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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 . S c , M.Sc, U n i v e r s i t y o f Poona M.Sc, U n i v e r s i t y o f 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 t h e s i s as conforming t o the required, standard  THE UNIVERSITY OF BRITISH COLUMBIA December, 1975  In  presenting  this  an a d v a n c e d d e g r e e the I  Library  further  for  shall  agree  thesis  in p a r t i a l  fulfilment  of  at  University  of  Columbia,  the  make  it  freely  that permission  available for  his  of  this  written  representatives. thesis  financial  gain  of  University  of  British  Columbia  2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  <&U~<  >1  by  the  is understood  permission.  Department  The  for  It  »qTS"  for  extensive  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  British  shall  requirements  reference copying of  Head o f  that  not  the  I  agree  and  be a l l o w e d  that  study.  this  thesis  my D e p a r t m e n t  copying or  for  or  publication  without  my  ii  ABSTRACT  A study i s described on the occurrence o f wetwood i n black cottonwood, found i n the Lower Fraser V a l l e y o f 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 t r e e s , wetwood i s always present. i s s i m i l a r to that o f a normal heartwood, with d i f f e r e n t 0^ requirements  Further extension o f wetwood A large number o f microorganisms  ( i . e . , aerobes as w e l l as f a c u l t a t i v e and  o b l i g a t e 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£ concent r a t i o n s (<0.1% or microaerobic c o n d i t i o n s , detected by a F i e l d - l a b 0^ analyzer) were found i n the wetwood during the summer.  Generally the 0^ concen-  t r a t i o n increased during the winter while the reverse was true f o r CO2 (detected by gas chromatography). t i t i e s , i n most t r e e s . in  CH^ was also present, but only i n small quan-  Mechanical wounding o f wetwood r e s u l t e d i n an increase  the 0^ concentration and decrease i n the CO2 and CH^ concentrations.  How-  ever, s e a l i n g of the wound r e - e s t a b l i s h e d the o r i g i n a l gas composition. Presumably the m i c r o b i a l f l o r a of wetwood i s p r i m a r i l y responsible f o r the microaerobic c o n d i t i o n s . The a b i l i t y o f 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 using a s o i l - b l o c k experimental technique,  determined,  Microaerobic conditions prevented  weight loss i n wood (average 0.1%) and therefore wood decay, whereas under  iii aerobic c o n d i t i o n s , the average weight loss was 41.7%.  S p e c i a l character-  i s t i c s o f wetwood such as high pH (average 7.8) or high moisture content (approximately ance.  160%) d i d not contribute s i g n i f i c a n t l y to the decay r e s i s t -  A l s o , microorganisms associated with wetwood showed no antagonism  to the growth of wood-destroying f u n g i . to microaerobic  Exposure o f wood-destroying fungi  conditions subsequent to aerobic conditions arrested t h e i r  growth and a b i l i t y to cause weight l o s s .  On the other hand, exposure o f  these fungi to aerobic conditions f o l l o w i n g 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 cons i d e r a b l y less than when the fungi were grown under aerobic conditions alone (average 41.7%). The 2 wood-destroying fungi survived 10 weeks exposure t o microaerobic conditions,  A 13 weeks exposure to anaerobic conditions (<0.002 % 0^), how-  ever, r e s u l t e d i n the death o f these, wood-destroying f u n g i .  Eight wood-  destroying fungi d i 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 cond i t i o n s ; generally brown-rot fungi t o l e r a t e d anaerobic conditions b e t t e r than the white-rot f u n g i .  Therefore, i f anaerobic conditions e x i s t i n the  tree trunks f o r 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 a v a i l a b l e . These findings l e d t o the concept that the microaerobic  conditions found  i n the wetwood o f black cottonwood may prevent the development o f decay.  iv  TABLE OF CONTENTS Page  TITLE PAGE  .....  ABSTRACT  i i i  TABLE OF CONTENTS  iv  LIST OF TABLES  v i i  LIST OF FIGURES  .....  ACKNOWLEDGEMENTS  v  iii xi  FRONTISPIECE  .....  x i i  GENERAL INTRODUCTION  .....  1  CHAPTER I - OCCURRENCE OF WETWOOD IN BLACK COTTONWOOD INTRODUCTION  6  ,  MATERIALS AND METHODS  6 .....  RESULTS AND DISCUSSION  10  CHAPTER I I - MICROORGANISMS ASSOCIATED WITH WETWOOD IN BLACK COTTONWOOD INTRODUCTION MATERIALS AND METHODS 1. 2. 3.  Tree samples Quantitative determinations Isolations 3.1 Aerobic b a c t e r i a 3.2 Fungi 3.3 Anaerobic b a c t e r i a ( f a c u l t a t i v e ) 3.4 Anaerobic b a c t e r i a (obligate)  29 .•  29  .....  34  ..... ..... ..... ..... .....  RESULTS 1. 2.  Quantitative determinations Isolations 2.1 Wetwood: Aerobic b a c t e r i a 2.2 Wetwood: fungi 2.3 Wetwood: Anaerobic b a c t e r i a ( f a c u l t a t i v e ) 2.4 Wetwood: Anaerobic b a c t e r i a (obligate) 2.5 Sapwood 2.6 Wound-initiated d i s c o l o r e d wood 2.7 Young trees and seedlings  DISCUSSION  8  34 35 36 37 37 37 38 38  ..... ..... ..... ..... .....  38 38 38 41 43 43 43 43 44 44  T a b l e o f C o n t e n t s (cont'd)  v  Page CHAPTER I I I - COMPOSITION OF GASES IN THE TRUNKS OF BLACK COTTONWOOD INTRODUCTION  51 .....  MATERIALS AND METHODS 1. 2. 3. 4.  L o c a t i o n o f sample t r e e s E x t r a c t i o n apparatus E x t r a c t i o n a p p a r a t u s : Assembly Method and o p e r a t i o n o f e x t r a c t i n g gas samples 4.1 B l a c k cottonwood: Sapwood 4.2 B l a c k cottonwood: From wetwood t h r o u g h i branch stubs 4.3 B l a c k cottonwood: ..Wetwood o f wounded t r e e s : Wetwood exposed 4.4 B l a c k cottonwood: ..Wetwood o f wounded t r e e s : Sapwood exposed Gas a n a l y s i s Measurement o f gas p r e s s u r e s i n t h e wetwood  5. 6. RESULTS 1. 2. 3. 4. 5. 6. 7. 8. 9.  B l a c k cottonwood: Wetwood: Examined a l l t h r o u g h the y e a r B l a c k cottonwood: Wetwood: Examined o n l y d u r i n g c e r t a i n months o f t h e y e a r B l a c k cottonwood: Sapwood B l a c k cottonwood: From wetwood t h r o u g h b r a n c h stubs ' B l a c k cottonwood: Wetwood o f wounded t r e e s : Wetwood exposed B l a c k cottonwood: Wetwood o f wounded t r e e s : Sapwood exposed Red a l d e r : Heartwood Lombardy p o p l a r : Heartwood ( p r o b a b l y wetwood) Measurement o f gas p r e s s u r e s i n t h e wetwood  DISCUSSION  51 60  ..... .....  60 64 70 75 80 80  .....  81  .....  81 87 88  .....  88 89  ..... .....  94 94  .....  94  .....  99  .....  102 105 105 105  ..... .....  105  .....  122  INTRODUCTION  .. ., .  122  MATERIALS AND METHODS  .....  127  CHAPTER IV - EFFECTS OF MICROAEROBIC CONDITIONS ON DEVELOPMENT OF DECAY  RESULTS AND DISCUSSION  132  CHAPTER V - SURVIVAL OF WOOD-DESTROYING FUNGI UNDER ANAEROBIC CONDITIONS INTRODUCTION  138 ......  138  MATERIALS AND METHODS  140  RESULTS AND DISCUSSION  143  vi Table o f Contents (cont'd)  Page  CHAPTER VI - SIGNIFICANCE OF WETWOOD IN BLACK COTTONWOOD (SUMMARY AND CONCLUSIONS) BIBLIOGRAPHY  ......  148  .  154  APPENDIX I s o l a t i o n o f anaerobic b a c t e r i a (obligate) from wetwood II-A. 1 . C a l i b r a t i o n curve of CO^ concentration 2 . C a l i b r a t i o n curve o f CH. concentration 4 II-B Gas composition i n the tree trunks  162  I.  ..•., .....  162 164 166 168  vii LIST OF TABLES Page 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 four trees obtained from the U.B.C. Research Forest, Maple Ridge, B.C. ......  15  The pH o f wetwood and sapwood samples c o l l e c t e d at two d i f f e r e n t heights from four t r e e s , obtained from the U.B.C. Research Forest, Maple Ridge, B.C....  17  Number o f 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 V a l l e y i n the Province of B r i t i s h Columbia  20  Number o f colonies o f b a c t e r i a i n the l i q u i d s ext r a c t e d from the wetwood (non-wounded trees) and d i s c o l o r e d wood (wounded trees) samples c o l l e c t e d at i n d i c a t e d heights. Counts are from 1:10,000 d i l u t i o n on n u t r i e n t agar p l a t e s  39  TABLE V:  Some morphological and p h y s i o l o g i c a l c h a r a c t e r i s t i c s o f b a c t e r i a i s o l a t e d from wetwood  40  TABLE VI:  Frequency and c h a r a c t e r i s t i c s o f fungi i s o l a t e d from wetwood on 2% malt agar  42  TABLE I I :  TABLE I I I :  TABLE IV:  TABLE V I I : D e s c r i p t i o n o f sample t r e e s , wood zones and duration o f sampling i n the year 1974 TABLE V I I I : Linear growth o f two wood-destroying fungi on 2% malt agar, a f t e r 16 days o f incubation under aerobic (control) and microaerobic conditions ...... TABLE IX:  TABLE X:  65  133  Mean % weight loss caused by two wood-destroying fungi i n black cottonwood blocks o f sapwood (S) and wetwood (W) under aerobic (A) and microaerobic (M) conditions ......  134  C h a r a c t e r i s t i c s o f 8 wood-destroying f u n g i , and t h e i r s u r v i v a l response to 13 weeks o f incubation under anaerobic conditions ......  141  viii  LIST OF FIGURES Page FIGURE 1:  Cross s e c t i o n o f a 29 year old black Cottonwood showing c e n t r a l l y located d i s c o l o r e d wood (wetwood) which i s surrounded by c o l o r l e s s wood (sapwood).  11  V e r t i c a l s e c t i o n o f the same t r e e p i c tured 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).  12  FIGURE 3:  D i s s e c t i o n o f a 45 year o l d black cottonwood at various heights. .....  13  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 o f a black cottonwood.  14  Cross s e c t i o n o f a wounded black cottonwood tree 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 rather i r r e g u l a r appearance.  19  V e r t i c a l s e c t i o n o f a 2 year o l d black cottonwood showing sapwood ( c o l o r l e s s ) and p i t h (dark).  23  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 o f wetwood development (bottom), sapwood c o l o r l e s s ) and p i t h (dark brown i n center) .  24  V e r t i c a l sections 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 piece 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 associated with a wound.  25  V e r t i c a l s e c t i o n o f a 1 year o l d black cottonwood showing wound-initiated d i s c o l o r a t i o n and sapwood.  27  V e r t i c a l sections o f a 2 year o l d black cottonwood showing wound-initiated d i s c o l o r a t i o n and sapwood.  27  FIGURE 2:  FIGURE 5:  FIGURE 6:  FIGURE 7:  FIGURE 8:  FIGURE 9:  FIGURE 10:  ix L i s t o f Figures (cont'd)  FIGURE 11:  FIGURE 12:  Page  Map o f the U n i v e r s i t y Endowment Lands showing l o c a t i o n of sample trees used f o r the gas composition work.  .....  62  Black cottonwood-red alder stand surrounding U n i v e r s i t y H i l l Church, Univ e r s i t y Endowment Lands (U.B.C).  63  FIGURE 13:  Schematic drawings o f the s p e c i a l l y designed apparatus.  69  FIGURE 14:  Portable e x t r a c t i o n apparatus mounted i n a plywood case.  72  Schematic drawing o f the e x t r a c t i o n apparatus assembly.  74  FIGURE 15: FIGURE 16:  FIGURE 17: FIGURE 18:  FIGURE 19:  FIGURE 20: FIGURE 21:  FIGURE 22:  A.. Schematic drawing o f the brass pipe in position. B. Brass pipe and shut-off valve i n p o s i t i o n i n the f i e l d C. V e r t i c a l section of a black cottonwood through the sampling hole (extending from l e f t to r i g h t )  77 77 .....  78  Complete gas e x t r a c t i o n set-up i n the field.  79  A. and B. Schematic drawing o f a tree (No. 23) showing set-up used to extract gases through a branch stub C. Set-up (as i n B) i n the f i e l d  83 83  A black cottonwood tree (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 ) .  84  Schematic drawing o f the two trees showing nature o f wounds.  86  Concentrations o f 0 , C0 and CH (monthl y averages) i n the wetwood o f a black cottonwood (Tree 15).  91  Concentrations o f 0 , CO and CH (monthl y averages) i n the wetwood o f a black cottonwood (Tree 6).  93  2  2  2  2  4  4  X  L i s t o f Figures (cont'd)  FIGURE 23:  FIGURE 24:  FIGURE 25:  FIGURE 26:  FIGURE 27: FIGURE 28:  FIGURE 29:  FIGURE 30:  FIGURE 31:  FIGURE 32:  FIGURE 33:  Page  Comparison of 0 and CO^ concentrations (monthly averages) i n the wetwood (Tree 8) and sapwood (several trees) o f black cottonwood. 2  .....  96  Concentrations of 0 , C0 and CH (monthly averages) i n the wetwood o f two black cottonwoods (Tree 19 and 23). .....  98  2  2  4  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 position o f the branch trace.  100  Cross-section o f a 19 year o l d black cottonwood (Tree 2) showing extent o f the wound.  101  Concentrations of 0 , C0 and CH i n the wetwood of a black cottonwood (Tree 21)  104  Concentrations of 0 and C0 (monthly averages) i n the heartwood of a red alder (Tree 42) .  . 1 0 7  2  2  2  4  2  Comparison o f 0 and CO^ concentrations (monthly averages) i n the wetwood o f a black cottonwood (Tree 11) and heartwood of a red alder (Tree 41). 2  109  Concentrations o f 0 , C0 and CH (monthly averages) i n the heartwood (probably wetwood) o f three Lombary poplar trees (Nos., 44, 45 and 46). 2  2  4  Percentage of black cottonwood trees (nonwounded) with microaerobic conditions present i n the wetwood from March u n t i l September, 1974.  Ill  •  114  Comparison of 0 and C0 concentrations (monthly averages) i n tne wetwood o f a nonwounded black cottonwood (tree 10) and a wounded black cottonwood (Tree 2).  119  Disposable Anaerobic System  129  2  2  xi  ACKNOWLEDGEMENTS.  I g r a t e f u l l y acknowledge Dr. B.J, van der Kamp f o r p r o v i d i n g guidance, advice and encouragement during t h i s research p r o j e c t .  I also wish t o ex-  tend my sincere appreciation to Dr. Roger S. Smith, Western Forest Products Laboratory, Vancouver, B.C. f o r extending the laboratory f a c i l i t i e s without which t h i s work might not have been completed. The t h e s i s was reviewed by Drs. R.J. Bandoni (Botany), K. Graham ( F o r e s t r y ) , B. M u l l i c k ( 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) f o r allowing me t o use the r e s u l t s o f a j o i n t p r o j e c t in this thesis.  I also wish t o thank Dr. A. Kozak (Forestry) f o r h i s advice  on the s t a t i s t i c a l a n a l y s i s , Mr. G. Bohnenkamp (Forestry) and Mr. A. Hoda (Plant Science) f o r t h e i r help i n constructing the e x t r a c t i o n apparatus and Mrs. P. Waldron f o r typing t h i s manuscript. Grateful appreciation i s due to the National Research Council o f Canada and to the U n i v e r s i t y o f B r i t i s h Columbia f o r 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 e n t i r e course o f t h i s study.  xii  WETWOOD IN BLACK COTTONWOOD  1  GENERAL INTRODUCTION  2  A casual look at any mature black cottonwood (Populus t r i c h o c a r p a Torrey and Gray) tree trunk i n cross s e c t i o n 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 d i s c o l o r e d wood i s not so obvious.  One.might con-  clude that t h i s inner wood i s heartwood because of i t s p o s i t i o n and d i s coloration.  However, i n contrast to normal heartwood, t h i s inner wood i s  unusually wet.  A f t e r a tree has been cut, water may continue to ooze from  the cut surface f o r hours.  The inner wood i s a l k a l i n e , and contains a  large number o f b a c t e r i a and some imperfect f u n g i .  Forest p a t h o l o g i s t s  t r a d i t i o n a l l y r e f e r to t h i s type o f wood as wetwood.  In t h i s t h e s i s ,  therefore, the term wetwood w i l l be used to designate the inner d i s c o l o r e d wood i n s p i t e of the f a c t that Wood Technology textbooks u s u a l l y r e f e r 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 d i s c o l o r a t i o n 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 o f wetwood has been reported or inferred^ i n more than 30 hardwood and 10 softwood species (Knutson 1970), i n c l u d i n g most elms (Ulmus L . ) , poplars (Populus L . ) , Willows ( S a l i x 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- v a r i a t i o n e x i s t s i n the c h a r a c t e r i s t i c s o f wetwood.  For example, the  colors a t t r i b u t e d to wetwoods vary w i t h i n a given species and also among  3 d i f f e r e n t species.  Aspen (Populus tremuloides Michx.) wetwood has been  described as having a darker c o l o r (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 c o l o r v a r i e s from brown to r u s t - r e d . d i f f e r s considerably.  S i m i l a r l y , p o s i t i o n of the wetwood w i t h i n trees  Wetwood can be present i n the t r e e as a 'zone be-  tween sapwood and heartwood' or can occupy 'most of the heartwood and wood' or 'only sapwood' or 'only heartwood'.  sap-  Wetwood can be associated with  branch stubs or wounds made by increment borer holes.  I t can also occur i n  a p o s i t i o n not associated with wounds or any other type o f 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 n i f i c a n c e i s not very c l e a r .  B a c t e r i a , i n p a r t i c u l a r , are often  considered  as causal organisms of wetwood formation (Carter 1945, S e l i s k a r ; 1950). Microorganisms are also implicated i n gas production  (Morani and Arru  as increased gas pressures have frequently been found i n the wetwood.  1958), Very  low oxygen (0^) and high carbon dioxide (C0 ) concentrations are often found 2  i n tree species showing a p a t t e r n of wetwood formation.  A reduction product  such as methane (CH^) has also been reported from a few poplar species, and r e c e n t l y , Zeikus and Ward (1974) i s o l a t e d anaerobic b a c t e r i a (obligate) r e sponsible f o r the production of CH^ from the wetwood of 2 eastern cottonwoods (Populus deltoides B a r t r . ) . Almost a l l studies of wetwood to date have been d e s c r i p t i v e i n nature, dealing with d i s t r i b u t i o n of wetwood w i t h i n trees and among various species,  4  gas composition o f wetwood, m i c r o f l o r a etc.  However, the e f f e c t o f wetwood  on the f u n c t i o n i n g o f the l i v i n g trees i s s t i l l poorly understood.  Those  i n v e s t i g a t o r s who have allowed themselves to speculate on the matter have generally taken the view that wetwood i s a p a t h o l o g i c a l condition  (e.g.  Smith 1970) which probably promotes decay o f heartwood by p r o v i d i n g more s u i t a b l e moisture conditions f o r fungal growth o r by prolonging  susceptibili-  ty by lengthening the time required f o r the wood to dry (Hartley et a l . 1961). The main hypothesis  examined i n my study opposes t h i s common view.  My preliminary i n v e s t i g a t i o n (parts o f i t included i n Chapters I and II) i n d i c a t e d that wetwood i n black cottonwood may not be a disease tion.  condi-  As a r e s u l t , rather than assuming wetwood t o be a d e l e t e r i o u s 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 t o the s u r v i v a l o f the tree because i t protects the tree from the development o f 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 d i t i o n s i n the wetwood which prevent establishment decay i n the wetwood.  or anaerobic con-  and/or development o f  Other c h a r a c t e r i s t i c s such as high pH o r high moisture  content o f wetwood also contribute to the decay r e s i s t a n c e . The hypothesis was tested i n four separate steps.  Firstly, isolation  studies were made to determine what kinds o f microorganisms are present i n the wetwood and i n what numbers (Chapter I I ) . Secondly, gas composition studies were made t o see i f microaerobic  or anaerobic conditions e x i s t i n the  wetwood o f l i v i n g trees (Chapter I I I ) . Then, based on observations made i n t h i s study, a p r o j e c t was undertaken to study the extent o f 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 s e t up t o study the e f f e c t s o f some inherent properties o f wetwood on i t s decay.  L a s t l y , the s u r v i v a l o f wood-destroy-  ing fungi under anaerobic conditions was studied (Chapter V) i n view o f the findings reported i n Chapter IV. Before undertaking these s p e c i f i c experiments, i t was e s s e n t i a l to give a d e s c r i p t i o n o f wetwood as i t occurs i n black cottonwood, i t s d i s t r i b u t i o n w i t h i n 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 s e l f - c o n t a i n e d u n i t and includes i t s own Introduction, M a t e r i a l and Methods, Results and the Discussion. the t e x t .  For convenience, a Bibliography has been presented at the end o f The l a s t chapter (Chapter VI) i s b a s i c a l l y a short discussion  of the r e s u l t s obtained during the course o f t h i s t h e s i s work.  6 CHAPTER I OCCURRENCE OF WETWOOD IN BLACK COTTONWOOD INTRODUCTION Water-soaked xylem t i s s u e , which i s generally r e f e r r e d to as wetwood, 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 B a r t r . or i t s segregates) and Lombardy poplars (Populus n i g r a 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 a d d i t i o n , they examined white  poplar (Populus alba L . ) , balsam poplar (Populus balsamifera L.) and black cottonwood trees from s i n g l e l o c a l i t i e s and reported that every tree contained wetwood.  W a l l i n (1954) mentioned common occurrence o f 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 s i z e without any  recognizable  wetwood, and i n Colorado, some e n t i r e study p l o t s were n e a r l y free o f wetwood (Hartley et a l . 1961).  A l s o , 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 o l d t r e e s , wetness and high pH are  u s u a l l y confined to the outer heartwood or to the t r a n s i t i o n zone between heartwood and sapwood. americana L . ) ,  In eastern cottonwood and American elm (Ulmus  Hartley et_ al_. (1961) found that wetwood extended throughout  the length of the bole and i n t o the l a r g e r 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 o l d seedlings.  In Lombardy poplar, wetwood was not only present but was regu-  l a r l y w e l l developed before the trees reached the age o f 3 years.  The  European aspen has been reported to be almost u n i v e r s a l l y a f f e c t e d by the age of 10 years with a non-decay.discoloration that was assumed to be wetwood (Ankudinov 1939),  Morani and Arru (1958) reported that a l l 17 year o l d  poplars growing i n the neighbourhood of P i s a , I t a l y that they studied, contained "symptoms" o f a b a c t e r i a l i n f e c t i o n i n the c e n t r a l part o f the trunk, s i m i l a r to those described f o r "wetwood disease". Wetwood has often.been found associated with increment borer holes (Davidson et_ al_. 1959), i n o c u l a t i o n holes (Riley 1952) or branch stubs (Hartley e t a l . 1961).  H a r t l e y and h i s co-workers quote Baker's study on  cottonwoods i n which he found wetwood o c c a s i o n a l l y i n " i n j u r e d " 1-year o l d seedlings and very commonly before the end o f the second year i n n a t u r a l reproduction at f i v e l o c a l i t i e s i n N, Dakota, Nebraska and Oklahoma.  He  a t t r i b u t e d the wetwood streaks i n less than 2-year o l d cottonwoods to b e e t l e i n j u r y near the s o i l surface.  Knutson (1970) d i s t i n g u i s h e d between two types  of wetwood i n aspen, i n a d d i t i o n 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 r e l a t e d to any wound or branch stub, and columns r a r e l y extended more than 4 or 5 feet up the bole. Thomas and Podmore (1953) f i r s t reported the occurrence of "watersoaked wood" i n black cottonwood.  Hartley et a l .(1961) also i n d i c a t e d the  presence o f wetwood i n black cottonwood.  However, none o f these authors gave  8 a d e s c r i p t i o n o f wetwood o r i t s d i s t r i b u t i o n w i t h i n the trees nor d i d they attempt to i s o l a t e microorganisms associated with i t .  