Antimicrobial Defences in the Wood of Living Trees

7/21/2019 Antimicrobial Defences in the Wood of Living Trees

1/32

Tansley Review No. 87. Antimicrobial Defences in the Wood of Living TreesAuthor(s): R. B. PearceSource: New Phytologist, Vol. 132, No. 2 (Feb., 1996), pp. 203-233Published by: Blackwell Publishingon behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2558445.

Accessed: 30/08/2011 13:58

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at.http://www.jstor.org/page/info/about/policies/terms.jsp

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of

content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms

of scholarship. For more information about JSTOR, please contact [email protected].

Blackwell PublishingandNew Phytologist Trustare collaborating with JSTOR to digitize, preserve and extend

access toNew Phytologist.

http://www.jstor.org
http://www.jstor.org/action/showPublisher?publisherCode=blackhttp://www.jstor.org/action/showPublisher?publisherCode=npthttp://www.jstor.org/stable/2558445?origin=JSTOR-pdfhttp://www.jstor.org/page/info/about/policies/terms.jsphttp://www.jstor.org/page/info/about/policies/terms.jsphttp://www.jstor.org/stable/2558445?origin=JSTOR-pdfhttp://www.jstor.org/action/showPublisher?publisherCode=npthttp://www.jstor.org/action/showPublisher?publisherCode=black


2/32

New Phytol. 1996), 132, 203-233

Tansley Review No. 87Antimicrobial defences in the wood ofliving treesBY R. B. PEARCESchool of Biological Sciences, University fBirmingham,Edgbaston,BirminghamB15 2TT, UK(Received 5 June 1995)CONTENTS (b) Nutritional actors 213Summary 203 (c) Anatomicalfeatures fnormalwood 213I. Introduction 204 (d) Interxylaryork 214II. Xylempathogens 205 (e) Constitutivenhibitoryompounds1. Wood decaying ungi 205 (phytoanticipins) 2142. Canker rots 205 (f) Resin 2153. Endophytes nd latent nfections 205 (g) Lyticenzymes 2164. Vascularwilts 206 (h) Cell wall alterations 2165. Xylem-inhabitingacteria 206 (i) Induced antimicrobialompounds6. Viruses 206 (phytoalexins) 218III. Bark the first ine of defence 206 (j) Necrotic nd hypersensitiveIV. Defence in functional apwood 207 responses 221

1. Models for hehost-pathogen V. Defence in heartwood 222interactionn the wood of iving rees 208 VI. Dynamic aspectsof thehost-pathogen(a) The heartrot oncept 208 interactionn wood 223(b) Compartmentalizationfdecay n VII. Genetic control f resistance n xylemtrees CODIT) 208 tissues 224(c) Dynamic decay development nd VIII. Antimicrobial efence n the ivingxylemreaction ones 209 of trees: towards consensusmodel 225(d) Environmental estriction ffungal Acknowledgements 227colonization 212 References 2272. Putativedefencemechanisms 212(a) Environmental onstraints npathogengrowth 212SUMMARYThe wood xylem) n a living ree s protected rommicrobial ttack y the secondary lant urface periderm ndrhytidome),which provides an effective arrierpreventing he entryof most potential pathogens,and byconstitutivend induced defencemechanisms n the bark cortex ndphloem).Although fewpathogens reabletopenetrate heseouter issuesdirectly,mostxylempathogens ainentry hroughwoundsthat xposethis issueand render t more vulnerable o attack. n functional apwood,microbialcolonizationmightbe restricted yactivedefencemechanisms, r by passive microenvironmentalestrictiononsequentupon the highwater ontentand low availability f02 inheal hy onductive ylem.These factors re notmutually xclusive: ndeed, heymayoperate in concert n many host-pathogen nteractions. apwood lesions made by wood-decayingfungi arecharacteristically ounded by multicellularwalls or barrier ones (compartmentalization all 4 barriers ndreaction ones columnboundary ayers)),whichmayfunction oth s inhibitory rdegradation-resistantarriersto further athogen pread, nd as seals to maintain ylem unctionnd prevent he drying nd aeration hat ouldpredispose to further nfection.A range of putative antimicrobial efence mechanismscontributing o theeffectivenessf uch barriers as been dentifiedn sapwood tissues.This includes ell wall alterations,onstitutiveand induced antimicrobial ompounds,necrotic esponses f iving ells and the deposition f gummymaterials,often esinous r polyphenolic,t thehost-pathogennterface. utritional, nvironmentalnd anatomical eaturesof ivingwood might lso contribute o pathogenrestriction. lthough heremightbe differencesn detail,thedefences perating ngymnospermsnd angiosperms re generally imilar.Defence responses gainst variety f


3/32

204 R. B. Pearcepathogen ategories lso have much n common.Dynamic studies re crucial n elucidating he rolesofthe variouscomponents fthe host-pathogennteractionn the wood of iving rees.A model for he protectionnd defenceof xylemtissues in woody angiosperms s suggested,based largely upon results fromdynamic studies ofhost-pathogen nteractionsn thewood of sycamore Acer pseudoplatanus .).Key words: Antimicrobial efence,disease resistance,wood, trees,decay.

I. INTRODUCTIONDespite the economic, environmental and ecologicalimportance of trees, our understanding of theirpathology has generally lagged behind that ofagricultural crop species, a fact that has been notedpreviously (e.g. Kemp & Burden, 1986; Pearce,1987; Duchesne, Hubbes & Jeng, 1992). Althoughthe host-pathogen interactions of woody perennialsexhibit a number of distinctive features, thesetypically receive scant mention in most general plantpathology texts, and the first book specificallyaddressing the physiological and biochemical aspectsof tree pathology has only recently been published(Blanchette & Biggs, 1992).This neglect of tree pathosystemscan be attributedto several factors. Until recently trees have beenregarded more as a natural resource to be exploitedthan as a managed crop, in which measures to reducedisease loss could be actively deployed. [In thiscontext it is to be noted that much of the earlyworkon host-pathogen interactions in trees is related toorchard species (Brooks & Storey, 1923; Swarbrick,1926).] Further,although the finalvalue ofa standingtree crop mightbe high, its annual increment is low.In natural and traditionally managed forests, thepopulation structure has not favoured the dev-astating disease epidemics that often appear to beproducts of intensive crop production (Schmidt,1978; Zadoks & Schein, 1979). However, whenconditions for disease have been favourable, dam-aging tree disease epidemics have occurred [e.g.Dutch elm disease, chestnutblight,whitepine blisterrust; see Manion (1991) for brief accounts], andcurrent trends towards the intensification of treeproduction favour an increase of disease problems inforestry (Evans, 1982). In addition to these es-sentially economic factors,technical difficulties avealso tended to discourage research on tree diseases.The size of trees, their slow development and thelong timescale ofmany of their diseases render themunattractive for experimental use. Further, thediversityof economically significantwoody species,coupled with the relatively small number of re-searchers active in treepathology, has meant that ourunderstanding, even ofmany common tree diseases,is often fragmentary nd superficial.

In consequence, many aspects of antimicrobialdefence in trees remain imperfectly understood.Whilst it is reasonable to suppose, a priori, that thedefence mechanisms operating in woody plants arelikelyto be similar to those better known (and more

readily tudied) n herbaceous pecies, thebulk andlongevity f trees impose important equirementsand constraintsfor the success of endogenousresistance. n the secondary tissues, that is bark,comprising eriderm, ortex, hloemand cambium,and wood (xylem),defencemay need to be effectivefor longtime.Whilstthe ability f a plantdefencemechanism o halt pathogendevelopment or fewdaysor weeks mightbe sufficiento protect phem-eral planttissues,the progressof a wood-decayingfungusmightneed to be restricted or decades, orperhapseven centuries f the tree host s not to dieprematurely.Additionally,many pathogens andpotentialpathogenscan establishextensive apro-trophic growth n dead or moribundwood. Suchbulky olonized issues,whichmight e still ttachedto an otherwisehealthy ree (for example decayedheartwood r suppressedbranches),provide a foodbase fromwhicha pathogen anmount prolongedassaulton the defencesof the host. The inoculumpotentialGarrett, 970) of such an attackmightbefar greaterthan that of a spore inoculum, forexample,and might hus requirea more extensiveand durable host defence o oppose it successfully.The abilityof mostwood-inhabiting athogenstoremain ctiveformanyyearswithin ong-establishedlesions similarly oses a continuing hreat. n theevent f anyfailure f defences tthe esion margins,thepathogenmayresume nvasion fhealthy issues.The resumption fdecaydevelopmentn the woodofEucalyptus pp. c. 14yrafterwoundinghas beenassociatedwith hedisruptionf a barrier one atthelesion margin t thistime White& Kile, 1994).Thus the need for long-termexclusion of apathogenwith potentially very high inoculumpotentialfrombulkyheterogeneous issues,withinwhich living and hence responsivecells are dis-continuously istributedcf. Esau, 1977; Carlquist,1988), imposesunusually tringent equirementsnthe defences f woodyplants.This reviewwill focuson mechanisms hat are believed importantn theprotectionf wood (xylem) issues gainstmicrobialattack n livingtrees.Wood is not only the mostabundant nd characteristicissueoftrees, t is alsothe principal productobtainedfrom reecrops. Arange of putative defencemechanismshas beenidentified n living wood, and models for theoperationof these mechanismsproposed,but ourunderstandingfmany spectsofthehost-pathogeninteractionsnvolvedremainsfragmentary.urrentoutstandingproblemswill be identified nd dis-cussedhere.


