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Mechanism of induced disease resistance in the bark and sapwood of western redcedar Parker, William Harrison 1972

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A MECHANISM OF INDUCED DISEASE RESISTANCE IN THE BARK AND SAPWOGD DF WESTERN REDCEDAR by WILLIAM HARRISON PARKER B.A., Reed College, 1?68 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Forestry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY GF BRITISH COLUMBIA January, 1972 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8, Canada Date J C V ^ ' 7; U? i i ABSTRACT Samples of sapwood and bark of western redcedar were collected at 3 day to 6 week intervals after injury and extracted with water, chloroform and acetone. Extracts were tested for the presence of some common heart-wood compounds and in vitro fungi toxic properties. Extracted samples col-lected 6 weeks after injury were inoculated with a decay fungus, and the resulting weight losses determined. No heartwood compounds were detected in any extracts, and no extracts were fungi toxic in vitro. Weight losses f o l -lowing decay of extracted chips indicated that decay resistance was i n i t i -ated in the bark and sapwood. Thus, these tissues possess a mechanism of disease resistance induced by injury. It i s concluded that this resistance results from the deposition of a toxic substance that i s unextractable with water, chloroform, or acetone. The alteration of sapwood, i f not the bark, is analogous in certain respects to the formation of reaction zones in the sapwood of various trees, since these zones are induced by injury and are char-acterized by abnormal toxin formation. However, the toxins formed in other trees are normal heartwood constituents, and in -this respect apparently not parallel to the toxic substance induced in western redcedar. i i i TABLE OF CONTENTS Abstract i i List of Tables i v List of Figures f v Acknowledgement i?vi Introduction 1 Literature Review 3 A. Induced Disease Resistance i n Herbaceous Plants 3 1. Hypersensitive Reaction 3 2. Phytoalexin Response 5 B. Disease and Decay Resistance of Trees 6 C. Induced Disease Resistance i n Trees 10 D. Comparison of Sapwood Reaction Zones with Heartwood lU E. Comparison of Reacted Sapwood with Mechanisms of Induced Disease Resistance 15 F. Summary of Investigations Made on Western Redcedar 17 Materials and Methods 23 Results 31 Discussion 37 Literature Cited 1(3 i v LIST OF TABLES TABLE I Composition of the V o l a t i l e O i l of Western Redcedar TABLE I I Summary of Sapwood and Bark Collections TABLE I I I Sample Weights, Extract Weights and Percentages of Extractives TABLE IV Weight Losses after Decay of 6 Week Samples TABLE V Comparison of Weight Losses after Decay V LIST OF FIGURES FIGURE I Methods Used to Detect Formation of Reaction Zones 25 v i ACKNOWLEDGEMENT I wish to thank: Dr. B.J. van der Kamp for guiding t h i s investigation with patience; Drs? R.J. Bandoni, J.A.F. Gardner, J.P. Kimmins and B. Mullich f o r thei r valuable assistance; My wife Jane f o r her encouragement and e d i t i n g . I INTRODUCTION When resistant plant tissue i s invaded by a pathogenic organism, i t commonly responds by producing one or more toxic compounds which bring about localization or death of the invading organism. This response involves alter-ation of normal metabolism resulting usually i n the death of the host cells involved. I f the toxic compounds formed are oxidation products of compounds existing normally i n the tissue, or i f the level of a preexisting toxic com-pound increases, the result i s termed a hypersensitive reaction. I f the tox-i c compounds formed are riot normally found i n the plant, the result i s a phy-toalexin response. Both classes of response are examples of induced chemi-cal disease resistance which provide protection against pathogens not pos-sessing specific counter mechanisms to avoid e l i c i t a t i b n or to degrade the toxins. Many tree species contain extractable substances i n the heartwood zone which are toxic to pathogenic fungi i n v i t r o . Decay resistance, and hence durability of wood i n use, corresponds roughly to the amounts and toxicity of the extractable toxins present. Sapwood possesses no toxic extractives, and as lumber i t i s much less decay resistant than most heartwood. However, even when exposed by injury, living sapwood i s often resistant to decay and dis-ease. This indicates that a mechanism which prevents disease and decay i s pre-sent i n resistant sapwood. The conversion of sapwood to heartwood i s somewhat analogous to the production of phytoalexins, i n that fungitoxic compounds are produced upon or after the death of ray bells. The sapwood of various angiosperm trees and species i n the Pihaceae forms reaction zones characterized by the synthesis of fungitoxic compounds when subjected to an irregular stimulus such as wound--2 ing and subsequent fungal inoculation (Hart & Johnson, 1970$ Jorgensen, 196lj Shain, 197i). In a l l investigated cases this altered sapwood contains toxic extractives normally found only i n the heartwood. The formation of reaction zones i n sapwood has been termed 'protection wood* by Jorgensen ( 1 9 6 I ) , •phytoalexin production' by Shain ( 1 9 6 7 ) , and i s an example of induced chemical disease resistance. This thesis attempts to determine whether disease resistance i n the sapwood and bark of western redcedar can be explained by phenomena similar to those lis t e d above for other trees. Western redcedar was selected for investigation because a great deal of work has been done on the chemistry of i t s heartwood extractives, certain of which have very high i n vitro fungitox-i c i t i e s . I t has already been shown that these toxic compounds do not normally occur i n the bark or sapwood (Gardner, x962), and a technique i s available for the detection of some of these extractives i n minute quantities (Maclean & Gardner, 1956). A demonstration of induced chemical resistance i n the bark or sapwood of western redcedar would indicate that such reactions occur i n gymnosperms outside the Pineaceae. A demonstrated absence of such a mechanism would indicate that these tissues r e s i s t disease by some other means. -3-LITERATURE REVIEW A. Induced Disease Resistance i n Herbaceous Plants 1. Hypersensitive Reaction s Interactions between host and pathogen are either susceptible or resistant. A susceptible interaction leads to disease, while a resistant interaction does not. Physical barriers or unfavorable conditions for pathogen survival lead to resistant interactions. In many cases unfavorable conditions are generated by the host after a pathogen becomes established. These changes result i n pathogen localization. This 'type of response i s dynamic and i s a 'hypersensitive reaction 1. I t i s extremely widespread and can be i n i t i a t e d i n most, i f not a l l , higher plants (Goodman et a l . , 1967). The Hypersensitive reaction, according.to Muller (19ii9), encompasses a l l morphological and histological changes 'that, when produced by an infectious agent, e l i c i t the premature dying off or necrosis of the infected tissue as well as inactivation and localization o f the infeectious agent." Once the reaction i s i n i t i a t e d , localized lesions develop which progressively darken. The reaction i s e l i c i t e d by mechanical stimuli as.well as by invasion. Both types of stimuli are somewhat similar i n a microscopic level, since individual c e l l s are damaged by mechanical wounding and hyphal intrusion. In either case, the reaction i s a disease resistance mechanism, since wounding results i n exposure of l i v i n g tissue. Exposure leads to inoculation by ubiquitous fungal spores. Since the hypersensitive response i s widespread, susceptibility to disease depends upon how quickly host metabolism alters after invasion. Alteration of normal systems i s apparently irreversible, since the affected host cells ultimately die. -h-Rahe et a l . (1969) have shown that e l i c i t a t i o n of a hypersensitive response by inoculation with non-pathogenic fungi of Phaseolus v u l g a r i s L. confers resistance to subsequent infections of bean anthracnose. This r e s u l t indicates that i n f e c t i o n success by i h i s pathogen depends upon i t s a b i l i t y to avoid or delay e l i c i t a t i o n of the hypersensitive reaction. Phenolic compound oxidation i s implicated i n the hypersensitive reac-tion since the a c t i v i t y of polyphenoloxidase correlates with i n t e n s i t y of reaction (Rubin et a l . , 1959). Also i t has been shown that oxidized phenolic compounds accumulate i n affected tissues of plants undergoing hypersensitive responses (Goodman e t a l . , 1967). Oxidized phenolic compounds, such as the aglycones phloretin and hydroquinone, are more toxic i n v i t r o than t h e i r r e -duced forms, the glycosides p h l o r i d z i n and arbutin (Ku^, 1967). However, even when present i n oxidized forms i n affected c e l l s , the concentrations of such compounds are only 1-10 percent of that required to produce an i n v i t r o fungi-s t a t i c e f f e c t (Tomiyama et a l . , 1967). This observation jbmplies one of three p o s s i b i l i t i e s : 1) additional factors contribute to l o c a l i z a t i o n ; 2) the i n vivo t o x i c i t i e s of the compounds are not the same as those i n v i t r o ; or 3) additional amounts of toxic compounds are transported from neighboring c e l l s a f t e r a c e r t a i n period of time. Tomiyama et a l . (1967) has calculated that transport of 30 percent of the phenolic compounds from adjacent c e l l s i n t o af-fected c e l l s would r e s u l t i n a f i v e - f o l d increase i n phenolics. He further calculated that transport from two c e l l layers bordering the affected c e l l s would y i e l d a twenty-fold increase, which would explain f u n g i s t a t i c r e s u l t s . Although not necessarily v a l i d a t i n g Tomiyama's theory, the following studies suggest that a transport system e x i s t s : 1) Sydow and Durbin (1962) have shown that "^ C from labeled phenolic compounds was transferred and accumulated at the s i t e of i n f e c t i o n i n rust-infected leaves of susceptible wheat plants. -5-2<)i Benda (1959) has observed nuclear movement and protoplasmic streaming t o infected c e l l s . Many phenolic compounds are involved i n hypersensitive reactions. Much of -the work demonstrates that amounts of preexisting phenolic substances i n -crease after the reaction has taken place. Examples of such compounds are caf-f e i c acid, chlorogenic a c i d , and the coumarins umbelliferone and scopoletin. A l l of these increase i n concentration a f t e r i n j u r y or inoculation of sweet potato leaves (Ku^, 196U). 