Tansley Review No. 87. Antimicrobial Defences in the Wood of Living Trees Author(s): R. B.

Pearce Source: New Phytologist, Vol. 132, No. 2 (Feb., 1996), pp. 203-233 Published by: Blackwell Publishing on behalf of the New Phytologist Trust Stable URL: http://www.jstor.org/stable/2558445 . Accessed: 30/08/2011 13:58
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New Phytol.(1996), 132, 203-233

Tansley Review No. 87 Antimicrobial defences in the wood of living trees

BY R. B. PEARCE
School of Biological Sciences, Universityof Birmingham,Edgbaston, BirminghamB15 2TT, UK (Received 5 June 1995)
CONTENTS

I. II.

III. IV.

Summary Introduction Xylem pathogens 1. Wood decayingfungi 2. Canker rots 3. Endophytesand latentinfections 4. Vascular wilts 5. Xylem-inhabiting bacteria 6. Viruses Bark- the first line of defence sapwood Defence in functional 1. Models forthe host-pathogen in the wood of livingtrees interaction concept (a) The heartrot of decay in (b) Compartmentalization trees(CODIT) (c) Dynamic decay developmentand reactionzones restriction of fungal (d) Environmental colonization 2. Putativedefencemechanisms constraints on (a) Environmental pathogengrowth

203 204 205 205 205 205 206 206 206 206 207 208 208 208 209 212 212 212

V. VI. VII. VIII.

factors (b) Nutritional of normalwood (c) Anatomicalfeatures cork (d) Interxylary compounds inhibitory (e) Constitutive (phytoanticipins) (f) Resin (g) Lytic enzymes (h) Cell wall alterations compounds (i) Induced antimicrobial (phytoalexins) (j) Necroticand hypersensitive responses Defence in heartwood Dynamic aspects of the host-pathogen interaction in wood Genetic controlof resistancein xylem tissues Antimicrobial defencein the livingxylem of trees: towardsa consensusmodel Acknowledgements References

213 213 214 214 215 216 216 218 221 222 223 224 225 227 227

SUMMARY

and plantsurface(periderm from microbialattackby thesecondary The wood (xylem)in a livingtreeis protected barrier preventingthe entryof most potential pathogens,and by which provides an effective rhytidome), and induceddefencemechanisms in thebark(cortexand phloem).Althougha fewpathogensare able constitutive wounds thatexpose thistissue theseoutertissuesdirectly, mostxylempathogensgain entry through to penetrate by and renderit more vulnerableto attack. In functional sapwood, microbialcolonizationmightbe restricted restriction consequentupon thehighwatercontent or by passive microenvironmental activedefencemechanisms, exclusive:indeed,theymay xylem.These factors are notmutually of 02 inhealthyconductive and low availability operate in concert in many host-pathogeninteractions.Sapwood lesions made by wood-decayingfungi are wall 4 barriersand bounded by multicellularwalls or barrierzones (compartmentalization characteristically barriers bothas inhibitory or degradation-resistant reaction zones (columnboundarylayers)),whichmayfunction and prevent thedrying and aerationthatcould xylemfunction to further pathogenspread,and as seals to maintain to the infection.A range of putative antimicrobialdefence mechanisms contributing predispose to further constitutive in sapwood tissues.This includescell wall alterations, ofsuch barriers has been identified effectiveness and induced antimicrobial compounds,necroticresponsesof livingcells and the depositionof gummymaterials, features environmental and anatomical at thehost-pathogen interface. Nutritional, often resinousor polyphenolic, in detail,the to pathogenrestriction. Althoughtheremightbe differences of livingwood mightalso contribute of similar.Defence responsesagainsta variety and angiosperms are generally defencesoperating in gymnosperms

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R. B. Pearce

therolesofthevarious also have muchin common.Dynamic studiesare crucialin elucidating pathogencategories and defence interaction in the wood of livingtrees.A model forthe protection components of the host-pathogen of xylem tissues in woody angiospermsis suggested, based largely upon results from dynamic studies of L.). interactions in the wood of sycamore(Acerpseudoplatanus host-pathogen wood, trees,decay. defence,disease resistance, Key words: Antimicrobial

readilystudied) in herbaceousspecies, the bulk and longevityof trees impose importantrequirements and constraints for the success of endogenous Despite the economic, environmental and ecological importance of trees, our understanding of their resistance. In the secondary tissues, that is bark, phloemand cambium, cortex, periderm, pathology has generally lagged behind that of comprising agricultural crop species, a fact that has been noted and wood (xylem),defencemay need to be effective previously (e.g. Kemp & Burden, 1986; Pearce, fora long time.Whilstthe abilityof a plant defence fora few 1987; Duchesne, Hubbes & Jeng, 1992). Although mechanismto halt pathogendevelopment to protectephemthe host-pathogen interactions of woody perennials days or weeks mightbe sufficient exhibit a number of distinctive features, these eral plant tissues,the progressof a wood-decaying for decades, or typically receive scant mention in most general plant fungusmightneed to be restricted pathology texts, and the first book specifically perhaps even centuriesif the tree host is not to die addressing the physiological and biochemical aspects prematurely.Additionally, many pathogens and of tree pathology has only recently been published potentialpathogens can establish extensivesaprotrophic growthin dead or moribund wood. Such (Blanchette & Biggs, 1992). be stillattached This neglect of tree pathosystems can be attributed bulkycolonizedtissues,whichmight to several factors. Until recently trees have been to an otherwisehealthytree (for example decayed regarded more as a natural resource to be exploited heartwoodor suppressedbranches),provide a food whicha pathogencan mounta prolonged than as a managed crop, in which measures to reduce base from disease loss could be actively deployed. [In this assault on the defencesof the host. The inoculum context it is to be noted that much of the early work potential(Garrett,1970) of such an attackmightbe on host-pathogen interactions in trees is related to far greater than that of a spore inoculum, for orchard species (Brooks & Storey, 1923; Swarbrick, example, and mightthus require a more extensive 1926).] Further, although the finalvalue of a standing and durable host defenceto oppose it successfully. pathogensto tree crop might be high, its annual increment is low. The abilityof most wood-inhabiting long-established In natural and traditionally managed forests, the remainactiveformanyyearswithin population structure has not favoured the dev- lesions similarlyposes a continuingthreat. In the at thelesionmargins, ofdefences astating disease epidemics that often appear to be eventof anyfailure tissues. mayresumeinvasionofhealthy products of intensive crop production (Schmidt, thepathogen of decay developmentin the wood 1978; Zadoks & Schein, 1979). However, when The resumption spp. c. 14 yr afterwoundinghas been conditions for disease have been favourable, dam- of Eucalyptus zone at the ofa barrier aging tree disease epidemics have occurred [e.g. associatedwiththedisruption Dutch elm disease, chestnut blight, white pine blister lesion marginat this time (White & Kile, 1994). Thus the need for long-term exclusion of a rust; see Manion (1991) for brief accounts], and current trends towards the intensification of tree pathogen with potentiallya very high inoculum tissues, within production favour an increase of disease problems in potentialfrombulky heterogeneous forestry (Evans, 1982). In addition to these es- which living and hence responsive cells are disdistributed (cf. Esau, 1977; Carlquist, sentially economic factors, technical difficultieshave continuously on requirements 1988), imposesunusuallystringent also tended to discourage research on tree diseases. of woodyplants.This reviewwill focus The size of trees, their slow development and the the defences in the long timescale of many of their diseases render them on mechanismsthat are believed important of wood (xylem)tissuesagainstmicrobial unattractive for experimental use. Further, the protection diversity of economically significant woody species, attack in living trees. Wood is not only the most tissue of trees,it is also coupled with the relatively small number of re- abundantand characteristic searchers active in tree pathology, has meant that our the principal product obtained fromtree crops. A understanding, even of many common tree diseases, range of putative defence mechanisms has been identified in living wood, and models for the is often fragmentaryand superficial. In consequence, many aspects of antimicrobial operation of these mechanismsproposed, but our ofmanyaspectsofthehost-pathogen defence in trees remain imperfectly understood. understanding Current involvedremainsfragmentary. Whilst it is reasonable to suppose, a priori, that the interactions defence mechanisms operating in woody plants are outstandingproblems will be identifiedand dislikely to be similar to those better known (and more cussed here.
I. INTRODUCTION

Antimicrobial defencesof living wood

205 (Pearce & Woodward, 1986; Pearce, 1991). Ultimately these fungi are capable of penetrating sapwood and bark from within to form fruitingbodies on living host trees (Burdekin, 1979). The desiccation tolerance strategy is associated with fungi decaying dead wood on trees (Rayner & Boddy, 1988). These exhibit a primarily saprotrophic habit, although in some cases they might be present as endophytes or latent infections in the living tree (see 11.3, below). 2. Canker rots

two botanicaldivisions, Trees includeplantsfrom and the angiosperms.Although the gymnosperms betweenthese divisions, thereare many differences fortheirdefencesby leading to separatetreatments & Biggs (1992), a high degreeof congruBlanchette To avoid ence is evidentin theseparalleltreatments. and angiosperms undue duplication,gymnosperms in thepresentreview,except will be treated together existbetweenthem.Tree wherematerialdifferences defences against insects have many parallels with antimicrobialdefences (cf. Mattson, Levieux & Bernard-Dagan,1988). Whilstdefenceagainstinsect pests (or other herbivores)will not be specifically it should be bornein mindthatthe same considered, processes may be involved in defenceagainst both and insects,and in wound healing micro-organisms to separatethese (Mullick, 1977), and thatattempts misleading. mightprove ultimately functions

Certain fungi, e.g. Inonotus obliquus on Betula spp. and Phellinus pini on Abies spp., can attack bark and xylem tissues simultaneously (Shigo, 1969; Blanchette, 1982). These pathogens are able to re-invade wood repeatedly from the bark, and may, by this means, evade containment by barrier zones formed within the wood (Blanchette, 1982). Strip cankers, PATHOGENS II. XYLEM where-infectiongives rise to axially elongated zones patho- of dead bark and underlying xylem, resemble canker pathogenicor potentially Micro-organisms, genic, in the xylemof trees can be subdivided into rots in that both bark and xylem are attacked, cankerrots, although development often occurs during a single six broad groups- wood decayingfungi, endophytes and latent infections,vascular wilts, year only and might be associated with the effectsof predisposing factors (Lonsdale, 1983; Rayner, 1986; bacteria,and viruses. xylem-inhabitating Boddy, 1994). These lesions can develop from fungi propagules latent in the wood and bark (Boddy, 1. Wood decaying 1994).

The frequent isolation of fungi from apparently normal, healthy wood (e.g. Roll-Hansen & RollHansen, 1979; Chapela, 1989) has kindled interestin the significance of these asymptomatic infections. These fungi can develop in declining wood under ized opportunism. Active pathogens are able to colonize healthy favourable environmental conditions, giving rise to sapwood, followingeither direct penetration(e.g. decay lesions (Chapela & Boddy, 1988b; Chapela, or infection 1989; Griffith& Boddy, 1990). This habit might Armillaria annosum) spp., Heterobasidion through wounds, which can provide a suitable confer on these fungi a selective advantage by purpureum, facilitating their early and rapid colonization of infection court (e.g. Chondrostereum Stereum gausapatum).These fungican cause rapidly dying stems and branches. Little is currentlyknown lesionsin thexylemoftheirhosts. of the route of entry of these fungi and of the form necrotic spreading Fungi adopting opportuniststrategiesare less taken by latent infections in the healthy tree (Boddy, clearly aggressive, initially colonizing only func- 1994), although infection processes for Hypoxylon tionally compromised sapwood, for example that fragiformehave been described (Chapela, Petrini & associatedwitha majorwound. Heart rotfungigrow Petrini, 1990; Chapela, Petrini & Hagmann, 1991). and cause decay within the heartwood,which is Evidence from controlled drying studies has indigenerallydevoid of livingcells and hence lacks the cated the importance of the high water content of capacityfor active response to infection.It might, functional xylem in arresting development of these however,be protectedby allelopathic compounds fungi (Chapela & Boddy, 1988b; Chapela, 1989; formation Boddy, 1994). The association of other putative thatare oftendepositedduringheartwood of sapwood defences with latent infections does not The opportunist pathogenicity (Hillis, 1987). to have been investigated to date. Immunoas the heartwood is such variable. appear Some, fungi and Sparassis crispa, logical methods provide a sensitive and specific rottersLaetiporussulphureus appear to have little or no ability to invade living host means of detecting and localizing fungi in woody tissues, whilst many others, such as Ganoderma spp., tissues (Blanchette & Abad, 1992) and have been Inonotus hispidus and Ustulina deusta function as used to detect endophytic hyphae in asymptomatic weak pathogens, slowly attacking living host tissues needles of Picea abies (Suske & Acker, 1989). Such

These comprise the most studied group of xylem inhabitantsin trees. Several ecological or pathological types can be distinguishedon the basis of their colonization strategies. Rayner (1986) and Rayner & Boddy (1988) recognizedfivedistinctive - heartrot,activepathogenesis, specialized strategies opportunism,desiccation tolerance and unspecial-