4/32

Antimicrobialdefences f living wood 205Trees includeplantsfrom wo botanicaldivisions,the gymnosperms nd the angiosperms.Althoughthere re manydifferencesetween hese divisions,leadingto separate reatmentsor heirdefencesby

Blanchette& Biggs 1992), a high degreeof congru-ence s evident n theseparallel reatments. o avoidundue duplication,gymnosperms nd angiospermswillbe treated ogethern the present eview, xceptwherematerial ifferencesxistbetween hem.Treedefences against insects have manyparallels withantimicrobialdefences (cf. Mattson, Levieux &Bernard-Dagan, 988). Whilstdefence gainst nsectpests (or other herbivores)will not be specificallyconsidered,tshould be borne nmindthat he sameprocessesmay be involved n defence gainstbothmicro-organismsnd insects, nd inwound healing(Mullick, 1977), and that ttempts o separate hesefunctionsmightprove ultimatelymisleading.II. XYLEM PATHOGENSMicro-organisms, athogenicor potentially atho-genic, n thexylemof treescan be subdivided ntosixbroad groups wood decaying ungi, anker ots,endophytesand latent infections,vascular wilts,xylem-inhabitatingacteria, nd viruses.1. Wood decaying ungiThese comprisethe most studied group of xyleminhabitants n trees. Several ecological or patho-logical types can be distinguished n the basis oftheir colonization strategies. Rayner (1986) andRayner & Boddy (1988) recognizedfivedistinctivestrategies heart ot, ctivepathogenesis,pecializedopportunism, esiccationtoleranceand unspecial-ized opportunism.Active pathogens are able to colonize healthysapwood, following ither direct penetration e.g.Armillaria pp.,Heterobasidionnnosum)r nfectionthroughwounds, which can provide a suitableinfection court (e.g. Chondrostereumurpureum,Stereum ausapatum).These fungi an cause rapidlyspreading ecroticesions nthexylem f heir osts.Fungi adopting opportuniststrategiesare lessclearly aggressive, nitially colonizing only func-tionally compromised sapwood, for example thatassociatedwith majorwound.Heart rotfungi rowand cause decay within the heartwood,which isgenerally evoidof livingcells and hence lacks thecapacityfor activeresponseto infection. t might,however,be protectedby allelopathic compoundsthat re often epositedduringheartwood ormation(Hillis, 1987). The pathogenicity f opportunistfungi is variable. Some, such as the heartwoodrottersLaetiporus ulphureusnd Sparassis crispa,appear to have little or no abilityto invade livinghosttissues, whilst many others, such as Ganoderma spp.,Inonotus hispidus and Ustulina deusta function asweak pathogens, slowly attacking living host tissues

(Pearce & Woodward, 1986; Pearce, 1991). Ulti-mately these fungi are capable of penetrating sap-wood and bark fromwithin to formfruitingbodieson living host trees (Burdekin, 1979).The desiccation tolerance strategy is associatedwith fungi decaying dead wood on trees (Rayner &Boddy, 1988). These exhibit a primarily sapro-trophic habit, although in some cases theymight bepresent as endophytes or latent infections in theliving tree (see 11.3, below).

2. Canker rotsCertain fungi, e.g. Inonotus obliquus on Betula spp.and Phellinus pini on Abies spp., can attack bark andxylem tissues simultaneously (Shigo, 1969; Blan-chette, 1982). These pathogens are able to re-invadewood repeatedly from the bark, and may, by thismeans, evade containment by barrier zones formedwithin the wood (Blanchette, 1982). Strip cankers,where-infectiongives rise to axially elongated zonesof dead bark and underlying xylem, resemble cankerrots in that both bark and xylem are attacked,although development often occurs during a singleyear only and might be associated with the effects fpredisposing factors Lonsdale, 1983; Rayner, 1986;Boddy, 1994). These lesions can develop frompropagules latent in the wood and bark (Boddy,1994).3. Endophytesand latent infectionsThe frequent isolation of fungi from apparentlynormal, healthy wood (e.g. Roll-Hansen & Roll-Hansen, 1979; Chapela, 1989) has kindled interest nthe significance of these asymptomatic infections.These fungi can develop in declining wood underfavourable environmental conditions, giving rise todecay lesions (Chapela & Boddy, 1988b; Chapela,1989; Griffith& Boddy, 1990). This habit mightconfer on these fungi a selective advantage byfacilitating their early and rapid colonization ofdying stems and branches. Little is currentlyknownof the route of entryof these fungi and of the formtaken by latent infections n the healthytree (Boddy,1994), although infection processes for Hypoxylonfragiformehave been described (Chapela, Petrini &Petrini, 1990; Chapela, Petrini & Hagmann, 1991).Evidence from controlled drying studies has indi-cated the importance of the high water content offunctional xylem in arresting development of thesefungi (Chapela & Boddy, 1988b; Chapela, 1989;Boddy, 1994). The association of other putativesapwood defences with latent infections does notappear to have been investigated to date. Immuno-logical methods provide a sensitive and specificmeans of detecting and localizing fungi in woodytissues (Blanchette & Abad, 1992) and have beenused to detect endophytic hyphae in asymptomaticneedles of Picea abies (Suske & Acker, 1989). Such


5/32

206 R. B. Pearcemethods should facilitate tudies of wood endo-phytes n situ nd permit urthernvestigation f thehost-pathogen nteractions f these fungi.4. Vascular wiltsIn vascular wilt diseases, microbialcolonizationofthe conductive ylemwithout ross disruption f tsstructure esults n impairedwater relations n thehost. Damage is mediated via vascular dysfunctionor translocated oxins produced by the pathogen.Propagules f hepathogen re disseminated hroughthe xylem, resulting n the rapid and extensivecolonization of conductive wood in infected us-ceptibletrees Mace, Bell & Beckman,1981).Dutch elm disease (Ophiostoma lmi,Ophiostomanovo-ulmi)s the best knownvascularwilt disease oftrees, lthoughVerticilliumpecies can cause diseasein a range of woody angiosperms.Typical vascularwilt syndromesdo not occur in gymnosperms,probably because their conductivexylem elements(tracheids) o notfavour heeasy and rapid preadofsuchpathogens Green, 1981). However, nfectionsthat impair vascular function o occur in gymno-sperms. The pine root pathogen Verticicladiellawagneri as manyof thecharacteristicsf a vascularwiltfungus Smith, 1967), and a nematode,Bursa-phalenchus xylophilus, causes a wilt syndrome(Mamiya, 1983).5. Xylem-inhabitingacteriaA few bacterial pathogens can colonize livingsapwood, causing gross symptomsthat resemblevascular wilts.Watermark iseaseofwillow,causedby Erwinia salicis, provides an example (Preece,1977). Also, fastidious xylem-limitedbacteria(Xylellafastidiosa) ave beenreported rom varietyof tree species in which they cause leaf scorchsymptoms Hopkins, 1989).Bacterial wetwood results from colonizationofheartwood and inner sapwood by populations ofbacteria apableofgrowing naerobically. olonizedwoodaccumulates luid, liphatic cids andmethane,often nderpressure, utdoes not becomedecayed(Ward& Zeikus, 1980). Although here ppears ittlecause todescribe hesebacteria s truepathogens,thasbeen stated hat he extent f colonizationwithinthe tree s limitedby plantdefences Pironeet al.,1988). Further,bacterial wetwood mightalso beassociated with other ree nfectionsBoddy, 1994).6. VirusesLittle is knownofthe interactions etweenvirusesand the living wood of trees (cf. Nienhaus &Castello, 1989). In one study the strength andspecific gravity of wood from poplars systemicallyinfected with poplar mosaic virus was reduced

(Biddle & Tinsley, 1971). As these tree infectionshave been little studied at the physiological level,defence against viruses will not be considered furtherin this review.Despite the range of pathogens attacking woodyxylem tissues, there is an underlying uniformity nthe responses of trees to these infections. Forexample, vessel-blocking responses observed inwood infected with the bacterium Xylella fastidiosa(Hopkins, 1989), are similar to those occurring at themargin of fungal decay lesions (Pearce, 1990).Separate treatmentsof defence against these diversedisease syndromes are not, therefore, required.

