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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 of B r i t i s h Columbia, F a c u l t y of 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 the Department of F o r e s t r y We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1961+. I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y , a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t , c o p y i n g o r p u b l i -c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n s . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r 8, C a n a d a - II -ABSTRACT I t was demonstrated t h a t the sound wood of two stems of l i v i n g western hemlock, Tsuga h e t e r o p h y l l a (Raf.) Sarg., was c o l o n i z e d by micro-organisms , which were i s o l a t e d by shaking samples of the wood i n s t e r i l e d i s t i l l e d water. The m i c r o f l o r a s i n h i b i t e d the growth of Fomes annosus , (Fr. ) Cke., and P o r i a monticola Murr. when added to one per cent malt 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 untreated surface s t e r i l i z e d wood incubated at one hundred per cent r e l a t i v e humidity a t room tem-per a t u r e . S i g n i f i c a n t v a r i a t i o n was observed, i n the r a t e of i n h i b i t i o n between the sapwood and heartwood and between the v a r i o u s s e c t i o n s of 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 of malt agar media, and lower r e l a t i v e humidity 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 decrease i n i n h i b i t i o n r a t e of F. annosus and P. monticola. A u t o c l a v i n g the experimental m a t e r i a l l e d to the l o s s of i n -h i b i t i o n w i t h both malt agar media and wood. Removal of the microorga-nisms from the 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 of growth of the t e s t f u n g i on autoclaved wood and i n malt agar c o n t a i n i n g the autoclaved p o r t i o n of the shake s o l u t i o n s . R e s u l t s of the experiment provide evidence t h a t the micro-f l o r a s i n h a b i t i n g the 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 of F. annosus and. P. monticola.. Furthermore, s u f f i c i e n t moisture l e v e l appeared necessary f o r m a i n t a i n i n g the i n h i b i t i n g power of these micro-organisms. - V I I -ACKNOWLEDGEMENTS I am indebted to Dr. John E. B i e r , P r o f e s s o r of Forest Pathology, f o r h i s encouragement, p r i o r to i n i t i a t i o n o f t h i s work, and h i s quidance, v a l u a b l e suggestions and generous help throughout the course of the study are g r a t e f u l l y acknowledged. I a l s o wish to extend my s i n c e r e thanks to Dr. P. G. Haddock, to Dr. R. W. Wellwood and to Dr. J . W. Wilson 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 are due to Dr. G. H. Smith 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. - I l l -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i TABLE OF TABLES i v TABLE OF ILLUSTRATIONS v.". ACKNOWLEDGEMENTS v i i I . INTRODUCTION 1 I I . MATERIALS AND METHODS 3 S e l e c t i o n of experimental m a t e r i a l s 3 P r e p a r a t i o n of shake s o l u t i o n s 3 P r e p a r a t i o n of malt agar media U Conditions f o r stud y i n g the e f f e c t s of v a r i o u s r e l a t i v e humidity l e v e l s on the r a t e of i n h i b i t i o n . . k Decay t e s t on wood cubes 6 I I I . EXPERIMENTAL RESULTS .• 10 C u l t u r a l studies on malt agar media 10 The e f f e c t of 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 M i l l i p o r e f i l t e r i n g 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 V I . BIBLIOGRAPHY hh - IV -TABLE OF TABLES Table Page 1. O u t l i n e of the experiment c a r r i e d out on three-quarter i n c h cubes of western hemlock sapwood and heartwood . . . . 8 2. A n a l y s i s of variance based on data from c u l t u r a l s t u d i e s on malt 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 of v a r i a n c e based on data from c u l t u r a l s t u d i e s on wood cubes 31 -. v -TABLE OF ILLUSTRATIONS Fig u r e Page 1. Growth chamber f o r s t u d y i n g e f f e c t s of v a r i o u s r e l a t i v e humidity 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 of F. annosus on one per cent malt agar 11 I;.- Growth of P. monticola on one per cent malt agar . . . . 12 5. Photomicrograph showing the microorganisms on one per cent malt agar 13 6. Graph showing the growth data of F. annosus and P. monticola on one per cent malt agar c o n t a i n i n g the v a r i o u s untreated and autoclaved shake s o l u t i o n s ... . . ±k 7. Grapft showing the growth data of F. annosus and P. monticola on f o u r per cent malt agar c o n t a i n i n g tne v a r i o u s u n treated and autoclaved shake s o l u t i o n s . . . . 15 8. Graph showing the growth data of F. annosus and monticola on s i x per cent malt agar c o n t a i n i n g the v a r i o u s untreated and autoclaved shake s o l u t i o n s . . . . 16 y. Growth of F. annosus and P. monticola on s i x per cent malt agar c o n t a i n i n g untreated shake s o l u t i o n s . . . 20 10. M y c e l i a l spread of F. annosus a. and b. At one hundred per cent and a t ninety-seven per cent r e l a t i v e humidity 21 11. M y c e l i a l spread of P. m o n t i c o l a a. and b. At one hundred per cent and at ninety-seven per cent r e l a t i v e humidity. . 22 - VI -Table of I l l u s t r a t i o n s continued Figure Page 12. Growth of F. annosus on one per cent malt agar c o n t a i n i n g no shake s o l u t i o n , untreated s o l u t i o n , and s o l u t i o n s autoclaved and M i l l i p o r e f i l t e r e d . 2 5 13. Growth o f P. monticola on one per cent malt agar c o n t a i n i n g no shake s o l u t i o n , untreated s o l u t i o n , and s o l u t i o n s autoclaved and M i l l i p o r e f i l t e r e d 26 l l i . Graph r e p r e s e n t i n g the growth r a t e of F. annosus on one per cent malt agar c o n t a i n i n g no shake s o l u t i o n , untreated s o l u t i o n , and s o l u t i o n s autoclaved and M i l l i p o r e f i l t e r e d 27 1 5 . Graph r e p r e s e n t i n g the growth r a t e of P. monticola on one per cent malt agar c o n t a i n i n g no shake s o l u t i o n , untreated s o l u t i o n , and s o l u t i o n s autoclaved and M i l l i p o r e f i l t e r e d 28 16. Growth of F. annosus on untreated, surface s t e r i l i z e d and autoclaved wood cubes 32 17- Growth of P. monticola on untreated, surface s t e r i l i z e d and autoclaved wood cubes 32 18. Growth of microorganisms on untreated, surface s t e r i l i z e d wood 33 19. Growth of F. annosus on sapwood and heartwood cubes 3h 20. Growth o f P. monticola on sapwood and heartwood cubes 3h - 1 -INTRODUCTION The importance of decay problems i n f o r e s t r y p r a c t i c e i s r e -cognized both 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 . A c c o r d i n g l y a great d e a l of work has been done i n these 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 the e f f e c t s of v a r i o u s wood decaying f u n g i on d i f f e r e n t h o s t s , s u b s t r a t a and host-organism r e l a t i o n s h i p s i n successive stages of decay. I t would appear t h a t most decay s t u d i e s undertaken 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 considered 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 , chemical composition and other pro-p e r t i e s . The development of a concept i n recent years ( B i e r 1958, l9i?9, i 9 6 0 , l°6l) has l e d t o the establishment of a r e l a t i o n s h i p between host r e s i s t a n c e and i t s water content expressed