2. Phytoalexin Response Phytoalexins are compounds conferring disease resistance which are syn-thesized de novo after i n j u r y or inoculation (sometimes only a f t e r inoculation) and are not present (within l i m i t s of detection) i n healthy plants (Cruick-shank, 1963). Although differences between phytoalexins and compounds involved i n hypersensitive reactions are present by d e f i n i t i o n , i n practice i t i s some-times impossible to c l e a r l y distinguish these two categories of compounds (Tomiyama et a l . , 1967). I n same cases, necrosis of host tissue may not occur a f t e r phytoalexin production (Cruickshank, 1963), whereas the development of necrotic lesions i s an inv a r i a b l e symptom of a hypersensitive reaction. The compounds p i s a t i n from peas, t r i f o l i r h i z i n from red clover, and phaseollin from beans are phytoalexins. A l l of these compounds are isoflavones. Cruickshank recognizes three other compounds as phytoalexins: orchinol, which i s apparently a stilbene and i s produced i n orchid leaves; isocoumarin, which i s apparently derived from acetate and i s produced i n carrot tissue} and i p o -meamarone, which i s synthesized by sweet potato roots and i s probably a sesqui-terpene. Ipomeamarone i s apparently the only terpenoid shown to have a r o l e i n disease resistance i n herbaceous plants. -6-Contention e x i s t s over the r e l a t i v e contribution of induced toxic sub-stances to disease resistance. This contention arises i n part from poorly-designed experimental work which does not j u s t i f y the conclusion drawn. For instance, the presence of greater amounts of phenolic compounds, or abnormal compounds, i n diseased plants does not necessarily demonstrate that these compounds are involved i n vivo i n disease resistance. Goodman et a l . (196?) has suggested that t h i s d i f f i c u l t y can be eliminated by testing plants with v i r u l e n t and avirulent strains of the same pathogen. Host int e r a c t i o n with the avirulent s t r a i n i n i t i a t e s the factors which impart host resistance, while i n f e c t i o n by the avirulent s t r a i n does not. A comparison of the two cases should disclose the nature of the resistance. The recipr o c a l s i t u a t i o n i s also u s e f u l . Isogenic l i n e s ( i s o l i n e s ) of a given plant d i f f e r i n g only i n a single gene f o r resistance are inoculated with a pathogen and i n f e c t i o n r e -s u l t s i n one case but not i n the other (Mace & Veech, 1971). In t h i s case also, the nature of the resistance should be disclosed by a comparison of the two interactions. B. Disease and Decay Resistance of Trees The mechanisms of disease resistance found i n herbaceous plants are also operative i n trees. However, because of the growth habit of trees, t i s -sue d i s i n t e g r a t i o n (decay) of wood i s an important part of disease, whereas i n herbaceous plants, tissue disintegration i s usually a secondary fa c t o r , not caused by the primary pathogen. Thus, i t has been important for f o r e s t pathologists to determine the factors which impart decay resistance to wood. Such investigations have been made f o r l i v i n g trees where decay causes disease and f o r wood used as lumber where decay causes f a i l u r e i n service. Due to economic factors, r e l a t i v e d u r a b i l i t i e s of woods have long been -7-recognized and taken i n t o consideration when determining t h e i r uses. Since d u r a b i l i t y i n use i s approximately synonymous with decay resistance, much i n -formation has been amassed r a t i n g r e l a t i v e decay resistances. Scheffer and Cowling (1966) rate trees as non-resistant to highly decay r e s i s t a n t . However, i t i s imprtant to note that t h i s scale i s misleading i f applied to a l l plant tissues. Actually non-resistant wood i s considerably more r e s i s t a n t to decay than other types of non-woody plant tissues because of the chemical structure of wood. Wood always consists of c e l l w a lls composed of a l i g n i n - c e l l u l o s e complex. This complex i s comparatively r e s i s t a n t to decay because few organ-isms possess enzymes which enable them to degrade l i g n i n . The composition of wood i s subdivided i n t o three a r t i f i c i a l categories based upon chemical properties. A certain amount of material i s removed from wood treated with polar solvents; additional material i s removed by treatment with strong acids leaving an unaltered residue. Although the correspondence i s not perfect, the following generalizations are made: fThe residue remaining after both treatments i s l i g n i n . The f r a c t i o n removed by strong acids i s c e l -l u l o s e . The material removed by polar solvents comprises nearly everything else found i n wood and i s defined as 'extractable substances 1 (Brown et a l . , 191*9). Although a l l woods have the same basic l i g n i n - c e l l u l o s i c structure, de-cay resistances of woods are variable. Thus, an addit i o n a l factor(s)&must be involved. Scheffer and Cowling (1966) state that "... toxic extractable sub-stances deposited during the formation of heartwood are the p r i n c i p l e source of decay resistance i n wood." This conclusion i s based upon three observations: 1) Extracts from heartwood often possess an i n v i t r o fungal t o x i c i t y , whereas sapwood extracts from the same trees do not. 2) Decay resistance of durable heartwood can be greatly reduced or destroyed by extraction with polar solvents. -8-3) The i n v i t r o t o x i c i t y of heartwood extractives of various species corre-lates roughly with the known d u r a b i l i t y i n service of these species. These generalizations apply to both angiosperms and gymnosperms. Specific heartwood extractive substances with recognized fungitoxic properties are nearly a l l aromatic. Most of these substances are phenolic compounds synthesized v i a the shikimic acid pathway} however, a c e r t a i n num-ber are terpenoid i n nature. Some of the compounds a r i s i n g v i a the shikimic acid pathway are dihydroquercetin or t a x i f o l i n (a flavonoid occurring i n Doug-l a s - f i r ) , pinosylvin and pinosylvinmonomethyl ether (stilbenes occurring i n pine). Many mono- and sesquiterpenes occurring throughout the Cupressaceae and c e r t a i n other gymnosperm taxa have high i n v i t r o fungitoxic properties. Heartwood extractives are synthesized by l i v i n g parenchymatous c e l l s at the sapwood-heartwood transformation ( H i l l i s , 1962). One school of thought i s that carbohydrates already present provide the materials for t h i s synthe-s i s . But the amount of extant carbohydrates i n some trees i s not s u f f i c i e n t to account for the observed amounts of deposited extractives ( H i l l i s , 1962). Therefore, additional carbohydrates may be transported to the sapwood-heart-wood t r a n s i t i o n zone. The contributions of s p e c i f i c heartwood extractives to decay resistance have been p a r t i a l l y determined by i n v i t r o t o x i c i t y t e s t s . The conclusions drawn from such tests are probably more v a l i d than those drawn from i n v i t r o t o x i c i t y tests of compounds l i n k e d to induced disease resistance i n l i v i n g plants, since heartwood i s not a true i n vivo s i t u a t i o n . Thus, these tests more nearly approximate conditions i n heartwood than i n l i v i n g t i s s u e . The type of t o x i c i t y t e s t employed has been shown to affect r e s u l t s i n the following case: The heartwood of Pseudotsuga menziesii (Mirb.) Franco (Douglas-fir) i s rated as moderately re s i s t a n t to decay (Scheffer & Cowling, -9-1966). Kennedy (1955) concluded that t a x i f o l i n i s primarily responsible for t h i s decay resistance on the basis of two types of t o x i c i y tests: 1) compar-ison of rates of growth of decay fungi on agar to which various d i f f e r e n t fractions of heartwood extractions had been added; and 2) comparison of weight losses of inoculated heartwood blocks from which different compounds had been removed by extraction with various solvents. Kennedy observed death of h i s te s t fungi at levels varying from O.U-0.6 percent t a x i f o l i n i n agar. Rudman (1962) also tested the t o x i c i t y of t a x i f o l i n by permeating non-decay re s i s t a n t wood meal with varying.concentrations of pure t a x i f o l i n , inoculating t h i s wood meal with decay fungi and then comparing weight "losses. He concludes that t a x i f o l i n i s only s l i g h t l y toxic since 1.0 percent concentration i n the meal, or that concentration which roughly equals the concentration i n heartwood, produced only s l i g h t l y i n h i b i t o r y e ffects upon decay. Rudman concludes that t a x i f o l i n i s not responsible f o r decay resistance of Douglas-fir heartwood and that Kennedy's conclusions are wrong due to a misinterpretation of h i s e v i -dence. Rudman surmises that the poisoned agar t e s t gives misleading r e s u l t s and that selective extraction of heartwood blocks increases water permeability, thereby enhancing decay. Although the poisoned agar t e s t used by Kennedy gives a v a l i d approximation of t o x i c i t y (Cruickshank & P e r r i n , 196U), Rudman's t o x i c i t y t e s t may provide a more v a l i d r e s u l t i n t h i s case since h i s t e s t more closely approximates actual heartwood conditions. However, the c o n f l i c t be-tween the two sets of r e s u l t s i s unresolvable since both t e s t s are contrived situations. I t i s also impossible to determine the v a l i d i t y of either Kenne-dy's or Rudman's conclusions, since one cannot distinguish the effects of water permeability from the t o x i c i t y of i n d i v i d u a l compounds. In addition, other extractives non-toxic to decay organisms i n v i t r o may contribute a synergistic effect i n the heartwood. Therefore, one may conclude that t a x i f o l i n i s toxic -10-t o decay organisms under some c o n d i t i o n s and imparts some decay r e s