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Saprophytes in the stem of living, healthy Tsuga heterophylla (Raf.) Sarg. and their role in decay resistance… Hudak, Janos 1964

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SAPROPHYTES IN THE STEM OF LIVING, HEALTHY TSUGA HETEROPHYLLA (RAF.) SARG. AND THEIR ROLE IN DECAY RESISTANCE OF THE WOOD.  by JANOS HUDAK B.S.F.j U n i v e r s i t y o f B r i t i s h C o l u m b i a , F a c u l t y o f F o r e s t r y j Sopron D i v i s i o n , 1959-  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF FORESTRY  i n t h e Department o f F o r e s t r y  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA March, 1961+.  In the  requirements  British  for extensive be  cation without  of my  Department  this  that  and  thesis in partial  by  degree at  the  study.  copying  granted  representatives.  this  advanced  agree  for reference  p u r p o s e s may his  f o r an  Columbia, I  available mission  presenting  the  of  this  Head  thesis for financial  written  permissions.  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  Columbia,  make i t  f u r t h e r agree  that  freely, per-  thesis for scholarly  o f my  I t i s understood  of  the U n i v e r s i t y of  Library shall I  fulfilment  Department  that,copying  gain  shall  not  or or  be  by publi-  allowed  - II ABSTRACT  I t was demonstrated t h a t t h e sound wood o f two stems o f l i v i n g w e s t e r n hemlock, Tsuga h e t e r o p h y l l a ( R a f . ) S a r g . , was c o l o n i z e d by m i c r o organisms , w h i c h were i s o l a t e d by s h a k i n g samples o f t h e wood i n s t e r i l e d i s t i l l e d water. (Fr.)  The m i c r o f l o r a s i n h i b i t e d t h e growth o f Fomes annosus  Cke., and P o r i a m o n t i c o l a Murr. when added t o one p e r c e n t m a l t  agar.  The t e s t f u n g i were a l s o i n h i b i t e d on u n t r e a t e d s u r f a c e s t e r i l i z e d  wood i n c u b a t e d a t one hundred p e r c e n t r e l a t i v e h u m i d i t y a t room temperature.  S i g n i f i c a n t v a r i a t i o n was o b s e r v e d , i n t h e r a t e o f i n h i b i t i o n  between t h e sapwood and heartwood and between t h e v a r i o u s s e c t i o n s o f the  two stems i n v e s t i g a t e d . A d r i e r environment ( h i g h e r agar c o n c e n t r a t i o n o f m a l t a g a r  media, and l o w e r r e l a t i v e h u m i d i t y w i t h wood) r e s u l t e d i n a s i g n i f i c a n t d e c r e a s e i n i n h i b i t i o n r a t e o f F. annosus and P. m o n t i c o l a . A u t o c l a v i n g the experimental m a t e r i a l l e d t o the l o s s of i n h i b i t i o n w i t h b o t h m a l t a g a r media and wood.  Removal o f t h e m i c r o o r g a -  nisms f r o m t h e shake s o l u t i o n s by M i l l i p o r e f i l t r a t i o n a l s o r e s u l t e d i n l o s s of the i n h i b i t i n g f a c t o r . The r e l a t i v e l y d r i e r environment d i d not e f f e c t the r a t e o f growth o f t h e t e s t f u n g i on a u t o c l a v e d wood and i n m a l t a g a r c o n t a i n i n g the  ,  a u t o c l a v e d p o r t i o n o f t h e shake  solutions.  R e s u l t s o f t h e experiment p r o v i d e e v i d e n c e t h a t t h e m i c r o f l o r a s i n h a b i t i n g t h e sound wood were r e s p o n s i b l e f o r i n h i b i t i o n o f F. annosus and. P. m o n t i c o l a . . F u r t h e r m o r e , s u f f i c i e n t m o i s t u r e l e v e l appeared n e c e s s a r y f o r m a i n t a i n i n g t h e i n h i b i t i n g power o f t h e s e m i c r o organisms.  -VII  -  ACKNOWLEDGEMENTS  I am i n d e b t e d t o D r . John E. B i e r , P r o f e s s o r o f F o r e s t P a t h o l o g y , f o r h i s encouragement, p r i o r t o i n i t i a t i o n o f t h i s work, and h i s q u i d a n c e , v a l u a b l e s u g g e s t i o n s and generous h e l p the course o f t h e study are g r a t e f u l l y  throughout  acknowledged.  I a l s o w i s h t o e x t e n d my s i n c e r e thanks t o Dr. P. G. Haddock, t o Dr. R. W. Wellwood a n d t o Dr. J . W. W i l s o n f o r t h e i r c r i t i c i s m and c r e a t i v e suggestion's. S p e c i a l thanks a r e due t o Dr. G. H. S m i t h and Dr. A. Kozak f o r t h e i r a i d i n the s t a t i s t i c a l analyses.  - Ill TABLE OF CONTENTS  Page ABSTRACT  i i  TABLE OF CONTENTS  i i i  TABLE OF TABLES  iv  TABLE OF ILLUSTRATIONS  v.".  ACKNOWLEDGEMENTS  v i i  I.  INTRODUCTION  1  II.  MATERIALS AND METHODS  3  S e l e c t i o n of experimental  materials  3  P r e p a r a t i o n o f shake s o l u t i o n s  3  P r e p a r a t i o n o f m a l t agar media  U  Conditions f o r studying the e f f e c t s of v a r i o u s r e l a t i v e h u m i d i t y l e v e l s on t h e r a t e o f i n h i b i t i o n Decay t e s t on wood cubes I I I . EXPERIMENTAL RESULTS .• C u l t u r a l s t u d i e s on m a l t agar media  .  .  k 6 10 10  The e f f e c t o f s t e r i l i z a t i o n by a u t o c l a v i n g and Millipore filtering  23  C u l t u r a l s t u d i e s on wood cubes  29  IV. ' DISCUSSION  3$  V.  CONCLUSIONS  hZ  VI.  BIBLIOGRAPHY  hh  - IV -  TABLE OF TABLES  Table 1.  O u t l i n e o f the experiment c a r r i e d  Page out on  three-quarter  i n c h cubes o f w e s t e r n hemlock sapwood and heartwood 2.  . . . .  8  A n a l y s i s o f v a r i a n c e b a s e d on d a t a from c u l t u r a l s t u d i e s on m a l t agar media  17  3.  Data from c u l t u r a l s t u d i e s on wood cubes  30  h'  A n a l y s i s o f v a r i a n c e based on d a t a from s t u d i e s on wood cubes  cultural 31  -. v TABLE OF ILLUSTRATIONS Figure 1.  Page  Growth chamber f o r s t u d y i n g e f f e c t s o f v a r i o u s r e l a t i v e h u m i d i t y l e v e l s on m y c e l i a l growth  5  2.  Growth chamber f o r decay t e s t on wood  7  3.  Growth o f F. annosus on one p e r c e n t m a l t agar  I;.-  Growth o f P. m o n t i c o l a on one p e r c e n t m a l t agar  5.  Photomicrograph  showing t h e microorganisms  on  11 . . . .  12  one  p e r cent malt agar 6.  13  Graph showing t h e growth d a t a o f F. annosus and P. m o n t i c o l a on one p e r c e n t m a l t a g a r c o n t a i n i n g t h e v a r i o u s u n t r e a t e d and a u t o c l a v e d shake s o l u t i o n s  7.  ...  .  .  ±k  Grapft showing t h e growth d a t a o f F. annosus and P. m o n t i c o l a on f o u r p e r c e n t m a l t a g a r c o n t a i n i n g t n e v a r i o u s u n t r e a t e d and a u t o c l a v e d shake s o l u t i o n s  8.  . . . .  15  Graph showing t h e growth d a t a o f F. annosus and m o n t i c o l a on s i x p e r c e n t m a l t a g a r c o n t a i n i n g t h e v a r i o u s u n t r e a t e d and a u t o c l a v e d shake s o l u t i o n s  y.  . . . .  Growth o f F. annosus and P. m o n t i c o l a on s i x p e r c e n t m a l t agar c o n t a i n i n g u n t r e a t e d shake s o l u t i o n s  10.  16  . . .  20  M y c e l i a l s p r e a d o f F. annosus a. and b. A t one hundred p e r c e n t and a t n i n e t y - s e v e n per cent r e l a t i v e humidity  11.  21  M y c e l i a l s p r e a d o f P. m o n t i c o l a a. and b. A t one hundred p e r c e n t and a t n i n e t y - s e v e n per cent r e l a t i v e humidity.  .  22  - VI -  Table o f I l l u s t r a t i o n s  continued  Figure 12.  Page  Growth o f F. annosus on one p e r c e n t m a l t a g a r c o n t a i n i n g no shake s o l u t i o n , u n t r e a t e d s o l u t i o n , and s o l u t i o n s 25  a u t o c l a v e d and M i l l i p o r e f i l t e r e d . 13.  Growth o f P. m o n t i c o l a on one p e r c e n t m a l t agar c o n t a i n i n g no shake s o l u t i o n , u n t r e a t e d s o l u t i o n , and s o l u t i o n s a u t o c l a v e d and M i l l i p o r e f i l t e r e d  lli.  26  Graph r e p r e s e n t i n g t h e growth r a t e o f F. annosus on one p e r c e n t m a l t agar c o n t a i n i n g no shake s o l u t i o n , u n t r e a t e d s o l u t i o n , and s o l u t i o n s a u t o c l a v e d and Millipore filtered  15.  27  Graph r e p r e s e n t i n g t h e growth r a t e o f P. m o n t i c o l a on one p e r c e n t m a l t agar c o n t a i n i n g no shake s o l u t i o n , u n t r e a t e d s o l u t i o n , and s o l u t i o n s a u t o c l a v e d and Millipore filtered  16.  28  Growth o f F. annosus on u n t r e a t e d , s u r f a c e s t e r i l i z e d and a u t o c l a v e d wood cubes  17-  32  Growth o f P. m o n t i c o l a on u n t r e a t e d , s u r f a c e  sterilized  and a u t o c l a v e d wood cubes 18.  Growth o f microorganisms  32 on u n t r e a t e d , s u r f a c e  s t e r i l i z e d wood  33  19.  Growth o f F. annosus on sapwood and heartwood cubes  3h  20.  Growth o f P. m o n t i c o l a on sapwood and heartwood cubes  3h  - 1 -  INTRODUCTION  The importance  o f decay problems i n f o r e s t r y p r a c t i c e i s r e -  c o g n i z e d b o t h i n l i v i n g t r e e s and i n wood i n s e r v i c e .  Accordingly a  g r e a t d e a l o f work has been done i n t h e s e f i e l d s , i n c l u d i n g d e t a i l e d d e s c r i p t i o n s o f t h e e f f e c t s o f v a r i o u s wood d e c a y i n g f u n g i on h o s t s , s u b s t r a t a and h o s t - o r g a n i s m o f decay.  