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UBC Theses and Dissertations

Wetwood in black cottonwood (Populus Trichocarpa Torrey and Gray): the effects of microaerobic conditions… Gokhale, Atulchandra Anant 1976

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WETWOOD IN BLACK COTTONWOOD (PQPULUS TRICHOCARPA TORREY AND GRAY): THE EFFECTS OF MICROAEROBIC CONDITIONS ON THE DEVELOPMENT OF DECAY by ATULCHANDRA ANANT GOKHALE B.Sc, M.Sc, University of Poona M.Sc, University of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF FORESTRY We accept t h i s thesis as conforming to the required, standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1975 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date <&U~< > 1 » q T S " i i ABSTRACT A study i s described on the occurrence of wetwood i n black cottonwood, found i n the Lower Fraser Valley of B r i t i s h Columbia, Canada. Wetwood f i r s t appears when trees are 8 to 10 years o l d , and i n 12 year and older trees, wetwood i s always present. Further extension of wetwood i s s i m i l a r to that of a normal heartwood, A large number of microorganisms with different 0^ requirements ( i . e . , aerobes as well as facu l t a t i v e and obligate anaerobes) were found i n the wetwood. Wood-destroying fungi were absent i n the wetwood. In the majority of the black cottonwoods examined, very low 0£ concen-trations (<0.1% or microaerobic conditions, detected by a Field-lab 0^ analy-zer) were found i n the wetwood during the summer. Generally the 0^ concen-tr a t i o n increased during the winter while the reverse was true for CO2 (de-tected by gas chromatography). CH^ was also present, but only i n small quan-t i t i e s , i n most trees. Mechanical wounding of wetwood resulted i n an increase i n the 0^ concentration and decrease i n the CO2 and CH^ concentrations. How-ever, sealing of the wound re-established the o r i g i n a l gas composition. Presumably the microbial f l o r a of wetwood i s primarily responsible for the microaerobic conditions. The a b i l i t y of 2 wood-destroying fungi (Polyporus delectans Peck and Ganoderma applanation [Pers.] Pat.) to cause a weight loss i n wood under microaerobic and aerobic conditions (10 weeks incubation each) was determined, using a soil-block experimental technique, Microaerobic conditions prevented weight loss i n wood (average 0.1%) and therefore wood decay, whereas under i i i aerobic conditions, the average weight loss was 41.7%. Special character-i s t i c s of wetwood such as high pH (average 7.8) or high moisture content (approximately 160%) did not contribute s i g n i f i c a n t l y to the decay r e s i s t -ance. Also, microorganisms associated with wetwood showed no antagonism to the growth of wood-destroying fungi. Exposure of wood-destroying fungi to microaerobic conditions subsequent to aerobic conditions arrested t h e i r growth and a b i l i t y to cause weight loss. On the other hand, exposure of these fungi to aerobic conditions following the microaerobic conditions, i n -duced a s i g n i f i c a n t weight loss i n wood (average 10.9%), but i t was con-siderably less than when the fungi were grown under aerobic conditions alone (average 41.7%). The 2 wood-destroying fungi survived 10 weeks exposure to microaerobic conditions, A 13 weeks exposure to anaerobic conditions (<0.002 % 0^), how-ever, resulted i n the death of these, wood-destroying fungi. Eight wood-destroying fungi di f f e r e d i n t h e i r a b i l i t i e s to survive under anaerobic con-di t i o n s ; generally brown-rot fungi tolerated anaerobic conditions better than the white-rot fungi. Therefore, i f anaerobic conditions exist i n the tree trunks for long durations, some fungi (probably white-rot fungi) may die during that period while others (probably brown-rot fungi) may survive such an exposure and resume decay a c t i v i t y when 0^ becomes available. These findings led to the concept that the microaerobic conditions found i n the wetwood of black cottonwood may prevent the development of decay. i v TABLE OF CONTENTS Page TITLE PAGE ..... i ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES ..... v i i i ACKNOWLEDGEMENTS x i FRONTISPIECE ..... x i i GENERAL INTRODUCTION ..... 1 CHAPTER I - OCCURRENCE OF WETWOOD IN BLACK COTTONWOOD 6 INTRODUCTION , 6 MATERIALS AND METHODS ..... 8 RESULTS AND DISCUSSION 10 CHAPTER II - MICROORGANISMS ASSOCIATED WITH WETWOOD IN BLACK COTTONWOOD 29 INTRODUCTION . • 29 MATERIALS AND METHODS ..... 34 1. Tree samples 34 2. Quantitative determinations ..... 35 3. Isolations ..... 36 3.1 Aerobic bacteria ..... 37 3.2 Fungi 37 3.3 Anaerobic bacteria (facultative) ..... 37 3.4 Anaerobic bacteria (obligate) ..... 38 RESULTS 38 1. Quantitative determinations ..... 38 2. Isolations 38 2.1 Wetwood: Aerobic bacteria ..... 38 2.2 Wetwood: fungi 41 2.3 Wetwood: Anaerobic bacteria (facultative) ..... 43 2.4 Wetwood: Anaerobic bacteria (obligate) ..... 43 2.5 Sapwood 43 2.6 Wound-initiated discolored wood ..... 43 2.7 Young trees and seedlings 44 DISCUSSION 44 Table o f Contents (cont'd) v Page CHAPTER I I I - COMPOSITION OF GASES IN THE TRUNKS OF BLACK COTTONWOOD 51 INTRODUCTION ..... 51 MATERIALS AND METHODS 60 1. Location o f sample tr e e s ..... 60 2. E x t r a c t i o n apparatus ..... 64 3. E x t r a c t i o n apparatus: Assembly 70 4. Method and operation o f e x t r a c t i n g gas samples 75 4.1 Black cottonwood: Sapwood 80 4.2 Black cottonwood: From wetwood through i branch stubs 80 4.3 Black cottonwood: ..Wetwood o f wounded t r e e s : Wetwood exposed ..... 81 4.4 Black cottonwood: ..Wetwood o f wounded t r e e s : Sapwood exposed 81 5. Gas a n a l y s i s 87 6. Measurement o f gas pressures i n the wetwood ..... 88 RESULTS ..... 88 1. Black cottonwood: Wetwood: Examined a l l through the year 89 2. Black cottonwood: Wetwood: Examined o n l y during c e r t a i n months o f the year ..... 94 3. Black cottonwood: Sapwood ..... 94 4. Black cottonwood: From wetwood through branch stubs ' ..... 94 5. Black cottonwood: Wetwood o f wounded t r e e s : Wetwood exposed ..... 99 6. Black cottonwood: Wetwood o f wounded t r e e s : Sapwood exposed ..... 102 7. Red a l d e r : Heartwood 105 8. Lombardy p o p l a r : Heartwood (probably wetwood) ..... 105 9. Measurement o f gas pressures i n the wetwood 105 DISCUSSION ..... 105 CHAPTER IV - EFFECTS OF MICROAEROBIC CONDITIONS ON DEVELOPMENT OF DECAY ..... 122 INTRODUCTION .. ., . 122 MATERIALS AND METHODS ..... 127 RESULTS AND DISCUSSION 132 CHAPTER V - SURVIVAL OF WOOD-DESTROYING FUNGI UNDER ANAEROBIC CONDITIONS 138 INTRODUCTION ...... 138 MATERIALS AND METHODS 140 RESULTS AND DISCUSSION 143 v i Table of Contents (cont'd) Page CHAPTER VI - SIGNIFICANCE OF WETWOOD IN BLACK COTTONWOOD (SUMMARY AND CONCLUSIONS) ...... 148 BIBLIOGRAPHY . 154 APPENDIX 162 I. Isolation of anaerobic bacteria (obligate) from wetwood ..•., 162 II-A. 1.Calibration curve of CO^ concentration 164 2.Calibration curve of CH. concentration ..... 166 4 II-B Gas composition i n the tree trunks 168 v i i LIST OF TABLES Page TABLE I: The moisture content (%) of wetwood and sapwood samples collected at three different heights from four trees obtained from the U.B.C. Research Forest, Maple Ridge, B.C. ...... 15 TABLE I I : The pH of wetwood and sapwood samples collected at two different heights from four trees, obtained from the U.B.C. Research Forest, Maple Ridge, B.C.... 17 TABLE I I I : Number of non-wounded black cottonwood trees and seedlings, with or without wetwood, from 5 separate l o c a l i t i e s i n Lower Fraser Valley i n the Province of B r i t i s h Columbia 20 TABLE IV: Number of colonies of bacteria i n the l i q u i d s ex-tracted from the wetwood (non-wounded trees) and discolored wood (wounded trees) samples collected at indicated heights. Counts are from 1:10,000 d i l u t i o n on nutrient agar plates 39 TABLE V: Some morphological and physiological characteris-t i c s of bacteria isolated from wetwood 40 TABLE VI: Frequency and characteristics of fungi isol a t e d from wetwood on 2% malt agar 42 TABLE VII: Description of sample trees, wood zones and dur-ation of sampling i n the year 1974 65 TABLE VIII: Linear growth of two wood-destroying fungi on 2% malt agar, after 16 days of incubation under aerobic (control) and microaerobic conditions ...... 133 TABLE IX: Mean % weight loss caused by two wood-destroying fungi i n black cottonwood blocks of sapwood (S) and wetwood (W) under aerobic (A) and micro-aerobic (M) conditions ...... 134 TABLE X: Characteristics of 8 wood-destroying fungi, and th e i r survival response to 13 weeks of incubation under anaerobic conditions ...... 141 v i i i LIST OF FIGURES Page FIGURE 1: Cross section of a 29 year old black Cottonwood showing centrally located discolored wood (wetwood) which i s sur-rounded by colorless wood (sapwood). 11 FIGURE 2: V e r t i c a l section of the same tree p i c -tured i n Figure 1, showing ce n t r a l l y located discolored wood (wetwood) and colorless wood (sapwood). 12 FIGURE 3: Dissection of a 45 year old black cotton-wood at various heights. ..... 13 FIGURE 4: V e r t i c a l section of a branch removed from the trunk of a black cottonwood. 14 FIGURE 5: Cross section of a wounded black cot-tonwood tree trunk showing discolora-tions associated with wounds merging into central wetwood column, giving i t a rather i r r e g u l a r appearance. 19 FIGURE 6: V e r t i c a l section of a 2 year old black cottonwood showing sapwood (colorless) and p i t h (dark). 23 FIGURE 7: V e r t i c a l section of an 8 year old black cottonwood showing early stage of wet-wood development (bottom), sapwood colorless) and p i t h (dark brown i n cen-ter) . 24 FIGURE 8: V e r t i c a l sections of a 7 year old black cottonwood showing sapwood (colorless) and p i t h (dark Brown) i n each piece and the t h i r d piece from l e f t also shows discoloration associated with a wound. 25 FIGURE 9: V e r t i c a l section of a 1 year old black cottonwood showing wound-initiated d i s -coloration and sapwood. 27 FIGURE 10: V e r t i c a l sections of a 2 year old black cottonwood showing wound-initiated d i s -coloration and sapwood. 27 i x L i s t of Figures (cont'd) Page FIGURE 11: Map of the University Endowment Lands showing location of sample trees used for the gas composition work. ..... 62 FIGURE 12: Black cottonwood-red alder stand sur-rounding University H i l l Church, Uni-v e r s i t y Endowment Lands (U.B.C). 63 FIGURE 13: Schematic drawings of the s p e c i a l l y de-signed apparatus. 69 FIGURE 14: Portable extraction apparatus mounted i n a plywood case. 72 FIGURE 15: Schematic drawing of the extraction apparatus assembly. 74 FIGURE 16: A.. Schematic drawing of the brass pipe i n position. 77 B. Brass pipe and shut-off valve i n position i n the f i e l d 77 C. V e r t i c a l section of a black cotton-wood through the sampling hole (extending from l e f t to right) ..... 78 FIGURE 17: Complete gas extraction set-up i n the f i e l d . 79 FIGURE 18: A. and B. Schematic drawing of a tree (No. 23) showing set-up used to extract gases through a branch stub 83 C. Set-up (as i n B) i n the f i e l d 83 FIGURE 19: A black cottonwood tree (No. 2) showing nature of the wound ( l e f t ) and normal extraction set-up ( r i g h t ) . 84 FIGURE 20: Schematic drawing of the two trees show-ing nature of wounds. 86 FIGURE 21: Concentrations of 0 2, C0 2 and CH4 (month-l y averages) i n the wetwood of a black cottonwood (Tree 15). 91 FIGURE 22: Concentrations of 0 2, CO2 and CH4 (month-l y averages) i n the wetwood of a black cottonwood (Tree 6). 93 X List of Figures (cont'd) Page FIGURE 23: Comparison of 0 2 and CO^ concentrations (monthly averages) in the wetwood (Tree 8) and sapwood (several trees) of black cottonwood. ..... 96 FIGURE 24: Concentrations of 0 2, C0 2 and CH4 (month-ly averages) in the wetwood of two black cottonwoods (Tree 19 and 23). ..... 98 FIGURE 25: Vertical section of Tree 23, through the branch stub showing where the brass pipe was placed (above) and position of the branch trace. 100 FIGURE 26: Cross-section of a 19 year old black cottonwood (Tree 2) showing extent of the wound. 101 FIGURE 27: Concentrations of 0 2, C0 2 and CH4 in the wetwood of a black cottonwood (Tree 21) 104 FIGURE 28: Concentrations of 0 2 and C0 2 (monthly averages) in the heartwood of a red alder (Tree 42) . . 1 0 7 FIGURE 29: Comparison of 0 2 and CO^ concentrations (monthly averages) in the wetwood of a black cottonwood (Tree 11) and heartwood of a red alder (Tree 41). 109 FIGURE 30: Concentrations of 0 2, C0 2 and CH4 (monthly averages) in the heartwood (probably wetwood) of three Lombary poplar trees (Nos., 44, 45 and 46). I l l FIGURE 31: Percentage of black cottonwood trees (non-wounded) with microaerobic conditions present in the wetwood from March unt i l September, 1974. • 114 FIGURE 32: Comparison of 0 2 and C0 2 concentrations (monthly averages) in tne wetwood of a non-wounded black cottonwood (tree 10) and a wounded black cottonwood (Tree 2). 119 FIGURE 33: Disposable Anaerobic System 129 x i ACKNOWLEDGEMENTS. I gra t e f u l l y acknowledge Dr. B.J, van der Kamp for providing guidance, advice and encouragement during t h i s research project. I also wish to ex-tend my sincere appreciation to Dr. Roger S. Smith, Western Forest Products Laboratory, Vancouver, B.C. for extending the laboratory f a c i l i t i e s with-out which this work might not have been completed. The thesis was reviewed by Drs. R.J. Bandoni (Botany), K. Graham (Forestry), B. Mullick ( P a c i f i c Forest Research Center, V i c t o r i a , B.C.), Roger S. Smith, J,V. Thirgood (Forestry) arid B.J. van der Kamp. For t h e i r counsel and c r i t i c i s m , 1 am much obliged. Special thanks are due to Dr. B. McBride (Microbiology) for allowing me to use the results of a j o i n t project i n t h i s thesis. I also wish to thank Dr. A. Kozak (Forestry) for his advice on the s t a t i s t i c a l analysis, Mr. G. Bohnenkamp (Forestry) and Mr. A. Hoda (Plant Science) for t h e i r help i n constructing the extraction apparatus and Mrs. P. Waldron for typing t h i s manuscript. Grateful appreciation i s due to the National Research Council of Canada and to the University of B r i t i s h Columbia for the f i n a n c i a l support extended throughout the academic program. Above a l l , I must acknowledge the continuous strength and encouragement given to me by my wife Veena during the entire course of t h i s study. x i i WETWOOD IN BLACK COTTONWOOD 1 GENERAL INTRODUCTION 2 A casual look at any mature black cottonwood (Populus trichocarpa Torrey and Gray) tree trunk i n cross section reveals that the inner wood i s dark, and the outer wood i s pale. The outer wood i s sapwood but the i d e n t i t y of the inner discolored wood i s not so obvious. One.might con-clude that this inner wood i s heartwood because of i t s position and d i s -coloration. However, i n contrast to normal heartwood, t h i s inner wood i s unusually wet. After a tree has been cut, water may continue to ooze from the cut surface for hours. The inner wood i s a l k a l i n e , and contains a large number of bacteria and some imperfect fungi. Forest pathologists t r a d i t i o n a l l y refer to t h i s type of wood as wetwood. In t h i s thesis, therefore, the term wetwood w i l l be used to designate the inner discolored wood i n spite of the fact that Wood Technology textbooks usually refer to i t as heartwood (Brown et a l . 1949). The term wetwood was f i r s t used by Lagerberg (1935) to describe condi-tions of high moisture content and discoloration found i n scots pine (Pinus  s y l v e s t r i s L.) and Norway spruce (Picea abies [L.] Karst.) i n Sweden. Since then, the occurrence of wetwood has been reported or inferred^ i n more than 30 hardwood and 10 softwood species (Knutson 1970), including most elms (Ulmus L.), poplars (Populus L . ) , Willows (Salix L.) and also some oaks (Quereus L.) and maples (Acer L.). Hartley et_ al_. (1961) and Knutson (1970) have reviewed the l i t e r a t u r e on wetwood, and i t i s apparent from t h e i r reviews that a considerable amount of- variation exists i n the characteristics of wetwood. For example, the colors attributed to wetwoods vary within a given species and also among 3 different species. Aspen (Populus tremuloides Michx.) wetwood has been described as having a darker color (Clausen et_ al_. 1949), or as being transluscent (Hartley et a l . 1961), or as being gray to grayish brown by Poluboyarinov (1963). Wetwood of European aspen (Populus tremula L.) has been described as yellow-pink to intense red-brown to green by Ankudinov (1939), while Borset and Haugen (1964) have stated that the color varies from brown to rust-red. S i m i l a r l y , position of the wetwood within trees d i f f e r s considerably. Wetwood can be present i n the tree as a 'zone be-tween sapwood and heartwood' or can occupy 'most of the heartwood and sap-wood' or 'only sapwood' or 'only heartwood'. Wetwood can be associated with branch stubs or wounds made by increment borer holes. It can also occur i n a position not associated with wounds or any other type of openings (Knutson 1970). As a r u l e , microorganisms are found i n the wetwood although t h e i r s i g -nificance i s not very clear. Bacteria, i n p a r t i c u l a r , are often considered as causal organisms of wetwood formation (Carter 1945, Seliskar ; 1950). Microorganisms are also implicated i n gas production (Morani and Arru 1958), as increased gas pressures have frequently been found i n the wetwood. Very low oxygen (0^) and high carbon dioxide (C0 2) concentrations are often found i n tree species showing a pattern of wetwood formation. A reduction product such as methane (CH^) has also been reported from a few poplar species, and recently, Zeikus and Ward (1974) isolated anaerobic bacteria (obligate) re-sponsible for the production of CH^ from the wetwood of 2 eastern cottonwoods (Populus deltoides Bartr.). Almost a l l studies of wetwood to date have been descriptive i n nature, dealing with d i s t r i b u t i o n of wetwood within trees and among various species, 4 gas composition of wetwood, microflora etc. However, the effect of wetwood on the functioning of the l i v i n g trees i s s t i l l poorly understood. Those investigators who have allowed themselves to speculate on the matter have generally taken the view that wetwood i s a pathological condition (e.g. Smith 1970) which probably promotes decay of heartwood by providing more suitable moisture conditions f o r fungal growth or by prolonging s u s c e p t i b i l i -ty by lengthening the time required for the wood to dry (Hartley et a l . 1961). The main hypothesis examined i n my study opposes this common view. My preliminary investigation (parts of i t included i n Chapters I and II) indicated that wetwood i n black cottonwood may not be a disease condi-t i o n . As a r e s u l t , rather than assuming wetwood to be a deleterious pheno-menon, i t i s suggested that i n black cottonwood wetwood i s b e n e f i c i a l to the survival of the tree because i t protects the tree from the development of decay. More s p e c i f i c a l l y , i t i s proposed that i n l i v i n g black cottonwoods, microorganisms associated with wetwood induce microaerobic or anaerobic con-ditions i n the wetwood which prevent establishment and/or development of decay i n the wetwood. Other characteristics such as high pH or high moisture content of wetwood also contribute to the decay resistance. The hypothesis was tested i n four separate steps. F i r s t l y , i s o l a t i o n studies were made to determine what kinds of microorganisms are present i n the wetwood and i n what numbers (Chapter I I ) . Secondly, gas composition studies were made to see i f microaerobic or anaerobic conditions exist i n the wetwood of l i v i n g trees (Chapter I I I ) . Then, based on observations made i n this study, a project was undertaken to study the extent of wood decay under a gaseous environment s i m i l a r to that found i n the wetwood (Chapter IV).• '5 Control experiments were set up to study the effects of some inherent properties of wetwood on i t s decay. Lastly, the survival of wood-destroy-ing fungi under anaerobic conditions was studied (Chapter V) i n view of the findings reported i n Chapter IV. Before undertaking these s p e c i f i c experiments, i t was essential to give a description of wetwood as i t occurs i n black cottonwood, i t s d i s t r i b u t i o n within trees and to know at what age wetwood begins to appear i n trees (Chapter I ) . Each of the above-mentioned chapters i s a self-contained unit and includes i t s own Introduction, Material and Methods, Results and the Dis-cussion. For convenience, a Bibliography has been presented at the end of the text. The last chapter (Chapter VI) i s b a s i c a l l y a short discussion of the results obtained during the course of t h i s thesis work. 6 CHAPTER I OCCURRENCE OF WETWOOD IN BLACK COTTONWOOD INTRODUCTION Water-soaked xylem tissue, which i s generally referred to as wet-wood, i s known to occur i n v i r t u a l l y every species of the genus Populus, Hartley et_ al_. (1961) reported that wetwood was present i n a l l trees of cottonwoods (Populus deltoides Bartr. or i t s segregates) and Lombardy poplars (Populus nigra var. i t a l i c a Muerchh.) that they sampled from various l o c a l i t i e s i n the United States. In addition, they examined white poplar (Populus alba L.), balsam poplar (Populus balsamifera L.) and black cottonwood trees from single l o c a l i t i e s and reported that every tree con-tained wetwood. Wallin (1954) mentioned common occurrence of wetwood i n balsam poplars i n Minnesota. On the other hand, some of the trees of east-ern cottonwood and bigtooth aspen (Populus grandidentata Michx.) from the midwestern United States, had reached large size without any recognizable wetwood, and i n Colorado, some entire study plots were nearly free of wet-wood (Hartley et a l . 1961). Also, Sachs et_al_. (1974) and Zeikus and Ward (1974) reported absence of wetwood i n white poplars from the Wisconsin area. Wetwood o r d i n a r i l y occupies the center of the stem i n very young trees and the upper parts of older ones. In old trees, wetness and high pH are usually confined to the outer heartwood or to the t r a n s i t i o n zone between heartwood and sapwood. In eastern cottonwood and American elm (Ulmus  americana L.), Hartley et_ al_. (1961) found that wetwood extended throughout the length of the bole and into the larger branches, and sometimes continued 7 through the roots below the s o i l surface. They stated that i n cottonwood (presumably eastern cottonwood), wetwood was commonly found i n 2-year old seedlings. In Lombardy poplar, wetwood was not only present but was regu-l a r l y well developed before the trees reached the age of 3 years. The European aspen has been reported to be almost universally affected by the age of 10 years with a non-decay.discoloration that was assumed to be wet-wood (Ankudinov 1939), Morani and Arru (1958) reported that a l l 17 year old poplars growing i n the neighbourhood of Pisa, I t a l y that they studied, con-tained "symptoms" of a b a c t e r i a l i n f e c t i o n i n the central part of the trunk, si m i l a r to those described for "wetwood disease". Wetwood has often.been found associated with increment borer holes (Davidson et_ al_. 1959), inoculation holes (Riley 1952) or branch stubs (Hartley et a l . 1961). Hartley and his co-workers quote Baker's study on cottonwoods i n which he found wetwood occasionally i n "injured" 1-year old seedlings and very commonly before the end of the second year i n natural reproduction at f i v e l o c a l i t i e s i n N, Dakota, Nebraska and Oklahoma. He attributed the wetwood streaks i n less than 2-year old cottonwoods to beetle injury near the s o i l surface. Knutson (1970) distinguished between two types of wetwood i n aspen, i n addition to normal heartwood and sapwood. The f i r s t could be traced to wounds and often formed long columns extending many feet up the trunk. The second type could not be related to any wound or branch stub, and columns rarely extended more than 4 or 5 feet up the bole. Thomas and Podmore (1953) f i r s t reported the occurrence of "water-soaked wood" i n black cottonwood. Hartley et a l .(1961) also indicated the presence of wetwood i n black cottonwood. However, none of these authors gave 8 a description of wetwood or i t s d i s t r i b u t i o n within the trees nor did they attempt to i s o l a t e microorganisms associated with i t . Consequently, I made a study with the following objectives: 1) to describe a few c h a r a c t e r i s t i c features of well developed wetwood (in mature trees) with emphasis on i t s d i s t r i b u t i o n within:.the trees. 2) to dissect a large number of trees to determine the age at which wetwood f i r s t appears, and 3) to search for wounds or special conditions that might be associated with wetwood i n i t i a t i o n . MATERIALS AND METHODS Tree samples came from 5 separate l o c a l i t i e s i n the Lower Fraser Valley i n the Province of B r i t i s h Columbia as shown i n Table I I I . The choice of l o c a l i t i e s was primarily based upon a v a i l a b i l i t y of black cottonwoods and not because of any s p e c i f i c environmental conditions of the l o c a l i t i e s . I n i t i a l l y , a large number of trees and seedlings of different ages was ex-amined for the presence of centrally-located, wet-appearing discolored wood. A very limited number of trees was then used to determine moisture content, pH (Chapter I) and microbial f l o r a (Chapter II) of this discolored wood. The findings of these studies provided the basis for considering the di s -colored wood to be a "wetwood" rather than a "heartwood". A l l the remaining trees i n which a detailed examination was not made, the presence or absence of centrally-located wet-appearing discolored wood was taken as presence or absence of wetwood, respectively. 