a r e l a t i v e t u r g i d i t y . Resistance was p o s i t i v e l y c o r r e l a t e d w i t h high r e l a t i v e t u r g i d i t y . I t was suggested, furthermore, t h a t h e a l t h y , l i v i n g host t i s s u e s may not be e n t i t i e s but b i o l o g i c a l communities comprised of host t i s s u e s and a saprophytic m i c r o f l o r a . I f t h i s i s t r u e , these microorganisms may have a s i g n i f i c a n t r o l e i n the degree of host r e -s i s t a n c e by i n h i b i t i n g the development of pathogens under c e r t a i n circumstances. The establishment of host r e s i s t a n c e - high 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 the p o s s i b l e r o l e of a m i c r o f l o r a r e -s u l t e d from work undertaken on a v a r i e t y of t r e e species i n connection w i t h canker diseases 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 1961, 1962). I t was of 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 the case of other types of pathogens, e.g., wood - 2 -decaying f u n g i . This study was undertaken to provide evidence i n support of t h i s hypothesis and p o s s i b l y e s t a b l i s h the d i s t r i b u t i o n of 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 the develop-ment of wood decaying f u n g i . Tsuga h e t e r o p h y l l a (Raf.) Sarg. was chosen as host t r e e , s i n c e t h i s s p e c i e s i s of major economic importance and was not 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 begin-ning of t h i s study. I t was considered of i n t e r e s t to use a represen-t a t i v e of both.types of decay organisms, namely a white r o t and a brown r o t fungus. Fomes annosus ( F r . ) Cke. and P o r i a monticola Murr. were chosen f o r t h i s purpose r e s p e c t i v e l y . Both f u n g i are pathogens of 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 which l e a d to t h e i r s e l e c t i o n f o r l a b o r a t o r y s t u d i e s . - 3 -MATERIALS AND METHODS The c u l t u r e s of F. annosus and P. monticola were s u p p l i e d by Dr. John E. B i e r and designated as No. 13 and No. 12UA r e s p e c t i v e l y . A vigorously-growing hemlock tre e e i g h t y years of age was f e l l e d i n the U n i v e r s i t y Campus Forest 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 the base of t r e e was termed the " b u t t s e c t i o n " ; the second was discar d e d ; the t h i r d was named the "middle s e c t i o n " ; the f o u r t h d i s c a r d e d , and the f i f t h was l a b e l l e d as the "top s e c t i o n " . This terminology - b u t t middle and top s e c t i o n - w i l l be used i n the t e x t . The three s e c t i o n s were taken immediately to the Vancouver Laboratory, Forest Products Research Branch, Fe d e r a l Department of F o r e s t -r y , where a one i n c h t h i c k board i n c l u d i n g the p i t h was sawn from each s e c t i o n . The boards were edged, trimmed and planed to three-quarter i n c h t h i c k n e s s . The lower f o u r - f o o t p o r t i o n of each board was used f o r sampling. Two three-quarter i n c h wide beams were cut from both edges of the f o u r - f o o t boards and were considered to represent sapwood. The next one-inch wide s t r i p of the edges was discard e d . The remainder of the boards was regarded as heartwood. Three-quarter i n c h cubes were cut from the sapwood and heartwood beams of the three 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 both sapwood and heartwood from each of the three s e c t i o n s . Every cube was surface s t r i l i z e d by fl 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 shaking f l a s k c o n t a i n i n g 75cc of s t e r i l e , d i s t i l l e d water. The f l a s k s were shaken con t i n u o u s l y on a low - h -speed shaker f o r f i v e days at room temperature. The f i n a l s o l u t i o n s obtained were b e l i e v e d t o contain a sample of the p o p u l a t i o n of micro-organisms present i n the 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 the p r e p a r a t i o n of the c u l t u r e media. One, f o u r and s i x per cent of agar i n three per cent malt e x t r a c t were prepared; - the shake s o l u t i o n represented t e n per cent of the water r e q u i r e d t o prepare the malt agar. 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 to the malt agar a f t e r i t had cooled down con-s i d e r a b l y (approximately U 0 - U 5°C), i n order to prevent the harmful e f f e c t of high temperature on the microorganisms. The s o l u t i o n obtained from each cube was used s e p a r a t e l y to prepare one, f o u r and s i x per cent malt agar, and f o u r P e t r i p l a t e s were poured from each concent-r a t i o n , two of which were i n o c u l a t e d w i t h F. annosus and the other two p l a t e s w i t h P. monticola. C o n t r o l p l a t e s were prepared f o r each con-c e n t r a t i o n of agar 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 incubated f o r a p e r i o d of two weeks at room temperature. M y c e l i a l growth was measured on each p l a t e on the 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 inocu-l a t i o n . Two diameter measurements a t r i g h t angle t o each other were taken on each p l a t e and the a r i t h m e t i c mean of these two measurements was recorded. At the end of the 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 the strongest i n h i b i t i o n to the wood de 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 diameter, were cut w i t h a s t e r i l i z e d cork borer from the malt agar medium where the growth of the s o l u t i o n microorganisms was evident. Each of 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 microscope s l i d e , i n a growth chamber, a d j a -cent to a di s c of the same s i z e cut from a pure c u l t u r e of P. annosus ( F i g . l ) . The distance between the 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 of 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 glass rods f o r the support of the microscope s l i d e . Twenty m i l l i l i t e r s of s t e r i l e d i s t i l l e d water was added to ten of the p l a t e s , so th a t the r e -l a t i v e humidity w i t h i n the p l a t e s had reached the one hundred per cent p r i o r to the i n t r o d u c t i o n of malt agar d i s c s . The s l i d e s w i t h the d i s c s always remained above water l e v e l . The other ten growth chambers con-t a i n e d 1.5 weight molar sucrose s o l u t i o n i n order to decrease the r e l a -t i v e humidity to a l e v e l of approximately ninety-seven per cent (Luther 1935). S i m i l a r s e r i e s of growth chambers were t e s t e d w i t h d i s c s con-Fi g u r e 1. Growth chamber f o r studying e f f e c t s of v a r i o u s r e l a t i v e humidity l e v e l s on m y c e l i a l spread. - 6 -t a i n i n g mycelium of P. monticola. The growth chambers were incubated at room temperature f o r twenty-one days during which the m y c e l i a l growth of the wood de s t r o y i n g f u n g i was observed. Another f o u r b l o c k s from both sapwood and heartwood of each o f the three s e c t i o n s were chosen randomly. Shake s o l u t i o n s were prepared. One, fo u r and s i x per cent malt agar c o n t a i n i n g t e n per cent s o l u t i o n were prepared u s i n g the same techinque. However, the s o l u t i o n s p r i o r to the a d d i t i o n t o the malt agar were autoclaved f o r twenty-five minu-tes a t f i f t e e n pounds gauge pressure. P e t r i p l a t e s were poured f o r each pathogen as w i t h the untreated, 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 incubated 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 the 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 -teenth day a f t e r i n o c u l a t i o n . Besides u s i n g v a r i o u s concentrations of malt agar, i t was f e l t t h a t i t would be of importance to demonstrate the i n h i b i t o r y e f f e c t of the saprophytes towards the wood decaying f u n g i on wood. F. annosus and P. monticola were t e s t e d on three-quarter i n c h wood cubes i n growth chambers which c o n s i s t e d of seventeen ounce mas-sage j a r s w i t h screw-on l i d s . A s h e l l v i a l of 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 at the center of each j a r and kept i n v e r t i c a l p o s i t i o n by a wire support ( F i g . 