3. Endophytes and latent infections

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R. B. Pearce

methods should facilitatestudies of wood endo- (Biddle & Tinsley, 1971). As these tree infections phytesin situand permitfurther investigation of the have been little studied at the physiological level, host-pathogen interactions of these fungi. defence against viruses will not be considered further 4. Vascularwilts In vascular wilt diseases, microbialcolonizationof the conductivexylemwithout grossdisruption of its structure resultsin impairedwater relationsin the host. Damage is mediated via vascular dysfunction or translocatedtoxins produced by the pathogen. Propagulesofthepathogenare disseminated through the xylem, resulting in the rapid and extensive colonization of conductive wood in infectedsusceptibletrees (Mace, Bell & Beckman,1981). Dutch elm disease (Ophiostoma ulmi,Ophiostoma novo-ulmi) is the best knownvascularwiltdisease of trees,althoughVerticillium species can cause disease in a range of woody angiosperms.Typical vascular wilt syndromes do not occur in gymnosperms, probably because theirconductivexylem elements (tracheids) do notfavour theeasyand rapidspreadof such pathogens(Green, 1981). However, infections that impair vascular functiondo occur in gymnosperms. The pine root pathogen Verticicladiella wagneri has manyof the characteristics of a vascular wilt fungus(Smith, 1967), and a nematode,Bursaphalenchus xylophilus, causes a wilt syndrome (Mamiya, 1983). bacteria 5. Xylem-inhabiting A few bacterial pathogens can colonize living sapwood, causing gross symptomsthat resemble vascularwilts.Watermark disease of willow,caused by Erwinia salicis, provides an example (Preece, 1977). Also, fastidious xylem-limited bacteria have been reported a variety from (Xylellafastidiosa) of tree species in which they cause leaf scorch symptoms (Hopkins, 1989). Bacterial wetwood results from colonization of heartwood and inner sapwood by populations of bacteriacapable ofgrowing Colonized anaerobically. wood accumulatesfluid, aliphaticacids and methane, oftenunder pressure,but does not become decayed thereappearslittle (Ward & Zeikus, 1980). Although it cause to describethesebacteriaas truepathogens, has been statedthattheextent of colonization within the tree is limitedby plant defences(Pirone et al., 1988). Further, bacterial wetwood might also be associatedwithothertree infections (Boddy, 1994). 6. Viruses
and the living wood of trees (cf. Nienhaus & Castello, 1989). In one study the strength and specific gravity of wood from poplars systemically infected with poplar mosaic virus was reduced in this review. Despite the range of pathogens attacking woody xylem tissues, there is an underlying uniformity in the responses of trees to these infections. For example, vessel-blocking responses observed in wood infected with the bacterium Xylella fastidiosa (Hopkins, 1989), are similar to those occurring at the margin of fungal decay lesions (Pearce, 1990). Separate treatments of defence against these diverse disease syndromes are not, therefore, required.
III. BARK-THE FIRST LINE OF DEFENCE

Little is known of the interactions betweenviruses

In the living tree the xylem is normally sheathed in bark, which comprises periderm, cortex, phloem and cambial tissues. Together, these present a formidable barrier to the ingress of most potential pathogens. Whilst it is not within the remit of this review to present a detailed treatment of defence in bark, it will be considered briefly,as bark defences serve to arrest many potential infections before they reach the wood. For a fuller consideration of disease resistance mechanisms in bark, see Biggs (1992a, b) and Woodward (1992). In a structurally intact living tree, the xylem is entirely encased within the bark, the only direct exposure being at sites of very recent leaf abscission. Any pathogen normally inhabiting the xylem must, therefore, cross these outer tissues. However, the xylem can be exposed by diverse wounding agents, which locally compromise the integrityof the plant surface. In nature such wounds occur abundantly. Although the infection biology of many woodinhabiting pathogens remains obscure, most probably enter through such wounds. Decay commonly develops behind major wounds that expose wood; these might occur naturally, e.g. as a result of wind damage, or may be the result of human activity (tree management operations) (Rayner & Boddy, 1988). Spores of the vascular pathogen Ophiostoma novoulmi are introduced into elm xylem through wounds made during maturation feeding by the bark beetle vectors of Dutch elm disease (cf. Ouellette & Rioux, 1992). A few fungi are capable of penetrating directly through an intact bark surface. Armillaria species provide the best understood examples; rhizomorphs of Armillaria growing from a bulky food base can mount a prolonged attack on the roots of a potential host and can ultimately break the surface barriers (Morrison, Williams & Whitney, 1991); some other cord-forming fungi behave similarly (Tourvieille de Labrouhe, 1982). Suberin-degrading enzymes may be important in the penetration of the highly resistant periderm surface by these fungi (Zimmerman & Seemuller, 1984; Ofong & Pearce, 1994).

Antimicrobial defencesof living wood Few, if any, fungi appear able to penetrate an intact bark surface directly from spore inoculum (Dickinson, 1976), although some can bypass the surface barrier by infecting through primary tissues, e.g. Verticillium dahliae, which enters through young roots (Schnathorst, 1981). The route of ingress of many wood-infecting fungi remains uncertain, although entry through wounds that expose the xylem, either above or below ground, seems likely. These wounds can result frommechanical damage, damage by herbivores, or might be a consequence of the natural turnover of small roots and shoots. In one interestingcase, the conifer heartrot fungus Phaeolus schweinitzii has been shown to gain entry into the wood of spruce trees via preexisting local Armillaria lesions on the roots. In the necrotic tissues of these lesions, P. schweinitzii was not hindered either by the hosts' normal defences or by interaction with Armillaria (Barrett, 1970). Attempted penetration by this fungus was swiftly arrested in healthy bark, even after wounding to remove the surface periderm (Woodward & Pearce, 1988b). Intact bark tissues thus appear to protect the xylem (and, perhaps most importantlyforthe overall health of the tree, the cambium) against potential pathogens. A range of putative defences which might confer this protection has been identified. At the surface the periderm is effective as a first line of defence. The suberized phellem (which can range in thickness from just a few cell layers to a multilayered rhytidome structure) is highly resistant to degradation, as evidenced by the persistence of bark surfaces over a period of several decades (Hepper, 1981). Suberin, a polymer composed primarily of long-chain aliphatic residues (interesterified hydroxy and epoxy-fattyacids, typically with a carbon chainlength of C18 or longer), together with domains comprising polymeric aromatic units (see Kolattukudy (1984) for an interpretation of the structure of suberin) is hydrophobic and highly durable. Those organisms able to degrade it do so only slowly (Swift, 1965; Zimmermann & Seemuller, 1984; Ofong & Pearce, 1994). In addition, accumulations of various constitutive antimicrobial compounds have been reported from periderm and rhytidome tissues. These include terpenes and polyphenols (Jensen et al., 1963). Despite, or perhaps because of, their ubiquity and effectiveness in protecting the underlying tissues, there have been few detailed studies of the constitutive protection provided by secondary plant surfaces (cf. Campbell, Huang & Payne, 1980; Pearce, 1987; Merrill, 1992). Non-specific periderm restoration responses are a common feature of wound repair, antimicrobial defence and response to insect damage (Mullick, 1977) in both woody angiosperms (Biggs, 1992 a, b) and gymnosperms (Woodward, 1992). These function to restore a refractory periderm beneath wounds

207

that breach the normal surface(Biggs, 1992a) and aroundrestricted lesionsofcankerpathogens(Biggs, Merrill & Davis, 1984; Biggs, 1992b) and unsuccessfulpenetration attemptsby such pathogens as Armillariaspp. (Pearce, 1989). Similar responses may also occur in the bulkystoragetissues of nonwoody species such as Armoraciarusticana(Pearce, 1989). Alterations to the walls of pre-existing cells may also be important in bark defence: lignification and suberization of barkcells were observedpriorto the formation of necrophylactic(wound) periderms (Biggs, 1985; Biggs & Stobbs, 1986; Woodward & Pearce, 1988b). These might contribute to protection during the differentiation of the more permanent periderm barrier. Aggressive pathogens have the abilityto penetrateor bypass these induced multicellular barriers (Biggs, Davis & Merrill, 1983; Biggs, 1986), and correlations have been reported betweenratesofaccumulation ofsuberinin wounded bark tissues and cultivar susceptibility to disease (Biggs & Miles, 1988). Other mechanismsthat might contribute to defencein bark tissues include high concentrations of pre-formed antimicrobial compounds (phytoinhibitins) such as the stilbenes present at high in healthy concentrations sprucebark(Woodward & Pearce,1988a); these mightact in concertwith cell wall alterationsto effect protection(Woodward & Pearce, 1988b). Induced, phytoalexin-like, antithebark from fungalcompoundshave been reported of a numberof woody species, e.g. Populus tremuloides(Flores & Hubbes, 1979, 1980) and Morusalba (Shirata, 1978; Takasugi et al., 1978b, 1979). Antifungal hydrolases(chitinaseand ,B-1,3-glucanfrom barktissuesin several ases) have been extracted woodyangiosperms (Wargo, 1975; Shain, 1993) and thejuvenile tissuesof certainPinus species (Bonello, 1991; Nsolomo & Woodward, 1994). Hypernecrosishas been reportedassociated sensitive-type with resistanceto stem and gall rusts in conifers (Kinloch, 1982). In gymnosperms, wounding or infection can stimulatethe productionof resins. A critical assessment of their importance in antimicrobialdefenceis rendereddifficult by conflicting reportsof theireffectiveness (cf. Woodward, 1992), but a consensussuggeststhatresinousexudatescan provide an effectivedefence mechanism (Hillis, 1987; Woodward, 1992). However,instanceswhere resinaccumulationdoes not appear to contribute to defence are well documented (e.g. BarrowsBroaddus & Dwinell, 1983).
IV. DEFENCE IN FUNCTIONAL SAPWOOD

Healthy sapwood comprises both living and nonliving cells. Living parenchyma cells which are capable of metabolic activity comprise a high proportion of the wood in some angiosperms, but,

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R. B. Pearce Wall 1 (b)

typically, only a small proportion of the cells in gymnosperm woods. In gymnosperms, parenchyma cells are present predominantly as xylem rays: axial parenchyma is generally poorly developed. Resin ducts, where present, are lined by living parenchyma cells. Conductive and mechanical functions are fulfilled by the tracheids which comprise the remainder of the wood in most species. Angiosperm xylem is anatomically more complex, and considerable variation in wood structure exists. Axial parenchyma is present, as well as ray parenchyma. Commonly, axial parenchyma is closely associated with vessels (paratracheal), but apotracheal parenchyma (distributed without relation to vessels) is often also present. Non-living cells comprise vessels, tracheids and fibres,which can be furthersubdivided and classified (see Esau, 1977; Fahn, 1982; Carlquist, 1988). By virtue of its viable cells, the sapwood of a living tree is capable of active responses to infection, unlike timber and other forest products, which only provide a passive substrate for microbial growth. The anatomy of wood, with an axial alignment of its principal structural elements (vessels, tracheids and fibres), and a discontinuous distribution of living cells, is reflected in the host-pathogen interactions observed in sapwood. These features impose constraints on both the spread of pathogens and the expression of tree responses to infection and damage. Since the pioneering work of Robert Hartig in the nineteenth century (Hartig, 1894), which has formed a basis of much subsequent tree pathology, four principal models have been advanced to describe the development of fungal lesions (staining or decay) in the wood of living trees - the heartrot concept, compartmentalization of decay in trees, the reaction zone model and the environmental restriction of fungal colonization. In three of these a close relationship is established between wounding and infection. In part this can be accounted for by the protection normally afforded by the bark which, when intact, denies most potential pathogens access to the xylem tissues (see III, above). The consequences of wounding on xylem function may also be important (see IV-1 (d) below). 1. Models for the host-pathogen interactionin the wood of living trees (a) The heartrotconcept. Until relatively recentlythe dominant model of decay in standing trees (see Boyce, 1961; Peace, 1962), the heartrot concept is now recognized as an over-simplification. Decay was perceived as a predominantly saprotrophic process, confined within the non-living heartwood. Wounds or dead organs exposing (directly or indirectly) the heartwood allowed the entry of fungal propagules. No killing of living tissue was envisaged and the fungus could only leave the living tree (normally by

.--Wall 2

(c)

Wall4
(d) WaIl 3

Figure 1. Compartmentalizationof decay in trees (CODIT). Location oftheproposedcompartmentalization walls 1-4 in relationto a wound-associateddecay lesion in the stem of a tree. (a) Longitudinal section. (b)-(d) Transversesectionsat threepositionsalong the lesion. In zone around reaction (b) wall 1 appearsas a circumferential the lesion.
basidiomes)) (usually fruiting bodies producing through interfaces between dead wood and the external environment. These could include the initial infection court or be created following mechanical failure of stems or branches weakened by internal important decay fungi (e.g. Laetiporus sulphureus, Phaeolus schweinitzii and Sparassis crispa) commonly behave according to this model (Burdekin, 1979; Greig, 1981). Whilst heartrot can cause severe economic loss in old and natural forests, this type of infection cannot account for the interactions between decay fungi and living tissues observed in many trees. (b) Compartmentalization of decay in trees (CODIT). The CODIT model was proposed and developed by Shigo and co-workers (Shigo & Marx, 1977; Shigo, 1979; 1984) to describe and account for the patterns and decay developing in living of discolouration wood behind tree wounds. According to the CODIT model, lesions in living wood are bounded and, by decay. Certain

inference, restricted by barriers laid down in the wood. These have been termed walls 1-4 (Fig. 1), and enclose any lesion or incipient lesion within a defined compartment. They were envisaged as

