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Fungicidal toxicity of certain extraneous components of Douglas fir heartwood Kennedy, Robert W. 1955

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FUNGICIDAL TOXICITY OF CERTAIN EXTRANEOUS COMPONENTS OF DOUGLAS FIR HEARTWOOD  by ROBERT W. KENNEDY  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF FORESTRY o  i n the Faculty of Forestry  We accept this thesis as conforming to the standard required from candidates f o r the degree of MASTER OF FORESTRY  Members of the Faculty of Forestry THE UNIVERSITY OF BRITISH COLUMBIA APRIL 1955  ABSTRACT  The h e a r t w o o d (Mirb.)  Franco) i s  has  fungi.  established  primary deterrents from Douglas activity the  i n order to  determine  ether  fraction.  a cellulosic  malt agar  (Fr.)  (B.&C.)  the  of  Cke.,  Curt,  The d e g r e e  of  effectiveness  expressed  numerically.  fungus  at  the  fungicidal influencing  of  Douglas  components,  namely:  as  these m a t e r i a l s  cellulosic  represented of  the  test  fungi.  were  substrates,  Fr. Both  employed. had  been  whereas  concentrations  of  the  the n o n - c e l l u l o s i c media.  e a c h component a s a  t h e most  i n h i b i t i n g growth o f  a concentration of  was  Lentinus lepideus  D i h y d r o q u e r c e t i n was f o u n d t o be completely  as  fractions  factor  substrate  impregnated w i t h v a r y i n g materials  fungicide,  for  extraction  separate  A bioassay  extraneous  was  extraneous  from which c e r t a i n e x t r a c t i v e s  removed were u s e d f o r  on o t h e r  extractives  precise  and w a t e r  and a n o n - c e l l u l o s i c  S m a l l wood b l o c k s  attack  a c i d , n e u t r a l , p h l o b a t a n n i n and  made u s i n g Fomes a n n o s u s incrassata  to  species.  a dihydroquercetin, free  and P o r i a  resistant  and e v a l u a t e d the  menziesii  investigations  Several  heartwood meal p r o v i d e d f i v e  carbohydrate  (Pseudotsuga  v a r i o u s heartwood  isolated  this  An a c e t o n e , fir  Previous  to decay.  f i r were  d u r a b i l i t y of  fir  known t o be r e l a t i v e l y  by w o o d - d e s t r o y i n g species  of Douglas  slightly  less  fungicide  potent  t h e most t h a n 0.5  sensitive per  cent.  T h i s value compares f a v o r a b l y w i t h experimental  results  p r e v i o u s l y r e p o r t e d w i t h p h e n o l i c e x t r a c t i v e s of the genus Pinus.  On the b a s i s of these data, timber  selected for i t s  h i g h d i h y d r o q u e r c e t i n content c o u l d be expected  to have an  extended s e r v i c e l i f e when used under c o n d i t i o n s f a v o r i n g decay.  The p o s s i b i l i t y of breeding h i g h l y r e s i s t a n t  types i s a l s o d i s c u s s e d  briefly.  genetic  ii  TABLE OF CONTENTS Page I. II.  III.  INTRODUCTION  1  CHEMICAL SEPARATION  8  A.  S e l e c t i o n and P r e p a r a t i o n o f M a t e r i a l . .  8  B.  I s o l a t i o n o f Chemical Components . . . .  11  1.  Acetone e x t r a c t i o n  11  2.  Ether e x t r a c t i o n  13  3.  Water e x t r a c t i o n  14  NATURE OF THE ISOLATED CHEMICAL COMPONENTS .  l6  A.  Acetone S o l u b l e s  l6  B.  Ether Solubles  19  1.  Neutrals  19  2.  Free a c i d s  20  3.  Phlobatannin  20  C. IV.  22  Water S o l u b l e s  BIOASSAY A.  24  Experimental 1.  Method  24  N o n - c e l l u l o s i c medium  24  a.  P r e p a r a t i o n of s u b s t r a t e  . . . .  26  b.  S e l e c t i o n o f f u n g a l organisms. .  29  c.  Inoculation of P e t r i dishes. . .  3°  d.  Incubation  and measurement of 3°  growth e.  Experimental  design  31  iii  Page 2.  C e l l u l o s i c medium  3 2  a.  P r e p a r a t i o n of s o i l j a r s  3 2  b.  S e l e c t i o n of f u n g a l organisms . .  3 3  c.  I n o c u l a t i o n and i n c u b a t i o n o f soil  B.  C.  D.  V. VI.  jars  3 3  d.  Preparation  of t e s t specimens . .  3 4  e.  Exposure o f t e s t b l o c k s  3 6  f.  Experimental d e s i g n  3 6  A n a l y t i c a l Method  3 7  1.  N o n - c e l l u l o s i c medium  3 7  2.  C e l l u l o s i c medium  40  Results  41  1.  N o n - c e l l u l o s i c medium  41  2.  C e l l u l o s i c medium  4 4  Discussion  4 5  1.  Taxifolin  4 5  2.  Phlobatannin  4 9  3.  Free a c i d s  5 1  4.  Neutrals  5 2  5.  Water s o l u b l e s  5 2  6.  Zinc chloride  5 3  CONCLUSION  5 4  LITERATURE CITED  5 6  iv  VII.  APPENDICES A.  Growth and t o t a l i n h i b i t i o n p o i n t curves o f Fomes annosus and Lentinus l e p i d e u s i n malt agar c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f extractives.  B.  S t a t i s t i c a l a n a l y s i s o f weight l o s s e s i n Douglas f i r b l o c k s exposed t o P o r i a i n c r a s s a t a and L e n t i n u s l e p i d e u s .  V  ILLUSTRATIONS Figure 1. 2. 3.  Page O u t l i n e o f e x t r a c t i o n procedure f o r i s o l a t i o n o f extraneous components Method of c a l c u l a t i n g percentage r e t a r d a t i o n of m y c e l i a l growth E f f e c t of v a r y i n g t a x i f o l i n c o n c e n t r a t i o n on growth o f Fomes annosus i n malt agar . .  9 3 9 42  TABLES Table 1.  2.  Page C o n c e n t r a t i o n of e x t r a c t i v e s and z i n c c h l o r i d e i n malt agar needed t o completely i n h i b i t growth of Fomes annosus and Lentinus l e p i d e u s a t 2 5 ° C. Average weight l o s s e s o f e x t r a c t e d Douglas f i r wood b l o c k s i n s o i l j a r s (2-month incubation)  4 3  44  vi  ACKNOWLEDGMENT The  author wishes t o acknowledge the encouragement  and h e l p f u l c r i t i c i s m s o f f e r e d by P r o f . J.W. W i l s o n , F a c u l t y of F o r e s t r y , under whose d i r e c t i o n t h i s work was undertaken. The  suggestions o f f e r e d by Dr. D.C. Buckland, F a c u l t y o f  F o r e s t r y , are a l s o The  appreciated.  f r e e use o f f a c i l i t i e s a t the Vancouver Branch,  F o r e s t Products L a b o r a t o r i e s acknowledged.  o f Canada, i s g r a t e f u l l y  S p e c i a l thanks are due t o Dr. J.A.F. Gardner,  Head, Wood Chemistry S e c t i o n , and the other members o f the S t a f f f o r t h e i r suggestions and a s s i s t a n c e .  1  I.  It total  has been e s t i m a t e d  destroyed  itself  wooden t i m b e r s  by'fungi.  fungal,  but  insect,  (6).  It  resistance  so t h a t  those  more e f f e c t i v e l y  that  ber  c o u l d be e x p e c t e d  manufactured the  within species  playing  to last  exhibiting  longer  by u s i n g  the highest  as a s e e d  degree  accounting  harvested  i n 1953  (11)•  be u n d e r -  timber  i n service  source  m i g h t be  exhibitacceptable  than  those  T h e r e w o u l d a l s o be genetic  only  of durability  f o r 39 p e r c e n t  species  the  made f r o m s u c h t i m -  resistance.  f i r (Pseudotsuga  States.  w o u l d be more  durable  types  T h i s m i g h t be those  trees  dis-  factors.  menziesii  the predominant commercial timber  bia,  that  material.  decay  forest  responsible for  Products  of breeding highly  Douglas is  known,  decay.  from u n s e l e c t e d  possibility  accomplished  were  i s due  high resistance  the f a c t o r s  o f these factors  favoring  been  influencing  timber  exhibiting  d u r a b i l i t y within a species  under c o n d i t i o n s  of the United  the factors  If  i n the  is  f o r Canada a r e u n a v a i l a b l e ,  o f o u r major  woods  have  d r a i n o f s i x per cent  those  utilized.  a preponderance  that  a n d w i n d damage  approximate  o f the  of the United States  statistics  i s important  n a t u r a l decay stood,  drought  nine per cent  i n service  A further  Comparable  undoubtedly  ing  that  annual d r a i n on the f o r e s t s  used t o r e p l a c e  to  INTRODUCTION  species  of the t o t a l  (Mirb.)  Franco)  of B r i t i s h Columcubic  Of a l l l u m b e r m a n u f a c t u r e d  volume i n the  2  P r o v i n c e i n 1952,  47 per cent was Douglas f i r ( 2 6 ) .  Virtually  a l l of the softwood plywood produced on the P a c i f i c Coast i s made from t h i s s i n g l e s p e c i e s .  Douglas f i r i s a l s o w i d e l y  u t i l i z e d f o r p o l e s and p i l i n g , and as a raw m a t e r i a l f o r s u l phate and mechanical p u l p i n g .  S i n c e the q u a n t i t y o f a c c e s s i -  b l e timber i n merchantable s i z e s i s being c o n t i n u a l l y reduced, it  i s expedient t h a t our e x i s t i n g supply of t h i s v a l u a b l e spe-  c i e s be extended as f a r as p o s s i b l e . The u s e f u l l i f e  of timber under c o n d i t i o n s f a v o r i n g  decay has been g r e a t l y extended through the use of wood p r e servatives.  These p r e s e r v a t i v e s g i v e maximum p r o t e c t i o n to  wood when they are a p p l i e d by p r e s s u r e methods.  The h e a r t -  wood of Douglas f i r grown on the Coast i s only moderately  dif-  f i c u l t to impregnate, but the Rocky Mountain form o f t h i s  spe-  c i e s i s very d i f f i c u l t t o p e n e t r a t e ( 3 0 ) .  Douglas f i r grown  i n the i n t e r i o r of B r i t i s h Columbia i s n o t o r i o u s i n t h i s respect.  S i n c e some forms of i t s heartwood  cannot be p r o p e r l y  t r e a t e d when d e s i r e d , Douglas f i r i s o f t e n used under  condi-  t i o n s f a v o r i n g r a p i d decay without f i r s t having r e c e i v e d a p r e s e r v a t i v e treatment.  The only a l t e r n a t i v e i n t h i s  i s t o s e l e c t n a t u r a l l y d u r a b l e timber f o r such u s e s .  instance This  cannot be done u n t i l the f a c t o r s i n f l u e n c i n g the r e l a t i v e l y h i g h d u r a b i l i t y of Douglas f i r are understood. I t i s apparent t h a t the e x i s t i n g s u p p l i e s of Dougl a s f i r c o u l d be extended i f the i n c i d e n c e and extent of decay i n t r e e s and timbers of t h i s s p e c i e s were reduced.  3  Wood decay i n g e n e r a l may  be d e f i n e d a s those chemical and  p h y s i c a l changes r e s u l t i n g from the a c t i v i t y of wooddestroying fungi.  The f u n g i capable of such d e s t r u c t i o n  r e q u i r e c e r t a i n c o n d i t i o n s i n order t o develop to t h e i r maximum  level.  One  f a c t o r of major importance  t h i s l e v e l i s a s u i t a b l e source of f o o d .  i n t h e i r reaching Wood substance i s  capable of s u p p l y i n g the necessary f o o d requirements.  Wood  decay occurs as these f u n g i s e c r e t e s p e c i f i c enzymes capable of h y d r o l y z i n g the wood substance absorbed by the f u n g i .  to forms t h a t can be  Separate enzymes a c t f u r t h e r upon  these h y d r o l y z e d products d u r i n g the process of f u n g a l metabolism. F i n h o l t , et a l (l6), have suggested two mechanisms by which i n t r o d u c e d p r e s e r v a t i v e s might prevent decay i n wood. T h e i r f i r s t h y p o t h e s i s proposes  t h a t these substances  actually  i n t e r f e r e w i t h the m e t a b o l i c processes of the fungus.  That i s ,  the o x i d a t i o n - r e d u c t i o n enzymes (endoenzymes) t h a t normally r e a c t to d i g e s t the w a t e r - s o l u b l e compounds are upset i n such a way  t h a t they are unable to f u n c t i o n .properly.  theory suggests t h a t the enzymes s e c r e t e d by the (exoenzymes) are denatured  T h e i r second fungus  so that they become i n c a p a b l e o f  h y d r o l y z i n g the wood complex.  T h e i r experimental  evidence  f a v o r s the second h y p o t h e s i s .  A h i g h - b o i l i n g creosote  was  found to be n o n - t o x i c when mixed w i t h malt agar, but h i g h l y t o x i c when used as a p r e s e r v a t i v e i n s m a l l wood b l o c k s . d e n t l y , the h y d r o l y t i c enzymes were denatured  Evi-  i n both c a s e s ,  4  but  t h i s a c t i o n had no e f f e c t i n malt agar, s i n c e the food  source (maltose) i s a l r e a d y l a t i o n by the fungus.  i n a form t h a t allows  On the other hand, i t i s w e l l known  t h a t f u n g i can be i n h i b i t e d and k i l l e d even a s m a l l c o n c e n t r a t i o n of t h i s f a c t , the f i r s t  i t s assimi-  i n malt agar  containing  of h i g h l y t o x i c chemicals.  theory  In view  seems most a p p r o p r i a t e .  It  appears t h a t both the endo- and exoenzymes a s s o c i a t e d w i t h a fungus may be d i s t u r b e d by the presence o f c e r t a i n chemical compounds. D i f f e r e n t species  of wood e x h i b i t a w i d e l y  varying  a b i l i t y t o r e s i s t the a c t i o n of wood-decaying organisms. Moreover, some v a r i a t i o n i n t h i s a b i l i t y e x i s t s from t r e e t o tree within a single species.  Attempts have been made t o  e x p l a i n t h i s v a r i a t i o n i n decay r e s i s t a n c e by c o n s i d e r i n g the g e n e r a l p h y s i c a l and chemical nature of wood.  Zabel  (57)  concluded from a l i t e r a t u r e survey t h a t no o v e r a l l r e l a t i o n s h i p e x i s t s between s p e c i f i c g r a v i t y and decay r e s i s t a n c e within a species.  When c o n s i d e r i n g  extremes w i t h i n a s p e c i e s ,  however, very dense wood may be more durable than the l i g h t e s t wood, s i n c e gaseous d i f f u s i o n i s a t a minimum i n the h e a v i e s t wood w i t h the s m a l l e s t v o i d volume.  