2 ) . Chrome-nickel r e s i s t a n c e wire was used f o r the reason of preventing any r u s t formation i n the j a r s which might i n t e r f e r e w i t h the growth of f u n g i . The growth chambers were autoclaved. The wood blocks were weighed to the nearest hundredth gram and seated i n d i v i d u a l l y on the top of the v i a l s i n the j a r s . - 7 -Figure 2 . Growth chamber for 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 prior to the inoculation as indicated i n Table 1. One set of four blocks from both sapwood and heartwood of the three sections for both pathogens was not subjected to any special treat-ment, except to surface s t e r i l i z a t i o n by flaming in order to exclude con-tamination from the atmosphere. The second set of four blocks from both sapwood and heartwood were autoclaved at fifteen pounds gauge pressure for forty minutes. Within each set of blocks a series of two blocks were kept at one hundred per cent relative 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 in the jar in order to keep i t s atmosp-here saturated. The l i d of the jar was not tight so as to provide sufficient aeration. In order to secure the one hundred per cent rela-- 8 -t i v e humidity the j a r s were placed on a s h e l f i n a t r a y w i t h tap water i n i t , and covered w i t h a transparent p l a s t i c cover. S u f f i c i e n t water supply was maintained i n the t r a y during the three-month i n c u b a t i o n pe-r i o d . The other s e r i e s i n each set was kept a t ninety-seven per cent r e l a t i v e humidity. This humidity was set by usin g 1.5 weight molar suc-rose s o l u t i o n . One hundred m i l l i l i t e r s sucrose s o l u t i o n was i n the growth chamber. 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 i n diameter cut from pure c u l t u r e s of the decay f u n g i and was incubated at room temperature f o r a p e r i o d of three months. Table 1. O u t l i n e of the experiment c a r r i e d out on three-quarter i n c h cubes of western hemlock sapwood and heartwood. Wood Sapwood Heartwood Treatment N a t u r a l Autoclaved N a t u r a l Autoclaved Test fungus Fomes P o r i a Fomes P o r i a Fomes P o r i a Fomes P o r i a R. humid. % 100 97 100 97 100 91 100 91 100 97 100 97 100 97 100 97 B u t t 1 cube sec'n. 2 Middle 1 cube sec'n. 2 Top 1 cube sec'n. 2 Simultaneously w i t h the drawing of the b l o c k s used i n the ex-periment, f o u r b l o c k s from both sapwood and heartwood of the three sec-- 9 -t i o n s were chosen randomly f o r the purpose of moisture content determi-n a t i o n . The b l o c k s were weighed when f r e s h , ovendried a t 105 ° C f o r 2J4. hours, reweighed and the moisture content was c a l c u l a t e d on ovendry b a s i s . The average of moisture content of f o u r b l o c k s of both sapwood and heartwood o f the three l e v e l s were considered as an approximation to t h a t o f the bloc k s used i n the experiment. On the b a s i s of t h i s mois-t u r e content and the f r e s h weight of the bloc k s used i n the experiment t h e i r ovendry weight was c a l c u l a t e d and termed as estimated ovendry weight. At the end of three months the s u p e r f i c i a l m y c e l i a l growth was removed from the surface of the blocks by a s o f t tooth-brush, e x e r c i z i n g care not to remove any wood p a r t i c l e s . The blocks were weighed, then ovendried a t 105° C and reweighed. Moisture content of the b l o c k s a t the end of the three-month p e r i o d and weight l o s s , i f any, were c a l c u -l a t e d . Both experiments ( t e s t on malt agar - and t e s t on wood) were repeated u s i n g the same technique, on m a t e r i a l from another v i g o r o u s l y growing h e a l t h y hemlock t r e e of about the same age as used i n the f i r s t experiments. S t a t i s t i c a l analyses were c a r r i e d out on the data from e x p e r i -ments on both t r e e s and the r e s u l t s are presented h e r e i n . Although the f i n a l growth measurements were made on the f o u r -teenth day a f t e r i n o c u l a t i o n , the s t a t i s t i c a l analyses are based on the data recorded on the eighth day, si 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 the diameter of the 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 malt agar media. Large numbers of v a r i o u s organisms were obtained from both the sapwood and heartwood of each s e c t i o n of the health y western hemlock stem. Every p l a t e c o n t a i n i n g untreated shake s o l u t i o n s showed evidence o f a micro-f l o r a being present i n the t i s s u e s . The organisms i n the m a j o r i t y of the instances formed s m a l l c o l o n i e s on the malt agar medium, and seemed to i n h i b i t the growth of F. annosus and P. monticola ( F i g . 3a, b, and Ua, b ) . Although no attempt was made to i d e n t i f y the organisms, i t was noted t h a t the p o p u l a t i o n c o n s i s t e d of b a c t e r i a , y e a s t s , moulds and im-p e r f e c t f u n g i . In a few cases the organisms produced no macroscopic, a e r i a l growth but microscopic examination of the media revealed t h e i r presence ( F i g . 5). The summarized experimental data are gi 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 study were not t r e a t e d i n the a n a l y s i s as tr u e r e p l i c a t e s (Table 2) f o r the reason t h a t s i g n i f i c a n t t r e e to t r e e v a r i a t i o n was expected. Variance was c a l c u l a t e d f o r the t r e e s and r e -moved from the experimental e r r o r thus making the a n a l y s i s more s e n s i t i v e . When a wet environment was provided - one per cent malt agar - the growth of both F. annosus and P. monticola was s i g n i f i c a n t l y i n h i -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 , untreated shake s o l u t i o n s . The t e s t 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 than on those w i t h s o l u t i o n s from 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 the three s e c t i o n s of the stem. The strongest i n h i b i -t i o n was exerted by the s o l u t i o n from sapwood of the b u t t s e c t i o n , f o l -lowed i n decreasing order by those from the top and middle 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. Note lack of mycelial growth from inoculum plug at center. Figure k b. Poria monticola on one per cent malt agar containing shake solution. Note lack of mycelial growth from inoculum plug at center. - 13 -Figure 5- Photomicrograph showing the microorganisms i n one per cent malt agar medium (approximately 600 x). Figure 6 a. Upper l e f t . Growth of F. annosus on one per cent malt agar c o n t a i n i n g the v a r i o u s untreated shake s o l u t i o n s . F i g ure 6 b. Upper r i g h t . Growth of P. monticola on one per cent malt agar c o n t a i n i n g the v a r i o u s untreated