9 A t o t a l of 224 trees, 213 non-wounded and 11 wounded, of various ages was examined to study the occurrence and d i s t r i b u t i o n of wetwood ( i n i t i a l l y , tree samples were collected at random although once the pat-tern of wetwood was known, some wounded trees were excluded, and there-fore are represented i n comparatively small number). In a l l of the trees examined, the stems were dissected transversely and/or v e r t i c a l l y i n the f i e l d . Only mature trees (with well developed wetwood) were used to study two important features of wetwood, namely the moisture content and pH. Four trees were f e l l e d on the U.B.C. Research Forest, Maple Ridge, B.C. and several 2 f t (61.0 cm)* long sections were removed at various heights (Tables I and II) and were brought back to the laboratory. To determine moisture content of the wood (wetwood and sapwood), test blocks (0.75 cm) were cut from the discs and, after weighing them imme-di a t e l y , were dried i n an oven at 102 C for about 24 hours. Next day, the blocks were cooled over dessiccant for one hour and weighed. Mois-ture content was expressed as a percentage of the dry weight of the wood. The method used to determine pH of the wood (wetwood and sapwood) was as described by Browning (1967). A f i n e l y divided sample (4.0 g) was soaked i n the d i s t i l l e d water (25 ml) for about 24 hours at approximately 25 C. A "Radiometer" pH-meter (PHM-28) was used to measure the pH of the aqueous extract,,,and was considered to represent the pH of the wood. Metric conversions have been given i n the brackets whenever actual measurements were made i n Imperial units. 10 RESULTS AND DISCUSSION The dissection studies revealed that i n mature black cottonwood trees, sapwood envelops a s o l i d column of discolored wood (wetwood) which extends from the roots through the trunk into the major branches (Figures 1, 2 and 3). The discolored column appears greenish-brown or rusty-brown upon cutting down the tree, and the color usually becomes even darker within a few hours of exposure to atmosphere. The boundary between the discolored wood and sapwood i s often i r r e g u l a r , the discolored wood ex-tending several annual rings closer to the cambium at some points than others. Generally sapwood consists of fewer rings (though usually wide ones) toward the crown than toward the butt. The central column extends into the major branches, although i t usually does not reach the branch t i p s . Frequently the discolored column originating from the trunk tapers o f f quickly as i t enters the branches (Figure 4). The discolored wood appears water-soaked and, when i t i s freshly cut, water oozes. The moisture content determinations (Table I) showed that the discolored wood had a r e l a t i v e l y high moisture content of approximately 160% (range: 134.0 - 192.8%), as compared with 125% (range: 116.1 -149.3%) of the sapwood. These values are perhaps s l i g h t l y less than the actual moisture content of the respective woods. Hartley et a l . (1961) point out that gravimetric comparisons of wetwood with sapwood may not precisely represent the r e l a t i v e moisture content as i t was before the tree was cut. A transverse cut i n a trunk, at least during the growing season, i s l i k e l y to result i n "instantaneous p u l l i n g away" of some of the water which might lead to a decrease i n the moisture content of the Figure 1: Cross s e c t i o n of a 29 year o l d black cottonwood showing c e n t r a l l y l o c a t e d d i s c o l o r e d wood (wetwood) which i s s u r -rounded by c o l o r l e s s wood (sapwood). This photograph was taken a f t e r the "darkening process" of d i s c o l o r a t i o n had taken place (3 hours a f t e r s e c t i o n c u t t i n g ) . Sample height-approximately 1 meter above the ground. Scale -X 0.25. 12 Figure 2: V e r t i c a l s e c t i o n o f the same tree p i c t u r e d i n Figure 1, showing c e n t r a l l y located d i s c o l o r e d wood (wetwood) and c o l o r l e s s wood (sapwood). Sample height - approximately 0.3 meters above the ground (bottom). Scale - X 0.17. 13 Figure 3: D i s s e c t i o n o f a 45 year o l d black cottonwood at various h e i g h t s , from bottom r i g h t to l e f t to top r i g h t ; 0.3, 1.2, 2.4, 4.5, 9.0, 13.5, 18.0, 22.5, 27.0 and 31.5 meters above the ground ( i n d i c a t e d on each d i s c i n f e e t ) . Note the presence o f wetwood throughout the b o l e . Section at 4.0 f e e t (1.2 meters) shows a wound and the d i s c o l o r a t i o n a s s o c i a t e d w i t h i t . 14 Figure 4: V e r t i c a l s e c t i o n o f a branch removed from the trunk of a black cottonwood. Note the r a p i d r e d u c t i o n i n width of the wetwood (bottom). Scale - X 0.17. 15 TABLE I: The moisture content (%) o f wetwood and sapwood samples c o l l e c t e d at three d i f f e r e n t heights from f o u r t r e e s ob-t a i n e d from the U.B.C. Research F o r e s t , Maple Ridge, B.C. Number Tree c h a r a c t e r i s t i c s Sample height Moisture content Age (Yr) DBM Height (in[cm]) (ft[m]) (ft[m] above ground) Wetwood Sapwood 1 37 25.0(63.5) 66.0(19.5) 3.0(0.9) 166.8 145.1 6.0(1.8) 192.8 149.3 12.0(3.6) 188.7 127.0 2 47 29.0(73.7) 70.0(21.3) 3.0(0.9) 160.2 119.7 6.0(1.8) 156.3 116.1 12.0(3.6) 155.0 116.6 3 41 31.0(78.8) 79.0(24.0) 3.0(0.9) 137.0 122.2 6.0(1.8) 138.0 124.3 12.0(3.6) 134.0 145.2 4 42 31.0(78.8) 79.0(24.0) 3.0(0.9) 148.5 121.1 6.0(1.8) 163.0 120.4 12.0(3.6) 171.3 115.4 Average 159.4 126.8 16 outer rings (sapwood).. At the same time, loss of some water from the f r e s h l y cut wetwood cannot be avoided. Even with many pos s i b l e v a r i a -t i o n s , i t i s generally agreed that wetwood has a higher moisture content than the sapwood. Moisture content of 160% (average) indicates that a sub s t a n t i a l part of the void space i n discolored wood i s s t i l l occupied by gases. Void space i n a given piece of wood represents the t o t a l volume of water and gases that wood can hold under normal pressure. In black cottonwood, the maximum moisture that the discolored wood can hold would be approxi-mately 270%, assuming that the s p e c i f i c gravity o f black cottonwood i s 0.3 and the true s p e c i f i c g r a v i t y of wood substance i s 1.5 (Stamm 1964). A simple subtraction of the actual moisture content (160%) from the maxi-mum pos s i b l e moisture content (270%) gives volume o f gases present i n the disco l o r e d wood (110%). Thus, approximately 1/3 of the void space i n the discolored wood i s occupied by the gases while the remaining 2/3 i s taken up by the water. Determination of pH values indicated the a l k a l i n e nature o f the discolored wood (Table I I ) . The pH o f discolored wood varied from 7.33 to 8.31 (average 7.84) while that of sapwood var i e d from 6.20 to 6.50 .(average 6.31). Thus these r e s u l t s are generally i n agreement with pre-v i o u s l y published r e s u l t s f o r many other Populus species (Hartley et a l . 1961). The i s o l a t i o n studies reported l a t e r i n d e t a i l (Chapter I I ) , showed that t h i s discolored wood i s also characterized by a high b a c t e r i a l popu-l a t i o n (average 7.3 x 10^/ml of the extracted l i q u i d ) . Therefore t h i s TABLE I I : The pH o f wetwood and sapwood samp les c o l l e c t e d a t two d i f f e r e n t h e i g h t s f r o m f o u r t r e e s , o b t a i n e d f r o m t h e U . B . C . R e s e a r c h F o r e s t , M a p l e R i d g e , B . C . T r e e c h a r a c t e r i s t i c s Sample h e i g h t " , pH v a l u e Number Age DBH H e i g h t ( f t [ m ] above Wetwood Sapwood ( y r ) ( i n [ c m ] ) ( f t [ m ] ) g round) 1 37" 2 5 . 0 ( 6 3 . 5 ) 6 6 . 0 ( 1 9 . 5 ) 3 . 0 ( 0 . 9 7 .92 6 . 2 1 1 2 . 0 ( 3 . 6 ) 7 . 3 3 6 . 2 4 2 47 2 9 . 0 ( 7 3 . 7 ) 7 0 . 0 ( 2 1 . 3 ) 3 . 0 ( 0 . 9 ) 8 .31 6 . 3 5 1 2 . 0 ( 3 . 6 ) 7 .70 6 . 2 0 3 41 3 1 . 0 ( 7 8 . 8 ) 7 9 . 0 ( 2 4 . 0 ) 3 . 0 ( 0 . 9 ) 7 .71 6 . 3 6 1 2 . 0 ( 3 . 6 ) 7 .75 6 . 2 5 4 42 3 1 . 0 ( 7 8 . 8 ) 7 9 . 0 ( 2 4 . 0 ) 3 . 0 ( 0 . 9 ) 8 . 30 6 . 3 7 1 2 . 0 ( 3 . 6 ) 7 . 7 0 6 . 5 0 A v e r a g e 7 .84 6 .31 18 discolored wood appears to possess a l l the ch a r a c t e r i s t i c features of a wetwood, but occupies normal position of a heartwood. For these rea-sons, I have used the term "wetwood" i n preference to the term "heartwood". Another type of discolored wood, which does not appear to be very wet, should also be mentioned b r i e f l y ; i t i s associated with wounds and branch stubs. Although i t s presence was recognized here, no detailed studies were done on t h i s type of discolored wood, except for some i s o -l a t i o n studies reported l a t e r (Chapter I I ) , The discoloration originating from the wounds, which looks different from the wetwood discoloration, spreads upwards and downwards i n the sapwood and often merges into central-ly-located wetwood giving i t a rather i r r e g u l a r appearance (Figure 5). New tissues that are formed after wounding are seldom affected and there i s usually a hard rim tissue between discolored wood and colorless wood. In a l l respects, this discolored wood appears s i m i l a r to wound-initiated discolored wood found i n several northern hardwoods (Shigo 1969) . Such wouhd-initiated discolored wood has been referred to as wetwood i n the past (see Introduction), but because of i t s different o r i g i n , appearance and development, I have chosen not to use the term wetwood. In order to understand wetwood i n i t i a t i o n , i t i s essential to know at what age wetwood begins to appear. Preliminary studies indicated that i n mature trees wetwood was always present whereas i t was absent i n the young seedlings. The results of the dissection study are summarized i n Table I I I . A d i s t i n c t relationship appears to exist between tree age and the occur-rence of wetwood. Wetwood formation evidently begins i n the age class of 8 to 10 years. Seedlings younger than age 7 were devoid of wetwood whereas Figure 5. Cross s e c t i o n o f a wounded black cottonwood t r e e trunk showing d i s c o l o r a t i o n s associated with wounds merging i n t o c e n t r a l wetwood column, g i v i n g i t a r a t h e r i r r e g u l a r appearance. Wounds were apparent on the d i s c about 0.3 meters below t h i s s e c t i o n . Scale - X 0.25. TABLE 111: Number of non-wounded black cottonwood trees and seedlings, with or without wetwood, from 5 separate l o c a l i t i e s i n Lower Fraser Valley i n the Province of B r i t i s h Columbia. L o c a l i t y West 16th Ave. George Massey South-West University U.B.C. Research Total No* of Percentage of U.B.C . Campus, Tunnel, Delta Marine Drive Boulevard Forest, Maple trees and t o t a l with Vancouver U.B.C. Campus U.B.C. Campus Ridge seedlings ex- wetwood Vancouver Vancouver ami ned Age Wetwood present absent present absent present absent present absent present absent 1 0 3 0 13 0 1 17 0, .0 2 0 4 0 9 0 5 18 0. .0 3 0 3 0 11 0 17 31 0. .0 4 0 7 0 7 0 • 7 21 0 .0 5 0 n 0 4 0 6 12 0. .0 6 0 11 0 1 0 5 17 0. .0 7 0 2 2 0. .0 S 0 7 1 9 17 5, .8 9 1 10 1 8 20 10. .0 10 3 2 4 4 13 53, .8 11 7 1 1 0 3 0 12 91 .6 12 3 0 6 0 9 100 .0 13 4 0 4 100 .0 14 7 0 2 0 9 100 .0 O TABLE I I I (cont'd) Age L o c a l i t y Wetwood West 16th Ave. U.B.C. Campus, Vancouver present absent George Massey Tunnel, Delta present absent South-West Marine Drive U.B.C. Campus Vancouver present absent University Boulevard U.B.C. Campus Vancouver U.B.C. Research Forest, Maple Ridge present absent present absent Total No.' of trees and seedlings examined Percentage of t o t a l with wetwood 20-25 26-30 31-35 36-50 11 15 6 11 16 9 5 100.0 100.0 100.0 100.0 Tn,aHili0V° 2 1 3 n ° n " w o u n d e d t r ^ t h a t w e r e dissected during t h i s i n v e s t i g a t i o n , 30 more trees were examined l a t e r f o r some other s p e c i f i c projects (Chapters I I I , IV and V). The occurrence of wetwood was detected i n a l l these trees ™1 T!rvef0re t t h e ^ a v e a l s ° b e e n included here. In 25 trees that were studied for t h e i r "gas composition", only cores taken out with an increment borer were examined; t h i s i s not unusual as Hartley et a l . (1961) i n t h e i r extensive study always inspected cores to detect presence of wetwood and did not dissect a l l the-freTs. extensive 22 i n 12 years or older ones, wetwood was invariably present. I t should be pointed out that only two of the f i v e l o c a l i t i e s show t h i s f u l l pattern, because of the d i f f i c u l t y i n obtaining tree samples of various ages, on any one s i t e . In spite of the differences i n s i t e between the f i v e samples, the pattern of wetwood appearance was consistent. Dissection of the young seedlings (ages 1 to 7) further revealed that the stem consisted almost e n t i r e l y of sapwood (c o l o r l e s s ) , which encompassed a darker appearing p i t h (Figure 6), In some trees of age 8 i n which the discoloration had just begun to appear i n the stem (Figure 7) , the roots were completely free from i t . It i s not known i f the absence of wetwood i n roots i s related to the absence of p i t h i n roots. In these trees, the stem just above the s o l i d level contained wetwood surrounding the p i t h and the sapwood i n turn enveloped both the wetwood and the p i t h . I t i s noteworthy that i n young seedlings i n which wetwood was absent, wound-ini-t i a t e d discoloration was often found spreading both upwards and downwards from the wound i n the sapwood (Figures 8, 9 and 10). In general, the developmental pattern of wetwood was very s i m i l a r to that of a t y p i c a l heartwood. The discoloration of wetwood always origina-ted i n the center of the stem (pith region) and then extended further to-wards the periphery of the stem. Wounding or any other external stimulus does not appear to be necessary for the i n i t i a t i o n of wetwood. With the increase i n tree age, wetwood gradually increased i n diameter, while sap-wood remained about the same thickness (except towards the crown). In case of a t y p i c a l heartwood, the inner part of the sapwood i s continuously transformed into heartwood as more sapwood i s formed by divisions of the F i g u r e 6: V e r t i c a l s e c t i o n o f a 2 y e a r o l d b l a c k cottonwood showing sapwood ( c o l o r l e s s ) and p i t h ( d a r k ) . Note t h e absence o f wetwood. The o l d e s t p i e c e i s on t h e l e f t and t h e r e m a i n -i n g ones f o l l o w base sequence. S c a l e -X 0.17. 2-1 Figure 7: V e r t i c a l s e c t i o n o f an 8 year o l d black cottonwood showing e a r l y stage of wetwood development (bottom), sapwood ( c o l o r l e s s ) and p i t h (dark brown i n c e n t e r ) . In the wet-wood column, note that the outer t i s s u e s are s l i g h t l y darker than the i n n e r t i s s u e s . Scale - X 0.25. 25 Figure 8: V e r t i c a l s e c t i o n s o f a 7 year o l d black cottonwood showing sapwood ( c o l o r l e s s ) and p i t h (dark brown) i n each pi e c e and the t h i r d piece from l e f t also shows d i s c o l o r a t i o n a s s o c i a -ted with a wound. Note the absence of wetwood. The piece on the l e f t i s o l d e s t and the remaining ones f o l l o w base sequence. Scale - X 0.17. 26 Figure 9: V e r t i c a l section of a 1 year old black cottonwood showing wound-initiated discoloration and sapwood. P i t h i s absent. Scale - X 0.33. Figure 10: V e r t i c a l sections of a 2 year old black cottonwood showing wound-initiated discoloration and sapwood. Pi t h i s present (barely v i s i b l e ) . Scale - X 0.25. 27 28 cambial c e l l s (Cronquist 1971). No external stimulus i s required f o r the heartwood formation. Heartwood increases i n diameter with tree age while the thickness of sapwood stays about the same throughout the l i f e of the tree. Another s i m i l a r i t y between wetwood and t y p i c a l heartwood concerns t h e i r "universal occurrence"; i n black cottonwood wetwood was always pre-sent i n a l l the trees of age 12 and over, and depending upon the species, heartwood i s also formed i n almost a l l of the trees at a certain given age (these points w i l l be considered again i n Chapter II which includes a d i s -cussion on the theories of i n i t i a t i o n of wetwood). In summary, i t appears that wetwood i n black cottonwood i s s i m i l a r i n some respects to the wetwoods reported from other tree species. Its d i s t r i b u t i o n within trees, pH and moisture content values are comparable to those found i n most poplar and elm species (Hartley et a l . 1961). Age of i n i t i a t i o n of wetwood was from 8 to 10 years and therefore different from most other poplar species. Generally, the pattern of occurrence of wet-wood i n black cottonwood was l i k e that of a " t y p i c a l heartwood". 29 CHAPTER IT MICROORGANISMS ASSOCIATED WITH WETWOOD IN  BLACK COTTONWOOD INTRODUCTION Numerous i s o l a t i o n studies have revealed the widespread occurrence of microorganisms i n the wetwood. Most of the e a r l i e r c u l t u r a l work was done i n connection with heart r o t s , and bacteria when encountered were often discarded as contaminants. Also i n many studies malt agar was used for i s o l a t i o n s and this agar i s on the acidic side and consequently, f a -vourable to most decay fungi and less favorable to most bacteria. Later when bacteria were isolated by use of alkaline media, t h e i r i d e n t i f i c a t i o n was not attempted i n a l l cases although they were characterized frequently. In recent years, various aerobic and anaerobic bacteria and fungi have been isola t e d and i d e n t i f i e d from the wetwood of elms, poplars and other hardwood species (Hartley et a l . 1961). Carter (1945) considered bacteria as the causal organisms of wetwood i n American elm. He isolated Erwiriia riimipressuaralis from 93% of the elm wetwood samples and also from 78% of the normal sapwood samples. He made 209 inoculations into different parts of l i v i n g trees. Inoculations made on branches, trunk phloem, trunk cambium, trunk current-season wood and some other tree parts did not produce t y p i c a l symptoms of wetwood. But, as Carter states, "Limited browning i n trunk wood was produced i n inoculated heartwood and older sapwood but was not at f i r s t believed to represent i n -fection. However, l a t e r experiments showed this browning i n trunk wood to represent i n f e c t i o n and early development of wetwood." He did not 30 state what distinguished this browning as early development of wetwood, and not merely the normal browning or staining of tissues o r i g i n a t i n g from wounds. Also, i f the bacteria are present i n sapwood, one would not expect inoculations with them to result i n wetwood unless some other conditions are changed simultaneously. In a green house study, Carter noted that a l l s i x trees inoculated with Erwinia nimipressuaralis showed "dark brown streaks i n the heartwood". This discoloration was comparable to streaking i n elms, naturally "infec-ted" with wetwood. An untreated tree and a tree injected with s t e r i l e nutrient broth plus dextrose had no dark brown discoloration i n the heart-wood, t y p i c a l of wetwood. Bacteria, s i m i l a r to the o r i g i n a l inoculum, were isolated from the s i x inoculated trees but not from the two control trees. Wetwood i n Lombardy poplar was regarded as a b a c t e r i a l disease by Seliskar (1950). He re covered Coryri eb acterium humi f e rum from the inocula-ted wood of poplar trees but not from the uninoculated trees. Both inocula-ted and control trees, however, had discolored zones around the inoculation holes. He stated that the infections "were sim i l a r to young wetwood infec-tion i n naturally diseased trees", without mentioning how he determined that the discolored tissue of the inoculated trees was wetwood while the discolored tissue of the control trees was not. Hartley et_ al_. (1961) noted frequent occurrence of a bacterium i n the wetwood of Lombardy poplars. This thick, "doubtfully gram-positive" rod was readily v i s i b l e i n unstained vessels i n recently formed wetwood. The bacterium grew well on malt agar (acidic) and produced abundant gas," promptly pushing part of the agar to the top of the tube". Based on t h e i r 31 extensive study and studies of others, they have advanced three hypo-theses to explain wetwood formation which w i l l be considered l a t e r i n view of the results presented here. Knutson (1970) isolated three types of bacteria from aspen and found that none of these were s p e c i f i c to wetwood. The bacteria were i d e n t i f i e d as species of B a c i l l u s , Cellulomonas and Erwiriia. Every sample of sapwood, wetwood and heartwood yielded bacteria. Sapwood sap consistently yielded low numbers of organisms while wetwood water generally had very large num-bers of bacteria. Heartwood populations were extremely variable, but generally low, with the high b a c t e r i a l populations present only i n deeply stained heartwood. Bacon and Mead (1971) isolated 9 species of bacteria belonging to 6 dif f e r e n t genera from 'non-distressed wood' of aspen, ponderosa pine (Pinus ponderosa Dougl. ex. Lawson) and mountain alder (Alrius tenuifola Nutt.). Five of the species were shown to be pectin digesters, and one, Cellulomonas acidula, u t i l i z e d c e l l u l o s e , while 6 species also showed pro-t e o l y s i s . They concluded, "The presence i n wood of microorganisms which digest pectin and cellulose suggests that wood from freshly cut trees or branches may contain the agents of i t s own decomposition." Wilcox and Oldham (1972) repeatedly isolated a bacterium species asso-ciated with wetwood i n white f i r (Abies concblor (Oord. and Glerid.) L i n d l . ) . The bacterium was a small, slow-growing, gram-variable f a c u l t a t i v e l y anaerobic rod which produced acid but no gas i n carbohydrate media. Its growth appeared to be stimulated by extracts from various plant tissues but not by extracts from white f i r wood, and the authors concluded that the bacterium was not s i g n i f i c a n t l y plant pathogenic.in white f i r . 32 Anaerobic bacteria have been isolated from healthy appearing trees containing some form of 'shake' (Ward et a l , 1969) and from the discolored tissues (Stankewich et_ al_. 1971). Zeikus and Ward (1974) isolated the bac-terium responsible for CH^ production from the wetwood of eastern cotton-woods and some other hardwoods, and i d e n t i f i e d i t as a member of the genus Methanobacterium. This s t r i c t anaerobe was a gram-positive, non-motile, curved rod that produced CH^ i n an inorganic sa l t s medium under an atmos-phere of hydrogen (rLp and CO^. The methanogenic organism was observed i n a l l enrichment cultures obtained from either f e t i d l i q u i d or wetwood cores from cottonwood, elm and willow trees containing CH^. Concerning the role of anaerobic bacteria i n trees, the authors stated, "The high number of methanogenic bacteria and other anaerobes found i n wetwood indicates vigor-ous microbial fermentation. I t i s d i f f i c u l t to ascertain whether the wood tissue i t s e l f i s being decomposed or whether other nutrients serve as the substrates for this CH^ fermentation." Sachs et a l . (1974) made Scanning Electron Microscopy (SEM) and micro-b i a l studies on the wetwood and sapwood of eastern cottonwoods and b i g -tooth aspens, and on the sapwood and normal heartwood of white poplars. The i s o l a t i o n studies yielded b a c t e r i a l populations that always contained an obligate anaerobe (Clostridium)and a microaerophilie bacterium from the inner rings of sapwood adjacent to wetwood. The wetwood was s i m i l a r l y "infected", but i t usually contained a mixed population of anaerobic, microaerophilie, and facultative bacteria plus occasional fungi. No micro-organisms were found i n the outer, or younger, rings of sapwood of cotton-woods, aspens or white poplars; neither were they found i n the inner sapwood 33 and adjacent heartwood of white poplars. Based on these and SEM studies, the authors concluded that wetwood i s formed i n l i v i n g trees primarily from the b a c t e r i a l action (this study w i l l be discussed l a t e r i n the chap-ter) . In many studies, special emphasis was given to i s o l a t i o n of bacteria and therefore the information on fungi from wetwood i s very fragmentary. F r i t z (1931) found Torula lignipefda i n the outer zone,of the wet red heart of paper birch (Betula papyrifera Marsh.) i n 25 of 28 trees examined. Her Torula inoculations on specimens i n v i t r o resulted i n discoloration of the tissues but not i n the wetness. Clausen et al. ;(1949) reported a general f a i l u r e to i s o l a t e decay fungi from wetwood and discolored heartwood of aspen. Knutson's examination of microbial population showed that a very few filamentous fungi were present i n the aspen wetwood, sapwood and heart-wood (1970). He found numerous colonies of P u l l u l a r i a i n two of his wetwood samples. Sachs ejt al_. (1974) also reported that fungi occurred r a r e l y i n the wetwood of bigtooth aspen and eastern cottonwood. While reviewing the studies on fungi, Hartley et a l . (1961) state, "For most other fungi, there has been l i t t l e indication of association with high moisture content i n trunks. ...A number of ascomycetes are known, i n the outer wood causing w i l t of various hosts, but no reports have been encountered of unusual wetness associated with them, and neither plant-inhabiting ascomycetes or hymenomy-cetes, so f a r as our experience goes, produce or thrive i n high pH condi-tions ." The l i t e r a t u r e thus suggests that numerous types of bacteria with different 0- requirements are frequently found associated with wetwood, 34 heartwood, discolored heartwood and also with sapwood. The primary ob-jec t i v e of these studies was tb search f o r a s p e c i f i c organism or organ-isms which could perhaps be implicated i n the wetwood formation. Some workers made inoculation studies to support t h e i r hypotheses. A few quan-t i t a t i v e studies were also made towards t h i s end. The purpose of my study was to get an estimate of number and kinds of microorganisms associated with wetwood which may have a bearing on the i n i t i a t i o n of wetwood and on i t s gas composition. The s p e c i f i c objectives were, 1) to i s o l a t e , characterize and possibly i d e n t i f y aerobic bacteria associated with wetwood and sapwood, 2) to detect the presence and possibly i s o l a t e f a c u l t a t i v e and obligate anaerobes i n the wetwood and sapwood. 