Consequently, I  made a study with the f o l l o w i n g o b j e c t i v e s : 1) to describe a few c h a r a c t e r i s t i c features o f w e l l developed wetwood ( i n 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 d i s s e c t a large number o f trees to determine the age at which wetwood f i r s t appears, and 3) to search f o r wounds or s p e c i a l 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 V a l l e y i n the Province o f B r i t i s h Columbia as shown i n Table I I I . The choice o f l o c a l i t i e s was p r i m a r i l y based upon a v a i l a b i l i t y o f black cottonwoods and not because o f any s p e c i f i c environmental conditions o f the l o c a l i t i e s . I n i t i a l l y , a large number o f trees and seedlings o f d i f f e r e n t ages was examined f o r the presence o f c e n t r a l l y - l o c a t e d , wet-appearing d i s c o l o r e d wood. A very l i m i t e d number o f trees was then used to determine moisture content, pH (Chapter I) and m i c r o b i a l f l o r a (Chapter I I ) o f t h i s d i s c o l o r e d wood. The findings of these studies provided the basis f o r considering the d i s colored wood to be a "wetwood" rather than a "heartwood".  A l l the remaining  trees i n which a d e t a i l e d examination was not made, the presence or absence of c e n t r a l l y - l o c a t e d wet-appearing d i s c o l o r e d wood was taken as presence o r absence o f wetwood, r e s p e c t i v e l y .  9 A t o t a l o f 224 t r e e s , 213 non-wounded and 11 wounded, o f various ages was examined to study the occurrence and d i s t r i b u t i o n o f wetwood ( i n i t i a l l y , tree samples were c o l l e c t e d at random although once the pattern o f wetwood was known, some wounded trees were excluded, and therefore are represented i n comparatively small number).  In a l l o f 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 w e l l developed wetwood) were used t o study two important features o f 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 o f the wood (wetwood and sapwood), t e s t blocks (0.75 cm) were cut from the discs and, a f t e r weighing them immed i a t e l y , were d r i e d i n an oven at 102 C f o r about 24 hours.  Next day,  the blocks were cooled over dessiccant f o r one hour and weighed.  Mois-  ture content was expressed as a percentage o f the dry weight o f the wood. The method used to determine pH o f 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) f o r about 24 hours at approximately 25 C. the  A "Radiometer" pH-meter (PHM-28) was used to measure the pH o f  aqueous extract,,,and was considered to represent the pH o f the wood.  M e t r i c conversions have been given i n the brackets whenever actual measurements were made i n Imperial u n i t s .  10  RESULTS AND DISCUSSION The d i s s e c t i o n studies revealed that i n mature black cottonwood t r e e s , sapwood envelops a s o l i d column o f d i s c o l o r e d wood (wetwood) which extends from the roots through the trunk i n t o the major branches 1, 2 and 3).  (Figures  The discolored column appears greenish-brown or rusty-brown  upon c u t t i n g down the t r e e , and the c o l o r u s u a l l y becomes even darker w i t h i n a few hours of exposure to atmosphere.  The boundary between the  d i s c o l o r e d wood and sapwood i s often i r r e g u l a r , the d i s c o l o r e d wood extending several annual r i n g s c l o s e r to the cambium at some points than others.  Generally sapwood consists o f fewer rings (though u s u a l l y wide  ones) toward the crown than toward the butt.  The c e n t r a l column extends  i n t o the major branches, although i t u s u a l l y does not reach the branch tips.  Frequently the d i s c o l o r e d column o r i g i n a t i n g from the trunk tapers  o f f q u i c k l y as i t enters the branches (Figure 4 ) . The d i s c o l o r e d wood appears water-soaked and, when i t i s f r e s h l y c u t , 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%) o f 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 p r e c i s e l y 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 l e a s t during the growing season, i s l i k e l y to r e s u l t i n "instantaneous p u l l i n g away" o f some o f the water which might lead to a decrease i n the moisture content o f the  F i g u r e 1:  Cross s e c t i o n o f a 29 y e a r o l d b l a c k cottonwood showing c e n t r a l l y located  discolored  wood (wetwood) w h i c h i s s u r -  rounded by c o l o r l e s s wood (sapwood).  T h i s photograph was  taken a f t e r the " d a r k e n i n g p r o c e s s " o f d i s c o l o r a t i o n had taken p l a c e (3 hours a f t e r s e c t i o n c u t t i n g ) . h e i g h t - a p p r o x i m a t e l y 1 meter above the ground. X 0.25.  Sample Scale -  12  Figure  2:  V e r t i c a l s e c t i o n o f t h e same t r e e p i c t u r e d i n F i g u r e  1,  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) and c o l o r l e s s wood (sapwood).  Sample h e i g h t  0.3 meters above t h e ground (bottom).  - approximately  Scale  - X 0.17.  13  F i g u r e 3:  D i s s e c t i o n o f a 45 y e a r o l d b l a c k cottonwood a t v a r i o u s h e i g h t s , from bottom r i g h t to l e f t t o t o p 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 t h e ground ( i n d i c a t e d on each d i s c i n f e e t ) . Note t h e p r e s e n c e o f wetwood throughout t h e b o l e .  Section  at 4.0 f e e t (1.2 meters) shows a wound and t h e d i s c o l o r a t i o n associated with i t .  14  F i g u r e 4:  V e r t i c a l s e c t i o n o f a branch removed from the t r u n k o f a b l a c k cottonwood.  Note the r a p i d r e d u c t i o n i n w i d t h  the wetwood (bottom).  Scale - X  0.17.  of  15  TABLE I : The m o i s t u r e c o n t e n t (%) o f wetwood and sapwood samples c o l l e c t e d a t t h r e e d i f f e r e n t h e i g h t s from f o u r t r e e s obt a i n e d from t h e U.B.C. Research F o r e s t , Maple R i d g e , B.C.  Tree c h a r a c t e r i s t i c s  Sample h e i g h t  Number  Age (Yr)  DBM (in[cm])  Height (ft[m])  ( f t [ m ] above ground)  1  37  25.0(63.5)  66.0(19.5)  2  3  4  47  41  42  29.0(73.7)  31.0(78.8)  31.0(78.8)  Average  70.0(21.3)  79.0(24.0)  79.0(24.0)  Moisture content Wetwood  Sapwood  3.0(0.9)  166.8  145.1  6.0(1.8)  192.8  149.3  12.0(3.6)  188.7  127.0  3.0(0.9)  160.2  119.7  6.0(1.8)  156.3  116.1  12.0(3.6)  155.0  116.6  3.0(0.9)  137.0  122.2  6.0(1.8)  138.0  124.3  12.0(3.6)  134.0  145.2  3.0(0.9)  148.5  121.1  6.0(1.8)  163.0  120.4  12.0(3.6)  171.3  115.4  159.4  126.8  16  outer rings  (sapwood).. At t h e same time, l o s s o f some water from t h e  f r e s h l y c u t wetwood cannot be avoided. t i o n s , i t i s g e n e r a l l y agreed  Even w i t h many p o s s i b l e v a r i a -  t h a t wetwood has a h i g h e r moisture  content  than the sapwood. M o i s t u r e content o f 160% (average) p a r t o f t h e v o i d space V o i d space  indicates that a substantial  i n d i s c o l o r e d wood i s s t i l l  i n a g i v e n p i e c e o f wood r e p r e s e n t s t h e t o t a l volume o f water  and gases t h a t wood can h o l d under normal p r e s s u r e . the maximum moisture mately  o c c u p i e d by gases.  In b l a c k cottonwood,  t h a t the d i s c o l o r e d wood can h o l d would be a p p r o x i -  270%, assuming t h a t t h e s p e c i f i c g r a v i t y o f b l a c k cottonwood i s  0.3 and the t r u e s p e c i f i c g r a v i t y o f wood substance  i s 1.5 (Stamm 1964).  A simple s u b t r a c t i o n o f the a c t u a l moisture  (160%) from t h e maxi-  mum p o s s i b l e m o i s t u r e  content  d i s c o l o r e d wood (110%).  content  (270%) g i v e s volume o f gases p r e s e n t i n t h e  Thus, a p p r o x i m a t e l y  1/3 o f t h e v o i d space i n the  d i s c o l o r e d wood i s o c c u p i e d by t h e gases w h i l e t h e r e m a i n i n g  2/3 i s taken  up by the water. Determination  o f pH v a l u e s i n d i c a t e d t h e a l k a l i n e n a t u r e o f t h e  d i s c o l o r e d wood (Table I I ) .  The pH o f d i s c o l o r e d wood v a r i e d from  7.33  to 8.31 (average  7.84) w h i l e t h a t o f sapwood v a r i e d from 6.20 t o 6.50  .(average 6.31).  Thus these r e s u l t s a r e g e n e r a l l y i n agreement w i t h p r e -  v i o u s l y p u b l i s h e d r e s u l t s f o r many o t h e r 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  t h a t t h i s d i s c o l o r e d wood i s a l s o c h a r a c t e r i z e d by a h i g h b a c t e r i a l population  (average  7.3 x 10^/ml o f the e x t r a c t e d l i q u i d ) .  Therefore  this  TABLE I I :  The p H o f w e t w o o d a n d s a p w o o d s a m p l e s 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 from f o u r t r e e s , o b t a i n e d from the U . B . C . Research F o r e s t , Maple R i d g e , B . C .  Tree c h a r a c t e r i s t i c s Number  1  2  3  4  Age (yr)  DBH (in[cm])  37"  25.0(63.5)  47  41  42  29.0(73.7)  31.0(78.8)  31.0(78.8)  Height (ft[m])  66.0(19.5)  70.0(21.3)  79.0(24.0)  79.0(24.0)  Sample h e i g h t " , ( f t [ m ] above ground)  pH v a l u e Wetwood  Sapwood  3.0(0.9  7.92  6.21  12.0(3.6)  7.33  6.24  3.0(0.9)  8.31  6.35  12.0(3.6)  7.70  6.20  3.0(0.9)  7.71  6.36  12.0(3.6)  7.75  6.25  3.0(0.9)  8.30  6.37  12.0(3.6)  7.70  6.50  Average  7.84  6.31  18 d i s c o l o r e d wood appears to possess a l l the c h a r a c t e r i s t i c features o f a wetwood, but occupies normal p o s i t i o n of a heartwood.  For these rea-  sons, I have used the term "wetwood" i n preference to the term  "heartwood".  Another type of d i s c o l o r e d 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 w i t h wounds and branch stubs.  Although i t s presence was recognized here, no d e t a i l e d  studies were done on t h i s type of d i s c o l o r e d wood, except f o r some i s o l a t i o n studies reported l a t e r (Chapter I I ) ,  The d i s c o l o r a t i o n o r i g i n a t i n g  from the wounds, which looks d i f f e r e n t from the wetwood d i s c o l o r a t i o n , spreads upwards and downwards i n the sapwood and often merges i n t o c e n t r a l l y - l o c a t e d wetwood g i v i n g i t a rather i r r e g u l a r appearance  (Figure 5).  New tissues that are formed a f t e r wounding are seldom affected and there i s u s u a l l y a hard rim t i s s u e between d i s c o l o r e d wood and c o l o r l e s s wood. In a l l respects, t h i s discolored wood appears s i m i l a r to wound-initiated d i s c o l o r e d wood found i n several northern hardwoods (Shigo 1969) .  Such  wouhd-initiated d i s c o l o r e d wood has been r e f e r r e d to as wetwood i n the past (see  I n t r o d u c t i o n ) , but because o f i t s d i f f e r e n t 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 e s s e n t i a l to know at what age wetwood begins to appear.  Preliminary studies i n d i c a t e d that i n  mature trees wetwood was always present whereas i t was absent i n the young seedlings.  The r e s u l t s of the d i s s e c t i o n study are summarized i n Table I I I .  A d i s t i n c t r e l a t i o n s h i p appears to e x i s t between tree age and the occurrence o f wetwood. 8 to 10 years.  Wetwood formation e v i d e n t l y begins i n the age class o f  Seedlings younger than age 7 were devoid o f wetwood whereas  F i g u r e 5.  Cross s e c t i o n o f a wounded b l a c k cottonwood t r e e t r u n k showing d i s c o l o r a t i o n s a s s o c i a t e d  w i t h 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 appearance.  irregular  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 .  S c a l e - X 0.25.  TABLE 111:  Age  Number o f non-wounded black cottonwood t r e e s and s e e d l i n g s , with o r without wetwood, from 5 separate l o c a l i t i e s i n Lower Fraser V a l l e y i n the Province o f B r i t i s h Columbia.  Locality  West 16th Ave. U.B.C . Campus, Vancouver  George Massey Tunnel, D e l t a  South-West Marine Drive U.B.C. Campus Vancouver  University Boulevard U.B.C. Campus Vancouver  U.B.C. Research F o r e s t , Maple Ridge  Wetwood  present absent  present absent  present absent  present absent  present absent  T o t a l No* o f t r e e s and seedlings exami ned  Percentage o f t o t a l with wetwood  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  0  91 .6  11  7  1  12  3  0  1  •  13 14  7  0  3  0  12  6  0  9  100 .0  4  0  4  100 .0  2  0  9  100 .0  O  TABLE I I I (cont'd)  Age  Locality  West 16th Ave. U.B.C. Campus, Vancouver  George Massey Tunnel, D e l t a  South-West Marine Drive U.B.C. Campus Vancouver  University Boulevard U.B.C. Campus Vancouver  U.B.C. Research Forest, Maple Ridge  Wetwood  present absent  present  present  present absent  present  20-25 26-30 31-35 36-50  Tn, Hil V° a  i0  absent  absent  T o t a l No.' o f t r e e s and seedlings examined  absent  11  11  15  16  100.0  6  9  100.0  5  100.0  ° " ^ d i s s e c t e d during t h i s i n v e s t i g a t i o n , 30 more t r e e s were examined l a t e r f o r some other s p e c i f i c p r o j e c t s (Chapters I I I , IV and V). The occurrence o f wetwood was detected i n a l l these t r e e s ™1 T! ^ ° i n c l u d e d here. In 25 trees that were s t u d i e d f o r t h e i r "gas composition", only cores taken out w i t h an increment borer were examined; t h i s i s not unusual as H a r t l e y et a l . (1961) i n t h e i r extensive study always inspected cores t o detect presence o f wetwood and d i d not d i s s e c t a l l the-freTs. extensive r  ef0re  v  2 1 3  t  t  h  e  n  n  a  v  w  e  o  u  a l s  n  d  e  d  t  b e e n  r  t h a t  w e r e  Percentage o f t o t a l with wetwood  100.0  22 i n 12 years or older ones, wetwood was i n v a r i a b l y 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 p a t t e r n , because of the d i f f i c u l t y i n obtaining tree samples of various ages, on one s i t e .  any  In s p i t e of the differences i n s i t e between the f i v e samples,  the pattern of wetwood appearance was  consistent.  D i s s e c t i o n of the young seedlings (ages 1 to 7) f u r t h e r 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  d i s c o l o r a t i o n had j u s t begun to appear i n the stem (Figure 7) , the roots were completely free from i t .  I t i s not known i f the absence of wetwood  i n roots i s r e l a t e d to the absence of p i t h i n roots.  In these t r e e s , the  stem j u s t above the s o l i d l e v e l contained wetwood surrounding the p i t h and the sapwood i n turn enveloped both the wetwood and the p i t h . noteworthy that i n young seedlings i n which wetwood was  It is  absent, wound-ini-  t i a t e d d i s c o l o r a t i o n 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 d i s c o l o r a t i o n of wetwood always o r i g i n a -  ted i n the center of the stem (pith region) and then extended f u r t h e r t o wards the periphery of the stem.  Wounding or any other e x t e r n a l stimulus  does not appear to be necessary f o r 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 sapwood 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  i n t o heartwood as more sapwood i s formed by d i v i s i o n s of the  F i g u r e 6:  Vertical sapwood wetwood. i n g ones  section of a 2 year o l d black ( c o l o r l e s s ) and p i t h The  oldest piece  (dark).  cottonwood  Note the absence  i s on t h e l e f t  f o l l o w base sequence.  Scale  showing  -X  of  and t h e r e m a i n 0.17.  2-1  F i g u r e 7:  V e r t i c a l s e c t i o n o f an 8 y e a r o l d b l a c k cottonwood showing e a r l y s t a g e o f 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 t h a t t h e o u t e r t i s s u e s a r e s l i g h t l y d a r k e r than the i n n e r t i s s u e s .  S c a l e - X 0.25.  25  F i g u r e 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 b l a c k cottonwood showing sapwood ( c o l o r l e s s ) and p i t h  (dark brown) i n each p i e c e and  the t h i r d p i e c e from l e f t a l s o shows 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 a wound.  Note t h e absence o f wetwood.  on t h e l e f t i s o l d e s t and the r e m a i n i n g sequence.  S c a l e - X 0.17.  The p i e c e  ones f o l l o w base  26  Figure 9:  V e r t i c a l s e c t i o n of a 1 year o l d black cottonwood showing wound-initiated d i s c o l o r a t i o n and sapwood.  P i t h i s absent.  Scale - X 0.33.  Figure 10:  V e r t i c a l sections o f a 2 year o l d black cottonwood showing wound-initiated d i s c o l o r a t i o n and sapwood. (barely v i s i b l e ) .  Scale - X 0.25.  P i t h i s present  27  28  cambial c e l l s (Cronquist 1971). heartwood formation.  No external stimulus i s required f o r the  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 " u n i v e r s a l 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 c e r t a i n given  age  (these points w i l l be considered again i n Chapter I I which includes a d i s cussion on the theories o f 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 w i t h i n t r e e s , 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 d i f f e r e n t from most other poplar species. wood i n black cottonwood was  Generally, the pattern of occurrence of wetl 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 o f microorganisms i n the wetwood.  Most o f 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 b a c t e r i a when encountered were often discarded as contaminants.  Also i n many studies malt agar was used  f o r i s o l a t i o n s and t h i s agar i s on the a c i d i c side and consequently, f a vourable t o most decay fungi and less favorable t o most b a c t e r i a .  Later  when b a c t e r i a were i s o l a t e d by use o f a l k a l i n e 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 b a c t e r i a and fungi have been i s o l a 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 b a c t e r i a as the causal organisms o f wetwood i n American elm. He i s o l a t e d Erwiriia  riimipressuaralis  from 93% o f the elm  wetwood samples and also from 78% o f the normal sapwood samples. 209 i n o c u l a t i o n s i n t o d i f f e r e n t parts o f l i v i n g t r e e s .  He made  Inoculations made  on branches, trunk phloem, trunk cambium, trunk current-season  wood and  some other tree parts d i d not produce t y p i c a l symptoms of wetwood. But, as Carter s t a t e s , "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 t h i s browning i n trunk wood  to represent i n f e c t i o n and e a r l y development o f wetwood."  He d i d not  30 state what distinguished t h i s browning as early development o f wetwood, and not merely the normal browning or s t a i n i n g o f t i s s u e s o r i g i n a t i n g from wounds.  A l s o , i f the b a c t e r i a are present i n sapwood, one would  not expect i n o c u l a t i o n s with them to r e s u l t 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 d i s c o l o r a t i o n was comparable to streaking i n elms, n a t u r a l l y " i n f e c ted" with wetwood.  An untreated tree and a tree i n j e c t e d with s t e r i l e  n u t r i e n t broth plus dextrose had no dark brown d i s c o l o r a t i o n i n the heartwood, t y p i c a l o f wetwood.  B a c t e r i a , s i m i l a r to the o r i g i n a l inoculum, were  i s o l a t e d from the s i x inoculated trees but not from the two c o n t r o l trees. Wetwood i n Lombardy poplar was regarded as a b a c t e r i a l disease by S e l i s k a r (1950).  He re covered Coryri eb acterium humi fe rum from the i n o c u l a -  ted wood o f poplar trees but not from the uninoculated  trees.  Both i n o c u l a -  ted and control t r e e s , however, had d i s c o l o r e d zones around the i n o c u l a t i o n holes.  He stated that the i n f e c t i o n s "were s i m i l a r t o young wetwood i n f e c -  t i o n i n n a t u r a l l y diseased t r e e s " , without mentioning how he determined that the discolored t i s s u e o f the inoculated trees was wetwood while the d i s c o l o r e d t i s s u e of the c o n t r o l trees was not. Hartley et_ al_. (1961) noted frequent occurrence o f a bacterium i n the wetwood o f Lombardy poplars.  This t h i c k , "doubtfully gram-positive" rod  was r e a d i l y v i s i b l e i n unstained vessels i n recently formed wetwood.  The  bacterium grew w e l l on malt agar ( a c i d i c ) and produced abundant gas," promptly pushing part o f the agar t o the top o f the tube".  Based on t h e i r  31 extensive study and studies of others, they have advanced three hypotheses to explain wetwood formation which w i l l be considered l a t e r i n view of the r e s u l t s presented here. Knutson (1970) i s o l a t e d three types o f b a c t e r i a from aspen and found that none of these were s p e c i f i c to wetwood.  The b a c t e r i a 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. wetwood and heartwood y i e l d e d b a c t e r i a .  Every sample of sapwood,  Sapwood sap c o n s i s t e n t l y y i e l d e d  low numbers of organisms while wetwood water generally had very large numbers of b a c t e r i a .  Heartwood populations were extremely v a r i a b l e , 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) i s o l a t e d 9 species o f b a c t e r i a belonging to 6 d i f f e r e n t genera from 'non-distressed wood' o f aspen, ponderosa pine (Pinus ponderosa Dougl. ex. Lawson) and mountain a l d e r (Alrius t e n u i f o l a Nutt.).  Five of the species were shown to be p e c t i n d i g e s t e r s , and one,  Cellulomonas a c i d u l a , u t i l i z e d c e l l u l o s e , while 6 species also showed proteolysis.  They concluded, "The presence i n wood of microorganisms  which  digest p e c t i n and c e l l u l o s e suggests that wood from f r e s h l y cut trees or branches may contain the agents of i t s own  decomposition."  Wilcox and Oldham (1972) repeatedly i s o l a t e d a bacterium species assoc i a t e d with wetwood i n white f i r (Abies concblor (Oord. and Glerid.) L i n d l . ) . The bacterium was a s m a l l , slow-growing, gram-variable f a c u l t a t i v e l y anaerobic rod which produced a c i d but no gas i n carbohydrate media.  Its  growth appeared to be stimulated by extracts from various plant t i s s u e s 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 b a c t e r i a have been i s o l a t e d from healthy appearing containing some form of 'shake' (Ward et a l , 1969) tissues (Stankewich et_ al_. 1971).  trees  and from the d i s c o l o r e d  Zeikus and Ward (1974) i s o l a t e d the bac-  terium responsible f o r CH^ production from the wetwood o f eastern cottonwoods 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 s a l t s medium under an atmosphere of hydrogen (rLp  and CO^.  The methanogenic organism was observed i n  a l l enrichment c u l t u r e s obtained from e i t h e r f e t i d l i q u i d or wetwood cores from cottonwood, elm and w i l l o w trees containing CH^.  Concerning the r o l e  of anaerobic b a c t e r i a i n t r e e s , the authors s t a t e d , "The high number of methanogenic b a c t e r i a and other anaerobes found i n wetwood i n d i c a t e s v i g o r ous m i c r o b i a l fermentation.  I t i s d i f f i c u l t to a s c e r t a i n whether the wood  t i s s u e i t s e l f i s being decomposed or whether other n u t r i e n t s serve as the substrates f o r t h i s 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 o f white poplars. The i s o l a t i o n studies y i e l d e d b a c t e r i a l populations that always contained an o b l i g a t e anaerobe (Clostridium)and a microaerophilie bacterium from the inner r i n g s of sapwood adjacent to wetwood.  The wetwood was  " i n f e c t e d " , but i t u s u a l l y contained a mixed population o f  similarly anaerobic,  m i c r o a e r o p h i l i e , and f a c u l t a t i v e b a c t e r i a plus occasional f u n g i .  No  micro-  organisms were found i n the outer, or younger, rings o f sapwood of cottonwoods, aspens or white poplars; n e i t h e r were they found i n the inner sapwood  33 and adjacent heartwood o f white poplars.  Based on these and SEM s t u d i e s ,  the authors concluded that wetwood i s formed i n l i v i n g trees p r i m a r i l y from the b a c t e r i a l action ( t h i s study w i l l be discussed l a t e r i n the chapter) . In many s t u d i e s , s p e c i a l emphasis was given to i s o l a t i o n of b a c t e r i a and therefore the information on fungi from wetwood i s very fragmentary. F r i t z (1931) found Torula l i g n i p e f d a i n the outer zone,of the wet red heart of paper b i r c h (Betula p a p y r i f e r a Marsh.) i n 25 of 28 trees examined.  Her  Torula i n o c u l a t i o n s on specimens i n v i t r o r e s u l t e d i n d i s c o l o r a t i o n 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 d i s c o l o r e d heartwood of aspen.  