i s t a n c e t o heartwood. Whether t a x i f o l i n i s the p r i n c i p l e source of decay r e s i s t a n c e i s an open question. C. Induced Disease R e s i s t a n c e i n Trees S t u d i e s of d i s e a s e r e s i s t a n c e i n t r e e s u s u a l l y have a d i f f e r e n t emphasis than s i m i l a r s t u d i e s of r e s i s t a n c e i n herbaceous p l a n t s . Thus, s t u d i e s on nori-l e t h a l f o l i a g e diseases o f t r e e s have produced l a r g e l y d e s c r i p t i v e r e p o r t s which do not apply h y p e r s e n s i t i v e and p h y t o a l e x i n concepts. I t i s c l e a r t h a t l a r g e numbers of angiosperm t r e e s produce h y p e r s e n s i t i v e r e a c t i o n s i n t h e i r f o l i a g e , s i n c e large numbers of 'shothole 1 d i s e a s e s are l i s t e d f o r these trees (Boyce, 1961). The cause of these symptoms becomes c l e a r o n l y when the concept o f the h y p e r s e n s i t i v e r e a c t i o n i s a p p l i e d . I t i s probable t h a t p a r a l l e l exam-p l e s o f induced d i s e a s e r e s i s t a n c e are c h a r a c t e r i s t i c i n the f o l i a g e o f some gymnosperms, but have escaped d e t e c t i o n due t o l a c k of i n v e s t i g a t i o n . Research on f o l i a r disease r e s i s t a n c e has been done on a few i n t e n s e l y * c u l t i v a t e d species. Chlorogehic a c i d has been i m p l i c a t e d i n disease r e s i s t a n c e i n c e r t a i n apple and pear v a r i e t i e s , p a r t i c u l a r l y i n the f r u i t (WJ, 1961*). A l s o , -the i n t e r a c t i o n between apple t r e e s and V e n t u r i a i n a e q u a l i s (Cooke) Wint. (apple scab) has.been c a r e f u l l y i n v e s t i g a t e d by W i l l i a m s and Rue* (1969). I t i s known t h a t r e s i s t a n t v a r i e t i e s of Malus spp. i s o l a t e the apple scab pathogen w i t h i n s m a l l l e s i o n s . These l e s i o n s c o n t a i n i n c r e a s e d c o n c e n t r a t i o n s of the u.v. f l o r e scent compound p h l o r e t i n , which i s f u n g i t o x i c i n v i t r o . A p p a r e n t l y the i n c r e a s e d concentrationeSf p h l o r e t i n e x p l a i n s the i s o l a t i o n . P h l o r e t i n i s a degradation product of p h l o r i d z i n , the most common g l y c o s i d e found i n apple f o l i a g e and f r u i t . Whereas a l l apple v a r i e t i e s contain p h l o r i d z i n , o n l y the -11-r e s i s t a n t v a r i e t i e s release phloretin upon i n f e c t i o n l o c a l i z i n g V. inaequalis to lesions. A number of investigations have focused attention on disease resistance i n the sapwood of trees. There are two types of sapwood resistance. In the f i r s t type immune sapwood i s added by growth a f t e r i n f e c t i o n has occurred. Shigo (1966) has shown by dissections of many forest-type hardwoods that, a l -though i n j u r i e s may lead to i n f e c t i o n and extensive decay, sapwood l a i d on a f t e r the time of wounding i n some way r e s i s t s further spread of i n f e c t i o n . Therefore some b a r r i e r (physical and/or chemical) i s produced by the immune sapwood. The second type of sapwood resistance i s a mechanism which a l t e r s extant sapwood so that the conditions for pathogenesis become unfavorable, and death or l o c a l i z a t i o n of the pathogen occurs. This type of resistance i s a dynamic response which i s induced i n affected zones of sapwood. Extreme abnormal metabolic changes occur, including premature death of parenchymatous c e l l s , and -the synthesis of metabolites which are not normally present i n the sapwood. y I t has been shown for the genus Prunus that induced host synthesis of toxins i n the sapijood leads to disease resistance. A r t i f i c i a l infections of P. persica (L.) Batsch (Braun & Helton, 1971) and P. domestica L. (Helton & Braun, 1971) by Cytospora c i n c t a Fr. l e d to resistance to subsequent i n f e c t i o n s by the same pathogen. I n the case of P. domestica the resistance does not appear to l o c a l i z e d but i s induced up to at least 120 cm from the s i t e of the o r i g i n a l i n f e c t i o n . This r e s u l t may indieaterithat sapwood metabolism i s changed throughout much, i f not a l l , of the tree. Hart and Johnson (1970) found that i n j u r i e s of the sapwood of Quercus  alba L., Robinia pseudoacacia L., and Madura pomifera (Raf.) Schn. lead to the production of abnormal zones of sapwood. These zones contain extractives -12-which are fungitoxic i n v i t r o to decay organisms. However, extracts from these zones are not as toxic as control extracts from normal heartwood of the same trees. Whether t h i s can be explained by quality or quantity of toxic extrac-t i v e s has not been established. I t has been shown conclusively that the sapwood of Pinus spp. responds to i n j u r y or inoculation with the production of toxins normally found only i n ithe heartwood. Jorgensen (1961) determined that an abnormal zone of sap-wood i n P. resinosa Sol. (Norway pine) e x i s t s i n advance of mycelial penetra-t i o n by Fomes annosus (Fr.) Cooke. He found both pinosylvin and pinosylvin-monomethyl ether present i n t h i s zone, neither of which i s detectable i n normal sapwood. Shain (1967), i n a study s i m i l a r to Jorgensen's, demonstrated that pinosylvin and i t s monomethyl ether are produced i n the sapwood of P. taeda L. ( l o b l o l l y pine) i n response to i n f e c t i o n by F. annosus. Lyr (1967) showed that pinosylvins are produced by the sapwood of P. s y l v e s t r i s L. (Scotch pine) i n response to cambial i n j u r y . The above three studies on three species of pine demonstrate that the abnormal synthesis of pinosylvins i s a dynamic response to inoculation and i n j u r y . In each case a time i n t e r v a l i s required before the pinosylvins ap-pear, although trees i n . the f i e l d require a longer i n t e r v a l than l i v i n g sapwood placed i n controlled conditions. Jorgensen found that trees wounded a r t i f i c -i a l l y reo^iired U to 9 weeks to produce the response. Lyr found pinosylvins present after 3 weeks, but they were present i n much greater quantities a f t e r 6 weeks. In controlled laboratory studies, Jorgensen found that sapwood could produce pinosylvins i n as l i t t l e as three days. Differences among species i n t h e i r pattern of formation of pinosylvins are present i n the studies of Jorgensen and Lyr. With respect to season of year, Jorgensen found that trees respond only i n the l a t t e r part of the growing -13-season and the dormant season, while Lyr found that trees do not respond i n the dormant season. Interestingly, both authors explain these phenomena by surmising that p i t c h flow controls exposure of the injured sapwood to a i r , and thus determines the rate of desiccation and subsequent death of affected cells.! Jorgensen fe e l s -that pinosylvins are not produced i n the early grow-ing season due to copious p i t c h flow which prevents desiccation. Reciprocally, Lyr f e e l s that pinosylvins are not produced i n the dormant season since l i t t l e r e s i n flow occurs, resulting i n rapid desiccation and c e l l death before syn-thesis of pinosylvins i s i n i t i a t e d . These conclusions do not necessarily con-f l i c t . They may suggest Wat extremes of desiccation of exposed sapwood lead to death rates which are either too f a s t or too slow; synthesis of pinosylvins w i l l not occur i n either case. Shain (1971) has observed the formation of reaction zones i n the sap-wood of Ficea abies Karst. (Norway spruce). These zones are formed i n advance of penetration by F. annosus and are s i m i l a r to those formed i n pines. He detected heartwood extractives i n the spruce reaction zone not normally found i n the sapwood; these extractives have a demonstrated i n v i t r o t o x i c i t y (Shain & H i l l i s , 1971)* which leads Shain to conclude that the reaction zone i n the sapwood contributes to the resistance of Norway spruce i n vivo. As for the cases of induced disease resistance i n herbaceous plants and decay resistance of heartwood, a strong implication e x i s t s that s p e c i f i c i s o -lated substances with known i n v i t r o t o x i c i t i e s , a r i s i n g from d i r e c t host-path-ogen interactions, make conditions untenable f o r the pathogen. At the present time, no available technique, such as the i n f e c t i o n of i s o l i n e s of cultivated plants, can prove t h i s i m p l ication. One piece of evidence suggests that con-clusions based on i n v i t r o t o x i c i t y tests of extracts of reaction zones can be misleading. Shain (1967) made extractions from portions of sapwood infected - l l i -with F. annosus which contained no pinosylvins. When tested by bioassay, these extracts proved as toxic to h i s t e s t organism as extracts from unin-fected reaction zones with pinosylvins present. Apparently i n t h i s case h i s t e s t i s not a v a l i d i n d i c a t o r of what happens i n vivo. However, the absence of pinosylvins i n the infected zone supports the theory that these compounds contribute to disease resistance, since i t i s probable that pathogenesis de-pends upon t h e i r degradation or absence. D. Comparison of Sapwood Reaction Zones with Heartwood There are s i m i l a r i t i e s between the formation of abnormal reaction zones i n sapwood and the formation of heartwood i n trees. The normal transformation of sapwood to heartwood involves a l t e r a t i o n of metabolism of parenchymatous c e l l s , synthesis of heartwood extractives, death of the parenchymatous c e l l s , and release of the extractives, some of which are deposited i n surrounding tracheid c e l l s . Metabolic a c t i v i t y decreases along a gradient from the cambium to the heartwood transformation zone (Frey-Wyssling & Bosshard, 1959$ D i e t r i c h s , 196U; Higuchi & Fukazawa, 1966j Higuchi,et a l . , 1967). The amounts of carb-ohydrates present i n the sapwood of cer t a i n tree species are represented by a sim i l a r gradient. I t i s known that once the heartwood zone i s reached, the parenchymatous c e l l s are dead, carbohydrates are generally no longer present, and heartwood extractives are present (Chattaway, 1952). Shain (1967) and Jorgensen (1962) have concluded that protection wood iwheny€;;initi'a;twi i n the sapwood of pine i s produced by a