3) to iso l a t e fungi (presumably aerobic - see Chapter IV) from the wetwood, 4) to i s o l a t e bacteria (aerobic) from young cottonwoods, with or without wetwood, and 5) to is o l a t e bacteria and fungi from the wound-initiated discolored wood. MATERIALS AND METHODS 1. Tree samples: Seven non-wounded and 2 wounded trees were f e l l e d on the U.B.C. Research Forest, Maple Ridge, B.C. Approximately 12 i n (30.5 cm) thick discs (2 from each tree) were then cut from the trees and were brought back to the laboratory on the same day. One disc per tree was used 35 to make quantitative determinations of the bac t e r i a l population while the other discs were used to i s o l a t e aerobic bacteria and fungi. Three addition-a l non-wounded trees from the Research Forest were also used (but not felled) to i s o l a t e facultative anaerobes. Cores of wood were obtained with an incre-ment borer for i s o l a t i o n s . Besides these, several young cottonwoods (approxi-mately 10) of ages varying from 1 to 15 from various l o c a l i t i e s (Chapter I) were also used to i s o l a t e aerobic bacteria. 2. Quantitative determinations: The viable count method (Collins and Lyne 1970) was used to determine the size of b a c t e r i a l population i n the wetwood (7 determinations - one per disc) and i n the discolored wood (2 determinations - one per disc) of the wounded trees. In p r i n c i p l e , the ma-t e r i a l containing bacteria i s s e r i a l l y diluted and aliquots of each d i l u t i o n are placed on suitable culture media. Each developing colony i s assumed to have grown from one viable unit representing one organism or a group of organ-isms. Peptone water (0.1%) was used as a diluent, and 9 ml of th i s diluent was dispensed into s t e r i l e test tubes. These " d i l u t i o n blanks" were auto-claved and allowed to cool before use. One ml of sample ( l i q u i d extracted from the indicated zones) was added to a d i l u t i o n blank and the liquids were mixed thoroughly. One ml was then removed from this tube and was added to another d i l u t i o n blank tube, and so on. In a l l , s i x d i l u t i o n s were made as follows: 36 Tube Number Di l u t i o n Volume of Original l i q u i d per ml  1 1/10 10" 1 2 1/100 10" 2 3 1/1000 - 10~ 3 4 1/10,000 10" 4 5 1/100,000 10" 5 6 1/1,000,000 10" 6 Standard nutrient agar (Difco) which had previously been tubed i n 10 ml amounts, was then melted and was allowed to cool to approximately 45 C i n a water bath. Two s t e r i l e p e t r i plates (100 x 15 mm) per d i l u t i o n were set out and 1 ml of each solution was pipetted into the center of the appro-p r i a t e l y marked dishes. Contents of each cooled agar tube were then added tb one plate and the plate was moved gently about s i x times i n a clock-wise c i r c l e and then i n an anti-clock-wise c i r c l e , After the medium was set, the plates were inverted and incubated at 25 C for 24-48 hours. After this period, plates showing between 30 to 300 colonies were selected and the ex-act number of colonies was counted. The 'colony count" was calculated by multiplying the average number of colonies counted per plate by the recipro-cal of the d i l u t i o n . 3. Isolation: Each disc that was stored at 4 C u n t i l the time of i s o -l a t i o n s , was s p l i t open longitudinally with a s t e r i l e axe (dipped i n 95% ethanol). Young cottonwood stems were also sectioned longitudinally using an axe. Small chips of wood (approximately 2 x 4 mm) were then removed asep-t i c a l l y from the wetwood and sapwood of the non-wounded trees and also from the discolored wood of the wounded trees. 37 3.1 Aerobic bacteria; About 10 chips were placed i n an Omnimixer (Ivan Sorvall Inc.) and were homogenized i n 25 ml of s t e r i l e saline solu-tion for about 5 minutes. The suspensions were plated out on nutrient agar medium and the plates were incubated at 25 C for 3.to 4 days. S o l i t a r y colonies were then isolated and pure cultures were maintained i n 1.0% pep-tone water at 4 C. Standard microbiological studies (Collins and Lyle 1970) were done i n search of the characters which could be used i n d i f f e r e n t i a t i o n of the isolat e d bacteria (Table V). I d e n t i f i c a t i o n was done by following the keys prepared by Bergey (1957) and Skerman- (1959). 3.2 Fungi: Standard 2% malt agar (Difco) was used to i s o l a t e fungi. Small chips were removed as described e a r l i e r and four such chips were placed i n one p e t r i dish. The plates were incubated at 23 C for a maximum of 3 weeks. The general i s o l a t i o n procedure was repeated u n t i l the pure c u l -tures were obtained. The fungi were i d e n t i f i e d by using the keys prepared by Barnett and Hunter (1972) and Nobles (1948). 3.3 Anaerobic bacteria ( f a c u l t a t i v e ) : Wood samples for this study came from three non-wounded trees; cores of wood were obtained by using a s t e r i l e increment borer (dipped i n 95% ethanol) and were quickly transferred to a GasPak Anaerobic System (for description, see Chapter I I I ) . An equal number of cores was then obtained from approximately the same position i n the trees and was placed i n a p l a s t i c bag (aerobic conditions) to avoid mois-ture loss. Anaerobically stored cores were fragmented into small pieces after they were brought back to the laboratory and these pieces were quickly trans-ferred oh to the nutrient agar plates (stored under anaerobic conditions). The inoculated plates were incubated i n the Anaerobic System at 30 C for about 14 days. Duplicate culture plates were also made from the aerobically 38 stored cores and were incubated under aerobic conditions to detect the pre-sence of the facultative anaerobes. 3.4 Anaerobic bacteria (obligate): A separate study was made, to i s o -late s t r i c t anaerobes, p a r t i c u l a r l y the methanogenic bacteria, under the supervision of Dr. B. McBride of the Department of Microbiology, U.B.C. As I was not the p r i n c i p a l investigator of the project, inclusion of those re-sults at this place would have been inappropriate, and therefore the details of t h i s study are presented separately i n Appendix I. RESULTS 1. Quantitative determinations: The viable count study (Table IV) revealed that colony count per ml of l i q u i d from wetwood was 7.3 x 10^ (average). The b a c t e r i a l count from the discolored wood of the wounded trees was also very high (average 7.0 x 10^/ml of l i q u i d ) . These estimates are perhaps on the conservative side as bacteria attached to the c e l l walls were not accounted for. In addition, anaerobic bacteria present i n the l i -quids would not survive under the aerobic conditions. Nevertheless i t i s clear that the wetwood and discolored wood of the wounded trees contain a high number of bacteria. 2. Isolations 2,1 Wetwood: Aerobic bacteria: From a t o t a l of 248 i s o l a t i o n s , 244 yielded bacteria (over 98%), Two genera, Erwinia and B a c i l l u s , were isolat e d consistently from a l l seven trees. Most of the isolates of Erwinia were motile rods, produced acid without gas and l i q u i f i e d gelatin (Table V). These cultures were i d e n t i f i e d as Erwinia amylovora as they f a i l e d to hydrolyze 39 TABLE IV. Number of colonies of b a c t e r i a i n the l i q u i d s extracted from the wetwood (non-wounded trees) and discolored wood (wounded trees) samples c o l l e c t e d at indicated heights. Counts are from 1:10,000- d i l u t i o n on nutrient agar p l a t e s . Tree c h a r a c t e r i s t i c s Sample height Colonies per plate  Number Age DBH Height Type of (ft[m] above (average of two (yr) (in[cm]) (ft[m]) Wood ground) plates) 1 37 25.0(63.5) 66.0(19.5) wetwood 12.0(3.6) 70 2 47 29.0(73.7) 70.0(21.3) 68 3 41 31.0(78.8) 79.0(24.0) M 80 4 42 31.0(78.8) 79.0(24.0) „ 61 5 45 25.9(66.0) 74.0(22.5) 75 6 41 30.0(76.2) 78.0(23.7) 75 7 38 25.0(63.5) 64.0(19.5) „ 81 8 49 32.0(81.3) 84.0(25.5) discolored 3.0(0.9) wood 68 9 45 27.9(71.1) 67.0(20.4) " 4.0(1.2) 72 73 Average 7.5 40 TABLE V: Some morphological and physiological characteristics of bacteria cultured from wetwood Tentative i d e n t i f i c a t i o n Characteristic Erwinia Erwinia Bacillus Protamino- Bacterium Entero-amylo- carne- sp. bacterium sp~] Hacte-vora gieana spT riaceae Colony colour Yellow- Yellow- White Red White Creamy ish ish Yellow C e l l shape Rod Rod Rod Rod Rod Rod M o t i l i t y + + + + + + Gram test G=- G=- G=+ G=- G=+ G=-Spores formed Gelatin lique- + faction Starch hydro- -l y s i s Acid from + - + +(?) sugars Acid plus gas - + from sugars Production of n i t r i t e s from + nitrates Production of hydrogen s u l - + - - + phide S p l i t t i n g of alky1amines 41 starch or to produce n i t r i t e s from n i t r a t e s . A few isolates produced acid plus gas i n sugar media, l i q u i f i e d g e l a t i n , produced n i t r i t e s from ni t r a t e s and also produced hydrogen sulphide. They are tent a t i v e l y i d e n t i f i e d as Erwinia carnegieana. Species of Erwinia are known to invade the tissues of l i v i n g plants and produce dry necrosis, w i l t s and soft r o t s . Several isolates were gram-positive and produced spores. These i s o l a t e s , according to Ske'rman's key (1959) belong to genus B a c i l l u s . These were motile rods, frequently i n chains, and produced acid i n sugar media. A num-ber of isolates were non-sporing rods which produced colonies with a red pigment and metabolized alkylamines. They were placed i n the genus Protamino- bacterium. There were two isolates which were gram-positive, non-sporing, motile rods and did not ferment carbohydrates. These probably belong to the genus Bacterium, previously known as Kurthia. There were several i s o l a t e s which did not produce acid or gas when supplied with various sugars. They produced non-transparent, round colonies which were creamy-yellow i n color. These have not been i d e n t i f i e d as yet but presumably belong to family Enterobacteriaceae. 2.2 Wetwood: Fungi: One yeast and several mycelial fungi were isolat e d from the wetwood. Colonies of the yeast were white to creamy-yellow i n color, and had small oval c e l l s which reproduced by budding. I d e n t i f i c a t i o n of the yeast i s o l a t e was not attempted. A l i s t of mycelial fungi i s o l a t e d , t h e i r frequency and general charac-t e r i s t i c s i s given i n Table VI; the i d e n t i f i c a t i o n was attempted only up to the generic l e v e l . Out of 140 i s o l a t i o n s , 86 yielded fungi. Per i i c i l l i u m was isolat e d most frequently while Trichbderma, Cephalosporium, Epicoccum and TABLE VI: Frequency and characteristics of fungi isolated from wetwood on 2% malt agar PeniciIlium Trichoderma Cephalbsporium Epicoccum Botrytis Isolate Isolate Isolate sp. sp. sp. sp. so. BC-1 BC-2 BC-3 Frequency 28.1% Culture color Greenish-gray Conidiophores conidia, etc. Conidiophores a r i s i n g singly, branched near apex ( p e n i d i -late) ending i n p h i a l i d s ; Conidia hyaline to b r i g h t - c o l -ored, 1-celled mostly globose, in chains 12.3% O r i g i n a l l y white,later green due to patches of conidia Conidiophores hyaline, branched; Conidia hyaline 1-celled, pro-duced i n cl u s t -ers 10.6% White Conidia hya-li n e 1-celled, c o l l e c t i n g i n slime-drope 8.4% Origi n a l l y white,later s l i g h t l y brown Conidiophores compact, dark, short; Conidia dark, 1-celled, globose 2.9% 4.3% 3.6% 2.9% White . White White White Conidiophores long, slender, hyaline, en-larged apical c e l l s Conidia hyaline to ash colored, 1-celled, black s c l e r o t i a pro-duced frequently Not produced Isolation Attempts -S t e r i l e -140 54 (38.6%) Botrytis occurred i n lesser frequency and i n that same order. Three types of fungi, a l l unidentified (isolates BC-1, BC-2, and BC-3) , were also i s o l a t e d , a l l of which produced white mycelia but f a i l e d to produce any kind of spores. 2.3 Wetwood: Anaerobic bacteria ( f a c u l t a t i v e ) : Examination of matched nutrient agar plates, incubated under aerobic and anaerobic con-ditions (18 attempts each) suggested presence of at least three types of facultative anaerobes (Types A, B and C). Type A bacteria were small, gram-negative rods believed to be a species of Erwinia. Types B and C, both unidentified, were gram-positive rods and produced opaque colonies. Type B produced pink, round colonies while Type C produced creamy colonies with irr e g u l a r margins. 2.4 Wetwood: Anaerobic bacteria (obligate): See Appendix I. 2.5 Sapwood: Bacteria were isolated very infrequently from sapwood; only 9% of the • c.Ul/tjirssw (4 out of 45) were pos i t i v e for bacteria. These isolates were gram-negative rods and produced acid on sugar media. A l -though t h e i r further i d e n t i f i c a t i o n was not attempted, they supposedly represent the family Enterobacteriaceae. Only 2 out of 40 i s o l a t i o n s yielded fungi, both representing the genus Periicillium. Attempts to i s o -late anaerobic bacteria j f a i l e d ; no growth was observed i n any of the i s o -lations made. 2.6 Wound-initiated discolored wood: Several types of aerobic bac-t e r i a were isolated from the discolored wood associated with wounds, a l -though t h e i r i d e n t i f i c a t i o n was not attempted. Morphological studies sug-gested that these bacteria were very s i m i l a r to the ones isolated for wet-wood. A substantial number of fungi, mostly Aspergillus and P e n i c i l l i u m , 44 was also isolated from the discolored wood (32 out of 40 i s o l a t i o n s y i e l d -ed fungi belonging to these two genera). Two decay fungi were found i n the close v i c i n i t y of the wounds and according to Noble's key (1948), one i s a species of Pholiota while the other i s tent a t i v e l y i d e n t i f i e d as Polyporus  adus tus. 2.7 Young trees and seedlings: In young (ages 1-7) black cottonwood seedlings, p i t h and sapwood regions were completely s t e r i l e (0 out of 45) while i n older ones (ages 7-15), some bacteria were present (9% of 45 at-tempts) i n the sapwood. The isolated bacteria were not i d e n t i f i e d . DISCUSSION Some early studies (Carter 1945,Seliskar 1950) suggested that bacteria are responsible for wetwood formation. Conclusions of these authors are primarily based on i s o l a t i o n studies and therefore are d i f f i c u l t to accept e n t i r e l y . The inoculation studies were not convincing because the authors did not state c l e a r l y what c r i t e r i a they used to i d e n t i f y discolorations as wetwood, which could have been merely browning o r i g i n a t i n g from the wounds. Knutson (1970) isolated Erwinia from sapwood, wetwood, normal heartwood and discolored heartwood of aspen. Because occurrence of the organism was 'non-s p e c i f i c ' and since his wounding experiments, not involving microorganisms, produced symptoms of wetwood, he concluded that there i s no reason to be-lieve that Erwinia causes wetwood. From Kriutson's experiments, i t appears that the evidence he presents i s inconclusive rather than negative. The control trees that he used for wounding experiments (not involving organisms) also contained Erwinia and therefore he cannot rule out the p o s s i b i l i t y that 45 Erwinia causes wetwood. Perhaps some sort of change i n conditions of wood i s necessary before Erwinia sp. (or other bacteria) can begin t h e i r action on wood. Three major hypotheses have been advanced to explain wetwood forma-ti o n (Hartley et a l . 1961). The f i r s t suggests that the parenchymatous elements at the inner l i m i t of the sapwood die of age or from impaired com-munication with cambium. The symptoms of wetwood condition are then pro-duced by the action of the trees'own enzymes on the contents of these af-fected c e l l s . The-second and t h i r d hypotheses assume involvement of bac-t e r i a i n the process of wetwood formation. In the second theory, the parenchyma c e l l s are presumed to die as suggested e a r l i e r but, a l l the sub-sequent changes are brought about by the a c t i v i t y of saprophytic bacteria. The t h i r d hypothesis i s that death of the parenchyma c e l l s i s caused or hastened by weakly p a r a s i t i c bacteria. These bacteria may or may not have been present i n small numbers i n the normal wood but could be assumed to be capable of infection only after the wood c e l l s have become senescent. The gas production and odor may be caused by secondary bacteria. This t h i r d hypothesis has been the one generally accepted as explaining wetwood for-mation i n elm and poplar species (Smith 1970) . Recently, Sachs and co-workers (1974) presented what appears to be a conclusive evidence of the active b a c t e r i a l p a r t i c i p a t i o n i n wetwood forma-t i o n . The observations were made by extracting woody tissues from standing trees and preparing matched samples f o r SEM examination and for i s o l a t i o n and culturing of the microorganisms present. Samples were obtained from sapwood and wetwood of eastern cottonwoods and bigtooth aspens, and from 46. sapwood and normal heartwood of white poplars. S t r i c t l y anaerobic bac-t e r i a , p a r t i c u l a r l y Clostridium sp., were consistently found i n wetwood and adjacent sapwood of the poplar species studied. No bacteria were found or isolated from normal heartwood and adjacent sapwood. SEM obser-vations suggested that wetwood occurs after an invasion of sapwood vessels by bacteria, presumably from i n i t i a l root infections. Also, SEM studies disclosed differences i n the c e l l u l a r condition of sapwood, normal heart-wood and wetwood. The main feature: of sapwood was the turgid smooth ap-pearance of the ray p i t membrane when viewed by SEM from the vessel lumen. The p i t membranes of a l l ray c e l l s i n the normal heartwood had a wrinkled appearance. Ray c e l l s with wrinkled p i t membranes also occur i n the sap-wood of a l l three species studied. However, an increase i n the number of ray parenchyma c e l l s with wrinkled p i t membranes was evident i n sapwood i n trees with wetwood. Also, the inner sapwood of the trees with wetwood was infested with bacteria and at the sap-to-wetwood t r a n s i t i o n zone, the vessel ray p i t membranes of most c e l l s were coated with b a c t e r i a l slime with a s l i g h t eroded appearance. Progressing inward toward/the p i t h region, most membranes of vessel to ray p i t s had been destroyed and bacteria were evident? i n the ray c e l l s . Therefore, these authors characterized wetwood as b a c t e r i a l degradation of the p i t membranes of vessel-to-ray p i t s . Nei-ther bacteria nor degraded p i t membranes were found i n the normal heartwood. Sachs et_ al_. (1974) also reported that i n eastern cottonwoods, only sapwood and wetwood were present and normal heartwood was not found i n any of the trees studied. However, the authors did not state at what age wetwood begins to appear i n these trees, nor did they attempt to i s o l a t e bacteria 47. from young trees, presumably without wetwood. I f bacteria were present i n the young cottonwoods without wetwood, i t would be worthwhile to know what type of 'stimulus' or change i n conditions i s required for i n i t i a t i o n of the bac t e r i a l action on the vessel ray p i t s . I f age of i n i t i a t i o n of wetwood was known then an examination of trees younger than that age group may provide some valuable information. Also l i k e many other investigators, these authors did not offer explanation of why b a c t e r i a l action should cause water accumulation or high pH, which are so cha r a c t e r i s t i c of wetwood. In black cottonwood, i t was observed that wetwood formation begins i n the age class 8 to 10 years, and i n 12 years or older trees, wetwood was always present (Chapter I ) . The dissection studies also showed that wet-wood always originated i n the center of the stem (pith region) and extend-ed r a d i a l l y i n a l l directions towards the sapwood. The increase i n the size of wetwood was i n fact very- s i m i l a r to that of a " t y p i c a l heartwood". Wetwood appeared as a progressively expanding core, the inner part of the sapwood being continuously transformed into wetwood as more sapwood was formed. Heartwood also constitutes such a continuously expanding core with-i n a tree, increasing i n diameter as the tree gets older. In general, the term heartwood refers to inner layers of wood which no longer contain; l i v -ing c e l l s and from which the reserve materials have been removed or conver-ted to more durable substances ( H i l l i s , 1971). Wetwood, located i n the inner layers of wood, also lacks viable parenchyma c e l l s (and reserve starch) and thus i s si m i l a r to a t y p i c a l heartwood. I t i s possible then that wetwood formation i n black cottonwood requires an in t e r n a l stimulus s i m i l a r to that needed for heartwood formation. The nature of the stimulus i s unknown but i t may well be related to tree age and could bring about 48 changes favorable to growth of bacteria. Isolation studies reported here indicated that the p i t h and sapwood regions i n young cottonwoods of ages 1-7 were completely s t e r i l e . Also, wetwood microorganisms from the. older cottonwoods were e s s e n t i a l l y non-pathogenic with the exception of Erwinia. How •<•. these microorganisms, p a r t i c u l a r l y bacteria, obtain entry into the wood i s not known but could be by transport from the s o i l by way of tree roots as suggested by Bacon and Mead (1971). When the conditions become suitable, such bacteria may multiply rapidly and inhabit the wood. Quantitative and frequency stu-dies showed that wetwood contained a large number of bacteria. Therefore, i t i s conceivable that an age-related stimulus together with bacteria play an important role i n wetwood i n i t i a t i o n , i n contrast to the concept of some workers (Carter 1945, Seliskar 1950, Sachs 'et'aJL 1974) that the ef-fects of bacteria alone may be responsible. Hartley and co-workers (1961) second hypothesis- stated that the parenchyma dies naturally and the other changes noted i n wetwood are due mainly to the action of saprophytic bac-t e r i a . This hypothesis appears plausible for the wetwood formation i n black cottonwood. The t h i r d hypothesis by Hartley et a l . (1961) states, "The death of the parenchyma i s caused or hastened by weakly p a r a s i t i c bacteria which may or may not have been present i n small numbers i n the nonliving elements of the sapwood but were unable to attack l i v i n g c e l l s and develop i n quantity u n t i l the c e l l s became senescent " F i r s t l y , the bacteria isolated from the wetwood of black cottonwood were essenti a l l y saprophytes. Second-l y , the young black cottonwood stems without wetwood were completely s t e r i l e . 49 It i s possible, of course, that some "weakly p a r a s i t i c bacteria" might have been present i n "very small numbers". In view of the work by Sachs et a l . (1974), use of modern techniques such as SEM may provide some v a l -uable information on presence of microorganisms i n wood. But to date, there i s no such evidence. In fact, Sivak and Person (1973) also found that i n 1 and 2 year old black cottonwoods, p i t h and wood (presumably sap-wood) were v i r t u a l l y free of any microorganisms. In consideration of these findings, I accept thesecond hypothesis (Hartley et a l . 1961) as a l i k e l y hypothesis to explain wetwood formation i n black cottonwood, as opposed to the t h i r d one which i s generally accepted for elms and poplars (Smith 1970). Assuming that wetwood i n i t i a t i o n i n black cottonwood i s more of a "natural process" than that caused by bacteria, i t i s st-i 11,not • known why the moisture content or pH are so high i n the wetwood. Accumulation of water may be due to low permeability of wetwood (Kemp., 1956). The high levels of calcium carbonate reported i n the wetwood (Hartley et a l . 1961) could explain the high pH values of wetwood. Also, i t may be due to ammonia released by the bacteria as suggested by Hartley et a l . (1961). Regarding the o r i g i n of the excess moisture i n the wetwood, Hartley ejt al_. (1961) state, "Whether wetwood i s the result of a natural process of the trees or induced by bacteria, no very plausible explanation occurs to the writers as to the source of the excess moisture i n the wetwood. I f water produced by the oxidation processes of microorganisms were the answer, more water should be produced by decay fungi than bacteria," The oxidation process i s ob-viously not the answer as action of decay fungi does not result i n accumula-tion of water. Knutson (1970) stated that the high moisture content could 50 be simply "an osmotic e f f e c t " , without giving any s p e c i f i c d e t a i l s why i t may occur. One of the most inte r e s t i n g features of the i s o l a t i o n studies was that wetwood yielded several species of aerobic bacteria and fungi (pre-sumably aerobic), i n addition to some f a c u l t a t i v e l y and obligately anaero-bi c bacteria. It i s not known how these various organisms with different ©2 requirements exist i n the same wetwood column under f i e l d conditions. I t has been demonstrated that the composition of gases within trees may d i f f e r widely from that of normal a i r (Chapter III) . It i s also known that the composition within trees changes with the seasons of the year. Therefore, at any given O2 concentratiom (or during a given season), any one group of organisms must fi n d i t d i f f i c u l t to carry out a l l the metabo-l i c processes. For example, O2 concentration of 20.0% would be generally favourable to aerobic bacteria and fungi, while anaerobic bacteria may survive i n the form of resting structures. On the other hand, under anaer-obic conditions, aerobic bacteria and fungi may go into dormancy, and may resume growth with the return of favourable conditions. A l t e r n a t i v e l y , i t i s possible that these microorganisms i n turn are responsible for de-termining the gas composition of tree trunks. These p o s s i b i l i t i e s w i l l be discussed l a t e r (Chapter III) i n view of the data obtained during the gas composition studies. 