shake s o l u t i o n s . Figure 6 c. Lower l e f t . Growth of F. annosus on one per cent malt agar c o n t a i n i n g the v a r i o u s autoclaved s o l u t i o n s . F i g u r e 6 d. Lower r i g h t . Growth of P. monticola on one per cent malt agar c o n t a i n i n g the v a r i o u s autoclaved s o l u t i o n s . Legend: B l a c k s o l i d l i n e - no shake s o l u t i o n added ( c o n t r o l ) 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 - middle 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 - middle 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 - top 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 represents the average of e i g h t experimental values w i t h each of the two t r e e s . Figure 7 a. Upper l e f t . Growth of F. annosus on f o u r per cent malt agar c o n t a i n i n g the v a r i o u s untreated 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 of P. monticola on f o u r per cent malt agar c o n t a i n i n g the v a r i o u s untreated shake s o l u t i o n s . F i g ure 7 c. Lower l e f t . Growth of F. annosus on f o u r per cent malt agar c o n t a i n i n g the v a r i o u s autoclaved s o l u t i o n s . F i g ure 7 d. Lower r i g h t . Growth of P. monticola on f o u r per cent malt agar c o n t a i n i n g the v a r i o u s autoclaved 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 ( c o n t r o l ) 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 - middle 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 - middle 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 - top s e c t i o n , heartwood s o l u t i o n Each p l o t t e d value represents the average of e i g h t experimental values w i t h each of the two t r e e s . -15 -TIMe - DAV^ F i g u r e 8 a. Upper l e f t . Growth of F. annosus on s i x per cent malt agar c o n t a i n i n g the v a r i o u s untreated shake s o l u t i o n s . Figure 8 b. Upper r i g h t . Growth of P. monticola on s i x per cent malt agar c o n t a i n i n g the v a r i o u s untreated shake s o l u t i o n s . F i g u r e 8 c. Lower l e f t . Growth of F. annosus on s i x per cent malt agar c o n t a i n i n g the v a r i o u s autoclaved s o l u t i o n s . F i g u r e 8 d. Lower r i g h t . Growth of P. monticola on s i x per cent malt agar c o n t a i n i n g the v a r i o u s autoclaved s o l u t i o n s . Legend: B l a c k s o l i d l i n e - no shake s o l u t i o n added ( c o n t r o l ) 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 - middle 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 - middle 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 - top s e c t i o n , heartwood s o l u t i o n Each p l o t t e d value represents the average of e i g h t experimental values w i t h each of the two t r e e s . 16 -- 17 -Table 2. A n a l y s i s of v a r i a n c e based on data recorded i n F i g u r e s 6, 7 and 8. Source of v a r i a t i o n DF. Sum square Mean square F. A Tree 1 367-3600 367.3600 15-10 ** B Sapw.-Heartw. 1 625.0000 625.0000 25.70 ** C Nat.-Autocl. 1 12731-3000 12731.3000 523.60 ** D Fomes-Poria 1 13963-3000 13963.3000 57k.27 E Agar % l-k-6 2 202k.5U00 1012.2700 la . 63 F S e c t i o n B-M-T. 2 150.U900 75.2U50 3-09 AB 1 132.2300 132.2300 5.k3 * AC 1 21.7700 21.7700 0.89 AD 1 5.U|00 5.UU00 0.22 AE 2 101.0000 50.5000 2.07 AF 2 193.5U00 96.7700 3.97 BC 1 616.6900 616.6900 25-36 ** BD 1 k.6900 k.6900 0.19 BE 2 266.6200 133.3100 5.k8 ** BF 2 90.U900 U5.2U50 1.86 CD 1 62k.9900 62k-9900 25.70 CE 2 5676.5000 2838.2500 116.72--:-** CF 2 713.5500 356.7750 lk.67 #* DE 2 58.5800 29-2900 1.20 DF 2 155.0500 77.5250 ' 3.18 * EF k 228.8300 57.2075 2.35 ABC 1 289.0200 289.0200 11.88 J-Hf ABD 1 75.1200 75.1200 3.08 ABE 2 , 67.5600 33-7800 1.38 ABF 2 11*3.2000 71.6000 2.9k ACD 1 - 26.6900 26.6900 I.09 ACE 2 52.1100 26.0550 1.07 ACF 2 215.L100 107.7050 k.k2 * ADE 2 67.3700 33-6850 1.38 ADF 2 11.0800 5.5kOO 0.22 AEF k 161.9600 k0.k900 1.66 BCD 1 93.kkOO 93.kkOO 3.8k BCE 2 153.1000 76.5500 3.1k BCF 2 132.0600 66.0300 2.71 BDE 2 8.5200 k-2600 0.17 BDF 2 26.0700 13.0350 0.53 BEF k 32.5000 8.1250 0.33 CDE 2 832.6500 kl6.3250 17.12 ** CDF 2 51.5200 25.7600 1.05 CEF k 93.U600 23.3650 0.96 DEF k 163.6200 kO.9050 1.68 ERROR 65 1580.L600 2k-3lU7 TOTAL Ik3 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 . - 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 from heartwood, i n decreasing order were as f o l l o w s : b u t t , middle and top s e c t i o n s , f o r both F. annosus and P. monticola. The best growth ( l e a s t i n h i b i t i o n ) of the two wood decay f u n g i occurred on p l a t e s with, s o l u t i o n s from the heart-wood of the top s e c t i o n , but 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 than the 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 per cent malt agar without the 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 of Duncan's new m u l t i p l e - r a n g e t e s t to these data showed t h a t an increase of the agar c o n c e n t r a t i o n to f o u r per cent ( d r i e r environment) brought about a h i g h l y s i g n i f i c a n t change i n the r a t e of growth of 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 decreased w i t h the i n -crease i n agar c o n c e n t r a t i o n . Both F. annosus and P. monticola produced b e t t e r m y c e l i a l growth on f o u r per cent malt agar than on one per cent malt agar r e g a r d l e s s of the source of shake s o l u t i o n s (sapwood or heart-wood, b u t t middle or top s e c t i o n ) used i n p r e p a r i n g the media ( F i g . 7 a, b ) . The i n h i b i t i o n of 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 shake s o l u t i o n s was found to be as f o l l o w s i n decreasing order: b u t t sapwood, top sapwood, b u t t heartwood, middle heartwood and sapwood, and top heart-wood ( F i g . 7 a ) . P. monticola produced the poorest growth i n p l a t e s con-t a i n i n g the s o l u t i o n from the b u t t sapwoodj f o l l o w e d by top sapwood, b u t t heartwood, top heartwood, middle sapwood and heartwood ( F i g . 7 b ) . Fur-thermore, the d i s t i n c t l y d i f f e r e n t degrees of i n h i b i t i o n between the so-l u t i o n s from sapwood and heartwood on one per cent malt agar, lessened when the s o l u t i o n s were added t o a d r i e r substratum ( f o u r per cent malt agar). F u r t h e r increase 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 the growth r a t e s . Both F. annosus and P. monticola seemed to 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 , producing c o l o n i e s of almost the same s i z e as those 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 ) . Although growth of the microorganisms occurred i n the dry medium, the i n h i b i t i o n f a c t o r was s i g -n i f i c a n t l y reduced. Both F. annosus and P. monticola has overgrown the s m a l l c o l o n i e s of the saprophytes on the dry medium - s i x per cent malt agar ( F i g . 9 a, b ) . The increase of the agar content of the media from one to f o u r and s i x per cent was considered to provide a d r i e r environment f o r the growth of t e s t f u n g i . However, i t was not p o s s i b l e to conclude t h a t the decrease i n the amount of a v a i l a b l e water was the 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 is r e l a t i o n s h i p was demonstrated i n another experiment when malt agar d i s c s of f i v e m i l l i m e t e r s i n diameter, c o l o n i z e d by the sap-rophytes, were placed i n growth chambers adjacent to a d i s c of the same s i z e c o n t a i n i n g mycelium of the decay f u n g i . The growth of both F. annosus and P. monticola was i n h i b i t e d a t one hundred per cent r e l a t i v e humidity. On the other hand, a decrease of r e l a t i v e humidity i n the growth chambers to approximately ninety-seven per cent r e s u l t e d i n f r e e growth of the mycelium of the t e s t f u n g i which covered the d i s c s of sap-rophytes ( F i g . 