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

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FUNGICIDAL TOXICITY OF CERTAIN EXTRANEOUS COMPONENTS OF DOUGLAS FIR HEARTWOOD by ROBERT W. KENNEDY A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF FORESTRY o in the Faculty of Forestry We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF FORESTRY Members of the Faculty of Forestry THE UNIVERSITY OF BRITISH COLUMBIA APRIL 1955 ABSTRACT The heartwood o f D o u g l a s f i r ( P s e u d o t s u g a m e n z i e s i i ( M i r b . ) F r a n c o ) i s known t o be r e l a t i v e l y r e s i s t a n t t o a t t a c k by w o o d - d e s t r o y i n g f u n g i . P r e v i o u s i n v e s t i g a t i o n s on o t h e r s p e c i e s has e s t a b l i s h e d v a r i o u s h e a r t w o o d e x t r a c t i v e s as the p r i m a r y d e t e r r e n t s t o d e c a y . S e v e r a l e x t r a n e o u s f r a c t i o n s from Douglas f i r were i s o l a t e d and e v a l u a t e d f o r f u n g i c i d a l a c t i v i t y i n o r d e r t o d e t e r m i n e the p r e c i s e f a c t o r i n f l u e n c i n g t h e d u r a b i l i t y o f t h i s s p e c i e s . An a c e t o n e , e t h e r and water e x t r a c t i o n o f D o u g l a s f i r heartwood meal p r o v i d e d f i v e s e p a r a t e components, n a m e l y : a d i h y d r o q u e r c e t i n , f r e e a c i d , n e u t r a l , p h l o b a t a n n i n and c a r b o h y d r a t e f r a c t i o n . A b i o a s s a y o f t h e s e m a t e r i a l s was made u s i n g Fomes annosus ( F r . ) C k e . , L e n t i n u s l e p i d e u s F r . and P o r i a i n c r a s s a t a ( B . & C . ) C u r t , as t h e t e s t f u n g i . B o t h a c e l l u l o s i c and a n o n - c e l l u l o s i c s u b s t r a t e were e m p l o y e d . S m a l l wood b l o c k s f r o m w h i c h c e r t a i n e x t r a c t i v e s had been removed were used f o r the c e l l u l o s i c s u b s t r a t e s , whereas m a l t agar i m p r e g n a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f the e x t r a n e o u s m a t e r i a l s r e p r e s e n t e d t h e n o n - c e l l u l o s i c m e d i a . The degree o f e f f e c t i v e n e s s o f e a c h component as a f u n g i c i d e was e x p r e s s e d n u m e r i c a l l y . D i h y d r o q u e r c e t i n was f o u n d t o be t h e most p o t e n t f u n g i c i d e , c o m p l e t e l y i n h i b i t i n g growth o f t h e most s e n s i t i v e fungus a t a c o n c e n t r a t i o n o f s l i g h t l y l e s s t h a n 0.5 p e r c e n t . This value compares favorably with experimental r e s u l t s previously reported with phenolic extractives of the genus Pinus. On the basis of these data, timber selected f o r i t s high dihydroquercetin content could be expected to have an extended service l i f e when used under conditions favoring decay. The p o s s i b i l i t y of breeding highly r e s i s t a n t genetic types i s also discussed b r i e f l y . i i TABLE OF CONTENTS Page I . INTRODUCTION 1 I I . CHEMICAL SEPARATION 8 A. Selection and Preparation of Material. . 8 B. I s o l a t i o n of Chemical Components . . . . 11 1. Acetone extraction 11 2. Ether extraction 13 3 . Water extraction 14 I I I . NATURE OF THE ISOLATED CHEMICAL COMPONENTS . l 6 A. Acetone Solubles l 6 B. Ether Solubles 19 1. Neutrals 19 2. Free acids 20 3 . Phlobatannin 20 C. Water Solubles 22 IV. BIOASSAY 24 A. Experimental Method 24 1. Non-cellulosic medium 24 a. Preparation of substrate . . . . 26 b. Selection of fungal organisms. . 29 c. Inoculation of P e t r i dishes. . . 3° d. Incubation and measurement of growth 3° e. Experimental design 31 i i i Page 2 . C e l l u l o s i c medium 3 2 a. Preparation of s o i l jars 3 2 b. Selection of fungal organisms . . 3 3 c. Inoculation and incubation of s o i l jars 3 3 d. Preparation of test specimens . . 3 4 e. Exposure of test blocks 3 6 f . Experimental design 3 6 B. A n a l y t i c a l Method 3 7 1 . Non-cellulosic medium 3 7 2 . C e l l u l o s i c medium 40 C. Results 41 1 . Non-cellulosic medium 41 2 . C e l l u l o s i c medium 4 4 D. Discussion 4 5 1 . T a x i f o l i n 4 5 2 . Phlobatannin 4 9 3 . Free acids 5 1 4 . Neutrals 5 2 5 . Water solubles 5 2 6 . Zinc chloride 5 3 V. CONCLUSION 5 4 VI. LITERATURE CITED 5 6 i v VII. APPENDICES A. Growth and t o t a l i n h i b i t i o n point curves of Fomes annosus and Lentinus lepideus i n malt agar containing various concentrations of extractives. B. S t a t i s t i c a l analysis of weight losses i n Douglas f i r blocks exposed to Poria incrassata and Lentinus lepideus. V ILLUSTRATIONS Figure Page 1. Outline of extraction procedure f o r i s o l a t i o n of extraneous components 9 2 . Method of ca l c u l a t i n g percentage retardation of mycelial growth 3 9 3 . E f f e c t of varying t a x i f o l i n concentration on growth of Fomes annosus i n malt agar . . 42 TABLES Table Page 1. Concentration of extractives and zinc chloride i n malt agar needed to completely i n h i b i t growth of Fomes annosus and Lentinus lepideus at 2 5 ° C. 4 3 2 . Average weight losses of extracted Douglas f i r wood blocks i n s o i l jars (2-month incubation) 44 v i ACKNOWLEDGMENT The author wishes to acknowledge the encouragement and h e l p f u l c r i t i c i s m s offered by Prof. J.W. Wilson, Faculty of Forestry, under whose d i r e c t i o n t h i s work was undertaken. The suggestions offered by Dr. D.C. Buckland, Faculty of Forestry, are also appreciated. The free use of f a c i l i t i e s at the Vancouver Branch, Forest Products Laboratories of Canada, i s g r a t e f u l l y acknowledged. Special thanks are due to Dr. J.A.F. Gardner, Head, Wood Chemistry Section, and the other members of the Sta f f for t h e i r suggestions and assistance. 1 I . INTRODUCTION I t has been e s t i m a t e d t h a t n i n e p e r c e n t o f t h e t o t a l a n n u a l d r a i n o n t h e f o r e s t s o f t h e U n i t e d S t a t e s i s used t o r e p l a c e wooden t i m b e r s i n s e r v i c e t h a t have been d e s t r o y e d b y ' f u n g i . A f u r t h e r d r a i n o f s i x p e r c e n t i s due t o f u n g a l , i n s e c t , d r o u g h t and wind damage i n t h e f o r e s t i t s e l f (6). Comparable s t a t i s t i c s f o r Canada a r e u n a v a i l a b l e , b u t u n d o u b t e d l y a p p r o x i m a t e t h o s e o f t h e U n i t e d S t a t e s . I t i s i m p o r t a n t t h a t t h e f a c t o r s i n f l u e n c i n g the n a t u r a l decay r e s i s t a n c e o f o u r major t i m b e r s p e c i e s be u n d e r -s t o o d , so t h a t t h o s e woods e x h i b i t i n g h i g h r e s i s t a n c e might be more e f f e c t i v e l y u t i l i z e d . I f t h e f a c t o r s r e s p o n s i b l e f o r d u r a b i l i t y w i t h i n a s p e c i e s were known, t h a t t i m b e r e x h i b i t -i n g a p r e p o n d e r a n c e o f t h e s e f a c t o r s would be more a c c e p t a b l e under c o n d i t i o n s f a v o r i n g d e c a y . P r o d u c t s made from s u c h t i m -b e r c o u l d be e x p e c t e d t o l a s t l o n g e r i n s e r v i c e t h a n t h o s e m a n u f a c t u r e d f r o m u n s e l e c t e d m a t e r i a l . T h e r e would a l s o be t h e p o s s i b i l i t y o f b r e e d i n g h i g h l y d u r a b l e g e n e t i c t y p e s w i t h i n s p e c i e s e x h i b i t i n g d e c a y r e s i s t a n c e . T h i s might be a c c o m p l i s h e d by u s i n g as a seed s o u r c e o n l y t h o s e t r e e s d i s -p l a y i n g the h i g h e s t degree o f d u r a b i l i t y f a c t o r s . D o u g l a s f i r ( P s e u d o t s u g a m e n z i e s i i ( M i r b . ) F r a n c o ) i s the p r e d o m i n a n t c o m m e r c i a l t i m b e r s p e c i e s o f B r i t i s h C o l u m -b i a , a c c o u n t i n g f o r 39 p e r c e n t o f the t o t a l c u b i c volume h a r v e s t e d i n 1953 (11)• Of a l l lumber m a n u f a c t u r e d i n the 2 Province i n 1952, 47 per cent was Douglas f i r ( 2 6 ) . V i r t u a l l y a l l of the softwood plywood produced on the P a c i f i c Coast i s made from th i s single species. Douglas f i r i s also widely u t i l i z e d for poles and p i l i n g , and as a raw material f o r s u l -phate and mechanical pulping. Since the quantity of accessi-ble timber i n merchantable sizes i s being continually reduced, i t i s expedient that our exi s t i n g supply of th i s valuable spe-cies be extended as f a r as possible. The useful l i f e of timber under conditions favoring decay has been greatly extended through the use of wood pre-servatives. These preservatives give maximum protection to wood when they are applied by pressure methods. The heart-wood of Douglas f i r grown on the Coast i s only moderately d i f -f i c u l t to impregnate, but the Rocky Mountain form of t h i s spe-cies i s very d i f f i c u l t to penetrate ( 3 0 ) . Douglas f i r grown i n the i n t e r i o r of B r i t i s h Columbia i s notorious i n t h i s respect. Since some forms of i t s heartwood cannot be properly treated when desired, Douglas f i r i s often used under condi-tions favoring rapid decay without f i r s t having received a preservative treatment. The only alternative i n t h i s instance i s to select n a t u r a l l y durable timber for such uses. This cannot be done u n t i l the factors influencing the r e l a t i v e l y high d u r a b i l i t y of Douglas f i r are understood. I t i s apparent that the existing supplies of Doug-las f i r could be extended i f the incidence and extent of decay i n trees and timbers of t h i s species were reduced. 3 Wood decay i n general may be defined a s those chemical and physical changes r e s u l t i n g from the a c t i v i t y of wood-destroying fungi. The fungi capable of such destruction require c e r t a i n conditions i n order to develop to t h e i r maxi-mum l e v e l . One factor of major importance i n t h e i r reaching th i s l e v e l i s a suitable source of food. Wood substance i s capable of supplying the necessary food requirements. Wood decay occurs as these fungi secrete s p e c i f i c enzymes capable of hydrolyzing the wood substance to forms that can be absorbed by the fungi. Separate enzymes act further upon these hydrolyzed products during the process of fungal metab-olism. F i n h o l t , et a l (l6), have suggested two mechanisms by which introduced preservatives might prevent decay i n wood. Their f i r s t hypothesis proposes that these substances a c t u a l l y i n t e r f e r e with the metabolic processes of the fungus. That i s , the oxidation-reduction enzymes (endoenzymes) that normally react to digest the water-soluble compounds are upset i n such a way that they are unable to function .properly. Their second theory suggests that the enzymes secreted by the fungus (exoenzymes) are denatured so that they become incapable of hydrolyzing the wood complex. Their experimental evidence favors the second hypothesis. A high-boiling creosote was found to be non-toxic when mixed with malt agar, but highly toxic when used as a preservative i n small wood blocks. E v i -dently, the hydrolytic enzymes were denatured i n both cases, 4 but t h i s action had no e f f e c t i n malt agar, since the food source (maltose) i s already i n a form that allows i t s assimi-l a t i o n by the fungus. On the other hand, i t i s well known that fungi can be i n h i b i t e d and k i l l e d i n malt agar containing even a small concentration of highly toxic chemicals. In view of t h i s f a c t , the f i r s t theory seems most appropriate. I t appears that both the endo- and exoenzymes associated with a fungus may be