51 CHAPTER I I I  COMPOSITION OF GASES IN THE TRUNKS  OF BLACK COTTONWOOD INTRODUCTION The composition of gases within tree trunks has been determined i n several species. In most of the e a r l i e r work, the primary goal of the investigators was to gather information for a better understanding of the pneumatic system of woody stems. I t was demonstrated that the gas composi-ti o n i n trees d i f f e r s widely from that of atmospheric a i r and that the com-position may vary i n different seasons of the year. Mostly, these studies formed only a small part of a major study dealing with growth and water conduction i n trees. In l a t e r investigations, special attention was given to the 0 2 concentrations and t h e i r possible significance to the a c t i v i t y of wood-destroying fungi. Most of the early studies have been adequately described by Chase (1934) and only those which are d i r e c t l y related to the present work w i l l be reviewed here. In t his century, Bushong (1907) was f i r s t to analyze gases collected from the heartwood (most probably wetwood) of a cottonwood tree. The com-position of gases was as follows: Q>2, 1.24%; C0 2, 7.21%; CH^, 60.90% and N 2, 30.65%. Ethylene and H 2 were found to be absent. Since then, several authors have reported the composition of gases i n the heartwood of various hardwood species. In some cases, gases were present under pressure, and occasionally they contained a flammable gas. For example, when a boring 52 was made i n a willow oak (Quercus phellos L.) stem, gases came ouf'with a bang" as described by one author (Reynolds 1931). Some others held a lighted match to the head of the increment borer and observed that the gas immediately caught f i r e . In order to interpret the more recent studies on gas compositions, i t i s essential to consider the methods used by different researchers i n ex-trac t i n g the gases and analyzing them. In p r i n c i p l e , the methodology used to c o l l e c t gases was very simple. In each case, i n i t i a l l y , a bore was made into the trunk (generally at breast height); the depth of the bore depended upon the tree zone under investigation. Immediately after d r i l l i n g the hole, a metal pipe or a probe was inserted through the hole. Each probe normally carried an on-off valve so that the gas samples could be obtained repeatedly. As regards the withdrawal of gases, some workers took advan-tage of the fact that gases within trees are sometimes under pressure. For example, Bushong (1907) used a piece of rubber tubing with one end connected to the gas pipe and the other inserted i n a w a t e r - f i l l e d bottle which i n turn was inverted over a dish containing water. As the stop-cock on the pipe was opened, the gases released from the tree displaced the water and thus were trapped i n the b o t t l e . The gases collected i n the f i r s t two bot-t l e s were discarded as they contained atmospheric a i r which was o r i g i n a l l y present i n the pipe, tubing etc. Chase (1934) connected a previously eva-cuated gas sampling tube to the steel tap already placed i n the tree. Then the valve between the pipe and the tube was opened and the tube was l e f t clamped on to the tree, After 48 hours, the gas sampling tube was removed from the tree and the collected gases were analysed. Most workers, however, applied suction i n order to obtain gases from the trees (MacDougal and Working 1933 , Thacker and Good 1952, Jensen 1967a). The methods included mercury displacement technique, as i s e v i -dent from this description of the method used by Thacker and Good (1952), "Gas was extracted by connecting a mercury-filled 25 ml pipette to the tap, Fig. 2. The pipette was connected at the bottom to. a mercury reservoir, the height of which could be varied. When a l l connections had been sealed, the reservoir was lowered, allowing the mercury to drain from the pipette. The resultant suction caused a flow of gas from the tree into the pipette. Two 25 ml samples were withdrawn from each hole, the f i r s t being discarded to eliminate traces of atmospheric a i r i n the borings." The collected gases were analyzed either by performing standard chemi-cal t i t r a t i o n s (MacDougal and Working, 1933) or by using commercially a v a i l -able analyzers (Thacker and Good 1952). For volumetric analysis, for ex-ample, Chase (1934) used potassium hydroxide to remove CC^, and alkaline pyrogallol to absorb C^, from the gas samples. The volume of each gas was then calculated and expressed i n terms of percentage of the t o t a l volume of the sample. Zeikus and Ward (1974) analyzed gases by a gas chromatograph equipped with a thermal conductivity detection system. Naturally, the sen-s i t i v i t y of the methods diffe r e d widely. Most workers were able to deter-mine gas concentrations of as low as 0.1% by the method they used, although some did not state c l e a r l y the s e n s i t i v i t y of t h e i r method, MacDougal and Working (1933) extracted gases from 6 hardwood and 2 softwood species at various times during a t o t a l period of 5 years. Their study s t i l l represents the most comprehensive work done on th i s subject. They surveyed pneumatic and hydrostatic systenfof trees and some of the con-54 elusions they arrived at are as follows , 1) Gases of the pneumatic system occur generally at pressures not very different from barometric, 2) The most common difference between the gases of the pneu-matic system and the (atmospheric) a i r i s the accumulation of CC>2 and the depletion of Oj. 3) The high proportions of CO^ i n the woody tissues could be a product of re s p i r a t i o n , or the amount dissolved i n the streams of the hydrostatic system, or di f f u s i o n into and out of l i v i n g c e l l s and of streaming movements to the outer a i r . 4) The low proportions (or occasionally none) of 0^ i n the pneumatic system may be ascribed to i t s combination i n res-p i r a t i o n and possibly i n connection with a c i d i f i c a t i o n . Chase (1934) studied composition of gases drawn from 4 hardwood and 1 softwood species, and a special emphasis was given to the seasonal fluctuations of gases. A d e f i n i t e relationship was found between metabo-lism and the internal gas content, CO^ concentration being higher i n a l l species during the growing season and lowest during the winter. The oppo-s i t e was true for 0^. In case of a single cottonwood (Populus delto.ides  virginiana [Castiglioni] Sudworth), CO^ reached a maximum over 25% on August 1 i n the heartwood and was above 15% for most of the time from August to November. During July and August, very low percentages of 0^ were present, and i n one sample none at a l l could be detected. In December, however, the percentage of 0^ rose rapidly to over 16, while the CO^  con-tent declined to a low l e v e l . Chase also found that rapidly growing species 55 such as cottonwood contained more C0*2 i n t h e i r stem gas than slowly-growing species such as bur oak (Quercus macrocarpa Michx.) or red oak (Quercus rubra L.). Generally there was more 0 2 and less CO,, i n the sapwood than i n the heartwood. Also, there appeared to be no r e l a t i o n -ship between gas compositions at different heights within a single tree. Thacker and Good (1952) found much smaller seasonal fluctuations i n the CO2 and 0^ contents of sugar maple (Acer saccharum Marsh.). C 0 2 con-tent i n the heartwood of 9 maples, i n June, ranged from 1.9 to 4.8%. Percentages from J u l y samples were also very s i m i l a r . They found that the CO2 content remained r e l a t i v e l y high through the summer and early autumn but decreased markedly during the winter; also that, i t was higher during the growing season i n the heartwood than i n the sapwood. These authors also investigated the composition ,of gases i n decayed as against sound heartwood. They found that the gas samples from decayed sugar maples averaged about 6% higher i n CO2 and a corresponding percent lower i n O2 than those from sound maples. Individual trees, however, showed wide v a r i -ations from these averages. Abnormally high pressures caused.by accumulated gas were reported by Carter (1945) i n the trunks of American elms affected with wetwood. The gas contained approximately 46% CH4, 34% N 2, 14% C 0 2 > 5% 0 2 and 1% H^. Morani and Arru (1958) studied vgas composition of some 17 year old poplars showing symptoms of a bac t e r i a l i n f e c t i o n i n the central part of th e i r trunk, "similar to those described by many authors for the wetwood disease." The composition of gases was, CH^, 53.6%; N 2, 28.4% and C 0 2 , 17.2%. They considered fermentation a c t i v i t i e s of methanogenic and deni-t r y f y i n g bacteria as the cause of the higher concentrations of CH. and CO-. 56 Jensen (1969) found that concentrations i n stems of red oak trees were usually less than 2% i n sound trees and less than 4% i n decayed trees. varied between 14 to 20% i n sound trees and 7 and 21% i n decayed trees. No clear-cut relationships were found between gas concentrations and d i f -ferent seasons, heights (within a tree) or depth (distance from cambium) i n the trees. Recently, Zeikus and Ward (1974) found CH^ as a p r i n c i p a l constituent i n the gases (61.7 and 55.2%) collected from 2 eastern cottonwoods. C0 2 was also present i n high proportions (14.1 and 16.3%), was detected i n both the trees while 0 2 was absent. Both of the trees contained wetwood. Analysis of gases from a white poplar heartwood showed high concentrations of N 2 (75.1%) and low proportions of 0 2 (6.4%) while CH^ was absent. In summary, very low 0 2 tensions were found i n fast growing hardwood species such as cottonwoods and, during the growing season, no 0 2 could be detected i n some analyses. In species showing a pattern of wetwood forma-t i o n , low 0 2 and high C0 2 concentrations were generally prevalent and i n some cases, abnormally high proportions of CH^ were also found i n the tree gases. In the tree species i n which high concentrations of CH^ were reported, the tree gases were always under pressure. When the gases were sampled at various heights within a single tree, the composition was very s i m i l a r throughout the length of the trunk. Usually sapwood exhibited higher 0 2 and lower C0 2 concentrations than the heartwood or the wetwood. L i t t l e i s known about how gases enter the tree trunks and how they exist i n proportions so-different from those of the atmospheric a i r . MacDougal and Working (1933) suggested that "some 0„ and more C0 9" probably 57 enter the roots dissolved i n the water of the sap stream. Another means of entrance of gases into the trunk may be through the stomata of the leaves (Chase 1934). However, so far as i s known, no experimental work has been done on movements of gases downwards from the leaves, and i t i s probable that the transpiration stream would prevent such a movement. Chase (1934) also suggested that gases may enter through the l e n t i c e l s of the bark into the i n t e r c e l l u l a r spaces of the cortex and phloem. This may account for the higher content generally found i n the sapwood. The abnormal proportion of gases i s sometimes attributed to the meta-bolism of the tree. Chase (1934) states that CG^ within the sapwood and heartwood of a tree may originate from the respiration of the l i v i n g c e l l s of the wood rays, v e r t i c a l parenchyma and cambium. During the summer months these c e l l s are very active and i s low and C0*2 i s high. MacDougal and Working (1933) also consider "respiration of l i v i n g c e l l s " as the p r i n c i p a l cause for low and high CO^ i n the trunks. Morani and Arru (1958), and Zeikus and Ward (1974) considered bacteria responsible for the gas contents of the tree trunks. High concentrations of CH^ were cert a i n l y produced by the a c t i v i t y of methanogenic bacteria. These concentrations of gases are apparently maintained within the trunk of trees because the l i v i n g cambium offers a good b a r r i e r to the dif f u s i o n of gases (Kramer and Kozlowski 1960). The role these gases play i n the actual metabolism of the tree i s not very clear, 0^ i s involved i n oxidation of materials within the l i v i n g c e l l s . C0 9 i s not used i n the xylem tissues of the stem, although some of be i t may diffuse through the cambium and/used i n photosynthesis i n the bark. 58 Numerous investigators have speculated upon the cause of heartwood for-mation. I t has been suggested that the large amounts of C0 2 and small amounts of 0 2 are responsible for the change from a l i v i n g to a dead con-d i t i o n (heartwood). After discussing the role of gases i n metabolism of the tree, Chase (1934) concluded, " I t appears that the varying proportions of these gases i n the trunk are a re s u l t of metabolism and probably have s l i g h t influence upon internal l i f e processes." As pointed out e a r l i e r , some of the studies dealing with composition of gases within tree trunks gave consideration to effects on decay. For example, Thacker and Good (1952) studied growth of 7 wood-destroying fungi i n v i t r o at various 0"2 and C0 2 concentrations, including those found i n the trunks of maples. 0 2, i n the percentages found by them i n maples, did not l i m i t growth of any of the fungi tested. Also, CO^ concentrations em-ployed generally favored growth of most fungi tested. The authors, there-fore, concluded that the composition of a i r commonly found i n maple trunks during the growing season i s nearly optimal for the development of wood-destroying fungi. Whether this view can be adopted as a v a l i d generaliza-tion for a l l tree species and a l l wood-destroying fungi seems open to ques-ti o n . A l i t e r a t u r e review shows that i n some tree species, 0 2 content was generally low, at least during the growing season, and occasionally none at a l l could be detected (MacDougal and Working 1933, Chase 1934, Zeikus and Ward 1974). The lowest 0 2 concentration found by Thacker and Good (1952) i n maples was 0.8%; the lowest l i m i t of detection i n t h e i r analyses was 0.4%. 0„ concentrations of 0.4% and less have already been recorded i n 59 some tree species. In addition, Thacker and Good's cu l t u r a l experiments involving wood-destroying fungi were done on agar medium as opposed to wood, and the growth response of fungi on these 2 media cannot be assumed to be the same. Also, many f i e l d experiments have indicated that decay . columns i n trees may become inactive after the entry point i s healed (Hepting. 1941, Toole 1965). These observations and the inferences that can be drawn from them suggest that additional basic data are required be-fore any generalizat ion, such as that of Thacker and Good (1952) , can be accepted. The present study was conducted to determine the gas composition of wetwood and sapwood of black cottonwood, and to see i f the gas levels were different from those i n tree species without wetwood. The underlying pur-pose was to see i f the concentrations of 0^, as they occur i n tree trunks, would have any effect on the growth of woodfedestroying fungi (Chapter IV). I f a relationship between gas composition and growth of fungi i s to be as-sumed, then i t i s important to know what effects wounding may have upon the gas composition. Also, i t i s essential to know i f branch stubs act as open-ings allowing gases to move i n and out. Therefore, the s p e c i f i c objectives of this study were, 1) to extract and analyze gases from the wetwood and also from the sapwood of several black cottonwood trees for a period of 1 year, 2) to examine effects of wounding on the gas composition of wetwood, 3) to determine i f exchange of gases takes place through the branch stubs, and 60 4) to study the gas composition of some other hardwood species growing i n an environment, s i m i l a r to that of black cottonwood, for comparative purposes. MATERIALS AND METHODS 1. Location of sample trees: A l l of the trees that were sampled during gas composition work are located on the University Endowment Lands (U.B.C), Vancouver, B.C. Black cottonwood and red alder (Alnus rubra Bong.) trees are situated north of the University Boulevard, surrounding the University H i l l Church (Figures 11 and 12). Two of the Lombardy poplar trees are located behind the University Health Center and the t h i r d one was behind the E l e c t r i c a l Engineering Department. This tree was cut down i n the summer of 1974 when the area was.cleared for construction projects. The details of climatic conditions, s o i l s and the h i s t o r y of the University Endowment Lands have been given by Norris (1971). The climate of t h i s area consists of r e l a t i v e l y mild winters and warm summers. The average monthly temperatures range from 4 C i n December to 17 C. i n J u l y , with -12 to 30 C as common extremes. The length of the growing period (above 5 C) i s 265 days. Sharp frosts l a s t i n g for. several days occur be-tween November and A p r i l . The average p r e c i p i t a t i o n i s about 115 cm with a t o t a l from May through September of only about 30 cm evenly distributed. There are 155 days per year with measurable r a i n f a l l . The s o i l of t h i s area i s classed as Gleyed Orthic Podzol, with pH ranging from 3.6 to 6.7 depending upon the horizon sampled. 61 Figure 11: Map of the University Endowment Lands showing location of sample trees used for the gas composition work. Xx*x% - Black cottonwood and red alder xxx °a>ft° " Lombardy poplar Figure 11 63 Figure 12: Black cottonwood-red a l d e r stand surrounding U n i v e r s i t y H i l l Church, U n i v e r s i t y Endowment Lands (U.B.C). 64 In 1951, a large area (about 300 acres) of forest extending from the Spanish Banks to the University Boulevard was cleared. This area i s now largely covered by a red alder stand. The area, s l i g h t l y north of Univer-s i t y Boulevard and near the University H i l l Church, consists of a f a i r l y even mixture of red alder, black cottonwood and maple, with very few coni-ferous species as an understorey. Vigorous growth of salmonberry (Rubus  spectabilis Pursh) and swordfern (Polystichum munitum) [Kaulf.] Presl.) i s also apparent i n this area. As indicated e a r l i e r , gas composition studies were done on black cottonwood and red alder trees growing i n this area. Generally, individual trees were selected at random although prefer-ence was given to the large diameter ones, and i n a few cases, further se-lec t i o n was necessary depending upon the purpose of the experiment. The relevant details of the trees such as age, diameter at breast height (dbh), height, wood zone investigated, etc. are given i n Table VII. A l l of the gas composition studies were made i n the year 1974. Some trees were examined a l l through the year while others were examined only during certain months of the year. In general, gas extraction and analysis were done on an i n d i -vidual tree every two weeks once the investigation began. 2. Extraction apparatus: The description of the i n d i v i d u a l apparatus used i s as follows, A. Brass pipe: Specially designed (Figure 13-A); length, 9.5 i n (24.1 cm); O.D., 0.37 i n (0.9 cm); I.D., 0.25 i n (0.6 cm); provided with an end-taper tb f a c i l i t a t e i n s e r t i o n and a heavy c o l l a r on the other end; heavy c o l l a r end carries fe-male thread, 1/8 NPT. B. Shut-off valve (Fairview F i t t i n g s and Manufacturing Ltd.): Hose barb to male pipe; hose O.D., 0.25 i n (0.6 cm). TABLE VII: Description of sample trees, wood zones and duration of sampling i n the year 1974 Species Tree Tree characteristics Wood zone Duration of Remarks number Age (yr) DBH (in [cm]) Height (ft[m]) extracted sampling Black cottonwood 10 23 18.1(46.0) 60.0(18.3) Wetwood Jan-Dec. 15 22 11.8(30.2) 51.0(15.5) Feb-Dec 6 21 10.0(25.4) 39.0(11.7) Jan-Dec 11 22 10.0(25.4) 41.0(12.3) Jan-Dec 8 20 10.3(26.3) 43.0(13.1) Jan-Dec 16 21 13.1(33.3) 60.0(18.3) Feb-Dec. 20 22 11.1(28.4) 56.0(17.1) May 21 23 21.2(54.1) 53.0(16.1) May-Jul 22 22 15.1(38.4) 59.0(18.0) May 27 21 12.7(32.3) 47.0(14.3) Jun-Oct. 28 23 16.2(41.4) 68.0(20.5) Jul-Oct 29 22 16.3(41.6) 36.0(11.0) Jul-Aug 5 18 8.9(21.8) 42.0(12.6) Jan 4 18 11.1(28.4) 44.0(13.4) Jan-Feb 9 19 11.4(29.0) 50.0(15.3) Jan-Feb 1 17 11.2(28.3) 49.0(14.9) Jan 7 17 9.7(24.6) 50.0(15.2) 11 Jan £ Table VII (cont'd) Species Tree Number Tree characteristics Age (yr) DBH (in [cm]) Height (ft[m]) Wood zone extracted Duration of sampling Remarks Black cottonwood 14 18 8.5(21.7) 41.0(12.5) Wetwood Feb. 17 20 15.1(38.5) 50.0(15.3) it Feb, 18 21 12.0(30.3) 60.0(18.2) II Apr 12 22 11.1(28.4) 44.0(13.4) Sapwood Feb -Mar 16 21 11.4(29.2) 58.0(16.0) II Mar. 32 20 15.3(39.0) 43.0(13.0) it Apr. 33 20 11.5(29.3) 43.0(13.0) it May 34 20 11.8(30.2) 52.0(15.7) ii May-June 35 21 10.3(26.3) 45.0(13.50 II Jun.-Jul 36 22 10.0(25.4) 54.0(16.3) it Aug 37 21 9.7(24.7) 52.0(15.7) M Sep-Oct 38 22 10.6(26.9) 59.0(17.9) II Oct 2 19 10.4(26.4) 43.0(13.1) Wetwood Feb-Dec 3 23 II Mar-Dec 24 22 16.8(42.7) 58.0(17.5) II May-Aug 30 23 10.5(26.7) 46.0(14.0) tt May-Aug 19 23 16.1(40.9) 55.0(16.9) H Apr-Aug 23 23 14.0(35.6) 49.0(14.8) M Apr-Jun Wounded n Wounded-Sapwood exposed Through branch stub ON Table VII (cont'd) Species Tree Number Tree characteristics Wood zone extracted Age (yr) DBH (in [cm]) Height (ft[m]) Duration of sampling Remarks Red alder Lombardy poplar 41 42 25 44 45 46 23 23 20 Data 10.0(25.4) 37.0(11.2) 9.9(23.1) 40.0(12.3) 9.0(23.0) 42.0(12.9.) not available 24 21.1(53.6) 56.0(17.0) 23 22.5(57.4) 59.0(18.1) Heartwood Heartwood (probably wetwood) I I Feb-Dec Feb.-Dec Jan May May-Jun 68 Figure 13: Schematic drawings of the s p e c i a l l y designed apparatus. A. Brass pipe. B. Large diameter glass tube. C. Small glass tube with a s l i g h t bend. "0.75" 8.00 Figure 13 70 C. Masterflex tubing pump (Cole-parmer Instruments Ltd.): Con-s i s t s of a high torque gear motor and a f u l l v i s i b i l i t y pump head; accepts a continuous length of tubing from and to the system; no p o s s i b i l i t y of any contamination of the c i r c u l a n t ; p r a c t i c a l l y pulseless and provides smooth suction; gases may be pumped at pressures up to 15 ps i (1.05 kg/sq cm), li q u i d s up to 40 p s i (2.8 kg/sq cm) intermittently; vacuum may be pulled up to 24 i n (60.9 cm) of Hg; tubing pump provided with a solid-state speed c o n t r o l l e r , 30 to 60 RPM; flow rates can be established accurately and main-tained; obtainable flow rates, from 1.8 ml/minute to 2280 ml/minute. D. Field-lab 0~ analyzer (Beckman's Instruments Co.): Analyzer equipped with a sensor; i n operation, the sensor i s placed i n the sample, a potential of 0.53 vo l t i s applied between the rhodium cathode and s i l v e r anode i n the sensor; 0^ i n the sample diffuses through a Teflon membrane, diffused 0^ ^ s re~ duced electrochemically at the cathode; t h i s reduction causes a current flow proportional to the p a r t i a l pressure of 0^ i n the sample, the flow i s amplified on a scale; i n gas, 0„ measurement l i m i t s are 0.05 to 25.0%, and i n l i q u i d s , tney are 1.0 to 25.0 ppm. E. Glass tubings: a. Specially designed (Figure 13-B); length, 8.0 i n (20.3 cm); large diameter, I.D., 1.5 i n (3.8 cm), re-duced to 0.12 i n (0.3 cm) I.D. and 0.25 i n (0.6 cm) O.D. with a short stem, length of the stem, 1.0 i n (2.5 cm). b. Specially designed (Figure 13-C); length, 3.5 i n (8.9 cm); s l i g h t l y bent (about 30°) at one end; I.D., 0.12 i n (0.3 cm); O.D., 0.25 i n (0.6 cm). F. Gas sampling tubes (Fisher S c i e n t i f i c Co.): Capacities to hold l i q u i d from 100 to 310 ml approximately. In addition some standard apparatus such as an increment borer (O.D., 0.25 i n [0.6 cm]), a vacuum gauge (range: 1-30 i n [2.5 to 76.2 cm] of Hg), 2 shut-off valves, a flow meter (range: 1.3-23 to 400 ml/minute), a thermometer (range:.-20 to 110 C), a Erlenmeyer flask (capacity 1.0 l i t e r ) , f l e x i b l e v i n y l tubing (I.D., 0.25 i n [o.6 cm]), and 2 rubber stoppers (No.s 8 and 9) was also used, 3. Extraction apparatus: Assembly: Gas sampling tube, large dia-meter glass tube, vacuum gauge and flow meter were mounted on a 23.0 x 19.0 i n (58.4 x 48.3 cm) piece of 0,5 i n (1,3 cm) plywood padded with 71 ordinary 0.5 i n (1.3 cm) foam rubber. Small cup-hooks were driven into the plywood and the rubber bands attached to the cup-hooks held the appa-ratus i n place. The plywood piece with clamped apparatus was then set . 11.5 i n (29.2 cm) back i n a 24.0 x 20.0 x 12.0 i n (61.1 x 51.0 x 30.5 cm) case of 0.5 i n (1,3 cm) plywood leaving enough space i n front to place the 0^ analyzer and the tubing pump (Figure 14). A p l a s t i c sheet was attached on the open front preventing water to enter the case while working i n the rai n . A handle fixed on the top of the case made the whole system portable. The general assembly of the extraction apparatus i s shown i n Figure 15. The sequence of the connections (made with v i n y l tubing) was as follows: (tree) tubing pump -* gas sampling tube -•-»- 0 2 analyzer Erlenmeyer flask ->• flow meter. The vacuum gauge was i n s t a l l e d between the tree and the tubing pump to record the suction developed. The shut-off valve was placed between the tubing pump and the 0 2 analyzer ( i . e . sensor i n the large diameter glass tube), running p a r a l l e l to the gas-sampling tube, i n case of excessive flow of liquids from the tree. The thermometer, the sensor (of the 0 2 analyzer) and the small diameter glass tube (with a s l i g h t bent) were inserted through 3 holes made i n a rubber stopper and the stopper was f i t t e d on the large diameter glass tube. This large diameter glass tube was clamped i n an angle of approximately 45° to the base of the case. The small d i a -meter glass tube was positioned at such a height and angle that i t s s l i g h t l y bent-end would be close to the rubber stopper touching inside of the large diameter glass tube. Both of these arrangements, i n addition to the slant-ing position of the large diameter glass tube, ensured maximum sw i r l i n g move-ment of the gases and at the same time, avoided l i q u i d from getting i n con-tact with the sensor-tip. The Erlenmeyer f l a s k , connected next to the large 72 Figure 14: Portable e x t r a c t i o n apparatus mounted i n a plywood case. 73 Figure 15: Schematic drawing of the extraction apparatus assembly. 1, Vacuum gauge; 2, Tubing pump; 3, Gas-sampling tube, 4, Shut-off valve; 5, Small glass tube; 6, Thermometer; 7, C>2 sensor; 8, Field-lab analyzer; 9, Large diameter glass tube; 10, 1 l i t e r Erlenmeyer f l a s k ; 11, Shut-off valve; 12, Flow meter. The flow of the gases and liqu i d s would be as follows: (Tree) •— Tubing pump Gas-sampling tube — 0 2 analyzer --- Erlenmeyer flask (trap for liquids) Flow meter (Atmosphere) . 75 diameter glass tube, trapped any l i q u i d while gases went ~ further through the flow meter to the atmosphere. A shut-off valve was placed between the Erlenmeyer flask and the flow meter, and opening of the valve was adjusted i n such a way that a steady flow of gases could be obtained without creating high gas pressures within the extraction apparatus. This was necessary to remove the pulsing effect of the tubing pump on the flow meter. 