Knutson's examination of m i c r o b i a l population showed that a very  few filamentous fungi were present i n the aspen wetwood, sapwood and heartwood (1970). samples.  He found numerous colonies of P u l l u l a r i a i n two of h i s wetwood  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 f u n g i , Hartley et a l . (1961) s t a t e , "For most other f u n g i , there has been l i t t l e i n d i c a t i o n of a s s o c i a t i o n with high moisture content i n trunks. ...A number of ascomycetes are known, i n the outer wood causing w i l t o f various hosts, but no reports have been encountered of unusual wetness associated with them, and n e i t h e r p l a n t - i n h a b i t i n g ascomycetes or hymenomycetes, so f a r as our experience goes, produce or t h r i v e i n high pH conditions ." The l i t e r a t u r e thus suggests that numerous types o f b a c t e r i a with d i f f e r e n t 0- requirements are frequently found associated with wetwood,  34  heartwood, d i s c o l o r e d heartwood and also with sapwood.  The primary ob-  j e c t i v e o f these studies was t b search f o r a s p e c i f i c organism o r organisms which could perhaps be implicated i n the wetwood formation. workers made i n o c u l a t i o n studies t o support t h e i r hypotheses.  Some  A few quan-  t i t a t i v e studies were also made towards t h i s end. The purpose o f my study was t o get an estimate o f number and kinds o f microorganisms associated with wetwood which may have a bearing on the i n i t i a t i o n o f wetwood and on i t s gas composition. The s p e c i f i c o b j e c t i v e s were, 1) t o i s o l a t e , characterize and p o s s i b l y i d e n t i f y  aerobic  b a c t e r i a associated with wetwood and sapwood, 2) to detect the presence and p o s s i b l y i s o l a t e  facultative  and o b l i g a t e anaerobes i n the wetwood and sapwood. 3) t o i s o l a t e fungi (presumably aerobic - see Chapter IV) from the wetwood, 4) to i s o l a t e b a c t e r i a (aerobic) from young cottonwoods, with or without wetwood, and 5) to i s o l a t e b a c t e r i a and fungi from the wound-initiated d i s c o l o r e d 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) t h i c k d i s c s (2 from each tree) were then cut from the trees and were brought back t o the laboratory on the same day. One d i s c per tree was used  35 to make q u a n t i t a t i v e determinations o f the b a c t e r i a l population while the other discs were used t o i s o l a t e aerobic b a c t e r i a and f u n g i .  Three a d d i t i o n -  a l non-wounded trees from the Research Forest were also used (but not f e l l e d ) to i s o l a t e f a c u l t a t i v e anaerobes. ment borer f o r i s o l a t i o n s .  Cores of wood were obtained with an i n c r e -  Besides these, several young cottonwoods (approxi-  mately 10) o f ages varying from 1 to 15 from various l o c a l i t i e s (Chapter I) were a l s o used t o i s o l a t e aerobic b a c t e r i a . 2.  Quantitative determinations:  The v i a b l e count method ( C o l l i n s  and Lyne 1970) was used t o determine the s i z e o f b a c t e r i a l population i n the wetwood (7 determinations - one per d i s c ) and i n the d i s c o l o r e d wood (2 determinations - one per d i s c ) o f the wounded trees.  In p r i n c i p l e , the ma-  t e r i a l containing b a c t e r i a i s s e r i a l l y d i l u t e d and a l i q u o t s o f each d i l u t i o n are placed on s u i t a b l e c u l t u r e media.  Each developing colony i s assumed to  have grown from one v i a b l e u n i t representing one organism or a group o f organisms. Peptone water (0.1%) was used as a d i l u e n t , and 9 ml of t h i s d i l u e n t was dispensed i n t o s t e r i l e t e s t tubes.  These " d i l u t i o n blanks" were auto-  claved and allowed to cool before use.  One ml o f sample ( l i q u i d extracted  from the i n d i c a t e d zones) was added to a d i l u t i o n blank and the l i q u i d s were mixed thoroughly.  One ml was then removed from t h i s tube and was added t o  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  Dilution  Volume o f O r i g i n a l 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 n u t r i e n t agar (Difco) which had p r e v i o u s l y 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 p l a t e s (100 x 15 mm) per d i l u t i o n were  set out and 1 ml o f each s o l u t i o n was p i p e t t e d i n t o the center o f the approp r i a t e l y marked dishes.  Contents o f each cooled agar tube were then added  tb one p l a t e and the p l a t e 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 ,  A f t e r the medium was s e t , the  p l a t e s were i n v e r t e d and incubated at 25 C f o r 24-48 hours.  After this  p e r i o d , plates showing between 30 to 300 colonies were s e l e c t e d and the exact number o f colonies was counted.  The 'colony count" was c a l c u l a t e d by  m u l t i p l y i n g the average number o f colonies counted per p l a t e by the r e c i p r o cal of the d i l u t i o n . 3.  I s o l a t i o n : Each d i s c that was stored at 4 C u n t i l the time o f i s o -  l a t i o n s , was s p l i t open l o n g i t u d i n a l l y with a s t e r i l e axe (dipped i n 95% ethanol).  Young cottonwood stems were also sectioned l o n g i t u d i n a l l y using  an axe. Small chips o f wood (approximately  2 x 4 mm) were then removed asep-  t i c a l l y from the wetwood and sapwood o f the non-wounded trees and also from the d i s c o l o r e d wood o f the wounded t r e e s .  37 3.1  Aerobic b a c t e r i a ;  About 10 chips were placed i n an Omnimixer  (Ivan S o r v a l l Inc.) and were homogenized i n 25 ml o f s t e r i l e s a l i n e s o l u t i o n f o r about 5 minutes.  The suspensions were p l a t e d out on n u t r i e n t agar  medium and the p l a t e s were incubated at 25 C f o r 3.to 4 days.  Solitary  colonies were then i s o l a t e d and pure cultures were maintained i n 1.0% peptone water at 4 C.  Standard m i c r o b i o l o g i c a l studies ( C o l l i n s and Lyle 1970)  were done i n search o f the characters which could be used i n d i f f e r e n t i a t i o n o f the i s o l a t e d b a c t e r i a (Table V).  I d e n t i f i c a t i o n was done by f o l l o w i n g  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 f u n g i .  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 d i s h . of 3 weeks.  The p l a t e s were incubated at 23 C f o r a maximum  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 b a c t e r i a ( f a c u l t a t i v e ) :  Wood samples f o r t h i s study  came from three non-wounded t r e e s ; cores o f wood were obtained by using a s t e r i l e increment borer (dipped i n 95% ethanol) and were q u i c k l y t r a n s f e r r e d to a GasPak Anaerobic System ( f o r d e s c r i p t i o n , see Chapter I I I ) .  An equal  number of cores was then obtained from approximately the same p o s i t i o n i n the trees and was placed i n a p l a s t i c bag (aerobic conditions) t o avoid moisture l o s s .  A n a e r o b i c a l l y stored cores were fragmented i n t o small pieces a f t e r  they were brought back t o the laboratory and these pieces were q u i c k l y transf e r r e d oh t o the n u t r i e n t agar p l a t e s (stored under anaerobic c o n d i t i o n s ) . The inoculated p l a t e s were incubated i n the Anaerobic System at 30 C f o r about 14 days.  Duplicate culture plates were also made from the a e r o b i c a l l y  38 stored cores and were incubated under aerobic conditions t o detect the presence of the f a c u l t a t i v e anaerobes. 3.4  Anaerobic b a c t e r i a ( o b l i g a t e ) :  A separate study was made, to i s o -  l a t e s t r i c t anaerobes, p a r t i c u l a r l y the methanogenic b a c t e r i a , 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 i n v e s t i g a t o r o f the p r o j e c t , i n c l u s i o n of those r e s u l t s at t h i s place would have been i n a p p r o p r i a t e , and therefore the d e t a i l s of t h i s study are presented separately i n Appendix I.  RESULTS 1.  Quantitative determinations:  The v i a b l e 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 d i s c o l o r e d wood o f 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 b a c t e r i a attached to the c e l l w a l l s were not accounted f o r .  In a d d i t i o n , anaerobic b a c t e r i a present i n the  li-  quids would not survive under the aerobic c o n d i t i o n s . Nevertheless i t i s c l e a r that the wetwood and d i s c o l o r e d wood o f the wounded trees contain a high number of b a c t e r i a . 2. 2,1  Isolations Wetwood: Aerobic b a c t e r i a :  y i e l d e d b a c t e r i a (over 98%),  From a t o t a l of 248 i s o l a t i o n s , 244  Two genera, Erwinia and B a c i l l u s , were i s o l a t e d  c o n s i s t e n t l y from a l l seven trees.  Most of the i s o l a t e s of Erwinia were  motile rods, produced a c i d without gas and l i q u i f i e d g e l a t i n (Table V). 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  These  39  TABLE IV.  Number o f c o l o n i e s o f b a c t e r i a i n the l i q u i d s e x t r a c t e d from the wetwood (non-wounded t r e e s ) and d i s c o l o r e d wood (wounded t r e e s ) samples c o l l e c t e d at i n d i c a t e d h e i g h t s . Counts are from 1:10,000d i l u t i o n on n u t r i e n t agar p l a t e s .  Tree c h a r a c t e r i s t i c s  Type o f Wood  Sample h e i g h t  Colonies per plate  (ft[m] above ground)  (average o f two plates)  Number  Age (yr)  DBH Height (in[cm]) (ft[m])  1  37  25.0(63.5) 66.0(19.5) wetwood  2  47  29.0(73.7) 70.0(21.3)  3  41  31.0(78.8) 79.0(24.0)  4  42  31.0(78.8) 79.0(24.0)  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)  8  49  32.0(81.3) 84.0(25.5) d i s c o l o r e d wood  3.0(0.9)  68  9  45  27.9(71.1) 67.0(20.4)  4.0(1.2)  72  12.0(3.6)  70 68  M  80  „  61  „  "  Average  81  7.5  73  40  TABLE V:  Some morphological and p h y s i o l o g i c a l c h a r a c t e r i s t i c s o f b a c t e r i a cultured from wetwood  Tentative i d e n t i f i c a t i o n Characteristic  E r w i n i a Erwinia amylocarnevora gieana  Colony colour  Yellow-  Yellow-  Bacillus sp.  White  Protaminobacterium spT Red  Bacterium sp~]  White  EnteroHacteriaceae Creamy  ish  ish  Rod  Rod  Rod  Rod  Rod  Rod  Motility  +  +  +  +  +  +  Gram t e s t Spores formed  G=-  G=-  G=+  G=-  G=+  +  C e l l shape  Gelatin liquefaction  +  Starch hydrolysis  -  A c i d from sugars  +  -  Acid plus gas from sugars  -  +  Production of n i t r i t e s from nitrates  +  Production o f hydrogen s u l phide  +  S p l i t t i n g of alky1amines  Yellow  G=-  +(?)  -  -  +  41 starch or to produce n i t r i t e s from n i t r a t e s .  A few i s o l a t e s 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 n i t r a t e s and also produced hydrogen sulphide.  They are t e n t a t i v e l y i d e n t i f i e d as  Erwinia carnegieana. Species o f Erwinia are known to invade the t i s s u e s o f l i v i n g plants and produce dry n e c r o s i s , w i l t s and s o f t r o t s . Several i s o l a t e s were gram-positive and produced spores. according to Ske'rman's key (1959) belong to genus B a c i l l u s .  These i s o l a t e s ,  These were  motile rods, frequently i n chains, and produced acid i n sugar media.  A num-  ber o f i s o l a t e s were non-sporing rods which produced colonies with a red pigment and metabolized alkylamines. bacterium.  They were placed i n the genus Protamino-  There were two i s o l a t e s which were gram-positive, non-sporing,  motile rods and d i d not ferment carbohydrates. These probably belong t o the genus Bacterium, p r e v i o u s l y known as K u r t h i a .  There were several i s o l a t e s  which d i d not produce a c i d or gas when supplied with various sugars.  They  produced non-transparent, round colonies which were creamy-yellow i n c o l o r . 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:  from the wetwood.  One yeast and several m y c e l i a l fungi were i s o l a t e d  Colonies o f the yeast were white to creamy-yellow i n c o l o r ,  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 o f the  yeast i s o l a t e was not attempted. A l i s t o f mycelial fungi i s o l a t e d , t h e i r frequency and general charact e r i s t i c s i s given i n Table V I ; 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 y i e l d e d f u n g i .  Periicillium  was i s o l a t e d most frequently while Trichbderma, Cephalosporium, Epicoccum and  TABLE VI:  Frequency and c h a r a c t e r i s t i c s o f fungi i s o l a t e d from wetwood on 2% malt agar  PeniciIlium sp.  Frequency  28.1%  Trichoderma sp.  12.3%  Cephalbsporium sp.  10.6%  Epicoccum sp.  8.4%  Botrytis so.  Isolate BC-1  Isolate BC-2  Isolate BC-3  2.9%  4.3%  3.6%  2.9%  Culture c o l o r  Greenishgray  Originally white,later green due to patches o f conidia  White  Originally white,later slightly brown  Conidiophores conidia, etc.  Conidiophores arising singly, branched near apex ( p e n i d i late) ending in phialids;  Conidiophores hyaline, branched;  Conidia hyal i n e 1-celled, collecting i n slime-drope  Conidiophores Conidiophores compact, dark, long, slender, short; h y a l i n e , enlarged a p i c a l cells Conidia dark, 1-celled, Conidia hyaline globose to ash colored, 1-celled, black s c l e r o t i a produced frequently  Conidia hyaline to b r i g h t - c o l ored, 1-celled mostly globose, i n chains I s o l a t i o n Attempts - 140 Sterile  -  54 (38.6%)  Conidia hyaline 1 - c e l l e d , produced i n c l u s t ers  White  . White  White  Not  White  produced  B o t r y t i s occurred i n l e s s e r frequency and i n that same order.  Three  types o f f u n g i , a l l u n i d e n t i f i e d ( i s o l a t e s BC-1, BC-2, and BC-3) , were also  i s o l a t e d , a l l o f which produced white mycelia but f a i l e d to produce  any k i n d o f spores. 2.3  Wetwood: Anaerobic b a c t e r i a ( f a c u l t a t i v e ) :  Examination o f  matched n u t r i e n t agar p l a t e s , incubated under aerobic and anaerobic cond i t i o n s (18 attempts each) suggested presence o f at l e a s t three types o f f a c u l t a t i v e anaerobes (Types A, B and C). Type A b a c t e r i a were s m a l l , gram-negative rods b e l i e v e d to be a species o f Erwinia.  Types B and C,  both u n i d e n t i f i e d , were gram-positive rods and produced opaque colonies. Type B produced pink, round colonies while Type C produced creamy colonies with i r r e g u l a r margins. 2.4  Wetwood: Anaerobic b a c t e r i a ( o b l i g a t e ) :  2.5  Sapwood:  See Appendix I .  B a c t e r i a were i s o l a t e d very i n f r e q u e n t l y from sapwood;  only 9% o f the • c.Ul/tjirssw (4 out o f 45) were p o s i t i v e f o r b a c t e r i a .  These  i s o l a t e s were gram-negative rods and produced acid on sugar media. A l though t h e i r f u r t h e r 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 o f 40 i s o l a t i o n s  y i e l d e d f u n g i , both representing the genus P e r i i c i l l i u m .  Attempts to i s o -  l a t e anaerobic b a c t e r i a j f a i l e d ; no growth was observed i n any o f the i s o l a t i o n s made. 2.6  Wound-initiated d i s c o l o r e d wood:  Several types o f aerobic bac-  t e r i a were i s o l a t e d from the d i s c o l o r e d 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 b a c t e r i a were very s i m i l a r t o the ones i s o l a t e d f o r wetwood.  A s u b s t a n t i a l number o f f u n g i , mostly A s p e r g i l l u s and P e n i c i l l i u m ,  44  was also i s o l a t e d from the d i s c o l o r e d wood (32 out o f 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 o f P h o l i o t a while the other i s t e n t 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 o f 45)  while i n older ones (ages 7-15), some b a c t e r i a were present (9% o f 45 a t tempts) i n the sapwood.  The i s o l a t e d b a c t e r i a were not i d e n t i f i e d .  DISCUSSION Some e a r l y studies (Carter 1945,Seliskar 1950) suggested that b a c t e r i a are responsible f o r wetwood formation.  Conclusions  o f these authors are  p r i m a r i l y 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 entirely.  The i n o c u l a t i o n 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 d i s c o l o r a t i o n s as wetwood, which could have been merely browning o r i g i n a t i n g from the wounds. Knutson (1970) i s o l a t e d Erwinia from sapwood, wetwood, normal heartwood and d i s c o l o r e d heartwood o f aspen.  Because occurrence o f the organism was 'non-  s p e c i f i c ' and since h i s wounding experiments, not i n v o l v i n g microorganisms, produced symptoms o f wetwood, he concluded that there i s no reason to bel i e v e that Erwinia causes wetwood.  From Kriutson's experiments, i t appears  that the evidence he presents i s inconclusive rather than negative.  The  c o n t r o l trees that he used f o r wounding experiments (not i n v o l v i n g organisms) also contained Erwinia and therefore he cannot r u l e out the p o s s i b i l i t y that  45 Erwinia causes wetwood.  Perhaps some sort o f change i n conditions o f  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 format i 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 d i e of age or from impaired communication with cambium.  The symptoms o f wetwood condition are then pro-  duced by the action of the trees'own enzymes on the contents o f these affected c e l l s .  The-second and t h i r d hypotheses assume involvement o f bac-  t e r i a i n the process o f 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 subsequent changes are brought about by the a c t i v i t y of saprophytic b a c t e r i a . 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 b a c t e r i a .  These b a c t e r i a may or may not have  been present i n small numbers i n the normal wood but could be assumed to be capable of i n f e c t i o n only a f t e r the wood c e l l s have become senescent. The gas production and odor may be caused by secondary b a c t e r i a .  This t h i r d  hypothesis has been the one generally accepted as e x p l a i n i n g wetwood f o r 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 formation.  The observations were made by e x t r a c t i n g woody t i s s u e s from standing  trees and preparing matched samples f o r SEM examination and f o r i s o l a t i o n and c u l t u r i n g o f the microorganisms present. Samples were obtained from sapwood and wetwood of eastern cottonwoods and bigtooth aspens, and from  46. sapwood and normal heartwood o f 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 C l o s t r i d i u m sp., were c o n s i s t e n t l y found i n wetwood and adjacent sapwood of the poplar species studied.  No b a c t e r i a were  found or i s o l a t e d from normal heartwood and adjacent sapwood.  SEM obser-  vations suggested that wetwood occurs a f t e r an invasion o f sapwood vessels by  b a c t e r i a , presumably from i n i t i a l root i n f e c t i o n s .  A l s o , SEM studies  d i s c l o s e d differences i n the c e l l u l a r condition o f sapwood, normal heartwood and wetwood.  The main feature: o f sapwood was the t u r g i d smooth ap-  pearance o f the ray p i t membrane when viewed by SEM from the vessel lumen. The p i t membranes o f 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 o f a l l three species studied.  However, an increase i n the number o f  ray parenchyma c e l l s with wrinkled p i t membranes was evident i n sapwood i n trees with wetwood.  A l s o , the inner sapwood o f the trees with wetwood  was i n f e s t e d with b a c t e r i a and at the sap-to-wetwood t r a n s i t i o n zone, the vessel ray p i t membranes o f 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 t o w a r d / t h e p i t h region,  most membranes o f vessel to ray p i t s had been destroyed evident? i n the ray c e l l s . as b a c t e r i a l degradation  and b a c t e r i a were  Therefore, these authors characterized wetwood  of the p i t membranes o f vessel-to-ray p i t s .  Nei-  ther b a c t e r i a 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 d i d not s t a t e at what age wetwood  begins to appear i n these t r e e s , nor d i d they attempt to i s o l a t e b a c t e r i a  47.  from young t r e e s , presumably without wetwood.  I f b a c t e r i a 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 f o r i n i t i a t i o n of the b a c t e r i a l action on the vessel ray p i t s .  I f age o f 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 i n v e s t i g a t o r s ,  these authors did not o f f e r explanation of why b a c t e r i a l a c t i o n should cause water accumulation or high pH, which are so c h a 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 c l a s s 8 to 10 years, and i n 12 years or older t r e e s , wetwood was always present  (Chapter I ) . The d i s s e c t i o n studies also showed that wet-  wood always o r i g i n a t e d i n the center of the stem ( p i t h region) and extended r a d i a l l y i n a l l d i r e c t i o n s towards the sapwood.  The increase i n the  s i z e of wetwood was i n f a c t very- s i m i l a r to that o f a " t y p i c a l heartwood". Wetwood appeared as a p r o g r e s s i v e l y expanding core, the inner part of the sapwood being continuously transformed formed.  i n t o wetwood as more sapwood was  Heartwood also c o n s t i t u t e s such a continuously expanding core with-  i n a t r e e , i n c r e a s i n g i n diameter as the tree gets o l d e r .  In general, the  term heartwood r e f e r s to inner layers of wood which no longer c o n t a i n ; l i v ing c e l l s and from which the reserve materials have been removed or converted to more durable substances ( H i l l i s , 1971).  Wetwood, located i n the  inner layers of wood, also lacks v i a b l e parenchyma c e l l s (and reserve starch) and thus i s s i m i l a r to a t y p i c a l heartwood.  I t i s p o s s i b l e then  that wetwood formation i n black cottonwood requires an i n t e r n a l stimulus s i m i l a r to that needed f o r heartwood formation.  The nature of the stimulus  i s unknown but i t may w e l l be r e l a t e d to t r e e age and could b r i n g about  48 changes favorable to growth of b a c t e r i a . I s o l a t i o n studies reported here i n d i c a t e d that the p i t h and sapwood regions i n young cottonwoods o f 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 nonpathogenic with the exception of Erwinia. How  •<•. these microorganisms,  p a r t i c u l a r l y b a c t e r i a , obtain entry i n t o 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). multiply  When the conditions become s u i t a b l e , such b a c t e r i a may  r a p i d l y and i n h a b i t the wood.  Quantitative and frequency s t u -  dies showed that wetwood contained a large number o f b a c t e r i a .  Therefore,  i t i s conceivable that an age-related stimulus together with b a c t e r i a play an important r o l e i n wetwood i n i t i a t i o n , i n contrast to the concept of some workers (Carter 1945, S e l i s k a r 1950, Sachs 'et'aJL 1974) f e c t s of b a c t e r i a alone may be responsible.  that the ef-  Hartley and co-workers (1961)  second hypothesis- stated that the parenchyma dies n a t u r a l l y and the other changes noted i n wetwood are due mainly to the a c t i o n of saprophytic bacteria.  This hypothesis appears p l a u s i b l e f o r the wetwood formation i n  black cottonwood. The t h i r d hypothesis by Hartley et a l . (1961) s t a t e s , "The  death o f  the parenchyma i s caused or hastened by weakly p a r a s i t i c b a c t e r i a which  may  or may not have been present i n small numbers i n the n o n l i v i n g 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 b a c t e r i a i s o l a t e d  from the wetwood of black cottonwood were e s s e n t i a l l y saprophytes. l y , the young black cottonwood stems without wetwood were completely  Secondsterile.  49 I t i s p o s s i b l e , of course, that some "weakly p a r a s i t i c b a c t e r i a " might have been present i n "very small numbers".  In view o f 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 o f microorganisms i n wood. there i s no such evidence.  