different  r e l a t i o n s h i p s i n successive stages  I t w o u l d appear t h a t most decay s t u d i e s u n d e r t a k e n  to date  were s i m i l a r i n t h a t woody t i s s u e s i n l i v i n g t r e e s were c o n s i d e r e d as e n t i t i e s , v a r y i n g i n s t r u c t u r e , c h e m i c a l c o m p o s i t i o n and o t h e r p r o perties. The  development o f a concept i n r e c e n t y e a r s ( B i e r  1958,  l 9 i ? 9 , i 9 6 0 , l°6l) has l e d t o the e s t a b l i s h m e n t o f a r e l a t i o n s h i p between h o s t r e s i s t a n c e and i t s w a t e r c o n t e n t e x p r e s s e d a r e l a t i v e turgidity.  R e s i s t a n c e was  p o s i t i v e l y correlated w i t h high r e l a t i v e  turgidity.  I t was  t i s s u e s may  n o t be e n t i t i e s b u t b i o l o g i c a l communities c o m p r i s e d  suggested, furthermore, t h a t h e a l t h y , l i v i n g host  h o s t t i s s u e s and a s a p r o p h y t i c m i c r o f l o r a . microorganisms  may  of  I f t h i s i s t r u e , these  have a s i g n i f i c a n t r o l e i n t h e degree o f h o s t r e -  s i s t a n c e by i n h i b i t i n g t h e development o f pathogens under c e r t a i n circumstances.  The e s t a b l i s h m e n t o f h o s t r e s i s t a n c e - h i g h r e l a t i v e  t u r g i d i t y r e l a t i o n s h i p and t h e p o s s i b l e r o l e o f a m i c r o f l o r a r e s u l t e d from work u n d e r t a k e n  on a v a r i e t y o f t r e e s p e c i e s i n c o n n e c t i o n  w i t h canker d i s e a s e s caused by n a t i v e f a c u l t a t i v e p a r a s i t e s ( B i e r 1962).  I t was  1961,  o f p a r t i c u l a r i n t e r e s t t o determine whether t h i s p r i n -  c i p l e would h o l d i n t h e case o f o t h e r t y p e s o f p a t h o g e n s , e.g., wood  - 2 -  decaying f u n g i . T h i s s t u d y was u n d e r t a k e n t o p r o v i d e e v i d e n c e i n s u p p o r t o f t h i s h y p o t h e s i s and p o s s i b l y e s t a b l i s h t h e d i s t r i b u t i o n o f microorganisms i n h e a l t h y wood, and t o determine t h e i r i n h i b i t o r y e f f e c t on t h e d e v e l o p ment o f wood d e c a y i n g f u n g i . Tsuga h e t e r o p h y l l a ( R a f . ) S a r g . was chosen as h o s t t r e e , s i n c e t h i s s p e c i e s i s o f m a j o r economic importance and was n o t under i n v e s t i g a t i o n i n the P a t h o l o g i c a l Laboratory i n the U n i v e r s i t y a t the beginning o f t h i s study.  I t was c o n s i d e r e d o f i n t e r e s t t o use a r e p r e s e n -  t a t i v e o f b o t h . t y p e s o f decay o r g a n i s m s , namely a w h i t e r o t and a brown r o t fungus.  Fomes annosus ( F r . ) Cke. and P o r i a m o n t i c o l a Murr. were  chosen f o r t h i s purpose  respectively.  Both f u n g i a r e pathogens o f i n -  t e r n a t i o n a l importance and grow r a p i d l y , c h a r a c t e r i s t i c s w h i c h l e a d t o their selection f o r laboratory studies.  - 3 MATERIALS AND METHODS  The c u l t u r e s o f F. annosus and P. m o n t i c o l a were s u p p l i e d by Dr. John E. B i e r and d e s i g n a t e d as No. 13 and No. 12UA r e s p e c t i v e l y . A v i g o r o u s l y - g r o w i n g hemlock t r e e e i g h t y y e a r s o f age was f e l l e d i n t h e U n i v e r s i t y Campus F o r e s t and bucked i n t o t e n - f o o t s e c t i o n s .  The  f i r s t commencing from t h e base o f t r e e was termed t h e " b u t t s e c t i o n " ; the  second was d i s c a r d e d ; t h e t h i r d was named t h e "middle s e c t i o n " ; t h e  f o u r t h d i s c a r d e d , and t h e f i f t h was l a b e l l e d as t h e " t o p s e c t i o n " . terminology - butt  middle  This  and t o p s e c t i o n - w i l l be used i n t h e  text. The t h r e e s e c t i o n s were t a k e n i m m e d i a t e l y t o t h e Vancouver L a b o r a t o r y , F o r e s t P r o d u c t s R e s e a r c h B r a n c h , F e d e r a l Department ry,  where a one i n c h t h i c k b o a r d i n c l u d i n g t h e p i t h was sawn from each  section.  The boards were edged, trimmed and p l a n e d t o t h r e e - q u a r t e r  inch thickness. sampling. the  The lower f o u r - f o o t p o r t i o n o f each b o a r d was used f o r  Two t h r e e - q u a r t e r i n c h wide beams were c u t from b o t h edges o f  f o u r - f o o t b o a r d s and were c o n s i d e r e d t o r e p r e s e n t sapwood.  o n e - i n c h wide s t r i p o f t h e edges was d i s c a r d e d . b o a r d s was r e g a r d e d as heartwood. the  of Forest-  The next  The remainder o f t h e  T h r e e - q u a r t e r i n c h cubes were c u t from  sapwood and heartwood beams o f t h e t h r e e s e c t i o n s .  The cubes were  p l a c e d i n p l a s t i c bags and s t o r e d a t 5° C. Four cubes were chosen randomly from b o t h sapwood and heartwood f r o m each o f t h e t h r e e s e c t i o n s .  E v e r y cube was s u r f a c e s t r i l i z e d  f l a m i n g and p l a c e d i n d i v i d u a l l y i n a s h a k i n g f l a s k c o n t a i n i n g sterile,  distilled  water.  75cc  by of  The f l a s k s were shaken c o n t i n u o u s l y on a low  -h speed s h a k e r f o r f i v e days a t room t e m p e r a t u r e .  The f i n a l s o l u t i o n s  o b t a i n e d were b e l i e v e d t o c o n t a i n a sample o f t h e p o p u l a t i o n o f m i c r o organisms p r e s e n t i n t h e cubes.  The s o l u t i o n s were used i n t h i s con-  d i t i o n f o r t h e p r e p a r a t i o n o f t h e c u l t u r e media. One, f o u r and s i x p e r c e n t o f agar i n t h r e e p e r c e n t m a l t e x t r a c t were p r e p a r e d ; - t h e shake s o l u t i o n r e p r e s e n t e d t e n p e r c e n t of t h e w a t e r r e q u i r e d t o p r e p a r e t h e m a l t a g a r .  The s o l u t i o n was added  by s t e r i l i z e d p i p e t t e t o t h e m a l t a g a r a f t e r i t had c o o l e d down cons i d e r a b l y (approximately U0-U5°C), i n order t o prevent the harmful e f f e c t o f h i g h temperature on t h e m i c r o o r g a n i s m s .  The s o l u t i o n o b t a i n e d  f r o m each cube was used s e p a r a t e l y t o p r e p a r e one, f o u r and s i x p e r c e n t m a l t a g a r , and f o u r P e t r i p l a t e s were poured from each c o n c e n t r a t i o n , two o f w h i c h were i n o c u l a t e d w i t h F. annosus and t h e o t h e r two p l a t e s w i t h P. m o n t i c o l a . C o n t r o l p l a t e s were p r e p a r e d f o r each conc e n t r a t i o n o f a g a r and i n o c u l a t e d w i t h each pathogen. The i n o c u l a t e d p l a t e s were i n c u b a t e d f o r a p e r i o d o f two weeks a t room t e m p e r a t u r e .  M y c e l i a l growth was measured on each p l a t e on t h e  f o u r t h , s i x t h , e i g h t h , t e n t h , t w e l f t h and f o u r t e e n t h day a f t e r i n o c u lation.  Two d i a m e t e r measurements a t r i g h t a n g l e t o each o t h e r were  t a k e n on each p l a t e and t h e a r i t h m e t i c mean o f t h e s e two measurements was r e c o r d e d . A t t h e end o f t h e i n c u b a t i o n p e r i o d s e v e r a l p l a t e s showing t h e s t r o n g e s t i n h i b i t i o n t o t h e wood d e s t r o y i n g f u n g i were s e l e c t e d f o r f u r t her study.  F o r t y d i s c s , f i v e m i l l i m e t e r s i n d i a m e t e r , were c u t w i t h a  s t e r i l i z e d c o r k b o r e r f r o m t h e m a l t agar medium where t h e growth o f t h e s o l u t i o n m i c r o o r g a n i s m s was e v i d e n t .  Each o f twenty d i s c s was p l a c e d  -5 i n d i v i d u a l l y on a s t e r i l e m i c r o s c o p e s l i d e , i n a growth chamber, a d j a c e n t t o a d i s c o f t h e same s i z e c u t from a pure c u l t u r e o f P. annosus (Fig.  l).  The d i s t a n c e between t h e two d i s c s was about f i v e m i l l i m e t e r .  The growth chamber c o n s i s t e d o f a s t e r i l e P e t r i p l a t e c o n t a i n i n g g l a s s rods f o r t h e s u p p o r t o f t h e m i c r o s c o p e s l i d e .  Twenty m i l l i l i t e r s o f  s t e r i l e d i s t i l l e d w a t e r was added t o t e n o f t h e p l a t e s , so t h a t t h e r e l a t i v e h u m i d i t y w i t h i n t h e p l a t e s had r e a c h e d t h e one hundred p e r c e n t p r i o r t o t h e i n t r o d u c t i o n o f m a l t agar d i s c s . always remained above w a t e r l e v e l .  The s l i d e s w i t h t h e d i s c s  The o t h e r t e n growth chambers c o n -  t a i n e d 1.5 w e i g h t m o l a r s u c r o s e s o l u t i o n i n o r d e r t o d e c r e a s e t h e r e l a t i v e humidity t o a l e v e l of approximately ninety-seven p e r cent (Luther 1935).  S i m i l a r s e r i e s o f growth chambers were t e s t e d w i t h d i s c s c o n -  F i g u r e 1.  Growth chamber f o r s t u d y i n g e f f e c t s o f v a r i o u s r e l a t i v e h u m i d i t y l e v e l s on m y c e l i a l s p r e a d .  - 6 t a i n i n g mycelium o f P. m o n t i c o l a .  The growth chambers were i n c u b a t e d  at  room t e m p e r a t u r e f o r twenty-one days d u r i n g w h i c h t h e m y c e l i a l growth  of  t h e wood d e s t r o y i n g f u n g i was o b s e r v e d . A n o t h e r f o u r b l o c k s from b o t h sapwood and heartwood o f each o f  the  t h r e e s e c t i o n s were chosen randomly.  