Between d i f f e r e n t spe-  c i e s , there i s no c o r r e l a t i o n between d e n s i t y and d u r a b i l i t y . Some f a c t o r other  than d e n s i t y i s t h e r e f o r e l a r g e l y r e s p o n s i -  b l e f o r the n a t u r a l decay r e s i s t a n c e e x h i b i t e d by some woods. Hawley, e t a l , ( 2 0 )  were the f i r s t t o note a r e l a -  t i o n between the d u r a b i l i t y and chemical composition o f wood.  5  When water e x t r a c t s from a number of d u r a b l e s p e c i e s were mixed w i t h malt agar s o l u t i o n s , they were found to exert  a  t o x i c e f f e c t on f u n g a l growth.  the  heartwood was  The  hot water e x t r a c t of  observed t o be the most t o x i c , w h i l e s i m i l a r  e x t r a c t s from the sapwood d i s p l a y e d very l i t t l e f u n g i c i d a l activity.  S e v e r a l other  i n v e s t i g a t o r s (38,  39,  40,  49,  57)  have s i n c e concluded t h a t the decay r e s i s t a n c e of d u r a b l e wood s p e c i e s can be best e x p l a i n e d  by c o n s i d e r i n g  the nature of  t h e i r heartwood e x t r a c t i v e s .  Sapwood of a l l s p e c i e s i s con-  s i d e r e d non-durable, except when i t e x i s t s i n the l i v i n g  tree.  In t h i s case, i t i s u s u a l l y more r e s i s t a n t to decay than heartwood, s i n c e the h i g h e r moisture content of sapwood present The  may  an u n f a v o r a b l e water-oxygen balance to most f u n g i .  i n a b i l i t y of most wood-destroying f u n g i to f u n c t i o n as  t r u e p a r a s i t e s a l s o prevents sapwood decay i n v i v o . Sherrard  and K u r t h (47)  showed t h a t the d u r a b i l i t y  o f redwood (Sequoia sempervirens (Lamb.) Endl.) was due  to i t s hot-water e x t r a c t , and  t i o n i n the stem. son  (43)  Southara and  that t h i s v a r i e d with p o s i -  Erlich  have concluded that v a r i o u s  (49)  oak  (57)  of p h e n o l i c He  Atkinof  are t o x i c to f u n g i .  suggested t h a t the w a t e r - s o l u b l e  (Quercus a l b a L.)  resistance.  and R o f f and  extractive portions  western red cedar (Thuja p l i c a t a D. Don) Zabel  largely  tannins  of white  are l a r g e l y r e s p o n s i b l e f o r i t s decay  Rennerfelt  (38,  39,  40)  e x t r a c t i v e s i n Scots pine  found t h a t p i n o s y l v i n , and  i n v e s t i g a t e d the e f f e c t (Pinus  silvestris  to a l e s s e r extent,  L.).  pinosylvin  6  monomethylether were t o x i c to f u n g i . In summary, woods n a t u r a l l y unfavorable t o f u n g i as a source of food are s a i d to be d u r a b l e , or to have a h i g h degree o f decay r e s i s t a n c e .  Species t h a t e x h i b i t t h i s p r o p e r t y  have n a t u r a l d u r a b i l i t y f a c t o r s t h a t a c t u a l l y i n h i b i t f u n g a l development. extracts.  These a r e u s u a l l y a t t r i b u t a b l e t o heartwood  The chemical nature o f heartwood e x t r a c t i v e s v a r i e s  between s p e c i e s ; those e x t r a c t s showing the h i g h e s t degree of t o x i c i t y are obtained from woods proven to be more durable i n service.  The chemicals i n the heartwood e x t r a c t probably  exert t h e i r t o x i c e f f e c t by i n t e r f e r i n g with the h i g h l y spec i a l i z e d and i m p e r f e c t l y understood  enzyme system o f the  fungus. The d u r a b i l i t y o f Douglas f i r i s i n t e r m e d i a t e between the very r e s i s t a n t and only moderately  durable  groups.  Redwood and western r e d cedar are examples f a l l i n g w i t h i n the former  c l a s s i f i c a t i o n , whereas the l a t t e r group i s r e p r e s e n t e d  by spruce and hemlock ( 5 1 ) .  A l i t e r a t u r e survey r e v e a l e d t h a t  no e x p l a n a t i o n had y e t been g i v e n f o r the comparatively h i g h n a t u r a l d u r a b i l i t y o f Douglas f i r .  Only one e x t r a c t i v e com-  ponent o f Douglas f i r , t a x i f o l i n , had been p r e v i o u s l y i n v e s t i gated f o r f u n g i c i d a l a c t i v i t y . weak f u n g i c i d e ( 5 4 ) .  I t was claimed t o be a very  The statement  correct bibliographical notation. r e c t i f i e d through correspondence  was n o t supported by the T h i s misunderstanding  w i t h the a u t h o r ' ( 1 2 ) ,  r e f e r r e d to unpublished r e s u l t s o f R e n n e r f e l t .  was  who  7  F u r t h e r correspondence  (37) r e v e a l e d t h a t o n l y a  c u r s o r y i n v e s t i g a t i o n had been made w i t h t a x i f o l i n i n malt agar.  P u l l u l a r i a < a genus p r i m a r i l y r e s p o n s i b l e f o r c o n i f e r -  ous f o l i a g e d i s e a s e s , was used as the t e s t fungus.  No numeri-  c a l or comparative e x p r e s s i o n f o r t o x i c i t y was o b t a i n e d , s i n c e the amounts o f t a x i f o l i n added t o the malt agar s o l u t i o n s were not determined. I t was the s p e c i f i c purpose o f t h i s work t o i n v e s t i gate chemical e x t r a c t s o f Douglas f i r heartwood  i n an attempt  to a s c r i b e the reason f o r i t s r e l a t i v e l y h i g h d u r a b i l i t y t o certain extractive materials.  In order t o do t h i s , i t was  necessary t o f i r s t prepare v a r i o u s Douglas f i r  heartwood  e x t r a c t s , and then t o d i v i d e these i n t o l e s s complex groups. F o l l o w i n g t h i s , an assessment  ( o r b i o a s s a y ) o f each of these  m a t e r i a l s as f u n g i c i d e s was made.  8  II.  CHEMICAL SEPARATION  Extraneous (or e x t r a c t i v e ) components have been defined  as o r g a n i c  by n e u t r a l s o l v e n t s  substances that may (23).  There i s no  be  extracted  single universal  vent t h a t w i l l remove a l l of these v a r i o u s nents.  In order to i n s u r e  from wood sol-  e x t r a c t i v e compo-  that the e x t r a c t i v e s have been  q u a n t i t a t i v e l y removed, a number of d i f f e r e n t s o l v e n t s must be employed.  Generally,  separate e x t r a c t i o n s w i t h a l c o h o l  acetone, ether, and water are s u f f i c i e n t . out the c o l o r i n g matter, tannins the p r o p e r t y  and  removes the s o l u b l e , short-chained some f r e e a c i d s and  salts  (28).  Acetone leaches  phlobaphenes.  of removing o i l s , f a t s and  or  resins.  Ether  Cold water  carbohydrates, as w e l l Accordingly,  has  these three  as sol-  vents were used i n t h i s study to prepare the major e x t r a c t i v e fractions. C e r t a i n extraneous components of Douglas f i r h e a r t wood have been i n v e s t i g a t e d by Schorger (46) A systematic  and  (33»34).  a n a l y s i s of the e n t i r e e x t r a c t i v e f r a c t i o n has  been made by Graham and Kurth (19). because i t provided  T h e i r method was  chosen  a complete e x t r a c t i o n procedure, w i t h sub-  sequent s u b d i v i s i o n i n t o known components.  An o u t l i n e of  t h i s e x t r a c t i o n procedure i s presented i n F i g . SELECTION AND  Pew  1.  PREPARATION OF MATERIALS  A s i n g l e codominant Douglas f i r t r e e was  felled  on  DOUGLAS FIR HEARTWOOD  extracted with  extracted with  ethyl ether  acetone and water  ETHER 2%  ACID SALTS  ACE" "ONE  EXTRACT No OH  wat er  NEUTRALS  WATER-INSOLUBLE  FRACTION  FRACTION  ethyl ether  H CI  WATER-  SOLUBLE  WATER - SOLUBLE  WATER-  ETHER-If (SOLUBLE  ETHER -SOLUBLE  ETHER-SOLUBLE  FRAC T I O N  FRAC TION  FRACTION  FRACTION  ethyl  acetate  IMPURITIES  I N S O L U B L E  W A T E R - I NS O L U B L E • T H E R -  ace tone  acetate  TANNIN  »S O L U B L E  F R A C T I O N  ethyl  PHLOBATANNIN  CARBOHYDRATES  ethyl ether  SOLI BLE  ethyl  EXTRACT  etha nol  WATER - SOLUBLE  ethyl ether  WA1 ER  WATER  EXTI ?ACT  petroleum  AMORPHOUS IMPURITIES  ether  PHLOBAPHENE  ether  CRUDE CRYSTALLINE MATERIAL recrys tallized wi th etha nol  TAXIFOLIN  Fig. I.  EXTRACTION  PROCEDURE FOR ISOLATION OF EXTRANEOUS  COMPOUNDS  10  October 1,  1954.  hemlock-cedar  T h i s t r e e had been growing on a  s i t e on the U n i v e r s i t y f o r e s t a t Haney,  I t measured 11.0  r e c t i o n f o r stump h e i g h t , was  I t s age, i n c l u d i n g a c o r -  approximately 83 y e a r s .  i n c h band o f apparent sapwood surrounded the Two  B.C.  inches i n diameter a t b r e a s t h e i g h t , and  had a t o t a l h e i g h t of 104 f e e t .  1.5  fir-  A  heartwood.  f o u r - f o o t b o l t s were s e l e c t e d from the b u t t end  of the t r e e a f t e r f e l l i n g .  The b u t t end was  preferentially  chosen, s i n c e t h e r e i s some evidence i n c e r t a i n other s p e c i e s to i n d i c a t e t h a t t h i s p o r t i o n i s more durable than the top (4,47).  A young, second-growth  work by Graham and K u r t h (19)  t r e e was  selected,  has i n d i c a t e d t h a t  because  heartwood  m a t e r i a l from t h i s source c o n t a i n s a h i g h e r percentage o f e x t r a c t i v e s than old-growth timber. A f t e r s l a b b i n g the apparent sapwood from the f i r s t b o l t , the heartwood  p o r t i o n was  sawn i n t o boards and  q u e n t l y reduced to p l a n e r s h a v i n g s .  subse-  These shavings were  f u r t h e r reduced .by g r i n d i n g i n a W i l e y m i l l u n t i l they would pass through a 20-mesh s c r e e n .  The r e s u l t i n g wood meal  was  then a i r seasoned u n t i l i t s moisture content dropped t o approximately 15 per c e n t .  An extended p e r i o d o f d r y i n g  was  avoided, s i n c e the v o l a t i l e p o r t i o n of the extraneous mater i a l s could be reduced by t h i s procedure.  I t has been  p o i n t e d out t h a t e x t e n s i v e a i r seasoning decreases the amount of e t h e r e x t r a c t o b t a i n e d from Douglas f i r wood  (19).  1 1  ISOLATION OF CHEMICAL COMPONENTS 1.  Acetone  extraction  A l a r g e g l a s s tank was used t o e x t r a c t 1 3 7 5 wood meal (oven-dry b a s i s ) w i t h acetone.  A f r e s h supply o f  s o l v e n t was i n t r o d u c e d i n t o the tank every two days, f i r s t removing  gms. o f  and s t o r i n g the p r e v i o u s e x t r a c t .  after  Removal  of the e x t r a c t from the wood meal was expedited by u s i n g a coarse g l a s s d i f f u s i o n tube, t o which s u c t i o n was a p p l i e d . The acetone was changed t w i c e , so t h a t a t o t a l o f t h r e e e x t r a c t i v e treatments was f i n a l l y o b t a i n e d . The b o i l i n g p o i n t of acetone it  ( 5 6 . 5 °  O  can e a s i l y be d i s t i l l e d a t low temperatures.  i s such t h a t Therefore,  the e x t r a c t was c o n c e n t r a t e d t o a volume of 6 3 5 ml. by d i s tilling  the acetone.  A 1 5 ml. a l i q u o t from t h i s c o n c e n t r a t e d  e x t r a c t was d r i e d t o constant weight under vacuum a t 3 5 °  C.  The t o t a l c a l c u l a t e d y i e l d o f s o l i d m a t e r i a l was 2 . 3 1 per cent o f the oven-dry weight of the wood. A milky-white suspension was o b t a i n e d upon a d d i t i o n of 2 5 0  ml. of water t o the 6 2 0  ml. of c o n c e n t r a t e d e x t r a c t .  The s o l u t i o n was then f u r t h e r c o n c e n t r a t e d t o a volume o f 2 5 0 ml. under a vacuum a t 4 5 ° was  C.  The c o l l o i d a l  suspension  f l o c c u l a t e d by adding a s m a l l q u a n t i t y o f sodium  sul-  phate w h i l e c o n s t a n t l y a g i t a t i n g the s o l u t i o n w i t h a magnetic s t i r r e r .  A f t e r the white p r e c i p i t a t e had s e t t l e d , the  c l e a r supernatant s o l u t i o n was decanted and saved.  12  Both the c l e a r l i q u i d and  the white p r e c i p i t a t e were  exhaustively extracted with e t h y l ether.  T h i s was  done to  i s o l a t e t h a t f r a c t i o n of the t o t a l wood e x t r a c t which i s r e a d i l y s o l u b l e i n ether a f t e r i n i t i a l tone.  e x t r a c t i o n with  ace-  I t would not have been p o s s i b l e to q u a n t i t a t i v e l y  remove t h i s m a t e r i a l d i r e c t l y from the wood meal w i t h (19>33>34)•  Four separate  procedure:  e x t r a c t s were obtained  a water-soluble,  by  To the two  this  ether-insoluble fraction;  water-and e t h e r - s o l u b l e f r a c t i o n ; a w a t e r - i n s o l u b l e , s o l u b l e f r a c t i o n , and  ether  a  ether-  a water-and e t h e r - i n s o l u b l e f r a c t i o n .  e t h y l e t h e r - s o l u b l e p o r t i o n s , petroleum ether  added to serve as a p r e c i p i t a t i n g agent. q u a n t i t y had  After a  sufficient  been added to the c o n s t a n t l y a g i t a t e d s o l u t i o n ,  a l i m i t e d amount of a red-brown, amorphous m a t e r i a l was c i p i t a t e d as i m p u r i t i e s and  discarded.  crude c r y s t a l l i n e mass was  pre-  A white p r e c i p i t a t e  f o l l o w e d a f t e r adding more petroleum e t h e r .  times.  was  The r e s u l t i n g  r e c r y s t a l l i z e d from ethanol  four  A f t e r the f o u r t h r e c r y s t a l l i z a t i o n , the m a t e r i a l  washed with water and sphere of burner gas  was  d r i e d under vacuum i n an i n e r t atmoto prevent o x i d a t i o n .  Slightly  over  three grams of creamy-white c r y s t a l s were produced, r e p r e s e n t i n g a y i e l d of 0.23  P r e  The w a t e r - s o l u b l e , exhaustively nin.  The  cent. ether-insoluble solution  was  e x t r a c t e d w i t h e t h y l a c e t a t e to i s o l a t e the  s o l v e n t was  of s o l i d s i s o l a t e d was  tan-  removed under vacuum, but the amount insignificant.  13  The water-and e t h e r - i n s o l u b l e f r a c t i o n was  redis-  solved i n acetone and r e p r e c i p i t a t e d w i t h e t h y l ether e r a l times i n order to o b t a i n a pure product. brown, amorphous powder was y i e l d of 0.014 2.  