.3 Results 243 3.3.3.1 Growth experiments — records 243 3.3.3.2 Enzyme assays — records 246 3.3.4 Discussion 248 IV GENERAL DISCUSSION AND CONCLUSIONS 261 4.1 General discussion 261 4.2 Conclusions 263 ACKNOWLEDGEMENTS 267 APPENDIX OF TABLES 268 REFERENCES 290 AUTHOR'S PUBLICATIONS 312 vii I INTRODUCTION 1.1 General The investigations reported in this thesis were carried out partly in the Botany Department, Imperial College of Science and Technology, and partly at Forest Products Research Laboratory (FPRL)*, Princes Risborough, Buckinghamshire. The author was registered under the University of London regulations for persons engaged in research at Public Research Institutions and Industrial Research Laboratories. The study was initiated as an extension of a research programme at FPRL on the location of extractive constituents in wood and its significance in the natural resistance of certain timbers to fungal decay. This research had shown that certain extractive chemicals could only be removed from the wood by using suitable solvents that could also •penetrate the cell walls whilst others could be extracted using non—wall penetrating solvents (Morgan and Orsler, 1969 a and b).
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