Shake s o l u t i o n s were p r e p a r e d .  One, f o u r and s i x p e r c e n t m a l t agar c o n t a i n i n g t e n p e r c e n t s o l u t i o n were p r e p a r e d u s i n g t h e same t e c h i n q u e . However, t h e s o l u t i o n s p r i o r to tes  t h e a d d i t i o n t o t h e m a l t a g a r were a u t o c l a v e d f o r t w e n t y - f i v e minua t f i f t e e n pounds gauge p r e s s u r e .  P e t r i p l a t e s were poured f o r  each pathogen as w i t h t h e u n t r e a t e d , n a t u r a l s o l u t i o n .  The p l a t e s were  i n o c u l a t e d and i n c u b a t e d f o r two weeks, and m y c e l i a l growth was measured on each p l a t e on t h e f o u r t h , s i x t h , e i g h t , t e n t h , t w e l f t h , and f o u r t e e n t h day a f t e r  inoculation.  B e s i d e s u s 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 m a l t a g a r , i t was f e l t t h a t i t would be o f i m p o r t a n c e t o demonstrate t h e i n h i b i t o r y e f f e c t of t h e s a p r o p h y t e s towards t h e wood d e c a y i n g f u n g i on wood. F. annosus and P. m o n t i c o l a were t e s t e d on t h r e e - q u a r t e r i n c h wood cubes i n growth chambers w h i c h c o n s i s t e d o f s e v e n t e e n ounce massage j a r s w i t h screw-on l i d s .  A s h e l l v i a l o f f i t e e n by f o r t y - f i v e  m i l l i m e t e r s was p l a c e d a t t h e c e n t e r o f each j a r and k e p t i n v e r t i c a l p o s i t i o n by a w i r e s u p p o r t ( F i g .  2 ) . C h r o m e - n i c k e l r e s i s t a n c e w i r e was  used f o r t h e r e a s o n o f p r e v e n t i n g any r u s t f o r m a t i o n i n t h e j a r s w h i c h might i n t e r f e r e w i t h t h e growth o f f u n g i . The growth chambers were a u t o c l a v e d .  The wood b l o c k s were  weighed t o t h e n e a r e s t hundredth gram and s e a t e d i n d i v i d u a l l y on t h e top  of the v i a l s i n the j a r s .  - 7 -  Figure 2 .  Growth chamber f o r decay test on wood. Two  sets of three-quarter inch cubes were drawn randomly from  both sapwood and heartwood of the three sections of the trunk and each was treated p r i o r to the inoculation as indicated i n Table  1.  One set of four blocks from both sapwood and heartwood of the three sections f o r both pathogens was not subjected to any s p e c i a l t r e a t ment, except to surface s t e r i l i z a t i o n by flaming i n order to exclude contamination from the atmosphere.  The second set of four blocks from both  sapwood and heartwood were autoclaved at f i f t e e n pounds gauge pressure f o r f o r t y minutes. Within each set of blocks a series of two blocks were kept at one hundred per cent r e l a t i v e humidity.  One hundred m i l l i l i t e r s of ste-  r i l e d i s t i l l e d water was poured i n the j a r i n order to keep i t s atmosphere saturated.  The l i d of the j a r was not t i g h t so as to provide  s u f f i c i e n t aeration.  In order to secure the one hundred per cent r e l a -  - 8 -  t i v e h u m i d i t y t h e j a r s were p l a c e d on a s h e l f i n a t r a y w i t h t a p w a t e r i n i t , and c o v e r e d w i t h a t r a n s p a r e n t p l a s t i c c o v e r .  S u f f i c i e n t water  s u p p l y was m a i n t a i n e d i n t h e t r a y d u r i n g t h e three-month i n c u b a t i o n p e riod.  The o t h e r s e r i e s i n each s e t was kept a t n i n e t y - s e v e n p e r cent  r e l a t i v e humidity. rose s o l u t i o n .  One hundred m i l l i l i t e r s s u c r o s e s o l u t i o n was i n t h e  growth chamber. i n diameter at  T h i s h u m i d i t y was s e t by u s i n g 1.5 w e i g h t molar s u c -  Each b l o c k was i n o c u l a t e d w i t h a d i s c f i v e m i l l i m e t e r s  c u t from p u r e c u l t u r e s o f t h e decay f u n g i and was i n c u b a t e d  room t e m p e r a t u r e f o r a p e r i o d o f t h r e e months.  T a b l e 1. O u t l i n e o f t h e experiment c a r r i e d o u t on t h r e e - q u a r t e r i n c h cubes o f w e s t e r n hemlock sapwood and heartwood.  Wood  Sapwood  Treatment  Natural  T e s t fungus  Fomes  R. humid. % 100 97 Butt  Poria  Heartwood  Autoclaved Fomes  Poria  1 0 0 97 1 0 0 91 1 0 0 91  Natural Fomes 100  Poria  97 1 0 0 97  Autoclaved Fomes  Poria  1 0 0 97 1 0 0 97  1 cube  sec'n.  2  Middle  1 cube  sec'n.  2  Top  1  cube sec'n.  2  S i m u l t a n e o u s l y w i t h t h e drawing o f t h e b l o c k s used i n t h e experiment,  f o u r b l o c k s from b o t h sapwood and heartwood o f t h e t h r e e s e c -  -  9  -  t i o n s were chosen randomly f o r t h e p u r p o s e o f m o i s t u r e c o n t e n t d e t e r m i nation.  The b l o c k s were weighed when f r e s h , o v e n d r i e d a t 105 ° C f o r  2J4. h o u r s , reweighed and t h e m o i s t u r e c o n t e n t was c a l c u l a t e d on ovendry basis.  The a v e r a g e o f m o i s t u r e c o n t e n t o f f o u r b l o c k s o f b o t h sapwood  and heartwood o f t h e t h r e e l e v e l s were c o n s i d e r e d as a n a p p r o x i m a t i o n t o t h a t o f t h e b l o c k s used i n t h e e x p e r i m e n t .  On t h e b a s i s o f t h i s mois-  t u r e c o n t e n t and t h e f r e s h w e i g h t o f t h e b l o c k s used i n t h e experiment t h e i r ovendry w e i g h t was c a l c u l a t e d and termed as e s t i m a t e d ovendry weight.  A t t h e end o f t h r e e months t h e s u p e r f i c i a l m y c e l i a l growth was  removed from t h e s u r f a c e o f t h e b l o c k s b y a s o f t t o o t h - b r u s h , e x e r c i z i n g c a r e n o t t o remove any wood p a r t i c l e s . o v e n d r i e d a t 105° C and reweighed.  The b l o c k s were w e i g h e d , t h e n  Moisture content of the blocks a t  t h e end o f t h e three-month p e r i o d and w e i g h t l o s s , i f any, were c a l c u lated. B o t h experiments  ( t e s t on m a l t agar - and t e s t on wood) were  r e p e a t e d u s i n g t h e same t e c h n i q u e , on m a t e r i a l f r o m a n o t h e r v i g o r o u s l y growing h e a l t h y hemlock t r e e o f about t h e same age as used i n t h e f i r s t experiments. S t a t i s t i c a l a n a l y s e s were c a r r i e d o u t on t h e d a t a from e x p e r i ments on b o t h t r e e s and t h e r e s u l t s a r e p r e s e n t e d h e r e i n . A l t h o u g h t h e f i n a l growth measurements were made on t h e f o u r t e e n t h day a f t e r i n o c u l a t i o n , t h e s t a t i s t i c a l a n a l y s e s a r e based on t h e d a t a r e c o r d e d on t h e e i g h t h d a y , s i n c e a t t h i s time some m y c e l i a l c o l o n i e s had covered t h e d i a m e t e r o f t h e p l a t e s .  - 10 -  EXPERIMENTAL RESULTS C u l t u r a l s t u d i e s on m a l t a g a r media. L a r g e numbers o f v a r i o u s organisms were o b t a i n e d from b o t h t h e sapwood and heartwood  o f each s e c t i o n o f t h e h e a l t h y w e s t e r n hemlock stem.  Every  p l a t e c o n t a i n i n g u n t r e a t e d shake s o l u t i o n s showed e v i d e n c e o f a m i c r o f l o r a being present i n the tissues.  The organisms i n t h e m a j o r i t y o f  t h e i n s t a n c e s formed s m a l l c o l o n i e s on t h e m a l t a g a r medium, and seemed t o i n h i b i t t h e growth o f F. annosus and P. m o n t i c o l a ( F i g . 3 a , b , and Ua, b ) .  A l t h o u g h no a t t e m p t was made t o i d e n t i f y t h e o r g a n i s m s , i t was  n o t e d t h a t t h e p o p u l a t i o n c o n s i s t e d o f b a c t e r i a , y e a s t s , moulds and i m perfect fungi.  I n a few c a s e s t h e organisms produced no m a c r o s c o p i c ,  a e r i a l growth b u t m i c r o s c o p i c e x a m i n a t i o n o f t h e media r e v e a l e d t h e i r p r e s e n c e ( F i g . 5). The summarized e x p e r i m e n t a l d a t a a r e g i v e n i n F i g u r e s 6, 7 and 8.  The two t r e e s used i n t h i s s t u d y were n o t t r e a t e d i n t h e a n a l y s i s as  t r u e r e p l i c a t e s ( T a b l e 2) f o r t h e r e a s o n t h a t s i g n i f i c a n t t r e e t o t r e e v a r i a t i o n was e x p e c t e d .  V a r i a n c e was c a l c u l a t e d f o r t h e t r e e s and r e -  moved from t h e e x p e r i m e n t a l e r r o r thus making t h e a n a l y s i s more s e n s i t i v e . When a wet environment was p r o v i d e d - one p e r c e n t m a l t agar - t h e growth o f b o t h F. annosus and P. m o n t i c o l a was s i g n i f i c a n t l y b i t e d i n p l a t e s c o n t a i n i n g n a t u r a l , u n t r e a t e d shake s o l u t i o n s . f u n g i grew b e t t e r on media c o n t a i n i n g s o l u t i o n s from heartwood  inhi-  The t e s t t h a n on  those w i t h s o l u t i o n s f r o m sapwood ( F i g . 6a, b ) , and t h i s growth p a t t e r n was c o n s i s t e n t i n t h e t h r e e s e c t i o n s o f t h e stem.  The s t r o n g e s t i n h i b i -  t i o n was e x e r t e d b y t h e s o l u t i o n from sapwood o f t h e b u t t s e c t i o n ,  fol-  lowed i n d e c r e a s i n g o r d e r b y t h o s e from t h e t o p and m i d d l e s e c t i o n s .  - 11 -  Figure 3 a. Fomes annosus on one per cent malt agar containing no shake solution (control).  