10 a, b, 11 a, b ) . I n another p a r t of the experiment the 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 to t h e i r being added to the malt agar. This treatment l e d to the l o s s of i n h i b i t i o n . - 20 -Figure 9 b. P. monticola on six per cent malt agar containing untreated shake solution. Figure 10 b. M y c e l i a l spread of F. annosus ( l e f t d i s c ) a t ninety-seven per cent r e l a t i v e humidity. Figure 11 b. M y c e l i a l spread of P. monticola ( l e f t d i s c ) at ninety-seven per cent r e l a t i v e humidity. - 23 -On media c o n t a i n i n g the autoclaved s o l u t i o n s , both F. annosus and P. monticola grew approximately as w e l l as on those without s o l u t i o n s , r e g a r d l e s s of the source o f s o l u t i o n s and the agar c o n c e n t r a t i o n o f the media ( F i g . 6 c, d, 7 c, d, 8 c, d ) . S t i m u l a t i o n occurred i n s t e a d of i n h i b i t i o n i n some cases. F. annosus produced b e t t e r growth than the cont-r o l i n the f o l l o w i n g i n s t a n c e s ; on wet medium - one per cent malt agar -c o n t a i n i n g the s o l u t i o n of b u t t and middle heartwood ( F i g . 6 c ) ; on d r i e r medium - f o u r per cent malt agar - w i t h the s o l u t i o n from the middle heartwood and top sapwood and heartwood ( F i g . 7 c ) ; on d r i e s t medium -s i x per cent malt agar - c o n t a i n i n g the s o l u t i o n obtained from b u t t sap-wood and heartwood ( F i g . 8 c ) . The a d d i t i o n o f the autoclaved s o l u t i o n s provided no s t i m u l a t i o n i n the growth of P. monticola. A u t o c l a v i n g i s a common technique 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 the hig h temperature (250 °F) reached during the process may have a l -tered the chemical composition o f the 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 the growth of the decay f u n g i . Therefore, i t was of prime importance to express the 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 the organisms which had shown the st r o n g e s t i n h i b i t i o n were added to each other and d i v i d e d i n t o three equal p o r t i o n s . One p a r t had no f u r t h e r treatment; the second was f i l -t e r e d through M i l l i p o r e of type HA, 0.U5 micron pore s i z e ; the t h i r d por-t i o n was autoclaved a t f i f t e e n pounds gauge pressure f o r twe n t y - f i v e mi-nutes. Each p o r t i o n of the s o l u t i o n was then used i n one per cent malt agar c u l t u r e media as before and t e s t e d w i t h the decay f u n g i . M y c e l i a l growth was recorded on the 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 -2k-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 . The growth of both F. annosus and P. monticola was s i g n i f i c a n t -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 the untreated, n a t u r a l shake s o l u t i o n . However, the growth of the decay f u n g i i n p l a t e s w i t h autoclaved 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 almost i d e n t i -c a l w i t h the growth produced i n the c o n t r o l p l a t e s having no s o l u t i o n ( F i g . 12 a, b, c, d, and 13 a, b, c, d ) . Both treatments-of s t e r i l i z a -t i o n , a u t o c l a v i n g 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 of the i n h i -b i t i n g f a c t o r ( F i g . lU and 15). Figure 12. F. annosus on one per cent malt agar, 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 ) v b. c o n t a i n i n g untreated s o l u t i o n ; note l a c k of m y c e l i a l growth from inoculum p l u g at center c. c o n t a i n i n g autoclaved s o l u t i o n 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. monticola on one per cent malt agar, 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 untreated s o l u t i o n ; note l a c k of m y c e l i a l growth from inoculum p l u g at c e n t e r . c. c o n t a i n i n g autoclaved s o l u t i o n 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 Figure 13 c. Figure 13 d. F i g u r e I k . Graph re p r e s e n t i n g the growth r a t e of F. annosus on one per cent malt agar c o n t a i n i n g no shake s o l u t i o n , untreated s o l u t i o n , and s o l u t i o n s autoclaved and M i l l i p o r e f i l t e r e d . Legend: S o l i d l i n e Dash - two dots l i n e Dash l i n e Dotted l i n e no shake s o l u t i o n added ( c o n t r o l ) u ntreated s o l u t i o n autoclaved s o l u t i o n 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 1$. Graph r e p r e s e n t i n g the growth r a t e of P. monticola on one per cent malt agar c o n t a i n i n g no shake s o l u t i o n , untreated s o l u t i o n , and s o l u t i o n s autoclaved and M i l l i p o r e f i l t e r e d . Legend: S o l i d l i n e Dash - two dots l i n e Dash l i n e Dotted l i n e no shake s o l u t i o n added ( c o n t r o l ) untreated s o l u t i o n autoclaved s o l u t i o n M i l l i p o r e f i l t e r e d s o l u t i o n - 28 -I x o CH o 40 -30 I 20 L 10 U - i 1 I 4 • 6 • 8 1 TIME - DAYS F i g u r e 1$. - 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 of F. annosus and P. monticola was s t u d i e s on three -quarter i n c h wood cubes incubated i n growth chambers. The growth of the decay f u n g i i s expressed as a percentage o f l o s s i n weight of the cubes during the i n c u b a t i o n p e r i o d of three months. The percentage l o s s i n weight was c a l c u l a t e d as a d i f f e r e n c e between the i n i t i a l es-timated ovendry weight and the f i n a l ovendry weight over the f i n a l oven-dry weight. ( F i n a l ovendry weight = ovendry weight of cube a t the end of i n c u b a t i o n p e r i o d ) . The r e s u l t s are summarized i n Table 3- A n a l y s i s of v a r i a n c e was made on the data and t h i s i s given i n Table k. The growth of F. annosus and P. monticola 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 , untreated w-ood ( F i g . 16 and 17). I t was p o s s i b l e to d e t e c t the microorganisms on n a t u r a l , untreated cubes. I n many i n -stances they produced v i s i b l e growth on the surface of the cubes i n v a d -i n g the inoculum p l u g ( F i g . 18 a, b ) . The weight l o s s o f untreated sapwood and heartwood cubes of three s e c t i o n s of the 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 than t h a t of autoclaved 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 weight l o s s - on sapwood cubes than on cubes of heartwood ( F i g . 19 and 20). 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 of the s t u d i e s on malt agar media. The' treatment of two l e v e l s of r e l a t i v e humidity y i e l d e d s i g -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. monticola on untreated wood. The t e s t f u n g i caused more weight l o s s i n b l o c k s a t ninety-seven per cent r e l a t i v e humidity than i n bloc k s kept a t one hundred per cent r e l a t i v e humidity. The d r i e r environment seemed to reduce c o n s i s t e n t l y the i n h i b i t i n g f a c t o r i n un t r e a t e d , n a t u r a l sapwood and heartwood. Table 3-Per cent weight losses o f three-quarter i n c h sapwood and heartwood cubes o f western hemlock, caused by F. annosus and P. monticola. Underlined f i g u r e s represent mean of v a l u e s recorded on m a t e r i a l s from the f i r s t and second t r e e s . Wood Sapwood Heartwood Treatment N a t u r a l Autoclaved N a t u r a l Autoclaved Test fungus Fomes P o r i a Fomes P o r i a Fomes P o r i a Fomes P o r i a R. humid. % 100 97 100 97 100 97 100 97 100 97 100 97' 100 97 100 97 B u t t 1 O.li* 1.3 13-2 10.8 6.1 5.5 23.3 16.5 0.3 1.5 15.3 1U.8 3.8 h.k 20.6 I i i . 