disturbed by the presence of ce r t a i n chemical compounds. Different species of wood exhibit a widely varying a b i l i t y to r e s i s t the action of wood-decaying organisms. Moreover, some v a r i a t i o n i n this a b i l i t y exists from tree to tree within a single species. Attempts have been made to explain t h i s v a r i a t i o n i n decay resistance by considering the general physical and chemical nature of wood. Zabel (57) concluded from a l i t e r a t u r e survey that no o v e r a l l r e l a t i o n -ship exists between s p e c i f i c gravity and decay resistance within a species. When considering extremes within a species, however, very dense wood may be more durable than the l i g h t e s t wood, since gaseous d i f f u s i o n i s at a minimum i n the heaviest wood with the smallest void volume. Between d i f f e r e n t spe-c i e s , there i s no c o r r e l a t i o n between density and d u r a b i l i t y . Some factor other than density i s therefore l a r g e l y responsi-ble f o r the natural decay resistance exhibited by some woods. Hawley, et a l , ( 2 0 ) were the f i r s t to note a r e l a -t i o n between the d u r a b i l i t y and chemical composition of wood. 5 When water extracts from a number of durable species were mixed with malt agar solutions, they were found to exert a toxic e f f e c t on fungal growth. The hot water extract of the heartwood was observed to be the most t o x i c , while s i m i l a r extracts from the sapwood displayed very l i t t l e f u n g i c i d a l a c t i v i t y . Several other investigators ( 3 8 , 39 , 40, 4 9 , 57) have since concluded that the decay resistance of durable wood species can be best explained by considering the nature of t h e i r heartwood extractives. Sapwood of a l l species i s con-sidered non-durable, except when i t exists i n the l i v i n g t ree. In t h i s case, i t i s usually more r e s i s t a n t to decay than heartwood, since the higher moisture content of sapwood may present an unfavorable water-oxygen balance to most fungi. The i n a b i l i t y of most wood-destroying fungi to function as true parasites also prevents sapwood decay i n vivo. Sherrard and Kurth (47) showed that the d u r a b i l i t y of redwood (Sequoia sempervirens (Lamb.) Endl.) was l a r g e l y due to i t s hot-water extract, and that t h i s varied with p o s i -t i o n i n the stem. Southara and E r l i c h (49) and Roff and Atkin-son (43) have concluded that various extractive portions of western red cedar (Thuja p l i c a t a D. Don) are toxic to fungi. Zabel (57) suggested that the water-soluble tannins of white oak (Quercus alba L.) are l a r g e l y responsible for i t s decay resistance. Rennerfelt (38, 3 9 , 40) investigated the e f f e c t of phenolic extractives i n Scots pine (Pinus s i l v e s t r i s L . ) . He found that pinosylvin, and to a lesser extent, pinosylvin 6 monomethylether were toxic to fungi. In summary, woods naturally unfavorable to fungi as a source of food are said to be durable, or to have a high degree of decay resistance. Species that exhibit t h i s property have natural d u r a b i l i t y factors that a c t u a l l y i n h i b i t fungal development. These are usually a t t r i b u t a b l e to heartwood extracts. The chemical nature of heartwood extractives varies between species; those extracts showing the highest degree of t o x i c i t y are obtained from woods proven to be more durable i n service. The chemicals i n the heartwood extract probably exert t h e i r toxic e f f e c t by i n t e r f e r i n g with the highly spe-c i a l i z e d and imperfectly understood enzyme system of the fungus. The d u r a b i l i t y of Douglas f i r i s intermediate between the very r e s i s t a n t and only moderately durable groups. Redwood and western red cedar are examples f a l l i n g within the former c l a s s i f i c a t i o n , whereas the l a t t e r group i s represented by spruce and hemlock ( 5 1 ) . A l i t e r a t u r e survey revealed that no explanation had yet been given for the comparatively high natural d u r a b i l i t y of Douglas f i r . Only one extractive com-ponent of Douglas f i r , t a x i f o l i n , had been previously i n v e s t i -gated for f u n g i c i d a l a c t i v i t y . I t was claimed to be a very weak fungicide ( 5 4 ) . The statement was not supported by the correct b i b l i o g r a p h i c a l notation. This misunderstanding was r e c t i f i e d through correspondence with the author ' ( 1 2 ) , who referred to unpublished r e s u l t s of Rennerfelt. 7 Further correspondence (37) revealed that only a cursory inves t i g a t i o n had been made with t a x i f o l i n i n malt agar. Pullularia< a genus primarily responsible for coni f e r -ous fo l i a g e diseases, was used as the test fungus. No numeri-c a l or comparative expression for t o x i c i t y was obtained, since the amounts of t a x i f o l i n added to the malt agar solutions were not determined. I t was the s p e c i f i c purpose of t h i s work to i n v e s t i -gate chemical extracts of Douglas f i r heartwood i n an attempt to ascribe the reason f o r i t s r e l a t i v e l y high d u r a b i l i t y to certai n extractive materials. In order to do t h i s , i t was necessary to f i r s t prepare various Douglas f i r heartwood extracts, and then to divide these into less complex groups. Following t h i s , an assessment (or bioassay) of each of these materials as fungicides was made. 8 I I . CHEMICAL SEPARATION Extraneous (or extractive) components have been defined as organic substances that may be extracted from wood by neutral solvents ( 2 3 ) . There i s no single universal s o l -vent that w i l l remove a l l of these various extractive compo-nents. In order to insure that the extractives have been quantit a t i v e l y removed, a number of d i f f e r e n t solvents must be employed. Generally, separate extractions with alcohol or acetone, ether, and water are s u f f i c i e n t . Acetone leaches out the coloring matter, tannins and phlobaphenes. Ether has the property of removing o i l s , fats and r e s i n s . Cold water removes the soluble, short-chained carbohydrates, as well as some free acids and s a l t s ( 2 8 ) . Accordingly, these three s o l -vents were used i n t h i s study to prepare the major extractive f r a c t i o n s . Certain extraneous components of Douglas f i r heart-wood have been investigated by Schorger (46) and Pew (33»34). A systematic analysis of the entire extractive f r a c t i o n has been made by Graham and Kurth (19). Their method was chosen because i t provided a complete extraction procedure, with sub-sequent subdivision into known components. An outline of t h i s extraction procedure i s presented i n F i g . 1. SELECTION AND PREPARATION OF MATERIALS A single codominant Douglas f i r tree was f e l l e d on DOUGLAS FIR HEARTWOOD extracted with ethyl ether extracted with acetone and water ETHER EXTRACT 2% No OH ACID SALTS NEUTRALS ethyl H ether CI ACE" EXTI "ONE ?ACT wat er WATER - SOLUBLE FRACTION WATER EXTRACT etha nol WATER-INSOLUBLE FRACTION IMPURITIES CARBOHYDRATES ethyl ether ethyl ether WA1 SOLI FRAC ER BLE T I O N ethyl acetate P H L O B A T A N N I N W A T E R -E T H E R - I f F R A C SOLUBLE ( S O L U B L E T I O N ethyl acetate TANNIN W A T E R - S O L U B L E E T H E R - S O L U B L E F R A C T I O N W A T E R - I N S O L U B L E E T H E R - S O L U B L E F R A C T I O N petroleum ether W A T E R - IN • T H E R - » F R A C S O L U B L E S O L U B L E T I O N ace ethyl tone ether PHLOBAPHENE AMORPHOUS IMPURITIES CRUDE CRYSTALLINE MATERIAL recrys wi etha tallized th nol TAXIFOLIN Fig. I. EXTRACTION PROCEDURE FOR ISOLATION OF EXTRANEOUS COMPOUNDS 10 October 1 , 1 9 5 4 . This tree had been growing on a f i r -hemlock-cedar s i t e on the University forest at Haney, B.C. I t measured 11.0 inches i n diameter at breast height, and had a t o t a l height of 104 f e e t . Its age, including a cor-r e c t i o n for stump height, was approximately 83 years. A 1 .5 inch band of apparent sapwood surrounded the heartwood. Two four-foot bolts were selected from the butt end of the tree af t e r f e l l i n g . The butt end was p r e f e r e n t i a l l y chosen, since there i s some evidence i n c e r t a i n other species to indicate that t h i s portion i s more durable than the top ( 4 , 4 7 ) . A young, second-growth tree was selected, because work by Graham and Kurth (19) has indicated that heartwood material from th i s source contains a higher percentage of extractives than old-growth timber. After slabbing the apparent sapwood from the f i r s t b o l t , the heartwood portion was sawn into boards and subse-quently reduced to planer shavings. These shavings were further reduced .by grinding i n a Wiley m i l l u n t i l they would pass through a 20-mesh screen. The r e s u l t i n g wood meal was then a i r seasoned u n t i l i t s moisture content dropped to approximately 15 per cent. An extended period of drying was avoided, since the v o l a t i l e portion of the extraneous mate-r i a l s could be reduced by t h i s procedure. It has been pointed out that extensive a i r seasoning decreases the amount of ether extract obtained from Douglas f i r wood ( 1 9 ) . 1 1 ISOLATION OF CHEMICAL COMPONENTS 1 . Acetone extraction A large glass tank was used to extract 1 3 7 5 gms. of wood meal (oven-dry basis) with acetone. A fresh supply of solvent was introduced into the tank every two days, a f t e r f i r s t removing and storing the previous extract. Removal of the extract from the wood meal was expedited by using a coarse glass d i f f u s i o n tube, to which suction was applied. The acetone was changed twice, so that a t o t a l of three extractive treatments was f i n a l l y obtained. The b o i l i n g point of acetone ( 5 6 . 5 ° O i s such that i t can e a s i l y be d i s t i l l e d at low temperatures. Therefore, the extract was concentrated to a volume of 6 3 5 ml. by d i s -t i l l i n g the acetone. A 1 5 ml. aliquot from t h i s concentrated extract was dried to constant weight under vacuum at 3 5 ° C. The t o t a l calculated y i e l d of s o l i d material was 2 . 3 1 per cent of the oven-dry weight of the wood. A milky-white suspension was obtained upon addition of 2 5 0 ml. of water to the 6 2 0 ml. of concentrated extract. The solution was then further concentrated to a volume of 2 5 0 ml. under a vacuum at 4 5 ° C. The c o l l o i d a l suspension was flocculated by adding a small quantity of sodium s u l -phate while constantly agitating the solution with a mag-netic s t i r r e r . After the white p r e c i p i t a t e had s e t t l e d , the clear supernatant solu t i o n was decanted and saved. 12 Both the clear l i q u i d and the white p r e c i p i t a t e were exhaustively extracted with ethyl ether. This was done to i s o l a t e that f r a c t i o n of the t o t a l wood extract which i s r e a d i l y soluble i n ether a f t e r i n i t i a l extraction with ace-tone. I t would not have been possible to q u a n t i t a t i v e l y remove this material d i r e c t l y from the wood meal with ether (19>33>34)• Four separate extracts were obtained by t h i s procedure: a water-soluble, ether-insoluble f r a c t i o n ; a water-and ether-soluble f r a c t i o n ; a water-insoluble, ether-soluble f r a c t i o n , and a water-and ether-insoluble f r a c t i o n . To the two ethyl ether-soluble portions, petroleum ether was added to serve as a p r e c i p i t a t i n g agent. After a s u f f i c i e n t quantity had been added to the constantly agitated solution, a l i m i t e d amount of a red-brown, amorphous material was pre-c i p i t a t e d as impurities and discarded. A white p r e c i p i t a t e followed a f t e r adding more petroleum ether. The r e s u l t i n g crude c r y s t a l l i n e mass was r e c r y s t a l l i z e d from ethanol four times. After the fourth r e c r y s t a l l i z a t i o n , the material was washed with water and dried under vacuum i n an i n e r t atmo-sphere of burner gas to prevent oxidation. S l i g h t l y over three grams of creamy-white c r y s t a l s were produced, repre-senting a y i e l d of 0 .23 P e r cent. The water-soluble, ether-insoluble solution was exhaustively extracted with ethyl acetate to i s o l a t e the tan-n i n . The solvent was removed under vacuum, but the amount of s o l i d s i s o l a t e d was i n s i g n i f i c a n t . 