sev-  Finally, a  i s o l a t e d , d r i e d and weighed.  per cent of the oven-dry wood was  A  obtained.  Ether e x t r a c t i o n A l a r g e S o x h l e t was  employed to e x t r a c t a f r e s h A t o t a l of 2.75  sample of wood meal w i t h e t h y l e t h e r . of wood meal (oven-dry weight) was  kg.  extracted i n consecutive  250  gm.  was  added between changes of wood meal to compensate f o r  p o r t i o n s f o r p e r i o d s of 24 hours.  t h a t which was  l o s t through  Only enough e t h e r  evaporation.  The r e s u l t i n g c l e a r yellow e x t r a c t was to 285 ml. by d i s t i l l i n g  the e t h e r .  concentrated  A 10-ml. a l i q u o t from  t h i s s o l u t i o n , d r i e d to constant weight under vacuum, i n d i cated t h a t a t o t a l s o l i d s y i e l d of 0.97  per cent of the  oven-dry weight of the wood had been o b t a i n e d . t h i s l i m i t e d amount of m a t e r i a l , i t was  In view of  i n a d v i s a b l e to  r e s o l v e i t i n t o a l l i t s separate compounds.  I f t h i s were  done, there would probably have been too l i t t l e of any  single  component to t e s t f o r f u n g i c i d a l p r o p e r t i e s .  Instead,  the  e x t r a c t was  classifications  separated i n t o three r a t h e r broad  f o r p r e l i m i n a r y study. t o x i c , a new  I f any group proved  e x t r a c t c o u l d be prepared  to be h i g h l y  and f u r t h e r sub-  d i v i d e d i n an attempt t o l o c a t e the exact source  of  14  fungicidal  activity.  The  concentrated ether s o l u t i o n was  w i t h a two per cent sodium hydroxide t o r y f u n n e l , thus forming  s o l u t i o n i n a separa-  s a l t s of the f r e e o r g a n i c a c i d s .  The n e u t r a l m a t e r i a l remained behind i n an ether l a y e r .  extracted  The ether s o l v e n t was  orange-colored  evaporated a t room  temperature, so t h a t as much of the v o l a t i l e o i l component as p o s s i b l e would remain behind. s o l v e n t was was  When no f u r t h e r l o s s of  noted, the h i g h l y v i s c o u s m a t e r i a l remaining  s e a l e d and s t o r e d . To the a l k a l i n e water s o l u t i o n , an equal volume of  e t h y l ether was  added.  The aqueous phase was  once a g a i n by the a d d i t i o n of HC1.  acidified  Thus a w a t e r - i n s o l u b l e :  a c i d f r a c t i o n ( e t h e r l a y e r ) and a w a t e r - s o l u b l e were o b t a i n e d .  fraction  A f t e r e v a p o r a t i n g the ether under  p r e s s u r e , a s o l i d a c i d p o r t i o n was  reduced  obtained.  The w a t e r - s o l u b l e phase l o s t most of i t s c o l o r upon exhaustive e x t r a c t i o n w i t h e t h y l a c e t a t e . powder was oven.  i s o l a t e d by e v a p o r a t i n g the s o l v e n t i n a vacuum  A y i e l d of 0 . 0 7  the wood meal was 3.  A red-brown  per cent of the oven-dry weight of  obtained.  Water e x t r a c t i o n A f t e r thoroughly a i r d r y i n g the a c e t o n e - e x t r a c t e d  sawdust, i t was meal was  leached w i t h d i s t i l l e d water.  The wood  e x t r a c t e d i n a l a r g e g l a s s j a r a t room temperature  15  for  a f i v e - d a y p e r i o d , w i t h one  days.  change of water a f t e r  E x t r a c t i o n a t e l e v a t e d temperatures  was  two  avoided,  as  t h i s c o u l d r e s u l t i n a g r a d u a l h y d r o l y s i s of the c e l l w a l l itself  (50).  The  e x t r a c t was  temperature water bath at 80° c i p i t a t e was nol  concentrated i n a constant C.  A f l o c c u l e n t white p r e -  formed on the a d d i t i o n of f o u r volumes of e t h a -  to the s o l u t i o n .  The p r e c i p i t a t e was  t r i f u g i n g , and then r e d i s s o l v e d i n water.  c o l l e c t e d by  cen-  Fractional  r e p r e c i p i t a t i o n w i t h ethanol removed f i r s t the i m p u r i t i e s , and a white p r e c i p i t a t e r e s u l t e d as more ethanol was  added.  The white p r e c i p i t a t e was  washed w i t h e t h a n o l and d r i e d i n  the a i r .  per cent was  A y i e l d of O.36  obtained.  16  III.  NATURE OF THE ISOLATED CHEMICAL COMPONENTS  ACETONE SOLUBLES The major p o r t i o n of the acetone  e x t r a c t was  obtained i n the form o f creamy-white c r y s t a l s , w h i l e the minor f r a c t i o n was i s o l a t e d as an amorphous powder. The c r y s t a l l i n e product o b t a i n e d from the e t h e r s o l u b l e p o r t i o n of the acetone e x t r a c t was f i r s t d e s c r i b e d by Pew  (33,34)  and subsequently by Graham and K u r t h  (19).  I t s s t r u c t u r e corresponds t o t h a t of a flavanone ( I ) , spec i f i c a l l y 3, (33,34). ( 5 , 7,  5, 7,  3/> 4 / - pentahydroxy  flavanone ( I I )  T h i s flavanone i s r e a d i l y o x i d i z e d t o q u e r c e t i n  3 / 4 / - t e t r a h y d r o x y flavanone) ( I I I ) ( 5 4 ) .  Since  the former flavanone d i f f e r s from q u e r c e t i n o n l y by two  I Flavanone S t r u c t u r e  II Taxifolin  ( 3 , 5 , 7 , 3 / , 4 / - pentahydroxy  flavanone)  17  OH  0  OH  0  III  IV  Quercetin  Eriodictyol  a d d i t i o n a l hydrogen atoms a t the 2 and 3 p o s i t i o n s w i t h a consequent  l o s s o f a double bond, i t i s commonly c a l l e d  dihydroquercetin.  Other e q u a l l y common names a r e t a x i -  f o l i n and Douglas f i r f l a v a n o n e . Eriodictyol  (IV) i s a r e d u c t i o n product o f t a x i -  f o l i n , formed on a d d i t i o n o f z i n c dust and h y d r o c h l o r i c a c i d t o an a l c o h o l i c s o l u t i o n of the f l a v a n o n e .  The d e v e l -  opment o f a d i s t i n c t i v e lavender c o l o r , r e p o r t e d l y charact e r i s t i c f o r 3 - hydroxy-flavanones, of  this reaction (33?34).  occurs i n the course  The deepness of the c o l o r p r o -  duced can probably be r e l i e d upon t o g i v e a q u a n t i t a t i v e estimate of the amount o f t a x i f o l i n p r e s e n t ( 3 ) .  Accord-  i n g l y , a s m a l l amount o f prepared c r y s t a l l i n e product was weighed out and d i s s o l v e d i n methanol.  The r e s u l t i n g  solu-  t i o n was then analyzed q u a n t i t a t i v e l y by the Wood Chemistry S e c t i o n o f the Vancouver Branch, F o r e s t Products t o r i e s of Canada.  The consequent  Labora-  d e t e r m i n a t i o n , which  agreed w e l l w i t h the c o n c e n t r a t i o n t h a t had p r e v i o u s l y been determined  g r a v i m e t r i c a l l y , i n d i c a t e d t h a t the i s o l a t e d  18  flavanone was  relatively  pure.  F u r t h e r evidence  obtained from a m e l t i n g - p o i n t d e t e r m i n a t i o n . crystals  prepared  from the acetone  a t 233°-236° C.  decomposition  the r e p o r t e d values of 240  was  The  taxifolin  e x t r a c t melted  with  T h i s compares f a v o r a b l y w i t h  - 242° C. (33,34) and 237°  C.  (19). A c o l o r i m e t r i c a n a l y s i s of a methanol e x t r a c t from the o r i g i n a l mixed heartwood meal showed the t a x i f o l i n c o n c e n t r a t i o n to be equal to 0.45 the i s o l a t e d was  y i e l d of 0.23  total  per c e n t .  per cent of c r y s t a l l i n e  s l i g h t l y more than 50 per cent of the t o t a l .  reported a y i e l d  (before r e c r y s t a l l i z a t i o n )  Graham and K u r t h  (19)  product Pew  of 0.62  obtained a y i e l d of 0.8 per  Thus  (33,34)  per  cent.  cent.  The brown amorphous powder obtained from the waterand e t h e r - i n s o l u b l e f r a c t i o n v i o u s l y been determined  of the acetone  e x t r a c t has  as a phlobaphene (19).  Phlobaphenes  are d e f i n e d as a l c o h o l - s o l u b l e , w a t e r - i n s o l u b l e products of t a n n i n s (37).  i n this reported  e x t r a c t was  study, whereas 3.7  isolated  Only  0.62  as phlobaphene  per cent had p r e v i o u s l y been  (19). T h i s amorphous m a t e r i a l was  acetone  and probably i s  a s s o c i a t e d w i t h the phlobaphenes (8,19).  per cent of the acetone  condensation  I n wood a n a l y s i s , n a t i v e l i g n i n  i s a l s o removed by the same treatment, closely  pre-  and dioxane  after  isolation.  explanations f o r t h i s i n s o l u b i l i t y :  insoluble i n alcohol, There are two p o s s i b l e f u r t h e r condensation  19  between molecules may  have o c c u r r e d d u r i n g p u r i f i c a t i o n , or  the i s o l a t i o n procedure may  have removed the a s s o c i a t e d  m a t e r i a l which normally e x e r t s a p e p t i z i n g e f f e c t The phlobaphene was  (53)•  not i n v e s t i g a t e d i n the f o l l o w i n g  toxi-  c i t y s t u d i e s , s i n c e the i n s o l u b i l i t y of t h i s m a t e r i a l p r e vented i t s proper impregnation i n t o the s u b s t r a t e s prepared for fungal attack. ETHER SOLUBLES Three f r a c t i o n s were obtained from the ether e x t r a c tion:  1 group of n e u t r a l m a t e r i a l s , a f r e e - a c i d  and a t a n n i n - l i k e substance.  portion,  Each of these are d i s c u s s e d  separately. 1.  Neutrals The n e u t r a l m a t e r i a l remaining i n the ether l a y e r  a f t e r e x t r a c t i o n w i t h d i l u t e a l k a l i probably c o n t a i n e d a heterogeneous  mixture of compounds.  T h i s product, which  r e p r e s e n t e d 10 per cent of the ether e x t r a c t , p r o b a b l y i n c l u d e d a c e r t a i n amount of combined a c i d s i n a d d i t i o n t o the u n s a p o n i f i a b l e v o l a t i l e o i l s and waxes (19).  Its highly  aromatic odor suggested the presence of the v o l a t i l e s a l l y a s s o c i a t e d w i t h Douglas f i r o l e o r e s i n .  Schorger  usu(46)  i n v e s t i g a t e d t h i s f r a c t i o n and found t h a t i t contained a mixture of terpenes, predominantly i t s d e r i v a t i v e , 1 - oC - t e r p i n e o l  1 - <K(VI).  pinene  (V) and  Johnson and C a i n  20  V  VI  o^-pinene  <X-terpineol The remaining  components  i n the n e u t r a l f r a c t i o n  have not been s p e c i f i c a l l y i d e n t i f i e d , but probably  consist  of a c i d s combined as e s t e r s , as w e l l as high-carbon a l c o h o l s (sterols). 2.  Free A c i d s The f r e e a c i d p o r t i o n o b t a i n e d by a l k a l i e x t r a c -  t i o n of the ether s o l u b l e s was i s o l a t e d as a dark-brown, tacky substance, having a c o n s i s t e n c y s i m i l a r to t a f f y candy.  T h i s f r a c t i o n has been r e p o r t e d to c o n s i s t o f an  a b i e t i c - t y p e r e s i n a c i d i n a d d i t i o n to s e v e r a l u n i d e n t i f i e d acids 3.  (19).  Phlobatannin The red-brown amorphous powder, which was  obtained  by e x t r a c t i n g the water phase w i t h e t h y l a c e t a t e , had the p r o p e r t i e s of a p h l o b a t a n n i n , s i n c e i t produced a red-brown p r e c i p i t a t e when heated w i t h d i l u t e m i n e r a l a c i d .  The  21  p r e c i p i t a t e from t h i s r e a c t i o n i s termed phlobaphene, and thus phlobatannins nins  are d e f i n e d as phlobaphene-producing t a n -  (54). Most n a t u r a l l y o c c u r r i n g phlobatannins  p h l o r o g l u c i n o l and p r o t o c a t e c h u i c  will  yield  a c i d on a l k a l i n e f u s i o n .  These same compounds are produced when c a t e c h i n (VII) i s s i m i l a r l y t r e a t e d (44).  C a t e c h i n may be r e l a t e d t o t a x i -  f o l i n inasmuch as i t i s thought t o be an u l t i m a t e product  of q u e r c e t i n .  reduction  Attempts t o convert t a x i f o l i n t o a  c r y s t a l l i n e c a t e c h i n have been u n s u c c e s s f u l , but the amorphous m a t e r i a l obtained by these experiments has shown q u a l i t a t i v e p r o p e r t i e s s i m i l a r t o c a t e c h i n (33)•  OH  OH  VII Catechin As p o i n t e d out p r e v i o u s l y , phlobaphenes may be s y n t h e s i z e d by h e a t i n g phlobatannins acids.  This conversion  with d i l u t e  mineral  i s accompanied by the l o s s of  water (18,44).  Thus phlobaphenes may be considered  s a t i o n products  of phlobatannins.  Similarly,  conden-  phlobatannins  22  are b e l i e v e d t o be condensed molecules, formed from c a t e c h i n nucleus  (44).  Moreover, c a t e c h i n r e p r e s e n t s a  reduced form of t a x i f o l i n . be a fundamental  the  T h e r e f o r e , d i h y d r o q u e r c e t i n may  molecular type from which the more complex  p h l o b a t a n n i n and phlobaphene a r i s e . been suggested by K u r t h , who  Such a r e l a t i o n  has  termed pigments such as  c e t i n " p r e c u r s o r s of the p h l o b a t a n n i n s " ( 2 7 ) .  quer-  Russell  a l s o i n d i c a t e d the p o s s i b i l i t y of t h i s r e l a t i o n s h i p  has  (44).  WATER SOLUBLES While d r y i n g i n the a i r , p a r t s of the white  granu-  l a r p r e c i p i t a t e o r i g i n a l l y i s o l a t e d from the water e x t r a c t turned s l i g h t l y brown i n c o l o r .  T h i s may  have been due  o x i d a t i o n of gums and p e c t i n - l i k e substances  to  that can occur  i n the w a t e r - s o l u b l e f r a c t i o n along w i t h the p o l y s a c c h a r i d e s . A s m a l l amount of the p r e c i p i t a t e d  carbohydrate  was  h y d r o l y z e d f o r one hour w i t h s u l p h u r i c a c i d i n a b o i l -  ing  water b a t h .  