2 t r e e 0.8 3.3 12.0 13.8 6.1 6.1 26.ii 26.3 0.8 1.5 11.9 12.ii 5.8 6.0 18.3 21.3 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 27.5 Middle 1 o.U 1.8 Iii.5 1U.8 8.8 8.6 27.7 23.ii 0.6 1.5 13.1 17.6 6.5 6.6 19.5 2U.5" t r e e 1.0 1.6 1U.9 15-6 8.9 8.3 23.5.21.7 0.8 1.7 1U.1 15-6 7.3 6.2 18.6 20.9 sec'n. 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 17.i; Top 1 0.8 5.3 16.U 18.7 y.U 9.6 36.0 U2.9 0.8 1.6 15.5 1U.8 6.U 6.3 25.5 27.3 t r e e 0.8 3.0 18.9 20.J4 7.9 8.0 39-5 Ul.7 1.1 1.7 16.9 1U.6 7.8 7.3 25-3 25-7 sec'n. 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 * Every f i g u r e i n each t r e e represents the mean of two experimental v a l u e s . - 31 -Table 1|. A n a l y s i s of va r i a n c e based on data shown i n Table 3. Source of v a r i a t i o n > DF. Sum square Mean square F>. A Tree 1 11.2620 11.2620 3.05 B Sapw.-Heartw. 1 98.1720 98.1720 26.59 ** C Nat.-Autocl. 1 1578.8500 1578.8500 1+27.72 ** D. Fomes-Poria 1 5757.9000 5757.9000 1559.85 ** E R.H.Jg 100-97 1 19.2320 19.2320 5.21 * F S e c t i o n B-M-T. 2 185-5820 92.7910 25.13 ** AB 1 0.8780 0.8780 0.23 AC 1 0.2580 0.2580 0.06 AD 1 0.8U60 0.81+60 0.22 AE 1 0.0000 0.0000 0.00 AF 2 1+0.3710 20.1855 5.1+6 ** BC 1 27.8UUO 27.81+1+0 7.51+ ** BD 1 36.52yo 36.5290 9.89 ** BE 1 0.7950 0.7950 0.21 BF 2 20.1+230 10.2115 2.76 CD 1 11.2180 11.2180 3.03 CE 1 15-7120 15.7120 U.25 * CF 2 30.2700 15.1350 1+.10 * DE 1 3.0170 3.0170 0.81 DF 2 102.1+120 51.2060 13.87 ** EF 2 2.3160 1.1580 6.31 ABC 1 5.1U50 5.11+50 1.39 ABD 1 36.0850 36.0850 9.77 ** ABE 1 1.1*710 1.1*710 0.39 ABF 2 20.0090 10.001+5 2.71 ACD 1 0.7680 0.7680 0.20 • ACE 1 lu 71+90 1+. 71+90 1.28 ACF 2 35.71+90 17.871+5 1+.81+ * ADE 1 11.7660 11.7660 3.18 ADF 2 19-8700 9.9350 2.69 AEF 2 51.2820 25.61+10 6.91+ **• BCD 1 20.5610 20.5610 5.57 * BCE. 1 2+.7000 1;.7000 1.27 BCF 2 3.5030 1.7515 0.1+7 BDE 1 1.9250 1.9250 0.52 BDF 2 33-01+80 16.521+0 U.l+7 * BEF 2 7.8770 3.9385 1.06 CDE 1 1U.8610 11+.8610 U.02 CDF 2 U7.6U20 • 23.8210 6.U5 ** CEF 2 1.3150 0.6575 0.17 DEF 2 1.8560 0.9280 0.25 ERROR 38 11+0.2700 3.6931 TOTAL 95 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 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 . - 32 -Figure 16. Growth of F. annosus on untreated ( l e f t ) and autoclaved ( r i g h t ) cubes. Figure 17. Growth of P. monticola on untreated ( l e f t ) and autoclaved ( r i g h t ) cubes. - 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 (left) 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 humidity, however, d i d not e f f e c t the growth of the t e s t f u n g i on autoclaved wood. Both F. annosus and P. monticola caused approximately the same l o s s i n weight of autoclaved cubes at one hundred per cent r e l a t i v e humidity as a t ninety-seven per cent r e l a t i v e 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 to the data 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 the best growth - most weight l o s s - on cubes from the top s e c t i o n of the t r e e . Poorest growth occurred on cubes from the b u t t s e c t i o n , but there was no s i g n i f i c a n t d i f f e r e n c e between the weight l o s s e s of cubes from the b u t t s e c t i o n and those of the cubes of the middle s e c t i o n . The growth of 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 e t t e r at 0.01 p r o b a b i l i t y l e v e l i n a l l instances than t h a t produced by F. annosus. This i s i n accordance w i t h the expected s i n c e the former i s known as a fungus of v e r y r a p i d growth (Cartwright et a l . , 1958). DISCUSSION In t h i s study samples were taken from sapwood and heartwood of the b u t t , middle and top s e c t i o n s of the stems of two h e a l t h y t r e e s of western hemlock. S o l u t i o n s were prepared by shaking three-quarter i n c h , surface 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 cubic centimetres of s t e r i l e d i s t i l l e d water. This technique was chosen to recover micro-organisms i n wood, si n c e i t was considered to provide b e t t e r coverage of the sample m a t e r i a l than other methods of i s o l a t i o n , e.g. the t r a n s -- 36 -f e r of small p o r t i o n s of wood i n t o p l a t e s of malt agar. The i n d i v i d u a l solutions" obtained from the samples of v a r i o u s p a r t s of the stem were used i n the p r e p a r a t i o n of malt agar, re p r e s e n t i n g t e n per cent of the water r e q u i r e d to prepare the malt agar. P e t r i p l a t e s were poured and one s e r i e s was i n o c u l a t e d w i t h F. annosus and the other w i t h P. 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 capable of s i g n i f i c a n t l y i n h i b i t i n g the growth of the t e s t f u n g i when added to one per cent malt agar, ( F i g . 6 a, b ) . The degree of i n h i b i t i o n of both t e s t f u n g i on one per cent malt agar was greater i n the p l a t e s w i t h so-l u t i o n s from sapwood than 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 from hea r t -wood. The malt agar provided uniform n u t r i t i o n f o r the t e s t f u n g i , and i t was considered t h a t the l a r g e numbers of microorganisms i n the sap-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 the greater degree of i n h i b i t i o n . The 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 r a t e of i n h i b i t i o n between the s o l u t i o n s from the three s e c t i o n s of the stem. The poorest growth on one per cent malt agar occurred i n the p l a t e s w i t h the s o l u t i o n s of the b u t t s e c t i o n s , and the best growth i n those p l a t e s which contained the s o l u t i o n s of the top s e c t i o n s . •' The growth of 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 d i f f e r s i g n i f i c a n t l y from 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 of the top s e c t i o n s . However, i t was p o s i t i v e l y greater than 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 of b u t t s e c t i o n s . The r e s u l t s of the c u l t u r a l s tudies on malt agar should not be r e l a t e d to those obtained on n a t u r a l s u b s t r a t a . Therefore, both F. annosus and P. monticola were t e s t e d on t h r e e - q u a r t e r i n c h s u r f a c e - 37 -s t e r i l i z e d wood cubes i n growth chambers a t one hundred per cent r e l a -t i v e humidity. The r e s u l t i n g weight l o s s e s of the cubes were c a l c u l a t e d as percentages of t h e i r i n i t i a l estimated ovendry weight. The estimated ovendry weight i n t u r n was c a l c u l a t e d by u s i n g the moisture content values determined on the ovendry b a s i s of a sample of f o u r blocks drawn from the same p o p u l a t i o n f o r t h i s purpose. This method i s known to be subject t o e r r o r , because of v a r i a t i o n i n d e n s i t y of the samples. This f a c t o r was not overlooked, but i t was not f e a s i b l e to apply other t e c h -niques such as described by E t h e r i d g e , (1957) where the moisture content i s expressed as a percentage of moisture present at s a t u r a t i o n . The growth of both F. annosus and P. monticola was s i g n i f i c a n t l y i n h i b i t e d on untreated, surface s t r i l i