13 The water-and ether-insoluble f r a c t i o n was r e d i s -solved i n acetone and reprecipitated with ethyl ether sev-e r a l times i n order to obtain a pure product. F i n a l l y , a brown, amorphous powder was i s o l a t e d , dried and weighed. A y i e l d of 0.014 per cent of the oven-dry wood was obtained. 2 . Ether extraction A large Soxhlet was employed to extract a fresh sample of wood meal with ethyl ether. A t o t a l of 2 .75 kg. of wood meal (oven-dry weight) was extracted i n consecutive 250 gm. portions f o r periods of 24 hours. Only enough ether was added between changes of wood meal to compensate fo r that which was l o s t through evaporation. The r e s u l t i n g clear yellow extract was concentrated to 285 ml. by d i s t i l l i n g the ether. A 10-ml. aliquot from t h i s solution, dried to constant weight under vacuum, i n d i -cated that a t o t a l s o l i d s y i e l d of 0 .97 per cent of the oven-dry weight of the wood had been obtained. In view of this l i m i t e d amount of material, i t was inadvisable to resolve i t into a l l i t s separate compounds. If t h i s were done, there would probably have been too l i t t l e of any single component to test f o r f u n g i c i d a l properties. Instead, the extract was separated into three rather broad c l a s s i f i c a t i o n s f o r preliminary study. I f any group proved to be highly to x i c , a new extract could be prepared and further sub-divided i n an attempt to locate the exact source of 14 f u n g i c i d a l a c t i v i t y . The concentrated ether solution was extracted with a two per cent sodium hydroxide solution i n a separa-tory funnel, thus forming s a l t s of the free organic acids. The neutral material remained behind i n an orange-colored ether layer. The ether solvent was evaporated at room temperature, so that as much of the v o l a t i l e o i l component as possible would remain behind. When no further loss of solvent was noted, the highly viscous material remaining was sealed and stored. To the a l k a l i n e water solution, an equal volume of ethyl ether was added. The aqueous phase was a c i d i f i e d once again by the addition of HC1. Thus a water-insoluble: acid f r a c t i o n (ether layer) and a water-soluble f r a c t i o n were obtained. After evaporating the ether under reduced pressure, a s o l i d acid portion was obtained. The water-soluble phase l o s t most of i t s color upon exhaustive extraction with ethyl acetate. A red-brown powder was i s o l a t e d by evaporating the solvent i n a vacuum oven. A y i e l d of 0 .07 per cent of the oven-dry weight of the wood meal was obtained. 3. Water extraction After thoroughly a i r drying the acetone-extracted sawdust, i t was leached with d i s t i l l e d water. The wood meal was extracted i n a large glass jar at room temperature 15 for a five-day period, with one change of water a f t e r two days. Extraction at elevated temperatures was avoided, as th i s could r e s u l t i n a gradual hydrolysis of the c e l l wall i t s e l f (50). The extract was concentrated i n a constant temperature water bath at 80° C. A f l o c c u l e n t white pre-c i p i t a t e was formed on the addition of four volumes of etha-nol to the sol u t i o n . The p r e c i p i t a t e was c o l l e c t e d by cen-t r i f u g i n g , and then redissolved i n water. Fr a c t i o n a l r e p r e c i p i t a t i o n with ethanol removed f i r s t the impurities, and a white p r e c i p i t a t e resulted as more ethanol was added. The white p r e c i p i t a t e was washed with ethanol and dried i n the a i r . A y i e l d of O.36 per cent was obtained. 16 I I I . NATURE OF THE ISOLATED CHEMICAL COMPONENTS ACETONE SOLUBLES The major portion of the acetone extract was obtained i n the form of creamy-white c r y s t a l s , while the minor f r a c t i o n was i s o l a t e d as an amorphous powder. The c r y s t a l l i n e product obtained from the ether-soluble portion of the acetone extract was f i r s t described by Pew (33,34) and subsequently by Graham and Kurth ( 1 9 ) . Its structure corresponds to that of a flavanone ( I ) , spe-c i f i c a l l y 3 , 5 , 7, 3/> 4/ - pentahydroxy flavanone (II) ( 3 3 , 3 4 ) . This flavanone i s r e a d i l y oxidized to quercetin ( 5 , 7, 3/ 4 / - tetrahydroxy flavanone) (III) ( 5 4 ) . Since the former flavanone d i f f e r s from quercetin only by two I Flavanone Structure ( 3 , 5 , 7 , 3 / , II T a x i f o l i n 4/ - pentahydroxy flavanone) 17 OH 0 OH 0 III IV Quercetin E r i o d i c t y o l a d d i t i o n a l hydrogen atoms at the 2 and 3 positions with a consequent loss of a double bond, i t i s commonly c a l l e d dihydroquercetin. Other equally common names are t a x i -f o l i n and Douglas f i r flavanone. E r i o d i c t y o l (IV) i s a reduction product of t a x i -f o l i n , formed on addition of zinc dust and hydrochloric acid to an alc o h o l i c solution of the flavanone. The devel-opment of a d i s t i n c t i v e lavender color, reportedly charac-t e r i s t i c f o r 3 - hydroxy-flavanones, occurs i n the course of t h i s reaction (33?34) . The deepness of the color pro-duced can probably be r e l i e d upon to give a quantitative estimate of the amount of t a x i f o l i n present ( 3 ) . Accord-in g l y , a small amount of prepared c r y s t a l l i n e product was weighed out and dissolved i n methanol. The r e s u l t i n g solu-t i o n was then analyzed quantitatively by the Wood Chemistry Section of the Vancouver Branch, Forest Products Labora-tories of Canada. The consequent determination, which agreed well with the concentration that had previously been determined gravimetrically, indicated that the is o l a t e d 18 flavanone was r e l a t i v e l y pure. Further evidence was obtained from a melting-point determination. The t a x i f o l i n c r y s t a l s prepared from the acetone extract melted with decomposition at 233°-236° C. This compares favorably with the reported values of 240 - 242° C. (33,34) and 237° C. (19). A colorimetric analysis of a methanol extract from the o r i g i n a l mixed heartwood meal showed the t o t a l t a x i f o l i n concentration to be equal to 0.45 per cent. Thus the i s o l a t e d y i e l d of 0.23 per cent of c r y s t a l l i n e product was s l i g h t l y more than 50 per cent of the t o t a l . Pew (33,34) reported a y i e l d (before r e c r y s t a l l i z a t i o n ) of 0.62 per cent. Graham and Kurth (19) obtained a y i e l d of 0.8 per cent. The brown amorphous powder obtained from the water-and ether-insoluble f r a c t i o n of the acetone extract has pre-viously been determined as a phlobaphene (19). Phlobaphenes are defined as alcohol-soluble, water-insoluble condensation products of tannins (37). In wood analysis, native l i g n i n i s also removed by the same treatment, and probably i s cl o s e l y associated with the phlobaphenes (8,19). Only 0.62 per cent of the acetone extract was i s o l a t e d as phlobaphene i n this study, whereas 3.7 per cent had previously been reported (19). This amorphous material was insoluble i n alcohol, acetone and dioxane aft e r i s o l a t i o n . There are two possible explanations f o r t h i s i n s o l u b i l i t y : further condensation 19 between molecules may have occurred during p u r i f i c a t i o n , or the i s o l a t i o n procedure may have removed the associated material which normally exerts a peptizing e f f e c t (53)• The phlobaphene was not investigated i n the following t o x i -c i t y studies, since the i n s o l u b i l i t y of t h i s material pre-vented i t s proper impregnation into the substrates prepared for fungal attack. ETHER SOLUBLES Three fract i o n s were obtained from the ether extrac-t i o n : 1 group of neutral materials, a free-acid portion, and a tannin-like substance. Each of these are discussed separately. 1. Neutrals The neutral material remaining i n the ether layer a f t e r extraction with d i l u t e a l k a l i probably contained a heterogeneous mixture of compounds. This product, which represented 10 per cent of the ether extract, probably included a c e r t a i n amount of combined acids i n addition to the unsaponifiable v o l a t i l e o i l s and waxes (19). Its highly aromatic odor suggested the presence of the v o l a t i l e s usu-a l l y associated with Douglas f i r oleoresin. Schorger (46) investigated t h i s f r a c t i o n and found that i t contained a mixture of terpenes, predominantly 1 - <K- pinene (V) and i t s d e r i v a t i v e , 1 - oC - terpineol (VI). Johnson and Cain 20 V VI o^-pinene <X-terpineol The remaining components i n the neutral f r a c t i o n have not been s p e c i f i c a l l y i d e n t i f i e d , but probably consist of acids combined as esters, as well as high-carbon alcohols ( s t e r o l s ) . 2. Free Acids The free acid portion obtained by a l k a l i extrac-t i o n of the ether solubles was is o l a t e d as a dark-brown, tacky substance, having a consistency si m i l a r to t a f f y candy. This f r a c t i o n has been reported to consist of an abietic-type r e s i n acid i n addition to several u n i d e n t i f i e d acids ( 1 9 ) . 3 . Phlobatannin The red-brown amorphous powder, which was obtained by extracting the water phase with ethyl acetate, had the properties of a phlobatannin, since i t produced a red-brown pr e c i p i t a t e when heated with d i l u t e mineral aci d . The 21 p r e c i p i t a t e from t h i s reaction i s termed phlobaphene, and thus phlobatannins are defined as phlobaphene-producing tan-nins ( 5 4 ) . Most n a t u r a l l y occurring phlobatannins w i l l y i e l d phloroglucinol and protocatechuic acid on a l k a l i n e fusion. These same compounds are produced when catechin (VII) i s s i m i l a r l y treated (44). Catechin may be related to t a x i -f o l i n inasmuch as i t i s thought to be an ultimate reduction product of quercetin. Attempts to convert t a x i f o l i n to a c r y s t a l l i n e catechin have been unsuccessful, but the amor-phous material obtained by these experiments has shown qual-i t a t i v e properties s i m i l a r to catechin (33)• O H OH VII Catechin As pointed out previously, phlobaphenes may be synthesized by heating phlobatannins with d i l u t e mineral acids. This conversion i s accompanied by the loss of water (18,44). Thus phlobaphenes may be considered conden-sation products of phlobatannins. S i m i l a r l y , phlobatannins 22 are believed to be condensed molecules, formed from the catechin nucleus (44). Moreover, catechin represents a reduced form of t a x i f o l i n . Therefore, dihydroquercetin may be a fundamental molecular type from which the more complex phlobatannin and phlobaphene a r i s e . Such a r e l a t i o n has been suggested by Kurth, who termed pigments such as quer-c e t i n "precursors of the phlobatannins" ( 2 7 ) . Russell has also indicated the p o s s i b i l i t y of t h i s r e l a t i o n s h i p (44). WATER SOLUBLES While drying i n the a i r , parts of the white granu-l a r p r e c i p i t a t e o r i g i n a l l y i s o l a t e d from the water extract turned s l i g h t l y brown i n color . This may have been due to oxidation of gums and p e c t i n - l i k e substances that can occur i n the water-soluble f r a c t i o n along with the polysaccharides. A small amount of the pre c i p i t a t e d carbohydrate was hydrolyzed for one hour with sulphuric acid i n a b o i l -ing water bath. After removing the acid by treating the solution with an ion-exchange r e s i n , a paper chromatographic analysis was made. The chromatogram was developed for 12 hours i n butanol-acetic acid-water i n the volume r a t i o 4:1:5, and then sprayed with a n i l i n e t r i c h l o r a c e t a t e ( 1 0 ) . Both the brown and white portions of the carbohydrate f r a c -t i o n were found to consist of polymers of arabinose and galactose since only these sugars were detected a f t e r hydro-l y s i s . A previous study of the hydrolyzate of the water-soluble polysaccharide of Douglas f i r has revealed the presence of galactose (90$), arabinose (<)%) and xylos OS) (50). Before t h i s time, the p r i n c i p a l carbohydrate had been regarded as galactan ( 1 9 ) . 