II FUNGAL COLONISATION OF EXTRACTION WOUNDS IN CONIFERS By HASHIM ALl EL-ATTA B.Sc. (Honours),University of Khartoum Submitted for the degree of Ph.D. University of Edinburgh, Department of Forestry and Natural I Resources, 1984. '4 U) ABSTRACT Four compartments of Norway spruce (Picea abies L. Karst) and Japanese larch (Larix kaempferi - Lamb. & Carr.) were examined for the occurrence of extraction damage on the stems and superficial roots and the associated microflora. The study was carried out in two forests (Glentress and lair Hill Forest) in the Tweed Valley in Scotland. Trees were dissected and the spread of discolouration and decay assosiated with extraction damage were recorded. Stereum sanguinolentum (Alb. and Schw. ex Fr.) Fr. was the most common species isolated from infected Norway spruce , whereas Chaetomium cochliodes Palliser was the dominant species in Japanese larch. Four strains of S.sanguinolentum were isolated during the course of experiments and a detailed study on these strains was carried out. The expansion of decay by S.sanguinolentum was much greater in Norway spruce than in Japanese larch. Extraction wound and tree vigour were - the most important factors that influenced the development of decay in Norway spruce. Fast growing trees showed the greatest expansion of decay as compared with slow growing trees. A number of correlations were demonstrated between wound , decay spread and tree vigour which might enable the forest manager to estimate the spread of decay by S.sanguinolentum with the knowledge of at least one variable. A total of 156 Norway spruce trees were artificially inoculated with 3.sanguinolentum to study the (ii) effect of inoculation site treatment. (open or sealed) , tree vigour and time of year at which the inoculations were made on the frequency, and development of decay. Open inoculation sites were more likely to be infected compared with sealed inoculation sites. Decay was much greater in trees inoculated in July as compared with trees inoculated in February. Most of the correlations derived from artificial inoculation results confirmed the results obtained from naturally infected trees. Trichoderma viride Fr. showed a greater tendency to replace and suppress S.sanguinolentum in living trees as well as in wood blocks in the laboratory. Wood samples from naturally and artificially inoculated trees as well as wood blocks artificially inoculated with S.sanguinolentum were analysed chemically to examine the effect of the fungus on cellulose and lignin. 8 years after natural infection with S.sanguinolentum an approximately 50% Loss of cellulose was recorded in dominant Norway spruce trees. The destruction of cellulose and lignin was consistently much greater in fast growing trees as compared with slow growing trees. (ii-') DECLARATION This thesis has been composed by myself and it has not been submitted for any previous degree. The work reported was executed by myself unless otherwise stated. (iv) ACKNOWLEDGEMENTS I wish to express my sincere appreciation for the people who have made this investigation possible. I am greatly indebted to my supervisor Dr. A.J. Hayes for his interest, encouragement and invaluable guidance during the course of this work. I would like to express my gratitude to Dr. W.E.S. Mutch, the Head, Department of Forestry and Natural Resources for allowing me to use the facilities in the Department. My warmest thanks to Mr. D. Scott for driving, arranging for transport facilities and his assistance in some measurements in the field. Many thanks are due to Mr. A. Harrower and Mr D. Mackenzie for their help in the workshop. I am also greatful to the Forestry Commission for providing me with the experimental sites. Many thanks to the Commonwealth Mycological Institute, Kew, for the identification of many fungi in culture. Many thanks to the staff at Edinburgh Regional Computing Centre (ERCC) for the invaluable computing facilities and guidance that enabled this thesis to be completed in the present form. I would like to thank the University of Khartoum for financing this study. Finally, I am deeply indebted to my family for their encouragement during my work. CONTENTS Page Abstract (i) Declaration (iii) Acknowledgements (iv) - CHAPTER 1 - Introduction 1 - CHAPTER 2 - Review of previous work 5 2.1. Extraction damage in 5 A. Conifers: 5 A.1. Britain 5 A.2. Europe g A.3. North America and Canada: 10 A.3.1. Douglas fir jpseudotsuga menziesii (Mirb. ) (Franco.)]. 11 A.3.2. Western hemlock (Tsuga heterophylla (Raf.)Sarg. 11 A.3.3. Sitka spruce and True firs (Picea sitchensis and 13 true firs) 16 A.3.4. Western hemlock . and Douglas fir (T.heterophvlla and P.menzjesii A.3.5. Englemann spruce (Picea enpelmanii (Parry). 