sequence of events par-a l l e l to those which produce normal heartwood; altered metabolism a r i s i n g from some in t e r n a l stimulus leads to the synthesis of heartwood extractives i n s i t u from l o c a l starch reserves by slowly dying parenchymatous c e l l s . -15-The differences between sapwood reaction zones and normal heartwood are i n s i t e of synthesis, i n circumstances of i n i t i a t i o n , and i n chemical composition. Although comparatively few compounds have been looked at i n the sapwood reaction zones, differences i n proportion of fungitoxic extractives have been observed i n both pine and spruce. Shain (1967) observed that the proportion of pinosylvin to pinosylvinmonomethyl ether i s greater i n reacted sapwood than i n normal heartwood. Since pinosylvin has a greater i n v i t r o t o x i c i t y than i t s monomethyl ether, t h i s possibly represents an-adaptation to enhance disease resistance. Shain and H i l l i s (1971) observed a p a r a l l e l s i t u a t i o n for Norway spruce. Among the compounds detected and measured, one lignan with a high i n v i t r o t o x i c i t y was found i n proportionally greater quantities i n reacted sapwood than i n cha r a c t e r i s t i c heartwood. E. Comparison of Reacted Sapwood with Mechanisms of Induced Disease Resistance The formation of sapwood reaction zones i n trees i s a response to ex-t e r n a l s t i m u l i and imparts disease r e s i s t a n t q u a l i t i e s s i m i l a r to those imparted to herbaceous plants by a phytoalexin or hypersensitive response. Reaction zone formation p a r t i a l l y f u l f i l l s the c r i t e r i a which characterize either of the l a t t e r responses. I t i s s i m i l a r to a phytoalexin response since i t con-s i s t s of induced de novo synthesis of fungitoxic metabolites. I t also resem-bles a hypersensitive response, since both involve death of affected c e l l s and subsequent l o c a l i z a t i o n of the pathogen. Certain authors have established the precedent of c l a s s i f y i n g sapwood reaction zone formation as a phytoalexin response (Shain, 1967j 1971; Smith, 1970). I t i s not e s s e n t i a l to equate sapwood reaction zone formation with e i t h e r a hypersensitive or phytoalexin response. The formation of reaction zones -16-represents a spe c i a l case of induced disease resistance i n l i v i n g wood tissue. This formation i s s t r i k i n g l y p a r a l l e l to mechanisms of induced disease r e s i s t -ance i n herbaceous plant tissues. Since decay fungi are ubiquitous, and the p r o b a b i l i t y i s high that i n -j u r i e s exposing l i v i n g sapwood w i l l occur i n a forest habitat over the long l i f e of i n d i v i d u a l trees, i t i s l i k e l y that a l l trees possess some mechanism of disease resistance i n the sapwood such as the formation of reaction zones. Since i t i s known that various pines and Norway spruce possess t h i s mechanism, i t i s probable that many other gymnosperms, and e s p e c i a l l y other taxa i n the Pinales, possess t h i s c a p a b i l i t y . To the best of t h i s author's knowledge no other investigations have been made to determine whether other genera of gymnosperms besides Pinus and Picea form abnormal reaction zones i n the sapwood. Therefore, due to the large number of gymnosperms with to x i c extractives occurring i n the heartwood, and the necessity of disease resistance, i t i s probable that the production of reac-t i o n zones i n sapwood i s a widespread phenomenon among many genera. The gymnosperm taxa i n which sapwood reaction zones are formed are a l l members of the Pinaceae. Members of the Gupressaceae belong to the same order, and many, such as western redcedar, have been shown to contain h i g h l y fungi-;®-toxic heartwood extractives. Therefore i t i s probable that western redcedar and perhaps other members of the Gupressaceae form sapwood reaction zones following the pattern of pine and spruce. This study i s designed to i n v e s t i -gate whether i n i t i a t i o n of r e a c t i o n zones i n the sapwood and bark of western redcedar i s induced by i n j u r y , and, i f so, whether the fungitoxic substances produced are normally occurring heartwood compounds. -17-F. Summary of Investigations Made on Western Redcedar Thuf!a p l i e at a Doim. (western redcedar) i s found from southern Alaska to northern California and commercially i s the most important of the North American cedars. Western redcedar i s used extensively for shingles, siding, poles, and fenceposts because of the large amounts of clear wood available and the high durability of the heartwood arising from i t s high decay resistance. I t has been recognized feor many years that the high decay resistance of western redcedar wood i s caused by the presence of heartwood extractives. The f i r s t recorded demonstration of this phenomenon was provided by Sowder (1929), who showed that hot water extraction of western, redcedar wood flour removes durability and ^ a t the water extracts are toxic to fungi i n vitr o . Anderson and Sherrard (1933) isolated two isomers of an acidic compound which they separated from the steam volatile o i l of western redcedar heartwood. These compounds proved ten times more toxic than creosote to F. annosus mdl-and Lenzites trabea (Pers.) Fr. They found that amounts of 0.006 percent by weight of either compound were fungicidal to both fungi. The indentification of these compounds as, p- and ir-thujaplicins and the demonstration of their fungicidal properties have been verified by various authors including Anderson and Gripenberg (19U8), Erdtman and Griperiberg (19U8), and Rennerfelt (19U8). In addition, Rennerfelt showed that a third isomer of thujaplicin (<*), also contained i n western redcedar heajptwood, possesses the same order of toxicity as the other two isomers. The observed high toxicities of the thujaplicins generated considerable interest i n western redcedar extractives. At least partly for this reason, a large number of investigations have been directed at discovering their chemical and compositional properties. Although western redcedar heartwood has a rela t i v e l y low density, i t - 18 -contains relatively large amounts of extractives. According to Gardner and Barton (195>8) the heartwood may contain amounts of extractives i n excess of twenty percent of the heartwood dry weight. Extraction with acetone or hot water of the bu t t heartwood y i e l d s a mixture of phenolic substances, arabinose, a s t e r o l , and a complex v o l a t i l e o i l . The composition of the v o l a t i l e o i l has proven i n t e r e s t i n g both because of the aforementioned t o x i c i t y of the ccmponeais and thei r novel chemical nature. Contained i n the o i l are t h u j i c acid, methyl thujate, various tropolones (including the thujaplicins) and a single tropone. A l l of these compounds contain ten carbon atoms possessing an unsaturated seven membered r i n g with quaslaromatic properties. Although the mode of biosynthesis of these compounds i s not proven, they are probably derived from two molecules of mevalonic acid. Loomis (1967) describes the thuj a p l i c i n s as unproven i r r e g u l a r monoterpenes. Methyl thujate i s the methyl ester of t h u j i c acid and contributes much of the odor associated with the heartwood. The tropo-lones comprise a family of compounds f i r s t recognized by Dewar (19U5) as derivatives of 2-hydroxy-2,U,6-cycloheptatriene-l-one. Five of the simplest naturally occurring tropolones are present i n western redcedar heartwood. The tree t h u j a p l i c i n s , and are isomers of isopropyl tropolone with the ary! group attached at the 3, U, and 5> positions respectively, fi - t h u j a p l i c i n o l i s s i m i l a r to p-thujaplicin i n structure d i f f e r i n g by the ad-d i t i o n of a hydroxyl group at the seven p o s i t i o n . £-dolabrin i s also similar to p-thujaplicin d i f f e r i n g by i t s reduction state so that the three carbon a r y l group i s isopropenyl. The one tropone present i n the heartwood i s nezukone (li-isopropyl-2,U,6-cycloheptatriene-l-one). This compound was f i r s t discov-ered i n the heartwood of T. s t a n d i s h i i Carr. by Hirose e t a l . (1966); i t s presence was l a t e r confirmed i n western redcedar (Hirose & Nakatsuka, {1967). -19-Table I l i s t s the composition of the v o l a t i l e o i l of western redcedar heart-wood. The tropolones have several i n t e r e s t i n g chemical properties due to the presence of adjacent carbonyl and hydroxy! groups at the 1 and 2 positions of the r i n g . This association makes them substantially more acidic than nor-mal phenols and also causes them to be steam v o l a t i l e . In addition, they form chelate complexes with heavy metals. These complexes absorb v i s i b l e l i g h t , enabling detection and quantification of extremely low concentrations of metal-thujaplicin complex by densitometric techniques (MacLean & Gardner, 1956). Besides the t o x i c i t y tests of the three t h u j a p l i c i n s , additional tests have been performed on other redcedar extractives showing that many of these are also t o x i c . Roff and Whittaker (1959) tested the t o x i c i t y of ^ - t h u j a p l i -c i n o l on various brown and white r o t types of decay fungi. They found that p - t h u j a p l i c i n o l i s approximately as toxic as the t h u j a p l i c i n s to the brown r o t fungi tested, but much less toxic t o the white r o t fungi tested. The same study showed s l i g h t differences i n t o x i c i t y of y-thujaplicin to the two types of decay fungi. Roff and Atkinson (195W made a t o x i c i t y test on the phenolic f r a c t i o n of water extracts of western redcedar heartwood. They describe t h i s f r a c t i o n as ' t h u j a p l l c i n free', which probably means tropolone f r e e . They found that concentrations of t h i s f r a c t i o n greater than 1.0 percent i n malt agar are fungi-s t a t i c , but concentrations as high as 8.0 percent are not f u n g i c i d a l . They conclude that although the t o x i c i t y of the phenolic f r a c t i o n i s not high, i t s t i l l plays an important role i n providing decay resistance since t h i s f r a c t i o n represents It to 5 percent of the oven-dry weight of the heartwood. Since riezukorie occurs i n r e l a t i v e l y large q u a n t i t i e s . i n western red-cedar heartwood (Jiang, 1968), i t may also contribute to decay resistance. -20-TABLE I Composition of the V o l a t i l e O i l of Western Redcedar Component Reference I Reference I I Methyl thujate (other neutrals) Thujic acid Tropolones p - t h u j a p l i c i n <x - t h u j a p l i c i n j - t h u j a p l i c i n p-dolabrin p - t h u j a p l i c i n o l Nezoukone 0.17 % 0.08 0.56 0.30 0.01 0.20 0.0003 0.07 0.03 % 0.30 0.15 0.22 *%—percent by weight of oven-dry heartwood After: I . Gardner & Barton (1958a) I I . Jiang (1968) -21-The extent of t h i s c o n t r i b u t i o n , i f any, i s unknown. However, i t i s p o s s i b l e t h a t nezukone may have c o n t r i b u t e d t o e x t r a c t t o x i c i t y , or a t l e a s t have been present, i n some of the s t u d i e s a l r e a d y mentioned. Raa and Goks/t<yr (1965; 1966) have t r i e d to determine the mechanism o f t o x i c i t y o f ^ - t h u j a p l i c i n t o Saccharomyces c e r e v i s i a e Meyen ex Hansen. Although they found t h a t ^ - t h u j a p l i c i n i n h i b i t s formation o f a c e t y l coenzyme A, they were unable t o d e l i n e a t e a mechanism of t o x i c i t y . Raa and Goks^yr d i d show t h a t the s t a b l e s o l u b l e copper chelate complex of t h u j a p l i c i n , formed from an excess of c u p r i c s u l f a t e added to a s o l u t i o n of / S - t h u j a p l i c i n , i s approximately two orders o f magnitude more t o x i c to baker's yeast i n l i q u i d c u l t u r e than p l a i n t h u j a p l i c i n . They showed t h a t f e r r i c complexes do not pro-duce t h i s a m p l i f i e d e f f e c t . Although copper i s a w e l l known f u n g i c i d e , the enhancement o f t o x i c i t y o f t h u j a p l i c i n by f o r m a t i o n of the copper c h e l a t e i s s u p r i s i n g . Before the importance o f t h i s d i s c o v e r y can be evaluated, f u r t h e r t e s t s are necessary to determine the t o x i c e f f e c t s on decay f u n g i w i t h i n cedar heartwood. Whereas decay r e s i s t a n c e o f western redcedar heartwood depends upon the amount o f normal e x t r a c t i v e s present, v a r i o u s studies have shown t h a t these e x t r a c t i v e s v a r y i n amounts between t r e e s and w i t h i n t r e e s . Gardner (1962; MacLean & Gardner, 1956a) found t h u j a p l i c i n s i n amountsiincreasing r a d i a l l y outward from the p i t h u n t i l they cease t o appear a t the sapwood-heartwood t r a n s i t i o n zone. I n a d d i t i o n these compounds occur i n decreasing amounts from b u t t t o apex. Ji a n g (1968) has v e r i f i e d t h i s p a t t e r n f o r t h u j a p l i c i n s and has shown t h a t i t holds f o r the d i s t r i b u t i o n of nezukone and v a r i o u s l i g n a n s . I n no cases have t h u j a p l i c i n s been shown to be present i n the sapwood or bark. Although the above p a t t e r n o f d i s t r i b u t i o n of heartwood e x t r a c t i v e s i s p h y s i o l o g i c a l l y normal i n western redcedar, m i c r o b i a l i n v a s i o n of the heartwood a l s o a f f e c t s the observed p a t t e r n o f d i s t r i b u t i o n . I n some tre e s w i t h b u t t -r o t p r e s e n t , c l e a r l y d e f i n e d c o n c e n t r i c c o l o r zones e x i s t which re p r e s e n t zones o f m i c r o b i a l succession (van der Kamp, 1971)• These zones move r a d i a l l y outward over time, a p p a r e n t l y dependent upon the speed a t which a pi o n e e r organism i s capable o f degrading t h u j a p l i c i n s . This degradation^changes the environment such t h a t other orgaMsmsj can invade, r e s u l t i n g u l t i m a t e l y i n wood decay. I t i s p o s s i b l e t h a t the absence o f t h u j a p l i c i n s i n the p i t h o f undecayed t r e e s a l l o w s the pioneer organism t o get a f o o t h o l d a t t h i s p o s i t i o n . Such a succession i s n ot known t o occur i n the opposite d i r e c t i o n — decay s t a r t i n g a t the p e r i p h e r y o f a t r e e and p r o g r e s s i n g i n toward the p i t h . - 23 -MATERIALS AND METHODS In the summer of 1970 an attempt was made to develop a t h i n l a y e r chromatography (TLC) s y s t e m ^ u s i n g s i l i c a - g e l as the adsorbent, which would r e s o l v e e x t r a c t s o f western redcedar heartwood i n t o components. I n l a t e summer and e a r l y f a l l of 1 9 7 0 , p o r t i o n s o f l i v i n g bark were exposed, i n o c u l a t e d , c o l l e c t e d a f t e r a time i n t e r v a l , and e x t r a c t e d w i t h h ot water. These e x t r a c t s were t e s t e d f o r the presence o f heartwood e x t r a c t i v e s . I n the summer of 1971> p o r t i o n s o f l i v i n g bark and sapwood of western redcedar were exposed, c o l l e c t e d a t v a r i o u s time i n t e r v a l s , and ex t r a c t e d w i t h various s o l v e n t s . These e x t r a c t s were t e s t e d f o r the presence o f t h u j a p l i c i n s and i n v i t r o f u n g i t o x i c i t y by s e v e r a l bioassay techniques. The e x t r a c t e d bark and sapwood c o l l e c t e d a f t e r the lon g e s t i n t e r v a l was t e s t e d f o r any unextracted t o x i c f a c t o r s by a weight-loss t e s t . Table I I i s . a complete l i s t of a l l -toe samples o f bark and sapwood c o l l e c t e d from August, 1970 t o August, 1 9 7 1 . I t summarizes the type o f treatment each sample underwent, the date o f the beginning o f l a b o r a t o r y p r o c e s s i n g , the numbers and ages o f tre e s from which each sample was taken, and the treatment time i n t e r v a l f o r each sample. Figure I: i s a g e n e r a l i z e d f l o w c h a r t of the methods u t i l i z e d i n f i e l d treatment and l a b o r a t o r y p r o c e s s i n g of samples. No i n d i v i d u a l sample was processed w i t h a l l the techniques l i s t e d , although a l l samples i n each c o l l e c t i o n block were t r e a t e d i n p r e c i s e l y the same manner. Samples o f l i v i n g bark which had been exposed and i n o c u l a t e d were c o l l e c t e d from t r e e s 2 and 3 i n August and October, 1970 r e s p e c t i v e l y ( C o l l e c t i o n b l o c k s 1 and 2). These tr e e s were l o c a t e d southeast o f Thunder-b i r d Stadium, U.B.C. Endowment Lands, Vancouver, B.C. Two squares, 1 0 cm on - 2 l i -TABLE I I Summary o f Sapwood and Bark C o l l e c t i o n s C o l l e c t i o n Sample Block Number 3?ree Approx. Number Age Treatment Treatment D e s c r i p t i o n I n t e r v a l Date Begin Process:: 1 101 X 102 2 50 y r s . 2 C o n t r o l bark 0 I n o c u l a t e d bark 3 days Aug. 2li Aug. 27 , 203 3 >o y r s . 3 C o n t r o l bark d I n o c u l a t e d bark 3 days Oct. lb Oct. 19 301 302 , 303 305 306 5 250 y r s . 5 Ii 25© y r s . k 5 5 E x c i s e d bark 1 wk. Exc i s e d sapwood 1 wk. Exposed bark 1 wk. C o n t r o l bark 0 Exposed sapwood 1 wk. C o n t r o l sapwood. 0 June 22 II II u it it h k07 5 E x c i s e d sapwood 3 wk. J u l y 5 508 509 5 510 5ii 512 7 250 y r s . 7 7 6 250 y r s . 5 Exposed bark k wk* Bark under 508 i i wk. C o n t r o l bark 0 E x c i s e d bark i i wk. Exposed sapwood k wk. J u l y 20 ii II II tt 613 , 6lU 6 615 616 k h k 6 C o n t r o l bark 0 C o n t r o l sapwood 0 Exposed bark 6 wk. Exposed sapwood 6 wk. Aug. U it ii it * — C o l l e c t i o n b l o c k s 1 & 2 processed i n 1970J c o l l e c t i o n b l o c k s 3-6 processed i n 1971. -25-FIGURE 1 Methods Used t o Detect Formation o f R e a c t i o n Zones TREATMENT: EXTRACTION: DETECTION: Exposure—Bark & Sapwood /Time I n t e r v a l E x c i s i o n — B a r k & Sapwood Time I n t e r v a l t C o l l e c t i o n C o n t r o l C o l l e c t i o n C o l l e c t i o n S o l v e n t E x t r a c t i o n s — w a t e r , acetone & chloroform E x t r a c t i v e Tests 1. Spore germination 2. M y c e l i a l growth r a t e 3. E x t r a c t a d d i t i o n Chemical Tests 1. Chromatography 2. T h u j a p l i c i n t e s t Decay Test o f E x t r a c t e d Chips -26-a s i d e , were l a i d out on each t r e e . The dead bark was removed from these squares exposing two 'windows' of l i v i n g b a r k . One of these windows was p a i n t e d w i t h a dense spore suspension of Trichoderma sp., while the outer lay«rjpof the other was cut away w i t h a s t a i n l e s s s t e e l k n i f e to serve as a c o n t r o l . The c o n t r o l s t r i p s , 2-h mm t h i c k , were chopped i n t o c h i p s about 6-10 mm square and e x t r a c t e d w i t h about 10 times t h e i r l i v i n g weight o f °0-95>GC water f o r h hours. Three days l a t e r the i n o c u l a t e d bark was c o l l e c t e d and e x t r a c t e d i n the same manner as the c o n t r o l bark. Each hot water e x t r a c t was simply d i s t i l l e d ; k a l i q u o t s ( t r e e 2— hO ml; t r e e 3—50 ml) were c o l l e c t e d of e x t r a c t d i s t i l l a t i o n s . These samples were e x t r a c t e d w i t h chloroform ( t r e e 2 samples with 20 ml chloroform; tree 3 samples f i r s t w i t h 20 ml and c o n s o l i d a t e d w i t h a subsequent 10 m l ) . D i s t i l l a t i o n r e s i d u e s were a l s o e x t r a c t e d , f i r s t w i t h 30 ml hexane and c o n s o l i d a t e d w i t h a subsequent 20 ml hexane. I n each case the aqueous phase was d i s c a r d e d , and the organic phases were evaporated, l e a v i n g crude o i l y r e s i d u e s . Each o f these r e s i d u e s was d i s s o l v e d i n 1 ml chloroform and t e s t e d f o r the presence o f known heartwood e x t r a c t i v e s by three techniques: 1) the a n a l y t i c a l method f o r t h u j a p l i c i n s d e s c r i b e d by MacLean and Gardner (1956); 2) TLC chromatograms u s i n g s i l i c a - g e l as an adsorbent developed w i t h 10:1 mixture o f chloroform to t - b u t a n o l ( v / v ) ; and 3) paper chromatograms prepared and developed a f t e r the method o f Zavarin and Anderson (1956). I n the summer o f 1971 i n v e s t i g a t i o n designed to d e t e c t induced f o r m a t i o n o f r e a c t i o n zones was c o n t i n u e d i n an expanded form. Sapwood was i n c l u d e d i n a d d i t i o n t o bark, and time i n t e r v a l s a f t e r treatment were extended. I n s t e a d of i n o c u l a t i o n w i t h Trichoderma sp., l i v i n g bark and sapwood exposed by treatment were l e f t exposed t o n a t u r a l sources o f inoculum. Various s o l v e n t s were used f o r e x t r a c t i