III. BARK-THE FIRST LINE OF DEFENCEIn the living tree the xylem is normally sheathed inbark,which comprises periderm, cortex, phloem andcambial tissues. Together, these present a formidablebarrier to the ingress of most potential pathogens.Whilst it is not within the remit of this review topresent a detailed treatment of defence in bark, itwill be considered briefly, s bark defences serve toarrest many potential infections before they reachthe wood. For a fuller consideration of diseaseresistance mechanisms in bark, see Biggs (1992a, b)and Woodward (1992).In a structurally intact living tree, the xylem isentirely encased within the bark, the only directexposure being at sites of veryrecent leaf abscission.Any pathogen normally inhabiting the xylem must,therefore, cross these outer tissues. However, thexylem can be exposed by diverse wounding agents,which locally compromise the integrityof the plantsurface. In nature such wounds occur abundantly.Although the infection biology of many wood-inhabiting pathogens remains obscure, most prob-ably enter through such wounds. Decay commonlydevelops behind major wounds that expose wood;these might occur naturally, e.g. as a result of winddamage, or may be the result of human activity treemanagement operations) (Rayner & Boddy, 1988).Spores of the vascular pathogen Ophiostoma novo-ulmi are introduced into elm xylem throughwoundsmade during maturation feeding by the bark beetlevectors of Dutch elm disease (cf. Ouellette & Rioux,1992).A few fungi are capable of penetrating directlythrough an intact bark surface. Armillaria speciesprovide the best understood examples; rhizomorphsof Armillaria growing from a bulky food base canmount a prolonged attack on the roots of a potentialhost and can ultimately break the surface barriers(Morrison, Williams & Whitney, 1991); some othercord-forming fungibehave similarly Tourvieille deLabrouhe, 1982). Suberin-degrading enzymes maybe important in the penetration of the highlyresistant periderm surface by these fungi (Zimmer-man & Seemuller, 1984; Ofong & Pearce, 1994).


6/32

Antimicrobialdefencesof livingwood 207Few, ifany, fungi appear able to penetrate an intactbark surface directly fromspore inoculum (Dickin-son, 1976), although some can bypass the surfacebarrier by infecting through primary tissues, e.g.Verticilliumdahliae, which enters through youngroots (Schnathorst, 1981).The route of ingress of many wood-infectingfungiremains uncertain, although entrythrough woundsthat expose thexylem,eitherabove or below ground,seems likely. These wounds can result frommechan-ical damage, damage by herbivores, or might be aconsequence of the natural turnover of small rootsand shoots. In one interesting ase, the coniferheart-rot fungus Phaeolus schweinitziihas been shown togain entry into the wood of spruce trees via pre-existing local Armillaria lesions on the roots. In thenecrotic tissues of these lesions, P. schweinitziiwasnot hindered eitherby the hosts' normal defences orby interaction with Armillaria (Barrett, 1970).Attempted penetration by this fungus was swiftlyarrested in healthy bark, even after wounding toremove the surface periderm (Woodward & Pearce,1988b).Intact bark tissues thus appear to protect thexylem (and, perhaps most importantlyforthe overallhealth of the tree, the cambium) against potentialpathogens. A range ofputative defences which mightconfer this protection has been identified. At thesurface the periderm is effective as a first ine ofdefence. The suberized phellem (which can range inthickness from just a few cell layers to a multi-layered rhytidome structure) is highly resistant todegradation, as evidenced by the persistence of barksurfaces over a period of several decades (Hepper,1981). Suberin, a polymer composed primarily oflong-chain aliphatic residues (interesterified ydroxyand epoxy-fatty cids, typicallywith a carbon chain-length of C18 or longer), together with domainscomprising polymeric aromatic units (see Kolat-tukudy (1984) for an interpretationof the structureof 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, accumulationsof various constitutive antimicrobial compoundshave been reported from periderm and rhytidometissues. These include terpenes and polyphenols(Jensen et al., 1963). Despite, or perhaps because of,their ubiquity and effectiveness in protecting theunderlying tissues, there have been few detailedstudies of the constitutive protection provided bysecondary plant surfaces (cf. Campbell, Huang &Payne, 1980; Pearce, 1987; Merrill, 1992).Non-specific periderm restorationresponses are acommon feature of wound repair, antimicrobialdefence and response to insect damage (Mullick,1977) in both woody angiosperms (Biggs, 1992 a, b)and gymnosperms (Woodward, 1992). These func-tion to restorea refractory eriderm beneath wounds

thatbreach the normal surface Biggs, 1992a) andaroundrestrictedesionsofcanker athogensBiggs,Merrill & Davis, 1984; Biggs, 1992b) and un-successfulpenetration ttempts y such pathogensas Armillaria pp. (Pearce, 1989). Similarresponsesmay also occurin the bulky torage issues of non-woody species such as Armoracia usticanaPearce,1989).Alterations o the walls ofpre-existing ells mayalso be important n bark defence: ignificationndsuberization f barkcells were observedprior otheformation of necrophylactic wound) periderms(Biggs, 1985; Biggs & Stobbs, 1986; Woodward&Pearce, 1988b).These might ontribute o protectionduring the differentiationf the more permanentperidermbarrier. Aggressivepathogenshave theability o penetrate r bypassthese inducedmulti-cellular barriers Biggs, Davis & Merrill, 1983;Biggs, 1986), and correlations ave been reportedbetween ates f ccumulation f uberin nwoundedbark tissues and cultivarsusceptibility o disease(Biggs& Miles, 1988).Othermechanisms hatmight contribute o de-fence n bark tissues ncludehigh concentrations fpre-formed antimicrobial compounds (phyto-inhibitins) such as the stilbenes present at highconcentrationsnhealthy prucebark Woodward &Pearce,1988a); these might ct in concertwith cellwall alterations o effect rotection Woodward &Pearce, 1988b). Induced, phytoalexin-like, nti-fungal ompoundshave beenreported rom hebarkof a numberofwoody species, e.g. Populustremu-loidesFlores& Hubbes, 1979,1980) andMorusalba(Shirata, 1978; Takasugi et al., 1978b, 1979).Antifungal ydrolases chitinase nd ,B-1,3-glucan-ases) havebeenextracted rom ark issues nseveralwoody angiospermsWargo, 1975; Shain, 1993) andthe uvenile tissuesofcertain inusspecies Bonello,1991; Nsolomo & Woodward, 1994). Hyper-sensitive-typeecrosishas been reported ssociatedwithresistance o stem and gall rusts in conifers(Kinloch, 1982). In gymnosperms,wounding orinfection an stimulate he production f resins. Acritical assessment of their importancein anti-microbialdefence s rendered ifficultyconflictingreports ftheir ffectivenesscf. Woodward,1992),but a consensus uggests hatresinous xudates canprovide an effectivedefence mechanism (Hillis,1987; Woodward, 1992). However, nstanceswhereresin accumulation oes notappearto contributeodefence are well documented (e.g. Barrows-Broaddus & Dwinell, 1983).IV. DEFENCE IN FUNCTIONAL SAPWOODHealthy sapwood comprises both living and non-living cells. Living parenchyma cells which arecapable of metabolic activity comprise a high pro-portion of the wood in some angiosperms, but,