4. Method and operation of extracting gas samples: An increment, bor-er, which was s t e r i l i z e d by dipping i n 95.0% ethanol, was used to bore a hole reaching the approximate center of the tree (in the case of wetwood and heartwood). The core was removed and retained to detect presence of wetwood, tree age and to study the microorganisms. The increment borer was then re-moved and a brass pipe was inserted immediately through the hole and hammered i n such a manner that the t i p of the pipe would be about 2-3 cm from the end of the bore (Figure 16). A shut-off valve was screwed on the. pipe making sure that a l l the connections were a i r t i g h t . The f i r s t gas sample was c o l -lected one week from the day pipe was placed i n the tree? allowing the gases in the tree and the pipe to reach an equilibrium. Before extracting gases, the 0 2 analyzer was calibrated according to the temperature of the a i r with the assumption that atmosphere contains 21.0% 0^. Vinyl tube leading to the tubing pump was then attached to the connect-ing hose f i t t e d on the bbrass pipe and the shut-off valve opened (Figure 17). The tubing pump connected to the power supply was switched on (with a setting of 4 on the controller) and the gases and liquids flowed out of the tree. During the extraction operation the vacuum gauge, thermometer, 0^ analyzer and flow meter were observed constantly. Any change i n the temperature was 76 Figure 16: A. Schematic drawing o f the brass pipe and s h u t - o f f valve i n p o s i t i o n . Dotted l i n e s i n d i c a t e extent o f the hole made with an increment borer. W -wetwood, S - sapwood. B. Brass pipe and s h u t - o f f valve i n p o s i t i o n i n the f i e l d . 7 7 Figure 16: C. V e r t i c a l s e c t i o n o f a black cottonwood through the sampling hole (extending from l e f t to r i g h t ) . 79 Figure 17: Complete gas e x t r a c t i o n set-up i n the f i e l d . P o r t a b l e gas e x t r a c t i o n apparatus on the ground, connected to the brass pipe (and tree) w i t h a v i n y l tubing. 80 adjusted for on the 0 2 analyzer. The actual time of c o l l e c t i n g gases for C0 2 and CH^ analysis, and recording temperature (C), suction developed (in [cm] of Hg) and flow rate (ml/min) of the gases was when the indicator on the 0 2 analyzer remained steady at a certain position for about 2 min-utes. The whole extraction procedure normally took about 10 minutes. Af-ter c o l l e c t i n g a sample from one tree and recording 0 2 concentration, the gas sampling tube (containing gas sample for C0 2 and CH^ analysis) was re-moved. The complete operation of extraction, including c a l i b r a t i o n of the 0 2 analyzer, was then repeated to c o l l e c t a gas sample from another tree. During the c a l i b r a t i o n , a l l of the gases present i n the extraction appara-tus from the previous tree are removed. The method of extracting gases from the sapwood, from the wetwood through branch stubs and from the wetwood of wounded trees was es s e n t i a l l y the same as described above. Some modifications were necessary and are pre sented below. 4.1 Black cottonwood: Sapwood; Generally, the sapwood offered re-sistance to extracting gases on a continuing basis from the same tree. About 4-6 weeks after placing the pipe i n theesapwood, no gases could be withdrawn therefore allowing only 2 or 3 sample collections per tree. Con-sequently, gases were collected from the sapwood of different trees from A p r i l u n t i l December. 4.2 Black:cottonwood: Wetwood through branch stubs: Two trees were used to obtain gases from the wetwood through the branch stubs. I n i t i a l l y , a brass pipe was placed i n each tree approximately 5.0 cm away, either hori zontally (as i n Tree 23) or v e r t i c a l l y (as i n Tree 19) from the dead branch 81 and the extraction and analysis were done as usual. Then, i n each case, the entire dead branch was broken o f f from the main trunk and another pipe was. hammered through the branch stub with same approximate angle as that of the dead branch (Figure 18). After a week, attempts were made to obtain gas samples from both the pipes (the normal entry point and the branch stub) on the same day. 4.3 Black cottonwood: Wetwood of wounded trees: Wetwood exposed: One tree (No. 2) was naturally wounded, and 2 trees were wounded during the gas composition work (No.s 3 and .21). The cause of the wound in Tree 2 was unknown. A v e r t i c a l crack was present s t a r t i n g from the ground level going upwards for about 1.5 m. In th i s tree, a pipe was placed about 15.0 cm away from the scar (Figure 19) and the gases were extracted and analyzed. Tree 3 was sampled regularly from March u n t i l the middle of May and then was wounded with an increment borer exposing the wetwood. The hole was made about 8.0 cm below the normal extraction point. Then the extraction and analyses were made for about 2 months. In the la s t week of Ju l y , the wound was plugged with a metal rod and the analysis was continued u n t i l December. Tree 21 was examined i n the s i m i l a r way; the tree gases were ana-lyzed i n May, then the tree was wounded exposing i t s wetwood and within 24 hours, the wound was sealed and the gas analysis was resumed. 4.4 Black cottonwood: Wetwood of wounded trees: Sapwood exposed: Two trees were sampled to examine possible effects of sapwood exposure on the gas composition of wetwood. Before wounding, gases were withdrawn and analyzed from the wetwood of both the trees for about 2 months. In Tree 24, the sapwood was exposed with an axe (Figure 20) while i n Tree 30, 4 holes 82 Figure 18: A, and B. Schematic drawing of a tree (No. 23) showing set-up used to extract gases through a branch.stub. A. Dead branch s t i l l present on the tree, about 5C0icm away from the normal extraction point. B. Dead branch removed and replaced with another brass pipe to extract gases. Figure 18. C. Set-up (as i n B) i n the f i e l d . Brass pipe on the l e f t i s the normal extraction point and the one on the right i s where the dead branch was present. Figure 18 84 Figure 19: A black cottonwood t r e e (No. 2) showing nature of the wound ( l e f t ) and normal e x t r a c t i o n set-up ( r i g h t ) . 85 Figure 20: Schematic drawing of the two trees showing nature of the wounds-. Gases- from wetwood were extracted and analysed. W - wetwood,.S - sapwood. A. Tree 24- Sapwood was exposed with an axe. B. Tree 30 - Sapwood cores were removed with an increment borer. 86 A .1.1 w Sapwood removed with an axe Tree 24 , r - l - l w ^_ Sapwood removed with an increment borer Tree 30 Figure 20 87 were made from a l l sides with an increment borer exposing i t s sapwood. Gas extraction and analysis were then continued for another 2 months. 5. Gas analysis: As indicated e a r l i e r , 0^ concentrations were mea-sured i n the f i e l d during actual extraction procedure with the f i e l d - l a b 0 2 analyzer. The analyzer reads d i r e c t l y the percentage of 0^ (weight/ volume) present i n the sample. The concentrations of C0 2 and CH^ were determined by gas chromatography as described by Smith (1973). This analysis work was done under the super-vi s i o n of Dr. Roger S. Smith at the Western Forest Products Laboratory, Vancouver, B.C. Using a gas chromatograph (Varian Aerograph, model 1740) with a flame-ionization detector, each gas sample was separated on a 10 f t (304.8 cm) by 3/16 i n (4.8 cm) stainless steel column, packed with Poropak Q (120 mesh). The C0 2 was reduced (after separation from any CH^ i n the sample) to CH 4 by passing i t through a 9 i n (22.9 cm) by 1/8 i n (3.2 mm) stainless steel column containing a ni c k e l catalyst. H 2 was used as a car-r i e r gas (flow rate 25 ml/min) and N 2 as the diluent i n the detector (25 ml/ min). The flow rate of the combustion a i r to the detector was 300 ml/min. Signals from the detector were passed through a d i g i t a l integrator (Hewlett Packard, Model 3370A), with a code converter board interface to a t e l e p r i n t -er (Teletype Corporation, No. 33-L), thereby providing a printed readout of peak-time and area. A ca l i b r a t i o n graph of C0 2 concentration and integrator response (micro-volts x seconds, uV.s) was prepared by taking 0.5 ml samples from known con-centrations of C0 2 i n a i r , and then i n j e c t i n g these samples d i r e c t l y into the gas chromatograph through the i n j e c t i o n port septum. The various gas 88 mixtures were prepared i n a glass vessel provided with a rubber septum. Another c a l i b r a t i o n graph was also prepared for CH^ concentrations using the same technique and both these c a l i b r a t i o n graphs are given i n Appendix I I . The s e n s i t i v i t y of the gas chromatograph unit was well below 0.1% for both the gases. Gas samples collected from the trees were usually analyzed within 2 hours from the c o l l e c t i o n time. Using a gas syringe, a gas sample of 0.5 ml was taken from the gas sampling tube and injected into the gas chroma-tograph. Each reading given i n the Appendix i s an average of a minimum of 3 determinations. 6. Measurement of gas pressures i n the wetwood: Two trees (22 and 27) were used to see i f gases within wetwood are present under pressure during summer. In each case, the brass pipe was placed i n the tree as usual and a pressure gauge (range: up to 30 p s i [2.1 kg/sq cm]) was immediately screwed on to the brass pipe. The gauge was observed every week for a per-iod of 2 months. RESULTS In the course of this study, more than 1200 individual gas analyses were made. Most of these determinations were made on gases extracted from the wetwood of black cottonwood trees. Some trees were examined only for a limited time. A l l of these determinations are given i n a complete form i n Appendix I I . Data from some representative trees have been selected to show the seasonal variations of the gases within a single tree, and also the gas compositions of 2 different tree species or tree zones for compara-89 tiv e purposes. They are presented graphically i n Figures 21-24, 27-32. 1. Black Cottonwood: Wetwood: Examined a l l through the year: General-l y the concentration i n the wetwood was lower during the summer than the winter iand the reverse was true for CO^. In 5 of the 6 regularly tested trees, the 0^ concentration dropped below the l i m i t of measurement (<0.05%) i n the summer. Concentrations of CH^ were consistently low throughout the year i n a l l regularly examined trees. In Tree 10, 0 2 was low a l l through the year and the presence of microaerobic conditions* persisted for about 10 weeks during the summer. In th i s tree, C0 2 was higher (13.1%) i n the summer than any other tree tested. From January u n t i l 1st week of June there was no CH^ present i n the tree while only traces were found thereaf-t e r . Flow of the gases was generally high (75-100 ml/min). 0 2 was also consistently low i n Tree 15 (Figure 21). In the summer, wetwood exhibited the presence of microaerobic conditions for almost 12 weeks and even during the winter, 0 2 did not reach 2.0% l e v e l . C0 2 concentrations were generally high. CH^ was present i n the tree throughout the year, although not i n any substantial quantities. Tree 6 was the only tree i n which wetwood never became microaerobic. Even during the summer, some O2 was always present i n the wetwood ( >0.1%). C0 2 was generally high, and CH4 was found i n the tree a l l through the year (Figure 22). Trees 11, 8 and 16 followed patterns 1 The term "microaerobic conditions" was used to describe "presence of very small concentrations of O2" i n a given system. This and 2 other commonly used terms, Aerobic and Anaerobic, are q u a l i t a t i v e terms, and . depending upon sophistication of the detection system, they are subject to include a wide range of O2 concentrations. In th i s t h e s i s , the f o l -lowing system has been adopted: Anaerobic conditions - <0.002% O2 Microaerobic conditions - < 0.,l%::r 0 2 Aerobic condition - ^0,1% 0„ 90 Figure 21: Concentrations of 0 2, C0 2 and CH^ (monthly averages) i n the wetwood of :h black cottonwood (Tree 15). Microaerobic conditions were f i r s t noted i n the 4th week of A p r i l and they persisted u n t i l the 3rd week of July, 1974 20.0 18.0 ° - ° 2 A - CO n - CH 16.0 14.0 Tree 15 2 o l -H E-i <. OS E-I Z W C_> O u c/} 12.0 10.0 8.0 6.0 4.0 2.0 O. • ^v—n a- . a--o- •O' > M A M J TIME O F YEAR J Figure 21 92 Figure 22. Concentrations of 0^, CO^ and CH4 (monthly average) i n the wetwood of a black cottonwood (Tree 6). In th i s tree, microaerobic conditions were not present even during the summer. 20.0 18.0 16.0 14.0 12.0 Tree 6 o - o 2 A - CO o - CH 10.0 8.0 6.0 4.0 2.0 J F M A M J J TIME OF YEAR Figure 22 94 of gas compositions s i m i l a r to that of Tree 1 0 , a l l becoming microaero-b i c during the summer. 2. Black cottonwood: Wetwood: Examined only during certain months of the year: Eighteen trees were examined occasionally for t h e i r gas con-tents during the year. Six out of 8 trees sampled during the summer had $2 concentrations below or close to 0.05%. The general trend of low 0^ and high CO2 was evident i n a l l 8 trees examined. CH^ concentrations, however, varied from 0.0 to 34.0%. Tree 21 which was examined for a longer period than others, contained high concentrations of CH^ (30.5 - 34.0%) during May. This tree also exhibited the effects of wounding on i t s gas composi-tion which w i l l be discussed l a t e r . In the remaining 10 trees examined dur-ing the winter, 0 2 concentrations were generally high and C0 2 concentrations were generally low. 3. Black cottonwood: Sapwood: The composition of gases i n the sap-wood was d i s t i n c t l y d ifferent from that of wetwood (Figure 23). 0^ contents were high throughout the year (11,4 - 19.0%) while C 0 2 contents were general-l y low (0 , 1 - 0.8%). There was no d i s t i n c t relationship between gas concen-trations and the season. Also, CH^ was absent i n the sapwood of a l l trees sampled. It should be noted that gases were withdrawn from dif f e r e n t trees for about 8 months of the year. This was due to the resistance offered by wood i n extracting the gases from a single tree on a continuing basis. 4. Black Cottonwood: Wetwood through branch stubs: Before placing pipes through the branch stubs, gas concentrations of both the trees were determined. 0^ was low and C0 2 was high and the 2 trees also contained some CH. (Figure 24). As described previously, i n Tree 19, o r i g i n a l pipe 95 Figure 23: Comparison of 0^ and C0 2 concentrations (monthly averages) i n the wetwood (Tree 8) and sapwood (several trees) of black cottonwood. 97 Figure 24: Concentrations of 0^,00^ and CH^ (monthly averages) i n the wetwood of two black cottonwoods (Tree 19 and 2?). Gases analysed were collected through normal extraction points. Attempts to withdraw gases through the branch stubs f a i l e d i n case of both the trees. 98 Figure 2.4 99 was about 5 cm away v e r t i c a l l y from the dead branch (which was l a t e r re-placed by another pipe), and i n Tree 23, i t was 5 cm away hori z o n t a l l y . The attempts to withdraw gases through the branch stubs f a i l e d i n both the cases. Later, Tree 23 was f e l l e d and a v e r t i c a l section through the branch stub revealed that the inserted pipe did not follow the exact orien-tation of the branch. The t i p of the pipe remained s l i g h t l y above the branch trace (Figure 25). Nevertheless, about 22.0 i n (55.0 cm) of suction developed during the extraction, and the flow rate was zero. When the ex-tractions were repeated on the same day using the o r i g i n a l pipes, the flow of the gases was usual (about 5.0 i n [12 cm] of suction). 5. Black Cottonwood: Wetwood of wounded trees: Wetwood exposed: In Tree 2 (naturally wounded), throughout the year, 0^ concentration was gener-a l l y high ranging from 16.5 to 19.0% while C0 2 was low, never reaching even 1,0% l e v e l . During a l l the extractions, suction never developed suggesting that the process of wound healing was perhaps incomplete and that the wet-wood was i n contact with atmosphere. At the end of the gas composition stu-dies,- the tree was f e l l e d to examine the nature and extent of the wound. In a cross section, i t can be seen that new growth had e f f e c t i v e l y covered the wound (Figure 26). However, when cross sections were taken repeatedly through the length of the wound, they revealed that a crack extended through the wound lengthwise, and towards the base of the tree, wetwood was connected to the outside through the crack. This explains the gas composition. CH^ was not found i n this tree i n either winter or summer. Before Tree 3 was wounded i n May, the 0 2 concentration was high, C0 2 was low while CH^ was present but only i n small quantities. When the wetwood was exposed, 0~ concentration generally remained unaltered although C0„ Figure 25: V e r t i c a l section of Tree 23, through the branch stub showing where the brass pipe was placed (above) and p o s i t i o n of the branch trace. 101 Figure 26: Cross s e c t i o n of a 19 year o l d black cottonwood (Tree 2) showing extent of the wound. A crack ( r i g h t ) extended through the e n t i r e length of the wound. 102 concentration dropped considerably and CH^ could not be detected any longer. After plugging the wound i n July, 0^ concentration dropped con-siderably, CO2 contents increased and CH^ re-appeared. In October and November, CO2 and CH^ concentrations increased substantially reaching higher levels than before the tree was wounded. As reported e a r l i e r , the gas composition of Tree 21 was unusual i n that wetwood of the tree contained over 30.0% CH^ i n the early summer. Attempts were made to i s o l a t e anaerobic bacteria from the wetwood (Appen-dix I ) . An increment borer (1.2 cm bore) was used to obtain a wooden core, and therefore i n e f f e c t , the tree was wounded. Before the tree was wounded, 3 previous determinations of the gas contents showed CH^ to be 34.0%, 30.5% and 32.6%. After wounding and then plugging the wound within 24 hours, a gas analysis was made oh June 27th, which showed that CH^ concen-t r a t i o n of the wetwood had dropped substantially reaching 3.2% l e v e l (Figure 27). Three weeks after t h i s , another analysis was made and i t re-vealed that CH4 was increasing again which at t h i s time had reached the 10% l e v e l . The tree also showed changes i n the composition of 0 2 C0 2 as anticipated; wetwood which was microaerobic before wounding showed a sharp increase i n i t s 0 2 concentration and C0 2 l e v e l f e l l after wounding. 6. Black Cottonwood: Wetwood of wounded trees: SapWood exposed: Two trees (No.s 24 and 30) were used to study i f exposure of sapwood had any effect on the gas composition of wetwood. These trees were examined during the summer and both of them continued the pattern of low 0 2 and high C0 2 even after the sapwood was wounded. 103 Figure 27. Concentrations of 0^, C0 2 and Crl^ i n the wetwood of a black cottonwood (Tree 21). Note the abrupt change i n the gas composition pattern after the wetwood was ex-posed. , i n the 1st week of June and covered within 24 hours (arrow). Also note the subsequent change i n the gas composition trend with-time, 104 o , — o > i i i i ' i. . t i _ 8 15 21 31 8 15 21 27 8 14 19 27 MAY JUNE JULY DATE OF SAMPLING Figure 27 105 7. Red alder: Heartwood: Two trees were studied a l l through the year and 1 tree was studied only during the summer. A l l of the determina-tions showed that 02 concentrations were generally high and C0^ concentra-tions were generally low (Figure 28). No seasonal variations of gases were evident from the analyses. CH^ was absent i n a l l 3 trees tested. In general, concentrations of 02 and CO 2 i n red alder heartwood were i n i n -verse proportions to those of black cottonwood wetwood (Figure 29). 8. Lombardy poplar: Heartwood (probably wetwood): A very limited number of extractions were done on Lombardy poplars; three trees were ex-amined and only during the summer. The wood cores removed suggested that i n a l l 3 trees, wetwood may have been present instead of heartwood; the i n -ner wood appeared wet and discolored. A further examination i s necessary to confirm the presence of wetwood. The gas analyses showed that 0^ was " present i n high proportions (except Tree 46 i n June), but and CH^ were present in generally low proportions (Figure 30), 9. Measurement of gas pressures i n the wetwood: Gases were never found under pressure i n both of the trees; the pressure gauge indicated zero pressure at a l l times. DISCUSSION A de f i n i t e seasonal variation occurred i n the concentrations of Q2 and CO2 i n the wetwood of black cottonwood. The percentage of O2 was lowest i n the summer and highest i n the winter. It varied inversely with the per-centage of CO2 a l l through the year. CO2 was highest during the growing season and lowest i n the winter. In the majority of the trees, tested 106 Figure 28: Concentrations of O2 and CO2 (monthly averages) i n the heartwood of a red alder (Tree 42). CH^ was never found, i n any of the red alders studied. 20.0 18.0 16.0 14.0 .<, 12.0 w u 10.0 z o u 8.0 6.0 4.0 2.0 Tree 42 • A — -JL . A-M A M J J TIME OF YEAR Figure 28 108 Figure 29: Comparison of 0^ and C0 2 concentrations (monthly averages) i n the wetwood of a black cottonwood (Tree 11) and heart-wood of a red alder (Tree 41). 109 Figure 29 110 Figure 30; Concentrations of 0 2, C0 2 and CH4 (monthly averages) i n the heartwood (probably wetwood) of three Lombardy poplar trees (Nos. 2, 44, 45 and -46) . I l l 2 0 . 0 O - 0, A - CO, 1 8 . 0 • - CH, 1 6 . 0 o 14.0 L < 1 2 . 0 : H • 2 : tq • 2 : o 1 0 . 0 6 . 0 T r e e 44 O T r e e 45 T r e e 46 4 . 0 A A 2 . 0 A M J • D H I 1 L _ M J TIME OF YEAR F i g u r e 30 -1 I ' L. M J J 112 regularly or occasionally, 0^ concentrations of <0.05% were found during the summer (Figure 31). Such microaerobic conditions lasted for prolonged periods (6-12 weeks) within trees. Some trees contained CH^ i n the wet-wood; i n regularly examined trees i t was consistently low while i n some trees occasionally tested, CH^ concentrations of as high as 34% were found during the summer. Gases drawn from the sapwood were always lower i n CO^ and higher i n than those obtained from the wetwood. No marked seasonal variations occurred i n the gas composition of sapwood, CH^ was never found i n the gases collected from sapwood. Gas composition of black cottonwood wetwood was quite d i f f e r e n t , both quantitatively and q u a l i t a t i v e l y , from that of the red alder heartwood despite the fact that both tree species were growing i n the same environment. Microaerobic conditions and the pre-sence of CH^ were evident i n the wetwood of many black cottonwoods studied. Both of these features, were absent i n the heartwood of a l l 3 red alders ex-amined . Occurrence of low 0^ high i n the heartwood or wetwood of pop-lars, has been reported previously. Bushong (1907) ..reported 1.24% 0^ from a cottonwood while MacDougal and Working (1933), Chase (1934) and Zeikus and Ward (1974) noted complete absence of O2 , but only during some analyses made, and these conditions did not persist f o r any length of time. The no-table feature here was that the microaerobic conditions continued to exist for long durations (6-12 weeks). Also, the data obtained here are based upon a large number of trees (20) as compared to Bushong's 1 tree, Chase's 1 tree, 3 trees of MacDougal and Working or 2 trees of Zeikus and Ward. Chase (1934) also reported C0~ concentration of over 25% from an eastern 113 Figure 31: Percentage of black cottonwood trees (non-wounded) with microaerobic conditions present i n the wetwood from March u n t i l September, 1974. Figures at the top of each bar represent number of trees examined during the indicated period. 