Antimicrobial defencesof living wood

209

essentially static barriers preventingthe further Intermediate decay spread of infection. Such putatively defensive zones zone Reaction Healthy wood of altered xylem had been reported many years Advanceddecay Transition zone previously(e.g. Swarbrick,1926; Hepting & Blaisdell, 1936), but had been largelyneglecteduntilthe formulation of the CODIT model. Walls 1-3 are formedwithinwood extantat the time of wounding. Wall 1 is formedtransversely acrossthexylem,and acts as a boundary or barrier to the axial spread of infection.It was envisaged as resulting from vessel-plugging responses in the (a) conductive tissuesand regardedas theweakestofthe compartmentalization walls. Wall 2, resistingthe radialspreadofa pathogeninwardsfrom thewound, has been attributed mainlyto anatomicalfeatures of the wood, and wall 3, resisting the lateralspread of infection, has been attributed to the activities of ray parenchyma cells. Walls 2 and 3 were perceivedas thanwall 1, but ultimately beingstronger capable of being defeatedby a pathogen,following whichthey (b) could be reinstated to containa lesion of increased volume (Shortle, 1979; Blanchette,1992). The wall 4 barrier differs fromwalls 1-3 in thatit is formed in the plane of the cambiumat the timeof wounding as a response to damage (and, perhaps, infection (Pearce, 1987)). It is thestrongest and most durable of the compartmentalization walls, comprisingcells laid down de novo, which can forma structurally homogeneousbarrier, rather thanbeing (c) formedafterdifferentiation in a tissue containing The Figure 2. reaction zone model as proposed by Shain diverse cell types as occurs for walls 1-3 (Shigo, (1967). The reaction zone retreats ahead of a continuously 1984; Blanchette, it is themost 1992). Functionally, advancing decay front, all sapwood becoming first a important barrier, protecting theyoungest wood and transition zone, then a reaction zone before becoming part the cambial tissues, both of which are vital to the of the spreading lesion. continuing growthand survivalof the tree. As originally formulated, little evidence was ized by the pathogen (Fig. 2). As originally conpresented for the physiological and biochemical ceived, reaction zones retreated ahead of a conmechanismsby which compartmentalization in the tinuously advancing lesion margin, sapwood passing treemightbe effected (Shigo & Marx, 1977; Shigo, through a reaction zone stage en route to becoming 1979). More recently, anatomical changes and cell decayed. Phytoalexin-like antifungal compounds wall alterations have been identified in accumulating in these regions were considered to (suberization) the wall 4 barrier zone in a number of species retard the advance of the colonizing fungus. Dis(Tippett & Shigo, 1980, 1981b; Pearce & Ruther- tinctive alterations at both tissue and cellular levels, ford, 1981; Pearce & Woodward, 1986; Pearce, regarded as representing reaction zone formation, 1990). These are discussed further below (IV 2(h) have been seen at the margins of decay lesions in (see Figs. 5, 6)). Inhibitory compounds mightalso many tree species (e.g. Pearce & Woodward, 1986; accumulate in these tissues (Pearce & Woodward, Pearce 1990) (Figs 3, 4). 1986). Compartmentalization walls 1-3 may be Since reaction zones were initially described equated with reactionzones (Shigo, 1984), see IV (Shain, 1967, 1971), evidence has accumulated from 1 (c) below, wherethe possible defencemechanisms several different angiosperm pathosystems to suggest involvedwill be considered. that these reaction zones are not the dynamic and reactionzones. static boundaries to infection (Pearce, 1987, 1991; (c) Dynamic decay development from Arising studiesoftheinvasivepathogen Hetero- Boddy, 1992). When reaction zone boundaries fail, a basidionannosum colonizing the stems of pine and volume of wood is colonized with little or no
structures initially envisaged, but normally form

spruce trees frominitial root infections, Shamn(1967, 1971, 1979) developed the concept of the reaction zone as a region of active host response at a dynamic interface between living sapwood and wood colon-

expression of characteristic reaction zone responses, until ultimately a new reaction-zone boundary is established (Pearce, 1991). This corresponds closely with the dynamic behaviour of CODIT walls 1-3