7/32

208 R. B. Pearcetypically, only a small proportion of the cells ingymnosperm woods. In gymnosperms, parenchymacells are present predominantly as xylem rays: axialparenchyma is generally poorly developed. Resinducts, where present, are lined by living parenchymacells. Conductive and mechanical functions arefulfilled by the tracheids which comprise the re-mainder of the wood in most species. Angiospermxylem is anatomically more complex, and consider-able variation in wood structure exists. Axial paren-chyma is present, as well as ray parenchyma.Commonly, axial parenchyma is closely associatedwith vessels (paratracheal), but apotracheal paren-chyma (distributed without relation to vessels) isoften also present. Non-living cells comprise vessels,tracheids and fibres,which can be further ubdividedand classified (see Esau, 1977; Fahn, 1982; Carl-quist, 1988). By virtue of ts viable cells, the sapwoodof a living tree is capable of active responses toinfection, unlike timber and other forest products,which only provide a passive substrate for microbialgrowth.The anatomy of wood, with an axial alignment ofits principal structural elements (vessels, tracheidsand fibres), and a discontinuous distribution ofliving cells, is reflectedin the host-pathogen inter-actions observed in sapwood. These features imposeconstraints on both the spread of pathogens and theexpression of treeresponses to infection and damage.Since the pioneering work of Robert Hartig in thenineteenth century Hartig, 1894), which has formeda basis of much subsequent tree pathology, fourprincipal models have been advanced to describe thedevelopment of fungal lesions (staining or decay) inthe wood of living trees - the heartrot concept,compartmentalization of decay in trees, the reactionzone model and the environmental restriction offungal colonization. In three of these a closerelationship is established between wounding andinfection. In part this can be accounted for by theprotection normally afforded by the bark which,when intact, denies most potential pathogens accessto the xylem tissues (see III, above). The conse-quences ofwounding on xylem functionmay also beimportant (see IV-1 (d) below).1. Models for thehost-pathogen nteraction n thewood of living trees(a) The heartrot oncept.Until relatively recentlythedominant model of decay in standing trees (seeBoyce, 1961; Peace, 1962), the heartrot concept isnow recognized as an over-simplification.Decay wasperceived as a predominantly saprotrophic process,confined within the non-living heartwood. Woundsor dead organs exposing (directly or indirectly) theheartwood allowed the entry of fungal propagules.No killing of living tissue was envisaged and thefungus could only leave the living tree (normally by

Wall1 (b)

.--Wall 2 (c)

Wall4(d) WaIl 3

Figure 1. Compartmentalization f decay in trees(CODIT). Location of heproposed ompartmentalizationwalls 1-4 in relation o a wound-associateddecay lesionin the stem of a tree. (a) Longitudinalsection. (b)-(d)Transverse ections t threepositions long the esion. n(b)wall 1appears s a circumferentialeaction one aroundthe esion.producing fruiting bodies (usually basidiomes))through interfaces between dead wood and theexternal environment. These could include the initialinfection court or be created following mechanicalfailure of stems or branches weakened by internaldecay.

Certain important decay fungi (e.g. Laetiporussulphureus, Phaeolus schweinitzii and Sparassis crispa)commonly behave according to this model (Bur-dekin, 1979; Greig, 1981). Whilst heartrot can causesevere economic loss in old and natural forests, thistype of infection cannot account for the interactionsbetween decay fungi and living tissues observed inmany trees.(b) Compartmentalization ofdecay in trees (CODIT).The CODIT model was proposed and developed byShigo and co-workers (Shigo & Marx, 1977; Shigo,1979; 1984) to describe and account for the patternsof discolouration and decay developing in livingwood behind tree wounds. According to the CODITmodel, lesions in living wood are bounded and, byinference, restricted by barriers laid down in thewood. These have been termed walls 1-4 (Fig. 1),and enclose any lesion or incipient lesion within adefined compartment. They were envisaged as


8/32

Antimicrobialdefences f living wood 209essentially static barrierspreventingthe furtherspread of nfection. uch putatively efensive onesof altered xylem had been reported many yearspreviously e.g. Swarbrick, 926; Hepting & Blais-dell, 1936), but had been largelyneglecteduntiltheformulation f theCODIT model.Walls 1-3 are formedwithinwood extant t thetime of wounding.Wall 1 is formedtransverselyacross hexylem, nd acts as a boundary r barrier othe axial spread of infection. t was envisaged asresultingfrom vessel-pluggingresponses in theconductive issues nd regarded s theweakest fthecompartmentalization alls. Wall 2, resisting heradial preadof a pathogen nwards rom hewound,has been attributedmainly o anatomical eatures fthe wood, and wall 3, resisting he ateral pread ofinfection, as been attributed o the activities frayparenchyma ells.Walls 2 and 3 wereperceivedasbeing trongerhanwall 1, butultimately apableofbeing defeated y a pathogen, ollowingwhichtheycould be reinstated o contain a lesionof increasedvolume Shortle,1979; Blanchette, 992).The wall4 barrier iffers romwalls 1-3 inthat tis formed nthe plane of thecambium t the timeofwoundingas a response to damage (and, perhaps,infectionPearce,1987)). It is the strongest nd mostdurable of the compartmentalization alls, com-prisingcells laid down de novo, which can formstructurallyomogeneous arrier, ather hanbeingformed fterdifferentiationn a tissue containingdiversecell types as occurs for walls 1-3 (Shigo,1984; Blanchette, 992). Functionally,t s the mostimportantarrier, rotecting heyoungestwoodandthe cambial tissues,both ofwhichare vital to thecontinuing rowth nd survival fthetree.As originallyformulated, ittle evidence waspresented for the physiologicaland biochemicalmechanismsbywhichcompartmentalizationn thetreemightbe effectedShigo & Marx, 1977; Shigo,1979). More recently, natomicalchanges and cellwall alterationssuberization) avebeen dentifiednthe wall 4 barrier zone in a number of species(Tippett & Shigo, 1980, 1981b; Pearce & Ruther-ford, 1981; Pearce & Woodward, 1986; Pearce,1990). These are discussed further elow (IV 2(h)(see Figs. 5, 6)). Inhibitory ompounds might lsoaccumulate n these tissues (Pearce & Woodward,1986). Compartmentalizationwalls 1-3 may beequated withreaction ones (Shigo, 1984), see IV1 c) below,wherethepossibledefencemechanismsinvolvedwill be considered.(c) Dynamic decaydevelopmentnd reaction ones.Arising romtudies f he nvasive athogenHetero-basidion nnosum olonizingthe stemsof pine andspruce trees from nitial root infections,Shamn1967,1971, 1979) developed the concept of the reactionzone as a region of active host response at a dynamicinterface between living sapwood and wood colon-

IntermediateecayReaction one Healthy oodAdvanceddecay Transitionone

(a)

(b)

(c)Figure 2. The reaction zone model as proposed by Shain(1967). The reaction zone retreatsahead of a continuouslyadvancing decay front, all sapwood becoming first atransitionzone, then a reaction zone before becoming partof the spreading lesion.ized by the pathogen (Fig. 2). As originally con-ceived, reaction zones retreated ahead of a con-tinuously advancing lesion margin, sapwood passingthrough a reaction zone stage en route to becomingdecayed. Phytoalexin-like antifungal compoundsaccumulating in these regions were considered toretard the advance of the colonizing fungus. Dis-tinctive alterations at both tissue and cellular levels,regarded as representing reaction zone formation,have been seen at the margins of decay lesions inmany tree species (e.g. Pearce & Woodward, 1986;Pearce 1990) (Figs 3, 4).Since reaction zones were initially described(Shain, 1967, 1971), evidence has accumulated fromseveral differentngiosperm pathosystemsto suggestthat these reaction zones are not the dynamicstructures initially envisaged, but normally formstatic boundaries to infection (Pearce, 1987, 1991;Boddy, 1992). When reaction zone boundaries fail, avolume of wood is colonized with little or noexpression of characteristic reaction zone responses,until ultimately a new reaction-zone boundary isestablished (Pearce, 1991). This corresponds closelywith the dynamic behaviour of CODIT walls 1-3


9/32

210 R. B. Pearce

3 w 4

5wg ~~~~~~~~6

7 eFigures 3-8. For legend see opposite.