o n i n place o f , or i n a d d i t i o n to, h o t water. And samples of l i v i n g bark and sapwood were excis e d from t r e e s and p l a c e d f o r v a r i o u s i n t e r n a l s i n an i n c u b a t i n g oven at room temperature, i n the dark, a t n e a r l y 100 p e r c e n t humidity. The depth o f r e a c t i o n zone format i o n , i f i t e x i s t s i n western redcedarj i s not known. Thus, a l l samples, i n c l u d i n g c o l l e c t i o n b o l c k s 1 and 2, were removed i n s t r i p s as t h i n as p r a c t i c a b l e , s i n c e t h i s procedure assures t h a t any abnormal m e t a b o l i t e s w i l l be m i n i m a l l y d i l u t e d w i t h unreacted p o r t i o n s o f t i s s u e . V a r i o u s b i o a s s a y s , as w e l l as chemical t e s t s used t o d e t e c t t r o p o l o n e s , were used t o i n d i c a t e any formation of r e a c t i o n zones i n the samples comprising c o l l e c t i o n b l o c k s 3-6. A l l the b i o a s s a y t e s t s were i n v i t r o . F. annosus (U.B.C. F o r e s t Pathology C u l t u r e #25) was chosen as the t e s t organism s i n c e t h i s fungus i s widely used i n p a r a l l e l s t u d i e s and i s adaptable to the f o l l o w -i n g four types of b i o a s s a y used i n t h i s i n v e s t i g a t i o n * 1) M y c e l i a l growth r a t e — a p l u g o f a c t i v e l y growing mycelium, taken w i t h a #U cork borer, i s de-p o s i t e d on a standard medium of 2 percent malt e x t r a c t and 2 percent agar t o which a s p e c i f i e d amount o f t e s t substance d i s s o l v e d i n e t h a n o l has been added, and the growth r a t e i s observed. 2) E x t r a c t a d d i t i o n s — t h e same as 1 except t h a t the t e s t substance i s added i n the form o f a crudely e x t r a c t e d t h i c k o i l to a spot on the medium, and i t i s noted whether the m y c e l i a l f r o n t w i l l grow over the e x t r a c t . 3) Spore g e r m i n a t i o n — s p o r e producing c o l o n i e s o f t e s t fungus are i n v e r t e d over p l a t e s c o n t a i n i n g standard medium to which t e s t sub-stances have been added, and the e f f e c t s on spofe germination are observed. h) Decay t e s t o f e x t r a c t e d c h i p s — e x t r a c t e d c h i p s are incubated on a c t i v e l y growing c u l t u r e s of decay fungus f o r a time i n t e r v a l , and weight l o s s e s are c a l c u l a t e d . Samples comprising c o l l e c t i o n b l o c k s 3-6 were taken from f o u r t r e e s (k-7) -28-l o c a t e d n o r t h of Blaney Lake, U.B.C. Research F o r e s t , Maple Ridge, B.C. Treatments were of three types: 1) Exposure by s t r i p p i n g away the dead bark or a l l o f the bark f o l l o w e d by a time i n t e r v a l . 2) Maintainence of sapwood or bark t i s s u e excised from the t r e e and kept i n the dark a t about 25°C a t n e a r l y 100 percent h u n i d i t y f o r a time i n t e r v a l . 3) C o l l e c t i o n o f c o n t r o l s i n the same manner used f o r the other samples without a time i n t e r v a l . Samples i n c o l l e c t i o n b l o c k s 3-6 were r e c t a n g l e s 17.5 by 35.0 cm. The outer 2-k mm of exposed bark were cut away w i t h a k n i f e . The sapwood samples were c o l l e c t e d t h i c k e r than the bark samples (3-6mm) since they were removed w i t h a c h i s e l n e c c e s s i t a t i n g a coarser cut.. E x c i s e d bark samples maintained under a r t i f i c i a l c o n d i t i o n s were c o l l e c t e d t h i c k e r than other bark samples to f a c i l i t a t e removal of samples i n one p i e c e . I n t h i s case a l l of the l i v i n g bark was removed r e s u l t i n g i n cambial exposure. Before e x t r a c t i o n a l l samples were cut i n t o s m a l l c h i p s 10-25 mm on a s i d e . .Samples i n c o l l e c t i o n b l ock 3 c o n s i s t e d o f bark and sapwood exposed f o r Mweek, excis e d bark and sapwood which had been incubated f o r 1 week, and bark and sapwood c o n t r o l s . The f o l l o w i n g procedure was f o l l o w e d f o r each of the 6 samples: Each was e x t r a c t e d w i t h about 3 times i t s weight of 90-95°C water f o r k hourss 2.0 g agar and 2.0 g malt e x t r a c t were added to a 100 ml a l i q u o t of each e x t r a c t before c o o l i n g , while the excess was f r o z e n . Each a l i -quot was poured i n t o 5 s t e r i l e p e t r i e p l a t e s so each contained 20 ml o f med-ium. A f t e r c o o l i n g , 2 of the 5 p l a t e s were t e s t e d w i t h the spore germination t e s t , and the remaining three p l a t e s were t e s t e d w i t h the m y c e l i a l growth r a t e t e s t . C o l l e c t i o n b l o c k h c o n s i s t e d o f one sample o f e x c i s e d sapwood k e p t under a r t i f i c i a l c o n d i t i o n s f o r 3 weeks. I t was e x t r a c t e d i n a Soxhlet e x t r a c -t i o n apparatus f o r U hours w i t h about 10 times i t s weight o f chloroform. -29-A 10 ml a l i q u o t of the chloroform s o l u t i o n was t e s t e d f o r the presence o f t h u j a p l i c i n s by the method of MacLean and Gardner (1956). The remainder o f the chloroform was flash-evaporated a t about 30°G to dryness; the r e s i d u e , d i s s o l v e d i n 1 ml e t h a n o l , was added t o 15 ml o f standard growth medium. Three c o n t r o l p l a t e s w i t h o u t t e s t substance c o n t a i n i n g the same percentage eth a n o l were a l s o made. These f o u r p l a t e s were t e s t e d by the m y c e l i a l growth r a t e t e s t . Samples comprising c o l l e c t i o n b lock 5 c o n s i s t e d o f bark and sapwood exposed f o r k weeks, e x c i s e d bark maintained f o r h weeks, and c o n t r o l b a r k . Each of these samples was a i r - d r i e d f o r 3 days and then e x t r a c t e d w i t h 10 times i t s a i r - d r i e d weight o f chloroform f o r 2 days. 10 ml a l i q u o t s o f c h l o r -oform e x t r a c t s from each sample were t e s t e d f o r the presence of t h u j a p l i c i n s . The remainder o f these e x t r a c t s were f i l t e r e d , flash-evaporated a t about 30°G, and the r e s i d u e wights determined. About 50 mg o f each r e s i d u e s were t e s t e d b y e x t r a c t a d d i t i o n t e s t s . The b u l k o f each r e s i d u e , d i s s o l v e d i n 1 ml ethan-o l , was added to 30 ml standard growth media and t e s t e d b y m y c e l i a l growth r a t e and spore germination t e s t s . C o n t r o l p l a t e s without t e s t substance c o n t a i n i n g e q u a l amounts o f ethanol were a l s o t e s t e d . Samples comprising c o l l e c t i o n b l o c k 6 c o n s i s t e d o f bark and sapwood exposed f o r 6 weeks and c o n t r o l bark and sapwood. Each sample was a i r - d r i e d f o r 6 days and then e x t r a c t e d f o r 2 days w i t h 10 volumes (w/v) o f acetone. A f t e r f i l t r a t i o n each sample was flash-ev a p o r a t e d a t about 30°C to dryness l e a v i n g o i l y r e s i d u e s . A f t e r weighing these r e s i d u e s were d i s s o l v e d i n 1 ml et h a n o l , added to 30 ml standard medium, and t e s t e d by m y c e l i a l growth r a t e and spore germination t e s t s . I n t h i s case i t was not p o s s i b l e t o d i s s o l v e a l l the r e s i d u e w i t h e t h a n o l , so p o r t i o n s o f u n d i s s o l v e d r e s i d u e were t e s t e d by e x t r a c t a d d i t i o n t e s t s . The acetone e x t r a c t e d c h i p s , a f t e r drying i n a -30-fume hood f o r 3 days, were f u r t h e r e x t r a c t e d w i t h 10 volumes (w/v) of 90-95°C water f o r h hours. These e x t r a c t s were flash-evaporated a t about 60°C t o about 1 ml, added t o 30 ml o f standard medium, and tes t e d by m y c e l i a l growth r a t e and spore germination t e s t s . Since previous experience had shown t h a t i t was u n d e s i r a b l e to evaporate the water e x t r a c t s to dryness, residue weights were c a l c u l a t e d i n d i r e c t l y by comparison of a i r - d r i e d chip weights b e f o r e and a f t e r the water e x t r a c t i o n . A f t e r water e x t r a c t i o n , the samples o f c o l l e c t i o n b l o c k 6 were a i r -d r i e d and then oven-dried f o r 3 days a t about 95>-100°C. Seven groups o f 10 c h i p s , randomly s e l e c t e d from each sample, were weighed and deposited on c u l t u r e s o f F. annosus growing on standard medium. Four groups from each san-p l e were p l a c e d on 11 week o l d c u l t u r e s o f F. annosus, w h i l e the remaining three groups were p l a c e d on 3 week o l d c u l t u r e s . Each o f these p l a t e s was incubated a t room temperature i n the dark a t n e a r l y 100 p e r c e n t humidity f o r 8 weeks. A f t e r removal the chips were oven-dried f o r 3 days and t h e i r weights determined. The percentage weight l o s s e s f o r each o f the 28 groups o f c h i p s were c a l c u l a t e d , and v a r i o u s t - t e s t s were performed on t h i s d ata. - 3 1 -RESULTS When l i v i n g bark of western redcedar i s exposed to a i r by s t r i p p i n g o f the dead outer bark, o r by e x c i s i o n from the t r e e , i t r a p i d l y darkens i n c o l o r from a p a l e cream t o a deep brown. This c o l o r change takes p l a c e w i t h i n a few minutes a f t e r exposure. A f t e r t h i s r a p i d change, the c o l o r continues t o darken a t a much slower r a t e f o r a day or so. Sapwood exposed to a i r also© darkens although much more slowly and w i t h a l e s s n o t i c e a b l e c o l o r c h a n g e — from cream to ye l l o w . T h i s change takes p l a c e over s e v e r a l days. The appear-ance o f c o l l e c t i o n s taken a t k weeks d i d not d i f f e r v i s i b l y from c o l l e c t i o n s taken a f t e r 3 days or 1 week. However, c o l l e c t i o n s of bark and sapwood ex-posed f o r 6 weeks e x h i b i t e d c e r t a i n d i f f e r e n c e s from samples c o l l e c t e d a f t e r s h o r t e r exposure i n t e r v a l s . Both the bark and sapwood had exuded r e s i n s which made the samples s t i c k y , a c h a r a c t e r i s t i c not noted f o r e a r l i e r c o l l e c t i o n s . I n a d d i t i o n , the presence of blue s t a i n i n g was macroscopic a l l y n o t i c e a b l e oh the sapwood surface. M i c r o s c o p i c examination showed t h a t hyphae were present, i n d i c a t i n g t h a t the s t a i n i n g was o f f u n g a l o r i g i n . This b l u e - s t a i n i n g fungus was not observed on any bark samples. Table I I I l i s t s sample weights (Column I I I ) , e x t r a c t e d residue weights (Column 1 7 ) , and m a t e r i a l e x t r a c t e d expressed as percentages of sample weights (Column V) and as percentages of the t e s t medium (Column f l ) . A comparison o f the v a l u e s i n Column V i n d i c a t e s notable d i f f e r e n c e s o n l y between p e r c e n t -ages of m a t e r i a l e x t r a c t e d w i t h acetone from c o n t r o l samples and samples exposed f o r 6 weeks. A comparison between the values i n Column V and VI i n d i c a t e s t h a t the c o n c e n t r a t i o n s a t which the e x t r a c t e d r e s i d u e s were tested,^»a^^[f |&essSffl^orderiasthe c o n c e n t r a t i o n s n a t u r a l l y present i n a i r - d r i e d samples. Attempts t o r e s o l v e heartwood e x t r a c t s by TLCswere u n s u c c e s s f u l . TABLE I I I Sample Weights, E x t r a c t Weights and Percentages o f E x t r a c t i v e s I . Samr-p l e # I I . Treatment Descrip. I I I . Sam-p l e wt. IV. Ex w< chlfm. tradted I sig h t acetone Residue H20 V. E x t as chlfm. racted R< % sample acetone ssidue wt. H 2 0 V I . Ex as chlfm. t r a c t e d I % t e s t i acetone Residue aedium H20 101 102 C o n t r o l bark Inoc. bark 20.0 g 15.6 ND* ND ND ND 203 201* C o n t r o l bark Inoc. bark_ 51*.5 2U.0 • • . •• ND ND ND ND 301 302 303 301* 305 306 E x c i s e d bark Exc i s e d sapwd. Exposed bark C o n t r o l bark Exposed sapwd. C o n t r o l sapwd. 