But to date,  In f a c t , Sivak and Person (1973) also found  that i n 1 and 2 year o l d black cottonwoods, p i t h and wood (presumably sapwood) were v i r t u a l l y free o f any microorganisms.  In consideration o f  these f i n d i n g s , I accept t h e s e c o n d hypothesis (Hartley et a l . 1961) as a l i k e l y hypothesis to e x p l a i n wetwood formation i n black cottonwood, as opposed to the t h i r d one which i s generally accepted f o r 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 o f a "natural process" than that caused by b a c t e r i a , i t i s st-i 11,not • known why the moisture content or pH are so high i n the wetwood. water may  Accumulation o f  be due to low permeability o f wetwood (Kemp., 1956).  The high  l e v e l s o f calcium carbonate reported i n the wetwood (Hartley et a l . could e x p l a i n the high pH values o f wetwood.  1961)  A l s o , i t may be due to ammonia  released by the b a c t e r i a 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)  s t a t e , "Whether wetwood i s the r e s u l t o f a n a t u r a l process o f the trees or induced by b a c t e r i a , no very p l a u s i b l e explanation occurs to the w r i t e r s as to the source of the excess moisture i n the wetwood.  I f water produced by  the o x i d a t i o n processes o f microorganisms were the answer, more water should be produced by decay fungi than b a c t e r i a , "  The oxidation process i s ob-  v i o u s l y not the answer as action o f decay fungi does not r e s u l t i n accumulat i o n of water.  Knutson (1970) stated that the high moisture content could  50 be simply "an osmotic e f f e c t " , without g i v i n g any s p e c i f i c d e t a i l s why i t may  occur.  One o f the most i n t e r e s t i n g features of the i s o l a t i o n studies  was  that wetwood y i e l d e d several species of aerobic b a c t e r i a and fungi (presumably a e r o b i c ) , i n a d d i t i o n to some f a c u l t a t i v e l y and o b l i g a t e l y anaerobic bacteria.  I t i s not known how  these various organisms with d i f f e r e n t  ©2 requirements e x i s t i n the same wetwood column under f i e l d conditions. I t has been demonstrated that the composition of gases w i t h i n trees d i f f e r widely from that of normal a i r (Chapter III) .  may  I t i s also known  that the composition w i t h i n trees changes with the seasons of the year. Therefore,  at any given O2 concentratiom (or during a given season), any  one group o f organisms must f i n d i t d i f f i c u l t to carry out a l l the metabol i c processes.  For example, O2 concentration of 20.0% would be generally  favourable to aerobic b a c t e r i a and f u n g i , while anaerobic survive i n the form of r e s t i n g s t r u c t u r e s .  b a c t e r i a may  On the other hand, under anaer-  obic c o n d i t i o n s , aerobic b a c t e r i a and fungi may  go i n t o 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 f o r determining the gas composition o f 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 I I I ) i n view of the data obtained during the composition studies.  gas  51  CHAPTER I I I COMPOSITION OF GASES IN THE TRUNKS OF BLACK COTTONWOOD  INTRODUCTION  The composition of gases w i t h i n tree trunks has been determined i n several species.  In most o f the e a r l i e r work, the primary goal o f the  i n v e s t i g a t o r s was to gather information f o r a b e t t e r understanding o f the pneumatic system of woody stems.  I t was demonstrated  that the gas composi-  t i o n i n trees d i f f e r s widely from that o f atmospheric a i r and that the comp o s i t i o n may vary i n d i f f e r e n t seasons o f the year.  Mostly, these studies  formed only a small p a r t o f a major study d e a l i n g with growth and water conduction i n trees. to the 0  2  In l a t e r i n v e s t i g a t i o n s , s p e c i a l a t t e n t i o n was given  concentrations and t h e i r p o s s i b l e s i g n i f i c a n c e to the a c t i v i t y  of wood-destroying  fungi.  Most o f the e a r l y studies have been adequately  described by Chase (1934) and only those which are d i r e c t l y r e l a t e d to the present work w i l l be reviewed here. In t h i s century, Bushong (1907) was f i r s t t o analyze gases c o l l e c t e d from the heartwood (most probably wetwood) o f a cottonwood t r e e .  The com-  p o s i t i o n of gases was as f o l l o w s : Q> , 1.24%; C0 , 7.21%; CH^, 60.90% and 2  N , 30.65%. 2  2  Ethylene and H were found to be absent. 2  Since then, several  authors have reported the composition of gases i n the heartwood o f various hardwood species.  In some cases, gases were present under pressure, and  o c c a s i o n a l l y they contained a flammable gas.  For example, when a b o r i n g  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  l i g h t e d match to the head o f the increment borer and observed that the gas immediately caught f i r e . In order to i n t e r p r e t the more recent studies on gas compositions, i t i s e s s e n t i a l t o consider the methods used by d i f f e r e n t researchers i n ext r a c t i n g the gases and analyzing them. to c o l l e c t gases was very simple.  In p r i n c i p l e , the methodology used  In each case, i n i t i a l l y , a bore was made  i n t o the trunk (generally at breast h e i g h t ) ; the depth o f the bore depended upon the tree zone under i n v e s t i g a t i o n .  Immediately a f t e r d r i l l i n g the  h o l e , a metal pipe or a probe was i n s e r t e d through the h o l e .  Each probe  normally c a r r i e d an on-off valve so that the gas samples could be obtained repeatedly.  As regards the withdrawal o f gases, some workers took advan-  tage o f the f a c t that gases w i t h i n trees are sometimes under pressure. For example, Bushong (1907) used a piece o f rubber tubing with one end connected to the gas pipe and the other i n s e r t e d i n a w a t e r - f i l l e d b o t t l e which i n turn was i n v e r t e d 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 c o l l e c t e d 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 e t c . Chase (1934) connected a p r e v i o u s l y evacuated gas sampling tube to the s t e e l tap already placed i n the tree. the  Then  valve between the pipe and the tube was opened and the tube was l e f t  clamped on t o the t r e e ,  A f t e r 48 hours, the gas sampling tube was removed  from the tree and the c o l l e c t e d 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 t h i s d e s c r i p t i o n o f the method used by Thacker and Good (1952), "Gas was extracted by connecting a m e r c u r y - f i l l e d 25 ml p i p e t t e t o the t a p , Fig.  2. The p i p e t t e was connected at the bottom to. a mercury r e s e r v o i r ,  the  height o f which could be v a r i e d .  When a l l connections had been sealed,  the  r e s e r v o i r was lowered, a l l o w i n g the mercury t o drain from the p i p e t t e .  The r e s u l t a n t suction caused a flow of gas from the tree i n t o the p i p e t t e . Two 25 ml samples were withdrawn from each h o l e , the f i r s t being discarded to eliminate traces o f atmospheric a i r i n the borings." The c o l l e c t e d gases were analyzed e i t h e r by performing standard chemical 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 a n a l y s i s , f o r ex-  ample, Chase (1934) used potassium hydroxide t o remove CC^, and a l k a l i n e p y r o g a l l o l t o absorb C^, from the gas samples.  The volume o f each gas was  then c a l c u l a t e d and expressed i n terms of percentage o f the t o t a l volume o f the  sample.  Zeikus and Ward (1974) analyzed gases by a gas chromatograph  equipped with a thermal c o n d u c t i v i t y detection system. s i t i v i t y o f the methods d i f f e r e d widely.  N a t u r a l l y , the sen-  Most workers were able to deter-  mine gas concentrations o f as low as 0.1% by the method they used, although some d i d not s t a t e 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 o f 5 years.  Their  study s t i l l represents the most comprehensive work done on t h i s subject. They surveyed pneumatic and h y d r o s t a t i c systenfof trees and some o f the con-  54  elusions they a r r i v e d at are as follows , 1)  Gases o f the pneumatic system occur generally at pressures not very d i f f e r e n t from barometric,  2)  The most common difference between the gases o f the pneumatic system and the (atmospheric) a i r i s the accumulation of CC>2 and the d e p l e t i o n o f Oj.  3)  The high proportions o f CO^ i n the woody tissues could be a product o f r e s p i r a t i o n , or the amount d i s s o l v e d i n the streams o f the h y d r o s t a t i c system, or d i f f u s i o n i n t o and out of l i v i n g c e l l s and o f streaming movements to the outer a i r .  4)  The low proportions (or o c c a s i o n a l l y none) o f 0^ i n the pneumatic system may be ascribed t o i t s combination i n resp i r a t i o n and p o s s i b l y i n connection with a c i d i f i c a t i o n .  Chase (1934) studied composition o f gases drawn from 4 hardwood and 1 softwood species, and a s p e c i a l emphasis was given t o the seasonal f l u c t u a t i o n s o f gases.  A d e f i n i t e r e l a t i o n s h i p was found between metabo-  lism and the i n t e r n a l gas content, CO^ concentration being higher i n a l l species during the growing season and lowest during the winter. s i t e was true f o r 0^. In case of a s i n g l e cottonwood  The oppo-  (Populus delto.ides  v i r g i n i a n a [ C a s t i g l i o n i ] Sudworth), CO^ reached a maximum over 25% on August 1 i n the heartwood and was above 15% f o r most o f the time from August to November.  During J u l y and August, very low percentages o f 0^  were present, and i n one sample none at a l l could be detected.  In December,  however, the percentage o f 0^ rose r a p i d l y to over 16, while the CO^ content d e c l i n e d to a low l e v e l .  Chase also found that r a p i d l y 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.) o r red oak (Quercus rubra L.). Generally there was more 0 sapwood than i n the heartwood.  2  and less CO,, i n the  A l s o , there appeared to be no r e l a t i o n -  ship between gas compositions at d i f f e r e n t heights w i t h i n a s i n g l e tree. Thacker and Good (1952) found much smaller seasonal f l u c t u a t i o n s i n the CO2 and 0^ contents o f sugar maple (Acer saccharum Marsh.).  C 0 con2  tent i n the heartwood o f 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 e a r l y 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 i n v e s t i g a t e d 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.  I n d i v i d u a l t r e e s , 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 w i t h wetwood. The gas contained approximately 46% CH , 34% N , 14% 4  2  C0 > 2  5%  0  2  and 1% H^.  Morani and Arru (1958) studied gas composition o f some 17 year o l d v  poplars showing symptoms o f a b a c t e r i a l i n f e c t i o n i n the c e n t r a l part o f t h e i r trunk, " s i m i l a r to those described by many authors f o r the wetwood disease." 17.2%.  The composition o f gases was, CH^, 53.6%; N , 28.4% and C 0 , 2  2  They considered fermentation a c t i v i t i e s o f methanogenic and deni-  t r y f y i n g b a c t e r i a as the cause of the higher concentrations o f CH. and CO-.  56  Jensen (1969) found that  concentrations i n stems of red oak trees  were u s u a l l y less than 2% i n sound trees and less than 4% i n decayed t r e e s . v a r i e d between 14 to 20% i n sound trees and 7 and 21% i n decayed t r e e s . No c l e a r - c u t r e l a t i o n s h i p s were found between gas concentrations and d i f ferent seasons, heights (within a tree) or depth (distance from cambium) i n the t r e e s . 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%) c o l l e c t e d from 2 eastern cottonwoods. was also present i n high proportions (14.1 and 16.3%), both the trees while 0  2  was absent.  C0  2  was detected i n  Both of the trees contained wetwood.  Analysis of gases from a white poplar heartwood showed high concentrations of N  (75.1%) and low proportions of 0  2  In summary, very low 0  2  2  (6.4%) while CH^ was  absent.  tensions were found i n f a s t growing hardwood  species such as cottonwoods and, during the growing season, no 0 detected i n some analyses. t i o n , low 0  and high C0  2  2  2  could be  In species showing a p a t t e r n of wetwood formaconcentrations 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 w i t h i n a s i n g l e t r e e , the composition was very s i m i l a r throughout the length of the trunk. and lower C0  2  Usually sapwood e x h i b i t e d higher 0  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  e x i s t i n proportions s o - d i f f e r e n t 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 d i s s o l v e d i n the water of the sap stream.  Another means  of entrance of gases i n t o the trunk may be through the stomata o f the leaves (Chase 1934).  However, so f a r 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 t r a n s p i r a t i o n 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 o f the bark i n t o the i n t e r c e l l u l a r spaces o f the cortex and phloem. account f o r the higher  This may  content generally found i n the sapwood.  The abnormal proportion o f gases i s sometimes a t t r i b u t e d to the metabolism of the t r e e .  Chase (1934) states that CG^ w i t h i n the sapwood and  heartwood of a tree may o r i g i n a t e from the r e s p i r a t i o n 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. months these c e l l s are very a c t i v e and  During the summer  i s low and C0*2 i s high.  MacDougal  and Working (1933) also consider " r e s p i r a t i o n o f l i v i n g c e l l s " as the p r i n c i p a l cause f o r low  and high CO^ i n the trunks.  Morani and Arru  (1958), and Zeikus and Ward (1974) considered b a c t e r i a responsible f o r the gas contents of the tree trunks.  High concentrations o f CH^ were c e r t a i n l y  produced by the a c t i v i t y o f methanogenic b a c t e r i a .  These concentrations  o f gases are apparently maintained w i t h i n the trunk of trees because the l i v i n g cambium o f f e r s a good b a r r i e r to the d i f f u s i o n o f gases (Kramer and Kozlowski 1960). The r o l e these gases play i n the actual metabolism of the tree i s not very c l e a r , c e l l s . C0  0^ i s involved i n oxidation o f materials w i t h i n the l i v i n g  i s not used i n the xylem tissues o f the stem, although some o f be i t may d i f f u s e through the cambium and/used i n photosynthesis i n the bark. 9  58 Numerous i n v e s t i g a t o r s have speculated upon the cause of heartwood f o r mation.  I t has been suggested that the large amounts of C0  amounts of 0  2  2  and small  are responsible f o r the change from a l i v i n g to a dead con-  d i t i o n (heartwood).  A f t e r discussing the r o l e of gases i n metabolism o f  the t r e e , Chase (1934) concluded, " I t appears that the varying  proportions  o f these gases i n the trunk are a r e s u l t o f metabolism and probably have s l i g h t influence upon i n t e r n a l l i f e  processes."  As pointed out e a r l i e r , some of the studies dealing with composition of gases w i t h i n tree trunks gave consideration to e f f e c t s 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" and C0 2  the trunks of maples.  2  concentrations, i n c l u d i n g those found i n  0 , i n the percentages found by them i n maples, d i d 2  not l i m i t growth of any of the fungi tested.  A l s o , CO^  ployed generally favored growth of most fungi tested.  concentrations The authors,  em-  there-  f o r e , concluded that the composition o f a i r commonly found i n maple trunks during the growing season i s nearly optimal f o r the development o f wooddestroying f u n g i .  Whether t h i s view can be adopted as a v a l i d g e n e r a l i z a -  t i o n f o r a l l tree species and a l l wood-destroying fungi seems open to question.  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 l e a s t during the growing season, and o c c a s i o n a l l y none at a l l could be detected Ward 1974). i n maples was 0.4%.  (MacDougal and Working 1933, Chase 1934,  The lowest 0 0.8%;  2  Zeikus and  concentration found by Thacker and Good (1952)  the lowest l i m i t of detection i n t h e i r analyses  0„ concentrations of 0.4%  was  and less have already been recorded i n  59 some tree species.  In a d d i t i o n , Thacker and Good's c u l t u r a l experiments  i n v o l v i n g wood-destroying fungi were done on agar medium as opposed to wood, and the growth response o f fungi on these 2 media cannot be assumed to be the same.  A l s o , many f i e l d experiments have i n d i c a t e d that decay .  columns i n trees may become i n a c t i v e a f t e r the entry p o i n t i s healed (Hepting. 1941, Toole 1965).  These observations and the inferences that  can be drawn from them suggest that a d d i t i o n a l b a s i c data are required before any generalizat i o n , such as that o f Thacker and Good (1952) , can be accepted. The present study was conducted to determine the gas composition o f wetwood and sapwood o f black cottonwood, and to see i f the gas l e v e l s were d i f f e r e n t 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 e f f e c t on the growth of woodfedestroying fungi (Chapter IV). I f a r e l a t i o n s h i p between gas composition and growth of fungi i s to be assumed, then i t i s important to know what e f f e c t s wounding may have upon the gas composition. A l s o , i t i s e s s e n t i a l to know i f branch stubs act as openings allowing gases to move i n and out.  Therefore, the s p e c i f i c objectives  of t h i s study were, 1)  to extract and analyze gases from the wetwood and also from the sapwood o f several black cottonwood trees f o r a period o f 1 year,  2)  to examine e f f e c t s of wounding on the gas composition of wetwood,  3)  to determine i f exchange o f 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, f o r comparative purposes.  MATERIALS AND METHODS  1.  Location of sample t r e e s :  A l l of the trees that were sampled  during gas composition work are located on the U n i v e r s i t y Endowment Lands (U.B.C), Vancouver, B.C.  Black cottonwood and red alder (Alnus rubra Bong.)  trees are s i t u a t e d north of the U n i v e r s i t y Boulevard, surrounding U n i v e r s i t y H i l l Church (Figures 11 and 12).  Two  the  of the Lombardy poplar  trees are located behind the U n i v e r s i t y Health Center and the t h i r d was behind the E l e c t r i c a l Engineering Department.  This tree was  one  cut down  i n the summer of 1974 when the area was.cleared f o r construction p r o j e c t s . The d e t a i l s of c l i m a t i c c o n d i t i o n s , s o i l s and the h i s t o r y of the U n i v e r s i t y 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. (above 5 C) i s 265 days. tween November and A p r i l .  The length o f the growing p e r i o d  Sharp f r o s t s l a s t i n g for. several days occur beThe 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 o f only about 30 cm evenly d i s t r i b u t e d . 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 depending upon the horizon sampled.  6.7  61  Figure 11: Map of the University Endowment Lands showing location of sample trees used for the gas composition work. Xx*x% xxx  °a>ft°  - Black cottonwood and red alder " Lombardy poplar  F i g u r e 11  63  F i g u r e 12:  B l a c k cottonwood-red a l d e r s t a n d s u r r o u n d i n g 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) o f f o r e s t extending from the Spanish Banks to the U n i v e r s i t y Boulevard was cleared. l a r g e l y covered by a red alder stand.  This area i s now  The area, s l i g h t l y north o f Univer-  s i t y Boulevard and near the U n i v e r s i t y H i l l Church, c o n s i s t s o f a f a i r l y even mixture o f red a l d e r , black cottonwood and maple, with very few coniferous species as an understorey.  Vigorous growth o f salmonberry (Rubus  s p e c t a b i l i s Pursh) and swordfern (Polystichum munitum) [Kaulf.] P r e s l . ) i s also  apparent i n t h i s area.  As i n d i c a t e d e a r l i e r , gas composition studies  were done on black cottonwood and red alder trees growing i n t h i s area. Generally, i n d i v i d u a l t r e e s were selected at random although p r e f e r ence was given t o the large diameter ones, and i n a few cases, f u r t h e r sel e c t i o n was necessary depending upon the purpose o f the experiment.  The  relevant d e t a i l s o f the trees such as age, diameter at breast height (dbh), height, wood zone i n v e s t i g a t e d , e t c . are given i n Table V I I . composition studies were made i n the year 1974.  A l l o f the gas  Some trees were examined  a l l through the year while others were examined only during c e r t a i n months of the year.  In general, gas e x t r a c t i o n and analysis were done on an i n d i -  v i d u a l tree every two weeks once the i n v e s t i g a t i o n began. 2.  E x t r a c t i o n apparatus:  The d e s c r i p t i o n o f the i n d i v i d u a l apparatus  used i s as f o l l o w s , A.  Brass pipe: S p e c i a l l y designed (Figure 13-A); l e n g t h , 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 c a r r i e s f e male thread, 1/8 NPT.  B.  Shut-off valve (Fairview F i t t i n g s and Manufacturing L t d . ) : Hose barb t o male pipe; hose O.D., 0.25 i n (0.6 cm).  TABLE V I I : Description o f sample t r e e s , wood zones and duration o f sampling i n the year 1974  Species  Black cottonwood  Tree number  Tree c h a r a c t e r i s t i c s  Wood zone extracted  Duration o f sampling  Age (yr)  DBH ( i n [cm])  10  23  18.1(46.0)  60.0(18.3)  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)  7  17  9.7(24.6)  50.0(15.2)  Height (ft[m])  Wetwood  Remarks  Jan-Dec.  Jan 11  Jan  £  Table VII (cont'd)  Species  Black cottonwood  Tree Number  14  Tree characteristics Age (yr)  18  DBH (in [cm])  8.5(21.7)  Height (ft[m])  41.0(12.5)  Wood zone extracted  Wetwood  17  20  15.1(38.5)  50.0(15.3)  it  18  21  12.0(30.3)  60.0(18.2)  II  12  22  11.1(28.4)  44.0(13.4)  16  21  11.4(29.2)  58.0(16.0)  Sapwood  Duration o f sampling  Feb. Feb, Apr Feb -Mar  II  Mar. Apr. May  32  20  15.3(39.0)  43.0(13.0)  it  33  20  11.5(29.3)  43.0(13.0)  it ii  34  20  11.8(30.2)  52.0(15.7)  35  21  10.3(26.3)  45.0(13.50  II  Aug  May-June Jun.-Jul  36  22  10.0(25.4)  54.0(16.3)  it  37  21  9.7(24.7)  52.0(15.7)  M  Sep-Oct  II  Oct  38  22  10.6(26.9)  59.0(17.9)  2  19  10.4(26.4)  43.0(13.1)  3  23  24  22  16.8(42.7)  58.0(17.5)  30  23  10.5(26.7)  46.0(14.0)  19  23  16.1(40.9)  23  23  14.0(35.6)  Wetwood  Remarks  Feb-Dec  II  Mar-Dec  II  May-Aug  tt  May-Aug  55.0(16.9)  H  Apr-Aug  49.0(14.8)  M  Apr-Jun  Wounded n  Wounded-Sapwood exposed  Through branch stub ON  Table VII (cont'd)  Species  Red alder  Lombardy poplar  Tree Number  Tree c h a r a c t e r i s t i c s Age (yr)  DBH ( i n [cm])  Height (ft[m])  41  23  10.0(25.4)  37.0(11.2)  42  23  9.9(23.1)  40.0(12.3)  25  20  9.0(23.0)  42.0(12.9.)  44  Data  45  24  21.1(53.6)  56.0(17.0)  46  23  22.5(57.4)  59.0(18.1)  not  available  Wood zone extracted  Duration of sampling  Heartwood  Feb-Dec Feb.-Dec Jan  Heartwood (probably wetwood) II  May May-Jun  Remarks  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.  glass tube with a s l i g h t bend.  C. Small  "0.75"  8.00  Figure 13  70  C. Masterflex tubing pump (Cole-parmer Instruments L t d . ) : Cons i s t s o f 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 o f tubing from and to the system; no p o s s i b i l i t y o f any contamination o f the c i r c u l a n t ; p r a c t i c a l l y p u l s e l e s s and provides smooth s u c t i o n ; gases may be pumped at pressures up t o 15 p s i (1.05 kg/sq cm), l i q u i d s up t o 40 p s i (2.8 kg/sq cm) i n t e r m i t t e n t l y ; vacuum may be p u l l e d up to 24 i n (60.9 cm) o f Hg; tubing pump provided with a s o l i d - s t a t e speed c o n t r o l l e r , 30 t o 60 RPM; flow rates can be e s t a b l i s h e d accurately and maint a i n e d ; obtainable flow r a t e s , from 1.8 ml/minute t o 2280 ml/minute. D.  