A f t e r removing the a c i d by t r e a t i n g  s o l u t i o n w i t h an ion-exchange a n a l y s i s was  made.  r e s i n , a paper  The chromatogram was  the  chromatographic  developed f o r 12  hours i n b u t a n o l - a c e t i c acid-water i n the volume r a t i o 4:1:5, and then sprayed w i t h a n i l i n e t r i c h l o r a c e t a t e  (10).  Both the brown and white p o r t i o n s of the carbohydrate  frac-  t i o n were found t o c o n s i s t of polymers  of arabinose and  g a l a c t o s e s i n c e o n l y these sugars were d e t e c t e d a f t e r lysis.  A p r e v i o u s study of the h y d r o l y z a t e of the  hydro-  water-soluble  p o l y s a c c h a r i d e of Douglas f i r has  the presence of g a l a c t o s e OS)  (50).  (90$), arabinose  Before t h i s time, the p r i n c i p a l  had been regarded  as g a l a c t a n  (19).  revealed  (<)%) and  xylos  carbohydrate  24  IV. The  BIOASSAY  i s o l a t e d components i n c l u d e d the  taxifolin,  n e u t r a l , f r e e a c i d , p h l o b a t a n n i n and carbohydrate These m a t e r i a l s had to be e v a l u a t e d f o r r e l a t i v e activity.  T h i s was  done by o b s e r v i n g how  between the substances  fungicidal  a g i v e n chemical  a f f e c t e d the growth of a f u n g a l organism. d i r e c t comparisons  fractions.  In order t h a t c o u l d be made,  n u m e r i c a l e x p r e s s i o n s f o r t h e i r e f f e c t i v e n e s s were  sought.  EXPERIMENTAL METHOD A measure of the t o x i c i t y of a chemical may  be  obtained by i n t r o d u c i n g the m a t e r i a l i n t o a s u b s t r a t e , which i s subsequently exposed to the a c t i o n of a f o r a s p e c i f i e d l e n g t h of time.  Two  fungus  g e n e r a l types of sub-  s t r a t e s have been employed t o e v a l u a t e t o x i c e f f e c t s wood-destroying  fungi.  One  on  type Involves the use o f some  n o n - c e l l u l o s i c medium such as malt agar or an aqueous n u t r i e n t s o l u t i o n t o which the chemical may  be added.  In  the second type, the chemical i n q u e s t i o n i s added d i r e c t l y to the c e l l u l o s e m a t e r i a l such as wood, pulp or sawdust. One  procedure  i n v o l v i n g each of these approaches  i n c l u d e d i n t h i s study.  is  These w i l l be c o n s i d e r e d sepa-  rately. 1.  N o n - c e l l u l o s i c Medium N o n - c e l l u l o s i c s u b s t r a t e s f o r e v a l u a t i n g wood  25  p r e s e r v a t i v e s have employed water or agar, to which n u t r i ents have been added (2,15,20,38,39,40,43,47,49,57). chemicals added as t o x i c a n t s may  The  not be present i n the same  form as they n a t u r a l l y occur i n the wood; thus, the  numeri-  c a l r e s u l t s obtained might not n e c e s s a r i l y apply t o a c e l l u l o s e - f u n g i c i d e system.  N e v e r t h e l e s s , the use of a non-  c e l l u l o s i c medium p r o v i d e s a s a t i s f a c t o r y method of d e t e r mining comparative  t o x i c i t y of d i f f e r e n t substances  (29).  I f an aqueous n u t r i e n t s o l u t i o n i s used, i t i s i n o c u l a t e d w i t h a fungus which i s then allowed t o grow f o r a s p e c i f i e d p e r i o d of time.  A f t e r the mycelium has been  removed by f i l t e r i n g the n u t r i e n t s o l u t i o n , i t i s ovend r i e d and weighed.  The amount of growth i n s o l u t i o n s of  v a r y i n g chemical c o n c e n t r a t i o n s , as r e f l e c t e d by the weight of the m y c e l i a , serves to i n d i c a t e the degree of f u n g i c i d a l toxicity.  T h i s method has one s e r i o u s disadvantage:  c a l s added i n the form of w a t e r - i n s o l u b l e s o l u t i o n s  chemicannot  be kept i n a d i s p e r s e d c o n d i t i o n without the a d d i t i o n of an e m u l s i f y i n g agent.  For t h i s reason, t h i s method was  s e r i o u s l y considered f o r t h i s  not  study.  When malt agar i s employed as a s u b s t r a t e , r a d i a l measurements of m y c e l i a l development are taken p e r i o d i c a l l y a f t e r i n o c u l a t i o n w i t h a fungus.  The r e t a r d e d r a t e of  growth on malt agar c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s of chemicals can be used to express t o x i c i t y .  T h i s technique  has been employed by s e v e r a l i n v e s t i g a t o r s  (15,20,38,39,43,  26  47>49j57).  T  h  e  m  a  l  t  agar method i s the most r a p i d one a v a i l -  able f o r the e v a l u a t i o n  of f u n g i c i d e s .  M y c e l i a l growth  begins soon a f t e r i n o c u l a t i o n so that d a t a can be c o l l e c t e d almost immediately, without w a i t i n g period.  f o r a long  incubation  I t s p r o p e r t y of g e l l i n g q u i c k l y on c o o l i n g makes i t  p o s s i b l e to r e t a i n water-insoluble state (29).  solutions i n a dispersed  Since two of the chemical f r a c t i o n s t h a t were  to be evaluated were i n s o l u b l e i n w a t e r - m i s c i b l e  solvents  such as a l c o h o l , malt agar was s e l e c t e d as the n o n - c e l l u l o s i c medium. Preparation  of s u b s t r a t e — D i f c o  malt agar was d i s s o l v e d i n  d i s t i l l e d water and s t e r i l i z e d i n 100-ml. q u a n t i t i e s .  In  order t o determine the e f f e c t o f chemical c o n c e n t r a t i o n t o x i c i t y , varying  amounts of the prepared e x t r a c t i v e s were  then added to the malt agar. and  on  Weighed amounts o f t a x i f o l i n  p h l o b a t a n n i n were added i n the form of a l c o h o l i c s o l u -  t i o n s , whereas the a c i d and n e u t r a l f r a c t i o n s were f i r s t solved  i n ether.  dis-  The amount of chemicals added ranged  between 0 . 0 1 gm. and 1.08 gm.  One ml. of s o l u t i o n was the  s m a l l e s t amount added, w h i l e h i g h e r volumes (up t o 4 ml.) were used to correspond t o l a r g e r amounts of m a t e r i a l . l i m i n a r y t e s t s had r e v e a l e d toxic effect. water-insoluble  Alchohol  that ether  exerted only a s l i g h t  had p r e v i o u s l y been used t o apply  substances t o malt agar ( 3 8 , 3 9 ) .  solutions containing  Pre-  both a l c o h o l and ether  Malt agar  of v a r i o u s  27  concentrations The  were prepared f o r c o n t r o l purposes.  c o n c e n t r a t i o n of m a t e r i a l i n the r e s u l t i n g  malt agar s o l u t i o n was meter. by 1 0 0 . cc,  T h i s was  expressed i n grams per c u b i c  converted  centi-  to a percentage by m u l t i p l y i n g  Since the volume of malt agar used was  100  always  the weight of m a t e r i a l added to the malt agar became a  d i r e c t measure of the percentage c o n c e n t r a t i o n . t r a t i o n of the chemical  s o l v e n t i n s o l u t i o n was  expressed as a percentage.  S i n c e between one  were added, the c o n c e n t r a t i o n ranged from one  to f o u r per  The  concen-  also  and  four  ml.  i n the malt-agar s o l u t i o n  cent.  When the f l a s k s c o n t a i n i n g the malt agar had  cooled  s u f f i c i e n t l y a f t e r s t e r i l i z a t i o n , the a l c o h o l i c chemical u t i o n s were a s e p t i c a l l y added and mixed w i t h g e n t l y s w i r l i n g the f l a s k s .  The  the malt agar by  m a t e r i a l i n the f l a s k s  then poured i n t o s m a l l P e t r i d i s h e s  (50 mm.).  sol-  Eight  was  dishes  c o u l d be poured w i t h each 100 ml. of prepared s o l u t i o n . s m a l l e r P e t r i d i s h e s were used i n p r e f e r e n c e  The  to l a r g e r ones,  so t h a t more r e p l i c a t i o n s w i t h i n a s i n g l e c o n c e n t r a t i o n would be obtained w i t h the l i m i t e d q u a n t i t y of e x t r a c t i v e s on hand. E s s e n t i a l l y the same method was P e t r i dishes  employed to prepare  t h a t would c o n t a i n the e t h e r - s o l u b l e groups.  S i n c e ether and water are i m m i s c i b l e ,  s p e c i a l precautions  were necessary to i n s u r e proper d i s p e r s a l of e x t r a c t i v e s i n the ether s o l u t i o n .  Ordinary  unsatisfactory dispersion.  s w i r l i n g or s t i r r i n g g i v e s  The  use of a Waring blender  an or  28  other high-speed mixer has been advocated  for similar  lems w i t h o i l - s o l u b l e wood p r e s e r v a t i v e s ( 1 5 ) .  prob-  This modifi-  c a t i o n d i v i d e s the i m m i s c i b l e m a t e r i a l i n t o very s m a l l dropl e t s so that a n e a r l y homogenized s o l u t i o n i s o b t a i n e d .  The  e t h e r - s o l u b l e chemical s o l u t i o n s were added to the malt  agar  and thoroughly mixed under a s e p t i c c o n d i t i o n s w i t h a stirrer.  J u s t before the s o l u t i o n g e l l e d , i t was  P e t r i d i s h e s r e s t i n g on an i c e bath. w i t h i n seconds,  magnetic  poured  into  The malt agar hardened  l e a v i n g the chemicals suspended as very  f i n e l y d i s p e r s e d d r o p l e t s , d i s c e r n a b l e o n l y w i t h a handlens. I t was  not a n t i c i p a t e d t h a t the w a t e r - s o l u b l e carbo-  hydrate f r a c t i o n would i n h i b i t f u n g a l growth.  This  was  added to a p l a i n agar s o l u t i o n i n c o n c e n t r a t i o n s up to per c e n t .  The carbohydrate was  sterilized  s e p a r a t e l y from  the agar s o l u t i o n t o a v o i d h y d r o l y s i s of the polymers simple sugars.  A f t e r c o o l i n g , the agar was  two  to  then added to  the dry g r a n u l a r carbohydrate f r a c t i o n , and the p l a t e s were poured  i n the u s u a l manner.  In t e s t i n g the w a t e r - s o l u b l e  f r a c t i o n , the carbohydrate m a t e r i a l r e p l a c e d the malt which was  used as a n u t r i e n t i n the p r e v i o u s t e s t s . I t was  d e s i r a b l e to compare the r e s u l t s o b t a i n e d  using the wood e x t r a c t s w i t h a w a t e r - s o l u b l e chemical commonly employed as a wood p r e s e r v a t i v e . T h e r e f o r e , a p a r a l lel  experiment,  was  s e t up.  c o n s i s t i n g of z i n c c h l o r i d e as the t o x i c a n t ,  29  S e l e c t i o n of f u n g a l o r g a n i s m s — A fungus must be a b l e t o grow reasonably w e l l i n c u l t u r e t o be o f v a l u e agar t e s t .  i n a malt-  Secondly, the f u n g i s e l e c t e d t o t e s t e x t r a c t i v e s  p e c u l i a r to a c e r t a i n species with t h i s species.  should  be commonly  associated  F i n a l l y , the t e s t organisms must n o t be  too s e n s i t i v e t o the a l c o h o l and ether  s o l v e n t s used.  A  combination o f these requirements l e d to the s e l e c t i o n o f Fomes annosus (Fr.) Cke. and Lentinus  lepideus F r .  P o r i a i n c r a s s a t a (B.&C.) C u r t , was chosen f o r use, low  concentration  I t was d i s c a r d e d  of a l c o h o l markedly r e t a r d e d  Initially but even  i t s growth.  i n f a v o r of Fomes annosus.  F. annosus i s common i n America, Europe, I n d i a and A u s t r a l i a , where i t causes a b u t t - r o t o f c o n i f e r s .  I t i s the  most important cause o f h e a r t r o t i n c o n i f e r s grown i n England where many p l a n t a t i o n s of Douglas f i r have been e s t a b l i s h e d (9). and  I t u s u a l l y enters  the heartwood through dead r o o t s ,  e v e n t u a l l y reduces the i n f e s t e d r o o t s and b u t t to a  s e r i e s of e l l i p t i c a l white p o c k e t s .  The fungus i s a l s o prev-  a l e n t on damp mine timbers i n both Europe and the U n i t e d States  (6,9).  Douglas f i r i s the most common source of mine  timbers i n the Western S t a t e s , and i s being for  the same purpose i n the East  (32).  used i n c r e a s i n g l y  F. annosus i s used  e x t e n s i v e l y i n l a b o r a t o r y t e s t s w i t h malt agar, s i n c e i t grows v i g o r o u s l y and i s g e n e r a l l y r e s i s t a n t t o f o r e i g n chemicals. Lentinus  lepideus  a l s o has a world-wide d i s t r i b u t i o n  30  I t i s r a r e l y found a t t a c k i n g a l i v i n g t r e e , but i s a very common f a c t o r i n d e t e r i o r a t i n g timbers i n s e r v i c e .  It  causes a t y p i c a l brown r o t , e v e n t u a l l y r e d u c i n g the wood t o a s e r i e s of brown cubes.  I n the U n i t e d S t a t e s , i t i s one  of the most s e r i o u s d e s t r o y e r s of c o n i f e r o u s r a i l r o a d and p o l e s ( 6 ) .  ties  Douglas f i r i s u t i l i z e d e x t e n s i v e l y f o r  both of these purposes, e s p e c i a l l y i n the Northwest and i s s u b j e c t to the a c t i o n o f t h i s fungus.  (32),  I n England i t  i s the most important wood-destroying fungus a t t a c k i n g paving b l o c k s and mine timbers ( 9 ) .  poles,  L_s. l e p i d e u s grows a t a  moderately f a s t r a t e i n malt agar, and i s commonly used when wood p r e s e r v a t i v e s a r e t o be e v a l u a t e d . I n o c u l a t i o n of P e t r i d i s h e s — P u r e c u l t u r e s of Fomes annosus and L e n t i n u s l e p i d e u s were o b t a i n e d from the Wood Pathology U n i t of the Vancouver Branch, F o r e s t Products L a b o r a t o r i e s of Canada.  From these o r i g i n a l c u l t u r e s , a number of t r a n s -  p l a n t s were made t o p l a i n malt agar.  A f t e r an l8-day p e r i o d  (± 3 d a y s ) , a p i e c e of inoculum was taken from the a c t i v e l y growing margin and t r a n s f e r r e d t o the outer edge of each Petri dish.  The i n o c u l a were a l l c u t w i t h a number one cork  borer (4 mm.  diam.) t o i n s u r e a uniform s i z e .  A l l inocula-  t i o n s were performed no l a t e r than 24 hours a f t e r p r e p a r a t i o n of the P e t r i  dishes.  