24 IV. BIOASSAY The i s o l a t e d components included the t a x i f o l i n , neutral, free acid, phlobatannin and carbohydrate f r a c t i o n s . These materials had to be evaluated for r e l a t i v e f u n g i c i d a l a c t i v i t y . This was done by observing how a given chemical affected the growth of a fungal organism. In order that d i r e c t comparisons between the substances could be made, numerical expressions for t h e i r effectiveness were sought. EXPERIMENTAL METHOD A measure of the t o x i c i t y of a chemical may be obtained by introducing the material into a substrate, which i s subsequently exposed to the action of a fungus for a sp e c i f i e d length of time. Two general types of sub-strates have been employed to evaluate toxic e f f e c t s on wood-destroying fungi. One type Involves the use of some non-cellulosic medium such as malt agar or an aqueous nutrient solution to which the chemical may be added. In the second type, the chemical i n question i s added d i r e c t l y to the c e l l u l o s e material such as wood, pulp or sawdust. One procedure involving each of these approaches i s included i n t h i s study. These w i l l be considered sepa-r a t e l y . 1. Non-cellulosic Medium Non-cellulosic substrates f o r evaluating wood 2 5 preservatives have employed water or agar, to which n u t r i -ents have been added (2,15,20,38,39,40,43,47,49,57). The chemicals added as toxicants may not be present i n the same form as they n a t u r a l l y occur i n the wood; thus, the numeri-c a l r e s u l t s obtained might not necessarily apply to a cellulose-fungicide system. Nevertheless, the use of a non-c e l l u l o s i c medium provides a s a t i s f a c t o r y method of deter-mining comparative t o x i c i t y of d i f f e r e n t substances ( 2 9 ) . I f an aqueous nutrient solution i s used, i t i s inoculated with a fungus which i s then allowed to grow for a spe c i f i e d period of time. After the mycelium has been removed by f i l t e r i n g the nutrient solution, i t i s oven-dried and weighed. The amount of growth i n solutions of varying chemical concentrations, as r e f l e c t e d by the weight of the mycelia, serves to indicate the degree of f u n g i c i d a l t o x i c i t y . This method has one serious disadvantage: chemi-cals added i n the form of water-insoluble solutions cannot be kept i n a dispersed condition without the addition of an emulsifying agent. For t h i s reason, t h i s method was not seriously considered for t h i s study. When malt agar i s employed as a substrate, r a d i a l measurements of mycelial development are taken p e r i o d i c a l l y a f t e r inoculation with a fungus. The retarded rate of growth on malt agar containing various concentrations of chemicals can be used to express t o x i c i t y . This technique has been employed by several investigators (15,20,38,39,43, 26 47>49j57). T h e m a l t agar method i s the most rapid one a v a i l -able f o r the evaluation of fungicides. Mycelial growth begins soon aft e r inoculation so that data can be coll e c t e d almost immediately, without waiting for a long incubation period. Its property of g e l l i n g quickly on cooling makes i t possible to r e t a i n water-insoluble solutions i n a dispersed state (29). Since two of the chemical fractions that were to be evaluated were insoluble i n water-miscible solvents such as alcohol, malt agar was selected as the non - c e l l u l o s i c medium. Preparation of s u b s t r a t e — D i f c o malt agar was dissolved i n d i s t i l l e d water and s t e r i l i z e d i n 100-ml. quantities. In order to determine the e f f e c t of chemical concentration on t o x i c i t y , varying amounts of the prepared extractives were then added to the malt agar. Weighed amounts of t a x i f o l i n and phlobatannin were added i n the form of a l c o h o l i c solu-tions, whereas the acid and neutral f r a c t i o n s were f i r s t d i s -solved i n ether. The amount of chemicals added ranged between 0 .01 gm. and 1.08 gm. One ml. of solution was the smallest amount added, while higher volumes (up to 4 ml.) were used to correspond to larger amounts of material. Pre-liminary tests had revealed that ether exerted only a s l i g h t toxic e f f e c t . Alchohol had previously been used to apply water-insoluble substances to malt agar ( 3 8 , 3 9 ) . Malt agar solutions containing both alcohol and ether of various 27 concentrations were prepared for control purposes. The concentration of material i n the r e s u l t i n g malt agar solution was expressed i n grams per cubic c e n t i -meter. This was converted to a percentage by multiplying by 1 0 0 . Since the volume of malt agar used was always 100 c c , the weight of material added to the malt agar became a d i r e c t measure of the percentage concentration. The concen-t r a t i o n of the chemical solvent i n s o l u t i o n was also expressed as a percentage. Since between one and four ml. were added, the concentration i n the malt-agar solution ranged from one to four per cent. When the flasks containing the malt agar had cooled s u f f i c i e n t l y after s t e r i l i z a t i o n , the a l c o h o l i c chemical s o l -utions were a s e p t i c a l l y added and mixed with the malt agar by gently swirling the f l a s k s . The material i n the f l a s k s was then poured into small P e t r i dishes (50 mm.). Eight dishes could be poured with each 100 ml. of prepared sol u t i o n . The smaller P e t r i dishes were used i n preference to larger ones, so that more r e p l i c a t i o n s within a single concentration would be obtained with the l i m i t e d quantity of extractives on hand. E s s e n t i a l l y the same method was employed to prepare P e t r i dishes that would contain the ether-soluble groups. Since ether and water are immiscible, s p e c i a l precautions were necessary to insure proper d i s p e r s a l of extractives i n the ether s o l u t i o n . Ordinary swirling or s t i r r i n g gives an unsatisfactory dispersion. The use of a Waring blender or 28 other high-speed mixer has been advocated f o r si m i l a r prob-lems with o i l - s o l u b l e wood preservatives ( 1 5 ) . This modifi-cation divides the immiscible material into very small drop-l e t s so that a nearly homogenized solution i s obtained. The ether-soluble chemical solutions were added to the malt agar and thoroughly mixed under aseptic conditions with a magnetic s t i r r e r . Just before the solu t i o n gelled, i t was poured in t o P e t r i dishes resting on an ice bath. The malt agar hardened within seconds, leaving the chemicals suspended as very f i n e l y dispersed droplets, discernable only with a handlens. It was not anticipated that the water-soluble carbo-hydrate f r a c t i o n would i n h i b i t fungal growth. This was added to a p l a i n agar solution i n concentrations up to two per cent. The carbohydrate was s t e r i l i z e d separately from the agar solution to avoid hydrolysis of the polymers to simple sugars. After cooling, the agar was then added to the dry granular carbohydrate f r a c t i o n , and the plates were poured i n the usual manner. In testing the water-soluble f r a c t i o n , the carbohydrate material replaced the malt which was used as a nutrient i n the previous t e s t s . I t was desirable to compare the re s u l t s obtained using the wood extracts with a water-soluble chemical com-monly employed as a wood preservative. Therefore, a p a r a l -l e l experiment, consisting of zinc chloride as the toxicant, was set up. 29 Selection of fungal organisms—A fungus must be able to grow reasonably well i n culture to be of value i n a malt-agar t e s t . Secondly, the fungi selected to test extractives peculiar to a ce r t a i n species should be commonly associated with t h i s species. F i n a l l y , the test organisms must not be too sensitive to the alcohol and ether solvents used. A combination of these requirements led to the selection of Fomes annosus (Fr.) Cke. and Lentinus lepideus Fr. I n i t i a l l y Poria incrassata (B.&C.) Curt, was chosen for use, but even low concentration of alcohol markedly retarded i t s growth. It was discarded i n favor of Fomes annosus. F. annosus i s common i n America, Europe, India and Au s t r a l i a , where i t causes a butt-rot of co n i f e r s . I t i s the most important cause of heartrot i n conifers grown i n England where many plantations of Douglas f i r have been established ( 9 ) . I t usually enters the heartwood through dead roots, and eventually reduces the infested roots and butt to a series of e l l i p t i c a l white pockets. The fungus i s also prev-alent on damp mine timbers i n both Europe and the United States ( 6 , 9 ) . Douglas f i r i s the most common source of mine timbers i n the Western States, and i s being used increasingly for the same purpose i n the East ( 3 2 ) . F. annosus i s used extensively i n laboratory tests with malt agar, since i t grows vigorously and i s generally r e s i s t a n t to foreign chemi-c a l s . Lentinus lepideus also has a world-wide d i s t r i b u t i o n 30 I t i s r a r e l y found attacking a l i v i n g tree, but i s a very common factor i n deteriorating timbers i n service. I t causes a t y p i c a l brown r o t , eventually reducing the wood to a series of brown cubes. In the United States, i t i s one of the most serious destroyers of coniferous r a i l r o a d t i e s and poles ( 6 ) . Douglas f i r i s u t i l i z e d extensively for both of these purposes, e s p e c i a l l y i n the Northwest ( 3 2 ) , and i s subject to the action of t h i s fungus. In England i t i s the most important wood-destroying fungus attacking poles, paving blocks and mine timbers ( 9 ) . L_s. lepideus grows at a moderately f a s t rate i n malt agar, and i s commonly used when wood preservatives are to be evaluated. Inoculation of P e t r i dishes—Pure cultures of Fomes annosus and Lentinus lepideus were obtained from the Wood Pathology Unit of the Vancouver Branch, Forest Products Laboratories of Canada. From these o r i g i n a l cultures, a number of trans-plants were made to p l a i n malt agar. After an l8-day period (± 3 days), a piece of inoculum was taken from the a c t i v e l y growing margin and transferred to the outer edge of each P e t r i dish. The inocula were a l l cut with a number one cork borer (4 mm. diam.) to