210

R. B. Pearce

5wg

~~~~~~~~6

7"e
Figures 3-8. For legend see opposite.

Antimicrobial defences of livingwood

211

zones are well which are not always entirely in componentsoccurs. Drier transition effective, resulting alternatingphases of 'breakout' and new com- documented in gymnosperm woods (Yamada, 1992). appears partment formation (Shortle & Smith, 1990; Accumulationof water,ratherthan drying, Blanchette,1992). The CODIT model does not in- more typical of reaction zones in angiosperms corporatea reactionzone boundary(formedin pre- (Pearce et al., 1994a; Pearce, unpublished),but has (Shain, 1971). from gymnosperms existing wood) at theoutercircumferential marginof also been reported lesions,the distinctive wall 4 barrieroccupyingthis Since clear evidence for chemical alterations in position.However, in transverse sectionsthrougha adjacent sapwood of normal appearance has been tree,a wall 1 boundaryforming a conical 'cap' to a obtained(Grime & Pearce, 1995), thisalteredtissue zone. decay column would appear as a circumferential may be consideredequivalent to a transition enzyme activity reactionzone (cf. Fig. 1). This equivalence allows Increased NAD-linked respiratory the unifi-cation of the COD IT and reaction zone in parenchymacells distal to incipientdiscoloured models for decay restriction in living trees. Since wood developingbehind wounds in Acer saccharum constraintsof wood anatomy dictate that fungal (Sharon, 1974) might also reflecttransitionzone colonization takes place most readily and rapidly activity. With the exceptionof the compartmentalization along the axis of a tree(Rayner& Boddy, 1988), the axial containment providedby these reactionzones wall 4 barrier, most host-pathogeninterfacesin is likely to be at least as significant as the radial living wood can be related to the reaction zone (suberization), component of any barrier function.The prepon- model. Induced cell wall alterations depositsand compounds,polyphenolic derant use of sections in the transverseplane in antimicrobial studiesofdecayin treeshas, perhaps,tendedto mask cell necrosis have been associated with reaction in sapwood zones, as discussed below (IV 2) (see Figs 3, 4). In thisfact.Reactionzones are also formed at thebase ofdyingsuppressedbranches, wherethey addition,variousotherchemicalchangesless readily may preventthe ingressof potentialpathogensinto relatedto defencehave been described,forexample the remainder of the tree(von Aufsess,1975; Shigo elevated pH, increased carbonate concentration, et al., 1979; Green, Shortle& Shigo, 1981). Again, increased metal ion concentrations(Shain, 1971; theprotection conferred is primarily againsttheaxial Shevenell& Shortle,1986; Smith& Houston, 1994). of thesein relationto reactionzone The significance spread of infection. in In the originalreactionzone model (Shain, 1967, function involvement remainsuncertain, although 1971, 1979) a transitionzone of morphologically defencecan be postulated(see IV 2(i)). normal but drier tissues, located between the To date, fewstudieshave addressedeventsat the in wood during phases of reaction zone cells and thenormalsapwood, host-pathogeninterface necrotic was described. This has been suggestedas the site activeinvasionand lesionexpansion(see VI, below). elements twocharacteristic where biosynthesisof characteristic reaction zone In Acerspecies,however,

margin of naturallyoccurringdecay caused by Figure 3. Transverse section throughthe circumferential colouredreactionzone (RZ) is presentat A broad, strongly Ustulinadeustain sycamore(Acerpseudoplatanus). exposurethis reactionzone appeared betweenhealthy(HW) and decayed (DW) wood. On first the interface Althoughreactionzones in thisphotograph. goldenyellow,but rapidlyoxidized to darkgreen,as represented much formedat the marginsof U. deustalesions are oftenbroad, as seen here,reactionzones are commonly narrowerthan the example illustrated.Scale bar = 1 cm. Figure 4. Radial longitudinalsection througha The decayfungusremainsundetermined. (Acerpseudoplatanus). zone in sycamore occurring reaction naturally across a stemthe distal part of which was dead (DW), green In this reactionzone (RZ), located transversely polyphenolicdeposits occluded vessels and the lumina of fibresand other cell types. Scale bar = 100 ,tm. wall 4 barrierin oak (Quercusrobur).The Figure 5. Radial longitudinalsection of a compartmentalization c. 12 cells wide, located betweenthe sheetof axial parenchyma, barrier zone (BZ) comprisesa circumferential decayed wood colonized by Stereumgausapatum (DW), which had been formed before wounding, and the healthysapwood (HW) formedafterwounding. Scale bar = 200 ,tm.Figure 6. Transversesection of a with extraction wall 4 barrierin poplar (Populussp.), stainedwith Sudan IV following compartmentalization (BZ) is stained,whereashealthy chlorine dioxideand acetone(Pearce, 1990). The suberizedwall 4 parenchyma Figures 7, 8. Radial longitudinal wood (HW) and decayedwood (DW) remainunstained.Scale bar = 200 /tm. ofnotchwounds byinoculation trees,challenged (Acerpseudoplatanus) sycamore sectionsofstemsof3-year-old were applied as mycelialcultures,c. 3-wk-old,on 3 % malt agar. Inoculatedwounds were sealed withplastic compounds phytoalexin-like film Camlab Ltd, Cambridge,UK). To visualizethe fluorescent ('Seal-On' film, light with long wave ultraviolet challengethe stem lengthswere illuminated induced in the xylemfollowing challenge.Figure 8: 48 h Figure7: 24 h after No. 2A filter. a Wratten through (A = 365 nm) and photographed shown, after challenge.Fluorescentcompoundswere induced aroundwounds within24 h. In the experiment wounds than accumulation at thistimewas less apparentarounduninoculatedand Chondrostereum-challenged wounds: however by 48 h accumulationwas similar in all treatments.Scale around Ustulina-challenged
bar = 10 mm. purpureum. The fungi cut into the stem with (a) sterile 3 % malt agar; (b) Ustulina deusta; or (c) Chondrostereum

212

R. B. Pearce formation of compartmentalization wall 4 barriers has been reported in several angiosperm trees as a response to vascular wilt pathogens (Shigo & Tippett, 1981; Tippett & Shigo, 1981 a).- However, it seems likely that none of these models is alone sufficient to describe adequately the interactions between living sapwood and potentially or actually pathogenic wood-inhabiting micro-organisms. Instead, elements of each might contribute to regulating the host-pathogen interaction, the role of individual components perhaps varying from pathosystem to pathosystem. Models for defence that invoke microenvironmental restriction and active host defence are not in any way mutually incompatible. This will be discussed below, in relation to the putative antimicrobial defences that have been identified in the xylem of woody plants. 2. Putative defencemechanisms

Within the context of the models above, a number of anatomical, physiological and biochemical features (d) Environmental restriction offungal colonization. of living sapwood have been identified that might As an alternative to the CODIT and reactionzone contribute to its ability to resist microbial attack. In models in which growth of wood decay fungi is general, these parallel the defence mechanisms which restricted by activehost defences,Boddy & Rayner are better known from herbaceous plants (see (1983) proposed, and subsequently developed Horsfall & Cowling, 1980; Callow, 1983; Pegg & (Rayner, 1986; Rayner & Boddy, 1988; Boddy, Ayres, 1987). However, for reasons of scale (both 1992), a model thatdoes not requirethe operationof physical size and the extended duration of hostactivehostdefences.They suggestedthathighwater pathogen interactions in woody tissues), clearly content and concomitant low 02tension in functional defined multicellular zones of altered host tissues are sapwood is itself sufficientto preclude fungal commonly associated with protection, and changes has come at a tissue level appear particularly important. development.Support forthis hypothesis from the patterns of functionallycompromised Although a number of putative defence mechxylem and fungalcolonizationassociated with tree anisms in living sapwood have been identified (see wounds (Boddy & Rayner, 1983; Rayner, 1986; below), evidence for their precise function in proRayner& Boddy, 1988; Boddy, 1992) and fromthe tection is mostly indirect, and an unequivocal role development of wood-inhabitingfungi in drying has rarelybeen proven. As in bark, protection against excised woody tissues (Chapela & Boddy, 1988b; microbial pathogens, insects and other pests, and wound repair processes are likely to share many Chapela, 1989). Accordingto thismodel,thelimitsofdecaywould common features, particularly since an important occur at the junction between microenvironmental function of wound repair might be to exclude conditionsconduciveto fungalcolonizationin wood potential pathogens. In consequence it may be aeratedas a resultof drying and dysfunction, e.g. as artificial to assign specific roles to these broadly a consequenceof wounding,and conditionsinimical defensive processes.

ofthereaction zone response- increasedtissuewater content(Pearce et al., 1994a; Pearce, unpublished) and depositionof coloured,insoluble,polyphenolic materials (Pearce & Woodward, 1986) - were not seen where the infection front appeared to be advancing,althoughsoluble phenolic phytoalexinlike compoundswere present(Pearce & Woodward, 1986; Pearce, unpublished). Such regions equate more closely with the 'reaction zone' as originally proposedby Shain (1967, 1971, 1979), thanthestatic lesion boundariesto which the termhas commonly been applied (cf. Shigo, 1984; Pearce, 1990, 1991; Blanchette, 1992). The term 'column boundary layer' has been used to describethe alteredtissue at static lesion margins (Shortle & Smith, 1990). Although this term has not yet achieved general the three-dimenacceptance,it conveys effectively sional context in which these putative defences operateand could be used to discriminate unequivocally between static and dynamic host-pathogen interfaces in livingwood.

to fungal growth,where the xylem remains functionaland water-filled. The cell wall alterations and polyphenolic deposits characteristic of column reactionzones (CBL reaction boundarylayer-type zones) would then have a wound repair function, theiroftenhydrophobic nature(see IV 2(h), below) servingto limitthe spread of dryingand cavitation and to maintain the hydraulic integrityof the than providingan adjacentfunctional xylem,rather barrier inhibitory per se.
These models have been advanced in relation to the colonization of living trees by wood-decay fungi. Similar processes are presumed to operate in respect of other classes of xylem pathogen; for example, the

(a) Environmental constraints on pathogen growth. Unsuitable microenvironmental conditions for fungal growth within functional living sapwood have been proposed as a major factorprotecting this tissue against infection and decay (Boddy & Rayner, 1983; Rayner, 1986; Rayner & Boddy, 1988; Boddy, 1992). High water contents are known to inhibit growth and decomposition by wood decay fungi,owing primarily to lack of 02 and build-up of CO2 in the waterlogged substrata. Also, if the water is mobile, extracellular enzymes important in decomposition may be removed from the vicinity of the secreting hyphae (Boddy, 1986, 1992).

Antimicrobial defences of livingwood

In intact functional sapwood, 02 may be present either in solution in the xylem water or in the gas phase occupying the tissues of dead cells (fibres, etc.). Elevated CO2 and reduced 02 concentrations are commonly reported from living wood (Rayner & Boddy, 1988): in living Acacia stems the gas composition was almost 10000 CO2 (Carrodus & Triffett, 1975). Oxygen concentration can also be low within decay lesions, with 0 8 00 02 reported in gases extracted from rotten heartwood of Acer saccharum (Thacker & Good, 1952). It should be noted, however, that many decay fungi can grow in a low 02/high CO2 environment (Jensen, 1967; Hintikka & Korhonen, 1970; Highley et al., 1983). Also, the highest 02 concentrations in a tree have been found in the youngest sapwood (Jensen, 1969), which is commonly the most resistant to fungal attack (Johansson & Stenlid, 1985; Rayner 1986). Oxygen concentrations in the youngest sapwood exhibit seasonal variation, being lowest at times when the cambium is active. This has been attributed to 02 consumption by the metabolism of the cambium (Eklund, 1993). Low rates of gas exchange in these tissues would ensure that 02 consumed by a pathogen attempting to grow in functional sapwood was not quickly replaced, resulting in a swift decline in its concentration to inhibitory levels. Damage disrupting the integrityof vascular tissues and resulting in cavitation would permit the ready ingress of air and allow increased gas exchange. Fungal growth would then be possible in these compromised tissues. Patterns of discoloration and decay observed behind wounds in living trees exhibiting 'compartmentalization' can also be explained in these microenvironmental terms (Boddy & Rayner, 1983; Rayner 1986; Rayner & Boddy, 1988; Boddy, 1992). In such an event, CBL reaction zones and the compartmentalization wall 4 barrier would have a repair function, preventing an increase in the volume of compromised wood by acting as an impermeable surface seal. Further evidence for the importance of reduced aeration in the restriction of fungal growth is provided by the observation that decay development behind wounds is often arrested if the wound face is occluded by healing callus growth. The main pathway for gas exchange, through the damaged wood, would be lost on wound closure (Jensen, 1967; Highley et al., 1983; Rayner & Boddy, 1988). For invasive pathogenesis, wood colonization would be preceded by the formation of a dry zone ahead of the infection front, which would permit better 02 ingress within these tissues. Such zones have been reported ahead of Heterobasidion annosum lesions in conifers (Coutts, 1976) and adjacent to Cryptostromacorticale lesions in Acer pseudoplatanus (Pearce et al., 1994a). Inimical environmental conditions in wood could be enhanced as an active defence. In Abies concolor,

213 decay-resistantwetwood might form as a host cell death,as well response,possiblyto parenchyma Low availas duringnormalheartwoodformation. abilityof 02 in wetwood might protectit-against decay, and thus act as an active defencemechanism (Worrall & Parmeter, 1982). In addition, NMR methods have demonstratedthat reaction zones L. stems chaldeveloping in Acer pseudoplatanus lenged with a weakly aggressive decay fungus (Ustulina deusta) accumulate water (measured as imageable protons) to 2 5-3 times the level in wood (Pearce et al., 1994a). Similar adjacenthealthy resultshave been obtainedfromNMR studiesusing juvenile trees wounded and challenged with U. deutsa (Pearce et al., 1994b). Gravimetricdeterminationsof the water contentof excised reaction zone tissues indicated an increase in total water of 60 0 comparedwithhealthy wood. Similarchanges were observed in young trees inoculated with Ganodermaadspersum (Pearce, unpublished). This accommodatedby the water,whichwas presumably displacementof any gases normallypresent,might enhance the protection imparted by the normal water content of the sapwood. An alternative, solvent, functioncan, however, be suggested (see VIII). (b) Nutritionalfactors. Although wood cell wall forthe growth materialprovidesthe main substrate of decay fungi,some of these, at least, have comrequirements.A definedbasal plex micronutrient contained many medium for Phaeolus schweinitzii organic supplements (Robbins & Hervey, 1969). the of such factorsin determining The significance susceptibilityof wood to fungal attack remains unclear, although the observed stimulationof the on spruce wood colonized growthof P. schweinitzii by Armillaria sp. (Barrett, 1970) could have a nutritional basis. in xylemsap The availability of soluble nutrients in determining ratesofcolonization be a factor might of the sapwood of fruittrees by Chondrostereum purpureum(Beever, 1970; Bielenin & Malewski, was found between 1982), althoughno correlation of sap extracted fromSalixfragilis the sugarcontent and susceptibilityto this pathogen (Stanislawek, Long & Davis, 1987). A reduction in starch reservesstored in xylem parenchymaof stressed trees has been associated to pathogensincluding withincreasedsusceptibility Armillariaspp. (Wargo, 1972). Althoughconcomitant changes in the broader nutritionalstatus of stressed tissueoccuralso (Wargo,1972), depletionof starchmightreducethe tree'scapacityfordefensive responses which would normallydraw upon these
reserves (cf. ~McLaughlin & Shriner, 1980). The normal (c) Anatomical features of normal wuood. structure and cell wall composition of wood imposes

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R. B. Pearce