I n c u b a t i o n and measurement of g r o w t h — T h e temperature t o which a f u n g a l organism i s exposed g r e a t l y I n f l u e n c e s i t s r a t e of growth.  The optimum temperature f o r Fomes annosus  31  i s 23° C, w h i l e 27° C i s most s u i t a b l e f o r L e n t i n u s l e p i d e u s (9).  Consequently, a l l the i n o c u l a t e d P e t r i d i s h e s were  incubated i n a constant temperature chamber a t 25° C.  The  p l a t e s were o r d i n a r i l y not s e a l e d because of the s h o r t durat i o n of the experiment.  However i n the case of the d i s h e s  c o n t a i n i n g the v o l a t i l e n e u t r a l f r a c t i o n , masking used t o s e a l the d i s h e s .  tape was  T h i s e f f e c t i v e l y prevented the  l o s s of p o s s i b l y t o x i c , v o l a t i l e m a t e r i a l s t o the surrounding chamber  atmosphere. Readings were taken a t approximately two-day i n t e r -  v a l s u n t i l the p l a t e s were covered w i t h mycelium,  or u n t i l  the agar s t a r t e d t o d r y and crack through d e s i c c a t i o n . Fomes annosus c o n t r o l p l a t e s were covered i n s i x days, w h i l e L e n t i n u s l e p i d e u s c o n t r o l s took a t o t a l of t e n days. The amount of r a d i a l growth between the c e n t e r of the inoculum and the margin of the mycelium was measured i n millimeters.  Under comparable  seldom v a r i e d more than f o u r  c o n d i t i o n s , the r e p l i c a t e s mm.  Experimental d e s i g n — A s has been p r e v i o u s l y s t a t e d , e i g h t p l a t e s were poured f o r each c o n c e n t r a t i o n o f a g i v e n chemical.  T h e r e f o r e , a t o t a l of f o u r r e p l i c a t i o n s were a v a i l a b l e  f o r t e s t i n g the r e a c t i o n s of each fungus.  Owing t o the  v a r y i n g amounts of chemicals a v a i l a b l e , the c o n c e n t r a t i o n s examined were not s t a n d a r d i z e d throughout the experiment. The c o n c e n t r a t i o n s t e s t e d f o r each g i v e n chemical may be read from the curves i n Appendix  A.  32  Malt-agar p l a t e s c o n t a i n i n g  one, two and f o u r per  cent ether and a l c o h o l were poured f o r c o n t r o l s .  These  sol-  vent volumes corresponded to those a c t u a l l y used t o d i s s o l v e the t a x i f o l i n , p h l o b a t a n n i n , n e u t r a l , and a c i d f r a c t i o n s . P e t r i dishes  containing  o n l y malt agar were a l s o  inoculated  to determine t o what degree the ether and a l c o h o l  solvents  themselves were t o x i c . 2.  C e l l u l o s e Medium N a t u r a l c o n d i t i o n s of wood decay can be more  c l o s e l y approximated by exposing wood b l o c k s isms under c o n t r o l l e d c o n d i t i o n s . t e s t i n g wood p r e s e r v a t i v e s wood block  t o decay organ-  The B r i t i s h  standard f o r  c o n s i s t s of exposing a t r e a t e d  t o the a c t i o n of a fungus growing on malt agar  i n a K o l l e f l a s k (9)•  British  and American workers now  f a v o r the s o i l - j a r method of L e u t r i t z (29)» where a t r e a t e d block has  i s p l a c e d on top of a s i m i l a r untreated  block  that  p r e v i o u s l y been b u r i e d i n s o i l i n o c u l a t e d w i t h a wood-  destroying  fungus.  I n both cases,  the degree of decay i s  determined by the weight l o s s s u f f e r e d by the wood a f t e r an i n c u b a t i o n p e r i o d o f three to nine months.  block The  American method was adopted f o r t h i s experiment, s i n c e i t requires l e s s manipulation, actual f i e l d Preparation  and more c l o s e l y  represents  conditions. of s o i l  . j a r s — P r e s e r v i n g j a r s (16 oz.) were  33  h a l f f i l l e d w i t h a h i g h - o r g a n i e content s o i l t h a t had p r e v i o u s l y been moistened 1.5-x  t o 45 per cent moisture c o n t e n t .  A  2.5-x 5»0-cm. feeder block o f Douglas f i r sapwood, c u t  so t h a t i t s long a x i s was p a r a l l e l t o the g r a i n , was b u r i e d i n the s o i l so t h a t o n l y i t s upper s u r f a c e was exposed.  The  j a r s were then s t e r i l i z e d f o r one hour a t 15 pounds steam pressure p r i o r to i n o c u l a t i o n . Selection of fungal organisms—In  a t e s t of t h i s nature, i t  i s d e s i r a b l e t o use f u n g i t h a t r a p i d l y decay timber i n s e r v ice,  thus causing a s i g n i f i c a n t l o s s i n weight over a s h o r t  p e r i o d of time.  L e n t i n u s l e p i d e u s has been found t o cause  severe weight l o s s e s w i t h i n f o u r months, but f o r the same p e r i o d o f time, Fomes annosus decomposed wood b l o c k s only f i v e t o seven per cent ( 9 ) . T h e r e f o r e , a s u b s t i t u t e f o r F. annosus seemed a d v i s a b l e f o r the wood-block t e s t . P o r i a i n c r a s s a t a a common cause o f d r y r o t , was s e l e c t e d f o r t h i s purpose.  T h i s fungus i s extremely  active  along the P a c i f i c Coast and i n the Southeastern U n i t e d S t a t e s , where the p r e v a i l i n g c l i m a t e presents an agreeable s i t u a t i o n f o r the decay of c o n i f e r o u s b u i l d i n g timbers ( 6 ) . I n o c u l a t i o n and i n c u b a t i o n o f s o i l j a r s — T h e i n o c u l a used were cut from the margin o f the fungus growing i n malt  agar,  and pressed down i n t o the s o i l d i r e c t l y adjacent t o the b u r i e d feeder b l o c k .  The j a r s were then incubated i n d i f f u s e  l i g h t a t room temperature  f o r 30 days.  At the end of t h i s  34  time, the m y c e l i a and  had  become w e l l - e s t a b l i s h e d i n the  on the feeder b l o c k s  j a r s had  i n most of the  An  excess of  been prepared so t h a t o n l y those p r o v i d i n g an  well-developed mycelium were continued Preparation ment had  jars.  soil  even,  i n the t e s t .  of t e s t s p e c i m e n s — I f the o b j e c t of t h i s  experi-  been the e v a l u a t i o n of a wood p r e s e r v a t i v e ,  of wood would have been impregnated w i t h the  blocks  preservative  and  then i n t r o d u c e d  The  t o x i c i t y of i n d i v i d u a l e x t r a c t i v e s cannot be t e s t e d i n  t h i s way,  i n t o the p r e v i o u s l y i n o c u l a t e d s o i l  s i n c e they are a l r e a d y  wood samples.  The  present  as a group i n the  t o x i c e f f e c t of an e x t r a c t i v e can  be  determined i n d i r e c t l y by l e a c h i n g i t from wood b l o c k s they are exposed to a fungus. t o x i c , the e x t r a c t e d faster rate.  The  blocks  jars.  before  I f the m a t e r i a l removed i s  can be expected to decay a t a  amount of decay t h a t occurs i n these  e x t r a c t e d b l o c k s w i l l be r e f l e c t e d by t h e i r weight l o s s e s . The  g r e a t e r weight l o s s e s s u f f e r e d by the e x t r a c t e d  as compared to the c o n t r o l samples serve importance of the e x t r a c t e d  blocks  to i n d i c a t e the  component as a n a t u r a l wood  preservative. Small samples were prepared from which e x t r a c t i v e s c o u l d be e a s i l y leached. p r o p o r t i o n of end penetration  I t was  n e c e s s a r y t h a t a major  g r a i n be exposed i n order  both f o r e x t r a c t i o n and  decay.  normally used i n s o i l - j a r t e s t s would not be  to The  facilitate i f - i n c h cubes  leashed  35  s a t i s f a c t o r i l y , nor would they undergo decay as r a p i d l y as s m a l l e r p i e c e s w i t h more exposed end g r a i n . has  Findlay  (14)  suggested that t h i n p i e c e s under 6 c c . w i l l give  sig-  n i f i c a n t weight l o s s e s over a short p e r i o d .  This i s  e x p l a i n e d by the i n c r e a s e d r a t e of gaseous d i f f u s i o n when surface-volume r a t i o s are  high.  A r a d i a l l y sawn board 5*0  cm.  i n thickness  was  taken from the o r i g i n a l t e s t b o l t f o r the p r e p a r a t i o n of wood-block samples.  The  amount and  t o x i c i t y of heartwood  e x t r a c t i v e s may  vary w i t h r a d i a l p o s i t i o n i n the stem of a  t r e e (4,9>57)»  Such v a r i a t i o n s can be n u l l i f i e d by making  a l l heartwood t e s t b l o c k s wood.  T h i s was  a uniform  d i s t a n c e from the  done by r i p p i n g a 2 . 5  cm.  band of  sap-  outer  heartwood from the e n t i r e l e n g t h of the r a d i a l l y sawn board. The r e s u l t i n g s t r i p was  dressed  to 1.5-x  5.0-cm.  reduced to s m a l l b l o c k s by c r o s s - c u t t i n g at 0 . 6 vals.  This cm.  1.5-x  Each sample b l o c k thus measured 0.6-x  was  inter4.0-cm.,  the s m a l l e s t dimension being p a r a l l e l to the l o n g i t u d i n a l a x i s of the wood f i b e r s . Ten randomly s e l e c t e d samples were then leached e i t h e r acetone, e t h e r , or water. preceding leached  i n i t i a l chemical  in  T h i s corresponded to the  analysis.  The  t e s t b l o c k s to be  i n acetone or water were p l a c e d i n g l a s s j a r s a t  room temperature f o r s i x days, to simulate  the o r i g i n a l con-  d i t i o n s of the acetone and water e x t r a c t i o n s . the r e s p e c t i v e s o l u t i o n s was  necessary  Aspiration i n  to i n s u r e complete  36  immersion of the b l o c k s .  The ether e x t r a c t i o n was  out i n a S o x h l e t u n t i l the c o l l e c t e d s o l v e n t was c o l o r e d , i n d i c a t i n g a q u a n t i t a t i v e removal materials.  carried  slightly  o f the extraneous  A one-week p e r i o d was s u f f i c i e n t f o r t h i s pur-  pose. In order t o e s t a b l i s h an i n i t i a l weight  of the e x t r a c t e d samples,  equilibrium  they were c o n d i t i o n e d to  a moisture content of 30 per cent i n a constant humidity chamber.  temperature-  T h i s was done i n p r e f e r e n c e t o oven-  d r y i n g , which might reduce t o x i c v o l a t i l e substances.  Heat-  i n g the b l o c k s may a l s o i n c r e a s e the r e s i n content a t the s u r f a c e of the acetone-and  w a t e r - e x t r a c t e d samples and so  i n c r e a s e decay r e s i s t a n c e  (52).  Exposure  of t e s t b l o c k s — W h e n the samples reached  equilib-  rium weight, they were each f i t t e d w i t h a s t r a i g h t p i n , and f l a m e - s t e r i l i z e d immediately The b l o c k s remained  before p l a c i n g i n the s o i l  jars.  i n the j a r s i n d i f f u s e l i g h t f o r two  months a t room temperature.  ?/hen t h i s i n c u b a t i o n p e r i o d had  e l a p s e d , the b l o c k s were removed, brushed mycelium and o v e n - d r i e d .  Oven-drying  case s i n c e s e v e r e l y decayed  f r e e of excess  i s necessary i n t h i s  samples w i l l come t o a h i g h e r  e q u i l i b r i u m moisture content i f r e c o n d i t i o n e d i n the same manner as i n i t i a l l y  (31).  A f t e r d r y i n g completely, the  b l o c k s were weighed t o determine  t h e i r l o s s i n weight.  Experimental d e s i g n — T e n b l o c k s were used i n each of the,  37  acetone, ether and water e x t r a c t i o n s . ten unextracted  samples was  Another group of  i n c l u d e d f o r c o n t r o l purposes.  F i v e b l o c k s from each treatment were s u b j e c t e d to decay by P. i n c r a s s a t a , w h i l e the remaining  h a l f were put i n t o s o i l  j a r s p r e v i o u s l y i n o c u l a t e d w i t h Lentinus  lepideus.  ANALYTICAL METHOD 1.  N o n - c e l l u l o s i c Medium I t has been w e l l e s t a b l i s h e d t h a t a s t e a d i l y  decreasing  amount of growth i s obtained as the  of f u n g a l i n h i b i t o r s i n malt agar i s i n c r e a s e d  concentration (5).  In  order to o b t a i n a d i r e c t comparison between the extraneous compounds t e s t e d , an estimate was c o n c e n t r a t i o n necessary  needed of the  to completely  chemical  i n h i b i t growth.  a c o n c e n t r a t i o n i s c a l l e d the T o t a l I n h i b i t i o n P o i n t and has been d e f i n e d by Schmitz (45) t r a t i o n a l l o w i n g no  Such (T.I.P.),  as the minimum concen-  signs of growth e i t h e r on the malt agar  or the inoculum plug i t s e l f .  T h i s should not be  confused  w i t h the k i l l i n g p o i n t , which i s t h a t c o n c e n t r a t i o n necessary to k i l l a fungus. necessarily k i l l e d .  At the T.I.P. the fungus i s not  Most f u n g i have the a b i l i t y to remain  dormant over long p e r i o d s of time without  v i s i b l e s i g n s of  growth. As  the chemical  c o n c e n t r a t i o n i n c r e a s e s , the amount  of m y c e l i a l growth decreases i n a c u r v i l i n e a r manner t h a t  38  can be represented The  curves  by a p a r a b o l i c or h y p e r b o l i c f u n c t i o n .  obtained  co-ordinate  by p l o t t i n g these p o i n t s on  paper c o u l d be  f i n d the T.I.P.  extrapolated  ordinary  to zero growth to  Such a procedure w i t h c u r v i l i n e a r Bateman (5)  o f t e n causes i n a c c u r a t e r e s u l t s . s t r a i g h t l i n e c o u l d be obtained cent r e t a r d a t i o n of growth was of per cent c o n c e n t r a t i o n  found t h a t a  i f the l o g a r i t h m  of the  p l o t t e d a g a i n s t the  of f u n g i c i d e .  i t d i d apply between the T.I.P. and  have been analyzed The  The  a concentration  This l i n e a r r e l a t i o n  data c o l l e c t e d i n t h i s  organism.  with study  The  chemical  resulting figures,  the means of f o u r measurements, were then  p l o t t e d on o r d i n a r y c o - o r d i n a t e respective controls.  