F i e l d - l a b 0~ analyzer (Beckman's Instruments Co.): Analyzer equipped with a sensor; i n operation, the sensor i s placed i n the sample, a p o t e n t i a l o f 0.53 v o 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 d i f f u s e s through a Teflon membrane, d i f f u s e d 0^ ^ ~ duced e l e c t r o c h e m i c a l l y at the cathode; t h i s reduction causes a current flow p r o p o r t i o n a l to the p a r t i a l pressure o f 0^ i n the sample, the flow i s a m p l i f i e d on a s c a l e ; i n gas, 0„ measurement l i m i t s are 0.05 t o 25.0%, and i n l i q u i d s , tney are 1.0 to 25.0 ppm. s  re  E.  Glass tubings: a. S p e c i a l l y designed (Figure 13-B); length, 8.0 i n (20.3 cm); large diameter, I.D., 1.5 i n (3.8 cm), r e 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 o f the stem, 1.0 i n (2.5 cm). b. S p e c i a l l y 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 t o hold l i q u i d from 100 to 310 ml approximately. In a d d i t i o n 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 t o 76.2 cm] o f Hg), 2 shut-off v a l v e s , a flow meter (range: 1.3-23 to 400 ml/minute), a thermometer (range:.-20 to 110 C), a Erlenmeyer f l a s k (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.  E x t r a c t i o n apparatus:  Assembly:  Gas sampling tube, l a r g e d i a -  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 o f 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 i n t o  the plywood and the rubber bands attached t o the cup-hooks h e l d the apparatus i n place.  The plywood piece w i t h 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 o f 0.5 i n (1,3 cm) plywood leaving enough space i n f r o n t t o 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 t o enter the case while working i n the rain.  A handle f i x e d on the top o f the case made the whole system portable. The general assembly o f the e x t r a c t i o n apparatus i s shown i n Figure  15.  The sequence o f the connections (made with v i n y l tubing) was as f o l l o w s :  (tree) flask  tubing pump -* gas sampling tube -•-»- 0 ->• flow meter.  2  analyzer  Erlenmeyer  The vacuum gauge was i n s t a l l e d between the tree and  the tubing pump t o record the suction developed. placed between the tubing pump and the 0  2  The s h u t - o f f valve was  analyzer ( i . e . sensor i n the large  diameter glass tube), running p a r a l l e l t o the gas-sampling tube, i n case o f excessive flow o f l i q u i d s from the t r e e . the 0  2  The thermometer, the sensor (of  analyzer) and the small diameter glass tube (with a s l i g h t bent) were  i n s e r t e d 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 o f approximately 45° t o the base o f the case.  The small d i a -  meter glass tube was p o s i t i o n e d 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 i n s i d e o f the large diameter glass tube.  Both o f these arrangements, i n a d d i t i o n t o the s l a n t -  i n g p o s i t i o n o f the large diameter glass tube, ensured maximum s w i r l i n g movement o f the gases and at the same time, avoided l i q u i d from g e t t i n g i n cont a c t with the sensor-tip.  The Erlenmeyer f l a s k , connected next to the large  72  F i g u r e 14:  P o r t a b l e e x t r a c t i o n apparatus mounted i n a plywood case.  73  Figure 15: Schematic drawing o f the e x t r a c t i o n 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, F i e l d - l a b  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 o f the gases and l i q u i d s  would be as f o l l o w s : (Tree) • — Tubing pump sampling tube — for liquids)  0  2  Gas-  analyzer --- Erlenmeyer f l a s k (trap  Flow meter  (Atmosphere) .  75  diameter glass tube, trapped any l i q u i d while gases went ~ f u r t h e r through the flow meter to the atmosphere.  A s h u t - o f f valve was placed between the  Erlenmeyer f l a s k and the flow meter, and opening o f the valve was adjusted i n such a way that a steady flow o f gases could be obtained without c r e a t i n g high gas pressures w i t h i n the e x t r a c t i o n apparatus.  This was necessary to  remove the p u l s i n g e f f e c t o f the tubing pump on the flow meter. 4. Method and operation o f e x t r a c t i n g gas samples:  An increment, bor-  e r , which was s t e r i l i z e d by dipping i n 95.0% ethanol, was used t o bore a hole reaching the approximate center o f the tree ( i n the case o f wetwood and heartwood).  The core was removed and r e t a i n e d to detect presence o f wetwood,  tree age and t o study the microorganisms.  The increment borer was then r e -  moved and a brass pipe was i n s e r t e d immediately through the hole and hammered i n such a manner that the t i p o f the pipe would be about 2-3 cm from the end of the bore (Figure 16).  A s h u t - o f f 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 -  l e c t e d one week from the day pipe was placed i n the tree? allowing the gases i n the tree and the pipe t o reach an e q u i l i b r i u m . Before e x t r a c t i n g gases, the 0  2  analyzer was c a l i b r a t e d according t o  the temperature o f the a i r with the assumption that atmosphere contains 21.0% 0^. V i n y l tube leading to the tubing pump was then attached t o the  connect-  ing hose f i t t e d on the bbrass pipe and the s h u t - o f f valve opened (Figure 17). The tubing pump connected to the power supply was switched on (with a s e t t i n g of 4 on the c o n t r o l l e r ) and the gases and l i q u i d s flowed out o f the t r e e . During the e x t r a c t i o n operation the vacuum gauge, thermometer, 0^ analyzer and flow meter were observed constantly.  Any change i n the temperature was  76  F i g u r e 16:  A.  Schematic drawing o f t h e b r a s s p i p e 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  o f t h e h o l e made w i t h an increment b o r e r .  extent W -  wetwood, S - sapwood. B.  Brass p i p e and s h u t - o f f v a l v e i n p o s i t i o n i n t h e field.  77  F i g u r e 16:  C.  V e r t i c a l s e c t i o n o f a b l a c k cottonwood t h r o u g h the sampling hole  ( e x t e n d i n g 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 .  Portable  e x t r a c t i o n apparatus on the ground, connected t o the pipe  (and t r e e ) w i t h a v i n y l t u b i n g .  gas  brass  80  adjusted f o r on the 0 f o r C0  2  analyzer.  The actual time o f c o l l e c t i n g gases  and CH^ a n a l y s i s , and recording temperature (C), suction developed  2  (in [cm] o f Hg) and flow rate (ml/min) o f the gases was when the i n d i c a t o r on the 0 utes.  2  analyzer remained steady at a c e r t a i n p o s i t i o n f o r about 2 min-  The whole e x t r a c t i o n procedure normally took about 10 minutes.  t e r c o l l e c t i n g a sample from one tree and recording 0 gas sampling tube (containing gas sample f o r C0 moved. 0  2  2  2  Af-  concentration, the  and CH^ analysis) was r e -  The complete operation of e x t r a c t i o n , i n c l u d i n g c a l i b r a t i o n of the  analyzer, was then repeated to c o l l e c t a gas sample from another t r e e .  During the c a l i b r a t i o n , a l l o f the gases present i n the e x t r a c t i o n apparatus from the previous tree are removed. The method of e x t r a c t i n g gases from the sapwood, from the wetwood through branch stubs and from the wetwood o f wounded trees was e s 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 r e -  sistance to e x t r a c t i n g gases on a continuing basis from the same t r e e . About 4-6 weeks a f t e r p l a c i n g the pipe i n theesapwood, no gases could be withdrawn therefore allowing only 2 or 3 sample c o l l e c t i o n s per t r e e .  Con-  sequently, gases were c o l l e c t e d from the sapwood o f d i f f e r e n t 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.  Initially,  a brass pipe was placed i n each tree approximately 5.0 cm away, e i t h e r h o r i z o n t a l l y (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 e x t r a c t i o n and analysis were done as usual.  Then, i n each case,  the e n t i r e 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 o f the dead branch (Figure 18).  A f t e r 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 o f wounded t r e e s : Wetwood exposed:  One tree (No. 2) was n a t u r a l l y wounded, and 2 trees were wounded during the gas composition work (No.s 3 and .21). unknown.  The cause o f the wound i n Tree 2 was  A v e r t i c a l crack was present s t a r t i n g from the ground l e v e l going  upwards f o r about 1.5 m.  In t h i s t r e e , 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 r e g u l a r l y from March u n t i l the middle o f May and then was wounded with an increment borer exposing the wetwood. made about 8.0 cm below the normal e x t r a c t i o n p o i n t . and analyses were made f o r about 2 months.  The hole was  Then the e x t r a c t i o n  In the l a s t week of J u 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 w i t h i n 24 hours, the wound was sealed and the gas analysis was resumed. 4.4  Black cottonwood: Wetwood o f wounded t r e e s : Sapwood exposed:  Two trees were sampled to examine p o s s i b l e e f f e c t s o f sapwood exposure on the gas composition of wetwood.  Before wounding, gases were withdrawn and  analyzed from the wetwood o f both the trees f o r 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 t r e e , about 5C0icm away from the normal e x t r a c t i o n p o i n t .  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 e x t r a c t i o n p o i n t and the one on the r i g h t i s where the dead branch was present.  F i g u r e 18  84  F i g u r e 19:  A b l a c k cottonwood t r e e (No. 2) showing n a t u r e o f t h e wound ( l e f t ) and normal e x t r a c t i o n s e t - u p  (right).  85  Figure 20:  Schematic drawing o f the two trees showing nature o f 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  .1.1 A Sapwood removed w i t h an axe  w  Tree 24  ,  r  - l - l  ^_  Sapwood removed with an increment b o r e r  w Tree 30  F i g u r e 20  87  were made from a l l sides with an increment borer exposing i t s sapwood. Gas e x t r a c t i o n and analysis were then continued f o r another 2 months. 5.  Gas a n a l y s i s :  As i n d i c a t e d e a r l i e r , 0^ concentrations were mea-  sured i n the f i e l d during actual e x t r a c t i o n 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 o f 0^ (weight/  volume) present i n the sample. The concentrations o f C 0 and CH^ were determined by gas chromatography 2  as described by Smith (1973).  This analysis work was done under the super-  v i 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 f l a m e - i o n i z a t i o n detector, each gas sample was separated on a 10 f t (304.8 cm) by 3/16 i n (4.8 cm) s t a i n l e s s s t e e l column, packed with Poropak Q (120 mesh).  The C0 was reduced ( a f t e r separation from any CH^ i n the 2  sample) to CH by passing i t through a 9 i n (22.9 cm) by 1/8 i n (3.2 mm) 4  s t a i n l e s s s t e e l column containing a n i c k e l c a t a l y s t . r i e r gas (flow rate 25 ml/min) and N min).  2  H  2  was used as a car-  as the d i l u e n t i n the detector (25 ml/  The flow rate o f the combustion a i r t o the detector was 300 ml/min.  Signals from the detector were passed through a d i g i t a l i n t e g r a t o r (Hewlett Packard, Model 3370A), with a code converter board i n t e r f a c e t o a t e l e p r i n t er (Teletype Corporation, No. 33-L), thereby providing a p r i n t e d readout o f peak-time and area. A c a l i b r a t i o n graph o f C 0 concentration and i n t e g r a t o r response (micro2  v o l t s x seconds, uV.s) was prepared by taking 0.5 ml samples from known concentrations o f C 0 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 i n t o 2  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 v e s s e l provided with a rubber septum. Another c a l i b r a t i o n graph was also prepared f o r 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 II.  The s e n s i t i v i t y of the gas chromatograph u n i t was w e l l below 0.1% f o r  both the gases. Gas samples c o l l e c t e d from the trees were u s u a l l y analyzed w i t h i n 2 hours from the c o l l e c t i o n time.  Using a gas s y r i n g e , a gas sample of 0.5  ml was taken from the gas sampling tube and i n j e c t e d i n t o the gas chromatograph.  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 w i t h i n wetwood are present under pressure during summer.  In each case, the brass pipe was placed i n the t r e e as usual  and a pressure gauge (range: up to 30 p s i [2.1 kg/sq cm]) was screwed on to the brass pipe.  immediately  The gauge was observed every week f o r a per-  iod of 2 months.  RESULTS In the course o f t h i s study, more than 1200 i n d i v i d u a l gas analyses were made. Most of these determinations were made on gases extracted from the wetwood o f black cottonwood trees. a l i m i t e d time.  Some trees were examined only f o r  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 s e l e c t e d to show the seasonal v a r i a t i o n s of the gases w i t h i n a s i n g l e t r e e , and also the gas compositions of 2 d i f f e r e n t tree species or tree zones f o r compara-  89 t i v e purposes. 1. l y the  They are presented g r a p h i c a l l y i n Figures 21-24, 27-32.  Black Cottonwood: Wetwood: Examined a l l through the year:  General-  concentration i n the wetwood was lower during the summer than  the winter and  the reverse was true f o r CO^.  i  In 5 o f the 6 r e g u l a r l y t e s t e d  t r e e s , the 0^ concentration dropped below the l i m i t o f measurement (<0.05%) i n the summer.  Concentrations  of CH^ were c o n s i s t e n t l y low throughout the  year i n a l l r e g u l a r l y examined t r e e s .  In Tree 10, 0  was low a l l through  2  the year and the presence o f microaerobic conditions* p e r s i s t e d f o r about 10 weeks during the summer.  In t h i s t r e e , C0 was higher (13.1%) i n the  summer than any other t r e e tested.  2  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 thereafter.  Flow o f the gases was generally high (75-100 ml/min).  c o n s i s t e n t l y low i n Tree 15 (Figure 21).  0  2  was also  In the summer, wetwood e x h i b i t e d  the presence o f microaerobic conditions f o r almost 12 weeks and even during the winter, 0 high.  2  d i d not reach 2.0% l e v e l .  C0 concentrations were g e n e r a l l y 2  CH^ was present i n the t r e e throughout the year, although not i n any  s u b s t a n t i a l q u a n t i t i e s . Tree 6 was the only tree i n which wetwood never became microaerobic. the wetwood ( >0.1%).  Even during the summer, some O2 was always present i n C0 was generally high, and CH was found i n the 2  tree a l l through the year (Figure 22).  4  Trees 11, 8 and 16 followed patterns  1 The term "microaerobic c o n d i t i o n s " was used to describe "presence o f very small concentrations o f 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 s o p h i s t i c a t i o n of the d e t e c t i o n system, they are subject to include a wide range o f O2 concentrations. In t h 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 Aerobic c o n d i t i o n ^0,1% 0„ :  2  90  Figure 21: Concentrations o f 0 , C0 and CH^ (monthly averages) i n 2  2  the wetwood o fh black cottonwood (Tree 15). :  Microaerobic  conditions were f i r s t noted i n the 4th week o f A p r i l and they p e r s i s t e d u n t i l the 3rd week o f J u l y , 1974  20.0  ° - ° 2 A  n -  18.0  16.0  T r e e 15 14.0  2 o l-H  12.0  E-i <. OS  EIZ  10.0  W C_>  O  u  c/}  - CO  8.0  6.0  4.0  2.0  O. • ^v—n M  A  . a-o-  aM  J  TIME O F YEAR F i g u r e 21  J  •O' >  CH  92  Figure 22.  Concentrations o f 0^, CO^ and CH  4  (monthly average)  i n the wetwood o f a black cottonwood (Tree 6 ) . In t h i s t r e e , microaerobic conditions were not present even during the summer.  20.0  o  -  o  A  - CO  2  o - CH  18.0  16.0 Tree 6 14.0  12.0  10.0  8.0  6.0  4.0  2.0  J  F  M  A  M  J  TIME OF YEAR Figure 22  J  94  of gas compositions s i m i l a r to that of Tree 1 0 , a l l becoming microaerob i c during the summer. 2.  Black cottonwood: Wetwood: Examined only during c e r t a i n months  of the year:  Eighteen trees were examined o c c a s i o n a l l y f o r t h e i r gas con-  tents during the year.  S i x out o f 8 trees sampled during the summer had  $2 concentrations below or close to 0.05%.  The general trend of low 0^  high CO2 was evident i n a l l 8 trees examined. v a r i e d from 0.0 to 34.0%.  CH^ concentrations, however,  Tree 21 which was examined f o r a longer p e r i o d  than others, contained high concentrations o f CH^ May.  and  (30.5 - 34.0%) during  This tree also e x h i b i t e d the e f f e c t s of wounding on i t s gas composi-  t i o n which w i l l be discussed l a t e r . i n g the w i n t e r , 0  2  In the remaining 10 trees examined dur-  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 i f f e r e n t from that of wetwood (Figure 23). were high throughout the year (11,4 l y low (0,1 - 0.8%).  2  contents  contents were general-  There was no d i s t i n c t r e l a t i o n s h i p between gas concen-  t r a t i o n s and the season. sampled.  - 19.0%) while C 0  0^  A l s o , CH^ was absent i n the sapwood of a l l trees  I t should be noted that gases were withdrawn from d i f f e r e n t trees  f o r about 8 months of the year.  This was due to the r e s i s t a n c e o f f e r e d by  wood i n e x t r a c t i n g the gases from a s i n g l e tree on a continuing b a s i s . 4.  Black Cottonwood: Wetwood through branch stubs:  Before p l a c i n g  pipes through the branch stubs, gas concentrations of both the trees were determined. some CH.  0^ was low and C0  (Figure 24).  2  was high and the 2 trees also contained  As described p r e v i o u s l y , i n Tree 19, o r i g i n a l pipe  95  Figure 23:  Comparison o f 0^ and C0  2  concentrations (monthly averages)  i n the wetwood (Tree 8) and sapwood (several trees) o f 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 c o l l e c t e d through normal e x t r a c t i o n p o i n t s . 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 r e placed by another p i p e ) , and i n Tree 23, i t was 5 cm away h o r i 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 s e c t i o n through the  branch stub revealed that the i n s e r t e d pipe d i d not f o l l o w the exact o r i e n t a t i o n o f the branch.  The t i p o f 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) o f suction  developed during the e x t r a c t i o n , and the flow rate was zero.  When the ex-  t r a c t i o n s were repeated on the same day using the o r i g i n a l p i p e s , the flow of the gases was usual (about 5.0 i n [12 cm] o f s u c t i o n ) . 5.  Black Cottonwood: Wetwood o f wounded t r e e s : Wetwood exposed: In  Tree 2 ( n a t u r a l l y wounded), throughout the year, 0^ concentration was genera l l y high ranging from 16.5 t o 19.0% while C 0 was low, never reaching even 2  1,0% l e v e l .  During a l l the e x t r a c t i o n s , suction never developed  suggesting  that the process o f wound h e a l i n g was perhaps incomplete and that the wetwood was i n contact with atmosphere.  At the end o f the gas composition s t u -  dies,- the tree was f e l l e d t o examine the nature and extent o f the wound. In a cross s e c t i o n , 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 o f the wound, they revealed that a crack extended through the wound lengthwise, and towards the base o f the t r e e , wetwood was connected to the outside through the crack.  This explains the gas composition.  CH^  was not found i n t h i s tree i n e i t h e r winter o r summer. Before Tree 3 was wounded i n May, the 0  2  concentration was h i g h , C 0  was low while CH^ was present but only i n small q u a n t i t i e s .  2  When the wetwood  was exposed, 0~ concentration generally remained unaltered although C0„  F i g u r e 25:  V e r t i c a l s e c t i o n o f Tree 23, through the branch stub showing where the b r a s s p i p e was p o s i t i o n o f t h e branch  trace.  p l a c e d (above)  and  101  F i g u r e 26:  Cross s e c t i o n o f a 19 y e a r o l d b l a c k cottonwood (Tree 2) showing e x t e n t o f t h e wound.  A crack  ( r i g h t ) extended through t h e e n t i r e l e n g t h o f t h e wound.  102  concentration dropped considerably and CH^ could not be detected any longer.  A f t e r plugging the wound i n J u l y , 0^ concentration dropped con-  s i d e r a b l y , CO2 contents increased and CH^ re-appeared. November, CO  In October and  and CH^ concentrations increased s u b s t a n t i a l l y reaching  2  higher l e v e l s 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 e a r l y summer. Attempts were made to i s o l a t e anaerobic b a c t e r i a from the wetwood (Appendix 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%.  A f t e r wounding and then plugging the wound w i t h i n 24  hours, a gas analysis was made oh June 27th, which showed that CH^ concent r a t i o n of the wetwood had dropped s u b s t a n t i a l l y reaching 3.2% (Figure 27).  Three weeks a f t e r t h i s , another a n a l y s i s was made and i t r e -  vealed that CH 10% l e v e l .  level  4  was i n c r e a s i n g again which at t h i s time had reached  The tree also showed changes i n the composition o f 0  2  the C0  2  as a n t i c i p a t e d ; wetwood which was microaerobic before wounding showed a sharp increase i n i t s 0 6.  2  concentration and C0  2  l e v e l f e l l a f t e r wounding.  Black Cottonwood: Wetwood of wounded trees: SapWood exposed:  trees (No.s 24 and 30) were used to study i f exposure o f sapwood had e f f e c t on the gas composition of wetwood.  any  These trees were examined during  the summer and both of them continued the p a t t e r n o f low 0 even a f t e r the sapwood was wounded.  Two  2  and high C0  2  103  Figure 27.  Concentrations o f 0^, C0  2  and Crl^ i n the wetwood o f a  black cottonwood (Tree 21). Note the abrupt change i n the gas composition p a t t e r n a f t e r the wetwood was exposed. , i n the 1st week o f June and covered w i t h i n 24 hours (arrow).  Also note the subsequent change i n the  gas composition trend with-time,  104  o  ,  8  15  —o  21 MAY  >  31  8  i  i  i  i  ' i. .  t  i_  15  21  27  8  14  19  27  JUNE DATE OF SAMPLING Figure 27  JULY  105  7.  Red a l d e r : Heartwood:  Two trees were studied a l l through the  year and 1 tree was studied only during the summer. tions showed that 0  2  A l l o f the determina-  concentrations were generally high and C0^ concentra-  t i o n s were generally low (Figure 28). No seasonal v a r i a t i o n s o f gases were evident from the analyses.  CH^ was absent i n a l l 3 trees t e s t e d .  general, concentrations o f 0  2  In  and CO 2 i n red a l d e r heartwood were i n i n -  verse proportions to those o f black cottonwood wetwood (Figure 29). 8.  Lombardy poplar:  Heartwood (probably wetwood):  A very l i m i t e d  number o f e x t r a c t i o n s were done on Lombardy poplars; three trees were examined and only during the summer.  The wood cores removed suggested  that  i n a l l 3 t r e e s , wetwood may have been present instead o f heartwood; the i n ner wood appeared wet and d i s c o l o r e d . A f u r t h e r examination i s necessary to confirm the presence o f wetwood.  The gas analyses showed that 0^ was "  present i n high proportions (except Tree 46 i n June), but present 9.  and CH^ were  in generally low proportions (Figure 30), Measurement o f gas pressures i n the wetwood:  Gases were never  found under pressure i n both o f the t r e e s ; the pressure gauge i n d i c a t e d zero pressure at a l l times.  DS ICUSSO IN A d e f i n i t e seasonal v a r i a t i o n occurred i n the concentrations o f Q and CO2 i n the wetwood o f black cottonwood. i n the summer and highest i n the winter. centage o f CO2 a l l through the year. season and lowest i n the winter.  2  The percentage o f O2 was lowest  I t v a r i e d i n v e r s e l y with the per-  CO2 was highest during the growing  In the majority o f the t r e e s , tested  106  Figure  28:  Concentrations  o f O2 and CO2 (monthly averages) i n the  heartwood o f a r e d a l d e r  (Tree 42).  i n any o f the r e d a l d e r s  studied.  