igure 3 b. Fomes annosus on one per cent malt agar containing shake solution. at center.  Note lack of mycelial growth from inoculum plug  Figure k b. Poria monticola on one per cent malt agar containing shake solution. at center.  Note lack of mycelial growth from inoculum plug  - 13 -  Figure 5- Photomicrograph showing the microorganisms i n one per cent malt agar medium (approximately 600  x).  F i g u r e 6 a. Upper l e f t . Growth o f F. annosus on one p e r c e n t m a l t a g a r containing  t h e v a r i o u s u n t r e a t e d shake s o l u t i o n s .  F i g u r e 6 b. Upper r i g h t . Growth o f P. m o n t i c o l a on one p e r c e n t m a l t agar containing  t h e v a r i o u s u n t r e a t e d shake s o l u t i o n s .  F i g u r e 6 c. Lower l e f t . Growth o f F. annosus on one p e r c e n t m a l t a g a r containing  the various autoclaved  solutions.  F i g u r e 6 d. Lower r i g h t . Growth o f P. m o n t i c o l a on one p e r c e n t m a l t a g a r containing  the v a r i o u s a u t o c l a v e d s o l u t i o n s .  Legend: Black s o l i d l i n e  - no shake s o l u t i o n added  (control)  Red dash l i n e  - b u t t s e c t i o n , sapwood s o l u t i o n  Red s o l i d l i n e  - b u t t s e c t i o n , heartwood s o l u t i o n  Orange dash l i n e  - m i d d l e s e c t i o n , sapwood s o l u t i o n  Orange s o l i d l i n e - m i d d l e s e c t i o n , heartwood s o l u t i o n Green dash l i n e  - t o p s e c t i o n , sapwood s o l u t i o n  Green s o l i d l i n e  - t o p s e c t i o n , heartwood s o l u t i o n  Each p l o t t e d v a l u e r e p r e s e n t s t h e average o f e i g h t e x p e r i m e n t a l v a l u e s w i t h each o f t h e two  trees.  F i g u r e 7 a. Upper l e f t . Growth o f F. annosus on f o u r p e r c e n t m a l t a g a r containing  t h e v a r i o u s u n t r e a t e d shake s o l u t i o n s .  F i g u r e 7 b. Upper r i g h t . Growth o f P. m o n t i c o l a on f o u r p e r c e n t m a l t a g a r containing  t h e v a r i o u s u n t r e a t e d shake s o l u t i o n s .  F i g u r e 7 c. Lower l e f t . Growth o f F. annosus on f o u r p e r c e n t m a l t a g a r containing  the v a r i o u s autoclaved  solutions.  F i g u r e 7 d. Lower r i g h t . Growth o f P. m o n t i c o l a on f o u r p e r c e n t m a l t agar containing  the v a r i o u s a u t o c l a v e d s o l u t i o n s .  Legend: Black s o l i d l i n e  - no shake s o l u t i o n added  (control)  Red dash l i n e  - b u t t s e c t i o n , sapwood s o l u t i o n  Red s o l i d l i n e  - b u t t s e c t i o n , heartwood s o l u t i o n  Orange dash l i n e  - m i d d l e s e c t i o n , sapwood s o l u t i o n  Orange s o l i d l i n e - m i d d l e s e c t i o n , heartwood s o l u t i o n Green dash l i n e  - top s e c t i o n , sapwood s o l u t i o n  Green s o l i d l i n e  - t o p s e c t i o n , heartwood s o l u t i o n  Each p l o t t e d v a l u e r e p r e s e n t s t h e average o f e i g h t e x p e r i m e n t a l v a l u e s w i t h each o f t h e two  trees.  -15 -  TIMe  -  DAV^  F i g u r e 8 a. Upper l e f t . Growth o f F. annosus on s i x p e r c e n t m a l t a g a r containing  t h e v a r i o u s u n t r e a t e d shake s o l u t i o n s .  F i g u r e 8 b. Upper r i g h t . Growth o f P. m o n t i c o l a on s i x p e r c e n t m a l t a g a r containing  t h e v a r i o u s u n t r e a t e d shake s o l u t i o n s .  F i g u r e 8 c. Lower l e f t . Growth o f F. annosus on s i x p e r c e n t m a l t a g a r containing  the v a r i o u s autoclaved  solutions.  F i g u r e 8 d. Lower r i g h t . Growth o f P. m o n t i c o l a on s i x p e r c e n t m a l t agar containing  the v a r i o u s autoclaved  solutions.  Legend: Black s o l i d l i n e  - no shake s o l u t i o n added  (control)  Red dash l i n e  - b u t t s e c t i o n , sapwood s o l u t i o n  Red s o l i d l i n e  - b u t t s e c t i o n , heartwood s o l u t i o n  Orange dash l i n e  - m i d d l e s e c t i o n , sapwood s o l u t i o n  ,  Orange s o l i d l i n e - m i d d l e s e c t i o n , heartwood s o l u t i o n Green dash l i n e  - t o p s e c t i o n , sapwood s o l u t i o n  Green s o l i d l i n e  - t o p s e c t i o n , heartwood s o l u t i o n  Each p l o t t e d v a l u e r e p r e s e n t s t h e average o f e i g h t e x p e r i m e n t a l v a l u e s w i t h each o f t h e two  trees.  16 -  - 17 T a b l e 2. A n a l y s i s o f v a r i a n c e based on d a t a r e c o r d e d i n F i g u r e s 6, 7 and 8. Source o f variation A Tree B Sapw.-Heartw. C Nat.-Autocl. D Fomes-Poria E Agar % l-k-6 F S e c t i o n B-M-T. AB AC AD AE AF BC BD BE BF CD CE CF DE DF EF ABC ABD ABE ABF ACD ACE ACF ADE ADF AEF BCD BCE BCF BDE BDF BEF CDE CDF CEF DEF ERROR TOTAL  DF. 1 1 1 1 2 2 1 1 1 2 2 1 1 2 2 1 2 2 2 2 k 1 1 2 2 1 2 2 2 2 k 1 2 2 2 2 k 2 2  k k 65 Ik3  Sum square  367-3600 625.0000 12731-3000 13963-3000 202k.5U00 150.U900 132.2300 21.7700 5.U|00 101.0000 193.5U00 616.6900 k.6900  Mean square  5676.5000 713.5500 58.5800 155.0500 228.8300 289.0200 75.1200 , 67.5600 11*3.2000 - 26.6900 52.1100 215.L100 67.3700 11.0800 161.9600  367.3600 625.0000 12731.3000 13963.3000 1012.2700 75.2U50 132.2300 21.7700 5.UU00 50.5000 96.7700 616.6900 k.6900 133.3100 U5.2U50 62k-9900 2838.2500 356.7750 29-2900 77.5250 57.2075 289.0200 75.1200 33-7800 71.6000 26.6900 26.0550 107.7050 33-6850 5.5kOO k0.k900  93.kkOO  93.kkOO  266.6200  90.U900  62k.9900  153.1000 132.0600 8.5200 26.0700  32.5000 832.6500 51.5200 93.U600 163.6200 1580.L600 U3029-0000  * S i g n i f i c a n t a t 0.05 p r o b a b i l i t y l e v e l . S i g n i f i c a n t a t 0.01 p r o b a b i l i t y l e v e l .  76.5500 66.0300 k-2600 13.0350 8.1250 kl6.3250 25.7600 23.3650 kO.9050 2k-3lU7  F.  15-10 ** 25.70 ** 523.60 ** 57k.27 l a . 63 3-09 5.k3 * 0.89 0.22  2.07 3.97 25-36 ** 0.19 5.k8 ** 1.86 25.70  116.72--:-**  lk.67 #* 1.20 ' 3.18 * 2.35 11.88-Hf 3.08 1.38 2.9k I.09 1.07 k.k2 * 1.38 J  0.22 1.66  3.8k 3.1k 2.71 0.17 0.53  0.33  17.12 ** 1.05 0.96 1.68  - 18 -  The i n h i b i t i o n on p l a t e s c o n t a i n i n g s o l u t i o n s f r o m heartwood, i n d e c r e a s i n g o r d e r were as f o l l o w s :  b u t t , m i d d l e and t o p s e c t i o n s , f o r  b o t h F. annosus and P. m o n t i c o l a . The b e s t growth ( l e a s t i n h i b i t i o n ) o f t h e two wood decay f u n g i o c c u r r e d on p l a t e s w i t h , s o l u t i o n s from t h e h e a r t wood o f t h e top s e c t i o n , b u t t h i s was s t i l l s i g n i f i c a n t l y l e s s t h a n t h e growth on c o n t r o l p l a t e s c o n t a i n i n g one p e r c e n t m a l t agar w i t h o u t t h e shake s o l u t i o n ( F i g . 6 a, b ) . A p p l i c a t i o n o f Duncan's new m u l t i p l e - r a n g e t e s t t o t h e s e d a t a showed t h a t an i n c r e a s e o f t h e agar c o n c e n t r a t i o n t o f o u r p e r c e n t ( d r i e r environment) b r o u g h t about a h i g h l y s i g n i f i c a n t change i n t h e r a t e o f growth o f the t e s t f u n g i .  The i n h i b i t i n g f a c t o r d e c r e a s e d w i t h t h e i n -  c r e a s e i n agar c o n c e n t r a t i o n .  Both F. annosus and P. m o n t i c o l a produced  b e t t e r m y c e l i a l growth on f o u r p e r c e n t m a l t agar t h a n on one p e r c e n t m a l t a g a r r e g a r d l e s s o f t h e s o u r c e o f shake s o l u t i o n s (sapwood  or heart-  wood, b u t t m i d d l e o r t o p s e c t i o n ) used i n p r e p a r i n g t h e media ( F i g . 7 a, b).  The i n h i b i t i o n o f F. annosus i n p l a t e s c o n t a i n i n g v a r i o u s  s o l u t i o n s was f o u n d t o be as f o l l o w s i n d e c r e a s i n g o r d e r :  shake  b u t t sapwood,  t o p sapwood, b u t t heartwood, m i d d l e heartwood and sapwood, and t o p h e a r t wood ( F i g . 7 a ) .  P. m o n t i c o l a produced t h e p o o r e s t growth i n p l a t e s con-  t a i n i n g t h e s o l u t i o n from t h e b u t t sapwoodj f o l l o w e d by t o p sapwood, b u t t heartwood, t o p heartwood, m i d d l e sapwood and heartwood ( F i g . 7 b ) .  Fur-  t h e r m o r e , t h e d i s t i n c t l y d i f f e r e n t degrees o f i n h i b i t i o n between t h e sol u t i o n s from sapwood and heartwood on one p e r c e n t m a l t a g a r , l e s s e n e d when t h e s o l u t i o n s were added t o a d r i e r s u b s t r a t u m ( f o u r p e r c e n t m a l t agar). F u r t h e r i n c r e a s e i n the agar c o n c e n t r a t i o n to s i x per cent  - 19 r e s u l t e d i n a more d r a s t i c change i n t h e growth r a t e s .  B o t h F. annosus  and P. m o n t i c o l a seemed t o grow f r e e l y i n the p l a t e s c o n t a i n i n g the shake s o l u t i o n s , p r o d u c i n g c o l o n i e s o f a l m o s t t h e same s i z e as t h o s e on c o n t r o l p l a t e s w i t h no shake s o l u t i o n s ( F i g . 8 a, b ) .  A l t h o u g h growth o f t h e  m i c r o o r g a n i s m s o c c u r r e d i n t h e d r y medium, the i n h i b i t i o n f a c t o r was n i f i c a n t l y reduced.  B o t h F. annosus and P. m o n t i c o l a has  sig-  overgrown  t h e s m a l l c o l o n i e s o f t h e s a p r o p h y t e s on t h e d r y medium - s i x p e r c e n t m a l t a g a r ( F i g . 