120.1; 191.8 56.1* 1*6.1* 67.2 109.3. ND ND SND ND ND ND ND ND ND ND m ND ND ND ND ND ND ND 1*07 E x c i s e d sapwd. 63.0 o.l*7g 0.75$ 3.0£ 508 509 510 511 512 Exposed bark Bk. under 508 C o n t r o l bark Excised bark, Exposed sapwd. 25.03 39.69 50.73 39.31* 52.83 0.09 0.13 0.17 0.11 0.11 0.36 0.33 0.31* 0.28 0.19 • /.i 1.2 1.1 ; 1.1 0.9 0.6 613 61U 615 616 C o n t r o l bark C o n t r o l sapwd. Exposed bark Exposed sapwd. 56.50 U5.6U 31.1*6 1*0.92 0.536g 0.131* 1.896 0.212 3.2l*g 2.75 1.75 0.30 " •' 0.952 0.29 6.01* 0.52 5.7k% 6.03 5.57 0.73 o.56£ 0.32 2.23 0.31 10.1$ 8.8 5.6 0.96 *ND—Not Determined P r e l i m i n a r y attempts i n d i c a t e d t h a t s e v e r a l heartwood compounds were separ-ated on s i l i c a - g e l developed w i t h a s o l v e n t o f chloroform:t-butanol (10:lj v / v ) . However, t h i s system d i d not separate tropolones, a l l o f which streaked. For t h i s reason, the system was abandoned. A l l t e s t s made to d e t e c t the presence of t h u j a p l i c i n s i n bark and sap-wood samples j a f t e r the method o f Maclean and Gardner (1956); produced negative r e s u l t s . This t e s t e a s i l y d e t e c t s lOGfg o f tropolone present i n a 10 ml s o l u t i o n of n-hexane. At t h i s range of s e n s i t i v i t y , assuming t h u j a p l i c i n s were e f f i c i e n t l y e x t r a c t e d , t h i s t e s t would have detected the presence of t h u j a p l i c i n s i n samples a t 0.01 percent by weight. B i o assays of e x t r a c t e d t e s t substances were of three t y p e s — m y c e l i a l growth r a t e , spore germination, and e x t r a c t a d d i t i o n s . I n a l l cases no d i f -ferences i n e f f e c t s were observed between t r e a t e d or c o n t r o l sample e x t r a c t s , e i t h e r f o r sapwood or bark. No t o x i c e f f e c t s were observed i n any i n s t a n c e , and water-extracted compounds from both the sapwood and bark enhanced m y c e l i a l growth r a t e over t h a t on unamended medium. Table IV l i s t s the r e s u l t s o f the c o n t r o l l e d decay t e s t performed on sapwood and bark samples exposed f o r 6 weeks and t h e i r c o n t r o l s , a l l p r e v i o u s -l y e x t r a c t e d w i t h acetone and h o t water. These r e s u l t s are expressed as a c t u a l weight l o s s e s and as percentages of weight l o s t from each group. Table V l i s t s the r e l e v a n t s t a t i s t i c a l parameters d e s c r i b i n g the percentages of weights l o s t from these samples. The c a l c u l a t e d means, v a r i a n c e s , and number o f observations are l i s t e d f o r each o f the b l o c k s o f the f o u r samples incubated on 11 week o l d c u l t u r e s o f F. annosus, f o r samples incubated on 3 week o l d c u l t u r e s , and f o r the combined data. Table V a l s o l i s t s c a l c u l a t e d t values which compare the mean weight l o s s e s of c o n t r o l bark and sapwood w i t h exposed bark and sapwood f o r each of the three b l o c k s o f o b s e r v a t i o n s . % WEIGHT LOSS P. WEIGHT LOSS I : £ ~ 0 OVn. ro H - 0 O V A OVEN DRY WEIGHT 4=-U> ro H H H H • • • vnvn. ro H ON H f H H H O N t - W ON • • • • O H O H M M o o o % • • fV) H ro ro vo - 0 - J r o vn. ro ONVO T l O O o o .% • • :' ro N> fo ro O H covn ~ j bo so ro TO H H H » • • ON ?NJ O ro • • • • ON £rvnvn ro ON NO CO CO NO pr TO NO ON NO NO GXJ O O %<-ON O' H H U) I H H NO O O • • • U> O f o H O NO NO • • •' • ro Vn. O VJO -o. —o NO -o O O O • • • O O O ON- J-J H U) - 0 CO-J v*> O O O o • • • • o o o o ON~0 ->J ON - J H ON ro Hvnvn. tr-o o o * • • ON~0 -O ONVJJ vn O fr-VA 4-T--0 VjJ o o o o • • * % ON—4 CO ON vn-p-.pr ON U> ON H V A VJJ vo vn—J TO ON I © ON •P~vn ro • • « V n f r v O H V O - J • • • • U) ro VOW vo vnW so o o o • • • o o o ON-O, r o trco.tr-O O O O % • • • o o o o -p-UJ *r-vn VA> —0 VA> —0 Vn H ON O TO H O H • • • O CO o ro ON CO CO NO —3 o fv> vn H O O H • • • • O CO CO o O -4 covn ir-H4=-ON Vn—3 N> fN5 o o i t * •fr-vnvn • • • ro NO U> O H to M H O H • • • • GO-0 NO ON •Cr-vo ro O O O O • • • O O O ON covn CO VA) CO O O O o • • • « o o o o ro ro O H v °-|r-vo TO H H O H O • • • VO ( - V O vn Co ro -o -a o NO vn-J O H H H • • • • NO H O H ,ON^" ON ON O ro ro O TO H1 CO S ft CO ON I ON H ON ts -35-TABLE V Comparison o f Weight Losses a f t e r Decay CULTURE BLOCK STATISTIC SAMPLE #613 #615 C o n t r o l bark 6-week bark #6lU #616 C o n t r o l spwd. 6-wk. spwd. 11 week X 2 s t (6 d f ) U U 15.06 % 9.58 % l . U i 0.25 8 . 5 l * t h k U.72 % 1.78 £ 0.29 0.63 6.13** 3 week n X 2 s t (U d f ) 3 3 15.U2 £ 9.87 % 0.69 0.21 11-. 71** 3 3 U.32 | 5.28 £ 1.62 0.8U 1.33 A l l — . 11 & 3 wk. combined n X 2 s t (12 d f j 7 7 15.21 $ • " • 9.70 % 0.97 0.22 13.37** 7 7 U.55 % 3.28 ^ 0.73 U.08 1.5U •sf*- S i g n i f i c a n t a t 0.01 p r o b a b i l i t y l e v e l . T a b l u l a t e d Values o f t df t(0.01) t ( 0 . 0 5 ) u U.60U 2.776 6 3.707 2.UU7 12 3.055 2.179 These v a l u e s i n d i c a t e t h a t there i s l e s s than a one p ercent chance t h a t the observed d i f f e r e n c e s arose due t o chance i n f o u r of the s i x comparisons. -37-DISCUSSION Thi s i n v e s t i g a t i o n t e s t e d whether the s y n t h e s i s o f f u n g i t o x i c com-pounds i s i n i t i a t e d upon i n j u r y and i n o c u l a t i o n o f sapwood or bark of west-ern redcedar and, i f so, whether these t o x i c compounds are heartwood e x t r a c r t i v e s normally not present i n the sapwood or b a r k . The experimental r e s u l t s i n d i c a t e f o u r conclusions which p r o v i d e the b a s i s f o r an e v a l u a t i o n of t h i s h y p o t hesis. This e v a l u a t i o n i n t u r n provides c e r t a i n i m p l i c a t i o n s concerning a mechanism o f disease r e s i s t a n c e i n western redcedar. I . P h y s i o l o g i c a l changes were observed i n exposed samples which a r e a n a l -ogous i n some resp e c t s t o those accompanying r e a c t i o n zone formation i n the sapwood of p i n e . The p h y s i c a l appearances of the 6 week samples o f bark and sapwood i n d i c a t e the i n i t i a t i o n o f changes. Along w i t h darkening o f c o l o r , a pronounced increase i n r e s i n exudation was noted over the exposed s u r f a c e s . This o b s e r v a t i o n may e x p l a i n the r e s u l t s o f Table I I I which shows t h a t g r e a t -er amounts o f acetone e x t r a c t a b l e substances were recovered f o r samples ex-posed f o r 6 weeks than f o r c o n t r o l s . The presence of r e s i n s on exposed surfaces i s unusual, since western redcedar does not c o n t a i n r e s i n c a n a l s nor i s i t capable o f forming traumatic r e s i n c a n a l s (Brown e t a l . , 191*9). The absence o f traumatic r e s i n c a n a l s i n exposed sapwood samples c o l l e c t e d i n t h i s i n v e s t i g a t i o n was v e r i f i e d by micro-scopic examination. Since a network o f r e s i n c a n a l s i s p r e s e n t i n pine (Brown e t a l . , 19l*9), r e s i n o s i s accompanying r e a c t i o n zone fo r m a t i o n can occur w i t h i n hours. A l -though the i n c r e a s e d amounts o f r e s i n s detected on samples i n t h i s study a r i s e a f t e r a much g r e a t e r i n t e r v a l and from a d i f f e r e n t t r a n s p o r t system, a b a s i c s i m i l a r i t y i n macroscopic changes accompanying i n j u r y e x i s t s between pine -38-and cedar. The presence of the blue-stain fungus on the surface of the sapwood seems to be inconsistent with the formation of a reaction zone. However, i t i s possible that thiseorganism became established before i n i t i a t i o n of the reaction. I t i s also possible that the increased r e s i n flow and other changes associated with reaction zone formation i n the sapwood were stimu-lated by the presence of the blue-stain fungus, although this i s u n l i k e l y since p a r a l l e l changes occurred i n the bark where no blue stain fungus was present. A t h i r d p o s s i b i l i t y regarding the significance of the presence of the blue stain fungus i s discussed under I I . I I . Toxic compounds which are unextractable by either hot water or acetone are formed within 6 weeks both i n the l i v i n g bark and sapwood exposed on a tree as a r e s u l t of i n j u r y . The r e s u l t s of Table V indicate that bark ex-posed f o r 6 weeks i s s i g n i f i c a n t l y more r e s i s t a n t to decay by F. annosus r e -gardless of culture age, while sapwood exposed f o r 6 weeks i s s i g n i f i c a n t l y more r e s i s t a n t to decay by 11 week old cultures of F. annosus than the control but not r e s i s t a n t to decay by 3 week old cultures of F. annosus. Since both controls and exposed samples were extracted f i r s t with acetone and then with 90-95°G water, i t i s probable that a large percentage of the normal extractable substances were