constraints on microbialcolonization.Induced ligni- in these plants appears functionally and, in some fication appearsto have a defensive function in those cases, morphologicallyequivalent to a compartplant tissues that are normallynon-lignified, pro- mentalizationwall 4 barrier (see IV 1(b) and IV tectingthem againstmicrobialdegradation(Vance, 2 (h)), and mightprotectthe vital cambium,phloem Kirk & Sherwood, 1980; Ride, 1983). Pathogens and, perhaps, the youngestxylem, frompotential normally inhabiting secondary xylemmustbe able to pathogens and desiccation (Moss, 1936; Moss & growin a highly lignified environment, althoughthe Gorham, 1953). ability to degrade lignin is not a prerequisitefor damaging woody tissues; brown rot fungi decay (e) Constitutiveinhibitorycompounds (phytoantiwood buthavelimitedeffects on itslignincomponent cipins).Phenolic compounds can be extractedfrom (Rayner & Boddy, 1988). For micro-organisms the healthy sapwood of many species. Examples unable to penetrate wood cell walls, pits and include gallic acid and catechinin Acer spp. (Tattar perforation plates provide points of weakness & Rich, 1973), saligenin,salicin,phloroglucinol and throughwhich spread fromcell to cell can occur (+)-catechin in Salix alba var. caerulea (Wong & (Tabirih & Seehann, 1984). Preece, 1978b) and ellagitanninsin Quercus and The gross anatomy of wood might constrain Castanea spp. (Peng, Scalbert & Monties, 1991). microbialcolonization,and be important in deter- Lignans (Shain & Hillis, 1971; Alcubilla et al., mining the ultimatedimensions of xylem lesions. 1974), long-chainfatty acids, resin acids and diterThe axial elongation ofmostxylemelements (vessels, pene alcohols and aldehydes (Ekman, 1979b) have tracheids, fibres, some parenchyma) means that been reportedfromthe sapwood of Picea abies,and colonizationoccurs most readilyalong the treeaxis: similar extractiveshave been reported from the fewerwalls have to be traversed by a pathogenthan sapwood of other conifers (e.g. Hergert, 1960). duringradial or tangentialspread. In addition,the Phenolic contents equivalent to c. 0 5-3 6 mg g-1 entry of oxygen is facilitatedaxially (Tabirih & f. wt have been reportedin these studies (Tattar & Seehann, 1984). Rays facilitate radial colonization, Rich, 1973; Wong & Preece, 1978b; Pearce, 1987; although densely packed latewood tissues might Stenlid & Johansson,1987). Typically,these compresent zones of increased resistance (Pearce & pounds occur at high concentrations in both bark Woodward, 1986; Boddy, 1992). However, no and heartwoodtissues,e.g. Ekman (1979 a). Indeed, convenientchannels of invasion exist tangentially, antifungal wood extractives are more characteristic in the direction of greatest passive resistance to ofheartwoods thanof sapwood (Hillis, 1987) (see V, infection. In the absence of other determinants, below). ofdecaydevelopment in dead wood (timber) patterns Wood extracts, and theircomponents,like gallic reflect these passive constraints constitutively acid and lignans, can be inhibitoryto woodpresentin wood (Wilcox, 1973). inhabitingfungi in culture, although the concenAnatomical features might also influence the trationsrequiredforactivity mightbe greaterthan effectiveness of active defence. For example, the those found in healthy sapwood (Shain & Hillis, expression of any response requiring metabolic 1971; Shortle, Tattar & Rich, 1971; Shaw, 1985; activitymay be dependent on the proportionof Stenlid & Johansson,1987). However, the levels of livingparenchyma cells presentin the wood. Also, the stilbenespinosylvin and pinosylvin monomethyl trees with fewer,smaller vessels might compart- etherin sapwood ofPinus densiflora werereported to mentalise morestrongly thantreeswithmany, to inhibitcompletelylinear growthof larger, be sufficient vessels which could allow easier pathogendissemi- Heterobasidion annosum (Dumas, Hubbes & Strunz, nation within the tree (Eckstein, Liese & Shigo, 1983). Associationshave been made betweenconsti1979). tutive forexample compoundsand diseaseresistance, in the differential and P. resistanceof P. densiflora cork. Interxylary (d) Interxylary cork comprises a rigidax radiata to H. annosum (Dumas & Hubbes, layerofsuberizedparenchyma cells laid downby the 1979), fromwhicha seriesof inhibitory compounds cambiumat theend ofa growth ring(Lemesle, 1928; was identified (Dumas et al., 1983). Moss, 1934; Carlquist, 1988). Similar interxylary Increasesin manyofthepre-formed antimicrobial periderms, formed by a phellogen arising in a compounds found in healthy sapwood have been zone of the xylem and ultimately reportedfromreactionzones at lesion marginsand parenchymatous joining the external surface periderm, are also heartwoodin both angiospermsand gymnosperms. formedin certainspecies (Moss, 1936). These are For example,catechinand salicinincreasedin willow normal anatomical featuresin a number of prin- wood infectedby Erwinia salicis (Wong & Preece, cipally shrub and herbaceous species with peren- 1978b), and in ash (Fraxinus excelsior), decayed by
nating woody rootstocks. In these plants there is a regular die-back of annual stems, which would allow micro-organisms colonizing these moribund stems easy access to the older xylem. The interxylarycork Inonotushispidus, amounts of a number of uncharacterized compounds were greater in reaction zone extracts than in extracts from healthy sapwood (Pearce, 1991). In Picea abies, lignans increased in

Antimicrobial defencesof living wood reaction zones and heartwood (Shain & Hillis, 1971) and norlignans normally present in very small amounts in the sapwood of Cryptomeria japonica were produced during heartwood formation and in reaction zones (Yamada, Tamura & Mineo, 1988). The stilbenes pinosylvin and pinosylvin monomethyl ether, constitutively present in the sapwood of Pinus spp. (Dumas et al., 1983), accumulated in reaction zones (Shain, 1967) and in heartwood (see Hart, 1981). These compounds exhibit many features in common with induced antimicrobial compounds (phytoalexins) and will be considered further in that context (see IV(h), below). Additionally, the gas phase in the wood of living conifers is probably saturated with terpene vapours, which may be

215

inhibitory (see IV (f), below).

In general, the role of preformed antimicrobial compounds in the defence of living sapwood remains little studied, and, in consequence, poorly understood. It has been suggested that these phytoanticipins might be important in determining the successions of micro-organisms colonizing wounds on trees. Pioneer colonizers can grow at concentrations of phenolic compounds such as gallic acid that are inhibitory to some decay fungi, and can modify these compounds, which might permit the subsequent development of decay (Tattar, Shortle & Rich, 1971; Shortle & Cowling, 1978). However, some caution is required in the interpretation of much of the literature on both constitutive and induced antimicrobial compounds in wood. Aggressive extraction has often been employed (e.g. Tattar et al. 1971; Tattar & Rich, 1973; Miller, Sutcliffe & Thauvette, 1990), or wood might have been dried and powdered before extraction (e.g. Shain, 1967): such methods might decompose or modify unstable compounds (cf. Wong & Preece, 1978 b). (f) Resin. Many gymnosperm trees have a welldeveloped resin canal system in both the bark and xylem tissues. The resin principally comprises terpenoid compounds (non-volatile resin acids and volatile terpenes which act as a solvent for the nonvolatile components and may amount to up to 50 0 of the total material); phenolic compounds (e.g. lignans) and fattyacids and their esters can also be present. Resin-like materials are also produced by some woody angiosperms: these are chemically more diverse, including carbohydrate gums (in some Prunus spp., Acacia senegal) and kino, a polyphenolic substance principally comprising polymerized proanthocyanidins, in some Eucalyptus species. Latex, e.g. from Hevea brasiliensis and Palaquium gutta, predominantly comprises a hydrocarbon, cis-1,4polyisoprene, stabilized by a thin absorbed film of protein and phospholipid (Hillis, 1987). Although these materials are generally considered to be involved in protection against pests, diseases and

damage (for example the ability of conifers to to withresistance produce resinhas been correlated annosum(Gibbs, 1968) the precise Heterobasidion is oftenuncertain. natureof theirinvolvement resinsarebyfarthemostintensively Gymnosperm studied of these tree exudates. These are produced by the epithelial cells lining resin ducts, and can to sitesof damage,wherethe flowin the duct system evaporateto leave a plug,whichcan volatileterpenes barrierto water to forma paint-like hardenfurther and the entryof externalagencies (Hillis, 1987). It has been suggested that this ability to effecta mechanical barrieris importantin the protection by resin, as the principal effectof tissue afforded of resinosisappearedto be the mechanicalinhibition hyphal growthby dried resin (Prior, 1976). Resin of xylem tissues could also act as a impregnation in function helpingto maintain barrier, hydrophobic adjacentconductivesapwood. Latex coagulationhas attackedby been noted in the bark of H. brasiliensis fungi,but has been considereda secondary root-rot eventin disease (Nicole, Geiger & Nandris, 1986). chemical The significance of resinas an inhibitory is less clear (cf. Yamada, 1992). defencein conifers The antimicrobial activities of volatile terpenes (Shrimpton& Whitney,1968; Cobb et al., 1968; Flodin, 1979; Schuck, 1982a) and resin acids (Henricks, Ekman & von Weissenberg, 1980; Franich, Gadgil & Shain, 1983) have been demonstrated in a number of studies. However, little inhibitionof growth,or even stimulationof tree in by thesecompounds,has been reported pathogens other investigations (Prior, 1976; Flodin & Fries, 1978; Franich et al., 1982; Schuck, 1982a). Correlations have been reportedbetween monoterpene annosum toHeterobasidion and resistance composition and certainotherpathogens(Forrest,1982; Schuck, are unclearand some1982a), but the relationships and some authorsreportfinding timescontradictory no such correlations(Ladejtschikova & Pasternak, 1982). Resin formed after trauma can differin composition from that present in the healthy tree (Schuck, 1982a, b; Paine et al., 1987). A better understanding of the role of resins and their individualcomponentsin resistanceis requiredbeofthisdifference can be properly forethesignificance resin ducts in of traumatic assessed. The formation after woundingis a thewood laid down immediately common response to damage and infection in coniferous trees,and zones of alteredwood anatomy containingmore parenchymacells and resin ducts than normal were associated with wounding or annosum lesionsof Armillariasp. and Heterobasidion in rootsof severalconifers(Tippett & Shigo, 1980,
1981 b; Blanch~ette, 1982; Tippett, Bogle & Shigo, 1982). These corresponded in location to wall 4 barriers of the COD IT model (see IV 1 (b)). Comparable formation of traumatic gum canals has

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R. B. Pearce

been reportedfollowingwounding in Liquidambar In the primarytissues of both herbaceous and styraciflua(Moore, 1978). Kino veins form in woody plants, cell wall appositions at sites of Eucalyptus wood afterwounding(Tippett, 1986) or attempted penetration are commonly observed. not afterprolonged fungal activityin the bark tissues These mayincorporate a numberof components walls, including (Tippett et al., 1983) and kino may exude like resin normally presentin the unmodified after wounding(White & Kile, 1993). lignin and other phenolic compounds, callose, and Like other tree defences,the elicitationof resin silicon, together with normal cell-wall materials accumulationhas been littlestudied. Chitosan, an (Aist, 1976, 1983). Such papillae have been foundin elicitor of defence responses in herbaceous plants both angiosperm (Edwards & Ayres, 1981) and (e.g. Barber & Ride, 1988), has been reportedto gymnosperm(Bonello et al., 1991) trees. Lignielicit both resin accumulationand hypersensitive- fication can also occur in the bark as an early type necrosis(see IV 2(j)) in pines. Similar results component response(see restoration oftheperiderm have been obtained with a proteinase inhibitor- III, above) (Biggs, 1992b; Woodward, 1992). Ininducingfactor derivedfrom might not be expected to be plantcell walls (Miller, duced lignification in defence in the highlylignifiedxylem. Berryman & Ryan, 1986; Croteau et al., 1987; effective Lieutier & Berryman, of However,therewas an increaseof 25-30 o in lignin1988a, b). The importance tapof like materialin the wood of Hevea brasiliensis such elicitorsin the host-pathogeninteractions treesremainsto be determined. roots close to the infectionfront of Rigidoporus (Geiger et al., 1986). There appears to be no lignosus on the monomer compositionof this (g) Lyticenzymes. Lytic enzymes,chitinasesand ,- information glucanases are an important component of the lignin (Nicole, Geiger & Nandris, 1992), and its remains in thehost-pathogen interaction pathogenesis-related proteinsinduced in plantsfol- significance can degrade lowing microbial challenge. They have been re- unclear, particularly since R. lignosus garded as having a potentiallydefensivefunction lignin(Geiger et al., 1986). The main cell wall polymer associated with (Boller, 1987; Scholtens-Toma,Joosten& De Wit, 1991). Chitinase enzymes (from a herbaceous host-pathogen interactions in xylem tissues is ofthe secondary component ofHeterobasidion suberin,a characteristic species)wereable to inhibit growth annosum (Susi et al., 1995). Similarhydrolases have plant surface, where its resistance to microbial been reportedfromthe bark tissues of angiosperm degradation and hydrophobic properties are imthe integrity of this planttrees. Enzymes from Quercus and Acer spp. were portantin maintaining interface (see III, above). It is not a capable of lysing hyphae of Armillaria (Wargo, environment 1975), and chitinases and ,-1,3-glucanases were normalcomponentof healthysapwood, althoughit induced in the bark of Castanea species after is presentin some heartwoods(Pearce & Holloway, in challenge with Cryphonectria parasitica (Shain, 1984), and is associatedwiththeresinduct system 1993). It has also been reportedthatthese enzymes Pinus spp. (Biggs, 1987; Pearce, 1990). in poplar leaves following accumulate systemically Xylem suberizationresponses at the marginsof recognizedin the compartwounding(Parsons,Bradshaw& Gordon, 1989) and decay lesions were first in thevicinity of wall 4 barrier formed in mycorrhizaland pathogen-challenged roots of mentalization Eucalyptus(Albrecht, Laurent & Lapeyrie, 1994; pruning wounds in oak (Quercus robur) where Albrechtet al., 1994). Hydrolases have also been sapwood was attacked by Stereum gausapatum 1981; Pearce & Holloway, reportedfrommycorrhizal (Sauter & Hager, 1989) (Pearce & Rutherford, distinct and pathogen-challenged (Nsolomo & Woodward, 1984). The wall 4 barrierwas anatomically 1994) conifer roots, and systemically in root- fromnormal wood and compriseda sheet of axial cells up to 30 cell layersthick(Fig. 5). challengedseedlings (Bonello, 1991). There do not parenchyma these Where adjacent to fungallycolonized wood, this appearto be anyreports specifically associating was suberized to a depth of withxylemtissuesofwoodyplants. traumaticparenchyma planthydrolases to However, the association of such enzymes with 3-20 cell layers.The suberizedcells wereresistant albo- degradation by S. gausapatum, but were readily responsesto a vascular pathogen(Verticillium thus atrum) in tomato (Young & Pegg, 1981) and the digestedifthesuberinwas removedchemically, that wood cell walls were protected cell walls in thexylemoftransgenic demonstrating damageto fungal a chitinase canola plantsexpressing gene (Benhamou against breakdownby decay fungiby suberization et al., 1993) providesevidence, albeit indirect, that (Pearce & Rutherford, 1981). Followingexperimencolontal wounding,only cells overlaying fungally lyticenzymesmightoperate in xylem. ized sapwood were suberized(Pearce, 1987; Pearce, (h) Cell wall alterations. Induced defences may unpublished). This suggested that, whilst the de
conveniently be divided into cell wall alterations and induced chemical defences, although this separation may be rather artificial, both biochemically and functionally. novo formation of the sheet of traumatic axial parenchyma was wound-induced, suberization of these cells was a response to fungal infection. However, the possibility that suberization was

Antimicrobial defences of livingwood

217