9 a, b ) . The i n c r e a s e o f t h e agar c o n t e n t o f t h e media from one t o f o u r and s i x p e r c e n t was c o n s i d e r e d t o p r o v i d e a d r i e r environment f o r the growth o f t e s t f u n g i .  However, i t was n o t p o s s i b l e t o c o n c l u d e t h a t t h e  d e c r e a s e i n t h e amount o f a v a i l a b l e w a t e r was t h e s o l e f a c t o r r e s p o n s i b l e f o r the d e c l i n e of i n h i b i t i o n . T h i s r e l a t i o n s h i p was demonstrated i n a n o t h e r experiment when m a l t agar d i s c s o f f i v e m i l l i m e t e r s i n d i a m e t e r , c o l o n i z e d by the sapr o p h y t e s , were p l a c e d i n growth chambers a d j a c e n t t o a d i s c o f the same s i z e c o n t a i n i n g mycelium o f t h e decay f u n g i .  The growth o f b o t h F.  annosus and P. m o n t i c o l a was i n h i b i t e d a t one hundred p e r c e n t r e l a t i v e humidity.  On t h e o t h e r hand, a d e c r e a s e o f r e l a t i v e h u m i d i t y i n t h e  growth chambers t o a p p r o x i m a t e l y n i n e t y - s e v e n p e r c e n t r e s u l t e d i n f r e e growth o f t h e mycelium o f t h e t e s t f u n g i w h i c h c o v e r e d t h e d i s c s o f s a p r o p h y t e s ( F i g . 10 a, b, 11 a, b ) . I n a n o t h e r p a r t o f t h e experiment t h e shake s o l u t i o n s were s t e r i l i z e d by a u t o c l a v i n g p r i o r t o t h e i r b e i n g added t o t h e m a l t a g a r . T h i s t r e a t m e n t l e d t o the l o s s o f i n h i b i t i o n .  - 20 -  Figure 9 b. P. monticola on s i x per cent malt agar containing untreated shake s o l u t i o n .  F i g u r e 10 b. M y c e l i a l s p r e a d o f F. annosus ( l e f t d i s c ) a t n i n e t y - s e v e n per cent r e l a t i v e humidity.  F i g u r e 11 b. M y c e l i a l spread o f P. m o n t i c o l a ( l e f t d i s c ) a t n i n e t y - s e v e n per cent r e l a t i v e humidity.  - 23 On media c o n t a i n i n g t h e a u t o c l a v e d s o l u t i o n s , b o t h F. annosus and P. m o n t i c o l a grew a p p r o x i m a t e l y as w e l l as on t h o s e w i t h o u t s o l u t i o n s , r e g a r d l e s s o f t h e s o u r c e o f s o l u t i o n s and t h e agar c o n c e n t r a t i o n o f t h e media ( F i g . 6 c , d , 7 c , d, 8 c , d ) . S t i m u l a t i o n o c c u r r e d i n s t e a d o f i n h i b i t i o n i n some c a s e s .  F. annosus p r o d u c e d b e t t e r growth t h a n t h e c o n t -  r o l i n t h e f o l l o w i n g i n s t a n c e s ; on wet medium - one p e r c e n t m a l t agar c o n t a i n i n g t h e s o l u t i o n o f b u t t and m i d d l e heartwood ( F i g . 6 c ) ;  on d r i e r  medium - f o u r p e r c e n t m a l t agar - w i t h t h e s o l u t i o n from t h e m i d d l e heartwood and t o p sapwood and heartwood ( F i g . 7 c ) ;  on d r i e s t medium -  s i x p e r c e n t m a l t a g a r - c o n t a i n i n g t h e s o l u t i o n o b t a i n e d from b u t t sapwood and heartwood ( F i g . 8 c ) .  The a d d i t i o n o f t h e a u t o c l a v e d s o l u t i o n s  p r o v i d e d no s t i m u l a t i o n i n t h e growth o f P. m o n t i c o l a . A u t o c l a v i n g i s a common t e c h n i q u e f o r s t e r i l i z a t i o n .  I t was f e l t  t h a t t h e h i g h t e m p e r a t u r e (250 ° F ) reached d u r i n g t h e p r o c e s s may have a l t e r e d t h e c h e m i c a l c o m p o s i t i o n o f t h e s o l u t i o n s , thus d e s t r o y i n g t h e i r i n h i b i t o r y e f f e c t on t h e growth o f t h e decay f u n g i .  T h e r e f o r e , i t was o f  prime i m p o r t a n c e t o e x p r e s s t h e i n h i b i t i n g f a c t o r i n a more e x p l i c i t form. The s o l u t i o n s c o n t a i n i n g t h e organisms w h i c h had shown t h e s t r o n g e s t i n h i b i t i o n were added t o each o t h e r and d i v i d e d i n t o t h r e e equal p o r t i o n s .  One p a r t had no f u r t h e r t r e a t m e n t ; t h e second was  fil-  t e r e d t h r o u g h M i l l i p o r e o f t y p e HA, 0.U5 m i c r o n p o r e s i z e ; t h e t h i r d p o r t i o n was a u t o c l a v e d a t f i f t e e n pounds gauge p r e s s u r e f o r t w e n t y - f i v e m i nutes.  Each p o r t i o n o f t h e s o l u t i o n was t h e n used i n one p e r c e n t m a l t  a g a r c u l t u r e media as b e f o r e and t e s t e d w i t h t h e decay f u n g i .  Mycelial  growth was r e c o r d e d on t h e f o u r t h , s i x t h , e i g h t h , t e n t h , t w e l f t h , and  -2kfourteenth  day The  after inoculation. growth o f b o t h F. annosus and P. m o n t i c o l a was  l y i n h i b i t e d on p l a t e s c o n t a i n i n g  significant-  the u n t r e a t e d , n a t u r a l shake s o l u t i o n .  However, the growth o f the decay f u n g i i n p l a t e s w i t h a u t o c l a v e d s o l u t i o n and  i n p l a t e s w i t h M i l l i p o r e f i l t e r e d s o l u t i o n were a l m o s t i d e n t i -  c a l w i t h the  growth p r o d u c e d i n the c o n t r o l p l a t e s h a v i n g no  ( F i g . 12 a, b, c, d, and t i o n , autoclaving  13 a, b, c, d ) .  Both t r e a t m e n t s - o f  solution steriliza-  and M i l l i p o r e f i l t e r i n g r e s u l t e d i n l o s s o f the  b i t i n g f a c t o r ( F i g . lU and  15).  inhi-  F i g u r e 12. F. annosus on one p e r c e n t m a l t a g a r , a. c o n t a i n i n g no shake s o l u t i o n ( c o n t r o l ) b. c o n t a i n i n g u n t r e a t e d from i n o c u l u m p l u g a t c. c o n t a i n i n g a u t o c l a v e d  v  s o l u t i o n ; n o t e l a c k o f m y c e l i a l growth center solution  d. c o n t a i n i n g M i l l i p o r e f i l t e r e d s o l u t i o n  F i g u r e 12  c.  F i g u r e 12  d.  F i g u r e 13. P. m o n t i c o l a  on one p e r c e n t m a l t a g a r ,  a. c o n t a i n i n g no shake s o l u t i o n ( c o n t r o l ) b. c o n t a i n i n g u n t r e a t e d from inoculum p l u g at c. c o n t a i n i n g a u t o c l a v e d  s o l u t i o n ; note l a c k o f m y c e l i a l growth center. solution  d. c o n t a i n i n g M i l l i p o r e f i l t e r e d s o l u t i o n  F i g u r e 13  c.  F i g u r e 13  d.  F i g u r e I k . Graph r e p r e s e n t i n g  the growth r a t e o f F. annosus on one  c e n t m a l t agar c o n t a i n i n g s o l u t i o n , and  no shake s o l u t i o n , u n t r e a t e d  s o l u t i o n s a u t o c l a v e d and M i l l i p o r e f i l t e r e d .  Legend: Solid  line  Dash - two  per  no dots l i n e  shake s o l u t i o n added  (control)  untreated solution  Dash l i n e  autoclaved s o l u t i o n  Dotted l i n e  Millipore f i l t e r e d solution  F i g u r e 1$.  Graph r e p r e s e n t i n g  the growth r a t e o f P. m o n t i c o l a on  per cent malt agar c o n t a i n i n g s o l u t i o n , and  one  no shake s o l u t i o n , u n t r e a t e d  s o l u t i o n s a u t o c l a v e d and M i l l i p o r e f i l t e r e d .  Legend: Solid  no shake s o l u t i o n added  line  Dash - two  dots l i n e  (control)  untreated solution  Dash l i n e  autoclaved s o l u t i o n  Dotted l i n e  Millipore f i l t e r e d solution  - 28 -  I  x o CH  o 40 -  30 I  20 L  10 U  -i  4  1  •  6  I  •  8 1 TIME - DAYS Figure  1$.  - 29 C u l t u r a l s t u d i e s on wood b l o c k s . The growth r a t e o f F. annosus and P. m o n t i c o l a was s t u d i e s on t h r e e - q u a r t e r i n c h wood cubes i n c u b a t e d i n growth chambers. the  The growth o f  decay f u n g i i s e x p r e s s e d as a p e r c e n t a g e o f l o s s i n w e i g h t o f t h e  cubes d u r i n g t h e i n c u b a t i o n p e r i o d o f t h r e e months.  The p e r c e n t a g e  l o s s i n w e i g h t was c a l c u l a t e d as a d i f f e r e n c e between t h e i n i t i a l e s t i m a t e d o v e n d r y w e i g h t and t h e f i n a l ovendry w e i g h t o v e r t h e f i n a l ovendry of  weight.  ( F i n a l o v e n d r y w e i g h t = ovendry w e i g h t o f cube a t t h e end  incubation period).  The r e s u l t s a r e summarized  i n T a b l e 3-  Analysis  of v a r i a n c e was made on t h e d a t a and t h i s i s g i v e n i n T a b l e k. The growth o f F. annosus and P. m o n t i c o l a was p a r t i a l l y i n h i b i t e d on n a t u r a l , u n t r e a t e d w-ood ( F i g .  16 and 1 7 ) .  I t was p o s s i b l e  t o d e t e c t t h e m i c r o o r g a n i s m s on n a t u r a l , u n t r e a t e d cubes.  I n many i n -  s t a n c e s t h e y p r o d u c e d v i s i b l e growth on t h e s u r f a c e o f t h e cubes i n v a d ing  t h e inoculum p l u g ( F i g .  18 a , b ) . The w e i g h t l o s s o f u n t r e a t e d  sapwood and heartwood cubes o f t h r e e s e c t i o n s o f t h e stem was s i g n i f i c a n t l y s m a l l e r a t 0.01 p r o b a b i l i t y l e v e l t h a n t h a t o f a u t o c l a v e d b l o c k s . Both t e s t f u n g i produced b e t t e r growth - more w e i g h t l o s s - on sapwood cubes t h a n on cubes o f heartwood ( F i g .  19 and 2 0 ) .  