removed by this treatment. Therefore percent weight losses probably represent a close approximation of percentage decay of l i g n i n and cellulose and do not represent metabolism of carbohydrates i n any of the samples. Since i t i s extremely u n l i k e l y that increases i n decay resistance arose due to host-mediated s t r u c t u r a l alterations of tissues formed before wounding, a toxic factor(s) was formed and deposited i n the bark and sapwood which caused the observed differences i n decay resistance between exposed samples and . controls. Any physical changes of samples that might arise from the extraction -39-procedure, such as p e r m e a b i l i t y changes, might q u i t e p o s s i b l y a l t e r suscept-i b i l i t y t o decay, b u t could not account f o r the observed d i f f e r e n c e s i n decay r e s i s t a n c e between samples and c o n t r o l s (See Tables IV & V ) . Bark samples exposed f o r 6 weeks r e s i s t e d decay r e g a r d l e s s o f colony age o f the decay fungus, w h i l e sapwood samples exposed f o r 6 weeks r e s i s t e d decay by 11 week o l d c o l o n i e s , b u t were l e s s able to r e s i s t decay by 3 week o l d c o l o n i e s . Thus, colony age of F. annosus i s a s i g n i f i c a n t f a c t o r i n determining weight l o s s e s i n the sapwood samples, b u t not i n the bark samples. A p o s s i b l e e x p l a n a t i o n f o r t h i s unexpected r e s u l t i s t h a t the c o n c e n t r a t i o n of t o x i c f a c t o r i n the sapwood samples was a t the t h r e s h o l d l e v e l needed to produce i n h i b i t i o n i n v i t r o , so t h a t the younger, more vigorous c o l o n i e s of F. annosus were ab l e to overcome the t o x i c e f f e c t s more q u i c k l y than the o l d e r , l e s s v i g o r o u s c o l o n i e s o f the same pathogen. Apparently the t o x i n c o n c e n t r a t i o n i n the bark samples was g r e a t e r than i n the sapwood samples, since colony age does not s i g n i f i c a n t l y a f f e c t decay i n the bark. Since r e a c t i o n zone f o r m a t i o n may be l i m i t e d t o o n l y a few c e l l l a y e r s below the exposed surface, samples were c u t away i n t h i n s t r i p s t o minimize d i l u t i o n w i t h u n a f f e c t e d p o r t i o n s of sapwood and b a r k . Since i t was p o s s i b l e t o c o l -l e c t t h i n n e r l a y e r s o f bark than sapwood due to the c u t t i n g c h a r a c t e r i s t i c s of each, i t h i s d i l u t i o n f a c t o r may be d i r e c t l y r e s p o n s i b l e f o r the l e s s e r t o x -i c i t y e x h i b i t e d by the sapwood sample. An a l t e r n a t e e x p l a n i t i o n o f the l e s s e r t o x i c i t y i s that the t o x i c f a c t o r was p r e s e n t i n v i v o a t a lower concentra-t i o n i n the sapwood than i n the bark. The presence o f b l u e - s t a i n fungus on the sapwood but n o t bark samples exposed f o r 6 weeks tends to support t h i s a l t e r n a t e e x p i i h a t i o n i f growth of the fungus was not i n h i b i t e d a f t e r i n i t i -a t i o n o f the r e a c t i o n zone i n the sapwood. I I I . No t o x i c compounds e x t r a c t a b l e by h o t water, chloroform, o r acetone were formed a f t e r any o f the experimental treatments. This c o n c l u s i o n i s based on the demonstrated complete l a c k o f t o x i c e f f e c t s e x h i b i t e d by a l l e x t r a c t s f o r a l l three types o f bioassay on amended standard medium. This c o n c l u s i o n f o l l o w s from these r e s u l t s p r o v i d e d the f o l l o w i n g four c o n d i t i o n s are v a l i d : 1) t h a t the e x t r a c t i o n procedure used would remove s i g n i f i c a n t amounts of normal e x t r a c t a b l e substances. T h i s c o n d i t i o n i s n e a r l y a d e f i n i t i o n and needs no f u r t h e r comment. 2) That F. annosus i s a good t e s t organism and would have responded t o any t o x i c e f f e c t s had they been present. This c o n d i t i o n i s probably j u s t i f i e d since F. annosus i s not a common pathogen o f western redcedar, even though the d i s t r i b u t i o n s of the two s p e c i e s o v e r l a p . Therefore, redcedar may possess some means of r e s i s t -ance to t h i s widespread t r e e pathogen. I n a d d i t i o n the d i f f e r e n c e s i n weight l o s s e s w i t h i n c o l l e c t i o n b l o c k 6 a f t e r decay by F. annosus i n d i c a t e t h a t F. annosus was s e n s i t i v e to the t o x i c f a c t o r p r e s e n t and would probably have responded to any e x t r a c t a b l e t o x i c e f f e c t s , i f they had been present. 3) That a h i g h p r o p o r t i o n of the exposed samples o f bark and sapwood under-went p h y s i o l o g i c a l changes a s s o c i a t e d w i t h formation of r e a c t i o n zones. This c o n d i t i o n i s p r o b a b l y j u s t i f i e d since the sampling technique was designed to minimize d i l u t i o n o f samples w i t h unreacted t i s s u e s . The weight l o s s r e s u l t s i n d i c a t e t h a t t h i s c o n d i t i o n was met f o r the bark and the sapwood a l s o , although perhaps to a l e s s e r degree i n the sapwood f o r the reasons d i s -cussed under I I . h) That the amount o f e x t r a c t a b l e m a t e r i a l s added t o the standard medium represented a l a r g e enough percentage such t h a t i f any t o x i c f a c t o r was e x t r a c t e d , i t would have been present i n the media a t concentrations roughly the same as those a t which i t occurs i n the r e a c t e d t i s s u e s . P r o -v i d i n g t h a t the former three c o n d i t i o n s are t r u e , t h i s c o n d i t i o n i s j u s t i f i e d f o r any m a t e r i a l s r e a d i l y s o l u b l e i n the e x t r a c t i n g s o l v e n t s , since the - i l l -v a l u e s l i s t e d f o r c o l l e c t i o n b l o c k s l*-6 i n Table I I I i n d i c a t e t h a t the p e r -centages bysweight o f e x t r a c t e d m a t e r i a l s present i n the t e s t media were e i t h e r greater o r of the same order o f magnitude as the corresponding p e r c e n t -ages i n a l l o f the samples from these b l o c k s . I ? . None of the normal e x t r a c t a b l e substances commonly recognized as con-t r i b u t o r s to heartwood decay r e s i s t a n c e are formed i n response t o exposure i n e i t h e r the bark or the sapwood. I n the case of tropolones, t h i s c o n c l u -s i o n i s very d e f i n i t e because the a n a l y t i c a l technique o f MacLean and Gardner (1956) i s h i g h l y s e n s i t i v e , and the e x t r a c t i o n procedures used would have removed a l a r g e percentage o f any tropolones present t h a t had been deposited i n samples p a r a l l e l to the manner o f d e p o s i t i o n i n heartwood. I f any h e a r t -wood compounds were formed and e x t r a c t e d from samples, these compounds were not f u n g i t o x i c i n v i t r o . P r o v i d i n g t h a t the above fo u r c o n c l u s i o n s are j u s t i f i e d , the f o l l o w i n g o v e r a l l c o n c l u s i o n may be drawn! there i s a mechanism o f disease r e s i s t a n c e i n western redcedar bark and sapwood which i s i n i t i a t e d i n response to exposure r e s u l t i n g from i n j u r y . This mechanism i n v o l v e s the formation o f r e a c t i o n zones i n the bark and sapwood c o n t a i n i n g a t o x i c substance which i s n e i t h e r a normal heartwood compound nor e x t r a c t a b l e by p o l a r s o l v e n t s . To the b e s t o f t h i s author's knowledge t h i s o b servation has not been made bef o r e f o r western redcedar, and no e x a c t l y p a r a l l e l observations have been made f o r any other t r e e . The presence i n r e a c t i o n zones o f t o x i n s i n s u f f i c i e n t c o n c e n t r a t i o n s to produce f u n g a l i n h i b i t i o n , a u t o m a t i c a l l y confers a c e r t a i n amount o f d i s -ease r e s i s t a n c e t o the h o s t . However, the formation o f to x i n s as demonstrated i n t h i s i n v e s t i g a t i o n does not provide the b a s i s f o r a q u a n t i t a t i v e assessment o f the c o n t r i b u t i o n o f t h i s component t o the o v e r a l l disease r e s i s t a n c e of -1|2-western redcedar. The weight l o s s e s observed a f t e r decay of samples from c o l l e c t i o n b l ock 6 i n d i c a t e the presence of a f a c t o r which reduced decay. Nevertheless, any i n f e r e n c e drawn from these r e s u l t s can only be a p p l i e d t e n t -a t i v e l y to n a t u r a l host-pathogen i n t e r a c t i o n s , since the r e s u l t s are d e r i v e d from a c o n t r i v e d s i t u a t i o n . Although o n l y f u r t h e r work w i l l r e v e a l the chemical nature of the t o x -i c f a c t o r and the reason i t i s u n e x t r a c t a b l e , experience i n pu^paaridgpaper research has shown t h a t some e x t r a c t i v e s are d i f f i c u l t t o e x t r a c t from p u l p . H i l l i s e t a l . (l!?66) r e p o r t s t h a t a s t i l b e n e ( 3> 3 1 -dimethoxy-U,k 1 -dihydroxy-s t i l b e n e ) , causing reddening of pine p u l p , i s s t r o n g l y adsorbed so t h a t i t s removal r e q u i r e s s p e c i a l e x t r a c t i o n procedures. Since other s t i l b e n e s are well-known f u n g i t o x i n s , t h i s case i s p a r t i c u l a r l y i n t e r e s t i n g i n l i g h t o f phenomena observed f o r western redcedar. I t i s p o s s i b l e t h a t a p a r a l l e l e x i s t s between these two s i t u a t i o n s . Determination of the s i g n i f i c a n c e o f - t h i s mechanism of induced disease r e s i s t a n c e i n the sapwood and bark i s dependent upon f u r t h e r knowledge of a v a r i e t y o f f a c t o r s . I s t h i s the only mechanism o f r e s i s t a n c e o r are there a d d i t i o n a l systems s u p e r i m p o s e d — e i t h e r chemical c r p h y s i c a l ? How important are h o s t and environmental c o n d i t i o n s i n deterndning the occurrence or ampli-tude o f t h i s r e a c t i o n ? What f a c t o r s or i n t e r a c M o n s o o c t m r g ^ and b i o c h e m i c a l s c a l e which c o n t r o l i n i t i a t i o n ? What i s the nature of the t o x i c f a c t o r ? Only c a r e f u l l y planned and executed experimentation can answer these questions. 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