z e d wood cubes incubated at one hundred per cent r e l a t i v e humidity. The degree of i n h i b i t i o n of both t e s t f u n g i was s i g n i f i c a n t l y greater on heartwood than on sapwood samples. These r e s u l t s were the r e -verse of those obtained when the s o l u t i o n s of sapwood and heartwood samples were added t o one per cent malt agar. I t i s p o s s i b l e that a l -though the sapwood contained g r e a t e r number of microorganisms capable of 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 of the sap-wood a f f o r d e d more favourable c o n d i t i o n s f o r the development of the wood de s t r o y i n g f u n g i . The r e s u l t s of 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 the r a t e of i n h i b i t i o n between the three sec-t i o n s of the stems. These r e s u l t s were s i m i l a r to those obtained on one per cent malt agar. The poorest growth and l e a s t weight l o s s e s occurred i n the cubes of 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 weight l o s s e s were observed i n cubes of the top s e c t i o n . The growth and weight l o s s e s i n the middle s e c t i o n d i d not d i f f e r s i g n i f i -c a n t l y from those i n the butt s e c t i o n , but they were p o s i t i v e l y below those recorded i n the top s e c t i o n . These d i f f e r e n c e s i n the r a t e of growth and weight l o s s e s between the three s e c t i o n s of the stem seem to 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 the age, s t r u c t u r e , n u t r i e n t and water content (Brown et a l . , I9h9 ), and populations of microorga-nisms of the s e c t i o n s . This study, however, was not designed to provide experimental evidence 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 the b a s i s of t r e e c h a r a c t e r i s t i c s . Environmental change brought about by i n c r e a s i n g the agar conc e n t r a t i o n t o f o u r and s i x per cent, i n malt agar, and by a drop i n the l e v e l of r e l a t i v e humidity to ninety-seven per cent i n the j a r s w i t h the 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 of i n h i b i t i o n . Both F. annosus and P. monticola produced b e t t e r growth on f o u r per cent than on one per cent malt agar c o n t a i n i n g samples of a l l s o l u t i o n s . This environmental change, however, d i d not seem t o a f f e c t the r a t e of growth i n the p l a t e s w i t h the v a r i o u s s o l u t i o n s to the same ext e n t . I t d i d become evident when the rank of the i n h i b i t i n g powers changed, e.g. P. monticola on one per cent malt agar produced the best growth i n the p l a t e s w i t h the s o l u t i o n from top heartwood ( F i g . 6 b ) ; on f o u r per cent malt agar the best growth of t h i s fungus occurred 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 of the middle heartwood ( F i g . 7 b ) . The reason may have been t h a t the organisms of the top heartwood were more t o l e r a n t to the change of environment while the organisms of the middle - 39 -heartwood were more s e n s i t i v e to the change, which r e s u l t e d i n a greater l o s s of i n h i b i t i n g power. The increase of agar c o n c e n t r a t i o n to f o u r per cent a l s o lessened the d i f f e r e n c e i n the degrees of i n h i b i t i o n bet-ween the s o l u t i o n s from sapwood and heartwood. A f u r t h e r increase of the agar c o n c e n t r a t i o n to s i x per cent l e d to almost complete l o s s of i n h i b i t i o n . The i n c r e a s e i n the agar concentrations of the media was b e l i -eved to r e s u l t i n a d r i e r environment f o r the growth of microorganisms, but a t the same time another v a r i a b l e was introduced represented by d i f -f e r e n t n u t r i t i o n . I t was r e a l i z e d t h a t the technique d i d not permit the se p a r a t i o n of the l i n k e d v a r i a b l e s , moisture content and n u t r i t i o n i n the media. Therefore, t h i s technique was supported by another method when agar d i s c s of the microorganisms were p a i r e d w i t h those of the t e s t f u n g i and kept i n growth chambers at 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 of F. annosus and P. monticola was i n h i b i t e d a t one hundred per cent r e l a t i v e humidity. A decrease i n the r e l a t i v e humidity to approxi-mately ninety-seven per cent l e d to the f r e e growth of the t e s t f u n g i , over-growing the 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 to i n d i c a t e that the moisture content of the en-vironment was an important f a c t o r i n the occurrence and degree of i n -h i b i t i o n . A decrease i n the l e v e l of r e l a t i v e humidity to approximately n i n e t y seven per cent i n the j a r s c o n t a i n i n g untreated wood brought about a s i g n i f i c a n t l o s s i n the degree of i n h i b i t i o n of F. annosus and P. monticola. Both t e s t f u n g i produced b e t t e r growth and caused greater weight l o s s e s i n the cubes incubated a t ninety-seven per cent r e l a t i v e - ho -humidity, than i n those i n a s a t u r a t e d atmosphere. The increase i n the weight losses a t the lower l e v e l of r e l a t i v e humidity was c o n s i s t e n t i n the sapwood and heartwood of a l l s e c t i o n s . These r e s u l t s appeared to f u r t h e r support the hypothesis t h a t the amount of a v a i l a b l e moisture i n the environment may p l a y an important r o l e i n the degree of i n h i b i t i o n . F. annosus and P. monticola were a l s o t e s t e d on s t e r i l i z e d media (one, f o u r and s i x per cent malt agar w i t h s t e r i l i z e d s o l u t i o n s , and s t e r i l i z e d wood). 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 of the experimental m a t e r i a l . The treatment l e d to the l o s s of i n h i b i t i n g f a c t o r i n both malt agar media and wood.' On malt agar there 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 of the t e s t f u n g i when the medium contained the autoclaved s o l u t i o n s . On the media c o n t a i n i n g the autoclaved s o l u t i o n s , both F. annosus and P. monticola grew approximately as w e l l as on those without s o l u t i o n s , r e g a r d l e s s of the source of s o l u t i o n and the agar c o n c e n t r a t i o n of the media. Rather, s t i m u l a t i o n occurred i n s t e a d of the i n h i b i t i o n i n some instances i n the growth of F. annosus e.g. on one per cent malt agar c o n t a i n i n g the s o l u t i o n from the middle heartwood. The r a t e of 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 induced by the nut-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 of the high temperature of auto-c l a v i n g on the p r o p e r t i e s of the s o l u t i o n s was considered of importance. Consequently the r e s u l t s of the treatment were compared w i t h those ob-t 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 served as another method of s t e -r i l i z a t i o n . The M i l l i p o r e f i l t r a t i o n served the purpose of removing microorganisms from the s o l u t i o n s , without s e r i o u s a l t e r a t i o n of the - k l -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 s o l u t i o n when added to one per cent malt agar had no i n h i b i t o r y e f f e c t on the growth of the t e s t f u n g i . Therefore, i t was b e l i e v e d t h a t the micro-f l o r a s were re s p o n s i b l e f o r the i n h i b i t i o n of the wood-decaying f u n g i . Both F. annosus and P. monticola were t e s t e d on autoclaved wood and