114 100.0 o Q o U -i—i ca o PS w < § w w OS H u.. o w < E-• Ui u OS w a, 90.0 80.0 70.0 60.0 L 50.0 40.0 30.0 20.0 10.0 15 1 15 1 15 1 15 1 15 1 15 1 15 M A M J J A S TIME OF YEAR Figure 31 115 cottonwood; the highest CO^ recorded during my study was 13.1%. CH^ concentrations reported i n the l i t e r a t u r e for poplar species were also much higher than those obtained here. No s p e c i f i c studies were made to determine how gases enter the trees and how they remain confined to the different zones i n such di f f e r e n t pro-portions within, trees . The fact that the gas compositions of wetwood and sapwood are so d i s t i n c t l y d i f f e r e n t , and that wetwood gas composition re-mains unaffected even i f the sapwood i s exposed to atmospheric a i r , and that gases i n the wetwood can be present under considerable pressure i n cottonwoods (Toole 1968, Zeikus and Ward 1974) suggest that wetwood and sapwood are two different systems. Therefore the factors determining t h e i r gas compositions may also be di f f e r e n t . I n i t i a l l y , gases may enter through the roots as suggested by MacDougal and Working (1933) but t h e i r l a t e r com-position might be determined by different factors: The i s o l a t i o n studies reported e a r l i e r (Chapter II) and those of others (Morani and Arru 1958, Zeikus and Ward 1974) suggest the p o s s i b i l i t y that microorganisms play an active role i n determining the gas composition of wetwood. My i s o l a t i o n studies showed that wetwood contains numerous types of microorganisms with different 0^ requirements. During t h i s study, i t was found that the temperature of the gases within trees generally f l u c t u -ates according to the outside temperatures. In the beginning of the grow-ing season when some i s present i n the wetwood and temperatures are r i s i n g , aerobic microorganisms (bacteria and presumably fungi) may show vigorous metabolici-activities, consuming the present i n the wetwood. During t h i s process a slow leakage of 0~ into wetwood may take place because of the difference i n the p a r t i a l pressures. Nevertheless, due to a high rate of metabolism, the amount of would decrease, and facultatively-anaerobic and microaerophilic organisms may become active and u t i l i z e most of the remaining O^ , thus creating microaerobic to anaerobic conditions. Occurrence of such a phenomenon i s quite conceivable; i n microbiological methods, acti v e l y growing cultures of aerobes are often used to remove 0^ from incubator a i r i n order to obtain growth of anaerobic microorganisms ( W i l l i s 1969). As i s a normal end product of aerobic r e s p i r a t i o n , i t s concentration would simultaneously increase i n the wetwood during the grow-ing season. In the near-absence of 0^ and presence of high CC^, anaerobes may begin physiological a c t i v i t i e s that are essential f o r t h e i r growth.and m u l t i p l i c a t i o n . Some anaerobes (methanogenic bacteria) may also produce CH^ i f free and more were available i n the wetwood. Therefore, as long as the temperatures are high enough during the summer, the developed microaerobic or anaerobic conditions would be maintained. In th i s study, i t was found that microaerobic conditions persisted for a minimum of 6 weeks i n most trees during the warm temperatures of summer. As the temperatures drop i n winter, the a c t i v i t y of a l l microorganisms w i l l decrease considerably. Consequently, because of differences i n the p a r t i a l pressures of gases and reduced a c t i v i t y (or possibly none) of microorganisms, 0^ m a v diffuse from outside to inside and C O 2 may diffuse out i n winter. Thus, the slow l a t e r a l movement of gases may exceed the gas consumption within wetwood and bring about the change i n the gas composition. Entrance of gases and the maintenance of t h e i r composition i n sapwood may be due to the exchange of gases through l e n t i c e l s and then cambium and/or 117 through the sapstream. The studies show that the gas composition of sap-wood i s usually s i m i l a r to that of the outside a i r . Also, during the l i f e of a tree, sapwood i s more vulnerable to wounds involving exposure to at-mosphere than wetwood which may contribute to the fact that sapwood gases are normally s i m i l a r to atmospheric a i r . Isolation studies showed that a very small number of microorganisms i s usually associated with sapwood of mature trees, and i n young cottonwoods, none could be detected. Presumably the microorganisms do not play any role i n determining the gas composition of sapwood. In the wounded trees, the gas composition of wetwood was different from that of non-wounded trees. Gases drawn from the wetwood of a wounded tree (No. 2) were lower i n CO^ and higher i n 0^ than those of a non-wounded tree (Figure 32). The trees that were wounded during the study showed marked changes i n t h e i r gas compositions after the wetwood was exposed. There was no further accumulation of CO^ i n the wetwood after wounding and 0^ showed a sharp increase i n concentration as anticipated. However, once the wounds were plugged, the trend i n the gas composition was reversed. The 0^ contents began decreasing, CC^ started increasing and CH^ which had disappeared after pounding, re-appeared. These fluctuations can be explained i f involvement of microorganisms i s assumed i n the gas composition. Once the wetwood was exposed, 0^ diffused from outside to inside making the tree more or less aerobic. This change would make i n t e r i o r conditions favorable to aerobes and unfavourable to anaerobes. Methanogenic bacteria being anaerobic would then survive i n a resting phase and therefore cease producing CH^. Aerobes under these suitable conditions, would show vigorous metabolic a c t i v i t i e s 118 Figure 32: Comparison of 0^ and C0 2 concentrations (monthly averages) i n the wetwood of a non-wounded black cottonwood (Tree 10) and a wounded black cottonwood (Tree 2). 120' u n t i l the conditions become unsuitable again when the wound i s covered completely. When the 0^ supply becomes limited because of metabolic pro-cesses of the aerobes, the microaerophilic and eventually anaerobic micro-organisms may become functional. Methanogenic bacteria may then produce CH^ and generally the o r i g i n a l gas composition would be resumed. Changes noted i n the concentrations of CH^ and 0^in Tree 21 strongly suggest such a phenomenon. A l l of the trees that were sampled during the gas composition studies had many branch stubes. The proportions i n which gases e x i s t i n the wetwood suggest that these branch stubs do not act as passages for the exchange of gases. During the present study, dead branches were broken o f f from 2 trees and attempts were made to extract gases through the branch stubs. These attempts were unsuccessful despite the use of an e l e c t r i c a l l y powered suc-t i o n pump. Up to 22 i n (55.0 cm) of suction developed i n both cases. Ap-parently an impermeable layer forms around the knot upon death of the branch (Mullickj personal communication), which may prevent wetwood gases from being extracted. The exact location of t h i s b a r r i e r or the mechanism of i t s formation are not known at the present time. I f the b a r r i e r i s host induced, wetwood cannot be assumed to play any role as i t does not contain any l i v -ing c e l l s . More anatomical studies are needed before this question of cause of apparent absence of gas exchange through branch stubs can be resolved. On the other hand, i f l i v i n g branches containing wetwood (Chapter I) are cut then presumably the gas composition of the trunk may be affected to some extent. However, most branches contain wetwood at the time of death. I f there are no l i v i n g host c e l l s i n the wetwood, i t i s d i f f i c u l t to imagine how the b a r r i e r forms. 121 The physiological significance of gases, i n the proportions they e x i s t , i s s t i l l very unclear although they may be a factor of some im-portance i n the growth of wood-destroying fungi. Apparently most f i l a -mentous fungi are aerobic ( i . e . they cannot grow i n the absence of O^)• Also, under very low pressures, fungal growth i s reduced considerably The studies reported here showed that i n black cottonwoods, very low levels of 0*2 (<0.05%) were present i n the wetwood for prolonged periods (6-12 weeks). Effects of such low levels on the growth and survival of wood-destroying fungi w i l l be discussed i n Chapters IV and V. 122 CHAPTER IV EFFECTS OF MICROAEROBIC CONDITIONS ON THE DEVELOPMENT OF DECAY INTRODUCTION "Fungi are probably a l l obligate aerobes, that i s , they are unable to grow i n the complete absence of oxygen, although the minimum concentration of the gas which w i l l permit s a t i s -factory growth may be very low." (Hawker 1950) "Apparently none of the fungi are obligate anaerobes. Many are s t r i c t l y aerobic, and some are f a c u l t a t i v e l y anaero-b i c . An aerobic organism requires uncombined oxygen, while a facu l t a t i v e anaerobe may use combined oxygen i n addition to free oxygen." ( L i l l y and Barnett 1951). "Fungi are commonly thought as s t r i c t l y aerobic, and t h i s opinion i s b a s i c a l l y correct. However, the quantitative r e l a -tions of growth and oxygen supply vary considerably among d i f -ferent forms." (Cochrane 1958) "Another factor important f o r fungal growth i s oxygen sup-ply. Most fungi are s t r i c t l y aerobic, and i n the complete ab-sence of oxygen growth ceases." (Ingold 1961) These 4 quotations are from Mycology textbooks which describe an im-portant physiological character of fungi. Numerous experiments have shown that most fungi are unable to grow i n the absence of free 0^' A. very limited number of fungi, however, are capable of growth i n the absence of free 0^ (Emerson and Held 1969, Held et_ al_. 1969). Gunner and Alexander (1964), f o r example, observed that Fusarium oxysporum, an alleged obligate aerobe, was able to grow under anaerobic conditions, provided the medium contained yeast extract, manganese oxide, n i t r a t e , s e l e n i t e , or f e r r i c ions. In general, the quantitative relations of filamentous fungi or yeasts and 0 ? supply d i f f e r considerably among different forms. The amount of 0,, 123 needed for optimum growth varies with the species, and for each species, other environmental factors influence the requirements for molecular O^ . Tabak and Cooke (1968) have reviewed much of the relevant l i t e r a t u r e on ®2 requirements of fungi, and therefore, a detailed review w i l l not be attempted here. To interpret the studies on effects of anaerobic conditions or re-duced 0 2 levels on wood-destroying fungi, i t i s necessary to consider i n general the methods used i n these studies. The term anaerobic only implies that i n a given system, there i s no free 0^ present. An atmosphere of pure N 2 would mean absence of free 0^ and, therefore, such a system could be considered anaerobic. On' this p r i n c i p l e , several workers used atmos-pheres of N 2, H 2 or C0 2 to create anaerobic conditions, Frequently, the gases were prepurified to eliminate even small traces of 0 2. On one occasion, the fungal growth was studied 'in vacuum' and thus', i n p r i n c i p l e , under anaerobic conditions. A variety of tanks, jars and incubators were used to establish and maintain these anaerobic conditions. In most cases, contin-uance of anaerobic environment i n the culture medium or i n an incubator was checked q u a l i t a t i v e l y by color indicators such as methylene blue. In some instances, growth of fungi was studied i n absolute terms, either positive or negative, <Bn others, quantitative determinations were made. Among these, a few workers made the growth measurements by weighing the dried mats, of mycelium from the l i q u i d medium and/or by measuring the li n e a r growth on a standard agar medium. Hirayama (1938) determined the a b i l i t y of fungi to respire under anaerobic conditions by using a fermentation appa-ratus. He measured the volume of gas produced, and computed the in t e n s i t y 124 of anaerobic respiration by the volume of CC^ produced by the mycelium of a unit weight. This anaerobic in t e n s i t y was considered as the measure of growth. I t i s noteworthy that i n a l l . growth studies, fungi were allowed to grow i n various a r t i f i c i a l culture media and none of the workers used wood as a medium, even when the investigations were concerned with wood-destroying fungi. Recently, i t has been pointed out that the culture medium used has a major influence on survival and growth of fungi under anaerobic conditions (Tabak and Cooke, 1968). Therefore, results of many of these studies are not.directly comparable. In 1910, Hoffman demonstrated that i n an atmosphere of (i.e.under anaerobic conditions), an act i v e l y growing culture of Merulius lacrymans died i n 4 days. Growth ceased immediately when the fungus was deprived of Q^- He also found that Coniophora cerebella and Pa x i l l u s panuoides did not grow under anaerobic conditions for 20 and 15 days respectively but were able to resume growth when exposed to 02--One of the early comprehensive studies of requirements of wood-decaying fungi was made by Bavendamm (1928). He found that p r a c t i c a l l y a l l of the 32 fungi tested could not only grow i n the presence of minute quanti-ties of ®2> k u t could survive the complete absence of O2 for a considerable length of time. No retardation of growth occurred u n t i l the pressure was reduced to 10 cm of Hg (approximately 13% O2) and only below this he found that growth of fungi l i k e Mefulius lacrymans and Coriophora cerebella was re-duced considerably. Scheffer and Livingston (1937) studied lin e a r growth and C0 2 production i n the fungus Polystictus (Pblyporus) versicolor (Davidson et_ al_. 1942) at 125 various temperatures (17.5 to 33,5 C) and 0 2 pressures (0.0 to 745 mm). The minimal 0 2 pressure for mycelial growth on malt agar was between 1.5 to 10 mm at a l l temperatures tested. They reported no growth when the fungus was exposed to <1.5 mm 0 2 pressure. Growth was most rapid at 29.5 C with 0 2 pressure varying from 16 to 745 mm. Higher concentrations of 0 2 usually lead to an increase i n the rate of evolution of C0 2, and conse-quently, i n the u t i l i z a t i o n of the substrate. They concluded that the rate of decay of wood would be i n proportion to the 0 2 concentration. Hirayama (1938) determined respiration of 10 wood-destroying fungi under anaerobic conditions and found that the anaerobic respiration inten-s i t y shown by 5 brown-rot fungi was generally greater than that shown by the 5 white-rot fungi. In t h e i r study of alcoholic fermentation and dehydrogenation of alco-hols by certain wood-destroying fungi, Nord and S c i a r i n i (1946) showed that Merulius and Fomes arinosus were able to ferment glucose, raffinose, and xylose under anaerobic conditions to ethanol, this fermentation being f o l -lowed by dehydrogenation to acetic acid and acetaldehyde. Thacker and Good (1952) exposed & wood-destroying fungi to 0 2 concen-tr a t i o n s , ranging from 0.8 to 35.0%. Growth of a l l fungi was "good" at both 0.8 and 35.0% of 0 2;growth tended to increase s l i g h t l y with increase of 0 2 up to 10.0%. Because the authors had found 0 2 concentrations ranging from 0.8 to 19.2% i n the heartwood of several maples, they concluded that aeration i s probably not an important factor i n the development of decay. Growth response of Fomes annosus to low 0 2 and high C0 2 was investigated by Gundersen (1961), Inoculated p e t r i plates were incubated at room 126 temperature i n Brewer's jars containing a i r , N 2, H 2 and C0 2. In the hydro-gen j a r s , a l l free 0 2 and 0 2 dissolved i n the agar was removed by c a t a l y t i c reaction with the H 2, the jars thus representing a s t r i c t anaerobic environ-ment. No growth was observed i n these j a r s . In the N 2 j a r s , the 0^ was removed by 3 consecutive evacuations to 40 mm mercury and by slow r e f i l l i n g s with N 2 which had been passed through alkaline pyrogallol (theoretically the jars contained 0.02 mm 0 2 ) . These bottles represented a microaerophilic (microaerobic) environment•in which ,the fungus grew unaffected. In jars with C0 2 ( f i l l e d in-the same way as with N 2, except that the washing i n pyrogal-l o l was omitted) and representing a microaerophilic habitat, with 760 mm C0 2, no growth occurred, whereas 23 mm of the gas i n combination with a i r or N 2 accelerated growth about 50 percent. I t was concluded that t h i s fungus i s able to grow equally well under aerobic or microaerophilic conditions, but not anaerobically. Jensen (1967b) found that i n the case of 4 wood-rotting fungi, dry weight production decreased with a decrease in' 0 2 concentration below 21.0% and with an increase i n C0 2 concentration from zero percent. No measurable growth was observed at 0.0% 0 2 > Since reduced levels of 0 2 and high levels of C0 2 can occur i n tree trunks, he suggested that these gases may influence the a c t i v i t y of wood-destroying organisms i n l i v i n g trees. This b r i e f l i t e r a t u r e survey indicates that wood-destroying fungi are unable to grow i n the absence of 0 2, although sometimes growth can be de-tected at very low 0 2 l e v e l s . The quantitative response of fungi to low 0 2 levels was variable and largely depended on the environmental conditions under which the test was done. As pointed out e a r l i e r , a l l growth studies were done on n u t r i t i o n a l media other than wood. I n i t i a l l y , I established 127 that on malt agar, the linea r growth of 2 wood-destroying fungi (Polyporus  delectans and Ganoderma applanatum) was reduced considerably at low tensions. Nothing i s known, however, about the relationship between growth of wood-destroying fungi on a r t i f i c i a l culture media and t h e i r a b i l i t y to grow on wood at very low lev e l s . This relationship i s p a r t i c u l a r l y i n -teresting because of the very low levels found i n the wetwood of black cottonwood during the summer. Therefore, I made a study to determine the a b i l i t y of wood-destroying fungi to cause a weight loss i n wood under micro-aerobic conditions. The experimental set-up also made i t possible to study effects of pH, moisture content or microbial population of wood on the ex-tent of decay. MATERIALS AND METHODS Figure 33 shows the disposable Anaerobic System (Gas Pak^-BBL) that was used to create microaerobic conditions. The system consists of a c a r r i e r assembly, an anaerobic container, a gas generator envelope, an anaerobic indicator and a p l a s t i c clamp. The c a r r i e r i s designed to accommodate the cultures, gas generator envelope and an anaerobic indicator. A f i l l e d cata-l y s t chamber i s fixed i n one wall of the c a r r i e r . The anaerobic container i s a f l e x i b l e disposable p l a s t i c bag with an open end provided with a spongy s t r i p across the width of the bag. The bag i t s e l f i s composed of t r i p l e laminated p l a s t i c films. Gas generator envelope produces and CO2 after adding 10 ml of water and the H 2 produced combines with atmospheric 0„ i n the bag, i n the presence of the platinum catalyst to form water. The 128 Figure 33: Disposable Anaerobic System. Yellow box i s the c a r r i e r assembly. A. Side view, Note the position of the methylene blue indicator (now c o l o r l e s s ) . B. Front view. A 3 Figure 33 130 end result i s that a microaerobic, .; atmosphere, containing 0.08% 0 2 (range: 0.06 to 0.1%) and 6.0% C0 2 (range: 4.5 to 7.0%), i s produced i n the bag. This 0 2 concentration-is s l i g h t l y higher than that found i n most black cottonwoods during the summer months. The procedure for use of the system i s as follows, 1) Set up the c a r r i e r by closing and locking the bottom f l a p s , then place the cultures i n the c a r r i e r and close the 2 top flaps. 2) Place the gas generator envelope and anaerobic i n -dicator i n the designed s l o t s i n the c a r r i e r , and place t h i s complete c a r r i e r i n the anaerobic con-tainer ( p l a s t i c bag) , 3) Cut o f f the marked corner (of the envelope) and d i s -pense 10 ml of water i n the envelope. 4) Immediately f o l d over the p l a s t i c bag i n such a way that maximum amount of a i r would escape the bag, then f o l d the top edge of the bag over the sponge s t r i p and f o l d i t again. 5) Beginning at one end, place the clamp over the f o l d -ed edge and s l i d e i t along u n t i l the entire top edge i s clamped. The system i s now i n operation. Polyporus delectans and Ganoderma applanatum, both white-rot fungi were used as test fungi. The former i s the most common wood-destroying fungus of l i v i n g black cottonwood i n B.C., while the l a t t e r i s common but v i r t u a l l y r e s t r i c t e d to dead cottonwood (Thomas and Podmore 1953). For l i n e a r growth studies, the fungi were grown on 2% malt agar plates for 7 days at 23 C, and these act i v e l y growing cultures served as the ino-culum source. The inoculum (4 mm discs of mycelium and agar) was placed i n a p e t r i dish containing malt agar and the growth of each fungus was studied under aerobic and microaerobic conditions. Each test was made i n t r i p l i c a t e . The cultures were incubated at 23 C for 16 days at which time the fungi 131 growing under aerobic conditions had covered the plates. The line a r growth of fungi '(under microaerobic conditions) was recorded by measuring the colony diameters (excluding colony diameters of the inoculum). After 16 days of incubation under microaerobic conditons, the fungi were allowed to grow under aerobic conditions to see i f they had survived. A modification of a soil-block test (ASTM D2017-71) was used to deter-mine the extent of decay of black cottonwood under microaerobic and aerobic conditions. Sapwood and wetwood blocks (0.75 i n [2.0 cm] cubes) from 4 black cottonwood trees (U.B.C. Research Forest) were cut and s t e r i l i z e d by either gamma radiation (Smith and Sharman 1971) at 2.5 x 10^ rads (complete s t e r i l i z a t i o n ) or by flame s t e r i l i z a t i o n (surface s t e r i l i z a t i o n ) . The mois-ture content of each test block was assumed to be equal to that measured from an end-matched adjacent block. The test was made i n 16 oz bottles which con-tained approximately 80.0 g of s o i l , with 27.2% moisture holding a b i l i t y by dry weight, and a black cottonwood sapwood feeder s t r i p (2.0 x 2.0 x 0.25 i n [5.0 x 5.0 x 0.6 cm]). After inoculation of the bottles with the required fungi and once the fungi were well established on the feeder s t r i p s , four test blocks, one from each tree, were placed i n each b o t t l e . Disposable Anaerobic System, as described previously, was used to establish microaerobic conditions. The experiment used a r e p l i c a t i o n of 12 blocks for each condition and was done i n 2 parts. In the f i r s t part, h a l f the bottles were subjected to microaerobic conditions while the other h a l f served as atmospheric controls (aerobic). At the end of this period (10 weeks), 2 blocks were removed from 132 each bottle and t h e i r weight loss determined (Part I ) . In the second part, the same bottles with the 2 remaining blocks were exposed to the environment opposite to i t s previous one, i . e . microaerobic to aerobic and aerobic to microaerobic for a further 10 weeks before weight loss assessment (Part I I ) . RESULTS AND DISCUSSION The results of growing fungi on malt agar under aerobic and micro-aerobic conditions are given i n Table VIII. Under microaerobic conditions, line a r growth of Polyporus delectans and Ganoderma applanatum was 4 and 12% respectively of that of controls grown under atmospheric 0 2 levels. Both of these fungi survived 16 days incubation under microaerobic conditions and resumed mycelial growth when returned to a normal atmosphere. These re-sults are generally i n agreement with many previously published r e s u l t s , i n that the growth of wood-destroying fungi i s reduced considerably under low 0 2 tensions. The results of 'soil-block experiment' are given i n Table IX (Parts I and I I ) . They show that there was no s t a t i s t i c a l l y s i g n i f i c a n t weight loss in test blocks exposed to microaerobic conditions for 10 weeks, while weight loss of control aerobic treatments varied from 29.5 to 47.6% (Part I ) . Therefore i t i s probable that under f i e l d conditions during summer, the con-centration of 0 2 found i n the wetwood i s too low to allow any development of decay, at least i n the 2 fungi tested. It should be pointed out, however, that i n t h i s experiment fungi survived the 10 week incubation under micro-aerobic conditions and presumably the same would happen i n the f i e l d . After TABLE VIII: Linear growth of two w6bd:-destrbying f*ungitons2-%smalt''ag'af,' after> 16- days of incubation under aerobic (control) and microaerobic conditions ji:'; Fungus Culture ident i f i -cation number Type of Colony diameters* rot (mm) aerobic microaerobic (control) conditionsc conditions Percentage growth under microaerobic.conditions of that of controls Polyporus delectans  Ganoderma applanation WFPL 84 A' WFPL 32 A $ White 75 75 3 9 4 12 - Average of 3 measurements $ - Cultures were obtained from the Western Forest Products Laboratory, Department of the Environment, Canadian Forestry Service, Vancouver, B.C.t -c;. h-' TABLE IX: Meanj^wei'ghtgl^^ cottonwood 'blocks of sapwood (S) and wetwood (W) under aerobic (A) and microaerobic. (M) conditions Fungus Polyporus delectans Ganoderma applanatum S t e r i l i z a t i o n Flame (surface) Gamma radiation (complete) Flame (surface) Gamma radiation (complete) method Wood zone S W S Wti S j- W I S W Growth Condi-.. -,A M A M A M A M A M A M A M A M tion Part I § 29.5 -0.1 38.7 0.8 47.6 0.3 41.8 -0.5 42.5 0.3 43.2 0.3 43.5 0.3 46.7 -0.1 (2.9)^ r(0.4) (3.9)(0.5) (2.8) (0.3) (3.0) (0.8) (4.0) (0.8) (3.3)(0.7) (2.9)(0.7) (3.1) (0.7) M A M A M A M A M A M A M A M A Part I I * 29.4 5.0 45.5 7.7 51.6 7.8 37.2 9.2 49.3 14.7 45.9 16.9 43.0 15.7 44.8 10.6 (4.6) (2.5) (3.6)(2.1) (4.7)(2.1) (5.6) (2.5) (2.8)(3.5) (1.0)(4.3) (2.7)(3.1) (4.6) (3.0) * Test bottles were incubated at 27 C and 70% r e l a t i v e humidity fi Weight loss after i n i t i a l 10 weeks exposure to the indicated conditions "^Weight loss after further 10 weeks exposure to the indicated conditions following exposure to the conditions w indicated i n Part I. ^Standard error i n brackets 135' the i n i t i a l 10 week aerobic incubation, there was no s i g n i f i c a n t d i f f e r -ence between the weight loss of surface s t e r i l i z e d sapwood and wetwood blocks, nor between surface s t e r i l i z e d and completely s t e r i l i z e d wetwood blocks. It i s apparent, therefore, that the lack of weight loss of a l l treatments under microaerobic conditions i s because of the low 0^ l e v e l s . High pH or moisture content, presence of microorganisms of wetwood have no detectable e f f e c t , nor do there appear to be any toxic chemicals i n the wet-wood, such as found by Hossfeld et_ al_. (1957) i n the wetwood of aspen. Table IX (Parts I and II) also shows that there was no s i g n i f i c a n t additional weight loss during 10 week exposure to microaerobic conditions following 10 weeks of aerobic conditions, while i n the reverse sequence, a s i g n i f i c a n t weight loss did occur during the second period. These results indicate that i f microaerobic conditions are created i n a tree after-the establishment of fungi, further advancement of decay would be arrested. In some trees, a phenomenon of i n a c t i v a t i o n of decay following healing of the inf e c t i o n court has been observed (Childs and Wright 1956, Toole 1965). For example, Toole observed that, decay did not advance s i g n i f i c a n t l y a f ter 2 years i n willow oak and nuttal oak (Quercus n u t t a l l i i Palm) trees inoculated with pure cultures of Poria ambigua, Pblypbrus f i s s i l i s and Polyporus hispidus. He also noted that most inoculation wounds were healed after 2 years. This lack of "advance" i n decay could have been due to the re-establishment of microaerobic conditions created after the wounds were healed. The results obtained here also indicate that wood-destroying fungi can resume t h e i r ac-t i v i t i e s when the 0 2 concentration increases, as i t does i n black cottonwood during the winter months (Chapter I I I ) , However, i t i s generally understood 136 that low temperatures of winter prevent or retard growth of most wood-destroying fungi (e.g. Wagener and Davidson 1954), and therefore, s i m i l a r i n h i b i t i o n of wood-destroying fungi may be observed i n black cottonwood despite the favorable 0^ concentrations present i n winter; It i s noteworthy that the weight loss of blocks exposed to microaero-b i c conditions followed by aerobic conditions was much lower (average 10.9%) than that of blocks exposed to aerobic conditions only (average 41.7%). Perhaps toxic metabolic by-products accumulate during the microaerobic conditions and t h e i r elimination i s required before normal growth resumes. Al t e r n a t i v e l y , i t i s possible that during the microaerobic period, l i v i n g mycelia of the fungus are transformed into special resting stages, thus substantially reducing t h e i r inoculum po t e n t i a l . A sim i l a r phenomenon might reduce the a c t i v i t y of fungi i n trees during winter months following microaerobic summer conditions. I expected that since Polypbrus delectans occurs i n l i v i n g trees, and Ganoderma applanatum i s normally r e s t r i c t e d to dead trees (Thomas and Podmore 1953), the former might be more tolerant to microaerobic conditions. But with both fungi, there was no s i g n i f i c a n t weight loss after 10 weeks under microaerobic conditions, therefore indicating no pre f e r e n t i a l a b i l i t y for Polyporus delectans to tolerate such conditions i n the laboratory. In the sequence of 10 weeks microaerobic followed by 10 weeks aerobic, however, the weight loss caused by Polyporus delectans was 7.4% (average) while that of Ganoderma applanatum was 14.5% (average), suggesting a more rapid recovery from microaerobic conditions by the l a t t e r . In the study of Thomas and 137 Podmore (1953), the sampled trees were old (60 years and above), and therefore, vulnerable to wounding'. As shown e a r l i e r , any wound or open-ing into the wetwood would admit 0 2 (Chapter III) and presumably negate decay i n h i b i t i o n (branch stubs do not appear to function as such openings). This may explain the d i s t r i b u t i o n of these fungi i n the f i e l d as re.ported by Thomas and Podmore. In summary, the microaerobic conditions immediately prevented weight loss i n wood and therefore wood decay, whereas on malt agar the lin e a r growth of fungi, although d r a s t i c a l l y reduced, was not completely prevented. The implication of this finding on many previously published results i s an obvious one. A l l the e a r l i e r c u l t u r a l work involving wood-destroying fungi was done on a r t i f i c i a l media (as opposed to wood) and t h e i r conclusions were that a very small amount of 0 2 i s adequate for the growth of wood-destroying fungi (e.g. Scheffer and Livingston 1937, Gundersen 1961). Under the f i e l d conditions s i m i l a r behavior of wood-destroying fungi was predicted by Thacker and Good (1952) and Gundersen (1961). Thacker and Good stated, "While a l l the effects of aeration on decay are s t i l l not understood, the data presented suggest that the idea of i n h i b i t i o n of decay fungi by 'poor' aeration cannot at present be entertained, aeration i n the tree appearing nearly optimal for decay,'! And Gundersen stated, " I t could be concluded that F. annosus i s a fungus able to grow equally well under aerobic as under microaerophilie condi-tions , but nO'fc'anaerobically,.... These observations seem to agree well with what was to be expected from a fungus p a r a s i t i z i n g the i n t e r i o r of roots and trunks." The results presented here strongly suggest limi t a t i o n s to the gen-eral a p p l i c a b i l i t y of the conclusions by Thacker and Good, and by Gundersen. 