CH^ was  n e v e r found,  20.0  18.0  16.0  14.0  Tree 42  .<, 12.0  w u z o u  10.0  8.0  6.0  4.0  2.0  • A — . AM  A  -JL M  J  TIME OF YEAR Figure 28  J  108  Figure 29:  Comparison o f 0^ and C0 concentrations (monthly averages) 2  i n the wetwood o f a black cottonwood (Tree 11) and heartwood o f a red alder (Tree 41).  109  F i g u r e 29  110  Figure 30;  Concentrations o f 0 , C0 2  2  and CH  4  (monthly averages) i n the  heartwood (probably wetwood) of three Lombardy poplar trees (Nos. 2, 44, 45 and -46) .  Ill  20.0  18.0  O  - 0,  A  - CO,  •  - CH,  16.0  T r e e 44  14.0  T r e e 45  Tree  46  L  o  <  12.0  H  :  •2:  O  tq 10.0 •2:  o  6.0  4.0 A  2.0  • A  M  J  HI  1  M TIME OF YEAR Figure  30  A  D  L_  J  -1I  ' M  L. J  J  112  r e g u l a r l y or o c c a s i o n a l l y , 0^ concentrations of <0.05% were found during the summer (Figure 31).  Such microaerobic conditions l a s t e d f o r prolonged  periods (6-12 weeks) w i t h i n t r e e s .  Some trees contained CH^ i n the wet-  wood; i n r e g u l a r l y examined trees i t was c o n s i s t e n t l y low while i n some trees o c c a s i o n a l l y t e s t e d , CH^ concentrations of as high as 34% were found during the summer. and higher i n  Gases drawn from the sapwood were always lower i n CO^  than those obtained from the wetwood.  v a r i a t i o n s occurred i n the gas composition of sapwood, i n the gases c o l l e c t e d from sapwood.  No marked seasonal CH^ was never found  Gas composition of black cottonwood  wetwood was q u i t e d i f f e r e n t , both q u a n t i t a t i v e l y and q u a l i t a t i v e l y , from that of the red alder heartwood despite the f a c t 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 o f many black cottonwoods studied. Both of these features, were absent i n the heartwood of a l l 3 red alders examined . Occurrence o f low 0^  high  i n the heartwood or wetwood of pop-  l a r s , has been reported p r e v i o u s l y .  Bushong (1907) ..reported 1.24%  a cottonwood while MacDougal and Working (1933), Chase (1934) and  0^ from Zeikus  and Ward (1974) noted complete absence o f O2, but only during some analyses made, and these conditions d i d not p e r s i s t f o r any length of time.  The  no-  table feature here was that the microaerobic conditions continued to e x i s t f o r long durations (6-12 weeks).  A l s o , the data obtained here are based  upon a large number of trees (20) as compared to Bushong's 1 t r e e , Chase's 1 t r e e , 3 trees of MacDougal and Working or 2 trees o f Zeikus and Ward. (1934) also reported C0~ concentration o f over 25% from an eastern  Chase  113  Figure 31: Percentage o f 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 o f each  bar represent number o f trees examined during the i n d i c a t e d period.  114  100.0  90.0  80.0  70.0 o  Q o  60.0 L  U-  i—i  ca o PS  w <  50.0  § 40.0 w w  OS H  u.. o  30.0  w  < E• Ui  u  20.0  OS  w a,  10.0  15 M  1  15 A  1  15  1  M  15 J  TIME OF YEAR F i g u r e 31  1  15 J  1  15 A  1  15 S  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 f o r 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 d i f f e r e n t zones i n such d i f f e r e n t pro-  portions within, trees . The fact that the gas compositions o f 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 r e mains unaffected even i f the sapwood i s exposed t o 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 t h a t wetwood and sapwood are two d i f f e r e n t systems.  Therefore the factors determining t h e i r  gas compositions may also be d i 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 comp o s i t i o n might be determined by d i f f e r e n t f a c t o r s : The i s o l a t i o n studies reported e a r l i e r (Chapter II) and those o f others (Morani and Arru 1958, Zeikus and Ward 1974) suggest the p o s s i b i l i t y that microorganisms p l a y an active r o l e 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 d i f f e r e n t 0^ requirements.  During t h i s study, i t  was found that the temperature o f the gases w i t h i n trees generally f l u c t u ates according to the outside temperatures. ing season when some  In the beginning of the grow-  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 m e t a b o l i c i - a c t i v i t i e s , consuming the  present i n the wetwood.  During t h i s process a slow leakage o f 0~ i n t o wetwood may take place because  o f the d i f f e r e n c e i n the p a r t i a l pressures. Nevertheless, due to a high rate o f metabolism, the amount o f  would decrease, and f a c u l t a t i v e l y -  anaerobic and m i c r o a e r o p h i l i c 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 m i c r o b i o l o g i c a l methods, a c t i v e l y growing cultures o f aerobes are often used to remove 0^ from incubator a i r i n order to obtain growth o f anaerobic microorganisms ( W i l l i s 1969).  As  i s a normal end product o f 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 growi n g season. may  In the near-absence o f 0^ and presence o f high CC^, anaerobes  begin p h y s i o l o g i c a l a c t i v i t i e s that are e s s e n t i a l f o r t h e i r growth.and  multiplication. CH^ i f free  Some anaerobes (methanogenic b a c t e r i a ) may also produce and more  were a v a i l a b l e 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 t h i s study, i t  was found that microaerobic conditions p e r s i s t e d f o r a minimum o f 6 weeks i n most  trees during the warm temperatures o f summer.  As the temperatures drop  i n winter, the a c t i v i t y o f a l l microorganisms w i l l decrease considerably. Consequently, because o f differences i n the p a r t i a l pressures o f gases and reduced a c t i v i t y (or p o s s i b l y none) o f microorganisms, 0^ outside to i n s i d e and C O 2 may d i f f u s e out i n winter.  m a v  d i f f u s e from  Thus, the slow l a t e r a l  movement of gases may exceed the gas consumption w i t h i n wetwood and b r i n g 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 o f sap-  wood i s u s u a l l y s i m i l a r to that o f the outside a i r . A l s o , during the l i f e of a t r e e , sapwood i s more vulnerable to wounds i n v o l v i n g exposure to a t mosphere than wetwood which may contribute to the f a c t t h a t sapwood gases are normally s i m i l a r to atmospheric a i r . I s o l a t i o n studies showed that a very small number o f microorganisms i s u s u a l l y associated with sapwood o f mature t r e e s , and i n young cottonwoods, none could be detected. the microorganisms do not play any r o l e i n determining  Presumably  the gas composition  of sapwood. In the wounded t r e e s , the gas composition o f wetwood was d i f f e r e n t from that o f non-wounded t r e e s .  Gases drawn from the wetwood o f a wounded tree  (No. 2) were lower i n CO^ and higher i n 0^ than those o f 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 a f t e r the wetwood was exposed.  There was  no f u r t h e r accumulation of CO^ i n the wetwood a f t e r wounding and 0^ showed a sharp increase i n concentration as a n t i c i p a t e d .  However, once the wounds  were plugged, the trend i n the gas composition was reversed.  The 0^ contents  began decreasing, CC^ s t a r t e d i n c r e a s i n g and CH^ which had disappeared a f t e r pounding, re-appeared.  These f l u c t u a t i o n s can be explained i f involvement  of microorganisms i s assumed i n the gas composition.  Once the wetwood was  exposed, 0^ d i f f u s e d from outside t o i n s i d e making the tree more or l e s s aerobic.  This change would make i n t e r i o r conditions favorable t o aerobes  and unfavourable to anaerobes.  Methanogenic b a c t e r i a being anaerobic would  then survive i n a r e s t i n g phase and therefore cease producing CH^. under these s u i t a b l e c o n d i t i o n s , would show vigorous metabolic  Aerobes  activities  118  Figure 32: Comparison o f 0^ and C0 concentrations (monthly averages) 2  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 l i m i t e d because o f metabolic pro-  cesses of the aerobes, the m i c r o a e r o p h i l i c and eventually anaerobic microorganisms may become f u n c t i o n a l .  Methanogenic b a c t e r i a may then produce  CH^ and generally the o r i g i n a l gas composition would be resumed. noted i n the concentrations of CH^ and 0^in  Changes  Tree 21 s t r o n g l y 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 f o r the exchange o f 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 suct i o n pump. Up to 22 i n (55.0 cm) of s u c t i o n developed i n both cases.  Ap-  p a r e n t l y an impermeable l a y e r forms around the knot upon death of the branch ( M u l l i c k j personal communication), which may prevent wetwood gases from being extracted. The exact l o c a t i o n 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 p l a y any r o l e as i t does not contain any l i v ing c e l l s .  More anatomical studies are needed before t h i s 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 a f f e c t e d to some extent.  However, most branches contain wetwood at the time of death.  If  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 p h y s i o l o g i c a l s i g n i f i c a n c e 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 f a c t o r o f some importance i n the growth o f wood-destroying f u n g i .  Apparently most f i l a -  mentous fungi are aerobic ( i . e . they cannot grow i n the absence o f O^)• A l s o , under very low  pressures, fungal growth i s reduced considerably  The studies reported here showed that i n black cottonwoods, very low l e v e l s o f 0*2 (<0.05%) were present i n the wetwood f o r prolonged periods (6-12 weeks).  E f f e c t s of such low  l e v e l s on the growth and s u r v i v a l  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 o b l i g a t e aerobes, that i s , they are unable to grow i n the complete absence o f oxygen, although the minimum concentration of the gas which w i l l permit s a t i s f a c t o r y growth may be very low." (Hawker 1950) "Apparently none of the fungi are o b l i g a t e 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 anaerobic. An aerobic organism requires uncombined oxygen, while a f a c u l t a t i v e anaerobe may use combined oxygen i n a d d i t i o n 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 q u a n t i t a t i v e r e l a t i o n s o f growth and oxygen supply vary considerably among d i f ferent forms." (Cochrane 1958) "Another f a c t o r important f o r fungal growth i s oxygen supply. Most fungi are s t r i c t l y aerobic, and i n the complete absence of oxygen growth ceases." (Ingold 1961) These 4 quotations are from Mycology textbooks which describe an important p h y s i o l o g i c a l character o f fungi.  Numerous experiments have shown  that most fungi are unable to grow i n the absence o f free 0^'  A. very  l i m i t e d number o f fungi, however, are capable of growth i n the absence o f free 0^ (Emerson and Held 1969, Held et_ al_. 1969).  Gunner and Alexander  (1964), f o r example, observed that Fusarium oxysporum, an alleged o b l i g a t e aerobe, was able to grow under anaerobic c o n d i t i o n s , provided the medium contained yeast e x t r a c t , 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 q u a n t i t a t i v e r e l a t i o n s of filamentous fungi or yeasts and 0  ?  supply  d i f f e r considerably among d i f f e r e n t forms.  The amount o f 0,,  123 needed f o r optimum growth v a r i e s with the species, and f o r each species, other environmental factors influence the requirements f o r molecular O^. Tabak and Cooke (1968) have reviewed much o f the relevant l i t e r a t u r e on ®2 requirements o f f u n g i , and t h e r e f o r e , a d e t a i l e d review w i l l not be attempted here. To i n t e r p r e t the studies on e f f e c t s o f anaerobic conditions o r r e duced 0  2  l e v e l s on wood-destroying f u n g i , i t i s necessary t o consider i n  general the methods used i n these s t u d i e s .  The term anaerobic only implies  that i n a given system, there i s no free 0^ present. pure N  2  An atmosphere o f  would mean absence o f free 0^ and, t h e r e f o r e , such a system could  be considered anaerobic. pheres o f N , H 2  2  On' t h i s p r i n c i p l e , several workers used atmos-  or C0 to create anaerobic conditions, 2  Frequently, the  gases were p r e p u r i f i e d to eliminate even small traces o f 0 . 2  On one occasion,  the fungal growth was studied ' i n vacuum' and thus', i n p r i n c i p l e , under anaerobic conditions.  A v a r i e t y o f tanks, j a r s and incubators were used t o  e s t a b l i s h and maintain these anaerobic conditions.  In most cases, c o n t i n -  uance o f anaerobic environment i n the c u l t u r e medium or i n an incubator was checked q u a l i t a t i v e l y by c o l o r i n d i c a t o r s such as methylene blue. In some instances, growth o f fungi was studied i n absolute terms, e i t h e r p o s i t i v e o r negative,  <Bn others, q u a n t i t a t i v e determinations  were made.  Among these, a few workers made the growth measurements by weighing the d r i e d mats, o f mycelium from the l i q u i d medium and/or by measuring the l i n e a r growth on a standard  agar medium. Hirayama (1938) determined the a b i l i t y  of fungi t o r e s p i r e under anaerobic conditions by using a fermentation ratus.  appa-  He measured the volume of gas produced, and computed the i n t e n s i t y  124  of anaerobic r e s p i r a t i o n by the volume o f CC^ produced by the mycelium of a u n i t weight. of growth.  This anaerobic i n t e n s i t y was considered as the measure  I t i s noteworthy that i n a l l . growth s t u d i e s , fungi were allowed  to grow i n various a r t i f i c i a l c u l t u r e media and none of the workers used wood as a medium, even when the i n v e s t i g a t i o n s were concerned with wooddestroying f u n g i .  Recently, i t has been pointed out that the c u l t u r e medium  used has a major influence on s u r v i v a l and growth of fungi under anaerobic conditions (Tabak and Cooke, 1968).  Therefore, r e s u l t s o f many of these  studies are n o t . d i r e c t l y comparable. In 1910, Hoffman demonstrated that i n an atmosphere of  (i.e.under  anaerobic c o n d i t i o n s ) , an a c t i v e l y growing c u l t u r e o f Merulius lacrymans died i n 4 days. Q^-  Growth ceased immediately when the fungus was deprived of  He also found that Coniophora c e r e b e l l a and P a x i l l u s panuoides d i d not  grow under anaerobic conditions f o r 20 and 15 days r e s p e c t i v e l y but were able to resume growth when exposed to 02-One o f the e a r l y comprehensive studies o f  requirements o f 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 o f minute quantit i e s of ®2> k  u t  could survive the complete absence of O2 f o r a considerable  length o f time. No r e t a r d a t i o n of growth occurred u n t i l the  pressure was  reduced to 10 cm o f Hg (approximately 13% O2) and only below t h i s he found that growth of fungi l i k e Mefulius lacrymans and Coriophora c e r e b e l l a was r e duced considerably. Scheffer and Livingston (1937) studied l i n e a r growth and C0  2  production  i n the fungus P o l y s t i c t u s (Pblyporus) v e r s i c o l o r (Davidson et_ al_. 1942) at  125 various temperatures (17.5 to 33,5 C) and 0 The minimal 0  2  fungus was exposed to <1.5 mm 0 2  pressures (0.0 to 745  mm).  pressure f o r mycelial growth on malt agar was between 1.5  to 10 mm at a l l temperatures tested.  C with 0  2  2  They reported no growth when the  pressure. Growth was most r a p i d at 29.5  pressure varying from 16 to 745 mm.  Higher concentrations o f 0  2  u s u a l l y lead to an increase i n the rate o f evolution o f C0 , and conse2  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 r e s p i r a t i o n of 10 wood-destroying fungi under anaerobic conditions and found that the anaerobic r e s p i r a t i o n i n t e n s i t y shown by 5 brown-rot fungi was generally greater than that shown by the 5 white-rot f u n g i . In t h e i r study of a l c o h o l i c fermentation and dehydrogenation o f alcohols by c e r t a i n wood-destroying f u n g i , Nord and S c i a r i n i (1946) showed that Merulius and Fomes arinosus were able to ferment glucose, r a f f i n o s e , and xylose under anaerobic conditions to ethanol, t h i s fermentation being f o l lowed by dehydrogenation to a c e t i c a c i d and acetaldehyde. Thacker and Good (1952) exposed & wood-destroying fungi to 0 t r a t i o n s , ranging from 0.8 to 35.0%.  2  concen-  Growth o f a l l fungi was "good" at  both 0.8 and 35.0% of 0 ;growth tended to increase s l i g h t l y with increase o f 2  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 o f several maples, they concluded that aeration i s probably not an important f a c t o r i n the development o f decay. Growth response of Fomes annosus to low 0 by Gundersen (1961),  2  and high C0  2  was i n v e s t i g a t e d  Inoculated p e t r i p l a t e s were incubated at room  126 temperature i n Brewer's j a r s containing a i r , N , H 2  gen j a r s , a l l free 0  2  r e a c t i o n with the H , 2  ment.  and 0  2  and C0 . 2  In the hydro-  d i s s o l v e d i n the agar was removed by c a t a l y t i c  2  the j a r s thus representing a s t r i c t anaerobic environ-  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 a l k a l i n e p y r o g a l l o l ( t h e o r e t i c a l l y  the j a r s contained 0.02 (microaerobic) C0  mm 0 ) . 2  These b o t t l e s represented  environment•in which ,the fungus grew unaffected.  ( f i l l e d in-the same way as with N ,  2  a microaerophilic  2  In j a r s with  except that the washing i n pyrogal-  l o l was omitted) and representing a m i c r o a e r o p h i l i c h a b i t a t , with 760  mm  C0 , no growth occurred, whereas 23 mm o f the gas i n combination with a i r 2  or N  accelerated growth about 50 percent.  2  I t was  concluded that t h i s fungus  i s able to grow equally w e l l under aerobic or m i c r o a e r o p h i l i c c o n d i t i o n s , but not anaerobically. Jensen (1967b) found that i n the case of 4 wood-rotting f u n g i , dry weight production decreased with a decrease in' 0 and with an increase i n C0  2  growth was observed at 0.0% of C0  2  2  concentration below  concentration from zero percent. 0  2>  Since reduced l e v e l s of 0  2  21.0%  No measurable and high l e v e l s  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 t r e e s . This b r i e f l i t e r a t u r e survey i n d i c a t e s that wood-destroying fungi are unable to grow i n the absence of 0 , although sometimes growth can be 2  tected at very low 0  2  levels.  de-  The q u a n t i t a t i v e response of fungi to low  0  l e v e l s was v a r i a b l e and l a r g e l y depended on the environmental conditions under which the t e s t 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  2  127  that on malt agar, the l i n e a r growth o f 2 wood-destroying fungi (Polyporus delectans and Ganoderma applanatum) was reduced considerably at low tensions.  Nothing i s known, however, about the r e l a t i o n s h i p 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 t o grow on wood at very low  levels.  t e r e s t i n g because o f the very low cottonwood during the summer.  This r e l a t i o n s h i p i s p a r t i c u l a r l y i n l e v e l s found i n the wetwood o f black  Therefore, I made a study t o determine the  a b i l i t y o f wood-destroying fungi to cause a weight loss i n wood under microaerobic conditions.  The experimental set-up also made i t p o s s i b l e t o study  e f f e c t s o f pH, moisture content or microbial population o f wood on the extent o f decay.  MATERIALS AND METHODS Figure 33 shows the disposable Anaerobic System (Gas Pak^-BBL) that was used to create microaerobic conditions.  The system consists o f a c a r r i e r  assembly, an anaerobic container, a gas generator envelope, an anaerobic i n d i c a t o r and a p l a s t i c clamp.  The c a r r i e r i s designed to accommodate the  c u l t u r e s , gas generator envelope and an anaerobic i n d i c a t o r . l y s t chamber i s f i x e d i n one w a l l o f the c a r r i e r .  A f i l l e d cata-  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 o f the bag. t r i p l e laminated p l a s t i c f i l m s .  The bag i t s e l f i s composed o f  Gas generator envelope produces  and CO2  a f t e r adding 10 ml o f water and the H produced combines with atmospheric 2  0„ i n the bag, i n the presence o f the platinum c a t a l y s t to form water.  The  128  Figure 33:  Disposable Anaerobic System. assembly.  A.  Side view,  methylene blue i n d i c a t o r  Yellow box i s the c a r r i e r  Note the p o s i t i o n of the (now c o l o r l e s s ) .  B.  Front view.  A  3  F i g u r e 33  130 end r e s u l t i s that a microaerobic, .; atmosphere, containing 0.08% 0 (range: 0.06 to 0.1%) and 6.0% C0 the bag.  This 0  2  2  2  (range: 4.5 to 7.0%), i s produced i n  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 f o r use o f the system i s as f o l l o w s , 1)  Set up the c a r r i e r by c l o s i n g and l o c k i n g 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 f l a p s .  2)  Place the gas generator envelope and anaerobic i n d i c a t o r 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 cont a i n e r ( 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 o f 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 o f 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 e n t i r e 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 t e s t f u n g i .  The former i s the most common wood-destroying  fungus o f 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 t o dead cottonwood (Thomas and Podmore 1953). For l i n e a r growth s t u d i e s , the fungi were grown on 2% malt agar p l a t e s f o r 7 days at 23 C, and these a c t i v e l y growing cultures served as the inoculum source.  The inoculum (4 mm discs o f mycelium and agar) was placed i n  a p e t r i dish containing malt agar and the growth o f each fungus was studied under aerobic and microaerobic conditions.  Each t e s t was made i n t r i p l i c a t e .  The cultures were incubated at 23 C f o r 16 days at which time the fungi  131  growing under aerobic conditions had covered the p l a t e s .  The l i n e a r growth  of fungi '(under microaerobic conditions) was recorded by measuring the colony diameters  (excluding colony diameters o f the inoculum).  A f t e r 16 days  of incubation under microaerobic conditons, the fungi were allowed to grow under aerobic conditions to see i f they had survived. A m o d i f i c a t i o n o f a s o i l - b l o c k t e s t (ASTM D2017-71) was used to determine the extent o f decay o f 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 e i t h e r gamma r a d i a t i o n (Smith and Sharman 1971) at 2.5 x 10^ rads  (complete  sterilization)  The mois-  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 ) .  ture content o f each t e s t block was assumed to be equal to that measured from an end-matched adjacent block.  The t e s t was made i n 16 oz b o t t l e s which con-  tained approximately 80.0 g o f 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]). A f t e r i n o c u l a t i o n o f the b o t t l e s with the required fungi and once the fungi were w e l l e s t a b l i s h e d on the feeder s t r i p s , four t e s t b l o c k s , one from each t r e e , were placed i n each b o t t l e .  Disposable  Anaerobic System, as described p r e v i o u s l y , was used to e s t a b l i s h microaerobic conditions. The experiment used a r e p l i c a t i o n o f 12 blocks f o r each condition and was done i n 2 p a r t s .  In the f i r s t p a r t , h a l f the b o t t l e s were subjected to  microaerobic conditions while the other h a l f served as atmospheric controls (aerobic).  At the end o f t h i s period (10 weeks), 2 blocks were removed from  132 each b o t t l e and t h e i r weight loss determined (Part I ) .  In the second p a r t ,  the same b o t t l e s 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 f o r a f u r t h e r 10 weeks before weight loss assessment (Part I I ) .  RESULTS AND DISCUSSION The r e s u l t s of growing fungi on malt agar under aerobic and aerobic conditions are given i n Table V I I I .  