10/32

Antimicrobialefences f ivingwood 211which are not always entirely ffective, esultingnalternatingphases of 'breakout' and new com-partment formation (Shortle & Smith, 1990;Blanchette, 992). The CODIT model does not n-corporate reaction one boundary formed n pre-existingwood) at theouter ircumferential argin flesions, he distinctivewall 4 barrier ccupying hisposition.However, n transverseections hroughtree, wall 1 boundaryforming conical cap' to adecay column would appear as a circumferentialreaction one (cf. Fig. 1). This equivalence allowsthe unifi-cationf the COD IT and reaction zonemodels fordecay restrictionn livingtrees. Sinceconstraints f wood anatomy dictate that fungalcolonization takes place most readilyand rapidlyalongthe axis of a tree Rayner& Boddy, 1988),theaxial containment rovided by these reaction onesis likely to be at least as significant s the radialcomponentof any barrier function.The prepon-derant use of sections in the transverseplane instudies fdecay ntreeshas, perhaps, ended o maskthisfact.Reaction ones arealso formednsapwoodatthebase ofdying uppressedbranches,where heymay prevent he ngress fpotentialpathogens ntothe remainder f the tree von Aufsess,1975; Shigoetal., 1979; Green,Shortle & Shigo, 1981). Again,theprotection onferreds primarilygainst he xialspreadof nfection.In the original eaction one model Shain, 1967,1971, 1979) a transition one of morphologicallynormal but drier tissues, located between thenecrotic eaction one cellsand thenormal apwood,was described. This has been suggested s the sitewhere biosynthesis f characteristic eaction zone

components ccurs. Drier transitionones are welldocumentedn gymnosperm oods Yamada, 1992).Accumulation f water,rather han drying, ppearsmore typical of reaction zones in angiosperms(Pearce et al., 1994a; Pearce, unpublished),but hasalso been reported rom ymnospermsShain, 1971).Since clear evidence for chemical alterations nadjacent sapwood of normal appearance has beenobtained Grime & Pearce, 1995), this altered issuemaybe consideredequivalentto a transition one.IncreasedNAD-linked respiratorynzyme activityin parenchyma ells distal to incipientdiscolouredwood developingbehindwounds n Acer saccharum(Sharon, 1974) might also reflect ransition oneactivity.With the exceptionof the compartmentalizationwall 4 barrier, most host-pathogen nterfaces nliving wood can be related to the reaction zonemodel. Induced cell wall alterations suberization),antimicrobialompounds, olyphenolic eposits ndcell necrosis have been associated with reactionzones, as discussed below (IV 2) (see Figs 3, 4). Inaddition, ariousother hemical hanges ess readilyrelated o defencehave been described, or xampleelevated pH, increased carbonate concentration,increased metal ion concentrationsShain, 1971;Shevenell& Shortle, 986; Smith& Houston, 1994).The significancef these n relation o reaction onefunctionemains ncertain, lthoughnvolvementndefence an be postulated see IV 2(i)).To date,few tudies have addressed events t thehost-pathogennterfacen wood during phases ofactive nvasion nd lesionexpansion see VI, below).In Acer pecies,however, wocharacteristiclements

Figure 3. Transverse section through he circumferential argin of naturally ccurringdecay caused byUstulina eusta n sycamoreAcerpseudoplatanus). broad, stronglyolouredreaction one (RZ) is present tthe nterface etweenhealthy HW) and decayed DW) wood. On first xposurethis reaction one appearedgoldenyellow,but rapidly xidized to darkgreen, s representedn thisphotograph.Although eaction onesformed t the margins f U. deusta esionsare often road, as seen here, reaction ones are commonlymuchnarrower hanthe example illustrated. cale bar = 1 cm. Figure 4. Radial longitudinal ectionthroughnaturally ccurring eaction one in sycamoreAcerpseudoplatanus). he decayfungus emains ndetermined.In this reaction one (RZ), located transverselycrossa stem thedistal part of which was dead (DW), greenpolyphenolicdepositsoccluded vessels and the lumina of fibres nd othercell types. Scale bar= 100,tm.Figure 5. Radial longitudinal ectionof a compartmentalizationall 4 barrier n oak (Quercusrobur).Thebarrier one (BZ) comprises circumferentialheetof axial parenchyma, . 12 cells wide, ocated between hedecayed wood colonized by Stereumgausapatum DW), which had been formed before wounding,andthe healthy apwood (HW) formed fterwounding.Scale bar= 200 ,tm.Figure 6. Transverse ectionof acompartmentalizationall 4 barriern poplar Populus sp.), stainedwith Sudan IV following xtractionwithchlorine ioxide and acetone Pearce,1990). The suberizedwall4 parenchymaBZ) is stained,whereashealthywood (HW) and decayedwood (DW) remain nstained. cale bar= 200/tm. igures 7, 8. Radial longitudinalsections f tems f 3-year-oldycamore Acerpseudoplatanus)rees, hallenged y noculation fnotchwoundscut into the stem with (a) sterile 3 % malt agar; (b) Ustulina deusta; or (c) Chondrostereum urpureum.The fungiwereapplied as mycelial ultures, . 3-wk-old, n 3% malt agar. Inoculated wounds weresealed withplasticfilm'Seal-On' film, amlab Ltd, Cambridge,UK). To visualizethe fluorescent hytoalexin-likeompoundsinduced n the xylemfollowing hallenge he stem engthswere lluminatedwith ong wave ultravioletight(A= 365nm)andphotographedhrough WrattenNo. 2A filter. igure7: 24h after hallenge.Figure 8: 48 hafter hallenge.Fluorescent ompoundswere nducedaroundwounds within 4 h. In theexperimenthown,accumulation tthis imewas lessapparent rounduninoculated nd Chondrostereum-challengedoundsthanaround Ustulina-challenged ounds: however by 48 h accumulationwas similar n all treatments. calebar = 10 mm.


11/32

212 R. B. Pearceof hereaction one response increased issuewatercontent Pearce et al., 1994 ; Pearce, unpublished)and depositionof coloured, nsoluble,polyphenolicmaterials Pearce & Woodward, 1986)- were notseen where the infection front appeared to beadvancing, although soluble phenolic phytoalexin-like compoundswere present Pearce & Woodward,1986; Pearce, unpublished). Such regions equatemore closely with the 'reaction zone' as originallyproposedby Shain 1967, 1971, 1979), than he taticlesion boundaries o which the termhas commonlybeen applied (cf. Shigo, 1984; Pearce, 1990, 1991;Blanchette, 1992). The term 'column boundarylayer' has been used to describe he altered issue atstatic lesion margins (Shortle & Smith, 1990).Although this term has not yet achieved generalacceptance, t conveys effectivelyhe three-dimen-sional context in which these putative defencesoperate ndcould be used todiscriminate nequivo-cally between static and dynamic host-pathogeninterfacesn livingwood.(d) Environmentalestrictionffungal colonization.As an alternative o the CODIT and reaction onemodels in which growthof wood decay fungi srestricted y active host defences,Boddy & Rayner(1983) proposed, and subsequently developed(Rayner, 1986; Rayner & Boddy, 1988; Boddy,1992),a model thatdoesnotrequire heoperation factivehost defences. hey suggested hathighwatercontent nd concomitantow02tension n functionalsapwood is itself sufficient o preclude fungaldevelopment. upportfor hishypothesis as comefrom the patterns of functionally ompromisedxylem and fungalcolonization ssociated withtreewounds (Boddy & Rayner, 1983; Rayner, 1986;Rayner& Boddy, 1988; Boddy, 1992) and from hedevelopmentof wood-inhabiting ungi in dryingexcised woody tissues (Chapela & Boddy, 1988b;Chapela, 1989).According o thismodel, he imits fdecaywouldoccur at the unctionbetween microenvironmentalconditions onducive ofungal olonizationnwoodaerated s a result fdrying nd dysfunction,.g. asa consequenceofwounding, nd conditions nimicalto fungal growth,where the xylemremains func-tional andwater-filled.he cellwall alterationsndpolyphenolic deposits characteristic of columnboundary ayer-type eaction ones (CBL reactionzones) would then have a wound repair function,their ftenhydrophobic ature see IV 2(h), below)serving o limit hespreadofdrying nd cavitationand to maintain the hydraulic integrity f theadjacentfunctional ylem, ather hanproviding ninhibitoryarrier erse.These models have been advanced in relation tothe colonization of living trees by wood-decay fungi.Similar processes are presumed to operate in respectof other classes of xylem pathogen; for example, the

formation of compartmentalization wall 4 barriershas been reported in several angiosperm trees as aresponse to vascular wilt pathogens (Shigo &Tippett, 1981; Tippett & Shigo, 1981 a).- However,it seems likely that none of these models is alonesufficient to describe adequately the interactionsbetween living sapwood and potentially or actuallypathogenic wood-inhabiting micro-organisms. In-stead, elements of each might contribute to regu-lating the host-pathogen interaction, the role ofindividual components perhaps varying from patho-system to pathosystem. Models for defence thatinvoke microenvironmental restriction and activehost defence are not in any way mutually incom-patible. This will be discussed below, in relation tothe putative antimicrobial defences that have beenidentified in the xylem of woody plants.2. Putative defencemechanismsWithin the context ofthe models above, a number ofanatomical, physiological and biochemical featuresof living sapwood have been identified that mightcontribute to its ability to resist microbial attack. Ingeneral, these parallel the defencemechanisms whichare better known from herbaceous plants (seeHorsfall & Cowling, 1980; Callow, 1983; Pegg &Ayres, 1987). However, for reasons of scale (bothphysical size and the extended duration of host-pathogen interactions in woody tissues), clearlydefinedmulticellular zones of altered host tissues arecommonly associated with protection, and changesat a tissue level appear particularly important.Although a number of putative defence mech-anisms in living sapwood have been identified (seebelow), evidence for their precise function in pro-tection is mostly indirect, and an unequivocal rolehas rarelybeen proven. As in bark, protection againstmicrobial pathogens, insects and other pests, andwound repair processes are likely to share manycommon features, particularly since an importantfunction of wound repair might be to excludepotential pathogens. In consequence it may beartificial to assign specific roles to these broadlydefensive processes.(a) Environmental constraints on pathogen growth.Unsuitable microenvironmental conditions forfungal growthwithinfunctional iving sapwood havebeen proposed as a major factorprotectingthis tissueagainst infectionand decay (Boddy & Rayner, 1983;Rayner, 1986; Rayner & Boddy, 1988; Boddy, 1992).High water contents are known to inhibitgrowthanddecomposition bywood decay fungi,owing primarilyto lack of 02 and build-up ofCO2 in thewaterloggedsubstrata. Also, if the water is mobile, extracellularenzymes important in decomposition may be re-moved from the vicinity of the secreting hyphae(Boddy, 1986, 1992).