insure a uniform s i z e . A l l inocula-tions were performed no l a t e r than 24 hours a f t e r preparation of the P e t r i dishes. Incubation and measurement of growth—The temperature to which a fungal organism i s exposed greatly Influences i t s rate of growth. The optimum temperature for Fomes annosus 31 i s 23° C, while 27° C i s most suitable for Lentinus lepideus (9). Consequently, a l l the inoculated P e t r i dishes were incubated i n a constant temperature chamber at 25° C. The plates were o r d i n a r i l y not sealed because of the short dura-t i o n of the experiment. However i n the case of the dishes containing the v o l a t i l e neutral f r a c t i o n , masking tape was used to seal the dishes. This e f f e c t i v e l y prevented the loss of possibly t o x i c , v o l a t i l e materials to the surrounding chamber atmosphere. Readings were taken at approximately two-day i n t e r -vals u n t i l the plates were covered with mycelium, or u n t i l the agar started to dry and crack through desiccation. Fomes annosus control plates were covered i n six days, while Lentinus lepideus controls took a t o t a l of ten days. The amount of r a d i a l growth between the center of the inoculum and the margin of the mycelium was measured i n millimeters. Under comparable conditions, the re p l i c a t e s seldom varied more than four mm. Experimental design—As has been previously stated, eight plates were poured f o r each concentration of a given chemi-c a l . Therefore, a t o t a l of four r e p l i c a t i o n s were available for testing the reactions of each fungus. Owing to the varying amounts of chemicals av a i l a b l e , the concentrations examined were not standardized throughout the experiment. The concentrations tested f o r each given chemical may be read from the curves i n Appendix A. 32 Malt-agar plates containing one, two and four per cent ether and alcohol were poured for controls. These s o l -vent volumes corresponded to those a c t u a l l y used to dissolve the t a x i f o l i n , phlobatannin, neutral, and acid f r a c t i o n s . P e t r i dishes containing only malt agar were also inoculated to determine to what degree the ether and alcohol solvents themselves were t o x i c . 2 . Cellulose Medium Natural conditions of wood decay can be more clo s e l y approximated by exposing wood blocks to decay organ-isms under controlled conditions. The B r i t i s h standard f o r testing wood preservatives consists of exposing a treated wood block to the action of a fungus growing on malt agar i n a Kolle f l a s k (9)• B r i t i s h and American workers now favor the s o i l - j a r method of L e u t r i t z (29)» where a treated block i s placed on top of a similar untreated block that has previously been buried i n s o i l inoculated with a wood-destroying fungus. In both cases, the degree of decay i s determined by the weight loss suffered by the wood block after an incubation period of three to nine months. The American method was adopted f o r t h i s experiment, since i t requires less manipulation, and more c l o s e l y represents actual f i e l d conditions. Preparation of s o i l .jars—Preserving jars (16 oz.) were 33 h a l f f i l l e d with a high-organie content s o i l that had p r e v i -ously been moistened to 45 per cent moisture content. A 1.5-x 2.5-x 5»0-cm. feeder block of Douglas f i r sapwood, cut so that i t s long axis was p a r a l l e l to the grain, was buried i n the s o i l so that only i t s upper surface was exposed. The jars were then s t e r i l i z e d f o r one hour at 15 pounds steam pressure p r i o r to inoculation. Selection of fungal organisms—In a test of t h i s nature, i t i s desirable to use fungi that r a p i d l y decay timber i n serv-i c e , thus causing a s i g n i f i c a n t loss i n weight over a short period of time. Lentinus lepideus has been found to cause severe weight losses within four months, but for the same period of time, Fomes annosus decomposed wood blocks only f i v e to seven per cent (9). Therefore, a substitute for F. annosus seemed advisable for the wood-block t e s t . Poria incrassata a common cause of dry r o t , was selected for thi s purpose. This fungus i s extremely active along the P a c i f i c Coast and i n the Southeastern United States, where the pre v a i l i n g climate presents an agreeable s i t u a t i o n f o r the decay of coniferous building timbers (6). Inoculation and incubation of s o i l j a r s — T h e inocula used were cut from the margin of the fungus growing i n malt agar, and pressed down into the s o i l d i r e c t l y adjacent to the buried feeder block. The jars were then incubated i n d i f f u s e l i g h t at room temperature f o r 30 days. At the end of thi s 34 time, the mycelia had become well-established i n the s o i l and on the feeder blocks i n most of the j a r s . An excess of jars had been prepared so that only those providing an even, well-developed mycelium were continued i n the t e s t . Preparation of test specimens—If the object of t h i s experi-ment had been the evaluation of a wood preservative, blocks of wood would have been impregnated with the preservative and then introduced into the previously inoculated s o i l j a r s . The t o x i c i t y of i n d i v i d u a l extractives cannot be tested i n t h i s way, since they are already present as a group i n the wood samples. The toxic e f f e c t of an extractive can be determined i n d i r e c t l y by leaching i t from wood blocks before they are exposed to a fungus. I f the material removed i s t o x i c , the extracted blocks can be expected to decay at a faster rate. The amount of decay that occurs i n these extracted blocks w i l l be r e f l e c t e d by t h e i r weight losses. The greater weight losses suffered by the extracted blocks as compared to the control samples serve to indicate the importance of the extracted component as a natural wood preservative. Small samples were prepared from which extractives could be e a s i l y leached. I t was necessary that a major proportion of end grain be exposed i n order to f a c i l i t a t e penetration both f o r extraction and decay. The i f - i n c h cubes normally used i n s o i l - j a r tests would not be leashed 35 s a t i s f a c t o r i l y , nor would they undergo decay as r a p i d l y as smaller pieces with more exposed end grain. Findlay (14) has suggested that t h i n pieces under 6 cc. w i l l give s i g -n i f i c a n t weight losses over a short period. This i s explained by the increased rate of gaseous d i f f u s i o n when surface-volume r a t i o s are high. A r a d i a l l y sawn board 5*0 cm. i n thickness was taken from the o r i g i n a l test bolt for the preparation of wood-block samples. The amount and t o x i c i t y of heartwood extractives may vary with r a d i a l p o s i t i o n i n the stem of a tree (4 ,9>57)» Such variations can be n u l l i f i e d by making a l l heartwood test blocks a uniform distance from the sap-wood. This was done by ripping a 2 . 5 cm. band of outer heartwood from the entire length of the r a d i a l l y sawn board. The r e s u l t i n g s t r i p was dressed to 1.5-x 5.0-cm. This was reduced to small blocks by cross-cutting at 0 . 6 cm. i n t e r -v a l s . Each sample block thus measured 0.6-x 1.5-x 4.0-cm., the smallest dimension being p a r a l l e l to the longitudinal axis of the wood f i b e r s . Ten randomly selected samples were then leached i n either acetone, ether, or water. This corresponded to the preceding i n i t i a l chemical a n a l y s i s . The test blocks to be leached i n acetone or water were placed i n glass jars at room temperature f o r six days, to simulate the o r i g i n a l con-diti o n s of the acetone and water extractions. Aspiration i n the respective solutions was necessary to insure complete 36 immersion of the blocks. The ether extraction was carried out i n a Soxhlet u n t i l the coll e c t e d solvent was s l i g h t l y colored, indicating a quantitative removal of the extraneous materials. A one-week period was s u f f i c i e n t for t h i s pur-pose. In order to establish an i n i t i a l equilibrium weight of the extracted samples, they were conditioned to a moisture content of 30 per cent i n a constant temperature-humidity chamber. This was done i n preference to oven-drying, which might reduce toxic v o l a t i l e substances. Heat-ing the blocks may also increase the r e s i n content at the surface of the acetone-and water-extracted samples and so increase decay resistance ( 5 2 ) . Exposure of te s t blocks—When the samples reached e q u i l i b -rium weight, they were each f i t t e d with a straight pin, and fl a m e - s t e r i l i z e d immediately before placing i n the s o i l j a r s . The blocks remained i n the jars i n d i f f u s e l i g h t for two months at room temperature. ?/hen t h i s incubation period had elapsed, the blocks were removed, brushed free of excess mycelium and oven-dried. Oven-drying i s necessary i n th i s case since severely decayed samples w i l l come to a higher equilibrium moisture content i f reconditioned i n the same manner as i n i t i a l l y ( 3 1 ) . After drying completely, the blocks were weighed to determine t h e i r loss i n weight. Experimental design—Ten blocks were used i n each of the, 3 7 acetone, ether and water extractions. Another group of ten unextracted samples was included for control purposes. Five blocks from each treatment were subjected to decay by P. incrassata, while the remaining h a l f were put into s o i l jars previously inoculated with Lentinus lepideus. ANALYTICAL METHOD 1. Non-cellulosic Medium It has been well established that a s t e a d i l y decreasing amount of growth i s obtained as the concentration of fungal i n h i b i t o r s i n malt agar i s increased ( 5 ) . In order to obtain a d i r e c t comparison between the extraneous compounds tested, an estimate was needed of the chemical concentration necessary to completely i n h i b i t growth. Such a concentration i s c a l l e d the Total I n h i b i t i o n Point (T.I.P.), and has been defined by Schmitz (45) as the minimum concen-t r a t i o n allowing no signs of growth either on the malt agar or the inoculum plug i t s e l f . This should not be confused with the k i l l i n g point, which i s that concentration neces-sary to k i l l a fungus. At the T.I.P. the fungus i s not necessarily k i l l e d . Most fungi have the a b i l i t y to remain dormant over long periods of time without v i s i b l e signs of growth. As the chemical concentration increases, the amount of mycelial growth decreases i n a c u r v i l i n e a r manner that 38 can be represented by a parabolic or hyperbolic function. The curves obtained by p l o t t i n g these points on ordinary co-ordinate paper could be extrapolated to zero growth to f i n d the T.I.P. Such a procedure with c u r v i l i n e a r functions often causes inaccurate r e s u l t s . Bateman (5) found that a straight l i n e could be obtained i f the logarithm of the per cent retardation of growth was plotted against the logarithm of per cent concentration of fungicide. While t h i s r e l a -tionship did not hold consistently at lower concentrations, i t did apply between the T.I.P. and a concentration approxi-mately one-third of that value. This l i n e a r r e l a t i o n was found to be v a l i d not only with Fomes anno.qnfi, but with green plants as w e l l . The data collected i n t h i s study have been analyzed af t e r the method of Bateman. The data obtained from measurements of fungal growth were f i r s t averaged for each i n d i v i d u a l chemical concentration, time, and organism. The r e s u l t i n g f i g u r e s , representing the means of four measurements, were then plotted on ordinary co-ordinate paper along with t h e i r respective controls. The ordinate represented r a d i a l growth i n millimeters, whereas time i n days was plotted on the abscissa. With few exceptions, the points were found to assume a nearly perfect s t r a i g h t - l i n e r e l a t i o n s h i p . Bateman had previously made the same observation ( 5 ) . A diagramatic sketch of such a graph i s shown i n F i g . 