micro-  Under microaerobic c o n d i t i o n s ,  l i n e a r growth of Polyporus delectans and Ganoderma applanatum was 4 12% r e s p e c t i v e l y of that of controls grown under atmospheric 0  2  and  levels.  Both of these fungi survived 16 days incubation under microaerobic conditions and resumed mycelial growth when returned to a normal atmosphere.  These r e -  s u l t s are generally i n agreement with many p r e v i o u s l y 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 r e s u l t s o f ' s o i l - b l o c k 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  i n t e s t blocks exposed to microaerobic conditions f o r 10 weeks, while weight loss of c o n t r o l aerobic treatments v a r i e d 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 concentration of 0  2  found i n the wetwood i s too low to allow any development  of decay, at l e a s t i n the 2 fungi tested.  I t should be pointed out, however,  that i n t h i s experiment fungi survived the 10 week incubation under microaerobic conditions and presumably the same would happen i n the f i e l d .  After  TABLE VIII:  Fungus  Linear growth of two w6bd:-destrbying f*ungitons2-% malt''ag'af,' after> 16- days of incubation under aerobic (control) and microaerobic conditions ji:'; s  Culture i d e n t i f i cation number  Type o f rot  Polyporus delectans  WFPL 84 A'$  White  Ganoderma applanation  WFPL 32 A  Colony diameters* (mm) aerobic microaerobic (control) conditionsc conditions  Percentage growth under microaerobic.conditions of that o f controls  75  3  4  75  9  12  - Average o f 3 measurements $ - Cultures were obtained from the Western Forest Products Laboratory, Canadian Forestry Service, Vancouver, B.C. -c;.  Department o f the Environment,  t  h-'  TABLE IX:  Meanj^wei'ghtgl^^ cottonwood 'blocks of sapwood (S) and wetwood (W) under aerobic (A) and microaerobic. (M) conditions  Fungus  Polyporus delectans  Sterilization method Wood zone  Flame (surface) S  Part I  §  M  29.5 -0.1 (2.9)^ (0.4) r  M Part I I *  29.4 (4.6)  Gamma r a d i a t i o n (complete)  W  Growth Condi-.. -,A tion  A 5.0  A  M  38.7 0.8  A  M  47.6 0.3  M A  Flame (surface)  Wti  S  (3.9)(0.5) (2.8) (0.3) M A  Ganoderma applanatum  A  M  A  S j-  W  M  A M  Gamma r a d i a t i o n  I  S A  M  (complete)  W A  M  41.8 -0.5  42.5 0.3  43.2 0.3  43.5 0.3  46.7 -0.1  (3.0) (0.8)  (4.0) (0.8) (3.3)(0.7)  (2.9)(0.7)  (3.1) (0.7)  M  A  45.5 7.7  51.6 7.8  37.2  9.2  (2.5) (3.6)(2.1)  (4.7)(2.1)  (5.6) (2.5)  M A  M A  M A  M  A  49.3 14.7  45.9 16.9  43.0 15.7  44.8 10.6  (2.8)(3.5)  (1.0)(4.3)  (2.7)(3.1)  (4.6) (3.0)  * Test b o t t l e s were incubated at 27 C and 70% r e l a t i v e humidity fi  Weight loss a f t e r i n i t i a l 10 weeks exposure to the indicated conditions "^Weight loss a f t e r f u r t h e r 10 weeks exposure to the indicated conditions f o l l o w i n g exposure to the conditions i n d i c a t e d i n Part I . ^Standard e r r o r i n brackets  w  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 b l o c k s , nor between surface s t e r i l i z e d and completely blocks.  s t e r i l i z e d wetwood  I t 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 o f wetwood have no detectable e f f e c t , nor do there appear to be any t o x i c chemicals i n the wetwood, 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 a d d i t i o n a l weight loss during 10 week exposure to microaerobic  conditions  f o l l o w i n g 10 weeks o f aerobic c o n d i t i o n s , 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 p e r i o d . i n d i c a t e that i f microaerobic establishment  These r e s u l t s  conditions are created i n a tree a f t e r - t h e  of f u n g i , f u r t h e r advancement of decay would be arrested.  In  some t r e e s , a phenomenon of i n a c t i v a t i o n o f decay f o l l o w i n g h e a l i n g of the i n f e c t i o n court has been observed (Childs and Wright 1956,  Toole 1965).  For  example, Toole observed t h a t , decay did not advance s i g n i f i c a n t l y a f t e r 2 years i n willow oak and n u t t a l oak  (Quercus n u t t a l l i i Palm) trees inoculated  with pure cultures of P o r i a ambigua, Pblypbrus f i s s i l i s and Polyporus h i s p i d u s . He also noted that most i n o c u l a t i o n wounds were healed a f t e r 2 years.  This  lack of "advance" i n decay could have been due to the re-establishment  of  microaerobic  conditions created a f t e r the wounds were healed.  The r e s u l t s  obtained here also i n d i c a t e that wood-destroying fungi can resume t h e i r act 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 o f winter prevent o r r e t a r d growth of most wooddestroying fungi (e.g. Wagener and Davidson 1954), and t h e r e f o r e , s i m i l a r i n h i b i t i o n o f wood-destroying fungi may be observed i n black cottonwood despite the favorable 0^ concentrations present i n winter; I t i s noteworthy that the weight loss o f blocks exposed t o microaerob i c conditions followed by aerobic conditions was much lower (average 10.9%) than that o f blocks exposed to aerobic conditions only (average 41.7%). Perhaps t o x i c metabolic by-products accumulate during the microaerobic conditions and t h e i r e l i m i n a t i o n i s required before normal growth resumes. A l t e r n a t i v e l y , i t i s p o s s i b l e that during the microaerobic p e r i o d , l i v i n g mycelia of the fungus are transformed  i n t o s p e c i a l r e s t i n g stages, thus  s u b s t a n t i a l l y reducing t h e i r inoculum p o t e n t i a l .  A s i m i l a r phenomenon  might reduce the a c t i v i t y o f fungi i n trees during winter months f o l l o w i n g microaerobic summer conditions. I expected that since Polypbrus delectans occurs i n l i v i n g t r e e s , 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 t o l e r a n t to microaerobic conditions.  But  with both f u n g i , there was no s i g n i f i c a n t weight loss a f t e r 10 weeks under microaerobic c o n d i t i o n s , therefore i n d i c a t i n g no p r e f e r e n t i a l a b i l i t y f o r Polyporus delectans to t o l e r a t e such conditions i n the laboratory.  In the  sequence o f 10 weeks microaerobic followed by 10 weeks aerobic, however, the weight loss caused by Polyporus delectans was 7.4% (average) while that o f Ganoderma applanatum was 14.5% (average), suggesting a more r a p i d recovery from microaerobic  conditions by the l a t t e r .  In the study o f Thomas and  137 Podmore (1953), the sampled trees were o l d (60 years and above), and therefore, vulnerable to wounding'. ing i n t o the wetwood would admit 0  As shown e a r l i e r , any wound or open2  (Chapter I I I ) 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 l i n e a r growth of f u n g i , although d r a s t i c a l l y reduced, was not completely prevented. The i m p l i c a t i o n of t h i s f i n d i n g on many p r e v i o u s l y published r e s u l t s 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 i n v o l v i n g 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 o f 0  2  i s adequate f o r the growth o f 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 o f wood-destroying fungi was p r e d i c t e d by Thacker and Good (1952) and Gundersen (1961).  Thacker and Good s t a t e d , "While a l l  the e f f e c t s 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 n e a r l y optimal f o r decay,'! And Gundersen s t a t e d , " I t could be concluded that F. annosus i s a fungus able to grow equally w e l l under aerobic as under m i c r o a e r o p h i l i e condit i o n s , but nO'fc'anaerobically,....  These observations seem to agree w e l l 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 r e s u l t s presented here s t r o n g l y suggest l i m i t a t i o n s to the gen-  e r a l 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 o f wood-destroying fungi has always been a f a s c i n a t i n g subject, and a r e l a t i v e l y large amount o f l i t e r a t u r e i s a v a i l a b l e d e s c r i b i n g studies on growth o f wood-destroying fungi on various n u t r i t i o n a l media, t h 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 r e -  searchers have studied the s u r v i v a l o f 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 t o contain low 0 C0  2  2  (and sometimes none) and high  concentrations, at l e a s t during c e r t a i n times o f the year l a s t i n g f o r  c e r t a i n durations  (Chapter I I I ) .  Hoffman (1910) found that a vigorously growing c u l t u r e o f Merulius lacrymans d i d not survive when exposed to anaerobic conditions ( i . e . i n an atmosphere o f H ) f o r 4 days. 2  A l s o , Coniophora c e r e b e l l a and P a x i l l u s  panuoides did not grow under anaerobic conditions f o r 20 and 15 days r e s p e c t i v e l y , but were able to continue growth i n the presence o f 0  2 >  Later,  Bavendamm (1928) confirmed Hoffman's r e s u l t s when he observed death o f Merulius lacrymans i n 2 to 3 days o f exposure t o anaerobic conditions.  He  also reported that i n the complete absence o f 0 , Stereum frustulosum was 2  "undamaged" a f t e r 10 days (and presumably grew when exposed t o aerobic  139 conditions).  From h i s extensive work i n v o l v i n g 32 species o f f u n g i ,  Bavendamm concluded that the t y p i c a l saprophytes were a f f e c t e d most under the anaerobic conditions whereas the heart r o t s were the most r e s i s t a n t . The s u r v i v a l 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  d i s s o l v e d 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 o f 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 s t r a i n s o f Fomes annbsus resumed growth i n aerobic conditions a f t e r the c u l t u r e s had been exposed t o anaerobic cond i t i o n s f o r 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 r e a c t i o n with H . 2  Boutelje and K i e s s l i n g (1964) i s o l a t e d several fungi from oak timber from 2 ships which sank i n the B a l t i c at the beginning o f the 17th century. One o f the i s o l a t e s , a species o f Phoma, showed d i s t i n c t wood-decaying a b i l i t y , causing a k i n d o f r o t resembling s o f t r o t .  Phoma and Phialophora are  often i s o l 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 cap a c i t y (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 In my previous study, 0  2  2  supply i s very minimal.  concentrations of 0.05% o r less were found t o  be present i n the wetwood o f black cottonwood during the summer (Chapter I I I ) ,  140 and these microaerobic  conditions e x i s t e d f o r up to 12 weeks i n some t r e e s .  My e a r l i e r study (Chapter IV) also showed that Polyporus delectans Ganoderma applanatum survived 10 weeks exposure to microaerobic  and  conditions,  although during that p e r i o d , 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 o f <0,05% 0^.  As the lowest l e v e l o f 0  0.05%, I could not have known how  2  measurement i n the 0^ analyzer  low these 0  2  was  concentrations were i n trees.  With the assumption that these 0^ l e v e l s may have been.low enough to be termed "anaerobic",  I made a study to see i f a c t i v e l y growing cultures of  wood-destroying fungi growing on wood can survive a long exposure t o "anaerob 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 f o r 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 f u n g i , i n c l u d i n g Polyporus delectans and Ganoderma applanatum, were used i n t h i s experiment (Table X).  The i n t e n t i o n was.to  s e l e c t an equal number o f white-rot and brown-rot f u n g i , and w i t h i n each c l a s s , some v a r i a t i o n wasaalso sought with respect t o t h e i r a b i l i t y to produce chlamydospores.  Here the objective was to see i f s u r v i v a l of fungi un-  der anaerobic conditions i s r e l a t e d 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:  Fungus  Poria monticola  Culture Identification Number  WFPL 120F  Lenzites trabea  WFPL 47D  Polyporus palustris  WFPL 227A  Fomes annosus  WFPL 19D  Polyporus delectans  WFPL 84A  Ganoderma applanatum  WFPL 32A  Polyporus versicolor  WFPL 105A  Polyporus hirsutus  WFPL 89B  C h a r a c t e r i s t i c s o f 8 wood-destroyingdfun>gi^ a n d y t h e i r r s u r v i v a l response t o -l^cweekseofuirieub conditions .  Type o f rot  Chlamydospore producing a b i l i t y  brown  Positive-numerous Chlamydospores  "  white  "  Negative  P o s i t i v e - f a i r to numerous chlamydospores Negative  Number o f blocks exposed  Culture condition ( a f t e r incubation)  Percentage o f Survival  11  All  living  100.0  12  All  living  100.0  12  All  living  100.0  13  A l l dead  0.0  A l l dead  0.0  12  A l l dead  0.0  11  5- l i v i n g 6- dead  12  All  living  45.4  100.0  ^Cultures were obtained from the Western Forest Products Laboratory, Department o f the Environment, Canadian Forestry S e r v i c e , Vancouver, B.C.,  142  w h i t e - r o t fungus was m i s t a k e n l y t a k e n as a brown-rot f u n g u s , and conseq u e n t l y , 1 i n i t i a t e d t h e experiment w i t h 3 brown-rot and 5 w h i t e - r o t f u n g i . A l l brown-rot f u n g i and one w h i t e - r o t f u n g u s , produced numerous chlamydospores w h i l e t h e r e m a i n i n g 4 w h i t e - r o t f u n g i l a c k e d t h e a b i l i t y t o produce chlamydospores.  Thus, I f a i l e d t o o b t a i n t h e d e s i r a b l e , and o r i g i n a l l y i n -  t e n d e d , v a r i a t i o n among t h e w o o d - d e s t r o y i n g f u n g i w i t h r e s p e c t t o t h e i r subs t r a t e s p e c i f i c i t y and a b i l i t y t o produce chlamydospores. A non-wounded b l a c k cottonwood t r e e , l o c a t e d on t h e U.B.C. Research F o r e s t , was f e l l e d and t h e r e q u i r e d number o f t e s t b l o c k s cut from t h e wetwood zone.  S t e r i l e blocks  (1 cm cubes) were  (gamma r a d i a t i o n ) were i n o c u l a t e d  a c c o r d i n g t o t h e s t a n d a r d s o i l - b l o c k t e s t p r o c e d u r e (ASTM: D2017-71) and were i n c u b a t e d f o r 8 weeks under a e r o b i c 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 o f each fungus was e v i d e n t , t h e b l o c k s 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 p a p e r (Whatman No. 3 ) . A p p r o x i m a t e l y 20 ml o f s t e r i l e d i s t i l l e d w a t e r was d i s pensed i n t o each p l a t e and t h e s e 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 A n a e r o b i c i n c u b a t o r ( N a t i o n a l A p p l i a n c e Co., USA).  Two methylene b l u e  i n d i c a t o r s were clamped from i n s i d e on t h e g l a s s door f o r easy i n s p e c t i o n o f incubator conditions at a l l times.  J u s t before c l o s i n g the incubator door,  the l i d s o f t h e 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 t h e b l o c k s would be i n d i r e c t c o n t a c t w i t h t h e environment i n t h e i n c u b a t o r .  A i r was t h e n w i t h -  drawn from t h e i n c u b a t o r w i t h a vacuum pump, and was i m m e d i a t e l y r e p l a c e d by L-grade N  2  gas.  The p r o c e d u r e o f e v a c u a t i o n * and r e p l a c e m e n t by N  2  was  A c c o r d i n g t o Zycha ( 1 9 3 8 ) , 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 f u n g i and, t h e r e f o r e , presumably on t h e 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  i n 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 f i l t e r papers revealed  that, although there was a certain loss of moisture from the f i l t e r papers, a l l 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 f i r s t 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. weeks exposure to anaerobic conditions.  endured 13  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 a l l 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 P o r i a monticola produce chlamydospores, whereas Polyporus hirsUtus c u l t u r e used i n t h i s study does not produce chlamydospores. Polyporus delectans, Ganoderma applanatum and Fomes annosus, a l l white r o t fungi which could not t o l e r a t e exposure to anaerobic c o n d i t i o n s , normally do not produce chlamydospores.  In the case o f Polyporus v e r s i c o l o r which  showed 45.4% s u r v i v a l , chlamydospores are produced i n f a i r number. These r e s u l t s suggest some trends regarding s u r v i v a l o f the fungi and the  types o f 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 r e l a t i o n s h i p s w i l l have t o be accepted with  some reservations.  Generally, brown-rot fungi were most r e s i s t a n t t o the  anaerobic conditions while white-rot fungi were most s e n s i t i v e . ' The 3 brownrot fungi produce numerous chlamydospores and perhaps have a b e t t e r chance of s u r v i v a l under the unfavorable conditions.  Among the white-rot f u n g i , 3  which did not survive are normally incapable o f producing : chlamydospores, Polyporus v e r s i c o l o r which produces a f a i r number o f chlamydospores under normal conditions, showed about 50% s u r v i v a l .  I t i s p o s s i b l e that f o r t h i s  fungus, the anaerobic incubation period o f 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% surv i v a l while with a longer exposure, i t might have shown 0.0% s u r v i v a l . Polyporus h i r s u t u s , a white-rot fungus, survived the anaerobic exposure o f 13 weeks and normally the fungus i s unable to produce chlamydospores.  It is  possible that the s u r v i v a l o f Polyporus h i r s u t u s depends on factors other than those considered here. There are 2 reports a v a i l a b l e f o r 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 f o r only 13 weeks, the response o f 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 a f t e r i t was incubated under anaerobic conditions f o r 6 days.  In my study, exposure  to anaerobic conditions r e s u l t e d i n death of a l l Fomes annbsus c u l t u r e s . This d i f f e r e n c e i n observed response i s e a s i l y a t t r i b u t a b l e to the d i f f e r ent methods used.  In my study, the fungus was exposed to anaerobic condi-  tions f o r 13 weeks and, therefore, i t i s p o s s i b l e that t h i s much longer exposure 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 d i f f e r e n t researchers and drawing general conclusions from i t , , i s that the d i f f e r e n t i n v e s t i g a t o r s use d i f f e r e n t 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 d i f f e r e n t methods. 0, 2  She found that i n an atmosphere of N  2  or i n a i r minus  the formation of chlamydospores i n Fusarium oxysporum f. cubense was  delayed, but eventually they were produced i n large numbers. fungus  under wateroor i n C0  2  was very d i f f e r e n t .  Behavior of the  Here, only occasional  chlamydospores were produced, whereas, the c o n i d i a were produced i n large numbers.  Conidia, however, are not known to be very l o n g - l i v e d i n s o i l .  She  therefore concluded that the production of chlamydospores which takes place i n the absence of 0 soil.  2  i s s i g n i f i c a n t i n respect of s u r v i v a l of the fungus i n  I m p l i c a t i o n o f 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 f u n g i .  Conclusions  based on r e s u l t s obtained i n s u r v i v a l s t u d i e s , done i n the presence of  CO^  and water (anaerobic c o n d i t i o n s ) , may vary from those when the studies were done i n an atmosphere o f N K  2  or i n a i r minus  O^.  Hirayama (1938) studied r e s p i r a t i o n o f 10 wood-destroying fungi under  anaerobic conditions.  A l l fungi survived and brown-rot fungi e x h i b i t e d  greater metabolic a c t i v i t y than the white-rot f u n g i .  However, the author  did not give d e t a i l s of h i s method concerning how he created and maintained the anaerobic c o n d i t i o n s , which could be very c r u c i a l i n i n t e r p r e t i n g the results.  For h i s study, Hirayama used 5 brown-rot and 5 white-rot f u n g i .  Again, he did not describe the c u l t u r a l c h a r a c t e r i s t i c s of the f u n g i , 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 v a r i e s from " r a r e " to "numerous" (Nobles 1948).  Despite t h i s v a r i a t i o n , he found that a l l brown-rot fungi  showed higher capacity f o r anaerobic r e s p i r a t i o n . Therefore, i t could be i n f e r r e d from Hirayama*s study that the kinds of organic substances that a fungus attacks and the enzymes i t produces f o r substrate breakdown determines whether the fungus i s going to survive under anaerobic c o n d i t i o n s , rather than the chlamydospore producing a b i l i t y of the fungus. Hepting (1941) i n v e s t i g a t e d decay that o r i g i n a t e s 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 p r e d i c t the cull.  He noted that much of the v a r i a t i o n i n amount o f c u l l among trees with  wounds o f s i m i l a r s i z e s and ages was due to the d i f f e r e n t fungi that became established.  In some cases, e s p e c i a l l y behind small wounds, the sap r o t  147  fungi progressed a short distance i n the heartwood and then there was f u r t h e r decay.  He s t a t e d , "Apparently  no  chance has much to do with which  heart r o t fungi become e s t a b l i s h e d behind any given wound i n oaks."  Al-  though no s p e c i f i c comparisons can be made, the r e s u l t s o f 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  a c t u a l l y be r e l a t e d to the v a r i a t i o n i n a b i l i t i e s o f fungi  to survive under anaerobic c o n d i t i o n s , created a f t e r the wounds were healed. The rate of wound h e a l i n g and i t s effectiveness i n c r e a t i n g anaerobic cond i t i o n s might also be r e l a t e d to the d i f f e r e n t behavior of d i f f e r e n t f u n g i . In summary, the s u r v i v a l or death o f 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 a t t a c k i n g subs t r a t e s , white-rot fungi being capable o f a t t a c k i n g both c e l l u l o s e and  lignin  and brown-rot fungi mainly attacking c e l l u l o s e . Such a r e l a t i o n s h i p would hold true since i t i s known that breakdown of l i g n i n involves o x i d a t i o n , whereas c e l l u l o s e can be broken down anaerobically with end-products such as a l c o h o l s , o x a l i c a c i d , etc.  The r e s u l t s reported e a r l i e r (Chapter IV)  suggested t h a t , i f microaerobic  conditions are created i n a tree a f t e r the  s  establishment  of f u n g i , f u r t h e r advancement o f decay would be prevented.  This study i n d i c a t e s that i f the conditions i n the wetwood o f black cottonwood 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  resume growth when CL becomes a v a i l a b l e again.  survive and  148  CHAPTER VI SIGNIFICANCE OF WETWOOD IN BLACK COTTONWOOD (SUMMARY AND CONCLUSIONS)  The c e n t r a l theme of t h i s t h e s i s was the demonstration that wetwood i n black cottonwood i s a phenomenon b e n e f i c i a l to the t r e e , p r o v i d i n g prot e c t i o n against decay.  The supposition that the microaerobic  conditions  found i n the wetwood prevent the development of decay, has been amply supported by the evidence presented.  Contrary to the o r i g i n a l concept, however,  microaerobic conditions do not p e r s i s t 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 p e r i o d .  Nevertheless,  the phenomenon i s s t i l l a very valuable one as i n most t r e e species of the Temperate Zone, almost a l l of the decay occurs during the warm summer months. During the w i n t e r , low temperatures are known to l i m i t the rate o f progress of wood-destroying fungi (Wagener and Davidson 1954). frequent i n j u r i e s , microaerobic  Therefore, b a r r i n g  conditions of the summer and low temperatures  of the winter may r e t a r d the development of decay i n black cottonwood. A large number o f microorganisms wasa found i n the wetwood of black cottonwood. failed.  Attempts to i s o l a t e wood-destroying fungi from the wetwood  The i n t e r e s t i n g feature of the i s o l a t i o n studies was that some of  the microorganisms were aerobic i . e . r e q u i r i n g 0^ f °  r  growth, some were f a 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 r e q u i r i n g i t , whereas some were o b l i g a t e l y anaerobic i . e . r e q u i r i n g absence of 0  ?  