12/32

Antimicrobialefences f ivingwood 213In intact functional sapwood, 02 may be presenteither in solution in the xylem water or in the gasphase occupying the tissues of dead cells (fibres,etc.). Elevated CO2 and reduced 02 concentrations

are commonly reported from iving wood (Rayner &Boddy, 1988): in living Acacia stems the gascomposition was almost 10000 CO2 (Carrodus &Triffett,1975). Oxygen concentration can also below within decay lesions, with 0 8 00 02 reported ingases extracted from rotten heartwood of Acersaccharum (Thacker & Good, 1952). It should benoted, however, that many decay fungi can grow ina low 02/high CO2 environment (Jensen, 1967;Hintikka & Korhonen, 1970; Highley et al., 1983).Also, the highest 02 concentrations in a tree havebeen found in the youngest sapwood (Jensen, 1969),which is commonly the most resistant to fungalattack (Johansson & Stenlid, 1985; Rayner 1986).Oxygen concentrations in the youngest sapwoodexhibit seasonal variation, being lowest at timeswhen thecambium is active. This has been attributedto 02 consumption by the metabolism of thecambium (Eklund, 1993). Low rates ofgas exchangein these tissues would ensure that 02 consumed by apathogen attemptingto grow in functional sapwoodwas not quickly replaced, resultingin a swiftdeclinein its concentration to inhibitory evels.Damage disrupting the integrity f vascular tissuesand resulting in cavitation would permit the readyingress of air and allow increased gas exchange.Fungal growth would then be possible in thesecompromised tissues. Patterns of discoloration anddecay observed behind wounds in living treesexhibiting 'compartmentalization' can also be ex-plained in these microenvironmental terms (Boddy& Rayner, 1983; Rayner 1986; Rayner & Boddy,1988; Boddy, 1992). In such an event, CBL reactionzones and the compartmentalization wall 4 barrierwould have a repair function, preventing an increasein the volume of compromised wood by acting as animpermeable surface seal.

Further evidence for the importance of reducedaeration in the restriction of fungal growth isprovided by the observation that decay developmentbehind wounds is oftenarrested ifthe wound face isoccluded by healing callus growth. The mainpathway for gas exchange, through the damagedwood, would be lost on wound closure (Jensen,1967; Highley et al., 1983; Rayner & Boddy, 1988).For invasive pathogenesis, wood colonizationwould be preceded by the formation of a dry zoneahead of the infection front,which would permitbetter 02 ingress within these tissues. Such zoneshave been reported ahead ofHeterobasidion annosumlesions in conifers (Coutts, 1976) and adjacent toCryptostroma orticale esions in Acerpseudoplatanus(Pearce et al., 1994a).Inimical environmental conditions in wood couldbe enhanced as an active defence. In Abies concolor,

decay-resistantwetwood might form as a hostresponse,possibly o parenchyma elldeath, s wellas duringnormal heartwoodformation. ow avail-ability of 02 in wetwood might protect t againstdecay, and thus act as an active defencemechanism(Worrall & Parmeter, 1982). In addition, NMRmethods have demonstrated hat reaction zonesdeveloping n Acer pseudoplatanus . stems chal-lenged with a weakly aggressive decay fungus(Ustulina deusta) accumulate water (measured asimageable protons) to 2 5-3 times the level inadjacenthealthywood (Pearceet al., 1994 ). Similarresultshave been obtainedfromNMR studiesusingjuvenile trees wounded and challenged with U.deutsa (Pearce et al., 1994b). Gravimetricdeter-minations f the watercontentof excised reactionzone tissues ndicated an increase n total water of60 0 comparedwithhealthywood. Similarchangeswere observed in young trees inoculated withGanoderma dspersum Pearce, unpublished).Thiswater,whichwas presumably ccommodated ythedisplacement f any gases normally resent,mightenhance the protection mparted by the normalwater content of the sapwood. An alternative,solvent,function an, however,be suggested seeVIII).(b) Nutritionalfactors. Although wood cell wallmaterialprovides hemainsubstrate or hegrowthof decay fungi, ome of these, at least,have com-plex micronutrient equirements.A defined basalmedium forPhaeolus schweinitzii ontained manyorganic supplements Robbins & Hervey, 1969).The significancef such factorsn determininghesusceptibility f wood to fungal attack remainsunclear, althoughthe observed stimulation f thegrowth f P. schweinitziin sprucewood colonizedby Armillaria sp. (Barrett, 1970) could have anutritional asis.The availability f solublenutrientsn xylem apmight e a factorndeterminingates fcolonizationof the sapwood of fruittrees by Chondrostereumpurpureum Beever, 1970; Bielenin & Malewski,1982), althoughno correlationwas found betweenthesugarcontent fsap extracted rom alixfragilisand susceptibility o this pathogen (Stanislawek,Long & Davis, 1987).A reduction n starchreserves stored in xylemparenchyma f stressedtrees has been associatedwith ncreased usceptibilityo pathogensncludingArmillaria pp. (Wargo, 1972). Although oncomi-tant changes in the broader nutritional tatus ofstressed issueoccur lso (Wargo,1972), depletion fstarchmight educethe tree'scapacity ordefensiveresponses which would normallydraw upon thesereserves (cf. ~McLaughlin & Shriner, 1980).(c) Anatomical features of normalwuood. he normalstructureand cell wall composition of wood imposes


13/32


14/32

Antimicrobial defences f living wood 215reaction zones and heartwood (Shain & Hillis, 1971)and norlignans normally present in very smallamounts in the sapwood of Cryptomeria aponicawere produced during heartwood formation and inreaction zones (Yamada, Tamura & Mineo, 1988).The stilbenes pinosylvin and pinosylvin mono-methyl ether, constitutively present in the sapwoodof Pinus spp. (Dumas et al., 1983), accumulated inreaction zones (Shain, 1967) and in heartwood (seeHart, 1981). These compounds exhibit many fea-tures in common with induced antimicrobial com-pounds (phytoalexins) and will be considered furtherin that context (see IV(h), below). Additionally, thegas phase in the wood of living conifers is probablysaturated with terpene vapours, which may beinhibitorysee IV (f), below).

In general, the role of preformed antimicrobialcompounds in the defence of iving sapwood remainslittle studied, and, in consequence, poorly under-stood. It has been suggested that these phyto-anticipins might be important in determining thesuccessions of micro-organisms colonizing woundson trees. Pioneer colonizers can grow at concen-trations of phenolic compounds such as gallic acidthat are inhibitory to some decay fungi, and canmodify these compounds, which might permit thesubsequent development of decay (Tattar, Shortle &Rich, 1971; Shortle & Cowling, 1978). However,some caution is required in the interpretation ofmuch of the literature on both constitutive andinduced antimicrobial compounds in wood. Ag-gressive extraction has often been employed (e.g.Tattar et al. 1971; Tattar & Rich, 1973; Miller,Sutcliffe & Thauvette, 1990), or wood might havebeen dried and powdered before extraction (e.g.Shain, 1967): such methods might decompose ormodify unstable compounds (cf. Wong & Preece,1978 b).(f) Resin. Many gymnosperm trees have a well-developed resin canal system in both the bark andxylem tissues. The resin principally comprisesterpenoid compounds (non-volatile resin acids andvolatile terpenes which act as a solvent for the non-volatile components and may amount to up to 50 0of the total material); phenolic compounds (e.g.lignans) and fatty cids and their esters can also bepresent. Resin-like materials are also produced bysome woody angiosperms: these are chemically morediverse, including carbohydrate gums (in somePrunus spp., Acacia senegal) and kino, a polyphenolicsubstance principally comprising polymerized pro-anthocyanidins, in some Eucalyptus species. Latex,e.g. from Hevea brasiliensis and Palaquium gutta,predominantly comprises a hydrocarbon, cis-1,4-polyisoprene, stabilized by a thin absorbed film ofprotein and phospholipid (Hillis, 1987). Althoughthese materials are generally considered to beinvolved in protection against pests, diseases and