The  paper along w i t h  ordinate represented  i n m i l l i m e t e r s , whereas time i n days was abscissa.  With few  exceptions,  their  r a d i a l growth  p l o t t e d on  the  the p o i n t s were found t o  assume a n e a r l y p e r f e c t s t r a i g h t - l i n e r e l a t i o n s h i p . had  was  from measurements of f u n g a l  averaged f o r each i n d i v i d u a l  c o n c e n t r a t i o n , time, and representing  approxi-  a f t e r the method of Bateman.  data obtained  growth were f i r s t  rela-  concentrations,  found to be v a l i d not o n l y with Fomes anno.qnfi, but green p l a n t s as w e l l .  per  logarithm  While t h i s  t i o n s h i p d i d not h o l d c o n s i s t e n t l y a t lower  mately o n e - t h i r d of t h a t v a l u e .  functions  p r e v i o u s l y made the same o b s e r v a t i o n  (5).  sketch of such a graph i s shown i n F i g . 2.  A  Bateman  diagramatic  /  /  ///  TIME  Figure 2 . a b b/ ab ab ax  -  a  X  Techniques of A n a l y s i s  t y p i c a l growth i n c o n t r o l dishes data of b/ c o r r e c t e d f o r r e s t p e r i o d t y p i c a l growth i n t o x i c a n t - c o n t a i n i n g amount of r e t a r d a t i o n per cent r e t a r d a t i o n  dishes  Throughout the course of the experiments, growth r a r e l y s t a r t e d immediately a f t e r t r a n s p l a n t i n g the even among the c o n t r o l s . before  A r e s t p e r i o d of a day  growth commenced was  p e r i o d was  not uncommon.  a fungal i n h i b i t o r .  f i n a l l y s t a r t e d , however, the r a t e was f a s t as that of the c o n t r o l s .  or more  A longer  o f t e n r e q u i r e d when an inoculum was  P e t r i dish containing  inoculum,  rest  planted  in a  When growth  sometimes n e a r l y  as  Bateman observed t h i s same  phenomenon, but added a c o r r e c t i o n f a c t o r f o r v a r i a b l e r e s t periods. work.  T h i s method was  I f a chemical was  a p p l i e d throughout the found to induce a longer  present rest  p e r i o d than the c o n t r o l , the p l o t t e d p o i n t s were f i r s t  40  connected i n the u s u a l manner t o get the best p o s s i b l e l i n e a r fit,  A l i n e was then drawn p a r a l l e l to t h i s , through the  p o i n t o f i n t e r s e c t i o n of the c o n t r o l l i n e and the a b s c i s s a . F i g . 2 exemplifies  t h i s case, where l i n e b / r e p r e s e n t s  the  o r i g i n a l d a t a , b i t s c o r r e c t i o n f o r r e s t p e r i o d , and a the control data. Percentage r e t a r d a t i o n of growth was c a l c u l a t e d i n the f o l l o w i n g manner: fungus i n c o v e r i n g  the r a d i a l growth a t t a i n e d by the  the c o n t r o l p l a t e was c o n s i d e r e d  per cent ( l i n e ax, F i g . 2 ) . i n a dish containing  as 100  F o r the same time p e r i o d ,  growth  a f u n g i c i d e reached only a f r a c t i o n of  the c o n t r o l ( l i n e bx, F i g . 2 ) .  Thus, the r e t a r d i n g e f f e c t -  could be r e p r e s e n t e d by l i n e ab, or the percentage  retarda-  t i o n by ab/ax. The  f i g u r e s obtained f o r per cent r e t a r d a t i o n were  plotted against l o g ;paper.  per cent c o n c e n t r a t i o n  of chemical on double-  The p o i n t s were f i t t e d t o a s t r a i g h t l i n e , and  the l i n e e x t r a p o l a t e d  t o 100 per cent r e t a r d a t i o n .  The con-  c e n t r a t i o n a t which r e t a r d a t i o n of growth equaled 100 per cent represented the T.I.P. 2.  C e l l u l o s i c Medium The  weight l o s s e s s u f f e r e d by the t e s t b l o c k s  undergoing decay i n the s o i l  while  j a r s were expressed as a per-  centage on the b a s i s of the o r i g i n a l oven-dry weight of the specimens.  Oven-dry v a l u e s were c a l c u l a t e d from the weights  41  o b t a i n e d a t an e q u i l i b r i u m moisture by d i v i d i n g by  content of 30 per  1.3.  The percentage  l o s s i n weight of the  e t h e r - and water- e x t r a c t e d wood blocks was the c o n t r o l s w i t h Student's for " t The  M  cent  " t " test.  acetone-,  compared a g a i n s t  The r e s u l t i n g  values  were evaluated at the f i v e per cent c o n f i d e n c e  level.  lower weight l o s s e s of 20 per cent have a s m a l l e r v a r i -  ance about t h e i r mean than the h i g h e r values of 55 P r e  I t was  t h e r e f o r e necessary  percentages  original  i n t o r a d i a n s , thereby making the data independent  of t h e i r r e s p e c t i v e means. /percentage  to f i r s t t r a n s f o r m the  cent.  was  The  t r a n s f o r m a t i o n arc s i n  used f o r t h i s purpose  (36).  RESULTS 1.  N o n - c e l l u l o s i c medium The  odd-numbered curves i n Appendix A show the  growth of the t e s t f u n g i i n malt agar c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s of e x t r a c t i v e s i n s o l u t i o n .  Curves obtained  w i t h some very low c o n c e n t r a t i o n s are not shown, s i n c e they are c o - i n c i d e n t w i t h the c o n t r o l l i n e s .  Higher  concentrations  of t o x i c e x t r a c t i v e s o b v i o u s l y had an e f f e c t on the growth r a t e of both f u n g i . 3,  An example of t h i s i s presented i n F i g .  where a s e r i e s of P e t r i d i s h e s c o n t a i n i n g s u c c e s s i v e l y  h i g h e r c o n c e n t r a t i o n s of t a x i f o l i n were exposed to the a c t i o n of F. annosus.  4-2  Figure 3 E f f e c t o f t a x i f o l i n c o n c e n t r a t i o n on growth of Fomes annosus i n malt agar A - control  D E F  B - 0.05$ C -  0.1%  - 0.2% - 0.4$ - 0.d%  Not o n l y d i d the r a d i a l growth o f the mycelium decrease w i t h c o n c e n t r a t i o n , but the c h a r a c t e r o f the f u n g a l mat changed as w e l l .  G e n e r a l l y speaking,  as concentra-  t i o n i n c r e a s e d , the hyphae became more a e r i a l i n n a t u r e , the mycelium being p a r t i c u l a r l y lum.  Mat  abundant around the i n o c u -  margins a l s o became more uniform,  s i n c e the  43  u s u a l advancing zone was The the  even-numbered curves i n Appendix A  logarithmic  against  per  r e l a t i o n of per  cent c o n c e n t r a t i o n .  l a t e d to 100 T.I.P.  lacking.  per  represent  cent r e t a r d a t i o n p l o t t e d These have been extrapo-  cent r e t a r d a t i o n i n order to determine  Values f o r T.I.P. are summarized i n Table Table  the  1.  1  C o n c e n t r a t i o n of Chemicals i n Malt Agar Needed to Completely I n h i b i t Fungal Growth a t 25°  1  Organism  Chemical or extractive tested  T.I.P.  taxifolin phlobatannin neutrals free acids zinc chloride  0.45 1.3  taxifolin phlobatannin neutrals free acids zinc chloride  0.70 1.6 4.7 2.0 0.08  Fomes annosus  Lentinus  1  lepideus  Total inhibition  T a x i f o l i n was investigated.  C  1.4 1.1  0.16  point  d e f i n i t e l y the most t o x i c wood e x t r a c t i v e However, none of the i s o l a t e d groups  approached the e f f e c t i v e n e s s of z i n c c h l o r i d e as a f u n g i c i d e .  44  . No c o n c e n t r a t i o n of ether or a l c o h o l a f f e c t e d the growth of Lentinus l e p i d e u s i n c u l t u r e .  However, Fomes  annosus grew s i g n i f i c a n t l y slower i n two per cent and f o u r per cent a l c o h o l .  ether  The curves obtained f o r these  s o l v e n t c o n c e n t r a t i o n s were used as c o n t r o l s f o r those e x t r a c t i v e groups r e q u i r i n g h i g h amounts t o p r o p e r l y d i s solve. 2.  C e l l u l o s i c Medium The weight l o s s e s of the e x t r a c t e d wood b l o c k s are  presented i n Appendix B .  The averages obtained f o r each  s e t of f i v e samples are i n c l u d e d i n Table  2.  Table 2 Average Weight Losses of E x t r a c t e d Wood B l o c k s i n S o i l J a r s (2-month i n c u b a t i o n )  Organism  Poria incrassata  Lentinus l e p i d e u s  Extractive treatment acetone ether water none acetone ether water none  Avg. wt. loss  1  25.3  24.7  29.7 23.0  52.2* 51.8* 44.7  37.6  1 Per cent of o r i g i n a l oven-dry weight • S i g n i f i c a n t l y d i f f e r e n t from u n e x t r a c t e d b l o c k s (5$ l e v e l )  45  The acetone- and e t h e r - e x t r a c t e d samples exposed to the a c t i o n of L e n t i n u s l e p i d e u s s u f f e r e d n e a r l y equal and s i g n i f i c a n t l y h i g h e r weight l o s s e s than the c o n t r o l s . The r e s u l t s suggest t h a t the three weakly t o x i c components i n v e s t i g a t e d as s u b - d i v i s i o n s o f the ether e x t r a c t may exert a combined f u n g i c i d a l a c t i o n equal t o t h a t of t a x i folin.  The w a t e r - e x t r a c t e d b l o c k s , although n o t v a r y i n g  s i g n i f i c a n t l y from the c o n t r o l s , n e v e r t h e l e s s show a somewhat h i g h e r weight l o s s .  T h i s was probably due t o the  removal of some w a t e r - s o l u b l e t a n n i n substances  along w i t h  the carbohydrate m a t e r i a l . The r e s u l t s obtained w i t h P o r i a i n c r a s s a t a were not s t a t i s t i c a l l y s i g n i f i c a n t when analyzed w i t h the " t " test.  A longer i n c u b a t i o n p e r i o d may be r e q u i r e d w i t h  fungus i n order t o o b t a i n meaningful  this  r e s u l t s , s i n c e the  weight l o s s e s amounted t o o n l y h a l f those of the samples i n f e c t e d with Lentinus l e p i d e u s . DISCUSSION 1.  Taxifolin Among the components t e s t e d i n malt agar, o n l y  t a x i f o l i n was i n c l u d e d i n s u f f i c i e n t q u a n t i t i e s t o comp l e t e l y i n h i b i t growth.  No growth of e i t h e r t e s t  occurred i n d i s h e s c o n t a i n i n g one per cent of t h i s none, and a c o n c e n t r a t i o n of as l i t t l e  organism flava-  as 0 . 6 per cent  46  prevented  L_g. l e p i d e u s from growing.  A f t e r three weeks,  the i n o c u l a from these t h r e e s e t s of d i s h e s were t r a n s f e r r e d to f r e s h malt agar. Was  concluded  Growth was  not renewed, so i t  t h a t these c o n c e n t r a t i o n s were s u f f i c i e n t  b r i n g about death of the f u n g i .  to  These p o i n t s of k i l l i n g  c o n c e n t r a t i o n are i n c l u d e d i n the t a x i f o l i n growth curves (1  and  3)  of Appendix A.  p o l a t i o n procedure,  They d i d not i n f l u e n c e the e x t r a -  s i n c e these values r e p r e s e n t a concen-  t r a t i o n above t h a t of the T.I.P. Rennerfelt  ( 3 8 , 3 9 ) has determined  p i n o s y l v i n (VIII) by the malt agar method. data f o r Fomes annosus, a T.I.P. of 0 . 0 6 obtained.  P i n o s y l v i n was  first  (Pinus s y l v e s t r i s L.) and has  the t o x i c i t y of By p l o t t i n g h i s  per cent i s  i s o l a t e d from Scots  s i n c e been found  of the hard pine ( D i p l o x y l o n ) group.  pine  i n a number  I t s monomethylether  (IX) has a l s o been i s o l a t e d , not only from the hard p i n e s ,  VIII Pinosylvin  IX P i n o s y l v i n monomethylether  but from the s o f t pine (Haploxylon)  group as w e l l (5"4).  T h i s monomethylether of Pinus a c t s as a weak f u n g i c i d e , s i n c e c o n c e n t r a t i o n s s l i g h t l y i n excess of one per cent are r e q u i r e d to completely  i n h i b i t growth of F. annosus (38>39).  47  In a d d i t i o n to both being p h e n o l i c i n c h a r a c t e r , p i n o s y l v i n and t a x i f o l i n have c e r t a i n other Both substances  are s o l u b l e i n e t h y l ether o n l y a f t e r  have been removed from wood w i t h acetone (54)  similarities. they  or a l c o h o l .  Erdtman  has a t t r i b u t e d t h i s phenomenon to e t h e r - i n s o l u b l e "mem-  brane substances" which envelop  the p h e n o l i c molecules  t h a t they are made i n a c c e s s i b l e to e t h e r . a l c o h o l or acetone  so  E x t r a c t i o n with  a p p a r e n t l y d i s s o l v e s both the p h e n o l i c  e t h e r - i n s o l u b l e substances, s y l v i n to be subsequently  a l l o w i n g the t a x i f o l i n or p i n o -  soluble i n ether.  T h e r e f o r e , there i s some q u e s t i o n as to whether f o l i n and  and  s i m i l a r p h e n o l i c compounds i s o l a t e d by  taxi-  chemical  means are i d e n t i c a l w i t h those o c c u r r i n g n a t u r a l l y i n the wood.  T h e i r chemical nature and p h y s i c a l a v a i l a b i l i t y  d i f f e r when i s o l a t e d .  F u r t h e r evidence  to support t h i s view  a r i s e s when the i n s e c t i c i d a l p r o p e r t i e s of t a x i f o l i n p i n o s y l v i n are c o n s i d e r e d .  may  and  Both compounds have been shown  to be h i g h l y t o x i c to the West I n d i a n dry-wood t e r m i t e (Cryptotermes  b r e v i s Walker) ( 5 6 ) .  Wood b l o c k s submerged i n  a 0 . 0 5 per cent t a x i f o l i n s o l u t i o n f o r t e n minutes have remained f r e e from a t t a c k f o r more than 42 months The  (55).  t a x i f o l i n present n a t u r a l l y i n Douglas f i r wood, however,  a p p a r e n t l y p r o v i d e s no p r o t e c t i o n whatsoever, s i n c e t h i s  spe-  c i e s i s r e a d i l y a t t a c k e d by the t r o p i c a l dry-wood t e r m i t e . The reason f o r t h i s d i s c r e p a n c y may  be one of  availability.  A wood block soaked i n a t a x i f o l i n s o l u t i o n , would present  an  48  even d i s t r i b u t i o n of pure compound t o an a t t a c k i n g  agency.  