induced by vessel cavitation,which subsequently cells and projectthrough pits to occlude the lumens permittedlocal fungal colonization of the com- of vessels (Koran & Cote, 1965), are commonly promisedtissues,could not be excluded. suberized(Pearce & Holloway,1984; Parameswaran, Similar suberized multicellularcompartmental- Knigge & Liese, 1985; Pearce, 1990; Rioux et al., ization wall 4 barriers have been reported from 1995). Such suberized tyloses are resistant to recognizably variousotherwoody angiosperms(Pearce & Wood- degradation by decay fungi,persisting ward, 1986; Pearce, 1990; Fig. 6). Althoughmostof after ofthewood has been completely the remainder the species examinedexhibiteda xylemsuberization degraded(Pearce, 1989). Togetherwiththe gummy ofphenolic thepolymerization response, two (Fraxinus excelsiorand Sophora ja- depositsformed from ponica) showedno suberization ofthetraumatic axial compoundsin the xylem(gummosis),tylosesmight parenchyma adjacent to decayed wood (Pearce, blocktheaxial spreadofpathogensalongthevessels, to in whichwall 4 barrier offer littleanatomicalrestriction 1990). In thoseconifers zones whichotherwise have been examined, a traumatictissue of paren- microbial spread (Rioux et al., 1995). Because of by largelyconferred chyma cells and resin ducts can be formed,but theirhydrophobicproperties, in maintaining suberizationof this tissue does not appear to have suberin, theymight also be important of cavitation theextent by restricting been reported (Tippett & Shigo, 1980, 1981b; xylemfunction Tippett et al., 1982; Pearce 1990; Blanchette, 1992). and air access (Rayner& Boddy, 1988). In contrast zone, whichoften Suberizationis not,therefore, a universalfeature of to the wall 4 barrier the compartmentalization wall 4. In addition to comprises a continuous sheet of suberized parensuberized, forming a decay-resistant zone around the site of a chyma, reaction zones are discontinuously cells capof parenchyma the distribution the youngestxylem,cam- reflecting major wound, protecting bium and bark from attack by micro-organisms able of expressing thisresponse.Althoughtracheids in woody angiosperms, via the wound, the suberized tissue could in conifers, and xylemfibres entering also act as an effective waterproofseal, reducing have been observed with suberized linings, these dryingand the entryof air around the lesion, and were near the cambium and appeared to have been at the timeof wounding differentiated helping to maintainan inimicalmicroenvironment incompletely forfungaldevelopment in new wood adjacentto the (Biggs, 1987; Pearce, 1990). Induced suberization wound (Boddy, 1992). render reaction zones realone cannot, therefore, Differentiated sapwood could exhibitsuberization sistant to penetration, sincethesuberizedtissuescan responsesas a resultofmechanicalwounding(Biggs, be bypassed.However,the principalaxial and radial 1987; Schmitt & Liese, 1991, 1993), or as a routes for fungalspread in wood - tyloses and ray - can be blocked by consequence of fungalcolonization(Pearce, 1990). parenchymacells, respectively The extent of this induced suberization differed degradation-resistantsuberized walls, thereby between species. In some, for example Fagus syl- slowing invasion along these key anatomicalpathvatica and Quercusspp., an extensiveresponsewas ways. has been interpreted Althoughxylemsuberization seen, in which xylem parenchyma cells of all (Biggs, categories, vessel linings and tyloses stained for as a responseto woundingand tissue drying in 1987; Schmitt& Liese, 1991, 1993) theexperimental suberin.In otherspeciestheextent ofsuberization CBL reactionzones was much reducedor absent.In systems used did not preclude the possibilityof and hence a role formicrobial general,strongreactionzone suberization responses fungalcontamination correlatedclosely with vessel occlusion by tyloses: infectionin the induction of xylem suberization. wheregummosiswas themain mechanism forvessel During active growthin summer,suberizationwas was more commonlyabsent, first apparent7-10 d afterwounding(Biggs, 1987), occlusion,suberization even in species with a suberized compart- or after3-6 wk (Schmitt & Liese, 1993). During no responsewas seen duringthe mentalization wall 4 barrier(Pearce & Woodward, winterdormancy, 1986; Pearce, 1990). In conifers,where the pro- first6 wk followingwounding (Schmitt & Liese, zone, induced portionof livingcells in the xylemis normally low, 1993). Suberizationin a wall 4 barrier suberization responses were generallypoorly de- in oak (Quercus robur) following wounding and (Pearce, 1987), withStereum gausapatum to note, inoculation veloped (Pearce, 1990). It is interesting however, that in a reaction zone in Picea abies was not observed until between 8 and 21 wk after suberin was associated only with ray parenchyma challenge(see Table 1). The time elapsingbetween cell end wall pits(Pearce, 1987). In a circumferential challenge and the expression of a suberization reactionzone this is preciselywhere suberinmight response appears long in comparison with that have the greatesteffectiveness, both in maintaining typicalofinduceddefenceresponsesin plants,which thehydraulic ofthefunctional integrity sapwood and can usually be detectedwithinhours of challenge
in blocking the major route of radial fungal spread (Pearce & Woodward, 1986; Schwarze, Lonsdale & Fink, 1995). Tyloses, which develop from xylem parenchyma (Yoshikawa, 1983). This suggests that suberization might not be important in the early restriction of fungal colonization in wounded tissues but may be more important in conferring durability to barriers

218 Table

R. B. Pearce 1. Development of a compartmentalizationwzall4 barrier in oak Quercus robur* Suberizationof wall 4 parenchyma Starchcontentof wall 4 parenchyma

Weeks after challenge Anatomicalfeatures 2 No new tissue of distinctive anatomy evidentbelow cambium Up to 7 cell layersof axial parenchyma formedby cambium Up to 25 cell layersof axial parenchyma formedby cambium Traumatic axial parenchyma up to c. 20 cell layersthick overlaidby wood reverting to more normalanatomy Traumatic axial parenchyma up to c. 20 cell layersthickoverlaid to by wood reverting more normalanatomy Traumatic axial parenchyma up to c. 20 cell layersthick, overlaidby wood reverting to more normalanatomy

Fungal colonization Few hyphaeevidentin wood

Traumaticparenchyma not suberized Traumatic parenchyma not suberized Inner 3-7 cell layersof traumatic parenchyma suberizedover discolouredwood Inner cell layersof traumatic parenchyma suberizedto a depth of c. 10 layersover discolouredwood Inner cell layersof traumatic parenchyma suberizedto a depthof 6-14 cell layers,over discolouredwood

Starch absent

8 21

Starch levels high in traumatic parenchyma Starch absent from suberizedcells, high in othertraumatic cells parenchyma Starch absent from suberizedcells high in othertraumatic cells parenchyma Starch absent from suberizedcells, high in othertraumatic cells parenchyma

Extensivefungal colonizationin of immediatevicinity inoculationonly Occasional hyphaein wood extantat the time of wounding Hyphae presentmainly in discolouredwood

32

Hyphae presentin discolouredwood

62

Hyphae presentin discolouredwood

* Quercusrobur a patch of bark trees,c. 50 yr old, were wounded on the trunkat a heightof c. 1-3m by removing withStereum gausapatum (Fr.) Fr. c. 85 x 100 mm,to expose thewood. A 15 mm dowel,inoculated3 monthspreviously was drivenintoa hole bored in a lowercornerof the wound to inoculate.Trees werechallengedin June,and felledand blockstakenc. 20 mm below theinoculation dissectedat intervals inoculation.Sections(20 am) werecut from following Suberinwas detectedusing Sudan IV (Pearce, 1990); starchwas detectedwiththe IKI site,using a slidingmicrotome. B: methyl greenmethod(Pearce, 1984). reagent(Jensen,1962) and fungalhyphaewerevisualizedusing the rhodamine

at wound or lesion margins. This could result fromthe intrinsic degradationresistanceof directly the suberized walls, or could be mediated through the drying the abilityof suberizedtissuesto prevent and aerationof adjacentxylem(cf. Rayner& Boddy, 1988; Boddy, 1992). Until dynamic studies of have been concolonizationand barrierformation ducted,the preciserole of these cell wall alterations in defencewill remainuncertain(cf. VI, below). Thin-walled suberized cells have been reported adjacent to the traumaticaxial parenchymaof the wall 4 in oak (Pearce & compartmentalization 1981). Similar tissues have been reRutherford, ported in other species (McGinnes, Chang & Wu, 1971; Tippett & Shigo, 1980), althoughsuberization in these species was not demonstrated. This tissue, which appears to resultfromthe local death of the vascularcambiumas a consequenceofwoundingand withinthe phloem, subsequent cambial restoration was termed the cambial-phloic zone by Mullick (1977). It createsa plane of physicalweaknessin the wood (McGinnes et al., 1971), but any defensive remainsunknown. significance

Little is knownof the abilityof wood-inhabiting fungi to degrade suberin. Armillaria species can slowly degrade this recalcitrantpolymer (Swift, 1965; Zimmermann& Seemuller, 1984), but the decayed persistenceof suberinin wood extensively (Pearce, 1989) indicates by Ganodermaadspersum thatthis fungusdid not readilydegrade it. (phytoalexins). compounds (i) Inducedantimicrobial Increased levels of phenolic compounds have been reportedfromlesion marginsin living sapwood in many tree species (e.g. Shain, 1967, 1971; Popoff, Theander & Johansson, 1975; Wong & Preece, 1978b; Pearce, 1987; Yamada, 1992). Often the compounds induced at sites of host-pathogeninor presentat onlyverylow are absentfrom, teraction levels in healthy sapwood (e.g. Aesculus hippo(Pearce, 1991), Pinus taeda Fagus sylvatica castanum, (Shain 1967)). These chemical changes often acand the depositionof companycell wall alterations materials(see IV 2(h), IV 2(j)) insolublepolymeric in CBL reaction zones and compartmentalization

Antimicrobial defences of livingwood

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Table 2. Phytoalexin-like compounds fromthesapwoodof trees:representative examples of themainchemical classes Class Phenols Stilbenes Tree Picea abies Pinusspp. Morusalba Lignans Picea abies Pathogen Heterobasidion annosum H. annosum Compound(s) 4-methylcatechol Reference Popoff etal., 1975

Liriodendron tulipifera domestica Phenylpropanoids Prunus

Pinosylvin Pinosylvin Shain,1967 monomethyl ether Fusarium solani f. sp. Oxyresveratrol mori 4-prenyl Takasugi etal., a 1978 oxyresveratrol H. annosum Hydroxymatairesinol Shain,1971; Shain & Hillis,1971; etal., 1975 Popoff Not determined Syringaresinol Chenetal., 1976 Scopoletin Hillis& Swain,1959

Chondrostereum purpureum Acer pseudoplatanus C. purpureum Acersaccharum Not determined

Platanus acerifolia Biaryls Dibenzofurans Maluspumila communis Pyrus

Fraxin 'Fraxetin-like coumarins' Ceratocystis fimbriata Scopoletin f. sp. platani Umbelliferone C. purpureum Aucuparin C. purpureum

M. alba Flavonoids Terpenoids Pinustaeda Broussonetia papyrifera Manyconifers Ulmus spp. Tilia europaea Alkaloids L. tulipifera

F. solani f. sp. mori H. annosum Not determined Various fungi ulmi Ophiostoma Ganoderma applanatum Not determined

Pearce, unpublished & Levitin, Manville 1974 El Modafar etal., 1993 & Kemp,Holloway 1985 Burden, & Kemp,Burden x,/3-,y-pyrufurans Loeffler, 1983; Kemp& Burden, 1984 M Takahashi & Shirata, Moracin 1982 Pinocembrin Shain,1967 C et al., Broussonin Takasugi a Broussin 1980 Volatile IV 2(f) 'resin' See Section monoterpenes Resinacids J & Overeem Mansonone E, F 1970 Elgersma, (-)-7Burden & Kemp, hydroxycalamenene 1984 (-)-7Burden & Kemp, hydroxycalamenene 1983 glaucine Chenetal., 1976

wall 4 barriers (Pearce & Woodward, 1986; Pearce, 1991). Some of the compounds accumulating de novo at lesion margins have antimicrobial activity, and may thus be considered phytoalexins (Kemp & Burden, 1986, and see Smith (1996) for a full account of origin, definitionand biochemistry of phytoalexins). These compounds belong to various chemical classes (Table 2). Although there are reports of the isolation and characterization of such compounds (cf. Table 2), there have been few in-depth studies. Indeed, the evidence for their involvement in resistance is often only circumstantial (see Kemp & Burden, 1986). Most phytoalexin-like compounds induced in living wood have not been quantified in the tissues where they accumulate. Crude determinations of total phenolic compounds in CBL reaction zones

sapwood: mightbe 5-10-foldhigherthanin healthy induced compounds can account for 1 5 %0 or more of the total materialin these reactionzones, on a freshweightbasis (Shain, 1967; Popoffet al., 1975; Pearce 1987; Yamada et al., 1988). Antifungal activityin reaction zone extracts,determinedby TLC plate bioassay (Homans & Fuchs, 1970; Woodward & Pearce, 1985), is, however, often low and can be relatedto onlya fewof the relatively compounds accumulatingat lesion margins.HPLC separations of extractsfrom green CBL reaction zones at the marginsof naturallyoccurringfungal revealed lesions in the xylemof Acerpseudoplatanus the presence of at least seven induced compounds (Pearce et al., 1994a). However, bioassay with the folcucumerinum, indicatororganismCladosporium lowing TLC, demonstratedrelativelyweak inhi-

220

R. B. Pearce (a) Leaves HO HO OH

1983; Burden & Kemp, 1984), belong to chemical groups that are known to include photosensitizers (Downum, 1992). Although the toxicity of photosensitizers has been associated with free radicals produced by light activation, some can alternatively OH HO O be activated by peroxidase- or polyphenol oxidaseHO mediated reactions in the dark (Leatham, King & Stahmann, 1980; Gommers & Bakker, 1988). Elevated peroxidase or polyphenoloxidase activity is a Chalcomoracin OH feature of many plant defence responses, including those in the functional wood of trees (Shain, 1971; (b) Cortex Wong & Preece, 1978a; Geiger et al., 1989), and OMe HO high levels of free radicals have been detected in CBL reaction zones in Acer pseudoplatanus (Pearce, OH OH Meo H MeO 0 Green & Edwards, unpublished). Activation could serve to reduce the autotoxicity of the induced OH OMe compounds which accumulate widely around inMoracin A Moracin B fection sites (see below; Figs 7, 8) by ensuring that (c) Xylem OH the biocidal form was restricted to the immediate OH OH OH of the host-pathogen interface. vicinity HO HO / < A furtherpossibility is that these compounds may function as precursors for the formation of physical OH OH barriers rather than as chemical inhibitors per se. Oxyresveratrol 4'-prenyloxyresveratrol Insoluble polyphenolic compounds are commonly Figure 9. Phytoalexins from(a) the leaves, (b) cortexand present in reaction zones (Pearce & Woodward, (c) xylem of mulberry(Morus alba) (Takasugi et al., 1980b; Takahashi & Shirata, 1982). The compounds 1986; Pearce, 1990; Blanchette, 1992; Yamada, accumulating in the different tissues were chemically 1992; Pearce et al., 1994a), where they might serve distinct. either as mechanical barriers to further fungal penetration (Pearce, 1987; Blanchette, 1992) or as permeability barriers limiting the spread of cavitation and drying, and hence susceptibility, in the bition of fungal growth by two components only underlying xylem (Boddy & Rayner, 1983; Rayner, (Pearce, unpublished). Similarly, hydroxymataires- 1986; Rayner & Boddy, 1988; Boddy, 1992) (see 2(j) inol, the predominant lignan from reaction zones in below). Picea abies, had no effecton Heterobasidion annosum Many of the phytoalexin-like compounds induced and five other wood-degrading fungi (Popoff et al., in damaged or challenged sapwood are also present 1975). Mansonones induced in Ulmus spp. following constitutively in heartwood. Examples include the challenge with Ophiostoma ulmi had little activity in stilbenes pinosylvin and pinosylvin monomethyl vitro against this fungus (Overeem & Elgersma, ether induced in the sapwood of pines following 1970; Elgersma & Overeem, 1971). wounding or fungal attack (Jorgensen, 1961; von Although such findings seem at variance with the Rudloff & Jorgensen, 1963; Shain, 1967; Hillis & presumed role of these compounds as phytoalexins, Inoue, 1968; Jorgensen & Balsillie, 1969; Prior, they do not discount their importance in defence. 1976), which are also present in pine heartwood The activities of plant allelochemicals might be (Erdtman & Misiorny, 1952), and the mansonones influenced by the environmental conditions under found in elm heartwood or accumulating in response which they are assayed; the activities of putative tree to wounding or infection (Duchesne, Hubbes & phytoalexins do not appear to have been determined Jeng, 1992). A number of other phytoalexin-like in a low-oxygen environment comparable with that compounds from sapwood have also been identified normally present within living sapwood where they as heartwood constituents in the same, or sometimes might be more active than in a fully aerobic different, species of angiosperm or gymnosperm environment. Indeed, photo-oxidation has been trees (see Kemp & Burden, 1986). Because of the similarities between the changes in suggested as a factor reducing the toxicityof phenols from tree xylem (Shortle & Smith, 1990). sapwood accompanying CBL reaction zone forIt should be noted also, that many of the mation and heartwood formation in certain species, phytoalexin-like compounds in trees, which include these processes can be difficult to separate. The coumarins (Manville & Levitin, 1974; Pearce & heartwood wings reported from decaying oak Arnold, unpublished), lignans (Shamn,1971; Popoff branches (Boddy & Rayner, 1981), are probably to et al., 1975; Chen et al., 1976) and sesquiterpenes be equated with reaction zones (Rayner & Boddy, (Overeem & Elgersma, 1970; Burden & Kemp, 1988). The ratio between individual constituents

Antimicrobial defences of livingwood

221