damage (for example the ability of coniferstoproduceresinhas been correlatedwith resistance oHeterobasidionnnosumGibbs, 1968) the precisenatureof their nvolvements oftenuncertain.Gymnosperm esins reby far hemost ntensivelystudied of thesetree exudates.These are producedby the epithelialcells lining resin ducts, and canflownthe ductsystem o sitesofdamage,where hevolatile erpenes vaporate o eave a plug,which anhardenfurther o form paint-like arrier o waterand the entry fexternal gencies Hillis, 1987). Ithas been suggested that this ability to effectmechanicalbarrier s important n the protectionafforded y resin,as the principaleffect f tissueresinosis ppearedto be the mechanical nhibition fhyphalgrowthby dried resin Prior, 1976). Resin

impregnation f xylemtissues could also act as ahydrophobic arrier, elping o maintain unctionnadjacentconductive apwood. Latex coagulationhasbeen noted n the bark of H. brasiliensisttacked yroot-rot ungi, ut has been considered secondaryevent n disease (Nicole, Geiger& Nandris, 1986).The significancef resin s an inhibitoryhemicaldefence n coniferss less clear cf. Yamada, 1992).The antimicrobial activities of volatile terpenes(Shrimpton& Whitney,1968; Cobb et al., 1968;Flodin, 1979; Schuck, 1982 ) and resin acids(Henricks, Ekman & von Weissenberg, 1980;Franich, Gadgil & Shain, 1983) have been demon-strated in a number of studies. However, littleinhibitionof growth,or even stimulation f treepathogens y these ompounds,hasbeen reportednother nvestigations Prior, 1976; Flodin & Fries,1978; Franichetal., 1982; Schuck, 1982a). Corre-lations have been reported between monoterpenecompositionndresistanceo Heterobasidionnnosumand certain therpathogensForrest, 982; Schuck,1982 ), buttherelationshipsre unclearand some-times ontradictorynd some authors eport indingno such correlations Ladejtschikova& Pasternak,1982).

Resin formed after trauma can differn com-position from that present in the healthy tree(Schuck, 1982a, b; Paine et al., 1987). A betterunderstandingof the role of resins and theirindividual omponents n resistance s requiredbe-fore he ignificancef hisdifferenceanbeproperlyassessed.The formation ftraumatic esin ducts nthe wood laid down mmediatelyfterwounding s acommon response to damage and infectioninconiferousrees, ndzonesof alteredwood anatomycontainingmoreparenchyma ells and resinductsthan normal were associated with wounding orlesions ofArmillaria p. and Heterobasidionnnosumin roots of severalconifersTippett& Shigo, 1980,1981 b; Blanch~ette,1982; Tippett, Bogle & Shigo,1982). These corresponded in location to wall 4barriers of the COD IT model (see IV 1 b)).Comparable formationof traumatic gum canals has


15/32

216 R. B. Pearcebeen reportedfollowingwounding n Liquidambarstyraciflua Moore, 1978). Kino veins form inEucalyptuswood afterwounding Tippett, 1986) orafterprolonged fungalactivity n the bark tissues(Tippettetal., 1983) and kino may exude like resinafterwounding White & Kile, 1993).Like other tree defences, he elicitation f resinaccumulationhas been little studied. Chitosan, anelicitor of defenceresponses n herbaceous plants(e.g. Barber & Ride, 1988), has been reported oelicit both resin accumulation nd hypersensitive-type necrosis see IV 2(j)) in pines. Similar resultshave been obtained with a proteinase inhibitor-inducing actor erivedfrom lant ell walls Miller,Berryman& Ryan, 1986; Croteau et al., 1987;Lieutier & Berryman, 988a, b). The importance fsuch elicitors n the host-pathogennteractions ftrees remains o be determined.(g) Lytic enzymes. yticenzymes, hitinases nd ,-glucanases are an important component of thepathogenesis-relatedroteins nduced n plantsfol-lowing microbial challenge. They have been re-garded as having a potentiallydefensivefunction(Boller, 1987; Scholtens-Toma,Joosten& De Wit,1991). Chitinase enzymes (from a herbaceousspecies)were ble to nhibit rowth fHeterobasidionannosumSusi et al., 1995). Similarhydrolases avebeen reported rom he bark tissues of angiospermtrees. Enzymes fromQuercusand Acer spp. werecapable of lysing hyphae of Armillaria (Wargo,1975), and chitinases and ,-1,3-glucanases wereinduced in the bark of Castanea species afterchallenge with Cryphonectria arasitica (Shain,1993). It has also been reported hattheseenzymesaccumulatesystemicallyn poplar leaves followingwounding Parsons,Bradshaw& Gordon, 1989) andin mycorrhizal nd pathogen-challenged oots ofEucalyptus Albrecht,Laurent & Lapeyrie, 1994;Albrechtet al., 1994). Hydrolaseshave also beenreported rommycorrhizalSauter & Hager, 1989)and pathogen-challengedNsolomo & Woodward,1994) conifer roots, and systemically n root-challenged eedlings Bonello, 1991). There do notappearto be anyreportspecificallyssociating heseplanthydrolaseswithxylem issuesofwoody plants.However, the association of such enzymes withresponses o a vascularpathogen Verticilliumlbo-atrum) n tomato (Young & Pegg, 1981) and thedamage ofungal ell walls nthexylem f ransgeniccanolaplants xpressing chitinase ene Benhamouetal., 1993) providesevidence,albeit ndirect, hatlytic nzymesmight perate nxylem.(h) Cell wall alterations. nduced defences mayconveniently be divided into cell wall alterations andinduced chemical defences, although this separationmay be rather artificial, both biochemically andfunctionally.

In the primary issues of both herbaceous andwoody plants, cell wall appositions at sites ofattempted penetration are commonly observed.These may ncorporate number fcomponents otnormally resent nthe unmodifiedwalls, ncludinglignin and other phenoliccompounds, callose, andsilicon, together with normal cell-wall materials(Aist, 1976, 1983). Such papillae have beenfound nboth angiosperm Edwards & Ayres, 1981) andgymnosperm Bonello et al., 1991) trees. Ligni-fication can also occur in the bark as an earlycomponent f the periderm estorationesponse seeIII, above) (Biggs, 1992b; Woodward, 1992). In-duced lignificationmight not be expected to beeffectiven defence n the highly ignified ylem.However, herewas an increase f 25-30 o in ignin-like material n thewood of Hevea brasiliensisap-roots close to the infectionfront of RigidoporuslignosusGeigeret al., 1986).There appearsto be noinformation n the monomer compositionof thislignin (Nicole, Geiger & Nandris, 1992), and itssignificancenthehost-pathogennteractionemainsunclear,particularly ince R. lignosus an degradelignin Geiger etal., 1986).The main cell wall polymer associated withhost-pathogen interactions n xylem tissues issuberin, characteristicomponent fthe secondaryplant surface,where its resistance to microbialdegradation and hydrophobicpropertiesare im-portant n maintaining he integrityf this plant-environmentnterfacesee III, above). It is not anormal component fhealthy apwood,although tis present n someheartwoods Pearce & Holloway,1984), and is associatedwith he resinduct systemnPinus spp. (Biggs, 1987; Pearce, 1990).Xylem suberizationresponsesat the marginsofdecaylesions were first ecognized n thecompart-mentalization all 4 barrier ormednthevicinity fpruning wounds in oak (Quercus robur) wheresapwood was attacked by Stereumgausapatum(Pearce & Rutherford, 981; Pearce & Holloway,1984). The wall 4 barrierwas anatomically istinctfromnormalwood and compriseda sheetof axialparenchymaells up to 30 cell layers hick Fig. 5).Where adjacent to fungally olonized wood, thistraumatic arenchymawas suberizedto a depthof3-20 cell ayers.The suberized ells wereresistant odegradation by S. gausapatum,but were readilydigested f he uberinwasremoved hemically,husdemonstratinghat wood cell walls were protectedagainstbreakdownby decay fungiby suberization(Pearce& Rutherford, 981). Followingexperimen-tal wounding,onlycells overlaying ungally olon-ized sapwoodwere suberized Pearce,1987; Pearce,unpublished). This suggestedthat,whilst the denovo formation of the sheet of traumatic axialparenchyma was wound-induced, suberization ofthese cells was a response to fungal infection.However, the possibility that suberization was