2. / a / /// TIME X Figure 2 . Techniques of Analysis a - t y p i c a l growth i n control dishes b - data of b/ corrected for rest period b/ - t y p i c a l growth i n toxicant-containing dishes ab - amount of retardation ab - per cent retardation ax Throughout the course of the experiments, growth ra r e l y started immediately af t e r transplanting the inoculum, even among the controls. A rest period of a day or more before growth commenced was not uncommon. A longer rest period was often required when an inoculum was planted i n a P e t r i dish containing a fungal i n h i b i t o r . When growth f i n a l l y started, however, the rate was sometimes nearly as fas t as that of the controls. Bateman observed t h i s same phenomenon, but added a correction factor f o r variable rest periods. This method was applied throughout the present work. I f a chemical was found to induce a longer rest period than the control, the plotted points were f i r s t 40 connected i n the usual manner to get the best possible l i n e a r f i t , A l i n e was then drawn p a r a l l e l to t h i s , through the point of i n t e r s e c t i o n of the control l i n e and the abscissa. F i g . 2 exemplifies t h i s case, where l i n e b/ represents the o r i g i n a l data, b i t s correction for re s t period, and a the control data. Percentage retardation of growth was calculated i n the following manner: the r a d i a l growth attained by the fungus i n covering the control plate was considered as 100 per cent ( l i n e ax, F i g . 2). For the same time period, growth i n a dish containing a fungicide reached only a f r a c t i o n of the control ( l i n e bx, F i g . 2). Thus, the retarding e f f e c t -could be represented by l i n e ab, or the percentage retarda-t i o n by ab/ax. The figures obtained for per cent retardation were plotted against per cent concentration of chemical on double-log ;paper. The points were f i t t e d to a straight l i n e , and the l i n e extrapolated to 100 per cent retardation. The con-centration at which retardation of growth equaled 100 per cent represented the T.I.P. 2. C e l l u l o s i c Medium The weight losses suffered by the test blocks while undergoing decay i n the s o i l jars were expressed as a per-centage on the basis of the o r i g i n a l oven-dry weight of the specimens. Oven-dry values were calculated from the weights 41 obtained at an equilibrium moisture content of 30 per cent by d i v i d i n g by 1 .3 . The percentage loss i n weight of the acetone-, ether- and water- extracted wood blocks was compared against the controls with Student's " t " t e s t . The r e s u l t i n g values for " t M were evaluated at the f i v e per cent confidence l e v e l . The lower weight losses of 20 per cent have a smaller v a r i -ance about t h e i r mean than the higher values of 55 P e r cent. It was therefore necessary to f i r s t transform the o r i g i n a l percentages into radians, thereby making the data independent of t h e i r respective means. The transformation arc s i n /percentage was used for t h i s purpose ( 3 6 ) . RESULTS 1. Non-cellulosic medium The odd-numbered curves i n Appendix A show the growth of the test fungi i n malt agar containing various concentrations of extractives i n sol u t i o n . Curves obtained with some very low concentrations are not shown, since they are co-incident with the control l i n e s . Higher concentrations of toxic extractives obviously had an e f f e c t on the growth rate of both fungi. An example of t h i s i s presented i n F i g . 3 , where a series of P e t r i dishes containing successively higher concentrations of t a x i f o l i n were exposed to the action of F. annosus. 4-2 Figure 3 E f f e c t of t a x i f o l i n concentration on growth of Fomes annosus i n malt agar A - control B - 0 .05$ C - 0.1% D - 0.2% E - 0.4$ F - 0.d% Not only did the r a d i a l growth of the mycelium decrease with concentration, but the character of the fun-gal mat changed as w e l l . Generally speaking, as concentra-t i o n increased, the hyphae became more a e r i a l i n nature, the mycelium being p a r t i c u l a r l y abundant around the inocu-lum. Mat margins also became more uniform, since the 43 usual advancing zone was lacking. The even-numbered curves i n Appendix A represent the logarithmic r e l a t i o n of per cent retardation plotted against per cent concentration. These have been extrapo-lated to 100 per cent retardation i n order to determine the T.I.P. Values for T.I.P. are summarized i n Table 1. Table 1 Concentration of Chemicals i n Malt Agar Needed to Completely I n h i b i t Fungal Growth at 25° C Organism Chemical or extractive tested 1 T.I.P. t a x i f o l i n 0 . 4 5 phlobatannin 1.3 Fomes annosus neutrals 1.4 free acids 1.1 zinc chloride 0.16 Lentinus lepideus t a x i f o l i n phlobatannin neutrals free acids zinc chloride 0.70 1 .6 4 .7 2 . 0 0 . 0 8 1 Total i n h i b i t i o n point T a x i f o l i n was d e f i n i t e l y the most toxic wood extractive investigated. However, none of the i s o l a t e d groups approached the effectiveness of zinc chloride as a fungicide. 44 . No concentration of ether or alcohol affected the growth of Lentinus lepideus i n culture. However, Fomes  annosus grew s i g n i f i c a n t l y slower i n two per cent ether and four per cent alcohol. The curves obtained f o r these solvent concentrations were used as controls for those extractive groups requiring high amounts to properly d i s -solve. 2. C e l l u l o s i c Medium The weight losses of the extracted wood blocks are presented i n Appendix B . The averages obtained f o r each set of f i v e samples are included i n Table 2. Table 2 Average Weight Losses of Extracted Wood Blocks i n S o i l Jars (2-month incubation) Organism Extractive treatment 1 Avg. wt. loss Poria incrassata acetone 25.3 ether 24.7 water 29.7 none 23.0 Lentinus lepideus acetone ether water none 52.2* 51.8* 44.7 37.6 1 Per cent of o r i g i n a l oven-dry weight • S i g n i f i c a n t l y d i f f e r e n t from unextracted blocks (5$ l e v e l ) 45 The acetone- and ether- extracted samples exposed to the action of Lentinus lepideus suffered nearly equal and s i g n i f i c a n t l y higher weight losses than the controls. The results suggest that the three weakly toxic components investigated as sub-divisions of the ether extract may exert a combined f u n g i c i d a l action equal to that of t a x i -f o l i n . The water-extracted blocks, although not varying s i g n i f i c a n t l y from the controls, nevertheless show a some-what higher weight l o s s . This was probably due to the removal of some water-soluble tannin substances along with the carbohydrate material. The re s u l t s obtained with Poria incrassata were not s t a t i s t i c a l l y s i g n i f i c a n t when analyzed with the " t " t e s t . A longer incubation period may be required with t h i s fungus i n order to obtain meaningful r e s u l t s , since the weight losses amounted to only h a l f those of the samples infected with Lentinus lepideus. DISCUSSION 1. T a x i f o l i n Among the components tested i n malt agar, only t a x i f o l i n was included i n s u f f i c i e n t quantities to com-p l e t e l y i n h i b i t growth. No growth of either test organism occurred i n dishes containing one per cent of t h i s f l a v a -none, and a concentration of as l i t t l e as 0 . 6 per cent 46 prevented L_g. lepideus from growing. After three weeks, the inocula from these three sets of dishes were trans-ferred to fresh malt agar. Growth was not renewed, so i t Was concluded that these concentrations were s u f f i c i e n t to bring about death of the fungi. These points of k i l l i n g concentration are included i n the t a x i f o l i n growth curves (1 and 3) of Appendix A. They did not influence the extra-polation procedure, since these values represent a concen-t r a t i o n above that of the T.I.P. Rennerfelt (38,39) has determined the t o x i c i t y of pinosylvin (VIII) by the malt agar method. By p l o t t i n g h i s data for Fomes annosus, a T.I.P. of 0 .06 per cent i s obtained. P i n o s y l v i n was f i r s t i s o l a t e d from Scots pine (Pinus s y l v e s t r i s L.) and has since been found i n a number of the hard pine (Diploxylon) group. Its monomethylether (IX) has also been i s o l a t e d , not only from the hard pines, VIII IX Pinosylvin P i n o s y l v i n monomethylether but from the soft pine (Haploxylon) group as well (5"4). This monomethylether of Pinus acts as a weak fungicide, since concentrations s l i g h t l y i n excess of one per cent are required to completely i n h i b i t growth of F. annosus (38>39). 4 7 In addition to both being phenolic i n character, pinosylvin and t a x i f o l i n have certain other s i m i l a r i t i e s . Both substances are soluble i n ethyl ether only a f t e r they have been removed from wood with acetone or alcohol. Erdtman ( 5 4 ) has attributed t h i s phenomenon to ether-insoluble "mem-brane substances" which envelop the phenolic molecules so that they are made inaccessible to ether. Extraction with alcohol or acetone apparently dissolves both the phenolic and ether-insoluble substances, allowing the t a x i f o l i n or pino-s y l v i n to be subsequently soluble i n ether. Therefore, there i s some question as to whether t a x i -f o l i n and similar phenolic compounds i s o l a t e d by chemical means are i d e n t i c a l with those occurring na t u r a l l y i n the wood. Their chemical nature and physical a v a i l a b i l i t y may d i f f e r when i s o l a t e d . Further evidence to support t h i s view arises when the i n s e c t i c i d a l properties of t a x i f o l i n and pinosylvin are considered. Both compounds have been shown to be highly toxic to the West Indian dry-wood termite (Cryptotermes brevis Walker) ( 5 6 ) . Wood blocks submerged i n a 0 . 0 5 per cent t a x i f o l i n solution for ten minutes have remained free from attack for more than 42 months ( 5 5 ) . The t a x i f o l i n present na t u r a l l y i n Douglas f i r wood, however, apparently provides no protection whatsoever, since t h i s spe-cies i s r e a d i l y attacked by the t r o p i c a l dry-wood termite. The reason for t h i s discrepancy may be one of a v a i l a b i l i t y . A wood block soaked i n a t a x i f o l i n solution, would present an 48 even d i s t r i b u t i o n of pure compound to an attacking agency. The n a t u r a l l y occurring substance i s probably d i s t r i b u t e d unevenly, and may be la r g e l y inaccessible to wood-destroying insects by exis t i n g i n an intimate form with other materials. T a x i f o l i n has been reported to exist n a t u r a l l y i n a average concentration of about one per cent in.Douglas f i r heartwood ( 3 3 > 3 4 ) . The combined concentration of pinosylvin and i t s monomethylether i n pine heartwood amounts to about 0 . 