often differsbetween reaction zones and normal reaction zone (Grime & Pearce, 1990), where it heartwood,e.g. pinosylvin:pinosylvin monomethyl might, perhaps, be equated with the transition zone etherin pine (Shain, 1967; Hillis & Inoue, 1968) and of the classical reaction zone model (Shain, 1967, sequirin-C: hinokiresinolin Cryptomeria japonica 1971, 1979) (see IV 1(c)). (Takahashi & Ogiyama, 1985a, b, 1986, in Yamada, It was extremely difficult to ensure that un1992), but it is difficult to assess the significance of inoculated wounds in these experimental systems these differences, which might merely reflectthe remained sterile and did not support the growth off circumstances of biosynthesis, althoughit has been opportunistic colonizers. This rendered problematic suggested that they might be of consequence in the unequivocal discrimination between the roles of defence,the ratios in reactionzones favouring the wounding and fungal infection in eliciting the moreantifungal components (Shain, 1967; Yamada, response. Although the differential effects on the 1992). accumulation of phytoalexin-like compounds seen in To datetherehave been fewstudiescomparing the some experiments suggest that the response can be phytoalexin-like compounds found in the different modulated by microbial colonizers of the xylen, the tissues of woody species, althoughthere are clear greatest differences between inoculations with tissue-specificdifferencesin the distributionof compatible and incompatible pathogens were seen at constitutive antimicrobial compounds (cf. Toscano the later stages of the host-pathogen interaction Underwood & Pearce, 1991). In Morus alba (mul- when polymeric materials were being deposited (cf. berry)the phytoalexin-like compoundsproduced in Pearce et al. 1994a). In wounded young sycamores induced fluorescent xylem tissues, bark and leaves following fungal challengewere chemicallydistinct.In the bark, 26 compounds in the xylem extended several cm above new antifungal compounds, named moracins A-Z and below the wound site within a few days of were reported. These had a 2-phenylbenzofuran challenge (Lenne & Pearce, unpublished; cf. Fig. 9). skeleton.In xylem,threeinduced compounds were This suggests the rapid propagation of an elicitor identified; moracin M and the stilbenes oxy- signal. Wound-associated hydraulic signals inducing resveratrol and 4'-prenyloxyresveratrol. In leaves, defensive responses have been described from herthe major phytoalexin-like compound was chalco- baceous plants (Malone, 1994; Malone, Alarcon & moracin (Fig. 9) (Takasugi et al., 1978a-c, 1979, Palumbo, 1994; Malone et al., 1994); their in1980b; Takahashi & Shirata, 1982). By contrast, volvement in the elicitation of widespread responses preliminary data have indicatedthatin Acerpseudo- in woody xylem could repay investigation. platanus the same major antifungal compound was Wood extractives are commonly located within the induced in xylem,bark and leaves following fungal cell walls, where there can be substantial free space and capillary water, as well as in cell lumina (Hillis, challenge(Arnold & Pearce, unpublished). The elicitation of phytoalexin-like compounds in 1987). This appears to be the case for these treexylemremainslargely uninvestigated. Although phytoalexin-like compounds in Acer (Pearce, unelicitationhas been reportedto be a non-specific published). Polymerization in situ could lead to process following injury or microbial attack (cf. blockage of water movement pathways in the Kemp & Burden, 1986; Duchesne, Hubbes & Jeng, apoplast and hence enhance the effectivenessof any 1992), a glycoprotein elicitorof the elm phytoalexin permeability barrier formed using these compounds mansonone F has been isolated using elm callus as precursors (see IV 2(j)). cultureas a model system(Yang, Jeng& Hubbes, Fungal components, including chitosan, can elicit the putative defence in the xylem of both angiosperm 1989; Yang et al., 1994). In A. pseudoplatanus, of phytoalexin-like accumulation compoundswas an and gymnosperm trees, paralleling the responses to and fungal challenge (Miller et al. 1986; Lieutier & integralpart of CBL reactionzone formation, was observed behind both fungally challengedand Berryman, 1988a, b; Nicole et al., 1991; Yamada, uninoculated wounds in an excised stem length 1992). Currently, however, information is very and the role of fungal elicitors and such reactionzone fragmentary, stemlengths, system.In unchallenged responses were delayed in comparison with those abiotic factors as vessel cavitation in the induction of challengedwiththe weaklyaggressivedecay fungus phytoalexin-like (and other) responses in the xylem Ustulina deusta (Pearce et al., 1994a). In young of living trees can be evaluated effectively. comLike other putative defences operating in living plants of A. pseudoplatanus, phytoalexin-like pounds commenced accumulationwithin 24 h. In sapwood, there is little direct evidence implicating some experiments morerapidaccumulation was seen phytoalexin-like compounds in the inhibition of around wounds inoculated with U. deusta. than pathogens or potential pathogens. As with cell-wall around uninoculatedwounds or wounds inoculated alterations, dynamic studies of plant-fungus interwith the aggressive pathogen Chondrostereumpurpureum (Figs 8, 9). Later, when CBL reaction zones were fully formed, the fluorescent wood was restricted to a narrow region adjacent to the coloured actions will be critical in resolving their significance: a start has been made in studies of the interactions between A. pseudoplatanus and wood-inhabiting fungi (see above; Pearce et al., 1994a).

222

R. B. Pearce

(j) Necroticand hypersensitive responses. Reaction and zinc in reactionzones. It has been reported that zone tissues in many trees are characterized by cell Mn was not readilyextracted fromthis tissue with wall alterationsand the accumulationof coloured water(Shevenall & Shortle,1986); however,micropolyphenolic deposits,whichocclude vesselsand cell analysisdata suggest thata majorpartofthiselement lumina (Shain, 1967, 1971, 1979; Pearce & Wood- is water-extractable (Pearce & Grime,unpublished). ward, 1986; Pearce, 1990; Pearce et al., 1994a). Although thisaccumulation could be associatedwith Although the cell necrosis accompanying this fungal Mn-dependent lignolyticenzymes, similar exhibitsfeatures in commonwith 'classical' hyper- Mn accumulationat the marginof non-inoculated sensitive necrosis(cf.Goodman & Novacky,1994), it control wounds(Smith& Houston,1994) encourages occurs relatively slowly, appearing 7-14 d after the suggestionthat changes in this ion may be of challenge, some time after the accumulation of plant originand involvedin the phenolic polymerphytoalexin-like compoundshas commenced(Pearce ization process. et al., 1994a; Pearce, unpublished). Since few sapwood pathogens,with the possible The formationof necrotic CBL reaction zones exception of certain fastidiousxylem-limited bacappears to be a key factorin functional barrieror teria,are biotrophic, cellular necrosis in the xylem boundaryformation. In Acer species, the extension (in which many cells are normallynon-living) is of naturallyoccurringdecay lesions appears to be unlikely to be effective by itselfas a defenceagainst prevented where there is a necrotic zone at the wood-inhabiting micro-organisms. Rapid cell necinterface betweenhealthyand infected wood. How- rosis has, however, been associated with the reever,fungaladvancemaytakeplace whenthereis no sponses of Ulmusspp. to Dutch elm disease, and it such tissue at the host-pathogeninterface,even has been suggested thatthesereactions, havingmuch though antifungalcompounds can be detected in in commonwithhypersensitivity, maybe important extracts from this region (Pearce & Woodward, in resistanceto this vascular pathogen(Ouellette & 1986). Similarly, observations on excised stem Rioux, 1992). lengths (Pearce et al., 1994a) and young trees (Pearce, unpublished) of A. pseudoplatanuschalV. DEFENCE IN HEARTWOOD lenged with eitherC. purpureum or U. deustahave revealedtheformation of a colourednecrotic zone in Heartwood has been definedas 'the innerlayersof advance of the static lesions of the latterfungus, thewood, which,in thegrowing tree,have ceased to whilst no such necrosis was seen ahead of the contain living cells, and in which the reserve front of C. purpureum. advancinginfection As noted materials(e.g. starch) have been removed or conabove(IV2. (i)),phytoalexin-likecompoundsaccumu-verted into heartwood substance' (Anon., 1957). lated in response to both fungi. Whilst cellular Typically,heartwoods containhigh levels of extracnecrosisper se is unlikelyto conferresistance,the tives, which are deposited withinthe cell walls or associated polymericdeposits mightbe important, as wall encrustations within the lumina of empty either directlyas a result of their resistance to cells (Hillis, 1987). These extractives, whichmay be degradation and/or fungitoxicity, or as a conse- present in relativelylarge amounts, often show quence of their abilityto forma seal and prevent antimicrobial For example,in Thuja plicata activity. dryingand aerationin adjacent uninfected wood. (Westernred cedar) heartwoodthe thujaplicinscan Detailed biochemical studies are required to constituteup to 120% of the wood (oven d. wt) elucidateprocessesleadingto thedepositionofthese (Hillis, 1962). They exhibit antifungalactivityat polyphenolicmaterialsat lesion margins.However, c. 0 015 00 in bioassay in malt agar media (Roff& the spontaneousformation of green colours during Whittaker,1959), and appear to be largely rethe handling of the normallyyellow material ex- sponsibleforthedurability ofthistimber.Similarly, tractedfromfungally challengedA. pseudoplatanus ellagitanninshave been identifiedas constitutive wood (Pearce, unpublished),suggeststhatthe green antifungal compounds in white oak (Quercus alba) of CBL reactionzones in this heartwood (Hart & Hillis, 1972). Many similar depositscharacteristic species might be polymerizationproducts of the examples are known(cf. Hillis, 1987). induced phytoalexin-like compounds. A primary Antifungalextractivesin heartwood commonly function forthese compounds as soluble precursors include compounds also found as inducible phytofor the formation of polymericbarrierswould be alexin-likeinhibitors in livingsapwood. Pinosylvin commensurate with their relativelylow antifungal and pinosylvinmonomethylether were found at activitiesdetermined in bioassay (see IV 2(i)). concentrations inhibitory (in the range 1-30 mg gAnalyses of reaction zones using either bulk d. wt) in pine heartwood(Hart & Shrimpton, 1979; methods (Shain, 1971; Blanchard et al., 1978; Hart, 1981): in culture, these compounds at
Shevenell & Shortle, 1986; Smith & Houston, 1994) or proton-induced X-ray emission (PIXE) microanalysis (Grime & Pearce, 1995) have shown increases in various elements including manganese inhibited hymenomycete fungi 50)200,ug ml(Rennerfelt, 1945; Hart, 198 1) . Similarly in elm, (- )-7-hydroxycalamenene has been reported as both a phytoalexin (Burden & Kemp, 1984) and a

Antimicrobial defences of livingwood

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heartwoodconstituent (Rowe et al., 1972). Tyloses, interactions typicallytake place at depth in bulky a common morphologicalfeatureof sites of host- tissues, their progresscannot readily be followed. pathogeninteraction in the sapwood (see IV 2(h)), Also, physicaldisturbance of the zone of interaction are also commonin heartwood (Bamber& Fukazawa, is likelyto perturbit, eitherby breachingdefensive 1985), where theymay be suberized,as in Quercus barriers or by disruptingthe integrity of xylem robur (Pearce & Holloway,1984). The highconstitu- tissues,allowingdrying and the ingressof air. Thus tive levels of allelochemicalsin heartwoodgenerally the investigation of dynamic aspects of hostconfer upon it a greater resistance to biodegradation pathogen interactions in xylem poses considerable -than sapwood in wood products(Kuc & Shain, 1977; practicaldifficulties and, in consequence,therehave Hart, 1981; Pearce, 1987). The hydrophobic nature been relativelyfew such studies. Three main apof many of these compounds, including suberin, proaches have been adopted to overcomethese exmightrenderthe heartwoodmicroenvironment less perimental the examinationof multiple difficulties: favourable for microbial growth because of its replicates at timeintervals withinan experiment, the reduced permeability.In Triplochiton scleroxylon, interpretation of 'historical' relics of the hostthe avoidance of heartwoodby Botryodiplodia theo- pathogeninteraction withininfected tissues,and the bromae was attributed to thetight closureofinfection use of non-invasive methodsof data acquisition. pathwaysin thistissue,rather than to restriction The first by approach,using replicatedsamples, has nutritional limitation or antimicrobial wood extrac- been the most widely adopted, and is, in any case, tives (Tabirih & Seehann, 1984). In the livingtree, requiredto supportotherapproaches (cf. Pearce et however,sapwood is typicallymore resistantthan al., 1994a). It has enabled the formulationof heartwoodto fungalattack.This greaterresistance integratedmodels for defence processes in bark mightresultfromthe abilityof sapwood to express tissues (Biggs, 1985; Woodward & Pearce, 1988b), active defenceresponsesand/or fromthe environ- and has allowed the early accumulationof phytomental restriction of microbial colonizationconse- alexin-likecompounds in Acerpseudoplatanus to be quent upon the inimicalmicroenvironment of func- demonstrated (see IV 2(i)). Althoughsuch destructional sapwood (see IV above). tivemethodsmay be successfully applied in longerAlthough events at lesion margins in decaying term experiments, forexampledetermining theonset heartwood in living trees have been discussed in of suberization in compartmentalization wall 4 relation to the CODIT model (Shigo & Shortle, barriersin oak (Pearce, 1987; Table 1), inter-tree 1979), the potential for active, host-mediatedre- variation mightseverely of complicateinterpretation sponses to infection must be extremely limitedin a the results. Studies followingthe developmentof tissuedevoid oflivingcells. However,thesurvivalof individuallesions are likelyto be more informative. active parenchyma cells has been reportedin some Important evidence for the significanceof the 'heartwoods' (Bottcher& Liese, 1975); such cells wood microenvironment in restricting fungalcoloncould contribute to more activedefensive processes. ization has been obtained using essentially this In the absence of living cells, chemical changes at approachto followdecaydevelopment in logs freshly lesion marginscould arise fromthe action of fungal cut fromlivingtreesand allowed to dryat different enzymeson heartwoodconstituents, or as a conse- rates.In severalangiosperm species,colonizationby quence of alteredoxygenand moisturelevels result- latent endophyteswas initiallymost rapid when ing fromfungalcolonization.Additionally, enzyme drying was mostrapid,although thegreatest ultimate activities have been shown to persistin the walls of development of decay occurredwhen a slow drying heartwood cells after protoplast death: theseinclude regimeallowedfungal to continue forlonger. activity phenol-oxidizingenzymes (Shain, 1971; Shain & Maintaining watersaturation in thewood prevented Mackay, 1973) and peroxidase(Ebermann & Stich, decay development (Chapela & Boddy, 1988b; 1982). These have both been implicated in the Chapela, 1989; Boddy, 1992). responsesof livingplant tissuesto microbialattack, Relics of CBL reaction zones that must have and, in the altered environment of a lesion, might formed at former lesionmargins have been identified catalyse changes in heartwood constituentsthat fromtheir characteristic visual, chemical, or anacould influence theplant-microorganism interaction. tomical features in the decayed wood of several Work is requiredto examinethis possibility. species of woody angiosperms(Cooke & Rayner, 1984; Rayner,1986; Pearce, 1987; Rayner& Boddy, 1988; Pearce, 1991; Boddy, 1992). These relicswere VI. DYNAMIC ASPECTS OF THE distributeddiscontinuouslyin the decayed wood, HOST-PATHOGEN INTERACTION IN WOOD and wereseparatedby wood in whichno evidenceof As indicated in preceding discussions, an under- past host responsescould be found (Pearce, 1991).
standing of dynamic aspects of the host-pathogen interaction in living wood is a key factor in resolving the roles of the various putative defensive and protective mechanisms. As these host-pathogen This indicated that, for these species at least, the widely accepted classical model of the reaction zone as a dynamic boundary receding ahead of a continuously advancing infection front (Shain, 1967;

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R. B. Pearce 2-3 wk of challenge. In contrast, stem lengths challenged with C. purpureum exhibited a continuously advancing cone of necrotic, infected tissue, with little visible evidence of plant response at the host-pathogen interface. In addition to illustrating the potential of this approach for dynamic studies of host-pathogen interactions, this study demonstrated changes in the imageable water content of the xylem (see IV 2(a) above). The extension of such dynamic studies should provide the means forestablishing spatial and temporal associations between microbial colonization, plant responses and tissue water relations. This understanding should do much to resolve the roles of the various components associated with the host-pathogen interactions in living wood.
VII. GENETIC TISSUES CONTROL OF RESISTANCE IN

1971, 1979; Kemp & Burden, 1986) could not be correct.Instead, reactionzones appear to be relatively durable static boundaries to lesions, which may ultimately be overcome or lose function, allowing the renewed colonizationof wood behind the reaction zone. Typical responses are not expressed in this tissue until fungalgrowthis again arrestedand a new reactionzone is formed(Pearce, 1991; Boddy, 1992). These anatomically and chemically distinct reaction zones are equivalent to column boundarylayers (see IV 1(c)), and will be referredto as CBL reaction zones to distinguish them from the little-known responses that might occur where the colonization of living wood is actuallytaking place (cf.Pearce & Woodward,1986). These zones would be truly equivalentto thereaction zone as originallyenvisaged (Shain, 1967, 1971, 1979). It should be noted, however, that since reaction zone relics do not appear to have been studied in any conifer, comparablelesion dynamics in gymnosperm treescannotbe assumed. Although analysis of reaction zone relics has provided strong evidence for discontinuity in the extension of decaylesionsin trees,it does not permit resolution of the mechanisms that regulate the nor does it provide any host-pathogeninteraction, indication of the timescale over which invasion occurred (Pearce, 1991). Careful examination of lesionsinvolving cambialdeathcould,perhaps,allow some calibrationof lesion spread against the time reference established by cambial activity. The of staticcolumn boundary observed preponderance layer reactionzones over regions of fungalspread when trees were sectioned (Pearce & Woodward, 1986) was indicative that periods of stasis were normallylong comparedwiththe time occupied by Non-invasive methods that can provide information on the host-pathogen interactionwithin that living wood, without themselves influencing have the potentialto provideanswersto interaction, these outstanding questions. Decay lesions have been imaged in wood using y-ray tomography (Dobos et al., 1991; Schwarze, 1995) and nuclear resonance(NMR) (Hall etal., 1986; Pearce magnetic et al., 1994a, b). Using NMR imaging methods, Pearce et al. (1994a) have followedthe interaction and two betweenlivingwood of Acerpseudoplatanus xylem pathogens of different aggressiveness, in Ustulina deusta and Chondrostereum purpureum, excised stem lengths.These provided a convenient model system in which wood parenchyma cells zone remained alive for3-4 wk and in whichreaction responses were expressed. Lesion development revealed by NMR closely paralleled that seen in