The n a t u r a l l y o c c u r r i n g substance i s p r o b a b l y d i s t r i b u t e d unevenly, and may be l a r g e l y i n a c c e s s i b l e to wood-destroying i n s e c t s by e x i s t i n g i n an i n t i m a t e form w i t h other m a t e r i a l s . T a x i f o l i n has been r e p o r t e d t o e x i s t n a t u r a l l y i n a average c o n c e n t r a t i o n of about one per cent in.Douglas f i r heartwood  (33>34).  The combined  and i t s monomethylether  c o n c e n t r a t i o n of p i n o s y l v i n  i n pine heartwood amounts t o about  0 . 8 per cent of the dry weight of the wood ( 3 8 , 3 9 ) . the l e s s t o x i c p i n o s y l v i n monomethylether  However,  i s present i n  amounts three t o f o u r times t h a t of the parent p i n o s y l v i n (54).  Thus, the combined  f u n g i c i d a l e f f e c t of these two p i n e  substances should be expected t o approach t h a t of t a x i f o l i n i n Douglas f i r heartwood.  Evidence t o s u b s t a n t i a t e  this  view has been p r o v i d e d by Smith ( 4 8 ) , who c l a s s e s Douglas f i r and Scots pine i n the same n a t u r a l d u r a b i l i t y group by service test experience. Another r e l a t e d p h e n o l i c molecule present i n the Pinaceae i s c o n i d e n d r i n ( X ) . T h i s has been found i n many s p e c i e s of the non-durable Tsuga, P i c e a and Abies genera. L i k e the p a r t i c u l a r phenols of the pines and Douglas  fir,  t h i s compound cannot be d i r e c t l y e x t r a c t e d w i t h e t h e r .  The  n o n - t o x i c nature of t h i s substance, as w e l l as the decreased potency of p i n o s y l v i n monomethylether,  may be e x p l a i n e d by  the s u b s t i t u t i o n of methoxyl groups f o r hydroxy r a d i c a l s . Both pine and Douglas f i r heartwoods  are r e s i s t a n t  49 o  H CO. 3  o HO  Y  0  C  H  3  OH  X Gonidendrin  (21,35*54).  t o p u l p i n g by t h e n o r m a l s u l p h i t e method formerly  thought t h a t  lignin  to  Recent  e v i d e n c e has  the  phenolic  f o r m an i n s o l u b l e  phenolic  indicated  that  may i n h i b i t p u l p i n g by d e c o m p o s i n g When t a x i f o l i n quercetin. ably  reacted  formed  decomposition  these phenolic the  cooking  of  this  it  can p r e c i p i t a t e  sulphuric acid the  (21).  calcium ions  as  (35>54).  (21).  oxidized is  to  prob-  further  bisul-  The a c i d a  with  liquor Is  was  compounds  reaction  w h i c h i n t u r n may c a t a l y z e to  reacted  l i g n i n complex  with bisulphite,  The r e d u c t i o n p r o d u c t  thiosulphate,  phite  2.  is  extractives  It  thus  sulphate.  Phlobatannin The p h l o b a t a n n i n f r a c t i o n d i s s o l v e d  more d i f f i c u l t y  than t a x i f o l i n .  i n a s m u c h as  phlobatannin molecule  taxifolin, flavanone.  the  T h i s was n o t is  and may e v e n be a c o n d e n s a t i o n Solubility  decreased  with  in alcohol  with  surprising,  more c o m p l e x product  increasing  of  than the  molecular  5 0  weights among the t a x i f o l i n , p h l o b a t a n n i n and fractions.  The  phlobaphene  d i f f e r e n c e s i n t o x i c i t y betv/een t a x i f o l i n  and p h l o b a t a n n i n i n d i c a t e d t h a t f u n g i c i d a l a c t i v i t y decreased w i t h i n c r e a s i n g molecular weights.  The  also  most h i g h l y  condensed molecule i n t h i s s e r i e s , the phlobaphene complex, was  not  tested for t o x i c i t y .  I t s h i g h degree of  insolubility  i n a l l s o l v e n t s suggested that i t would have very l i m i t e d fungicidal properties.  T h i s i s f u r t h e r evidenced by  the  f a c t that n a t i v e l i g n i n i s u s u a l l y a s s o c i a t e d w i t h the baphene complex ( 8 , 1 9 ) .  A n a t i v e l i g n i n f r a c t i o n has  i s o l a t e d from most woods, i n c l u d i n g those having low durability.  phlobeen  natural  This implies that i n a l l p r o b a b i l i t y , native  l i g n i n does not f u n c t i o n as a f u n g i c i d e . An had  independent i n v e s t i g a t i o n on a Douglas f i r sample  revealed  greater (25).  four  times  i n the outer heartwood than i n the c e n t r a l p i t h area The  p o s s i b l e r e l a t i o n s h i p between t a x i f o l i n and  b a t a n n i n has low  t h a t the t a x i f o l i n c o n c e n t r a t i o n was  been p r e v i o u s l y d i s c u s s e d .  concentration  I t was  phlo-  f e l t that  the  of t a x i f o l i n i n the i n n e r heartwood might  have been the r e s u l t of i t s being  slowly  b a t a n n i n over a p e r i o d of y e a r s .  The  transformed t o  phlo-  t a x i f o l i n produced i n  the outer heartwood might have been formed too r e c e n t l y to condense to any  extent.  t e s t f o r p h l o b a t a n n i n was the  Consequently, a rough q u a n t i t a t i v e made to see  i f the above was,  case. Samples from the outer  and  i n n e r heartwood were  indeed  51  e x t r a c t e d w i t h e t h e r , and the p h l o b a t a n n i n was e t h y l a c e t a t e s o l u t i o n i n the customary way.  isolated i n Douglas f i r and  redwood p h l o b a t a n n i n had p r e v i o u s l y been shown to absorb v i o l e t l i g h t s t r o n g l y i n a wave l e n g t h o f 280 mu Spectophotometric subsequently  ultra-  (8,19).  a n a l y s i s of the two p h l o b a t a n n i n e x t r a c t s  showed the r a t i o of t a n n i n i n the outer  inner heartwood t o be 5'3»  and  From these r e s u l t s , i t would  appear t h a t t a x i f o l i n i s not converted t o t a n n i n , i f i t i s assumed t h a t the amount of t a x i f o l i n o r i g i n a l l y produced i s constant throughout  the l i f e  of the t r e e .  Unfortunately,  there are no p r e c i s e q u a n t i t a t i v e methods a v a i l a b l e f o r b e t t e r t a n n i n comparisons. The f o r e g o i n g o b s e r v a t i o n s are based on o n l y  one  sample, and thus p r o v i d e o n l y an i n d i c a t i o n at b e s t . still  q u i t e p o s s i b l e t h a t t a x i f o l i n may  t a n n i n , but was to  phlobaphene.  It is  condense t o phloba-  not d e t e c t e d because of f u r t h e r  condensation  Fundamental work i s needed i n t h i s f i e l d  d e f i n i t e l y e s t a b l i s h the d i s t r i b u t i o n p a t t e r n of these related 3.  to  three  substances.  Free A c i d s Growth curves of Fomes annosus i n malt agar w i t h  a c i d s i n c l u d e d are presented importance  Appendix A.  of c o r r e c t i n g f o r r e s t p e r i o d i s f o r c i b l y  t r a t e d here. did  in Fig. 13,  Growth i n a c o n c e n t r a t i o n of 0.8  not s t a r t u n t i l two  the  The illus-  per cent  days a f t e r growth began i n the  acid one  52  per cent d i s h e s .  Once s t a r t e d , however, i t s f a s t e r r a t e o f 7  growth produced a curve of h i g h e r s l o p e .  Each of these con-  c e n t r a t i o n s were c o r r e c t e d f o r r e s t p e r i o d i n the u s u a l  way.  The  found  curve r e p r e s e n t i n g the lower c o n c e n t r a t i o n was  to f a l l  above t h a t of the h i g h e r .  These c o r r e c t e d curves  i l l u s t r a t e d by d o t t e d l i n e s i n F i g . 13, 4.  then  Appendix  are  A.  Neutrals The  c o n c e n t r a t i o n of the n e u t r a l ether  r e q u i r e d to completely  fraction  i n h i b i t growth v a r i e d w i d e l y , depend-  i n g on the organism i n v o l v e d .  F. annosus i s only  r e s i s t a n t , s i n c e the T.I.P. was  determined  as 1.4  Lentinus l e p i d e u s a p p a r e n t l y i s very r e s i s t a n t , a c a l c u l a t e d c o n c e n t r a t i o n of almost  moderately per  cent.  requiring  f i v e per cent (Table  1.)  T h i s organism i s a l s o u n u s u a l l y r e s i s t a n t to o i l - s o l u b l e p r e s e r v a t i v e s such as c r e o s o t e ( 9 ) .  L i k e the n e u t r a l f r a c -  t i o n , c r e o s o t e c o n t a i n s a heterogeneous mixture i n s o l u b l e , h i g h molecular-weight 5.  hydrocarbons  of water-  (42).  Water S o l u b l e s The  carbohydrate  f r a c t i o n apparently d i d neither  support nor hinder f u n g a l development i n p l a i n agar.  There-  f o r e , the w a t e r - s o l u b l e f r a c t i o n from Douglas f i r seems to f u n c t i o n n e i t h e r as a food source nor as a t o x i c a n t to f u n g i . S i n c e the h y d r o l y z a t e was concluded  90 per cent g a l a c t o s e , i t can  t h a t the carbohydrate  i s largely a galactan  be (50).  53  Galactans,  u n l i k e mannans and  pentosans are q u i t e r e s i s t a n t  to a t t a c k by wood-destroying f u n g i 6.  Zinc  Chloride The  inhibit  (7).  concentrations  of z i n c c h l o r i d e n e c e s s a r y to ,  growth agree w e l l w i t h data p r e v i o u s l y r e p o r t e d  R i c h a r d s (41).  L_. l e p i d e u s  chloride; a concentration f i c i e n t to k i l l  i s extremely s e n s i t i v e to z i n c  of l e s s than 0 . 1  the fungus.  The  times t h i s l a t t e r  inhibit  of the other  A t a x i f o l i n con-  Even h i g h e r  extraneous m a t e r i a l s are r e q u i r e d  f u n g a l growth.  m a t e r i a l s , however, may  The  to  e v e n t u a l l y prove s u p e r i o r to z i n c  p r o p e r t i e s , the s a l t leaches exposure c o n d i t i o n s  mul-  naturally occurring extractive  chloride i n c e r t a i n instances.  wet  cent  amount i s needed to  prevent growth o f F. annosus i n malt agar. tiples  per  the average s e n s i v i t y  of wood-destroying f u n g i .  c e n t r a t i o n of three  per cent i s s u f -  T.I.P. of 0 . 1 6  a t t a i n e d w i t h Fomes annosus r e p r e s e n t s of the m a j o r i t y  by  (22).  Due  to i t s water s o l u b l e  out of t r e a t e d timbers under The w a t e r - i n s o l u b l e  nature  of the f u n g i c i d a l Douglas f i r e x t r a c t i v e s permits more permanent p r o t e c t i o n than i s a f f o r d e d by z i n c c h l o r i d e .  54  V. The  CONCLUSION  s p e c i f i c c o n c e n t r a t i o n s of wood e x t r a c t i v e s  needed to i n h i b i t growth i n malt agar should not be  inter-  p r e t e d as r e p r e s e n t i n g the amount of m a t e r i a l t h a t must n e c e s s a r i l y be present i n wood t o prevent f u n g a l growth. Rather,  they serve to i n d i c a t e the r e l a t i v e importance t h a t  a component may  p l a y i n p r o t e c t i n g wood from decay.  On  the  b a s i s of the data c o l l e c t e d i n t h i s study, i t appears t h a t t a x i f o l i n may  be l a r g e l y r e s p o n s i b l e f o r the moderate decay  r e s i s t a n c e of Douglas f i r . S i n c e the d i s c o v e r y of the f u n g i c i d a l p r o p e r t i e s of p i n o s y l v i n and i t s monomethylether, i t has been  suggested  t h a t t r e e s c o n t a i n i n g h i g h c o n c e n t r a t i o n s of these evenly d i s t r i b u t e d throughout for  breeding purposes ( 1 3 ) .  the heartwood should be used The  lumber produced from  s t r a i n s would be i n h e r e n t l y more d u r a b l e . work to proceed,  substances  such  I n order f o r t h i s  a simple c o l o r i m e t r i c t e s t has been d e v e l -  oped f o r the s e m i - q u a n t i t a t i v e e s t i m a t i o n of p i n o s y l v i n i n the f i e l d  ( 1 3 ) .  I f a comparable t e s t were a v a i l a b l e f o r the  q u a n t i t a t i v e a n a l y s i s of t a x i f o l i n , Douglas f i r t r e e s w i t h h i g h amounts of t h i s flavanone c o u l d be s e l e c t e d f o r r a c i a l improvement.  Work i s now  i n progress to estimate  taxifolin  q u a n t i t a t i v e l y from methanol e x t r a c t s of Douglas f i r ( 3 ) . N e v e r t h e l e s s , a more r a p i d means of e s t i m a t i o n i n the is  field  s t i l l needed, s i n c e the above method i s i n a p p l i c a b l e to .  55 wood i n t o t o . Western l a r c h ( L a r i x o c c i d e n t a l i s Nutt.) has been r e p o r t e d to c o n t a i n t a x i f o l i n  (17).  Heartwood of  s p e c i e s , although  somewhat e a s i e r to penetrate  type Douglas f i r ,  s t i l l does not accept p r e s e r v a t i v e s  (30).  i n both I n t e r i o r Douglas f i r and  Therefore,  also this  than I n t e r i o r easily  western  l a r c h , t a x i f o l i n w i l l most o f t e n be the main d e t e r r e n t to decay.  T a x i f o l i n c o n c e n t r a t i o n should be c o n s i d e r e d when  lumber d e r i v e d from these  s p e c i e s i s to be used under c o n d i -  t i o n s where wood-destroying f u n g i might be a c t i v e . Chemical s t i m u l a t i o n i s being r e s i n p r o d u c t i o n i n the southern  pines  employed to i n c r e a s e (32).  A s e r i e s of  papers w i l l soon be p u b l i s h e d on other methods of s t i m u l a t i n g t r e e s to produce g r e a t e r amounts of extraneous m a t e r i a l s From such s t u d i e s , economical methods may  be developed to  grow t r e e s w i t h g r e a t e r p r o p o r t i o n s of f u n g i c i d a l e x t r a c tives.  (.1).  56  LITERATURE CITED 1.  Anderson, A.B.  2.  Ascorbe, F.J. The i n h i b i t o r y action of organic chemicals on a blue-stain fungus. Caribbean For. 14: I 3 6 - I 3 9 . 1953.  3.  Barton, G.M., and J.A.F. Gardner. Work i n progress. Vancouver Branch, Forest Products Laboratory of Canada. 1955.  4.  Barton, G.M., and J.A.F. Gardner. The chemical nature of the acetone extractive of western red cedar. Pulp and Pap. Mag. of Can. 55: I 3 2 - I 3 7 . 1954.  5.  