8 per cent of the dry weight of the wood ( 3 8 , 3 9 ) . However, the less toxic p i n o s y l v i n monomethylether i s present i n amounts three to four times that of the parent pinosylvin ( 5 4 ) . Thus, the combined f u n g i c i d a l e f f e c t of these two pine substances should be expected to approach that of t a x i f o l i n i n Douglas f i r heartwood. Evidence to substantiate t h i s view has been provided by Smith ( 4 8 ), who classes Douglas f i r and Scots pine i n the same natural d u r a b i l i t y group by service test experience. Another related phenolic molecule present i n the Pinaceae i s conidendrin (X). This has been found i n many species of the non-durable Tsuga, Picea and Abies genera. Like the p a r t i c u l a r phenols of the pines and Douglas f i r , t h i s compound cannot be d i r e c t l y extracted with ether. The non-toxic nature of this substance, as well as the decreased potency of pinosylvin monomethylether, may be explained by the substitution of methoxyl groups f o r hydroxy r a d i c a l s . Both pine and Douglas f i r heartwoods are r e s i s t a n t 49 H 3 C O . o o H O Y 0 C H 3 OH X G o n i d e n d r i n t o p u l p i n g by t h e n o r m a l sulphite method (21,35*54). I t was f o r m e r l y t h o u g h t t h a t t h e p h e n o l i c e x t r a c t i v e s r e a c t e d w i t h l i g n i n t o form an i n s o l u b l e p h e n o l i c l i g n i n complex (35>54). R e c e n t e v i d e n c e has i n d i c a t e d t h a t t h e s e p h e n o l i c compounds may i n h i b i t p u l p i n g by decomposing t h e c o o k i n g l i q u o r (21). When t a x i f o l i n i s r e a c t e d w i t h b i s u l p h i t e , i t Is o x i d i z e d t o q u e r c e t i n . The r e d u c t i o n p r o d u c t o f t h i s r e a c t i o n i s p r o b -a b l y t h i o s u l p h a t e , w h i c h i n t u r n may c a t a l y z e f u r t h e r b i s u l -p h i t e d e c o m p o s i t i o n t o s u l p h u r i c a c i d (21). The a c i d thus formed c a n p r e c i p i t a t e t h e c a l c i u m i o n s as a s u l p h a t e . 2. P h l o b a t a n n i n more d i f f i c u l t y t h a n t a x i f o l i n . T h i s was n o t s u r p r i s i n g , inasmuch as t h e p h l o b a t a n n i n m o l e c u l e i s more complex t h a n t a x i f o l i n , and may even be a c o n d e n s a t i o n p r o d u c t o f t h e f l a v a n o n e . S o l u b i l i t y d e c r e a s e d w i t h i n c r e a s i n g m o l e c u l a r The p h l o b a t a n n i n f r a c t i o n d i s s o l v e d i n a l c o h o l w i t h 5 0 weights among the t a x i f o l i n , phlobatannin and phlobaphene f r a c t i o n s . The differences i n t o x i c i t y betv/een t a x i f o l i n and phlobatannin indicated that f u n g i c i d a l a c t i v i t y also decreased with increasing molecular weights. The most highly condensed molecule i n t h i s s e r i e s , the phlobaphene complex, was not tested for t o x i c i t y . Its high degree of i n s o l u b i l i t y i n a l l solvents suggested that i t would have very limited f u n g i c i d a l properties. This i s further evidenced by the f a c t that native l i g n i n i s usually associated with the phlo-baphene complex ( 8 , 1 9 ) . A native l i g n i n f r a c t i o n has been is o l a t e d from most woods, including those having low natural d u r a b i l i t y . This implies that i n a l l p r o b a b i l i t y , native l i g n i n does not function as a fungicide. An independent inves t i g a t i o n on a Douglas f i r sample had revealed that the t a x i f o l i n concentration was four times greater i n the outer heartwood than i n the central p i t h area ( 2 5 ) . The possible r e l a t i o n s h i p between t a x i f o l i n and phlo-batannin has been previously discussed. I t was f e l t that the low concentration of t a x i f o l i n i n the inner heartwood might have been the r e s u l t of i t s being slowly transformed to phlo-batannin over a period of years. The t a x i f o l i n produced i n the outer heartwood might have been formed too recently to condense to any extent. Consequently, a rough quantitative t e s t f o r phlobatannin was made to see i f the above was, indeed the case. Samples from the outer and inner heartwood were 51 extracted with ether, and the phlobatannin was i s o l a t e d i n ethyl acetate solution i n the customary way. Douglas f i r and redwood phlobatannin had previously been shown to absorb u l t r a -v i o l e t l i g h t strongly i n a wave length of 280 mu ( 8 , 1 9 ) . Spectophotometric analysis of the two phlobatannin extracts subsequently showed the r a t i o of tannin i n the outer and inner heartwood to be 5'3» From these r e s u l t s , i t would appear that t a x i f o l i n i s not converted to tannin, i f i t i s assumed that the amount of t a x i f o l i n o r i g i n a l l y produced i s constant throughout the l i f e of the tree. Unfortunately, there are no precise quantitative methods available f o r better tannin comparisons. The foregoing observations are based on only one sample, and thus provide only an i n d i c a t i o n at best. I t i s s t i l l quite possible that t a x i f o l i n may condense to phloba-tannin, but was not detected because of further condensation to phlobaphene. Fundamental work i s needed i n t h i s f i e l d to d e f i n i t e l y e s t a b l i s h the d i s t r i b u t i o n pattern of these three related substances. 3 . Free Acids Growth curves of Fomes annosus i n malt agar with the acids included are presented i n F i g . 1 3 , Appendix A. The importance of correcting f o r r e s t period i s f o r c i b l y i l l u s -trated here. Growth i n a concentration of 0.8 per cent acid did not s t a r t u n t i l two days afte r growth began i n the one 52 per cent dishes. Once started, however, i t s faster rate of 7 growth produced a curve of higher slope. Each of these con-centrations were corrected for rest period i n the usual way. The curve representing the lower concentration was then found to f a l l above that of the higher. These corrected curves are i l l u s t r a t e d by dotted l i n e s i n F i g . 13, Appendix A. 4. Neutrals The concentration of the neutral ether f r a c t i o n required to completely i n h i b i t growth varied widely, depend-ing on the organism involved. F. annosus i s only moderately r e s i s t a n t , since the T.I.P. was determined as 1 .4 per cent. Lentinus lepideus apparently i s very r e s i s t a n t , requiring a calculated concentration of almost f i v e per cent (Table 1.) This organism i s also unusually r e s i s t a n t to o i l - s o l u b l e preservatives such as creosote ( 9 ). Like the neutral f r a c -t i o n , creosote contains a heterogeneous mixture of water-insoluble, high molecular-weight hydrocarbons (42). 5. Water Solubles The carbohydrate f r a c t i o n apparently did neither support nor hinder fungal development i n p l a i n agar. There-fore, the water-soluble f r a c t i o n from Douglas f i r seems to function neither as a food source nor as a toxicant to fungi. Since the hydrolyzate was 90 per cent galactose, i t can be concluded that the carbohydrate i s largely a galactan ( 5 0 ) . 53 Galactans, unlike mannans and pentosans are quite r e s i s t a n t to attack by wood-destroying fungi ( 7 ) . 6 . Zinc Chloride The concentrations of zinc chloride necessary to , i n h i b i t growth agree well with data previously reported by Richards (41). L_. lepideus i s extremely sensitive to zinc chloride; a concentration of less than 0 . 1 per cent i s suf-f i c i e n t to k i l l the fungus. The T.I.P. of 0 . 1 6 per cent attained with Fomes annosus represents the average sensivity of the majority of wood-destroying fungi. A t a x i f o l i n con-centration of three times this l a t t e r amount i s needed to prevent growth of F. annosus i n malt agar. Even higher mul-t i p l e s of the other extraneous materials are required to i n h i b i t fungal growth. The n a t u r a l l y occurring extractive materials, however, may eventually prove superior to zinc chloride i n c e r t a i n instances. Due to i t s water soluble properties, the s a l t leaches out of treated timbers under wet exposure conditions (22). The water-insoluble nature of the f u n g i c i d a l Douglas f i r extractives permits more perm-anent protection than i s afforded by zinc chloride. 54 V. CONCLUSION The s p e c i f i c concentrations of wood extractives needed to i n h i b i t growth i n malt agar should not be i n t e r -preted as representing the amount of material that must necessarily be present i n wood to prevent fungal growth. Rather, they serve to indicate the r e l a t i v e importance that a component may play i n protecting wood from decay. On the basis of the data c o l l e c t e d i n t h i s study, i t appears that t a x i f o l i n may be l a r g e l y responsible for the moderate decay resistance of Douglas f i r . Since the discovery of the f u n g i c i d a l properties of pinosylvin and i t s monomethylether, i t has been suggested that trees containing high concentrations of these substances evenly d i s t r i b u t e d throughout the heartwood should be used for breeding purposes ( 1 3 ) . The lumber produced from such strains would be inherently more durable. In order for t h i s work to proceed, a simple colorimetric test has been devel-oped f o r the semi-quantitative estimation of pinosylvin i n the f i e l d ( 1 3 ) . I f a comparable test were available for the quantitative analysis of t a x i f o l i n , Douglas f i r trees with high amounts of t h i s flavanone could be selected f o r r a c i a l improvement. Work i s now i n progress to estimate t a x i f o l i n q uantitatively from methanol extracts of Douglas f i r ( 3 ) . Nevertheless, a more rapid means of estimation i n the f i e l d i s s t i l l needed, since the above method i s inapplicable to . 55 wood i n toto. Western lar c h (Larix occidentalis Nutt.) has also been reported to contain t a x i f o l i n ( 1 7 ) . Heartwood of this species, although somewhat easier to penetrate than I n t e r i o r -type Douglas f i r , s t i l l does not accept preservatives e a s i l y ( 3 0 ) . Therefore, i n both I n t e r i o r Douglas f i r and western la r c h , t a x i f o l i n w i l l most often be the main deterrent to decay. T a x i f o l i n concentration should be considered when lumber derived from these species i s to be used under condi-tions where wood-destroying fungi might be a c t i v e . Chemical stimulation i s being employed to increase r e s i n production i n the southern pines (32). A series of papers w i l l soon be published on other methods of stimulating trees to produce greater amounts of extraneous materials (.1). From such studies, economical methods may be developed to grow trees with greater proportions of f u n g i c i d a l extrac-t i v e s . 56 LITERATURE CITED 1. Anderson, A.B. Personal correspondence. Feb. 16, 1955« 2. Ascorbe, F.J. The inhibitory action of organic chemicals on a blue-stain fungus. Caribbean For. 14: I 3 6 - I 3 9 . 1953. 3. Barton, G.M., and J.A.F. Gardner. Work in progress. Vancouver Branch, Forest Products Laboratory of Canada. 1955. 4 . Barton, G.M., and J.A.F. Gardner. The chemical nature of the acetone extractive of western red cedar. Pulp and Pap. Mag. of Can. 55: I 3 2 - I 3 7 . 1954. 5 . Bateman, E. The effect of concentration on the toxicity of chemicals to living organisms. U.S. Dept. Agr. Tech. Bul l . 346. 1933. 6. Boyce, J.S. Forest pathology, 2nd edition. McGraw-Hill Book Co., Inc., New York. 1948. 7. Brown, H.P., A.J. Panshin, and C.C. Forsaith. Textbook of wood technology, Vol. II. McGraw-Hill Book Co. Inc., New York. 1952. 8. Buchanan, M.A., H.F. Lewis, and E.F. Kurth. Chemical nature of redwood tannin and phlobaphene. Ind. and Eng. Chem. 36: 907-910. 1944. 9. Cartwright, K. St. G., and W.P.K. Findlay. Decay of timber and i t s prevention. His Majesty's Stationery Office, London. 