for  \  149 growth.  The f a c t that these microorganisms  wood column despite the v a r i a t i o n i n t h e i r  s u c c e s s f u l l y i n h a b i t the wetrequirements suggests that  they l i v e i n the wetwood as a s t a b l e community and/or are able to adapt w e l l to the occasional unfavourable  conditions.  Although no s p e c i f i c experiments were done to show that  microorganisms  a c t u a l l y induce microaerobic conditions i n the wetwood, c i r c u m s t a n t i a l e v i dence s t r o n g l y suggests t h i s mechanism. to consume  Due to t h e i r r e s p i r a t o r y a c t i v i t y ,  the wetwood would decrease and at the same time,  increase.  would have  present i n the wetwood i n order to carry out the e s s e n t i a l  metabolic processes. in  Aerobic microorganisms  concentration concentration would  The m i c r o b i a l a c t i v i t y would be at maximum when the  are high during the summer.  temperatures  Therefore, one would expect 0^ concentration i n  the wetwood to be lowest, and CC>2 concentration to be h i g h e s t , during the summer.  The gas composition studies revealed e x a c t l y t h a t ; very low propor-  t i o n s of O2  (<0.1% or microaerobic conditions) and high proportions of CO^  were observed i n the wetwood during the warm summer months.  L a t e r , the  wounding experiments showed that the exposure of wetwood causes changes i n i t s gas composition as one would expect; i f present, decreases or disappears.  increases, CO^ decreases and  However, a f t e r plugging the wound, the  gas composition of wetwood changes again; O2 decreases, CO^ increases and increases or re-appears.  The r e s p i r a t o r y a c t i v i t y of microorganisms  t h e i r a b i l i t y to adapt to d i f f e r e n t changes i n gas composition.  CH^,  environments  CH^  and  would e x p l a i n the observed  In a d d i t i o n , studies made here (Appendix I) and  those of Zeikus and Ward (1974) showed that methanogenic b a c t e r i a were present i n trees containing s u b s t a n t i a l q u a n t i t i e s o f CH . 4  F i n a l l y , very low l e v e l s  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  p r i m a r i l y responsible f o r the gas composition and that they induce microaerobic conditions i n the wetwood during the summer. The s o i l - b l o c k 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 o f wood-destroying fungi can be expected i n the f i e l d as microaerobic conditions do occur i n the wetwood o f black cottonwood.  Furthermore,  when the wood blocks were exposed to aerobic conditions f o l l o w i n g an incubat i o n imder the microaerobic  c o n d i t i o n s , 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 a d d i t i o n a l weight loss.  I t 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 t o increased 0^ conc e n t r a t i o n s , and when the wound i s healed, the a c t i v i t y o f wood-destroying fungi would decrease as the 0 And i f microaerobic  2  concentration w i t h i n wetwood would decrease.  conditions are created a f t e r the wound h e a l i n g then the  a c t i v i t y .of wood-destroying fungi would be h a l t e d (no s i g n i f i c a n t a d d i t i o n a l weight l o s s ) .  The s o i l - b l o c k experiment also showed that inherent properties  of wetwood such as high pH or high moisture content d i d not contribute to the decay r e s i s t a n c e ,  A l s o , the microorganisms d i d not e x h i b i t any a n t a g o n i s t i c  e f f e c t s on the growth o f wood-destroying f u n g i . The 2 wood-destroying fungi used i n the s o i l - b l o c k 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  s e n s i t i v e to anaerobic conditions than the brown-rot f u n g i .  Therefore, i f  151 anaerobic conditions e x i s t i n the tree trunks f o r long durations, white-rot fungi may not show any decay a c t i v i t y , and quite p o s s i b l y d i e , 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 l e a s t 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  i n d i c a t e s that i t could w e l l be a b e n e f i c i a l phenomenon p r o t e c t i n g 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 f o r e s t 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-  t r o y i n g fungi enter through wounds exposing wood, and eventually destroy heartwood.  the  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 l o s s e s . t h i s suggestion.  I support  The r e s u l t s obtained here i n d i c a t e that i n j u r i e s not only  serve as the entry points f o r the wood-destroying fungi but also admit 0^ to the wetwood (or heartwood), thereby destroying the mechanism o f decay r e s i s t ance. ly.  C e r t a i n l y , deep and large wounds would destroy the mechanism permanentOn the other hand, shallow and small wounds, such as those caused by  branch pruning, heal q u i c k l y and have no permanent e f f e c t on the gas composit i o n of wetwood. Some i n v e s t i g a t o r s dealing with wetwood have regarded i t as a p a t h o l o g i c a l phenomenon.  The term p a t h o l o g i c a l (Pathology-Science  of diseases, Fowler  152 and Fowler 1964)  i n i t s common usage implies that i n a given c l a s s , some  b i 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 p h y s i o l o g i c a l functioning i s severely affected which almost i n v a r i a b l y r e s u l t s i n reduced growth or e a r l y m o r t a l i t y of the host.  Hartley et a l . (1961) have suggested that occurrence of wetwood  may be r e l a t e d to e a r l y m o r t a l i t y of Lombardy poplar  (trees frequently die  before age 20), and also i n an unexplained m o r t a l i t y often observed i n balsam firs.  In view of these i m p l i c a t i o n s , l e t us consider i f the occurrence of  wetwood i n black cottonwood can be classed as a p a t h o l o g i c a l phenomenon. F i r s t l y , the occurrence of wetwood i s u n i v e r s a l , 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 s p e c i a l conditions other than tree age.  Generally, the developmental pattern  of wetwood and i t s p o s i t i o n w i t h i n 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 u s e f u l functions (mechanical support, f o r example) that are normally c a r r i e d out by heartwood.  Secondly, many growth and y i e l d studies (summarized by Maini  Cayford 1968)  and  i n d i c a t e that black cottonwood i s the l a r g e s t o f the North Ameri-  can poplars, and l o n g e s t - l i v e d 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 t h i s species grows w e l l up to 200 years (Thomas 1949) , and there i s no evidence of e a r l y m o r t a l i t y ( i . e . without i n v o l v i n g known b i o l o g i c a l or physi c a l agents) associated with the species.  T h i r d l y , the wetwood of black  cottonwood together with i t s m i c r o b i a l community appears to o f f e r decay r e sistance to the tree and does not appear to have any d e l e t e r i o u s e f f e c t s 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 p a t h o l o g i c a l .  153 This concept of wetwood can perhaps be extended to the wetwood o f 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 o f  wetwood appears to be a u n i v e r s a l phenomenon i n t h i s species as w e l l .  The  microbial studies o f the same authors showed that aerobic, m i c r o a e r o p h i l i c and anaerobic b a c t e r i a were associated with wetwood.  The study o f Sachs et a l .  (1974) also showed that filamentous fungi were r a r e l y found i n the wetwood. The gas composition s t u d i e s (Chase 1934, Zeikus and Ward 1974) showed that low 0^ (and sometimes none) and high CO^ were the c h a r a c t e r i s t i c features of wetwood gases.  These f i n d i n g s together describe a s i t u a t i o n which i s quite  s i m i l a r to that found i n the case o f black cottonwood.  I t i s possible then  that wetwood i n eastern cottonwood may also i n h i b i t decay development. In summary, I suggest that i n l i v i n g black cottonwood t r e e s , the occurrence o f wetwood i s best regarded as a normal phenomenon which imparts a considerable degree of decay r e s i s t a n c e t o the inner wood.  154 BIBLIOGRAPHY  American Society f o r Testing and M a t e r i a l s . D 2017-71. * Ankudinov, A.M.  1971. A.S_.T.M. Designation  P h i l a d e l p h i a , Pa. 1939. Serdtsevinaia G n i l ' O s i n i I Mery Bor* by S. Neiu.  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Inoculation o f bottom-land red oaks with P o r i a aiiiblgua,  Polyporus f i s s i l i s and Polyporus h i s p i d u s . .  1968. Wetwood i n cottonwood.  Plant Disea. Rept. 49: 823-  Plant Disea. Rept. 52: 822-823.  161 Wagener, W.W.  and R.S. Davidson.  1954.  Heart rots i n l i v i n g t r e e s .  Bot.  Re, 20: 61-134. W a l l i n , W.B.  1954.  Wetwood i n balsam poplar.  Minn. Forestry Notes No. 28,  Agr. Expt. Sta. S c i . Jour. Series Paper 3118: 1-2. Ward, J.C., J.E. Kuntz, and E. McCoy. i n broadleaf t r e e s . Wilcox, W.W.,  (Abstract).  and N.D. Oldham.  1969.  B a c t e r i a associated with "shake"  Phytopathology. 59: 1056.  1972.  Bacterium associated with wetwood  i n white f i r . Phytopathology 62: 384-385. W i l l i s , A.T.  1969.  bacteria. and D.W. Wolfe, R.S.  Techniques f o r the study of anaerobic, spore-forming  Chapter I I I . Ribbons.  1971.  In: Methods i n Microbiology (ed.) N o r r i s , J.R.,  Academic Press, New York, N.Y.  M i c r o b i a l formation of methane.  Advan. M i c r o b i o l .  P h y s i o l . 6: 107-146. Zeikus, J.G., and Ward, J.C. microbial o r i g i n . Zycha, H.  1937.  1974.  Methane formation i n l i v i n g trees: A  Science 184: 1181-1183.  Uber das Wachstun zweier holzzerstorender P i l z e und i h r  V e r h a l t n i s zur kohlensaure.  Z e n t r a l b l . Bakt. I I . 97: 224-244.  162 APPENDIX I ISOLATION OF ANAEROBIC BACTERIA (OBLIGATE) FROM WETWOOD Attempts were made to see i f methanogenic b a c t e r i a ( s t r i c t anaerobes) were present i n the wetwood o f Tree 21 as t h i s tree contained over 30% CH^ during the month o f May.  In the f i r s t week o f 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 o f 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 t o the laboratory (Department o f Microb i o l o g y , U.B.C.) w i t h i n an hour.  Core was then cut i n t o 5 mm sections i n  an anaerobic growth chamber and the pieces were t r a n s f e r r e d i n t o tubes cont a i n i n g standard Bryant medium supplemented with 30% rumen f l u i d .  A small  quantity o f l i q u i d (about 0.5 ml) c o l l e c t e d 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 i n t o three groups: tubes belonging to f i r s t group were supplied with H^, the second group with N t h i r d group with a mixture o f rh, and C0 (80:20, v o l : v o l ) . 2  2  and the  Then, the produc-  t i o n o f CH^ by the microorganisms placed i n the tubes was taken as an i n d i cation of the presence o f methanogenic b a c t e r i a .  CH^ was detected with a  Bendix (model 2500) gas chromatograph using a s i l i c a g e l column.  Gas samples  from the tubes were analyzed p e r i o d i c a l l y f o r a t o t a l p e r i o d o f 36 to 48 hours.  Wood sections belonging to group I I tubes (gassed with N ) d i d not 2  produce any CH. while those belonging t o groups I and I I I tubes produced CH..  163  These r e s u l t s show that presence o f  and CO^ aids microorganisms i n  production of CH^. D e t a i l s of techniques used i n i s o l a t i o n of methanogenic b a c t e r i a are given by Edwards and McBride (1974).  A review a r t i c l e by Wolfe (1971) i s  also a v a i l a b l e which provides much of the relevant information regarding these microorganisms.  164  APPENDIX I I - A . 1:  C a l i b r a t i o n curve o f CO-  concentration.  16.0  14.0  o  12.0  I—I  < OC  10.0  E2  UJ  u  8.0  O  u  6.0  CN  o u  4.0  2.0  ON  200,000  400,000  600,000  MICROVOLTS APPENDIX  - SEC  II-A.l  800,000  1,000,000  166  APPENDIX IT - A, 2:  C a l i b r a t i o n curve of CH.  concentration.  _  40.0  400,000  800,000  1,200,000  1,800,000  MICROVOLTS - S E C APPENDIX II-A. 2  2,000,000  2,400,000 ^  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 f i r s t 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 i n the text and are stated here only briefly. with a field-lab 0  2  Concentrations of 0^ were measured  analyzer (in the field) while C0  2  and CH  4  concentra-  tions were determined by gas chromatography (in the laboratory).  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 Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration Flow rate (ml/min)  0_  CO.  (%) CH  2  B l a c k 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  traces  13  June 26  16  7(17.5)  75  0,05  13.0  traces  ON  APPENDIX I I ( c o n t ' d ) :  Gas composition i n the t r e e t r u n k s .  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed ( i n [cm])  Gas Concentration Flow rate (ml/min)  0 2  CO 2  CH 4  Black cottonwood - Wetwood - Tree 10 (continued) 14  Jul.; 10 i c  18  5(12.5)  100  <0.05  12.4  traces  15  J u l . 2 4 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 I I ( c o n t ' d ) :  Gas c o m p o s i t i o n i n the t r e e trunks  Gas sample c h a r a c t e r i s t i c s Number  Date o f Collection  Temperature (C)  S u c t i o n developed (infcm])  Gas C o n c e n t r a t i o n (%) Flow r a t e (ml/min)  0  CO, 2  CH. *  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  Jul 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  Gas composition i n the tree trunks.  APPENDIX I I (cont'd):  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  0„ 2  CO  CH  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 c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) 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 I I (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 Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  0-  CO.  CH  2  Black cottonwood - Wetwood - Tree 6 (cont'd) 14  Jul  15  7  21  7(17.5)  50  0.90  7.5  1.0  J u l y 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 c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration Flow rate (ml/min)  0  C0„  CH  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 i n the tree trunks  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (infcm])  Gas Concentration Flow rate (ml/min)  0, 2  CO  (%) CH  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  Gas composition i n the tree trunks  APPENDIX II (cont'd):  Gas sample c h a r a c t e r i s t i c s Number  Date o f Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  0„ 2  CO 2  CH *  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  Jul 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 I I ( c o n t ' 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 Number  Date o f Collection  Temperature (C)  Suction developed (infcmj)  Gas Concentration Flow rate (ml/min)  0 2  CO 2  CH 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 c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (infcm])  Gas Concentration (%) Flow rate (ml/min)  CO,  CH,  Black cottonwood - Wetwood - Tree 16 1  Feb 25  11  9(22.5)  2  Mar 15  12  16(40.0)  3  Mar 28  12  8(20.0)  4  Apr 12  17  5  Apr 24  6  50  6.20  5.9  0.0  Low  5.60  7.0  o.o  50  14.00  2.7  0.0  10(25.0)  50  10.50  2.3  0.0  16  9(22.5)  50  11.00  3.5  0.0  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 I I (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 Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  0.  C0„  CH  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 I I (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 Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration Flow rate (ml/min)  (%)  CO,  CH,  Black cottonwood - Wetwood - Tree 20 May 7  15  9(22.5)  50  <0.05  9.9  0.0  May 28  17  6(15.0)  50  <0.05  7.5  0.0  Tree 21 1  May 8  15  12(30.0)  25  <0.05  8.9  34.0  2  May 21  18  7(17.5)  75  <0.05  8.8  30.5  3  May 31  17  7(17.5)  75  <0.05  8.9  32.6  3.2  1st week o f June, tree wounded, wetwood exposed; wound plugged w i t h i n 24 hours a f t e r wounding 4  Jun 27  17  5(12.5)  100  6.50  1.0  5  J u l 19  20  8(20.0)  50  1.20  2.4  10.  Tree 22 May 8  15  10(25.0)  50  1.20  4.1  0.5  May 21  18  9(22.5)  50  1.00  4.9  0.5 oo  APPENDIX II (cont'd):  Gas composition in the tree trunks  Gas sample characteristics Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  0  CO 2  CH. 4  Black cottonwood - Wetwood Tree 27 1  Jun 5  18  10(25.0)  50  <0.05  4.7  4.0  2  Jun 18  20  11(27.5)  50  <0.05  5.5  4.0  3  Jul 9  21  7(17.5)  50  <0.05  6.0  4.5  4  Aug 20  22  7(17.5)  75  <0.05  5.0  6.0  5  Oct 11  20  7(17.5)  50  1.20  7.2  5.5  Tree 28 1  Jul 30  22  7(17.5)  50  <0.05  8.9  19.5  2  Aug 27  23  6(15.0)  75  <0.05  8.8  19.0  3  Oct 3  20  6(15.0)  75  2.60  5.8  0.5  Tree 29 Jul 30  22  6(15.0)  75  0.20  8.3  15.0  Aug 27  23  6(15.0)  75  0.20  8.4  16.0  Gas composition i n the tree trunks  APPENDIX II (cont'd):  Gas sample Number  Date o f Collection  Temperature (C)  characteristics S u c t i o n developed (infcm])  Gas C o n c e n t r a t i o n (%) Flow r a t e (ml/min)  CO,  CH,  Black cottonwood - Wetwood Tree 5 8(20.0)  Jan 9  50  2.00  6.4  0.0  Tree 4 1  Jan 16.  14  9(23.5)  50  3.00  5.1  0.0  2  Feb 13  5  7(17.5)  50  2.80  4.2  0.0  Tree 9 1  Jan 4  4  9(22.5)  50  2.50  4.2  0.0  2  Jan 16  14  7(17.5)  50  2.00  3.7  0.0  3  Feb 13  5  4(10.0)  75  3.80  4.0  0.0  75  4.00  3.0  0.0  25  1.80  3.9  0,0  50  11.00  3.4  0.0  3(7.5)  75  8.00  3.7  0.0  Tree 18 8(20.0)  50  8.00  3.4  0.0  iTreeU Jan 18  10  7(17.5) Tree 7 10(25.0)  Jan 30  Tree 14 Feb 25  10  7(7.5) Tree 17  Feb 27 Apr 22  8 14  APPENDIX I I (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 Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  CO,  CH,  BI ack cottonwood - Sapwood Tree 12 1  Feb 8  9  8(20.0)  2  Feb 15  11  12(30.0)  3  Mar 1  developed 20 i n (50.0 cm) o f s u c t i o n , no sample  4  Mar 27  developed 20 i n (50.0 cm) o f s u c t i o n , no sample  25  11.40  0.2  0.0  Low  17.00  0.2  0.0  15.00  0.2  0.0  Tree 16 1  Mar 6  12  2  Mar 15  developed 20 i n (50.0 cm) o f s u c t i o n , no sample  3  Mar 29  developed 20 i n (50.0 cm) of s u c t i o n , no sample  16(40.0)  Low  Tree 32 1  Apr 10  16  9(22.5)  50  18.00  0.6  0.0  2  Apr 26  15  11(27.5)  25  11.40  0.2  0.0  50  13.00  0.6  0.0  Tree 33 May 10  14  9(22.5)  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 Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  CO,  CH,  Black cottonwood - Sapwood Tree 34 May 24  17  9(22.5)  50  16.50  0.2  0.0  Jun .4  18  10(25.0)  25  13.00  0.8  0.0  Tree 35 Jun 21  18  10(25.0)  50  12.60  0.4  0.0  Jul 5 .  21  12(30.0)  25  18.00  0.4  0.0  Jul 19  20  13(32.5)  25  17.50  0.4  0.0  Tree 36 Aug 2  22  13(32.5)  25  19.00  0.2  0.0  Aug 16  21  12(30.0)  25  18.60  0.2  0.0  Aug 30  developed 20 i n (50.0 cm) of suction, no sample Tree 37  Sep  13  Oct 11  21  12(30.0)  25  18.00  0.1  0.0  20  13(32.5)  25  16.00  0.1  0.0  Tree 38 Oct 25  19  13(32.5)  oo  Low  18.00  0.2  0.0  APPENDIX I I (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 mber  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) 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  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  .  125  APPENDIX I I (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 Number  Date of Collection  Temperature (C)  Suction developed ( i n [cm])  Gas Concentration Flow rate (ml/min)  0„ 2  CO. 2  CH, 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  Gas composition in the tree trunks  APPENDIX II: (cont'd):  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  0„  CO.  CH  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  Gas  (cont'd):  c o m p o s i t i o n i n the  tree  trunks  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C) Black  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  c o t t o n w o o d - Wetwood - T r e e 3 - Wounded  0„  C0_  CH  (cont'd)  14  Oct  2  20  6(15.0)  100  4.00  4.2  2.0  15  Oct  16  19  7(17.5)  75  3.50  5.0  1.5  16  Oct  30  17  4(10.0)  100  5.50  5.1  2.0  17  Nov  13  14  7(17.5)  75  5.00  5.1  2.0  18  N o v 27  14  7(17.5)  75  5.00  6.1  2.0  19  Dec  14  6(15.0)  75  4.00  6.0  .'.2.5  11  Gas composition i n the tree trunks  APPENDIX I I (cont'd):  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration 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 J u l 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  '  Gas composition i n the tree trunks  APPENDIX I I (cont'd):  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  0^  CO^  CH^  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 o f J u l y , t r e e 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 I I (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 Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  0_  CO-  CH  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 i n (50.0 cm) o f s u c t i o n , no sample  3-B  May 28  17  flow o f gas,but no analysis  4-A  Jun 5  18  developed 20 i n (50.0 cm) o fs u c t i o n , no sample  4-B  Jun 5  18  9.9  2.0  5-A  Jun 11  20  5-B  Jun 11  20  9.8  3.0  6-A  Jul 9  21  6-B  Jul 9  21  9.9  2.0  7-A  Aug 20  22 •  7-B  Aug 20  22  2.6  2.0  8(20.0)  75  <0.05  developed 20 i n (50.0 cm) o fs u c t i o n , no sample 9(22.5)  75  <0.05  developed 20 i n (50.0 cm) o fsuction,lino sample 8(20.0)  75  <0.05  developed 20 i n (50.0 cm) o f s u c t i o n , no sample 9(22.5)  75  1.20  Gas composition i n the tree trunks  APPENDIX I I (cont'd):  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration Flow rate (ml/min)  CO,  CH,  11.7  0.5  6.0  0.5  Black cottonwood - Wetwood - Tree 23 A - through branch stub B - normal sampling point 1- A  Apr 29  15  developed 20 i n (50.0 cm) o f s u c t i o n , no sample  2- A  May 9  15  developed 20 i n (50.0 cm) o f s u c t i o n , no sample  3- A  May 28  17  developed 20 i n (50.0 cm) o f s u c t i o n , no sample  3- B  May 28  17  4.-A  Jun 11  20  4- B  Jun 11  20  5(12.5)  75  <0.05  developed 20 i n (50.0 cm) u f s u c t i o n , no sample 3(7.5)  100  0.05  Gas composition in the tree trunks  APPENDIX II (cont'd):  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  irature Temperature (C)  Suction developed (in[cm])  Gas Concentration (%) Flow rate (ml/min)  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  Gas composition i n the tree trunks  APPENDIX I I (cont'd):  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Concentration Flow rate (ml/min)  (%)  CO,  CH,  Red alder - Heartwood - Tree 41 (cont'd) 14  Oct 18  20  8(20.0)  50  17.00  1.4  0.0  15  Nov 1  15  14(35.0)  25  18.00  0.8  0.0  16  Nov 29  14  13(32.5)  50  17.50  0.8  0.0  17  Dec 13  14  12(30.0)  50  17.00  0.8  0.0  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 Number  Date of Collection  Temperature (C)  Suction developed (infcm])  Gas Concentration 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  Gas composition in the tree trunks  APPENDIX II (cont'd):  Gas sample characteristics Number  Date of Collection  irature Temperature (C)  Suction developed (in[cm])  Gas Concentration Flow rate rate (ml/min)  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  Gas composition i n the tree trunks  APPENDIX I I (cont'd):  Gas Concentration  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Flow rate (ml/min)  (%)  CO,  CH,  Red alder - Heartwood - Tree 25 Jun 5  18  8(20.0)  75  18.00  0.2  0.0  Jun 18  20  17(17.5)  75  17.00  0.2  0.0  Gas composition i n the tree trunks  APPENDIX I I (cont'd):  Gas sample c h a r a c t e r i s t i c s Number  Date of Collection  Temperature (C)  Suction developed (in[cm])  Gas Flow rate (ml/min)  Concentration CO,  CH,  2.8  0.6  Lombardy poplar - Heartwood (probably wetwood) Tree 44 May 6  17  7(17.5)  50  10.50  No f u r t h e r a n a l y s i s , tree f e l l e d by the Physical P l a n t , U.B.C, Tree 45 1  May 6  16  7(17.5)  50  10.00  3.6  1.5  2  May 21  19  7(17.5)  75  11.50  3.7  1.5  3  Jan 4  18  ;7(17.5)  75  10.50  3.6  1.5  Tree 46 1  May 6  15  15(37.5)  Low  11.00  1.8  0.6  2  May 21  18  15(37.5)  Low  11.00  2.1  0.6  3  Jun 6  18  16(40.0)  Low  1;50  6.5  2.0  

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