This r e s u l t i s i n  c o n t r a d i c t i o n w i t h t h a t o f t h e s t u d i e s on m a l t agar media. The' t r e a t m e n t o f two l e v e l s o f r e l a t i v e h u m i d i t y y i e l d e d  sig-  n i f i c a n t l y d i f f e r e n t growth f o r F. annosus and P. m o n t i c o l a on u n t r e a t e d wood. per  The t e s t f u n g i caused more w e i g h t l o s s i n b l o c k s a t n i n e t y - s e v e n  c e n t r e l a t i v e h u m i d i t y t h a n i n b l o c k s k e p t a t one hundred p e r c e n t  r e l a t i v e humidity. the  The d r i e r environment seemed t o reduce c o n s i s t e n t l y  i n h i b i t i n g f a c t o r i n u n t r e a t e d , n a t u r a l sapwood and heartwood.  Table 3P e r c e n t w e i g h t l o s s e s o f t h r e e - q u a r t e r i n c h sapwood and heartwood cubes o f w e s t e r n hemlock, caused by F. annosus and P. m o n t i c o l a .  U n d e r l i n e d f i g u r e s r e p r e s e n t mean o f v a l u e s r e c o r d e d on m a t e r i a l s  from t h e f i r s t and second t r e e s .  Wood  Sapwood Natural  Treatment Test fungus R. humid. % Butt  Heartwood  Fomes  100  1 O.li* 0.8 tree  Autoclaved  Poria  Fomes  Poria  100  97  100  6.1  5.5  3.3  13-2 10.8 12.0 13.8  6.1  97  100  1.3  97  Natural Fomes  Autoclaved  Poria  Fomes  Poria  100  97  100  97'  100  97  0.3 0.8  1.5 1.5  15.3 1U.8 11.9 12.ii  3.8  h.k 20.6  6.1  23.3 16.5 26.ii 26.3  5.8  6.0 18.3 21.3  97  100  97 Iii. 2  sec'n.  2 •1.2  5.U  10.9 16.9  6.2  6.7  29-5  31.6  1.3  1.6  8.6 10.1  7.9  7.6 16.1  Middle  1 o.U  Iii.5  1U.8  8.8  8.6  27.7 23.ii  0.6  1U.9 15-6  8.9  8.3  23.5.21.7  0.8  1.5 1.7  13.1 17.6 1U.1 15-6  6.5  1.0  1.8 1.6  7.3  6.6 19.5 2U.5" 6.2 18.6 20.9  2 1.6  1.3  l5.ii 16. k  9.0  8.0  19.3  20.0  1.0  1.9  15.1 lU.6  8.1  5.9 17.8  1 0.8 tree 0.8  5.3 3.0  16.U 18.7  y.U  9.6  36.0 U2.9  0.8  1.6  6.3 25.5 27.3  7.9  8.0  39-5 Ul.7  1.1  1.7  15.5 1U.8 16.9 1U.6  6.U  18.9 20.J4  7.8  7.3 25-3 25-7  2 0.7  0.8  21.5 22.1  6.5  b.h U3.2 U0.5  l.U  1.7  18.h 1U.5  9.2  8.3 25.1 2U-2  tree sec'n. Top  sec'n.  * E v e r y f i g u r e i n each t r e e r e p r e s e n t s t h e mean o f two e x p e r i m e n t a l v a l u e s .  27.5  17.i;  - 31 T a b l e 1|. A n a l y s i s o f v a r i a n c e b a s e d on d a t a shown i n T a b l e 3. Source o f variation A B C D.  Tree Sapw.-Heartw. Nat.-Autocl. Fomes-Poria  E  R.H.Jg 100-97  F S e c t i o n B-M-T. AB AC AD AE AF BC BD BE BF CD CE CF DE DF EF ABC ABD ABE ABF ACD ACE ACF ADE ADF AEF BCD BCE. BCF BDE BDF BEF CDE CDF CEF DEF ERROR TOTAL  >  DF.  1 1 1 1 1 2 1 1 1 1 2 1 1 1 2 1 1 2 1 2 2 1 1 1 2 1 1 2 1 2 2 1 1 2 1 2 2 1 2 2 2 38 95  Sum square  11.2620  98.1720 1578.8500 5757.9000 19.2320 185-5820 0.8780 0.2580 0.8U60 0.0000 1+0.3710 27.8UUO 36.52yo 0.7950 20.1+230 11.2180 15-7120 30.2700 3.0170 102.1+120 2.3160 5.1U50 36.0850 1.1*710 20.0090 0.7680 lu 71+90 35.71+90 11.7660 19-8700 51.2820 20.5610 2+.7000 3.5030 1.9250 33-01+80 7.8770 1U.8610 U7.6U20 1.3150 1.8560 11+0.2700 81+08.3300  * S i g n i f i c a n t a t 0.05 p r o b a b i l i t y S i g n i f i c a n t a t 0.01 p r o b a b i l i t y  level. level.  Mean square  F>.  11.2620  3.05 26.59 ** 1+27.72 * * 1559.85 ** 5.21 * 25.13 ** 0.23  98.1720 1578.8500 5757.9000 19.2320 92.7910 0.8780 0.2580 0.81+60 0.0000 20.1855 27.81+1+0 36.5290 0.7950 10.2115 11.2180 15.7120 15.1350 3.0170 51.2060 1.1580 5.11+50 36.0850 1.1*710 10.001+5 0.7680 1+. 71+90 17.871+5 11.7660 9.9350 25.61+10 20.5610 1;.7000 1.7515 1.9250 16.521+0 3.9385 11+.8610 • 23.8210 0.6575 0.9280 3.6931  0.06  0.22 0.00 5.1+6 ** 7.51+ **  9.89 ** 0.21 2.76 3.03 U.25 * 1+.10 * 0.81 13.87 ** 6.31 1.39 9.77 ** 0.39 2.71 0.20 • 1.28 1+.81+ *  3.18 2.69  6.91+ **•  5.57 * 1.27 0.1+7 0.52 U.l+7 * 1.06 U.02 6.U5 ** 0.17 0.25  - 32 -  F i g u r e 16. Growth o f F. annosus on u n t r e a t e d  ( l e f t ) and a u t o c l a v e d  (right)  cubes.  F i g u r e 17. Growth o f P. m o n t i c o l a on u n t r e a t e d ( r i g h t ) cubes.  ( l e f t ) and a u t o c l a v e d  -  33 -  Figure 18 a. Untreated wood cube inoculated with F. annosus.  Figure 18 b. Showing the inoculum invaded by the microorganisms of Figure 18 a.  Figure 19. Growth of F. annosus on sapwood ( l e f t ) and heartwood (right) cubes.  - 35 The d i f f e r e n c e i n r e l a t i v e h u m i d i t y , however, d i d not e f f e c t t h e growth o f t h e t e s t f u n g i on a u t o c l a v e d wood.  B o t h F. annosus and P.  monticola  caused a p p r o x i m a t e l y t h e same l o s s i n w e i g h t o f a u t o c l a v e d cubes a t hundred p e r c e n t r e l a t i v e h u m i d i t y as a t n i n e t y - s e v e n p e r c e n t  one  relative  humidity. A p p l i c a t i o n o f Duncan's new m u l t i p l e range t e s t t o t h e d a t a showed t h a t the t e s t f u n g i produced s i g n i f i c a n t l y t h e b e s t growth - most weight l o s s - on cubes f r o m the t o p s e c t i o n o f t h e t r e e .  P o o r e s t growth  o c c u r r e d on cubes f r o m t h e b u t t s e c t i o n , b u t t h e r e was  significant  no  d i f f e r e n c e between t h e w e i g h t l o s s e s o f cubes f r o m t h e b u t t s e c t i o n and t h o s e o f t h e cubes o f t h e middle  section.  The growth o f P. m o n t i c o l a was  s i g n i f i c a n t l y better at  0.01  p r o b a b i l i t y l e v e l i n a l l i n s t a n c e s t h a n t h a t p r o d u c e d by F. annosus. T h i s i s i n accordance w i t h the e x p e c t e d s i n c e t h e former i s known as a fungus o f v e r y r a p i d growth ( C a r t w r i g h t e t a l . ,  1958).  DISCUSSION  I n t h i s s t u d y samples were t a k e n from sapwood and heartwood o f t h e b u t t , m i d d l e and top s e c t i o n s o f t h e stems o f two h e a l t h y t r e e s o f w e s t e r n hemlock.  S o l u t i o n s were p r e p a r e d by s h a k i n g t h r e e - q u a r t e r  i n c h , s u r f a c e s t e r i l i z e d wood cubes i n s e v e n t y - f i v e c u b i c c e n t i m e t r e s o f s t e r i l e d i s t i l l e d water.  T h i s t e c h n i q u e was  organisms i n wood, s i n c e i t was  chosen t o r e c o v e r m i c r o -  c o n s i d e r e d t o p r o v i d e b e t t e r coverage  o f t h e sample m a t e r i a l than o t h e r methods o f i s o l a t i o n , e.g. t h e t r a n s -  - 36 -  f e r o f s m a l l p o r t i o n s o f wood i n t o p l a t e s o f m a l t a g a r . The  i n d i v i d u a l solutions" obtained  f r o m the samples o f v a r i o u s  p a r t s o f the stem were u s e d i n the p r e p a r a t i o n o f m a l t a g a r , t e n p e r c e n t o f t h e water r e q u i r e d t o p r e p a r e the m a l t agar. p l a t e s were p o u r e d and one o t h e r w i t h P.  s e r i e s was  representing Petri  i n o c u l a t e d w i t h F. annosus and  the  monticola.  A l l untreated  s o l u t i o n s y i e l d e d a v a r i e t y of organisms c a p a b l e  o f s i g n i f i c a n t l y i n h i b i t i n g t h e growth o f the t e s t f u n g i when added t o one p e r c e n t m a l t a g a r , ( F i g . 6 a, b ) . t e s t f u n g i on one p e r c e n t m a l t a g a r was  The  degree o f i n h i b i t i o n o f b o t h  greater i n the p l a t e s w i t h  so-  l u t i o n s f r o m sapwood t h a n i n p l a t e s c o n t a i n i n g t h e s o l u t i o n s f r o m h e a r t wood.  The m a l t agar p r o v i d e d u n i f o r m n u t r i t i o n f o r the t e s t f u n g i ,  i t was  considered  t h a t t h e l a r g e numbers o f m i c r o o r g a n i s m s i n the  wood s o l u t i o n s were r e s p o n s i b l e f o r t h e g r e a t e r degree o f The  and  sap-  inhibition.  r e s u l t s a l s o i n d i c a t e d a s i g n i f i c a n t v a r i a t i o n i n the  rate  o f i n h i b i t i o n between t h e s o l u t i o n s f r o m the t h r e e s e c t i o n s o f the stem. The p o o r e s t growth on one p e r c e n t m a l t agar o c c u r r e d the s o l u t i o n s o f the b u t t s e c t i o n s , and which contained  i n the p l a t e s w i t h  the b e s t growth i n t h o s e p l a t e s  the s o l u t i o n s o f t h e top s e c t i o n s . •' The  growth o f t e s t  f u n g i i n p l a t e s having the s o l u t i o n s of middle s e c t i o n s d i d not  differ  s i g n i f i c a n t l y f r o m t h a t i n p l a t e s c o n t a i n i n g the s o l u t i o n s o f t h e sections.  However, i t was  top  p o s i t i v e l y g r e a t e r t h a n the growth i n p l a t e s  c o n t a i n i n g the s o l u t i o n s o f b u t t s e c t i o n s . The  r e s u l t s o f the c u l t u r a l s t u d i e s on m a l t a g a r s h o u l d  be r e l a t e d t o those o b t a i n e d on n a t u r a l s u b s t r a t a .  Therefore,  F. annosus and P. m o n t i c o l a were t e s t e d on t h r e e - q u a r t e r  inch  not  both surface  -  37  -  s t e r i l i z e d wood cubes i n growth chambers a t one hundred p e r cent r e l a t i v e humidity. as p e r c e n t a g e s  The r e s u l t i n g w e i g h t l o s s e s o f t h e cubes were c a l c u l a t e d o f t h e i r i n i t i a l e s t i m a t e d ovendry w e i g h t .  ovendry weight i n t u r n was v a l u e s determined  The  c a l c u l a t e d by u s i n g t h e m o i s t u r e  estimated  content  on the ovendry b a s i s o f a sample o f f o u r b l o c k s drawn  from the same p o p u l a t i o n f o r t h i s p u r p o s e .  T h i s method i s known t o be  s u b j e c t t o e r r o r , because o f v a r i a t i o n i n d e n s i t y o f t h e samples. f a c t o r was  not o v e r l o o k e d , but i t was  This  not f e a s i b l e t o a p p l y o t h e r t e c h -  niques such as d e s c r i b e d by E t h e r i d g e , ( 1 9 5 7 ) where t h e m o i s t u r e  content  i s e x p r e s s e d as a p e r c e n t a g e  The  of moisture present at s a t u r a t i o n .  