similar stem lengths examined destructively: in stem lengths challenged with U. deusta little invasion occurred during the first16 d following inoculation, and reaction zone responses were apparent within active invasion.

XYLEM

There have been few genetic studies of resistance in the xylem of woody plants, at either the molecular or physiological level. Genotype-dependent differences in the ability of trees to 'compartmentalize' discoloration and decay have been demonstrated (Shigo, Shortle & Garrett, 1977; Garrett et al., 1979; Santamour, 1979; Lowerts & Kellison, 1981). In most cases, the underlying mechanisms remain unknown, although in hybrid popular trees a correlation was established between wood structure and the ability to compartmentalize decay. Strongly compartmentalizing trees had fewer and smaller vessels, and fewer connections to other vessels than weakly compartmentalizing trees (Eckstein et al., 1979). Comparisons between host-pathogen interactions in relatively resistant and susceptible genotypes have also been made in elm species infected with Ophiostoma ulmi and 0. novo-ulmi. Despite the examination of a wide range of putative defences (see Ouellette & Rioux (1992) for review), no single factor has yet been shown to be of key importance. Although comparison of host-pathogen interactions in resistant and susceptible genotypes might facilitate identification of the determinants of resistance or susceptibility, it seems likely that more sophisticated experiments allowing temporal analysis of the interaction will be required to establish the roles of potential determinants. For example, differences in vessel anatomy associated with weak compartmentalization (Eckstein et al., 1979) or susceptibility to Ophiostoma spp. (Elgersma, 1970; McNabb, Heybroek & MacDonald, 1970; Sinclair, Zahand & Melching, 1975) could function by favouring vessel dysfunction (and hence reducing the potential for environmental restriction of infection), or by facilitating pathogen spread within the tree. Vessel-occluding tyloses and gummosis responses could be less effective in larger, more

Antimicrobial defences of livingwood interconnected vessels. Alternatively, easier passage of the pathogenthroughthe xylem could enable a pathogento keep ahead of induced host responses, which would then be unable to containthe lesion. Whilst correlationsbetween resistanceand tree characters (for example terpene composition in conifers(Schuck, 1982a)), may be helpfulto plant breedersseekingto selectforresistance, theyare not, in themselves, indicativeof a causal role in defence.

225

Rayner, 1983; Rayner, 1986; Rayner & Boddy, 1988; Boddy, 1992), providing further indirect evidence to support the microenvironmental restriction hypothesis. Alternatively, the formation of

morphologicallyand chemically defined zones at the margins of lesions, which can be resistantto degradationby decay fungi(Pearce & Rutherford, 1981), or which contain antimicrobial compounds (Shain, 1967, 1971; Kemp & Burden, 1986; Pearce & Woodward, 1986) encouragesthe contention that lesions are restricted by activedefencemechanisms. Most of the evidenceadvanced in supportof each hypothesis is open to alternative interpretations, and little, if any, is entirelyincompatiblewith either model. Althoughsuberized or polyphenolicbarrier VIII. ANTIMICROBIAL DEFENCE IN THE LIVING zones are formed,they could functionby sealing XYLEM OF TREES: TOWARDS A CONSENSUS wounds againstwaterloss and air entry, rather than MODEL by providingan antimicrobial barrier,thus having Despite our fragmentary understandingof anti- onlyan indirect role in protection (Rayner& Boddy, microbialprotection and defencein livingwood, it is 1988). This proposal has been supported by the clear thatthesetissuesare capable of restricting the claim that these zones are readilybreached if the developmentof pathogensfor periods of time that wood behindthembecomes dried(Rayner& Boddy, are withoutequal elsewherein the plant kingdom. 1988). Althoughpatternsof decay developmentin Althougha rangeof different pathogentypesattack attachedbranches(Chapela & Boddy, 1988a) can be sapwood, the trees' responses to all exhibit many interpreted in this way, criticaldynamicmeasurecommonfeatures.Similarlythereis a close congru- mentsof watercontentand fungalinvasionhave not ence between the responses of gymnosperms and been made. Whilst the antimicrobialactivity of woody angiospermsto infection. The anatomyand manyof the compoundsinduced in xylemfollowing chemistryof reaction zones and compartmental- fungalchallenge is oftenrelatively low (Rayner & izationwall 4 barriers mightdiffer in detailbetween Boddy, 1988) theiractivity mightbe greaterin the these two divisions, but these responses, which tree than in the assay systems hithertoused to together encompass the key elements of active measurethis(see IV 2 (i)). Some degradation ofCBL defencein livingwood, are fundamentally similarin reaction zone materialdoes occurwhentheybecome both. Defence in barktissuesalso showscomparable non-functional;however, distinctivecomponents parallels(Biggs, 1992b; Woodward, 1992). persist in decayed wood (Pearce, 1991). The inThe mechanism by whichmicrobialgrowth in the creased resistanceof CBL reaction zone wood to living wood of trees is restricted remains contro- fungalattackhas been demonstrated in degradation versial,protection being variouslyattributed either studies (Hepting & Blaisdell, 1936). Probably the to passive microenvironmental restriction of poten- most compelling evidence for the importance of tial pathogens (Boddy & Rayner, 1983; Rayner, aeration in allowing lesion developmentin living 1986; Rayner & Boddy, 1988; Boddy, 1992) or to wood is the common observationthat decay deactive antimicrobial defence(Shigo, 1984; Kemp & velopmentbehind wounds usually ceases when the Burden, 1986; Pearce, 1987, 1989; Yamada, 1992). wound has been sealed completely by callus growth These mechanisms are not mutually exclusive: (cf. Shigo, 1986; Biggs, 1992a). indeed,bothmight operatein concertin many,ifnot Much of the evidence that argues for the imall, infectionsof living sapwood. It is, however, portanceof active defenceis similarly inconclusive. difficult to resolve the precise functions of the The mere demonstrationof antimicrobial comseparate components of the host-pathogen inte- pounds or structural barrierswithinthe wood does raction to provide an integrated description of not in itself prove their role in host-pathogen defence. interactions.In general, correlationsthat link the Studies of decay developmentin freshly cut logs activitiesof putativedefencemechanismswith the under differing dryingregimeshave demonstrated expression of resistanceremain to be established, that the high water contentof functionalsapwood althoughdifferential accumulationsof phytoalexincan suppress fungaldevelopmentby rendering the like compounds in compatible and incompatible wood microenvironment inimical to fungalgrowth interactions in Dutch elm disease have been reported (Chapela & Boddy, 1988b; Chapela, 1989). Patterns (see Ouellette & Rioux, 1992) and in Acer pseudoof fungal developmentassociated with damage or platanuschallengedwithwood-inhabiting pathogens stressin livingtreesequate withthe distribution of ofdifferent aggressiveness (see IV 2 (i) above; Figs 7, drier wood resultingfrom this trauma (Boddy & 8). As previously of manyof the noted,the activity
proposed protective compounds isolated from wood appears low, and 'defensive' compounds might be metabolized by pathogens (e.g. Armillaria sp. can degrade suberin (Zimmermann & Seemuller, 1984)).

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R. B. Pearce

Table 3. Factors contributing to the outcomeof hdst-pathogeninteractionsin the living sapwood of angiosperm trees Event Xylem tissues become drierand aeratedfollowing dysfunction, commonlyconsequentupon wounding Possible role References

Removal of passive Boddy & Rayner,1983; Rayner, microenvironmental of inhibition 1986; Rayner& Boddy, 1988; microbialgrowthpermitting Boddy, 1992 establishment and easy colonization of disruptedtissues. Early accumulationof phytoalexin- Creationof an inhibitory Kemp & Burden, 1986; Pearce, compoundswithin24 h of fungal environment withincompromised unpublished(see above) challenge wood; accumulationof precursors forthe polymerization of structural barriers Increased watercontentof xylem Reinforcement of Pearce et al., 1994a, b; Pearce, tissues at lesion margins, microenvironmental restrictions to unpublished presumably by the replacement of microbialcolonization.Infusionof gases withinfibrelumina etc., induced phytoalexin-like 4-7 d afterchallenge commencing compoundsthroughout tissues at the lesion margin Deposition of insolublepolyphenolic Formationof a degradation-resistant Hepting & Blaisdell, 1936; Boddy & compoundsand/orsuberinat lesion barrierto microbialspread at the Rayner; 1983; Rayner,1986; marginsto forma CBL reaction lesion margin.Formationof a Rayner& Boddy, 1988; Pearce, 7-12 d after zone, commencing water-and gas-impermeable seal 1989, 1990; Boddy, 1992; Pearce et challenge betweenfunctional and al., 1994a compromised xylemto maintainan inimicalmicroenvironment for microbialcolonization Accumulationof phytoalexin-like Maintenanceof reactionzone Grime & Pearce, 1995; Pearce, compounds in healthywood function by chemicalinhibition or unpublished of barriers adjacent to CBL reactionzones continuing augmentation (long-term phenomena)

The heterogeneity ofnormalwood structure renders the formation of continuous barrierzones in preexisting xylemproblematic, and might thuslimitthe efficacyof reaction zones as physical barriers. Although the more rapid fungal colonization of wood afterkillingcan be taken as evidence for the ofactivedefencein livingtissues(Pearce et operation al., 1994a), changes in the cellular distribution of water, notwithstanding the absence of any gross change in water content, could influenceease of

static restriction at lesion margins (Pearce, 1991). Where the host-pathogen interface is static, comto reconcile prising a CBL reaction zone, it is difficult this with restriction by microenvironmental factors alone. In the absence of antimicrobial activity of some kind, which could be mediated through inhibitory compounds, cell wall alterations or gummy occluding deposits, a dynamic lesion margin, slowly but continuously retreating ahead of the infection frontwould be anticipated, as in the initial invasion. model of Shain (1967, 1971, 1979), as the pathogen The formation of well-definedcoloured bound- slowly eroded into the functional xylem by cell-byaries behind pruning wounds in Acer rubrum has cell attrition. However, in angiosperms at least, this been associated with effective restriction of fungal pattern of lesion development is not seen (Pearce, colonization:where such a boundarywas not seen, 1987, 1991; Boddy, 1992). decay and discolorationextended further In A. pseudoplatanus, non-invasive NMR imaging into the stem (Green et al., 1981). Such evidence was again studies (Pearce et al., 1994a) have not revealed any indicativeof an association between reactionzone reduction in xylem water content ahead of the formationand resistance, but this interpretation infection front of the invasive pathogen Chondromust remain equivocal in the absence of any stereumpurpureum in excised stem lengths, which information on the dynamicsof lesion development were used as a model system for the host-pathogen and host response. interaction. By contrast, there was an increase in Detailed studiesenablingthe spatialand temporal imageable water at the lesion margin in stem lengths resolutionof all componentsof the host-pathogen challenged with the weakly invasive Ustulina deusta. interactionin living sapwood are required con- Similar increases in water at the margins of lesions the rolesof thesecomponents resulting fromthe inoculation of young trees with U. clusivelyto determine in defence.Most critically, it is stillunclearwhether deusta or Ganoderma adspersum have also been active defence or microenvironmental limitation observed (Pearce et al., 1994b; Pearce, unpublished), effectthe transitionfrom invasive colonization to suggesting the active enhancement of water-medi-

Antimicrobial defences of livingwood

227

ated tissue protectionin the incompatible inter- lesion margin,possibly contributing to the microaction,rather thandrying-mediated in environmental susceptibility restriction of infection (Pearce et al., the compatible. Additionally,the accumulationof 1994a) or serving as a solvent, saturating thewood in phytoalexin-like compounds in the compatible in- thisregionwitha solutionoftheinducedcompounds teraction was sometimes delayedin comparisonwith and allowingthem to accumulatein the non-living the incompatible(see IV 2 (i)) paralleling results tissuesas well as in the immediatevicinity of living reported fromDutch elm disease (Duchesne, Jeng& cells, permittingthe formationof a continuous Hubbes, 1985). Similarresultshave been obtainedin reaction zone response, despite the anatomical non-woody species where phytoalexinshave been heterogeneity of thewood. The inducedcompounds ascribeda key role in resistance(Mansfield,1983). mightthemselves be antimicrobial, or theirtoxicity Irrespectiveof mechanism,static lesion margins might be enhanced by activation. [Photoactivated are delimitedin the livingsapwood by multicellular biocides can be activatedby peroxidase and polyboundary zones - compartmentalizationwall 4 phenoloxidaseenzymes(Leatham etal., 1980; Swain barriers or CBL reactionzones. It seems likelythat & Downum, 1991), which are knownto accumulate the durability requiredforthe long-term protection at reactionzones (Shain, 1971; Geiger et al., 1989; of the bulky secondarytissues of trees (xylem or Pearce, unpublished), as well as by light.] The phloem) can be provided only by these relatively induced compounds might also polymerizein the large-scaleresponsesthatfunction at a tissue level. cell walls and lumina of vessels and fibresto form On the basis of the evidence discussed in this insoluble gummy deposits that could act both as review, a model for lesion development and re- physicalbarriersand hydraulicsealants. In species striction in the living wood can be suggested (see with a xylem suberizationresponse (Pearce, 1990) Table 3). This model,based on studiesin Acer spp., this response might also contribute to barrier relatesprimarily to decay in angiosperms, but major function. elements might also be applicable to protection The dynamic studies that would permitidentiagainst other categoriesof pathogen,and to host- ficationof the mechanism of CBL reaction zone in gymnosperms. pathogeninteractions failureand subsequent fungalspread remainto be Access to the wood, commonlythrougha wound done. The importanceof directbarrierpenetration exposingthis tissue,is a pre-requisite forinfection. and active pathogenesis in lesion expansion, as A fewpathogens, e.g. Armillaria spp, Roselliniaspp., distinct fromthe colonizationonlyof wood whichis penetratethroughintactbark (Tourvieille de Lab- predisposed by xylem dysfunction (cf. Rayner & rouhe, 1982; Morrisonet al., 1991). In additionto Boddy, 1988; Boddy, 1992) also remainsunresolved. access to thexylem,barkdamage disrupts providing A volume of sapwood, the dimenxylemfunction. sions of which are, in part, determined by the size ACKNOWLEDGEMENTS and locationof the wound, becomes non-functional, I thank thestaff ofthePlantSciences Library, University drier, and in consequence aerated. This com- of Oxford, fortheir assistance withlocating someof the cited. Much of the workon host-pathogen promised wood is intrinsically susceptible to mi- literature in Acer spp. discussedin this reviewwas crobial colonization,since it is no longerprotected interactions theLeverhulme from Trust. bya research grant by the high water content and low oxygen en- supported vironmentof functionalxylem. This vulnerable wood becomes colonized by micro-organisms causing stain or decay. Ultimately,fungal invasion is REFERENCES arrested eitherby defencemechanisms inducedafter Aist JR, 1976. Papillae and relatedwound plugs of plant cells. 14: 145-163. Annual ReviewofPhytopathology or because theadvancing woundingand/orinfection, mechanisms.In: responsesas resistance infectionfront has reached intrinsically resistant Aist JR. 1983. Structural New ofHost Defence. BaileyJA,Deverall BJ,eds. TheDynamics functional xylem,which slows invasion sufficiently York: Academic Press, 33-70. to permit theformation ofa CBL reaction zone at the Albrecht C, Burgess T, Dell B, Lapeyrie F. 1994. Chitinaseand Aggressive pathogens like Chondrostereum purpureumwould eitherinvade functionalxylem rapahead of the idly, or propagatexylem dysfunction infection front, whilst avoiding, detoxifyingor suppressinghost defensiveresponses. Constitutive defencein the xylemcould slow the colonizationof compromised tissue, perhaps reducing the size
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