17 A.4. Africa,New Zealand and Australia 17 B. Hardwoods: . 18 8.1. Sugar maple (Acer saccharum March.), red maple 18 (A.rubrurn L.), yellow birch (Betula afleghaniensis Britt.), paper birch (apapyrifera Marsh.), American beech (Pagus grandifolia Ehrh.) and white ash (Fraxinus americana L.) B.2. Oak (Ouercus spp) 20 B.3. Aspen (Populus tremuloides Michx.) 21 B.4. Yellow poplar (Liriodendron tulipifera L.), black gum 22 (Nyssa sylvatica Marsh.) and ash (Fraxinus spp). 22 8.5. Hickory (Carya spp). 3.6. Southern and Appalachian hardwoods 23 2. 2. Relationships between different parameters in the process 23 of decay 2.2.1. wound parameters 23 2.2.2. tree parameters 29 2.3. Artificial wounding and inoculation experiments . 32 2.4. Stereum spncruinpj.entum [Alb. & Schw. a Fr.] Fr. as 36 a saprophyte and a wound pathogen 2.5. Fomes annosus [FrjCke. as a wound pathogen 41 2.6. Physiological studies on S.sanguinolentum 42 2.7. Effect of insect boring 44 2.8. Succession and interaction of microorganisms 48 - CHAPTER 3 Materials and methods 56 Results CHAPTER 4 Investigation of the frequency of extraction damage and decay 76 4.1. Frequency of injured and infected trees 4.1.1. Younger Norway spruce (Picea abies (L.) Karst. 4.1.2. older Norway spruce 4.1.3. : L:src ~ Japanese larch (Larix kaempferi (Lamb. and Carr.) cp5;) 4.1.4. *Ejp Japanese larch £?5 4) 4.2. Description of the infection column 4.2.1. Norway spruce 4.2.2. Japanese larch 4.3. Rate of progress of decay 4.4. Factors affecting the development of decay 4.4.1. Norway spruce Younger Norway spruce Older Norway spruce 4.4.2. Japanese larch 3) Ci) •:.'L 2 Japanese larch C Japanese larch t 6o vp4vtMct CO 4.5. Frequency of microorganisms isolated A. Younger Norway spruce A.1. Isolations from the surface inwards at the wound site A.2. Isolations above and below the wound site 109. Older Norway spruce 111 B.1. Isolations from the surface inwards at the wound site 111 5.2. Isolations above and below the wound site 113 Isolations above and below the wouhd site 113 Younger Japanese larch 113 Older Japanese larch 115 - CHAPTER 5 - 5. Artificial inoculation experiments 117 5.1. Description of the infection column 117 5.2. Frequency of successful infection 117 5.2.1. Trees inoculated in July 1982 117 5.2.2. Trees inoculated in February 1983 120 5.3. Comparison between July and February inoculations 124 5.3.1. Frequency of infection 124 5.3.2. Development of incipient decay 127 5.4. Factors affecting the development of decay 132 5.5. Frequency of microorganisms isolated 148 A. Sealed inoculation sites 148 A.I. Isolations at the site of inoculation inwards 148 Isolations above the inoculation site 150 Isolations below the inoculation site 152 Isolations from the distal parts of the decay columns 152 B. Open inoculation sites 152 51. Isolations at the site of inoculation inwards 155 5.2. Isolations above the inoculation site 155 5.3. Isolations below the inoculation site 158 B.4. Isolations from the distal parts of the decay columns 158 C. Naturally infected controls 158 5.6. Effect of inoculation site treatment on the 161 frequency of Stereum sanguinolentum (Mb. and Schw. ex Fr.) Fr. - CHAPTER 6 6. Growth studies Growth of strains 1,23 and 4 on 6.1. 3% malt extract agar (MEA) 168,179,190,199 6.2. 3% corn meal agar (CMA) 169,179,190,199 169,182,190,199 6.3. spruce-sawdust agar 6.4. Utilization of different carbon sources in combination with 172,182, 192 199 6.4.1.tnorganic nitrogen sources - 172,182,192,199 (j.) Sodium nitrate , 172,182,192,201 Ammonium sulphate 172,183,192,201 Comparison between sodium nitrate and ammonium sulphate 172,83,92,20: Effect of carbon and nitrogen sources on optimum growth 177,88,206 177,188,196,206 6.4.2. organic nitrogen sources : 6.4.3. optimum temperature for growth 179,188,196,208 6.4.4. Effect of pH (U Strain 1 208 (ii) Strain 2 208 (iii)Strain 3 208 211 (iv) Strain 4 - CHAPTER 7 7. cellulose and lignin decomposition 214 7.1. Introduction 214 Chemical structure of wood 214 Action of decay fungi on wood constituents 214 Differences between white and brown rot fungi 215 7.2. Analysis of artificially inoculated standing trees 217 Initial lipidce1lulose and lignin content 217 Effect of S.sanguinolentum six months after inoculation 217 7.3. Analysis of naturally infected trees 224 A. Initial lipid,cellulose and lignin content 224 B. Effect of S.sanguinolentum eight years after natural 229 infection 8.1. Dominants 229 8.2. Codominants 229 8.3. Subdominants 230 C. Comparison between younger and older crops 236 Dominants . 236 Codominants 236 Subdominants - 238 7.4. Analysis of artificially inoculated wood blocks 238 7.4.1. Analysis of control samples 238 7.4.2. Effect of S.spnguinolentum 240 240 (i) One month after inoculation (ii) Two months after inoculation 240 a.