138 CHAPTER V SURVIVAL OF WOOD-DESTROYING FUNGI UNDER  ANAEROBIC CONDITIONS INTRODUCTION Physiology of wood-destroying fungi has always been a fascinating subject, and a r e l a t i v e l y large amount of l i t e r a t u r e i s available describing studies on growth of wood-destroying fungi on various n u t r i t i o n a l media, th e i r enzyme systems, vitamin requirements, and f i n a l l y t h e i r growth under anaerobic\or microaerobic conditions (Chapter IV). However, very few re-searchers have studied the survival of wood-destroying fungi under s t r i c t anaerobic conditions. This i s p a r t i c u l a r l y i n t e r e s t i n g , as i n a few tree species, trunks are known to contain low 0 2 (and sometimes none) and high C0 2 concentrations, at least during certain times of the year las t i n g for certain durations (Chapter I I I ) . Hoffman (1910) found that a vigorously growing culture of Merulius  lacrymans did not survive when exposed to anaerobic conditions ( i . e . i n an atmosphere of H2) for 4 days. Also, Coniophora cerebella and Pa x i l l u s panuoides did not grow under anaerobic conditions for 20 and 15 days re-spectively, but were able to continue growth i n the presence of 0 2 > Later, Bavendamm (1928) confirmed Hoffman's results when he observed death of Merulius lacrymans i n 2 to 3 days of exposure to anaerobic conditions. He also reported that i n the complete absence of 0 2, Stereum frustulosum was "undamaged" after 10 days (and presumably grew when exposed to aerobic 139 conditions). From his extensive work involving 32 species of fungi, Bavendamm concluded that the ty p i c a l saprophytes were affected most under the anaerobic conditions whereas the heart rots were the most resistant. The survival of wood-destroying fungi in;,-wood, which i s submerged i n water, was examined by Schmitz and Kaufert (1938). They immersed the wood blocks i n water and evacuated a i r present i n wood which was replaced by water, and by the same method, they removed the 0 2 dissolved i n the water and created anaerobic conditions. Trametes s e r i a l i s , Lentinus lepideus and Lenzites trabea growing i n Norway pine sapwood blocks survived 38 weeks of immersion, but Polyporus anceps under the same conditions was k i l l e d i n about 6 weeks. Gundersen (1961) observed that 9 strains of Fomes annbsus resumed growth i n aerobic conditions after the cultures had been exposed to anaerobic con-ditions for 6 days. He used 2% malt agar as n u t r i t i o n a l medium, and created anaerobic conditions through a c a t a l y t i c reaction with H 2. Boutelje and Kiessling (1964) isolated several fungi from oak timber from 2 ships which sank i n the B a l t i c at the beginning of the 17th century. One of the i s o l a t e s , a species of Phoma, showed d i s t i n c t wood-decaying a b i l i -t y , causing a kind of rot resembling soft rot. Phoma and Phialophora are often isol a t e d from wood submerged i n water (Duncan 1960, Seipmann and Johnson 1960) , and these genera contain some species with wood-decaying ca-pacity (Duncan 1960), Therefore, these fungi with wood-decaying a b i l i t y are found i n a medium ( i . e . water), where 0 2 supply i s very minimal. In my previous study, 0 2 concentrations of 0.05% or less were found to be present i n the wetwood of black cottonwood during the summer (Chapter I I I ) , 140 and these microaerobic conditions existed for up to 12 weeks i n some trees. My e a r l i e r study (Chapter IV) also showed that Polyporus delectans and Ganoderma applanatum survived 10 weeks exposure to microaerobic conditions, although during that period, they f a i l e d to cause s i g n i f i c a n t weight loss i n wood. The 0^ concentration i n the Anaerobic Systems was 0.08% (average) and therefore above those found i n the wetwood of black cottonwoods. I t was not known i f wood-destroying fungi would be able to survive under atmospheres of <0,05% 0^. As the lowest le v e l of 0 2 measurement i n the 0^ analyzer was 0.05%, I could not have known how low these 0 2 concentrations were i n trees. With the assumption that these 0^ levels may have been.low enough to be termed "anaerobic", I made a study to see i f actively growing cultures of wood-destroying fungi growing on wood can survive a long exposure to "anaero-b i c " conditions. Wetwood blocks were inoculated with desired wood-destroying fungi and incubated under aerobic conditions; the blocks were then exposed to anaerobic conditions for 13 weeks and f i n a l l y a check was made to see i f the fungi had survived such an exposure by allowing them to grow under aerobic conditions. MATERIALS AND METHODS Eight wood-destroying fungi, including Polyporus delectans and Ganoderma  applanatum, were used i n this experiment (Table X). The intention was.to select an equal number of white-rot and brown-rot fungi, and within each class, some var i a t i o n wasaalso sought with respect to t h e i r a b i l i t y to pro-duce chlamydospores. Here the objective was to see i f survival of fungi un-der anaerobic conditions i s related to the kinds of substrates fungi u t i l i z e or to t h e i r a b i l i t y to produce chlamydospores or both. Unfortunately, one » TABLE X: Characteristics of 8 wood-destroyingdfun>gi^ andytheirrsurvival response to -l^cweekseofuirieub conditions . Fungus Culture Type of Ide n t i f i c a t i o n rot Number Chlamydospore Number of producing a b i l i t y blocks exposed Culture condition (after incubation) Percentage of Survival Poria monticola Lenzites trabea WFPL 120F brown Positive-numerous Chlamydospores WFPL 47D " " 11 12 A l l l i v i n g A l l l i v i n g 100.0 100.0 Polyporus  palustris Fomes annosus Polyporus delectans Polyporus versicolor Polyporus hirsutus WFPL 227A WFPL 19D WFPL 84A Ganoderma WFPL 32A applanatum WFPL 105A WFPL 89B white Negative P o s i t i v e - f a i r to numerous chlamydo-spores Negative 12 13 12 11 12 A l l l i v i n g A l l dead A l l dead A l l dead 5- l i v i n g 6- dead A l l l i v i n g 100.0 0.0 0.0 0.0 45.4 100.0 ^Cultures were obtained from the Western Forest Products Laboratory, Department of the Environment, Canadian Forestry Service, Vancouver, B.C., 142 wh i t e - r o t fungus was mistakenly taken as a brown-rot fungus, and conse-quently, 1 i n i t i a t e d the experiment w i t h 3 brown-rot and 5 wh i t e - r o t f u n g i . A l l brown-rot f u n g i and one wh i t e - r o t fungus, produced numerous chlamydo-spores while the remaining 4 w h i t e - r o t fungi lacked the a b i l i t y to produce chlamydospores. Thus, I f a i l e d to o b t a i n the d e s i r a b l e , and o r i g i n a l l y i n -tended, v a r i a t i o n among the wood-destroying f u n g i w i t h respect to t h e i r sub-s t r a t e s p e c i f i c i t y and a b i l i t y to produce chlamydospores. A non-wounded black cottonwood t r e e , l o c a t e d on the U.B.C. Research F o r e s t , was f e l l e d and the re q u i r e d number o f t e s t blocks (1 cm cubes) were cut from the wetwood zone. S t e r i l e b l o c k s (gamma r a d i a t i o n ) were i n o c u l a t e d according to the standard s o i l - b l o c k t e s t procedure (ASTM: D2017-71) and were incubated f o r 8 weeks under aerobic c o n d i t i o n s . A f t e r v t h i s p e r i o d when the growth of each fungus was evident, the blocks were a s e p t i c a l l y p l a c e d i n a s t e r i l e p e t r i d i s h (100 x 25 mm) c o n t a i n i n g a s t e r i l e f i l t e r paper (Whatman No. 3). Approximately 20 ml o f s t e r i l e d i s t i l l e d water was d i s -pensed i n t o each p l a t e and these p e t r i c p l a t e s w i t h t e s t b l o c k s were p l a c e d i n an Anaerobic incubator (National Appliance Co., USA). Two methylene blue i n d i c a t o r s were clamped from i n s i d e on the glass door f o r easy i n s p e c t i o n o f incubator c o n d i t i o n s at a l l times. J u s t before c l o s i n g the inc u b a t o r door, the l i d s of the p e t r i p l a t e s were r a i s e d s l i g h t l y so t h a t the blocks would be i n d i r e c t contact w i t h the environment i n the incubator. A i r was then w i t h -drawn from the incubator with a vacuum pump, and was immediately rep l a c e d by L-grade N 2 gas. The procedure o f evacuation* and replacement by N 2 was According to Zycha (1938), and H i n t i k k a and Korhonen (1970) , a b r i e f vacuum has no d e t e c t a b l e e f f e c t on growth r a t e o f fu n g i and, t h e r e f o r e , presumably on the s u r v i v a l o f f u n g i . 143 carried out 7 consecutive times which resulted in an atmosphere of <0.002% 0 2 in the incubator. Blocks were exposed to these anaerobic con-ditions for 13 weeks at 23 C (+2 C).' At the end of this period, blocks were removed from the incubator. Examination of fil t e r papers revealed that, although there was a certain loss of moisture from the fi l t e r papers, all of them were s t i l l moist at the end of the 13 week period. Each re-moved block was then placed aseptically on a 2% malt agar plate. These plates with blocks were examined daily for the first 3 weeks, and after that,.weekly for a total period of 10 weeks. Any new growth on the agar medium was considered as survival of the fungus. RESULTS AND DISCUSSION Table X shows that the ability to survive under anaerobic environment varied with fungi; 4 showed 100% survival, one showed 45.4% while 3 showed 0.0% survival. AH 3 brown-rot fungi, Lenzites trabea Pers. ex Fries, Polyporus palustris Berk, and Curt, and Poria monticola Murr. endured 13 weeks exposure to anaerobic conditions. These fungi not only survived the exposure but the new growth was evident after 2 days of their return to aerobic conditions. Among the white-rot fungi, only one (Polyporus hirsutus Wulf. ex Fries) survived and commenced growth within 3 days of incubation under aerobic conditions. Polyporus versicolor L. ex. Fries showed 45.4% survival, with new growth occurring within 11 days. The remaining 3 white-rot fungi, Polyporus delectans, Ganoderma applanatum and Fomes annosus (Fries) Cooke all died under anaerobic conditions. 144 Among the fungi which showed 100% s u r v i v a l , Lenzites trabea, Polyporus . pal lis t r i s and Poria monticola produce chlamydospores, whereas Polyporus  hirsUtus culture used i n this study does not produce chlamydospores. Polyporus delectans, Ganoderma applanatum and Fomes annosus, a l l white rot fungi which could not tolerate exposure to anaerobic conditions, normally do not produce chlamydospores. In the case of Polyporus versicolor which showed 45.4% s u r v i v a l , chlamydospores are produced i n f a i r number. These results suggest some trends regarding survival of the fungi and the types of substrates they u t i l i z e and/or t h e i r a b i l i t i e s to produce chlamydospores. However, these relationships w i l l have to be accepted with some reservations. Generally, brown-rot fungi were most resistant to the anaerobic conditions while white-rot fungi were most sensitive. ' The 3 brown-rot fungi produce numerous chlamydospores and perhaps have a better chance of survival under the unfavorable conditions. Among the white-rot fungi, 3 which did not survive are normally incapable of producing : chlamydospores, Polyporus versicolor which produces a f a i r number of chlamydospores under normal conditions, showed about 50% su r v i v a l . I t i s possible that for t h i s fungus, the anaerobic incubation period of 13 weeks was a c r i t i c a l one and that with a s l i g h t l y shorter exposure, the fungus could have shown 100% sur-v i v a l while with a longer exposure, i t might have shown 0.0% su r v i v a l . Polyporus hirsutus, a white-rot fungus, survived the anaerobic exposure of 13 weeks and normally the fungus i s unable to produce chlamydospores. I t i s possible that the survival of Polyporus hirsutus depends on factors other than those considered here. There are 2 reports available for comparison i n which other authors had worked with 2 wood-destroying fungi included i n my study. Schmitz and 145 Kaufert (1938) found that Lenzites trabea survived 38 weeks of anaerobic exposure when inoculated blocks were immersed i n water. Although a d i f -ferent method was used to create anaerobic conditions i n my study, and the fungus was exposed to anaerobic environment for only 13 weeks, the response of Lenzites trabea was e s s e n t i a l l y the same. Gundersen (1961) ob-served that Fomes anribsus was able to grow i n aerobic conditions after i t was incubated under anaerobic conditions for 6 days. In my study, exposure to anaerobic conditions resulted i n death of a l l Fomes annbsus cultures. This difference i n observed response i s e a s i l y attributable to the d i f f e r -ent methods used. In my study, the fungus was exposed to anaerobic condi-tions for 13 weeks and, therefore, i t i s possible that t h i s much longer ex-posure k i l l e d the fungus. Also i n Gundersen's study, the fungus was grown on malt agar whereas i n my study, i t was allowed to grow on wood. The major d i f f i c u l t y i n r e l a t i n g the work of different researchers and drawing general conclusions from i t , , i s that the different investigators use different methods. This can be elaborated by c i t i n g a study made by Newcombe (1960) on growth of a s o i l fungus under the anaerobic conditions created by different methods. She found that i n an atmosphere of N 2 or i n a i r minus 0 2, the formation of chlamydospores i n Fusarium oxysporum f. cubense was delayed, but eventually they were produced i n large numbers. Behavior of the fungus under wateroor i n C0 2 was very d i f f e r e n t . Here, only occasional chlamydospores were produced, whereas, the conidia were produced i n large numbers. Conidia, however, are not known to be very long-lived i n s o i l . She therefore concluded that the production of chlamydospores which takes place i n the absence of 0 2 i s s i g n i f i c a n t i n respect of survival of the fungus i n s o i l . Implication of Newcombe's study i s obvious, although no such observa-146 t i o n has yet been made on any of the wood-destroying fungi. Conclusions based on results obtained i n survival studies, done i n the presence of CO^ and water (anaerobic conditions), may vary from those when the studies were done i n an atmosphere of N 2 or i n a i r minus O^ . K Hirayama (1938) studied respiration of 10 wood-destroying fungi under anaerobic conditions. A l l fungi survived and brown-rot fungi exhibited greater metabolic a c t i v i t y than the white-rot fungi. However, the author did not give details of his method concerning how he created and maintained the anaerobic conditions, which could be very c r u c i a l i n interpreting the r e s u l t s . For his study, Hirayama used 5 brown-rot and 5 white-rot fungi. Again, he did not describe the c u l t u r a l characteristics of the fungi, i n p a r t i c u l a r t h e i r a b i l i t i e s to produce chlamydospores. Among the brown-rot fungi that he used, Polyporus sulphureus i s known to produce chlamydospores, Polyporus betulinus lacks chlamydospores and i n the case of Polyporus  s c h w e i r i i t z i i , the chlamydospore production varies from "rare" to "numerous" (Nobles 1948). Despite this v a r i a t i o n , he found that a l l brown-rot fungi showed higher capacity for anaerobic respiration. Therefore, i t could be inferred from Hirayama*s study that the kinds of organic substances that a fungus attacks and the enzymes i t produces for substrate breakdown determines whether the fungus i s going to survive under anaerobic conditions, rather than the chlamydospore producing a b i l i t y of the fungus. Hepting (1941) investigated decay that originates from basal f i r e wounds i n oaks and then developed a " s t a t i s t i c a l mechanism" to predict the c u l l . He noted that much of the variation i n amount of c u l l among trees with wounds of similar sizes and ages was due to the different fungi that became established. In some cases, especially behind small wounds, the sap rot 147 fungi progressed a short distance i n the heartwood and then there was no further decay. He stated, "Apparently chance has much to do with which heart rot fungi become established behind any given wound i n oaks." A l -though no s p e c i f i c comparisons can be made, the results of the present study and studies made by others suggest a p o s s i b i l i t y that what Hepting termed as a "chance" may actually be related to the variation i n a b i l i t i e s of fungi to survive under anaerobic conditions, created after the wounds were healed. The rate of wound healing and i t s effectiveness i n creating anaerobic con-ditions might also be related to the different behavior of different fungi. In summary, the survival or death of wood-destroying fungi under anaerobic conditions might depend upon t h e i r s p e c i f i c i t y i n attacking sub-strates, white-rot fungi being capable of attacking both cellulose and l i g n i n and brown-rot fungi mainly attacking c e l l u l o s e . Such a relationship would hold true since i t i s known that breakdown of l i g n i n involves oxidation, whereas cellulose can be broken down anaerobically with end-products such as alcohols, o x a l i c acid, etc. The results reported e a r l i e r (Chapter IV) suggested t h a t , s i f microaerobic conditions are created i n a tree after the establishment of fungi, further advancement of decay would be prevented. This study indicates that i f the conditions i n the wetwood of black cotton-wood are t r u l y anaerobic, some wood-destroying fungi ( p a r t i c u l a r l y white-rot fungi) may die whereas others ( p a r t i c u l a r l y brown-rot fungi) may survive and resume growth when CL becomes available again. 148 CHAPTER VI  SIGNIFICANCE OF WETWOOD IN BLACK  COTTONWOOD (SUMMARY AND CONCLUSIONS) The central theme of t h i s thesis was the demonstration that wetwood i n black cottonwood i s a phenomenon be n e f i c i a l to the tree, providing pro-tection against decay. The supposition that the microaerobic conditions found i n the wetwood prevent the development of decay, has been amply sup-ported by the evidence presented. Contrary to the o r i g i n a l concept, however, microaerobic conditions do not persist i n the trees a l l through the year, but occur only during the summer months, and therefore, the phenomenon of decay resistance would only be operative during that period. Nevertheless, the phenomenon i s s t i l l a very valuable one as i n most tree species of the Temperate Zone, almost a l l of the decay occurs during the warm summer months. During the winter, low temperatures are known to l i m i t the rate of progress of wood-destroying fungi (Wagener and Davidson 1954). Therefore, barring frequent i n j u r i e s , microaerobic conditions of the summer and low temperatures of the winter may retard the development of decay i n black cottonwood. A large number of microorganisms wasa found i n the wetwood of black cottonwood. Attempts to isol a t e wood-destroying fungi from the wetwood f a i l e d . The interesting feature of the i s o l a t i o n studies was that some of the microorganisms were aerobic i . e . requiring 0^ f° r growth, some were fa c u l -t a t i v e l y anaerobic i . e . able to grow i n the presence of 0^ but not requiring i t , whereas some were obligately anaerobic i . e . requiring absence of 0 ? for \ 149 growth. The fact that these microorganisms successfully inhabit the wet-wood column despite the variation i n t h e i r requirements suggests that they l i v e i n the wetwood as a stable community and/or are able to adapt well to the occasional unfavourable conditions. Although no s p e c i f i c experiments were done to show that microorganisms actually induce microaerobic conditions i n the wetwood, circumstantial e v i -dence strongly suggests this mechanism. Aerobic microorganisms would have to consume present i n the wetwood i n order to carry out the essential metabolic processes. Due to t h e i r respiratory a c t i v i t y , concentration i n the wetwood would decrease and at the same time, concentration would increase. The microbial a c t i v i t y would be at maximum when the temperatures are high during the summer. Therefore, one would expect 0^ concentration i n the wetwood to be lowest, and CC>2 concentration to be highest, during the summer. The gas composition studies revealed exactly that; very low propor-tions of O2 (<0.1% or microaerobic conditions) and high proportions of CO^ were observed i n the wetwood during the warm summer months. Later, the wounding experiments showed that the exposure of wetwood causes changes i n i t s gas composition as one would expect; increases, CO^ decreases and CH^, i f present, decreases or disappears. However, after plugging the wound, the gas composition of wetwood changes again; O2 decreases, CO^ increases and CH^ increases or re-appears. The respiratory a c t i v i t y of microorganisms and t h e i r a b i l i t y to adapt to different environments would explain the observed changes i n gas composition. In addition, studies made here (Appendix I) and those of Zeikus and Ward (1974) showed that methanogenic bacteria were present i n trees containing substantial quantities of CH4. F i n a l l y , very low levels 150 of 0 2 have only been reported i n trees with large b a c t e r i a l populations i n t h e i r trunks. In summary, therefore, i t appears that microorganisms are primarily responsible for the gas composition and that they induce micro-aerobic conditions i n the wetwood during the summer. The soil-block experiment showed that the 2 wood-destroying fungi used did not cause s i g n i f i c a n t weight loss under microaerobic conditions. Similar response of wood-destroying fungi can be expected i n the f i e l d as micro-aerobic conditions do occur i n the wetwood of black cottonwood. Furthermore, when the wood blocks were exposed to aerobic conditions following an incuba-t i o n imder the microaerobic conditions, there was a s i g n i f i c a n t weight loss i n wood, whereas i n the reverse sequence, there was no additional weight loss. It follows then that i f the tree i s wounded and wetwood exposed, the wood-destroying fungi would resume t h e i r a c t i v i t y due to increased 0^ con-centrations, and when the wound i s healed, the a c t i v i t y of wood-destroying fungi would decrease as the 0 2 concentration within wetwood would decrease. And i f microaerobic conditions are created after the wound healing then the a c t i v i t y .of wood-destroying fungi would be halted (no s i g n i f i c a n t additional weight l o s s ) . The soil-block experiment also showed that inherent properties of wetwood such as high pH or high moisture content did not contribute to the decay resistance, Also, the microorganisms did not exhibit any antagonistic effects on the growth of wood-destroying fungi. The 2 wood-destroying fungi used i n the soil-block experiment survived 10 week exposure to microaerobic conditions but were unable to survive 13 week exposure to anaerobic conditions. Generally, white-rot fungi were more sensitive to anaerobic conditions