growth o f b o t h F. annosus and P. m o n t i c o l a was  significantly  inhibited  on u n t r e a t e d , s u r f a c e s t r i l i z e d wood cubes i n c u b a t e d a t one hundred p e r cent r e l a t i v e humidity. The degree o f i n h i b i t i o n o f b o t h t e s t f u n g i was g r e a t e r on heartwood t h a n on sapwood samples.  significantly  These r e s u l t s were t h e r e -  v e r s e o f t h o s e o b t a i n e d when the s o l u t i o n s o f sapwood and heartwood samples were added t o one p e r c e n t m a l t a g a r .  I t i s possible that a l -  though the sapwood c o n t a i n e d g r e a t e r number o f microorganisms  capable  o f i n h i b i t i n g the t e s t f u n g i , b e t t e r n u t r i t i o n a l p r o p e r t i e s o f t h e  sap-  wood a f f o r d e d more f a v o u r a b l e c o n d i t i o n s f o r t h e development o f t h e wood destroying fungi. The r e s u l t s o f the c u l t u r a l s t u d i e s on wood a l s o i n d i c a t e d a s i g n i f i c a n t v a r i a t i o n i n t h e r a t e o f i n h i b i t i o n between t h e t h r e e s e c t i o n s o f t h e stems.  These r e s u l t s were s i m i l a r t o t h o s e o b t a i n e d  one p e r c e n t m a l t a g a r .  on  The p o o r e s t growth and l e a s t w e i g h t l o s s e s  o c c u r r e d i n the cubes o f b u t t s e c t i o n s .  S i g n i f i c a n t l y b e t t e r growth and  - 38 g r e a t e r w e i g h t l o s s e s were o b s e r v e d i n cubes o f t h e top s e c t i o n . growth and w e i g h t l o s s e s i n t h e m i d d l e s e c t i o n d i d n o t d i f f e r  The  signifi-  c a n t l y f r o m those i n t h e b u t t s e c t i o n , b u t t h e y were p o s i t i v e l y below those r e c o r d e d i n t h e top s e c t i o n .  These d i f f e r e n c e s i n t h e r a t e o f  growth and w e i g h t l o s s e s between t h e t h r e e s e c t i o n s o f t h e stem seem t o conform w i t h the v a r i a t i o n s t h a t e x i s t i n t h e age, s t r u c t u r e , n u t r i e n t and w a t e r c o n t e n t (Brown e t a l . , I9h9 nisms o f t h e s e c t i o n s .  ), and p o p u l a t i o n s o f m i c r o o r g a -  T h i s s t u d y , however, was n o t d e s i g n e d t o p r o v i d e  e x p e r i m e n t a l e v i d e n c e f o r f u r t h e r e x p l a n a t i o n o f the v a r i a t i o n on t h e basis of tree c h a r a c t e r i s t i c s . E n v i r o n m e n t a l change b r o u g h t about by i n c r e a s i n g the agar c o n c e n t r a t i o n t o f o u r and s i x p e r c e n t , i n m a l t a g a r , and by a drop i n the l e v e l of r e l a t i v e h u m i d i t y t o n i n e t y - s e v e n p e r c e n t i n the j a r s w i t h t h e wood b l o c k s , r e s u l t e d i n a s i g n i f i c a n t change i n the degree o f inhibition. Both F. annosus and P. m o n t i c o l a p r o d u c e d b e t t e r growth on f o u r p e r c e n t t h a n on one p e r c e n t m a l t agar c o n t a i n i n g samples o f a l l solutions.  T h i s e n v i r o n m e n t a l change, however, d i d n o t seem t o a f f e c t  t h e r a t e o f growth i n t h e p l a t e s w i t h t h e v a r i o u s s o l u t i o n s t o t h e same extent.  I t d i d become e v i d e n t when t h e r a n k o f t h e i n h i b i t i n g powers  changed, e.g. P. m o n t i c o l a on one p e r c e n t m a l t a g a r produced t h e b e s t growth i n the p l a t e s w i t h t h e s o l u t i o n f r o m t o p heartwood ( F i g . 6 b ) ; on f o u r p e r c e n t m a l t a g a r the b e s t growth o f t h i s fungus o c c u r r e d i n p l a t e s c o n t a i n i n g t h e s o l u t i o n o f the m i d d l e heartwood ( F i g . 7 b ) .  The  r e a s o n may have been t h a t t h e organisms o f t h e t o p heartwood were more t o l e r a n t t o the change o f environment w h i l e t h e organisms o f the m i d d l e  - 39 heartwood were more s e n s i t i v e t o t h e change, w h i c h r e s u l t e d i n a g r e a t e r l o s s o f i n h i b i t i n g power.  The i n c r e a s e o f a g a r c o n c e n t r a t i o n t o f o u r  p e r c e n t a l s o l e s s e n e d t h e d i f f e r e n c e i n t h e degrees o f i n h i b i t i o n b e t ween t h e s o l u t i o n s from sapwood and heartwood.  A further increase of  t h e a g a r c o n c e n t r a t i o n t o s i x p e r cent l e d t o almost complete l o s s o f inhibition. The i n c r e a s e i n t h e agar c o n c e n t r a t i o n s o f t h e media was b e l i eved t o r e s u l t i n a d r i e r environment  f o r t h e growth o f m i c r o o r g a n i s m s ,  b u t a t t h e same t i m e a n o t h e r v a r i a b l e was i n t r o d u c e d r e p r e s e n t e d b y d i f ferent nutrition.  I t was r e a l i z e d t h a t t h e t e c h n i q u e d i d n o t p e r m i t t h e  s e p a r a t i o n o f t h e l i n k e d v a r i a b l e s , m o i s t u r e c o n t e n t and n u t r i t i o n i n the media.  T h e r e f o r e , t h i s t e c h n i q u e was s u p p o r t e d b y a n o t h e r method  when agar d i s c s o f t h e microorganisms  were p a i r e d w i t h t h o s e o f t h e t e s t  f u n g i and k e p t i n growth chambers a t d i f f e r e n t r e l a t i v e h u m i d i t i e s . The growth o f F. annosus and P. m o n t i c o l a was i n h i b i t e d a t one hundred p e r cent r e l a t i v e humidity.  A decrease i n the r e l a t i v e humidity t o a p p r o x i -  m a t e l y n i n e t y - s e v e n p e r c e n t l e d t o t h e f r e e growth o f t h e t e s t f u n g i , over-growing t h e d i s c s c o n t a i n i n g microorganisms  ( F i g . 10 a , b , c , d ) .  These r e s u l t s appear t o i n d i c a t e t h a t t h e m o i s t u r e c o n t e n t o f t h e env i r o n m e n t was an i m p o r t a n t f a c t o r i n t h e o c c u r r e n c e and degree o f i n hibition. A decrease i n t h e l e v e l o f r e l a t i v e h u m i d i t y t o a p p r o x i m a t e l y n i n e t y seven p e r cent i n t h e j a r s  c o n t a i n i n g u n t r e a t e d wood brought  about a s i g n i f i c a n t l o s s i n t h e degree o f i n h i b i t i o n o f F. annosus and P. m o n t i c o l a .  B o t h t e s t f u n g i produced b e t t e r growth and caused g r e a t e r  w e i g h t l o s s e s i n t h e cubes i n c u b a t e d a t n i n e t y - s e v e n p e r c e n t r e l a t i v e  - ho h u m i d i t y , t h a n i n those i n a s a t u r a t e d atmosphere.  The i n c r e a s e i n t h e  w e i g h t l o s s e s a t t h e l o w e r l e v e l o f r e l a t i v e h u m i d i t y was c o n s i s t e n t i n t h e sapwood and heartwood o f a l l s e c t i o n s .  These r e s u l t s appeared t o  f u r t h e r s u p p o r t t h e h y p o t h e s i s t h a t t h e amount o f a v a i l a b l e m o i s t u r e i n the environment may p l a y an i m p o r t a n t r o l e i n t h e degree o f i n h i b i t i o n . F. annosus and P. m o n t i c o l a were a l s o t e s t e d on media (one, f o u r and s i x p e r c e n t m a l t agar w i t h s t e r i l i z e d and s t e r i l i z e d wood).  sterilized solutions,  A u t o c l a v i n g was used f o r the s t e r i l i z a t i o n o f  the experimental m a t e r i a l .  The t r e a t m e n t l e d t o t h e l o s s o f i n h i b i t i n g  f a c t o r i n both m a l t a g a r media and wood.' On malt a g a r t h e r e was no s i g n i f i c a n t i n h i b i t i o n i n the  growth  o f the t e s t f u n g i when t h e medium c o n t a i n e d t h e a u t o c l a v e d s o l u t i o n s . On the media c o n t a i n i n g t h e a u t o c l a v e d s o l u t i o n s , b o t h F. annosus and P. m o n t i c o l a grew a p p r o x i m a t e l y as w e l l as on t h o s e w i t h o u t s o l u t i o n s , r e g a r d l e s s o f the s o u r c e o f s o l u t i o n and t h e a g a r c o n c e n t r a t i o n o f t h e media.  R a t h e r , s t i m u l a t i o n o c c u r r e d i n s t e a d o f the i n h i b i t i o n i n  some i n s t a n c e s i n t h e growth o f F. annosus e.g. on one p e r c e n t m a l t agar c o n t a i n i n g t h e s o l u t i o n from the m i d d l e heartwood.  The r a t e o f  s t i m u l a t i o n was not s i g n i f i c a n t , and may have been i n d u c e d by the n u t r i t i o n a l p r o p e r t i e s o f the s o l u t i o n s . The p o s s i b l e h a r m f u l . e f f e c t o f t h e h i g h t e m p e r a t u r e o f a u t o c l a v i n g on the p r o p e r t i e s o f t h e s o l u t i o n s was  considered of importance.  C o n s e q u e n t l y the r e s u l t s o f t h e t r e a t m e n t were compared w i t h those obt a i n e d from M i l l i p o r e f i l t r a t i o n , which s e r v e d as a n o t h e r method o f s t e rilization.  The M i l l i p o r e f i l t r a t i o n s e r v e d t h e purpose o f removing  microorganisms from t h e s o l u t i o n s , w i t h o u t s e r i o u s a l t e r a t i o n o f the  - kl -  chemical c o n s t i t u e n t s of the f i l t r a t e .  The M i l l i p o r e f U t e r e s  solution  when added t o one p e r c e n t m a l t a g a r had no i n h i b i t o r y e f f e c t on growth o f the t e s t f u n g i .  