both f u n g i produced s i g n i f i c a n t l y b e t t e r growth and greater weight l o s s i n autoclaved than i n untreated wood cubes. Both F. annosus and P. monticola caused more weight loss i n the sapwood than i n the heartwood, probably because the sapwood provided 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 the r e l a t i v e l y d r i e r environments (higher agar c o n c e n t r a t i o n of the malt agar media and the wood blocks 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 to a f f e c t the r a t e of growth of the t e s t f u n g i on autoclaved m a t e r i a l . There was 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 weight losses between the autoclaved cubes kept a t one hundred per cent r e l a t i v e hu-m i d i t y and those incubated at ninety-seven per cent r e l a t i v e 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 tw o f o l d , namely, the presence of microorganisms, and s u f f i c i e n t amount of moisture to maintain t h e i r a c t i v i t y . The pathway, time of entrance of the microorganisms 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 k i n d w i t h the s i t e and age of the t r e e are some questions which remain to be answered by f u t u r e i n v e s t i g a t i o n . - U2 -CONCLUSIONS The r e s u l t s of t h i s study provide a b a s i s f o r the f o l l o w i n g c o n c l u s i o n s : The h e a l t h y , normal wood of two l i v i n g western hemlock tr e e s sampled was c o l o n i z e d by microorganisms, c o n s i s t i n g of b a c t e r i a , y e a s t s , moulds, and members of imperfect fungi., "i I t was demonstrated on malt agar media and wood, t h a t the microorganisms 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 of F. annosus and P. monticola. The moisture content of the environment seemed to be an im-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 . D r i e r environments s i g n i -f i c a n t l y decreased the i n h i b i t i n g power of the microorganisms. S i g n i f i c a n t v a r i a t i o n was found i n the degrees of i n h i b i t i o n between the three s e c t i o n s of the stems. Greatest i n h i b i t i o n occurred i n the b u t t s e c t i o n s , and the l e a s t i n the top s e c t i o n s . On malt agar media the s o l u t i o n s of the sapwood samples provided g r e a t e r i n h i b i t i o n than those from the heartwood. Using wood as substratum the i n h i b i t i o n was g r e a t e r i n heartwood than t h a t i n sapwood. I t i s b e l i e v e d that t h i s may have been due 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 of the sapwood. The d i f f e r e n c e i n the behaviour of the t e s t f u n g i on the two substrates emphasises the importance of using n a t u r a l s u b s t r a t e s i n a d d i t i o n to a r t i f i c i a l media to a r r i v e at more r e l i a b l e c o n c l u s i o n s . A u t o c l a v i n g the experimental m a t e r i a l r e s u l t e d i n the l o s s of 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 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 of growth of F'." annosus and P. monticola on - U 3 -autoclaved m a t e r i a l . Consequently, 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 the presence of the microorganisms and s u f f i c i e n t moisture to maintain t h e i r a c t i v i t y . M i l l i p o r e f i l t r a t i o n of the shake s o l u t i o n s l e d to the l o s s of i n h i b i t i n g power. This method of 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 the chemical c o n s t i t u e n t s of the s o l u t i o n s , which provided f u r t h e r evidence i n support of the hypothesis t h a t the micro-organisms played a p o s i t i v e r o l e i n the i n h i b i t i o n of F. annosus and P. monticola. - hh -BIBLIOGRAPHY B i e r , J . E. 1959- The r e l a t i o n of bark moisture to the development of canker diseases 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 . 1. Cryptodiaporthe canker on w i l l o w . Can. J . Botany. 37 : 229-238. 1959. The r e l a t i o n of bark moisture to the development of canker diseases 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 . 2. Fusarium 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 bark moisture to the development of canker diseases 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 . 3 . Cephalosporium canker on western hemlock. Can. J . Botany. 37 : 11U0-11U2. 1961. The r e l a t i o n of bark moisture to the development of canker diseases 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 . k. P a t h o g e n i c i t y studies of Cryptodiaporthe s a l i c e l l a (Fr.) Petrak, and Fusarium l a t e r i t u m Nees., on Populus t r i c h o c a r p a . Torrey and Gray, P. 1 r o b u s t a 1 , P. tremuloides Michx., and S a l i x sp. Can. J . Botany. 3 9 : 139-lUU-1961. The r e l a t i o n of bark moisture to the development of canker diseases 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 . 5. Rooting behaviour and disease v u l n e r a b i l i t y i n c u t t i n g s of Populus t r i c h o c a r p a . Torrey and Gray, and P. 'ro b u s t a 1 . Can. J . Botany. 39 : LL5-15U. 1961. The r e l a t i o n of bark moisture to the development of canker diseases 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 . - US -6. P a t h o g e n i c i t y s t u d i e s of Hypoxylon pruinatum ( K l o t z s c h ) Cke., and S e p t o r i a musciva Pk. on species of Acer, Populus and S a l i x . Can. J . Botany. 39: 1555-1561. 1963- Tissue saprophytes and the p o s s i b i l i t y of b i o l o g i c a l c o n t r o l of some t r e e diseases. For. C h r o n i c l e . 39 ( l ) : 81-8U. and M. H. Rowat, 1962. The r e l a t i o n o f bark moisture to the development of canker diseases 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 . 7- Some e f f e c t s o f the saprophytes on the bark of Poplar and Willow on the incidence o f Hypoxylon canker. Can. J . Botany. UO: 61-69 . and M. H. Rowat, 1963- Further e f f e c t s of the bark saprop-hytes on Hypoxylon canker. For. Science. 9 (3)2 263-270. Bourc h i e r , R. J . 1961. Laboratory s t u d i e s on mic r o f u n g i i s o l a t e d from the stems of l i v i n g lodgepole p i n e , Pinus c o n t o r t a Dougl. *• Can. J . Botany. 39: 1373-1385-Brown, H. P., A. J . Panshin and C. C. F o r s a i t h , 19U9- Textbook of wood technology. F i r s t Ed. V o l . 1. McGraw-Hill Book Co. Cart w r i g h t , K. S. G. and W. P. K. F i n d l a y , 1958. Decay of timber and i t s prevention. Second Ed. Her Majesty's S t a t i o n a r y O f f i c e , London. Cowling, E. B. 1961. Comparative biochemistry of the decay of Sweetgum sapwood by 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. Etheridge, D. E. 1957- A method f o r the study of 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 moisture c o n d i t i o n s . Can. J . Botany. 35: 615-618. - U6 -Gundersen, K. i960. The ph y s i o l o g y of Fomes annosus. Paper presented at the Conference and Study tour 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 of Forest Research Organizations. Keener, P. D. 1950. M i c r o f l o r a of buds. 1. Re s u l t s of 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 of c e r t a i n woody p l a n t s . Am. J . Botany. 37: 7, 520-527. Keener, P. D. I y 5 l . M i c r o f l o r a of buds. 2. Re s u l t s of h i s t o l o g i c a l s t u d i e s of n o n - i r r i t a t e d buds of c e r t a i n woody p l a n t s . Am. J . Botany. 38: 2, 105-110. Luther, 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 apple and pear. C o r n e l l Agr. Exp. S t a . , Memoirs, 181-209, pp. 3-39-

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