Bateman, E. The effect of concentration on the t o x i c i t y of chemicals to l i v i n g organisms. U.S. Dept. Agr. Tech. B u l l . 346. 1933.  6.  Boyce, J.S. Forest pathology, 2nd e d i t i o n . Book Co., Inc., New York. 1948.  7.  Brown, H.P., A.J. Panshin, and C.C. Forsaith. Textbook of wood technology, V o l . I I . McGraw-Hill Book Co. Inc., New York. 1952.  8.  Buchanan, M.A., H.F. Lewis, and E.F. Kurth. Chemical nature of redwood tannin and phlobaphene. Ind. and Eng. Chem. 36: 907-910. 1944.  9.  Cartwright, K. St. G., and W.P.K. Findlay. Decay of timber and i t s prevention. His Majesty's Stationery O f f i c e , London. 1946.  Personal correspondence.  Feb. 16,  1955«  McGraw-Hill  10.  Cramer, F. Paper chromatography. London. 1954.  11.  Dept. of Lands and Forests, Province of B r i t i s h Columbia. Report of the forest service, year ended Dec. 31?  1953.  MacMillan & Co., Ltd.,  1954.  12.  Erdtman, H.  13.  Erdtman, H., A. Frank, and G. Linstedt. Constituents of pine heartwood XXVII: The content of pinosylvin phenols i n Swedish pines. Svensk Papperstidn.  Personal correspondence.  54: 275-279.  14.  Oct. 20,  1954.  1951.  Findlay, W.P.K. Influence of sample size on decay rate of wood i n culture. Timber Tech. 6 l : 160-162. 1953.  57  15.  F i n h o l t , R.W. Improved t o x i m e t r i c a g a r - d i s h t e s t f o r e v a l u a t i o n of wood p r e s e r v a t i v e s . A n a l . Chem. 2 3 : IO38-IO39. 1951.  16.  F i n h o l t , R.W., M. Weeks, and C. Hathaway. New theory on wood p r e s e r v a t i o n . Ind. and Eng. Chem. 4 4 : 101-105. 1952.  17.  Gardner, J.A.F.  18.  Gortner, R.A. Outlines of biochemistry, 3 ^ e d i t i o n . John Wiley & Sons, Inc., New York. 1949.  19.  Graham, H.M., and E.F. K u r t h . C o n s t i t u e n t s of e x t r a c t i v e s from Douglas f i r . Ind. and Eng. Chem. 41: 400-414. 1949.  20.  Hawley, L.F., L.C. F l e c k , and C.A. R i c h a r d s . The r e l a t i o n between d u r a b i l i t y and chemical composition i n wood. Ind. and Eng. Chem. 16: 699-70O. 1924.  21.  Hoge, W.M. The r e s i s t a n c e of Douglas f i r to s u l p h i t e p u l p i n g . Tappi 37: 3 6 9 - 3 7 4 . 1954.  22.  Hunt, G.M., and G.A. G a r r a t t . Wood p r e s e r v a t i o n . McGraw-Hill Book Co., Inc., New York. 1938.  23.  Isenberg, I.H., M.A. Buchanan, and L.E. Wise. Extraneous components of American pulpwood. Pap. Ind. and Pap. Trade 2 8 : 8 1 6 - 8 2 2 . 1946.  24.  Johnson, C.H., and R.A. C a i n . The wood o i l o f Douglas fir. J . Am. Pharm. Assoc. 2 6 : 6 2 3 - 6 2 5 . 1937.  25.  Kennedy, R.W.  26.  K i n g s t o n , J.T.E. S t a t i s t i c a l r e c o r d o f the lumber i n d u s t r y i n B r i t i s h Columbia. Bur. of Econ. and S t a t . , Dept. of Trade and Ind. 1955.  27.  K u r t h , E.F. S e p a r a t i o n of wood e x t r a c t i v e s i n t o simpler components. Ind. and Eng. Chem., A n a l . Ed. 11: 2 0 3 - 2 0 5 . 1939-.  28.  K u r t h , E.F. Chemical a n a l y s i s of western woods, p a r t I . Pap. Trade J . 1 2 6 : 56-571948.  29.  L e u t r i t z , J . A w o o d - s o i l c o n t a c t c u l t u r e technique f o r l a b o r a t o r y study of wood-destroying f u n g i , wood decay and wood p r e s e r v a t i o n . B e l l System Tech. J . 25: 1 0 2 - 1 3 5 . 1946.  Unpublished o b s e r v a t i o n . r  Unpublished d a t a .  58  30.  31.  MacLean, J.D. P r e s e r v a t i v e treatment of wood, by pressure methods. U.S. Dept. Agr. Handb. no. 1952.  40.  Mulholland, J.R. Changes i n weight and s t r e n g t h of S i t k a spruce a s s o c i a t e d w i t h decay by a brown-rot fungus, P o r i a m o n t i c o l a . J . F o r . Prod. Res. Soc.  4: 410-4lrST  1954.  32.  Panshin, A.J., E.S. H a r r a r , W.J. Baker, and P.B. P r o c t o r , Forest products. McGraw-Hill Book Co., Inc., New York. 1950.  33*  Pew, J.C. A flavanone from Douglas f i r heartwood. F o r . Prod. Lab. Rept. no. R1692. 1947.  34.  Pew, J.C. A flavanone from Douglas f i r heartwood. Am. Chem. Soc. 7 0 : 3 0 3 I - 3 0 3 4 . 1948.  35*  Pew, J.C. Douglas f i r heartwood flavanone: Its p r o p e r t i e s and i n f l u e n c e on s u l p h i t e p u l p i n g . Tappi 3 2 : 39-44. 1949.  36.  Q u e n o u i l l e , M.H.  Introductory s t a t i s t i c s .  S p r i n g e r L t d . , London.  U.S. J.  Butterworth-  1950.  37.  R e n n e r f e l t , E.  38.  R e n n e r f e l t , E. Die T o x i z i t a t der p h e n o l i s c h e n I n h a l t s s t o f f e das K i e f e r n k e r n h o l z e s gegenliber e i n i g e h F a u l n i s p i l z e n . Svensk Bot. T i d s k r . 3 7 : 83-93. 1943. R e n n e r f e l t , E. The t o x i c i t y of the p h e n o l i c e x t r a c t i v e s of pine heartwood i n regard to some decay f u n g i . T r a n s l a t i o n No. 8 , F a c u l t y of F o r e s t r y , U n i v e r s i t y of B r i t i s h Columbia. 1953.  39.  P e r s o n a l correspondence.  March 2,  1955.  40.  R e n n e r f e l t , E . The i n f l u e n c e of the p h e n o l i c compounds i n the heartwood of Scots pine (Pinus s i l v e s t r i s L.) on the growth of some decay f u n g i i n n u t r i e n t s o l u t i o n . Svensk Bot. Tidskr. 39: 3II-318. 1945.  41.  R i c h a r d s , C.A. Comparative r e s i s t a n c e of eighteen species of wood d e s t r o y i n g f u n g i to z i n c c h l o r i d e . P r o c . Am. Wood P r e s e r v e r s Assoc., 21st Ann. Meeting. 18-22. 1925. 1  42.  Roche, J.N. Coal t a r creosote: I t s composition and how i t f u n c t i o n s as a wood p r e s e r v a t i v e . Koppers Co., Inc., P i t t s b u r g h . 1952.  59  43.  R o f f , J.W., and J.M. A t k i n s o n . T o x i c i t y t e s t s o f a water-soluble phenolic f r a c t i o n ( t h u j a p l i c i n - f r e e ) of western r e d cedar. Can. J . B o t . 32: 308-309. 1954.  44.  R u s s e l l , A.  The n a t u r a l t a n n i n s .  Chem. Revs.  17:  155-186. 1 9 3 5 . 45.  Schmitz, H. A suggested t o x i m e t r i c method f o r wood p r e s e r v a t i v e s . Ind. and Eng. Chem., A n a l . E d . 2 :  36I-363. 1930.  46.  Schorger, A.W. The o l e o r e s i n o f Douglas f i r . Chem. Soc. 39: 1040-1044. 1917.  47.  S h e r r a r d , E.C., and E.F. K u r t h . The d i s t r i b u t i o n of e x t r a c t i v e i n redwood: I t s r e l a t i o n t o d u r a b i l i t y . U.S. F o r . Prod. Lab. Rept. R 9 8 8 . 1 9 3 3 .  48.  Smith, D.N. The n a t u r a l d u r a b i l i t y of timber. F o r . Prod. Res. Rec. No. 3 C H i s Majesty's S t a t i o n e r y O f f i c e , London. 1 9 4 9 .  49.  Southam, CM., and J . E h r l i e h . E f f e c t s o f e x t r a c t of western r e d cedar heartwood on c e r t a i n wood-decaying f u n g i i n c u l t u r e . Phytopath. 33: 5 1 7 - 5 2 4 . 1 9 4 3 .  50.  Thompson, J.O., J . J . Becher, and L.E. Wise. A p h y s i o chemical study o f a w a t e r - s o l u b l e p o l y s a c c h a r i d e from Douglas f i r (Pseudotsuga t a x i f o l i a ) . Tappi 3 6 : 319-324. 1953.  51.  U.S. Dept. Agr., F o r . Prod. Lab. Wood handbook. Govt. P r i n t i n g O f f i c e , Washington. 1940.  52.  Varner, R.W., and R.L. Krause. Agar-block and s o i l block methods f o r t e s t i n g wood p r e s e r v a t i v e s . I n d . and Eng. Chem. 4 3 : 1 1 0 2 - 1 1 0 7 . 195.1.  53.  Wise, L.E. Wood c h e m i s t r y . New York. 1 9 4 6 .  54.  Wise, L.E., and E.C. Jahn. I and I I .  J . Am.  Reinhold Publishing  U.S.  Corp.,  Wood chemistry, 2nd ed. V o l .  R e i n h o l d P u b l i s h i n g Corp., New York.  1952,  55*  W o l c o t t , G.N.  56.  Wolcott, G.N. S t i l b e n e and comparable m a t e r i a l s f o r dry-wood t e r m i t e c o n t r o l . J . Econ. E n t . 4 6 : 3 7 4 - 3 7 5 . 1953.  P e r s o n a l correspondence.  Feb. 7, 1 9 5 5 .  Zabel, R.A. Variations i n the decay resistance of white oak. N.Y. State C o l l . of For. Tech. Pub. 68.  1948.  APPENDIX A  Growth and t o t a l i n h i b i t i o n p o i n t  curves  °f Forces annosus and L e n t i n u s l e p i d e u s i n malt agar c o n t a i n i n g various  concentrations  of e x t r a c t i v e s  Note:  The l i n e a r r e l a t i o n s h i p between l o g per cent r e t a r d a t i o n of growth and l o g per cent c o n c e n t r a t i o n h o l d s o n l y between the T.I.P. and o n e - t h i r d of t h a t c o n c e n t r a t i o n . Hence p o i n t s r e p r e s e n t i n g some lower c o n c e n t r a t i o n s were not c o n s i d e r e d i n f i t t i n g the s t r a i g h t lines.  F i g u r e 1. Rate of growth of Fomes annosus containing different  on malt agar  concentrations  of  taxifolin.  (1% a l c o h o l = c o n t r o l (0.00$ t a x i f o l i n )  F i g u r e 2. R e t a r d a t i o n of growth of Fomes annosus on malt agar c o n t a i n i n g d i f f e r e n t of  taxifolin.  concentrations  T I M E  IN  DAYS  Figure 3 . Rate of growth of Lentinus lepideus on malt agar containing d i f f e r e n t concentrations of taxifolin. 1% alcohol = control (0.00$ t a x i f o l i n )  Figure  4.  Retardation of growth of Lentinus  lepideus  on malt agar containing d i f f e r e n t concentrations of t a x i f o l i n .  Figure 5. Rate of growth of Fomes annosus on malt agar containing d i f f e r e n t concentrations of phlobatannin. 1% alcohol = control f o r 0.2% and 0.5% phlobatannin 4% alcohol = control f o r 1% phlobatannin  Figure 6 . Retardation of growth of Fomes annosus on malt agar containing d i f f e r e n t concentrations of phlobatannin.  3-A  TIME  IN DAYS  Figure 7. Rate of growth of Lentinus lepideus on malt agar containing different concentrations of phlobatannin. 1% alcohol = control  Figure  8.  Retardation of growth of Lentinus lepideus on malt agar containing d i f f e r e n t tions of phlobatannin.  concentra-  •2  . 4  PER  CENT  .6  .8 1.0  1.5 2.0  CONCENTRATION  OF  PHLOBATANNIN  Figure 9. Rate of growth of Fomes annosus on malt agar containing d i f f e r e n t concentrations of neutrals. 1% ether = control  Figure 10. Retardation of growth of Fomes annosus on malt agar containing d i f f e r e n t concentrations of neutrals.  3-A  0  2  6  4  8 T I M E  PER  CENT  10 IN  12  14  DAYS  CONCENTRATION  OF  NEUTRALS  16  18  Figure 11. Rate of growth of Lentinus lepideus on malt agar containing d i f f e r e n t concentrations of neutrals. 1% ether = control  Figure 12. Retardation of growth of Lentinus lepideus on malt agar containing d i f f e r e n t concentrations of neutrals.  TIME  IN  DAYS  Figure 13. Rate of growth of Fomes annosus on malt agar containing d i f f e r e n t concentrations of free acids. 1% ether = control f o r 0.05$, .1% and .2% acids 2% ether = control f o r .4%, .6%, .8% and 1.0% acids Broken l i n e represents data corrected f o r rest period.  Figure 14. Retardation of growth of Fomes annosus on malt agar containing different concentrations of free acids.  0  2  4  6  8 TIME  PER  CENT  10 IN  12  14  16  DAYS  CONCENTRATION  OF  FREE  ACIDS  18  Figure 15. Rate of growth of Lentinus lepideus on malt agar containing d i f f e r e n t concentrations of free acids. 1% ether = control  Figure 16. Retardation of growth of Lentinus lepideus on malt agar containing d i f f e r e n t concentrations of free acids.  3  TIME  IN  DAYS  -  ,  Figure 17. Rate of growth of Fomes annosus on malt agar containing d i f f e r e n t concentrations of zinc chloride.  Figure 18. Retardation of growth of Fomes annosus on malt agar containing different concentrations of zinc chloride.  9-A  TIME  IN  DAYS  Figure 19. Rate of growth of Lentinus lepideus on malt agar containing d i f f e r e n t concentrations of zinc chloride.  Figure 20. Retardation of growth of Lentinus lepideus on malt agar containing d i f f e r e n t of zinc chloride.  concentrations  10-A  APPENDIX B S t a t i s t i c a l analysis of weight losses i n Douglas f i r blocks exposed to Poria incrassata and Lentinus lepideus.  Per cent weight losses of extracted wood blocks exposed to action of Lentinus lepideus; Ether  Acetone  Water  Control  54.58 51.68 43.78 53.12 55.79  49.01 55.29 57.40 52.36 47.05  36.01 44.33 50.80 50.90 41.28  39.24 43.93 43.63 23.43 37.70  Transforming  these percentages  to arc s i n j[ percentage,  they become: Ether  Acetone  Water  Control  0.84 0.81 0.72 0.82 0.85  0.78 0.84 0.86 0.81 0.76  0.64 0.73 0.80 0.80 0.69  0.67 0.73 0.73 0.50 0.66  = 0.81  0.81  0.73  0.66  = 0.0009 0.0000 0.0081 0.0001 0.0016  0.0009 0.0009 0.0025 0.0000 0.0025  0.0081 0.0000 0.0049 0.0049 0.0016  0.0001 0.0049 0.0049 0.0256 0.0000  0.0107  0.0068  0.0195  0.0355  2-B  t =  x  - x  x  —  2  2  —  (x - x )  +  x  n  l  +  n  2 -  2  1_ + 1_ nn,  (x - x ) 2  2  Between ether and control: t = 0.81 - 0.66 i— —  .0107 + .0355 8 —  2 5  3.12'  =  T..05 = 2.31  _  t  ,01  Between acetone and control: t = 0.81 - 0.66 .0068 + .0355 8  2 5  3.26'  Between water and control: t = 0.73 - 0.66 .0195 + .0355 8  -  2 5  =  1,33  =  3  '  3 6  3-B  Per cent weight losses of extracted wood blocks exposed to action of Poria incrassata:  Ether  Acetone  Water  Control  30.19 23.85 23.89 20.78 24.72  20.78 24.79 29.58 26.70 24.51  24.93 29.85 32.81 40.64 20.49  12.25 36.22 15.52 26.46 24.48  Transforming these percentages to arc s i n ypercentage, they become:  Ether  Acetone  Water  0.58 0.51 0.51 0.48 0.52  0.48 0.52 0.58 0.55 0.52  0.52 0.58 0.61 0.69 0.46  0.35 0.64 0.41 0.54 0.51  x = 0.52  0.53  0.57  0.49  (x-x)  2  Control  = 0.0036 0.0001 0.0001 0.0016 0.0000  0.0025 0.0001 0.0025 0.0004 0.0001  .0025 .0001 .0016 .0144 .0121  0.0196 0.0225 0.0064 0.0025 0.0004  0.0054  0.0056  .0307  0.0514  4-B  Between ether and control:  t =  0.52 - 0.49 =  <1  =  < 1  .0054 + .0514 8  Between acetone and control::  t =  0.53 - 0.49 .0056 + .0514 8  Between water and control;  t =  0.57 - 0.49 .0307 + .0514 8  1.25  

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