1946. 10. Cramer, F. Paper chromatography. MacMillan & Co., Ltd., London. 1954. 11. Dept. of Lands and Forests, Province of Bri t i s h Columbia. Report of the forest service, year ended Dec. 31? 1953. 1954. 12. Erdtman, H. Personal correspondence. Oct. 20, 1954. 13. Erdtman, H., A. Frank, and G. Linstedt. Constituents of pine heartwood XXVII: The content of pinosylvin phenols in Swedish pines. Svensk Papperstidn. 54: 275-279. 1951. 14. Findlay, W.P.K. Influence of sample size on decay rate of wood in culture. Timber Tech. 6 l : 160-162. 1953. 57 15. F i n h o l t , R.W. Improved toximetric agar-dish test for evaluation of wood preservatives. Anal. Chem. 2 3 : I O 3 8 - I O 3 9 . 1951. 16. F i n h o l t , R.W., M. Weeks, and C. Hathaway. New theory on wood preservation. Ind. and Eng. Chem. 4 4 : 101-105. 1 9 5 2 . 17. Gardner, J.A.F. Unpublished observation. 18. Gortner, R.A. Outlines of biochemistry, 3 r ^ e d i t i o n . John Wiley & Sons, Inc., New York. 1 9 4 9 . 19. Graham, H.M., and E.F. Kurth. Constituents of extractives from Douglas f i r . Ind. and Eng. Chem. 41: 400-414. 1 9 4 9 . 2 0 . Hawley, L.F., L.C. Fleck, and C.A. Richards. The r e l a t i o n between d u r a b i l i t y and chemical composition i n wood. Ind. and Eng. Chem. 16: 699-70O. 1 9 2 4 . 2 1 . Hoge, W.M. The resistance of Douglas f i r to sulphite pulping. Tappi 37: 3 6 9 - 3 7 4 . 1 9 5 4 . 2 2 . Hunt, G.M., and G.A. Garratt. Wood preservation. McGraw-Hill Book Co., Inc., New York. 1 9 3 8 . 2 3 . Isenberg, I.H., M.A. Buchanan, and L.E. Wise. Extraneous components of American pulpwood. Pap. Ind. and Pap. Trade 28: 8 1 6 - 8 2 2 . 1 9 4 6 . 24. Johnson, C.H., and R.A. Cain. The wood o i l of Douglas f i r . J . Am. Pharm. Assoc. 26: 6 2 3 - 6 2 5 . 1 9 3 7 . 2 5 . Kennedy, R.W. Unpublished data. 2 6 . Kingston, J.T.E. S t a t i s t i c a l record of the lumber industry i n B r i t i s h Columbia. Bur. of Econ. and Stat., Dept. of Trade and Ind. 1 9 5 5 . 27. Kurth, E.F. Separation of wood extractives into simpler components. Ind. and Eng. Chem., Anal. Ed. 1 1 : 2 0 3 - 2 0 5 . 1939-. 2 8 . Kurth, E.F. Chemical analysis of western woods, part I. Pap. Trade J . 1 2 6 : 56-57- 1 9 4 8 . 2 9 . L e u t r i t z , J . A wood-soil contact culture technique for laboratory study of wood-destroying fungi, wood decay and wood preservation. B e l l System Tech. J . 25: 1 0 2 - 1 3 5 . 1 9 4 6 . 58 3 0 . MacLean, J.D. Preservative treatment of wood, by pressure methods. U.S. Dept. Agr. Handb. no. 40. 1 9 5 2 . 3 1 . Mulholland, J.R. Changes i n weight and strength of Sitka spruce associated with decay by a brown-rot fungus, Poria monticola. J . For. Prod. Res. Soc. 4: 410-4lrST 1954. 3 2 . Panshin, A.J., E.S. Harrar, W.J. Baker, and P.B. Proctor, Forest products. McGraw-Hill Book Co., Inc., New York. 1 9 5 0 . 33* Pew, J.C. A flavanone from Douglas f i r heartwood. U.S. For. Prod. Lab. Rept. no. R1692. 1 9 4 7 . 34. Pew, J.C. A flavanone from Douglas f i r heartwood. J . Am. Chem. Soc. 7 0 : 3 0 3 I - 3 0 3 4 . 1948. 35* Pew, J.C. Douglas f i r heartwood flavanone: Its properties and influence on sulphite pulping. Tappi 3 2 : 39-44. 1 9 4 9 . 3 6 . Quenouille, M.H. Introductory s t a t i s t i c s . Butterworth-Springer Ltd., London. 1 9 5 0 . 3 7 . Rennerfelt, E. Personal correspondence. March 2, 1 9 5 5 . 3 8 . Rennerfelt, E. Die T o x i z i t a t der phenolischen Inhaltsstoffe das Kiefernkernholzes gegenliber einigeh F a u l n i s p i l z e n . Svensk Bot. Tidskr. 3 7 : 8 3 - 9 3 . 1 9 4 3 . 3 9 . Rennerfelt, E. The t o x i c i t y of the phenolic extractives of pine heartwood i n regard to some decay fungi. Translation No. 8 , Faculty of Forestry, University of B r i t i s h Columbia. 1 9 5 3 . 40. Rennerfelt, E. The influence of the phenolic compounds i n the heartwood of Scots pine (Pinus s i l v e s t r i s L.) on the growth of some decay fungi i n nutrient solution. Svensk Bot. Tidskr. 3 9 : 3II - 3 1 8 . 1 9 4 5 . 41. Richards, C.A. Comparative resistance of eighteen species of wood destroying fungi to zinc chloride. Proc. Am. Wood Preservers 1 Assoc., 21st Ann. Meeting. 18-22. 1 9 2 5 . 42. Roche, J.N. Coal tar creosote: Its composition and how i t functions as a wood preservative. Koppers Co., Inc., Pittsburgh. 1 9 5 2 . 59 4 3 . Roff, J.W., and J.M. Atkinson. T o x i c i t y tests of a water-soluble phenolic f r a c t i o n ( t h u j a p l i c i n - f r e e ) of western red cedar. Can. J . Bot. 32: 308-309. 1 9 5 4 . 44. Russell, A. The natural tannins. Chem. Revs. 17: 155-186. 1 9 3 5 . 4 5 . Schmitz, H. A suggested toximetric method f o r wood preservatives. Ind. and Eng. Chem., Anal. Ed. 2 : 3 6 I - 3 6 3 . 1 9 3 0 . 4 6 . Schorger, A.W. The oleoresin of Douglas f i r . J . Am. Chem. Soc. 39: 1040-1044. 1917. 4 7 . Sherrard, E.C., and E.F. Kurth. The d i s t r i b u t i o n of extractive i n redwood: Its r e l a t i o n to d u r a b i l i t y . U.S. For. Prod. Lab. Rept. R 9 8 8 . 1 9 3 3 . 48. Smith, D.N. The natural d u r a b i l i t y of timber. For. Prod. Res. Rec. No. 3 C His Majesty's Stationery O f f i c e , London. 1 9 4 9 . 49. Southam, CM., and J . E h r l i e h . E f f e c t s of extract of western red cedar heartwood on ce r t a i n wood-decaying fungi i n culture. Phytopath. 33: 5 1 7 - 5 2 4 . 1 9 4 3 . 50. Thompson, J.O., J . J . Becher, and L.E. Wise. A physio-chemical study of a water-soluble polysaccharide from Douglas f i r (Pseudotsuga t a x i f o l i a ) . Tappi 36: 3 1 9 - 3 2 4 . 1 9 5 3 . 51. U.S. Dept. Agr., For. Prod. Lab. Wood handbook. U.S. Govt. P r i n t i n g O f f i c e , Washington. 1940. 52. Varner, R.W., and R.L. Krause. Agar-block and s o i l -block methods for testing wood preservatives. Ind. and Eng. Chem. 4 3 : 1 1 0 2 - 1 1 0 7 . 195.1. 53. Wise, L.E. Wood chemistry. Reinhold Publishing Corp., New York. 1 9 4 6 . 5 4 . Wise, L.E., and E.C. Jahn. Wood chemistry, 2nd ed. Vol. I and I I . Reinhold Publishing Corp., New York. 1952, 55* Wolcott, G.N. Personal correspondence. Feb. 7, 1 9 5 5 . 56. Wolcott, G.N. Stilbene and comparable materials f o r dry-wood termite cont r o l . J . Econ. Ent. 4 6 : 3 7 4 - 3 7 5 . 1 9 5 3 . Zabel, R.A. Variations i n the decay resistance of white oak. N.Y. State Co l l . of For. Tech. Pub. 6 8 . 1 9 4 8 . APPENDIX A Growth and t o t a l i n h i b i t i o n point curves °f Forces annosus and Lentinus lepideus i n malt agar containing various concentrations of extractives Note: The l i n e a r r e l a t i o n s h i p between log per cent retardation of growth and log per cent concentration holds only between the T.I.P. and one-third of that concentration. Hence points representing some lower concentrations were not considered i n f i t t i n g the s t r a i g h t l i n e s . Figure 1. Rate of growth of Fomes annosus on malt agar containing d i f f e rent concentrations of t a x i f o l i n . (1% a l cohol = cont ro l (0.00$ t a x i f o l i n ) Figure 2. Retardation of growth of Fomes annosus on malt agar containing d i f fe rent concentrations of t a x i f o l i n . T I M E I N D A Y S Figure 3. Rate of growth of Lentinus lepideus on malt agar containing different concentrations of taxifo l i n . 1% alcohol = control (0.00$ taxifolin) Figure 4. Retardation of growth of Lentinus lepideus on malt agar containing different concen-trations of tax i f o l i n . Figure 5. Rate of growth of Fomes annosus on malt agar con-taining different concentrations of phlobatannin. 1% alcohol = control for 0.2% and 0.5% phlobatannin 4% alcohol = control for 1% phlobatannin Figure 6 . Retardation of growth of Fomes annosus on malt agar containing different concentrations of phlobatannin. 3-A TIME IN DAYS Figure 7. Rate of growth of Lentinus lepideus on malt agar containing different concentrations of phlobatannin. 1% alcohol = control Figure 8. Retardation of growth of Lentinus lepideus on malt agar containing different concentra-tions of phlobatannin. • 2 . 4 .6 .8 1.0 1.5 2.0 PER CENT CONCENTRATION OF PHLOBATANNIN Figure 9. Rate of growth of Fomes annosus on malt agar containing different concentrations of neutrals. 1% ether = control Figure 10. Retardation of growth of Fomes annosus on malt agar containing different concentrations of neutrals. 3-A 0 2 4 6 8 10 12 14 16 18 T I M E IN D A Y S P E R C E N T CONCENTRATION OF N E U T R A L S Figure 11. Rate of growth of Lentinus lepideus on malt agar containing different concentrations of neutrals. 1% ether = control Figure 12. Retardation of growth of Lentinus lepideus on malt agar containing different concentrations of neutrals. TIME IN DAYS Figure 13. Rate of growth of Fomes annosus on malt agar containing different concentrations of free acids. 1% ether = control for 0.05$, .1% and .2% acids 2% ether = control for .4%, .6%, .8% and 1.0% acids Broken line represents data corrected for rest period. Figure 14. Retardation of growth of Fomes annosus on malt agar containing different concentrations of free acids. 0 2 4 6 8 10 12 14 16 18 T I M E IN DAYS PER C E N T CONCENTRATION OF F R E E ACIDS Figure 15. Rate of growth of Lentinus lepideus on malt agar containing different concentrations of free acids. 1% ether = control Figure 16. Retardation of growth of Lentinus lepideus on malt agar containing different concentrations of free acids. 3 - , TIME IN DAYS Figure 17. Rate of growth of Fomes annosus on malt agar containing different concentrations of zinc chloride. Figure 18. Retardation of growth of Fomes annosus on malt agar containing different concentrations of zinc chloride. 9-A T I M E IN DAYS Figure 19. Rate of growth of Lentinus lepideus on malt agar containing different concentrations of zinc chloride. Figure 20. Retardation of growth of Lentinus lepideus on malt agar containing different concentrations of zinc chloride. 10-A APPENDIX B Stat i s t i c a l analysis of weight losses in Douglas f i r blocks exposed to Poria incrassata and Lentinus lepideus. Per cent weight losses of extracted wood blocks exposed to action of Lentinus lepideus; Ether 54.58 51.68 43.78 53.12 55.79 Acetone 49.01 55.29 57.40 52.36 47.05 Water 36.01 44.33 50.80 50.90 41.28 Control 39.24 43.93 43.63 23.43 37.70 Transforming these percentages to arc sin j[ percentage, they become: Ether Acetone Water Control 0.84 0.78 0.64 0.67 0.81 0.84 0.73 0.73 0.72 0.86 0.80 0.73 0.82 0.81 0.80 0.50 0.85 0.76 0.69 0.66 = 0.81 0.81 0.73 0.66 = 0.0009 0.0000 0.0081 0.0001 0.0016 0.0009 0.0009 0.0025 0.0000 0.0025 0.0081 0.0000 0.0049 0.0049 0.0016 0.0001 0.0049 0.0049 0.0256 0.0000 0.0107 0.0068 0.0195 0.0355 2-B t = x x - x 2 — 2 — 2 (x - x x) + (x - x 2) n l + n2 - 2 1_ + 1_ n- n, Between ether and control: t = 0.81 - 0.66 i— — .0107 + .0355 2 = 8 5 — _ 3.12' T..05 = 2.31 t,01 = 3 ' 3 6 Between acetone and control: t = 0.81 - 0.66 .0068 + .0355 8 2 5 Between water and control: t = 0.73 - 0.66 3.26' .0195 + .0355 2 = 1,33 8 5 -3-B Per cent weight losses of extracted wood blocks exposed to action of Poria incrassata: Ether Acetone Water Control 30.19 23.85 23.89 20.78 24.72 20.78 24.79 29.58 26.70 24.51 24.93 29.85 32.81 40.64 20.49 12.25 36.22 15.52 26.46 24.48 Transforming these percentages to arc sin ypercentage, they become: Ether 0.58 0.51 0.51 0.48 0.52 x = 0.52 (x-x) 2 = 0.0036 0.0001 0.0001 0.0016 0.0000 Acetone 0.48 0.52 0.58 0.55 0.52 0.53 0.0025 0.0001 0.0025 0.0004 0.0001 Water 0.52 0.58 0.61 0.69 0.46 0.57 .0025 .0001 .0016 .0144 .0121 Control 0.35 0.64 0.41 0.54 0.51 0.49 0.0196 0.0225 0.0064 0.0025 0.0004 0.0054 0.0056 .0307 0.0514 Between ether and control: 4-B t = 0.52 - 0.49 = <1 .0054 + .0514 8 Between acetone and control:: t = 0.53 - 0.49 = < 1 .0056 + .0514 8 Between water and control; t = 0.57 - 0.49 1.25 .0307 + .0514 8 

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