than the brown-rot fungi. Therefore, i f 151 anaerobic conditions exist i n the tree trunks for long durations, white-rot fungi may not show any decay a c t i v i t y , and quite possibly die, whereas the brown-rot fungi may survive but may or may not show any decay a c t i v i t y . These findings and inferences suggest that at least i n the case of black cottonwood, occurrence of wetwood cannot be assumed to be a deleterious phenomenon i n terms of tree's own s u r v i v a l . On the contrary, the evidence indicates that i t could well be a b e n e f i c i a l phenomenon protecting the tree from advancement of decay. However, i t i s obvious that t h i s does not ex-clude problems i n u t i l i z a t i o n that wetwood. presents to the forest industry. For example, i t i s known that wetwood i s d i f f i c u l t to k i l n - d r y during lumber processing as the drying time required i s longer than that of normal wood, and therefore, more energy i s used to achieve the proper moisture content. Decay of heartwood by fungi i s one of the important causes of f i b e r loss i n l i v i n g poplars (Davidson and Prentice 1968). Generally the wood-des-troying fungi enter through wounds exposing wood, and eventually destroy the heartwood. Therefore, i t has been suggested that i n j u r i e s should be avoided during s i l v i c u l t u r a l operations'in order to reduce such losses. I support this suggestion. The results obtained here indicate that i n j u r i e s not only serve as the entry points for the wood-destroying fungi but also admit 0^ to the wetwood (or heartwood), thereby destroying the mechanism of decay r e s i s t -ance. Certainly, deep and large wounds would destroy the mechanism permanent-l y . On the other hand, shallow and small wounds, such as those caused by branch pruning, heal quickly and have no permanent effect on the gas composi-tion of wetwood. Some investigators dealing with wetwood have regarded i t as a pathologi-cal phenomenon. The term pathological (Pathology-Science of diseases, Fowler 152 and Fowler 1964) i n i t s common usage implies that i n a given class, some bi o l o g i c a l e n t i t i e s are healthy and some are affected by a disease. I t also implies that i n the diseased ones, the physiological functioning i s severely affected which almost invariably results i n reduced growth or early mortality of the host. Hartley et a l . (1961) have suggested that occurrence of wetwood may be related to early mortality of Lombardy poplar (trees frequently die before age 20), and also i n an unexplained mortality often observed i n balsam f i r s . In view of these implications, l e t us consider i f the occurrence of wetwood i n black cottonwood can be classed as a pathological phenomenon. F i r s t l y , the occurrence of wetwood i s universal, i . e . a l l trees of age 12 and over contain wetwood, and t h i s phenomenon appears to be independent of any special conditions other than tree age. Generally, the developmental pattern of wetwood and i t s position within trees i s quite s i m i l a r to that of normal heartwood and therefore i t can be assumed that wetwood also performs useful functions (mechanical support, for example) that are normally carried out by heartwood. Secondly, many growth and y i e l d studies (summarized by Maini and Cayford 1968) indicate that black cottonwood i s the largest of the North Ameri-can poplars, and longest-lived and fastest-growing hardwood species i n the P a c i f i c Northwest. Studies made in.the Quesnel region,.B.C. show that gener-a l l y this species grows well up to 200 years (Thomas 1949) , and there i s no evidence of early mortality ( i . e . without involving known b i o l o g i c a l or phys-i c a l agents) associated with the species. Thirdly, the wetwood of black cottonwood together with i t s microbial community appears to o f f e r decay re-sistance to the tree and does not appear to have any deleterious effects on the tree growth i n general. Therefore, i t i s my b e l i e f that such a phenomenon should not be termed pathological. 153 This concept of wetwood can perhaps be extended to the wetwood of eastern cottonwood. Sachs eJTal_. (1974), and Zeikus and Ward (1974) reported that normal heartwood was absent i n a l l eastern cottonwoods examined and that only wetwood and sapwood were present. Therefore, the occurrence of wetwood appears to be a universal phenomenon i n this species as we l l . The microbial studies of the same authors showed that aerobic, microaerophilic and anaerobic bacteria were associated with wetwood. The study of Sachs et a l . (1974) also showed that filamentous fungi were rarely found i n the wetwood. 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I I . 97: 224-244. 162 APPENDIX I ISOLATION OF ANAEROBIC BACTERIA (OBLIGATE) FROM WETWOOD Attempts were made to see i f methanogenic bacteria ( s t r i c t anaerobes) were present i n the wetwood of Tree 21 as this tree contained over 30% CH^ during the month of May. In the f i r s t week of June, a core sample (length 4 i n [10.0 cm] excluding sapwood, diameter, 0.5 i n [1.2 cm] was taken out with an increment borer and was placed immediately i n an anaerobic system (Brewer's j a r ) . Before removing the core, about 25 ml of l i q u i d was extracted from the wetwood and was stored i n a gas sampling tube (Chapter I I I ) . This l i q u i d and the core were taken back to the laboratory (Department of Micro-biology, U.B.C.) within an hour. Core was then cut into 5 mm sections i n an anaerobic growth chamber and the pieces were transferred into tubes con-taining standard Bryant medium supplemented with 30% rumen f l u i d . A small quantity of l i q u i d (about 0.5 ml) collected from the wetwood was also added to these tubes. The tubes were stoppered with s t e r i l e rubber stoppers. For gassing purposes, the tubes were divided into three groups: tubes belong-ing to f i r s t group were supplied with H^, the second group with N 2 and the t h i r d group with a mixture of rh, and C0 2 (80:20, v o l : v o l ) . Then, the produc-ti o n of CH^ by the microorganisms placed i n the tubes was taken as an i n d i -cation of the presence of methanogenic bacteria. CH^ was detected with a Bendix (model 2500) gas chromatograph using a s i l i c a gel column. Gas samples from the tubes were analyzed p e r i o d i c a l l y for a t o t a l period of 36 to 48 hours. Wood sections belonging to group II tubes (gassed with N 2) did not produce any CH. while those belonging to groups I and I I I tubes produced CH.. 163 These results show that presence of and CO^  aids microorganisms i n production of CH^. Details of techniques used i n i s o l a t i o n of methanogenic bacteria are given by Edwards and McBride (1974). A review a r t i c l e by Wolfe (1971) i s also available which provides much of the relevant information regarding these microorganisms. 164 APPENDIX II-A. 1: C a l i b r a t i o n curve o f CO- concentration. o I—I < OC E -2 UJ u O u CN o u 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 200,000 400,000 600,000 800,000 ON 1,000,000 MICROVOLTS - SEC APPENDIX I I - A . l 166 APPENDIX IT - A, 2: Calibration curve of CH. concentration. _ 40.0 400,000 800,000 1,200,000 1,800,000 2,000,000 2,400,000 MICROVOLTS - S E C ^ APPENDIX II-A. 2 168 APPENDIX II-B  GAS COMPOSITION IN THE TREE TRUNKS Details of the gas analysis work are presented here. The last 3 columns indicate the concentrations of individual gases, expressed as percentages while the first 5 columns provide other relevant sample characteristics. Column 3 (from left) refers to temperature of the tree gases and not of the outside air. Column 4 represents developed suction during the gas extractions while Column 5 represents flow rate as the gases were being extracted. Description of sample trees, wood zones and any variations from the normal procedure have been given elsewhere in the text and are stated here only briefly. Concentrations of 0^ were measured with a field-lab 0 2 analyzer (in the field) while C02 and CH4 concentra-tions were determined by gas chromatography (in the laboratory). APPENDIX I I (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0_ CO. CH Collection (C) (in[cm]) (ml/min) 2 Black cottonwood - Wetwood - Tree 10 1 Jan 11 2 7(17.5) 50 1.10 8.7 0.0 2 Jan 23 12 7(17.5) 100 1.50 10.2 0.0 3 Feb 1 15 '5(12,5) 50 1.70 10.0 0.0 4 Feb 15 11 6(15.0) 75 0.10 10.7 0.0 5 Mar 1 11 6(15.0) 75 0.15 10.4 0.0 6 Mar 20 15 5(12.5) 100 0.20 10.3 0.0 7 Apr 3 16 5(12.5) 100 1.50 10.5 0.0 8 Apr 17 18 7(17,5) 75 0.55 10.6 0.0 9 May 7 15 7(17.5) 75 0.05 11.8 0.0 10 May 15 14 6(15.0) 75 <0.05 12.4 0.0 11 May 29 16 7(17.5) 75 <0.05 12,6 0.0 12 June 12 20 7(17.5) 75 0.05 13.1 tra c e s 13 June 26 16 7(17.5) 75 0,05 13.0 traces ON APPENDIX II (cont'd): Gas composition i n the tree trunks. Gas sample characteristics Gas Concentration Number Date of Temperature Suction developed Flow rate 0 CO CH Collection (C) (in [cm]) (ml/min) 2 2 4 Black cottonwood - Wetwood - Tree 10 (continued) 14 Jul.; 10 i c 18 5(12.5) 100 <0.05 12.4 traces 15 Jul.24 24 22 6(15.0) 100 0.30 10.5 traces 16 Aug 7 22 7(17.5) 100 1.40 9.9 traces 17 Aug 21 22 5(12.5) 100 2.00 10.6 traces 18 Sept 4 22 6(15.0) 100 2.00 9.8 traces 19 Oct 2 20 6(15.0) 100 2.80 11.8 traces 20 Oct 16 19 6(15.0) 100 3.10 13.3 traces 21 Oct 30 17 6(15.0) 100 4.0 11.9 traces 22 Nov 13 14 7(17.5) 100 4.20 13.1 traces 23 Nov 2 7 14 6(15.0) 100 3.80 12.1 traces 24 Dec 11 15 6(15.0) 100 2.40 12.0 traces APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample c h a r a c t e r i s t i c s Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0 CO, CH. Co l l e c t i o n (C) (infcm]) (ml/min) 2 * Black cottonwood - Wetwood - Tree 15 1 Feb 20 10 5(12.5) 125 1.80 4.4 0.0 2 Mar 15 11 7(17.5) 50 1.00 5.8 0.5 3 Mar 28 12 5(12.5) S50 0.05 5.6 0.5 4 Apr 12 18 5(12.5) 50 0.05 5'\ 6 0.5 5 Apr 24 16 5(12.5) 50 <0.05 7.2 0.5 6 May 8 15 7(17.5) 50 <0.05 6.9 0.5 7 May 24 17 5(12.5) 100 <0.05 7.4 0.5 8 June 4 18 6(15.0 75 <0.05 7.2 0.5 9 June 19 20 7(17.5) u75o <0.05 7.9 0.5 10 J u l 3 20 6(15.0) 75 < 0.0 5 8.8 0.5 11 J u l 17 18 7(17.5) 100 <0.05 7.9 0.5 12 J u l 31 21 6(15.0) 100 <0.50 9.8 0.5 13 Aug 14 20 4(10.0) 100 0.60 10.7 0.5 APPENDIX II (cont'd): Gas composition i n the tree trunks. Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0„ CO CH Collection (C) (in[cm]) (ml/min) 2 2 Black cottonwood - Wetwood - Tree 15 (cont'd) 14 Aug'28 23 4(10.0) 100 0.40 10.8 1.0 15 Sept 11 23 4(10.0) 100 0.40 10.2 1.0 16 Oct 9 20 4(10.0) 100 0.60 10.7 0.7 17 Oct 23 19 4(10.0) - 100 1.20 9.4 0.7 18 Nov 6 14 3(7.5) 100 0.25 8.9 0.7 19 Nov 20 14 3(7.5) 100 0.20 7.2 0.5 20 Dec 4 14 4(10.0) 100 0.20 7.2 0.7 21 Dec 19 13 3(7.5) 100 0.80 7.2 0.7 APPENDIX II (cont'd): Gas composition in the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Collection Temperature (C) Suction developed (in[cm]) Flow rate (ml/min) CO, CH, Black cottonwood - Wetwood - Tree 6 1 Jan 19 6 10(25.0) 75 4.90 6.2 1.2 2 Jan 25 11 9(22.5) 50 3.00 4.1 0.7 3 Feb 1 10 9(22.5) 50 4.00 4.8 0.7 4 Feb 8 11 10(27.5) 50 2.50 4.5 0.5 5 Feb 22 10 16(40.0) 50 1.00 5.2 0.5 6 Mar 3 12 16(40,0) ?5 2.00 5.8 0.7 7 Mar 27 16 18(32.5) 25 2.40 4.0 0.5 8 Apr 10 16 9(22.5) 25 2.00 4.1 0.5 9 Apr 26 15 9(22.5) 25 1.00 2.9 0.9 10 May 10 14 9(22.5) 25 1.50 4.8 0.6 11 May 24 17 9(22.5) 25 0.60 8.2 1.2 12 Jun 4 18 9(22.5) 25 0.10 8.8 1.0 13 Jun 21 18 9(22.5) 25 0.10 7.9 0.7 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0- CO. CH Collection (C) (in[cm]) (ml/min) 2 Black cottonwood - Wetwood - Tree 6 (cont'd) 14 Jul 7 21 7 (17 .5 ) 50 0 .90 7.5 1.0 15 July 19 20 7 (17 .5 ) 50 0 .90 7.5 1.0 16 Aug 2 22 6 (15 .0 ) 75 0 .50 8 .0 1.0 17 Aug 16 21 9 (22 .5 ) 75 1.20 6 .4 2 .0 18 Aug 30 28 8 (20 .0 ) 75 1.80 7 .0 1.6 19 Sept 13 21 7 (17 .5 ) . 75 2 .40 6 .4 2 .0 20 Oct 11 20 7 (17 .5 ) 75 2 .00 5 .0 2 . 5 21 Oct 25 19 8 (20 .0 ) 50 2 .00 5 .0 2 . 5 22 Nov 8 15 8 (20 .0 ) 50 3.40 4 . 8 2 . 5 23 Nov 22 10 9 (22 .5 ) 50 3.40 4 .2 2 . 0 24 Dec 21 10 7 (17 .5 ) 50 3.50 4 .2 2 . 5 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration Number Date of Temperature Suction developed Flow rate 0 C0„ CH Collection (C) (in[cm]) (ml/min) 2 ""4 Black cottonwood - Wetwood - Tree 11 1 Jan 11 2 10(25.0) 50 0.90 8.8 0.0 2 Jan 23 10 7(17.5) 75 1.00 6.0 0.0 3 Feb 6 12 55(12.5) 75 1.20 4.8 0.0 4 Feb 15 11 7(17.5) 75 0.20 5.5 0.0 5 Mar 3 12 6(15.0) 75 0.20 5.7 0.0 6 Mar 20 14 5(12.5) 125 0.15 5.3 0.0 7 Apr 3 14 5(12.5) 100 1.00 6.5 0.0 8 Apr 17 16 6(15.0) 75 0.05 5.5 0.0 9 May 7 15 7(17.5) 75 <0.05 8.5 0.0 10 May 15 14 7(17.5) 75 <0.05 8.8 0.0 11 May 29 16 7(17.5) 75 <0.05 9.3 0.0 12 Jun 12 20 5(12.5) 100 <0.05 9.3 0.0 13 Jun 26 16 5(12.5) 75 <0.05 9.4 0.0 APPENDIX II (cont'd): Gas composition in the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0, CO CH Collection (C) (infcm]) (ml/min) 2 2 Black cottonwood - Wetwood - Tree 11 (cont'd) 14 Jul 10 18 5(12.5) 100 <0.05 8.9 0.0 15 Jul 24 22 6(15.0) 100 0.80 8.4 0.0 16 Aug 7 22 7(17.5) 100 1.00 8.9 0.0 17 Aug 21 22 • 5(12.5) 100 1.80 8.8 0.0 18 Sept 4 22 4(10.0) 100 2.00 9.2 0.0 19 Oct 2 20 4(10.0) 100 2.40 8.9 0.0 20 Oct 16 19 5(12.5) 100 2.60 8.9 0.0 21 Oct 30 17 4(10.0) 100 2.60 8.4 0.0 22 Nov 13 14 4(10.0) 100 0.75 8.8 0.0 23 Nov 27 14 4(10.0) 100 2.10 6.6 0.0 24 Dec 11 14 4(10.0) 100 0.40 7.1 0.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0„ CO CH Collection (C) (in[cm]) (ml/min) 2 2 * Black cottonwood - Wetwood - Tree 8 1 Jan 25 12 10(25.0) Low 1.20 6.3 0.0 2 Feb 6 10 8(20.0) 25 2.00 4.8 0.0 3 Feb 27 11 13(32.5) 25 1.40 6.0 0.0 4 Mar 13 11 13(32.5) 25 1.00 7.1 0.0 5 Mar 27 14 14(35.0) 25 1.20 6.8 0.0 6 Apr 10 15 15(37.0) 25 1.00 7.1 0.0 7 Apr 26 15 13(32.5) 20 1.50 5.4 0.0 8 May 10 14 13(32.5) 25 1.00 6.6 0.0 9 May 24 17 12(30.0) 25 0.40 8.6 0.0 10 Jun 7 18 12(30.0) 25 0.10 8.3 0.0 11 Jun 21 18 7(17.5) 50 <0.05 8,6 0.0 12 J u l 5 21 6(15.0) 50 0.05 8.7 0.0 13 J u l 19 20 7(17.5) 50 0.05 8.6 0.0 APPENDIX II (cont 'd) : Gas composition i n the tree trunks Gas sample characteristics Gas Concentration Number Date of Temperature Suction developed Flow rate 0 CO CH Collection (C) (infcmj) (ml/min) 2 2 4 Black cottonwood - Wetwood - Tree 8 (cont'd) 14 Aug 2 22 6(15.0) 50 0.50 8.2 0.0 15 Aug 16 21 8(20.0) 75 1.60 6.3 0.0 16 Aug 30 23 6(15.0) 75 1.90 6.8 0.0 17 Sept 13 21 6(15.0) 50 2.00 6.8 0.0 18 Oct 11 20 7(17.5) 75 2.40 4.9 0.0 19 Oct 25 19 8(20.0) 75 2.40 6.2 0.0 20 Nov 8 15 7(17.5) 50 3.00 5.4 0.0 21 Nov 22 10 8(20.0) 50 4.50 4.0 0.0 22 Dec 21 10 7(17.5) 50 5.00 4.1 0.0 APPENDIX II (cont'd): Gas composition in the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Collection Temperature (C) Suction developed (infcm]) Flow rate (ml/min) CO, CH, Black cottonwood - Wetwood - Tree 16 1 Feb 25 11 9(22.5) 50 6.20 5.9 0.0 2 Mar 15 12 16(40.0) Low 5.60 7.0 o . o 3 Mar 28 12 8(20.0) 50 14.00 2.7 0.0 4 Apr 12 17 10(25.0) 50 10.50 2.3 0.0 5 Apr 24 16 9(22.5) 50 11.00 3.5 0.0 6 May 8 15 8(20.0) 50 14.00 2.6 0.0 7 May 24 17 10(25.0) 50 0.80 6.4 0.0 8 Jun 4 18 9(22.5) 50 0.05 6.9 0.0 9 Jun 19 20 6(15.0) . 50 <0.05 6.9 0.0 10 Jul 3 20 5(12.5) 75 <0.05 7.2 0.0 11 Jul 17 18 6(15.0) 75 <0.05 7.0 0.0 12 Jul 31 20 6(15.0) 75 0.90 7.4 0.0 13 Aug 14 20 5(12.5) 75 0.80 7.4 0.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0. C0„ CH Collection (C) (in[cm]) (ml/min) 2 Black cottonwood - Wetwood - Tree 16 (cont'd) 14 Aug 28 23 10(25.0) 50 1.90 5.0 0.0 15 Sept 11 20 7(17.5) 50 2.00 5.2 0.0 16 Oct 10 20 6(15.0) 50 2.00 5.0 0.0 17 Oct 23 19 6(15.0) 50 2.00 5.6 0.0 18 Nov 6 14 7(17.5) 50 1.80 7.6 0.0 19 Nov 20 14 8(20.0) 50 3.40 4.0 0.0 20 Dec 4 14 6(15.0) 50 4.00 4.8 0.0 21 Dec 19 13 6(15.0) 50 4.20 4.8 0.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Collection Temperature (C) Suction developed (in[cm]) Flow rate (ml/min) CO, CH, Black cottonwood - Wetwood - Tree 20 1 2 3 May 7 May 28 May 8 May 21 May 31 15 17 15 18 17 9(22.5) 6(15.0) Tree 21 12(30.0) 7(17.5) 7(17.5) 50 50 25 75 75 <0.05 <0.05 <0.05 <0.05 <0.05 1st week of June, tree wounded, wetwood exposed; wound plugged within 24 hours after wounding 4 5 Jun 27 Ju l 19 May 8 May 21 17 20 15 18 5(12.5) 8(20.0) Tree 22 10(25.0) 9(22.5) 100 50 50 50 6.50 1.20 1.20 1.00 9.9 7.5 8.9 8.8 8.9 1.0 2.4 4.1 4.9 0.0 0.0 34.0 30.5 32.6 3.2 10. 0.5 0.5 oo APPENDIX II (cont'd): Gas composition in the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0 CO CH. Collection (C) (in[cm]) (ml/min) 2 4 Black cottonwood - Wetwood Tree 27 1 2 3 4 5 1 2 3 Jun 5 Jun 18 Jul 9 Aug 20 Oct 11 Jul 30 Aug 27 Oct 3 Jul 30 Aug 27 18 20 21 22 20 22 23 20 22 23 10(25.0) 11(27.5) 7(17.5) 7(17.5) 7(17.5) Tree 28 7(17.5) 6(15.0) 6(15.0) Tree 29 6(15.0) 6(15.0) 50 50 50 75 50 50 75 75 75 75 <0.05 <0.05 <0.05 <0.05 1.20 <0.05 <0.05 2.60 0.20 0.20 4.7 5.5 6.0 5.0 7.2 8.9 8.8 5.8 8.3 8.4 4.0 4.0 4.5 6.0 5.5 19.5 19.0 0.5 15.0 16.0 APPENDIX II (cont'd): Gas composition in the tree trunks Gas sample c h a r a c t e r i s t i c s Gas Concentration (%) Number Date of C o l l e c t i o n Temperature (C) Suction developed (infcm]) Flow rate (ml/min) CO, CH, 1 2 1 2 3 Jan 9 Jan 16. Feb 13 Jan 4 Jan 16 Feb 13 Jan 18 Jan 30 Feb 25 Feb 27 Apr 22 14 5 4 14 5 10 10 8 14 Black cottonwood - Wetwood Tree 5 8(20.0) Tree 4 9(23.5) 7(17.5) Tree 9 9(22.5) 7(17.5) 4(10.0) iTreeU 7(17.5) Tree 7 10(25.0) Tree 14 7(7.5) Tree 17 3(7.5) Tree 18 8(20.0) 50 50 50 50 50 75 75 25 50 75 50 2.00 3.00 2.80 2.50 2.00 3.80 4.00 1.80 11.00 8.00 8.00 6.4 5.1 4.2 4.2 3.7 4.0 3.0 3.9 3.4 3.7 3.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0,0 0.0 0.0 0.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Collection Temperature (C) Suction developed (in[cm]) Flow rate (ml/min) CO, CH, 1 2 3 4 1 2 3 1 2 Feb 8 Feb 15 Mar 1 Mar 27 Mar 6 Mar 15 Mar 29 Apr 10 Apr 26 May 10 BI ack cottonwood - Sapwood  Tree 12 9 8(20.0) 25 11 12(30.0) Low developed 20 i n (50.0 cm) of suction, no sample developed 20 i n (50.0 cm) of suction, no sample Tree 16 12 16(40.0) Low developed 20 i n (50.0 cm) of suction, no sample developed 20 i n (50.0 cm) of suction, no sample Tree 32 16 9(22.5) 50 15 11(27.5) 25 14 Tree 33 9(22.5) 11.40 17.00 15.00 50 18.00 11.40 13.00 0.2 0.2 0.2 0.6 0.2 0.6 0.0 0.0 0.0 0.0 0.0 0.0 APPENDIX II (cont'd): Gas composition in the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Collection Temperature (C) Suction developed (in[cm]) Flow rate (ml/min) CO, CH, May 24 Jun .4 Jun 21 Jul 5 . Jul 19 Aug 2 Aug 16 Aug 30 Sep 13 Oct 11 Oct 25 17 18 18 21 20 Black cottonwood - Sapwood  Tree 34 9(22.5) 10(25.0) Tree 35 10(25.0) 12(30.0) 13(32.5) Tree 36 50 25 50 25 25 22 13(32.5) 25 21 12(30.0) 25 developed 20 in (50.0 cm) of suction, no sample Tree 37 21 20 19 12(30.0) 13(32.5) Tree 38 13(32.5) 25 25 Low 16.50 13.00 12.60 18.00 17.50 19.00 18.60 18.00 16.00 18.00 0.2 0.8 0.4 0.4 0.4 0.2 0.2 0.1 0.1 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 oo APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) mber Date of Collection Temperature (C) Suction developed (in[cm]) Flow rate (ml/min) °2 co 2 CH. 4 Black cottonwood - Wetwood - Tree 2 - Wounded 1 Feb 27 11 0 125 16.50 0.5 0.0 2 Mar 13 11 0 100 17.00 0.5 0.0 3 Mar 22 17 0 125 18.00 0.6 0.0 4 Apr 5 15 0 125 17.00 0.4 0.0 5 Apr 19 16 0 125 18.50 0.5 0.0 6 May 9 16 0 125 18.00 0.6 0.0 7 May 31 17 0 . 125 18.00 0.6 0.0 8 Jun 14 21 0 125 18.50 0.6 0.0 9 Jun 28 18 0 125 19.00 0.6 0.0 10 J u l 12 20 0 125 18.50 0.6 0.0 11 J u l 26 20 0 125 18.00 0.3 0.0 12 Aug 9 23 0 125 17.50 0.3 0.0 13 Aug 23 22 0 125 18.00 0.4 0.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration Number Date of Temperature Suction developed Flow rate 0„ CO. CH, Collection (C) (in [cm]) (ml/min) 2 2 4 Black cottonwood - Wetwood - Tree 2-Wounded (cont'd) 14 Sept 6 23 0 125 18,00 0.3 0.0 15 Oct 4 20 0 125 18.00 0.8 0.0 16 Oct 18 20 0 125 17.00 0.6 0.0 17 Nov 1 15 0 125 13.00 0.6 0.0 18 Nov 15 14 0 125 13;. 00 0.6 0.0 19 Nov 29 14 0 125 14.00 0.6 0.0 20 Dec 13 14 0 125 15.00 0.6 0.0 APPENDIX II: (cont'd): Gas composition in the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0„ CO. CH Collection (C) (in[cm]) (ml/min) 2 Black cottonwood - Wetwood - Tree 3 - Wounded 1 Mar 20 12 4(10.0) 125 16.00 4.4 0.5 2 Apr 3 14 5(12.5) 75 17.50 4.2 0.5 3 Apr 17 16 8(20.0) 75 18.00 4.4 0.5 4 May 7 15 9(22.5) S50 16.50 4.6 1.0 5 May 15 14 8(20.0) 550 18.00 4.4 1.0 May 17 tree wounded, wetwood exposed 6 May 29 16 7(17.5) 50 19.00 0.8 0.0 7 Jun 12 20 8(20.0) 75 18.50 0.9 0.0 8 Jun 26 166 7(17.5) 75 17.00 0.9 0.0 9 Jul 10 18 7(17.5) 50 18.00 1.2 0.0 10 Jul 24 22 7(17.5) 75 17.00 1.4 0.0 Jul 27, wound plugged 11 Aug 7 22 6(15.0) 75 10.50 1.6 0.0 12 Aug 21 22 6(15.0) 75 11.50 1.4 0.0 13 Sept : 4 22 5(12.5) 75 9.20 3.0 0.0 APPENDIX I I ( c o n t ' d ) : Gas c o m p o s i t i o n i n t h e t r e e t r u n k s Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0„ C0_ CH Collection (C) (in[cm]) (ml/min) B l a c k c o t t o n w o o d - Wetwood - T r e e 3 - Wounded ( c o n t ' d ) 14 O c t 2 20 6 ( 1 5 . 0 ) 100 4 . 0 0 4 . 2 2 . 0 15 O c t 16 19 7 ( 1 7 . 5 ) 75 3 .50 5 . 0 1.5 16 O c t 30 17 4 ( 1 0 . 0 ) 100 5 .50 5 .1 2 . 0 17 Nov 13 14 7 ( 1 7 . 5 ) 75 5 .00 5 .1 2 . 0 18 Nov 27 14 7 ( 1 7 . 5 ) 75 5 .00 6 .1 2 . 0 19 Dec 11 14 6 ( 1 5 . 0 ) 75 4 . 0 0 6 . 0 .'.2.5 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration Number Date of Collection Temperature (C) Suction developed (in[cm]) Flow rate (ml/min) CO, CH, Black cottonwood - Wetwood - Tree 24 - Wounded 1 May 5 15 7(17.5) 75 0.90 7.2 0.0 2 May 31 17 5(12.5) 100 0.10 7.2 0.0 3 Jun 14 21 5(12.5) 100 0.05 7.5 0.0 4 Jun 28 18 7(17.5) 75 0.05 7.4 0.0 1st week of Jul y , tree wounded, sapwood exposed 5 J u l 12 19 6(15.0) 75 0.10 7.4 0.0 6 J u l 26 20 6(15.0) 75 0.05 7.5 0.0 7 Aug 23 22 ' 7(17.5) 50 0.05 8.0 0.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0^ CO^ CH^ Collection (C) (in[cm]) (ml/min) Black cottonwood - Wetwood - Tree 30 - Wounded 1 May 5 15 7(17.5) 75 0.10 6.4 0.0 2 May 31 17 7(17.5) 75 0.20. 6.4 0.0 3 Jun 13 20 8(20.0) 50 0.05 6.2 0.0 4 Jun 28 18 7(17.5) 75 0.10 7.0 0.0 1st week of July, tree wounded, sapwood exposed 5 J u l 12 19 6(15.0) 75 0.10 6.9 0.0 6 J u l 26 21 7(17.5) 75 0.10 7.2 0.0 7 Aug 23 22 8(20.0) 50 0.20 7.1 0.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Temperature Suction developed Flow rate 0_ CO- CH Collection (C) (in[cm]) (ml/min) Black cottonwood - Wetwood - Tree 19 A - through branch stub B - normal sample point 1-B Apr 22 14 9(22.5) 75 <0.05 8.3 1.5 2-B May 9 15 9(22.5) 75 <0.05 7.8 1.2 3-A May 28 17 developed 20 in (50.0 cm) of suction, no sample 3-B May 28 17 flow of gas, but no analysis 4-A Jun 5 18 developed 20 i n (50.0 cm) of suction, no sample 4-B Jun 5 18 8(20.0) 75 <0.05 9.9 2.0 5-A Jun 11 20 developed 20 i n (50.0 cm) of suction, no sample 5-B Jun 11 20 9(22.5) 75 <0.05 9.8 3.0 6-A Ju l 9 21 developed 20 in (50.0 cm) of suction,lino sample 6-B Jul 9 21 8(20.0) 75 <0.05 9.9 2.0 7-A Aug 20 22 • developed 20 in (50.0 cm) of suction, no sample 7-B Aug 20 22 9(22.5) 75 1.20 2.6 2.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration Number Date of Collection Temperature (C) Suction developed (in[cm]) Flow rate (ml/min) CO, CH, 1- A 2- A 3- A 3- B 4.-A 4- B Apr 29 May 9 May 28 May 28 Jun 11 Jun 11 15 15 17 17 20 20 Black cottonwood - Wetwood - Tree 23 A - through branch stub B - normal sampling point developed 20 i n (50.0 cm) of suction, no sample developed 20 i n (50.0 cm) of suction, no sample developed 20 i n (50.0 cm) of suction, no sample 5(12.5) 75 <0.05 developed 20 i n (50.0 cm) uf suction, no sample 3(7.5) 100 0.05 11.7 6.0 0.5 0.5 APPENDIX II (cont'd): Gas composition in the tree trunks Gas sample characteristics Gas Concentration (%) irature Suction developed Flow rate Collection (C) (in[cm]) (ml/min) Number Date of Tempera 0^ CG^ CH^ Red alder - Heartwood - Tree 41 1 Feb 13 6 12(30.0) Low 18.00 0.3 0.0 2 Mar 22 18 8(20.0) 50 20.00 0.3 0.0 3 Apr 5 15 13(32.5) 25 18.00 0.4 0.0 4 Apr 19 14 7(17.5) 50 20.50 0.2 0.0 5 May 9 15 8(20.0) 25 19.00 0.2 0.0 6 May 31 17 9(22.5) 25 18.50 0.2 0.0 7 Jun 14, 20 9(22.5) 25 19.00 0.3 0.0 8 Jun 28 18 7(17.5) 50 18.50 0.3 0.0 9 Jul 12 20 6(15.0) 50 17.50 0.4 0.0 10 Jul 26 20 7(17.5) 50 18.00 0.4 0.0 11 Aug 23 22 8(20.0) 50 16.50 0.4 0.0 12 Sep-" 6 23 7(17.5) 50 14.00 0.4 0.0 13 Oct 4 20 11(27.5) 50 17.00 0.4 0.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Collection Temperature (C) Suction developed (in[cm]) Flow rate (ml/min) CO, CH, Red alder - Heartwood - Tree 41 (cont'd) 14 15 16 17 Oct 18 Nov 1 Nov 29 Dec 13 20 15 14 14 8(20.0) 14(35.0) 13(32.5) 12(30.0) 50 25 50 50 17.00 18.00 17.50 17.00 1.4 0.8 0.8 0.8 0.0 0.0 0.0 0.0 APPENDIX II (cont'd): Gas composition in the tree trunks Gas sample characteristics Gas Concentration Number Date of Collection Temperature (C) Suction developed (infcm]) Flow rate (ml/min) CO, CH, Red alder - Heartwood - Tree 42 1 Feb 15 10 12(30.0) Low 17. 50 0.8 0.0 2 Mar 3 10 11(27.5) Low 17. 00 1.0 0.0 3 Mar 22 17 12(30.0) 25 19. ,00 0.8 0.0 4 Apr 5 15 14(35.0) Low 19. ,00 0.6 0.0 5 Apr 19 14 10(25.0)) 25 20. ,00 0.5 0.0 6 May 9 15 11(27.5) 25 19. ,50 0.5 0.0 7 May 31 17 11(27.5) 25 19, .00 0.5 0.0 8 Jun 14 21 10(25.0) 25 18, .50 0.6 0.0 9 Jun 28 18 10(25.0) 25 17, .00 0.6 0.0 10 Jul 12 19 10(25.0) 25 17 .50 0.6 0.0 11 Jul 26 20 10(25.0) 50 18 .00 0.8 0.0 12 Aug 23 22 7(17.5) 75 17 .50 0.9 0.0 13 Sept 6 23 9(22.5) 50 17 .80 0.6 0.0 APPENDIX II (cont'd): Gas composition in the tree trunks Gas sample characteristics Gas Concentration irature Suction developed Flow rate Collection (C) (in[cm]) (ml/min) Number Date of Tempera  0^ CC^  CH^  Red alder - Heartwood - Tree 42 (cont'd) 14 Oct 4 20 9(22.5) 50 16.00 0.8 0.0 15 Oct 18 20 8(20.6) 50 18.00 0.9 0.0 16 Nov 1 15 13(32.5) Low 19.00 0.8 0.0 17 Nov 15 14 8(20.0) 50 17.50 0.8 0.0 18 Nov 29 14 7(17.5) 50 18.00 0.8 0.0 19 . Dec 13 14 8(20.0) 50 17.20 0.6 0.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration (%) Number Date of Collection Temperature (C) Suction developed (in[cm]) Flow rate (ml/min) CO, CH, Red alder - Heartwood - Tree 25 Jun 5 Jun 18 18 20 8(20.0) 17(17.5) 75 75 18.00 17.00 0.2 0.2 0.0 0.0 APPENDIX II (cont'd): Gas composition i n the tree trunks Gas sample characteristics Gas Concentration Number Date of Collection Temperature (C) Suction developed (in[cm]) Flow rate (ml/min) CO, CH, 1 2 3 1 2 3 May 6 May 6 May 21 Jan 4 May 6 May 21 Jun 6 Lombardy poplar - Heartwood (probably wetwood)  Tree 44 17 7(17.5) 50 10.50 No further analysis, tree f e l l e d by the Physical Plant, U.B.C, Tree 45 16 19 18 15 18 18 7(17.5) 7(17.5) ;7(17.5) Tree 46 15(37.5) 15(37.5) 16(40.0) 50 75 75 Low Low Low 10.00 11.50 10.50 11.00 11.00 1;50 2.8 3.6 3.7 3.6 1.8 2.1 6.5 0.6 1.5 1.5 1.5 0.6 0.6 2.0 

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