16/32


17/32

218 R. B. PearceTable 1. Development of a compartmentalizationwzall4 barrier n oak Quercus robur*Weeksafter Suberization f wall 4 Starch content f wall4challenge Anatomicalfeatures parenchyma parenchyma Fungal colonization2 No new tissue of Few hyphaeevident ndistinctive natomy woodevidentbelowcambium4 Up to 7 cell layersof Traumaticparenchyma Starchabsent Extensivefungalaxial parenchyma notsuberized colonization nformed y cambium immediate icinity finoculation nly8 Up to 25 cell layersof Traumaticparenchyma Starch evelshigh n Occasional hyphae naxial parenchyma not suberized traumatic arenchyma wood extant t theformed y cambium timeofwounding21 Traumatic axial Inner 3-7 cell layersof Starch absent from Hyphae presentmainlyparenchyma p to c. 20 traumatic arenchyma suberized cells, high n in discolouredwoodcell layers hick suberizedover other raumaticoverlaidby wood discolouredwood parenchyma ellsreverting o morenormal natomy32 Traumatic axial Inner cell layersof Starch absentfrom Hyphae present nparenchyma p to c. 20 traumatic arenchyma suberizedcellshigh n discolouredwoodcell layers hick verlaid suberized to a depthof other raumaticbywood reverting o c. 10 layersover parenchyma ellsmorenormal natomy discolouredwood62 Traumatic axial Inner cell layersof Starch absent from Hyphae present nparenchyma p to c. 20 traumatic arenchyma suberized cells, high n discolouredwoodcell layers hick, suberized to a depthof other raumaticoverlaidbywood 6-14 cell layers, ver parenchyma ellsreverting o more discolouredwoodnormal natomy* Quercus obur rees, . 50yr old, were wounded on thetrunk t a height f c. 1-3m byremoving patchof barkc. 85x 100 mm, o expose thewood. A 15 mmdowel, noculated months reviouslywith tereum ausapatum Fr.) Fr.was driven nto hole bored na lowercorner f the wound to inoculate.Trees werechallenged nJune, nd felled nddissected t ntervals ollowingnoculation. ections 20 am) werecutfrom locks aken . 20 mm below the noculationsite,usinga slidingmicrotome. uberin was detected singSudan IV (Pearce, 1990); starchwas detectedwith he KIreagent Jensen, 962) and fungalhyphaewere visualizedusing the rhodamine : methyl reenmethod Pearce, 1984).

at wound or lesion margins. This could resultdirectly rom heintrinsic egradation esistance fthe suberized walls, or could be mediated throughthe ability f suberized issuesto prevent he dryingand aeration fadjacentxylem cf. Rayner& Boddy,1988; Boddy, 1992). Until dynamic studies ofcolonization nd barrierformation ave been con-ducted, hepreciserole of these cell wall alterationsin defencewill remainuncertain cf.VI, below).Thin-walled suberized cells have been reportedadjacent to the traumatic xial parenchyma f thecompartmentalizationwall 4 in oak (Pearce &Rutherford, 981). Similar tissues have been re-ported n otherspecies (McGinnes, Chang & Wu,1971; Tippett & Shigo, 1980), althoughuberizationin these specieswas not demonstrated. his tissue,which appears to resultfrom he local deathofthevascular ambium s a consequenceofwounding ndsubsequentcambial restorationwithin hephloem,was termed the cambial-phloic zone by Mullick(1977). It creates plane of physicalweakness n thewood (McGinnes et al., 1971), but any defensivesignificanceemainsunknown.

Little is knownof the ability fwood-inhabitingfungito degrade suberin. Armillaria species canslowly degrade this recalcitrantpolymer (Swift,1965; Zimmermann& Seemuller, 1984), but thepersistence f suberin n wood extensively ecayedby Ganoderma dspersum Pearce, 1989) indicatesthat this fungusdid notreadilydegrade t.(i) Induced ntimicrobialompoundsphytoalexins).Increasedlevels of phenoliccompounds have beenreportedfrom esion margins n living sapwood inmanytreespecies (e.g. Shain, 1967, 1971; Popoff,Theander & Johansson,1975; Wong & Preece,1978b; Pearce, 1987; Yamada, 1992). Often thecompounds induced at sites of host-pathogenn-teraction re absentfrom, r present t onlyvery owlevels in healthy sapwood (e.g. Aesculus hippo-castanum, agussylvatica Pearce,1991),Pinus taeda(Shain 1967)). These chemical changes often ac-companycell wall alterations nd the depositionofinsolublepolymericmaterials see IV 2(h), IV 2(j))in CBL reactionzones and compartmentalization


18/32

Antimicrobialefences f ivingwood 219Table 2. Phytoalexin-likeompoundsrom he apwood f trees: representativexamples f themain chemicalclassesClass Tree Pathogen Compound(s) ReferencePhenols Piceaabies Heterobasidion 4-methylcatechol Popoff t l., 1975annosumStilbenes Pinus pp. H. annosum PinosylvinPinosylvin Shain, 967monomethyltherMorus lba Fusariumolani . sp. Oxyresveratrolmori 4-prenyl Takasugi t al.,oxyresveratrol 1978Lignans Picea abies H. annosum Hydroxymatairesinolhain, 971;Shain& Hillis, 971;Popofft l., 1975Liriodendron Not determined Syringaresinol Chen tal., 1976tulipiferaPhenylpropanoids Prunus omestica Chondrostereum Scopoletin Hillis& Swain, 959

purpureumAcer seudoplatanus C. purpureum Fraxin Pearce, npublishedAcer accharum Not determined 'Fraxetin-like Manville Levitin,coumarins' 1974Platanus cerifolia Ceratocystisimbriata Scopoletin El Modafar t l.,f.sp. platani Umbelliferone 1993Biaryls Maluspumila C. purpureum Aucuparin Kemp,HollowayBurden, 985Dibenzofurans Pyrus ommunis C. purpureum x,/3-,y-pyrufuransKemp,BurdenLoeffler,983;Kemp& Burden,1984M. alba F. solani . sp. mori MoracinM Takahashi Shirata,1982Flavonoids Pinus aeda H. annosum Pinocembrin Shain, 967Broussonetia Not determined Broussonin Takasugi tal.,papyrifera Broussin 1980Terpenoids Many onifers Various ungi Volatilemonoterpenesresin' See SectionV 2(f)Resin cids JUlmuspp. Ophiostomalmi Mansonone , F Overeem(-)-7- Elgersma,970hydroxycalameneneurden Kemp,Tiliaeuropaea Ganoderma (-)-7- 1984applanatum hydroxycalameneneurden Kemp,1983Alkaloids L. tulipifera Not determined glaucine Chen t l., 1976

wall 4 barriers (Pearce & Woodward, 1986; Pearce,1991).Some of the compounds accumulating de novo atlesion margins have antimicrobial activity, and maythus be considered phytoalexins (Kemp & Burden,1986, and see Smith (1996) for a full account oforigin, definitionand biochemistryof phytoalexins).These compounds belong to various chemical classes(Table 2). Although there are reports of the isolationand characterization of such compounds (cf. Table2), there have been few n-depth studies. Indeed, theevidence for their involvement in resistance is oftenonly circumstantial (see Kemp & Burden, 1986).Most phytoalexin-like compounds induced inliving wood have not been quantified in the tissueswhere they accumulate. Crude determinations oftotal phenolic compounds in CBL reaction zones

might e 5-10-foldhigher han n healthy apwood:induced compounds can account for 1 5%0or moreof the total material n these reactionzones, on afreshweightbasis (Shain, 1967; Popoff t al., 1975;Pearce 1987; Yamada et al., 1988). Antifungalactivity n reaction zone extracts,determinedbyTLC plate bioassay (Homans & Fuchs, 1970;Woodward & Pearce, 1985), is, however, oftenrelativelyow and can be related oonly a fewof thecompoundsaccumulating t lesion margins.HPLCseparations of extracts from green CBL reactionzones at the marginsof naturally ccurring ungallesions n the xylem fAcerpseudoplatanus evealedthe presenceof at least seven induced compounds(Pearce et al., 1994a). However,bioassay with theindicator rganismCladosporiumucumerinum,ol-lowing TLC, demonstrated elativelyweak inhi-


19/32

220 R. B. Pearce(a) Leaves HO OHHO

HO O OHHO

Chalcomoracin OH(b) CortexOMe HO

OH OHMeo H MeO 0OH OMe

Moracin Moracin(c) Xylem OHOH OH OHHO / HO

Documents

Antimicrobial Defences in the Wood of Living Trees