Therefore,  i t was  the  b e l i e v e d that the micro-  f l o r a s were r e s p o n s i b l e f o r the i n h i b i t i o n o f the wood-decaying f u n g i . B o t h F. annosus and P. m o n t i c o l a were t e s t e d on  autoclaved  wood and b o t h f u n g i p r o d u c e d s i g n i f i c a n t l y b e t t e r growth and weight l o s s i n autoclaved and  P.  t h a n i n u n t r e a t e d wood cubes.  m o n t i c o l a caused more weight l o s s i n the  greater  B o t h F. annosus  sapwood t h a n i n  the  heartwood, p r o b a b l y because the sapwood p r o v i d e d b e t t e r n u t r i t i o n f o r t h e i r growth. I t was  i n f o r m a t i v e t h a t t h e r e l a t i v e l y d r i e r environments  ( h i g h e r a g a r c o n c e n t r a t i o n o f the m a l t agar media and the wood b l o c k s incubated  i n j a r s at a r e l a t i v e humidity of ninety-seven  per cent) d i d  not seem t o a f f e c t t h e r a t e o f growth o f the t e s t f u n g i on material.  There was  autoclaved  no s i g n i f i c a n t d i f f e r e n c e i n the w e i g h t l o s s e s  between the a u t o c l a v e d  cubes kept a t one hundred p e r cent r e l a t i v e at ninety-seven  per cent r e l a t i v e  hu-  m i d i t y and  those i n c u b a t e d  humidity.  Therefore,  the r e s u l t s suggested t h a t the f a c t o r s r e s p o n s i b l e f o r the  i n h i b i t i o n were t w o f o l d , namely, t h e p r e s e n c e o f m i c r o o r g a n i s m s , and s u f f i c i e n t amount o f m o i s t u r e t o m a i n t a i n t h e i r  activity.  The pathway, time o f e n t r a n c e o f t h e m i c r o o r g a n i s m s i n t o the t r e e , t h e i r i d e n t i f i c a t i o n , the v a r i a t i o n i n t h e i r d e n s i t y and w i t h the s i t e and age  kind  o f the t r e e a r e some q u e s t i o n s w h i c h remain t o  answered by f u t u r e i n v e s t i g a t i o n .  be  - U2 -  CONCLUSIONS  The  r e s u l t s o f t h i s s t u d y p r o v i d e a b a s i s f o r the f o l l o w i n g  conclusions: The sampled was  h e a l t h y , n o r m a l wood o f two  l i v i n g w e s t e r n hemlock t r e e s  c o l o n i z e d by m i c r o o r g a n i s m s , c o n s i s t i n g o f b a c t e r i a , y e a s t s ,  moulds, and members o f i m p e r f e c t f u n g i . , "i  I t was  demonstrated on m a l t a g a r media and wood, t h a t  the  m i c r o o r g a n i s m s had a s i g n i f i c a n t r o l e i n the i n h i b i t i o n o f F. annosus and P.  monticola. The  m o i s t u r e c o n t e n t o f t h e environment seemed t o be an  p o r t a n t f a c t o r i n the degree of i n h i b i t i o n .  im-  D r i e r environments s i g n i -  f i c a n t l y d e c r e a s e d the i n h i b i t i n g power o f the m i c r o o r g a n i s m s . S i g n i f i c a n t v a r i a t i o n was  found i n t h e degrees o f  between the t h r e e s e c t i o n s o f the stems.  Greatest  inhibition  i n the b u t t s e c t i o n s , and t h e l e a s t i n the top s e c t i o n s . media the s o l u t i o n s o f the sapwood samples p r o v i d e d t h a n t h o s e from the heartwood. was  t h i s may sapwood.  have been due The  occurred  On m a l t a g a r  greater  U s i n g wood as s u b s t r a t u m t h e  g r e a t e r i n heartwood t h a n t h a t i n sapwood.  inhibition  inhibition inhibition  I t i s believed that  t o the s u p e r i o r n u t r i t i o n a l p r o p e r t i e s o f  the  d i f f e r e n c e i n the b e h a v i o u r o f t h e t e s t f u n g i on the  s u b s t r a t e s emphasises the importance o f u s i n g n a t u r a l s u b s t r a t e s a d d i t i o n t o a r t i f i c i a l media t o a r r i v e a t more r e l i a b l e A u t o c l a v i n g the experimental of i n h i b i t i n g f a c t o r .  The  two  in  conclusions.  m a t e r i a l r e s u l t e d i n the l o s s  r e l a t i v e l y d r i e r environments d i d not a f f e c t  s i g n i f i c a n t l y the r a t e o f growth o f F'." annosus and P. m o n t i c o l a  on  -  autoclaved material.  U3  -  C o n s e q u e n t l y , i t was  concluded t h a t the occurrence  of i n h i b i t i o n r e q u i r e d t h e p r e s e n c e o f the microorganisms  and  sufficient  moisture to maintain t h e i r a c t i v i t y . Millipore of i n h i b i t i n g power.  f i l t r a t i o n o f t h e shake s o l u t i o n s l e d t o the l o s s T h i s method o f s t e r i l i z a t i o n was  considered to  have l i t t l e e f f e c t on t h e c h e m i c a l c o n s t i t u e n t s o f the s o l u t i o n s , w h i c h p r o v i d e d f u r t h e r evidence i n support of the hypothesis t h a t the microorganisms p l a y e d a p o s i t i v e r o l e i n t h e i n h i b i t i o n o f F. annosus and P. m o n t i c o l a .  - hh BIBLIOGRAPHY  B i e r , J . E. 1959-  The r e l a t i o n o f b a r k m o i s t u r e t o t h e development o f  c a n k e r d i s e a s e s caused b y n a t i v e , f a c u l t a t i v e 1.  parasites.  C r y p t o d i a p o r t h e c a n k e r on w i l l o w . Can. J . Botany.  37:  229-238. 1959.  The r e l a t i o n o f b a r k m o i s t u r e t o t h e development o f  c a n k e r d i s e a s e s caused b y n a t i v e , f a c u l t a t i v e 2.  parasites.  F u s a r i u m canker on b l a c k cottonwood. Can. J . Botany.  37s  781-788. 1959.  The r e l a t i o n o f b a r k m o i s t u r e t o t h e development o f  canker d i s e a s e s caused b y n a t i v e , f a c u l t a t i v e 3.  parasites.  C e p h a l o s p o r i u m c a n k e r on w e s t e r n hemlock. Can. J . Botany.  37: 11U0-11U2. 1961.  The r e l a t i o n o f b a r k m o i s t u r e t o t h e development o f  canker d i s e a s e s caused b y n a t i v e , f a c u l t a t i v e k.  parasites.  Pathogenicity studies of Cryptodiaporthe s a l i c e l l a (Fr.)  P e t r a k , and F u s a r i u m l a t e r i t u m Nees., on P o p u l u s t r i c h o c a r p a . T o r r e y and Gray, P. r o b u s t a , P. t r e m u l o i d e s M i c h x . , and 1  1  S a l i x sp. Can. J . Botany. 1961.  3 9 : 139-lUU-  The r e l a t i o n o f b a r k m o i s t u r e t o t h e development o f  canker d i s e a s e s caused b y n a t i v e , f a c u l t a t i v e 5.  parasites.  R o o t i n g b e h a v i o u r and d i s e a s e v u l n e r a b i l i t y i n c u t t i n g s o f  Populus t r i c h o c a r p a . Can. J . Botany. 1961.  T o r r e y and Gray, and P. ' r o b u s t a . 1  3 9 : LL5-15U.  The r e l a t i o n o f b a r k m o i s t u r e t o t h e development o f  c a n k e r d i s e a s e s caused b y n a t i v e , f a c u l t a t i v e  parasites.  - US 6.  P a t h o g e n i c i t y s t u d i e s o f Hypoxylon p r u i n a t u m ( K l o t z s c h )  Cke., and S e p t o r i a musciva Pk. on s p e c i e s o f A c e r , Populus and S a l i x . 1963-  Can. J . Botany.  3 9 : 1555-1561.  T i s s u e s a p r o p h y t e s and t h e p o s s i b i l i t y o f b i o l o g i c a l  c o n t r o l o f some t r e e d i s e a s e s . and M. H. Rowat, 1962.  F o r . C h r o n i c l e . 39 ( l ) :  81-8U.  The r e l a t i o n o f b a r k m o i s t u r e t o t h e  development o f canker d i s e a s e s caused b y n a t i v e , f a c u l t a t i v e parasites.  7- Some e f f e c t s o f t h e s a p r o p h y t e s on t h e b a r k o f  P o p l a r and W i l l o w on t h e i n c i d e n c e o f Hypoxylon Can. J . Botany.  UO: 6 1 - 6 9 .  and M. H. Rowat, 1963h y t e s on Hypoxylon B o u r c h i e r , R. J . 1961.  canker.  Further e f f e c t s of the bark  canker.  saprop-  9 (3)2 263-270.  For. Science.  L a b o r a t o r y s t u d i e s on m i c r o f u n g i i s o l a t e d from  t h e stems o f l i v i n g l o d g e p o l e p i n e , P i n u s c o n t o r t a Dougl.  Can. J . Botany.  *•  39: 1373-1385-  Brown, H. P., A. J . P a n s h i n and C. C. F o r s a i t h , 19U9wood t e c h n o l o g y .  F i r s t Ed. V o l . 1. M c G r a w - H i l l Book Co.  C a r t w r i g h t , K. S. G. and W. P. K. F i n d l a y , 1958. i t s prevention.  Textbook o f  Decay o f t i m b e r and  Second Ed. H e r M a j e s t y ' s S t a t i o n a r y O f f i c e ,  London. C o w l i n g , E. B. 1961.  Comparative  b i o c h e m i s t r y o f t h e decay o f Sweetgum  sapwood b y w h i t e - r o t and brown-rot f u n g i .  U. S. D. A. Tech.  B u l l . No. 1258. E t h e r i d g e , D. E. 1957-  A method f o r t h e s t u d y o f decay r e s i s t a n c e i n  wood under c o n t r o l l e d m o i s t u r e c o n d i t i o n s . 35:  615-618.  Can. J .  Botany.  - U6 Gundersen, K. i 9 6 0 .  The p h y s i o l o g y o f Fomes annosus.  Paper p r e s e n t e d  a t t h e Conference and S t u d y t o u r on F. annosus, S c o t l a n d , I960.  S e c t i o n 2k.  I n t e r n a t i o n a l Union o f Forest Research  Organizations. Keener, P. D. 1950. M i c r o f l o r a o f buds.  1. R e s u l t s o f c u l t u r e s from  n o n - i r r i t a t e d m a t e r i a l s o f c e r t a i n woody p l a n t s . Botany.  Am. J .  37: 7, 520-527.  Keener, P. D. I y 5 l .  M i c r o f l o r a o f buds.  2. R e s u l t s o f h i s t o l o g i c a l  s t u d i e s o f n o n - i r r i t a t e d buds o f c e r t a i n woody p l a n t s .  Am.  J . Botany. 38: 2, 105-110. L u t h e r , S. 1935.  I n t e r c e l l u l a r humidity i n r e l a t i o n to f i r e - l i g h t  s u s c e p t i b i l i t y i n a p p l e and p e a r . Memoirs, 181-209, pp. 3-39-  C o r n e l l A g r . Exp. S t a . ,  

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